Energy and Protein Metabolism and Nutrition: EAAP Publication No. 124, 2007 Vichy, France 9-13 September, 2007

Energy and protein
metabolism and
nutrition
EAAP publication No. 124, 2007

Vichy, France
9-13 September, 2007
ningen Academic
b l i s h e r s
Energy and protein metabolism and nutrition
EAAP – European Federation of Animal Science
ISEP – 2nd International Symposium on Energy and Protein metabolism and nutrition
INRA – Institut National de la Recherche Agronomique
The European Association for Animal Production wishes to express its appreciation to the Ministero
per le Politiche Agricole e Forestali and the Associazione Italiana Allevatori for their valuable support of
its activities
Energy and protein
metabolism and nutrition
EAAP publication No. 124
Scientific editor:
I. Ortigues-Marty
with the contribution of the National Scientific Committee
Managing editor:
N. Miraux
with the contribution of the Managing Editorial Committee
Language editor:
W. Brand-Williams
Wageningen Academic
P u b l i s h e r s
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publisher, Wageningen Academic Publishers,
P.O. Box 220, 6700 AE Wageningen, the
Netherlands,
www.WageningenAcademic.com
The individual contributions in this

publication and any liabilities arising from
them remain the responsibility of the
authors.
ISBN: 978-90-8686-041-8 The designations employed and the

e-ISBN: 978-90-8686-613-7 presentation of material in this publication
DOI: 10.3920/978-90-8686-613-7 do not imply the expression of any opinion
whatsoever on the part of the European
Association for Animal Production
ISSN 0071-2477 concerning the legal status of any country,
territory, city or area or of its authorities,
or concerning the delimitation of its
First published, 2007 frontiers or boundaries.
The publisher is not responsible for

© Wageningen Academic Publishers possible damages, which could be a result of
The Netherlands, 2007 content derived from this publication.
The 2nd EAAP International Symposium on Energy and Protein Metabolism
and Nutrition 2007 was organised by:
Institut National de la Recherche Agronomique
Department of Animal Physiology and Livestock Systems and Department of Food and Human Nutrition
Centre de Recherches de Clermont-Ferrand-Theix-Lyon
F-63122 Saint Genès Champanelle, France
The scientific programme was organised by the National Scientific Committee:

• Dr. Isabelle Ortigues-Marty (INRA Clermont-Ferrand-Theix, President of the National
Scientific Committee)
• Dr. Yves Chilliard (INRA Clermont-Ferrand-Theix)
• Dr. Patrick Herpin (INRA Rennes)
• Dr. Jean-François Hocquette (INRA Clermont-Ferrand-Theix)
• Dr. Jean-Paul Lallès (INRA Rennes)
• Dr. Béatrice Morio (INRA Clermont-Ferrand-Theix)
• Dr. Didier Rémond (INRA Clermont-Ferrand-Theix)
• Dr. Sophie Tesseraud (INRA Tours)
• Dr. Jaap van Milgen (INRA Rennes)
• Dr. Chantal Wrutniak-Cabello (INRA Montpellier)
In interaction with the International Scientific Committee:

• Dr. Matteo Crovetto (President of Nutrition Commission of EAAP, Italy)
• Dr. Mickael Kreuzer (Secretary, Switzerland)
• Dr. Isabelle Ortigues-Marty (Organiser of the 2007 Symposium on Energy and Protein
Metabolism and Nutrition, France)
• Dr. Wolfgang Souffrant (Organiser of the 2003 Symposium on Energy and Protein Metabolism
and Nutrition, Germany)
• Dr. Andre Chwalibog (Organiser of the 2000 Symposium on Energy Metabolism in Animals,
Denmark)
• Dr. John MacRae (Organiser of the 1999 Symposium on Protein Metabolism and Nutrition,
United Kingdom)
• Dr. Hélène Lapierre (Canada representative, Canada)
• Dr. James Oltjen (USA representative, USA)
• Dr. Kunio Sugahara (Australasia representative, Japan)
All communications were reviewed by a Reviewing Board:

Agabriel J., Aguilera J., Bequette B., Boudon A., Caton J., Chavatte-Palmer P., Chwalibog A.,
Combaret L., Dardevet D., de Lange C.F.M., Glasser F., Gondret F., Guinard-Flament J., Gruffat
D., Herpin P., Hocquette J.-F., Kreuzer M., Lallès J.-P., Lapierre H., Le Floc’h N., Lescoat P., Marini
J., McNabb W., McNamara J., MacRae J., Médale F., Morio B., Niewold T., Noblet J., Nozière
P., Oltjen J., Ortigues-Marty I., Orzechowski A., Rémond D., Reynolds C., Sainz R.D., Savary-
Auzeloux I., Souffrant W., Sugahara K., Tesseraud S., van Milgen J., van Straalen M.W., Verstegen
M., Wenk C., Wrutniak-Cabello C. and additional scientists.
6 Energy and protein metabolism and nutrition

The Managing Editorial Committee was in charge of these proceedings:
Brand-Williams W., Dandurand C. and Poirel D. under the responsibility of Miraux N.
Locally, the Symposium was organised by the National Organising Committee:

I. Ortigues-Marty (President), Brun J.-P. (General Secretary), Espinasse C., Rocher E. & Monségu
M.-J. (ISEP secretaries), Farce M.-H. (ISEP website), Buffière C. & Rudel S. (links with the
Congress Hall of Vichy), Brand-Williams W. (translations), Bernard O., Cassar-Malek I. & Gruffat
D. (organisation of visits), Vernet J. (transport), Vincent C. (accountant), J. Espinasse (logo) with
the contribution of additional INRA staff members.
The Coordinators in charge of the plenary sessions and workshops were:

B. Morio and C. Wrutniak-Cabello (Plenary session 1), S. Tesseraud (Plenary session 2); F.
Gondret (Workshop 2A), J. Guinard-Flament (Workshop 2B), L. Combaret (Workshop 2C), P.
Chavatte-Palmer (Workshop 2D), F. Médale (Workshop 2E), I. Savary-Auzeloux and D. Gruffat
(Workshop 2F), J.-F. Hocquette (Plenary session 3), D. Rémond (Plenary session 4), D. Dardevet
(Workshop 4A), J.-P. Lallès (Workshop 4B), P. Nozière (Workshop 4C), J. van Milgen (Plenary
session 5), J. Noblet (Workshop 5A), N. Le Floc’h (Workshop 5B), J. Agabriel (Workshop 5C), P.
Lescoat and F. Glasser (Workshop 5D), A. Boudon (Workshop 5E).
With the contribution of the General Discussion Leaders:

M. Toyomizu (Plenary session 1), N. Scollan (Plenary session 2), J.P. McNamara (Workshop 2A),
W.C. McNabb (Workshop 2B), A. Orzechowski (Workshop 2C), J. Caton (Workshop 2D), J.F.
Aguilera (Workshop 2E), B.J. Bequette (Workshop 2F), A.-M. Mullen (Plenary session 3), C.J.
Seal (Plenary session 4); C.K. Reynolds (Workshop 4A); T.A. Niewold (Workshop 4B); J.C.
Marini (Workshop 4C), D. Sauvant (Plenary session 5); M. Verstegen (Workshop 5A); K. de
Lange (Workshop 5B); C. Wenk (Workshop 5C); R.D. Sainz (Workshop 5D); W.M. van Straalen
(Workshop 5E).
Energy and protein metabolism and nutrition 7

Table of contents
Preface 31
Part 1. Nutrition and mitochondrial functions
On-going and prospective research on nutrition and mitochondrial functions 37

M.P. Mollica, L. Lionetti, A. Lombardi, E. Silvestri, F. Goglia and A. Barletta
Insulin modulates the decrease of oxidative phosphorylation induced by ingested butyrate.

NMR study on the isolated and perfused rat liver 51
M.-C. Beauvieux, H. Roumes, V. Rigalleau, H. Gin and J.-L. Gallis
Effects of a diet enriched in trans fatty acids (trans MUFA) on muscle mitochondrial
functions and development of insulin resistance in rodents 53
A.-L. Tardy, P. Rousset, C. Giraudet, J.M. Chardigny and B. Morio
Up-regulation or activation of avian UCP attenuates mitochondrial ROS production and

oxidative damage in broiler chickens exposed to acute heat stress 55
A. Mujahid, Y. Akiba, M. Kikusato and M. Toyomizu
Relationships between hepatic mitochondrial function and residual feed intake in growing
beef calves 57
P.A. Lancaster, G.E. Carstens, J. Michal, K.M. Brennan, K.A. Johnson, L.J. Slay, L.O.
Tedeschi and M.E. Davis
Interaction between adipose tissue and skeletal muscle in the control of muscle
mitochondrial functions 59
E. Chanséaume, P. Rousset, C. Gryson, S. Walrand, Y. Boirie and B. Morio
Influence of protein-free diet on morphology of rat hepatocytes’ mitochondrium 61

E. Sawosz, A. Chwalibog, M. Grodzik, T. Niemiec, J. Skomiał and I. Kosieradzka
Expression of uncoupling proteins and mitochondrial activity are dependent on muscular

fibre type in rabbits and chickens 63
R. Joubert, A. Collin, C. Berri, L. Lefaucheur, A. Vincent, M. Fillaut, P. Ecolan, L. Mur, E.
Godet, S. Crochet, T. Bordeau, S. Skiba-Cassy, S. Tesseraud, P. Herpin and M. Damon
Regulation of mitochondrial and tissue oxidations by thyroid hormones in chicken muscle 65

A. Collin, Q. Swennen, S. Métayer Coustard, S. Skiba-Cassy, R. Joubert, G. Briclot, S.
Crochet, E. Decuypere, J. Buyse and S. Tesseraud
Variation in animal energy expenditure and mitochondrial function and protein expression 67
J. Michal, K. Brennan, K. Ross and K.A. Johnson
Cardiac muscle UCP-3 expression in relation to nutrient intake and beef cattle metabolism 69
G.K. Murdoch, W.T. Dixon, J. Moibi, B.T. Li, M. Vinskyand R.J. Christopherson
Inter-muscular variability of metabolic properties of bovine muscle fibres 71

M.P. Oury, C. Jurie, J.F. Hocquette and B. Picard

Part 2. Regulation of body composition and/or product quality by tissue metabolism
2. Body composition and/or product quality
Regulation of marbling and body composition - Growth and development, gene markers
and nutritional biochemistry 75
D.W. Pethick, W. Barendse, J.F. Hocquette, J.M. Thompson and Y.H. Wang
Protein and selected mineral deposition in the body of pigs during compensatory growth 89
D. Weremko, G. Skiba, S. Raj and H. Fandrejewski
Meal feeding regulates S6K1 phosphorylation but not that of 4E-BP1 in rainbow trout
(Oncorhynchus mykiss) muscle 91
I. Seiliez, J.C. Gabillard, S. Panserat, S. Cassy, J. Gutiérrez, S. Kaushikand S. Tesseraud
Dietary methionine supply affects the amino acid composition of body proteins 93
J. van Milgen, L. Brossard, N. Le Floc’h and M. Rademacher
Does a time interval for methionine supplementation favour weight gain and body
composition? A model study with growing rats 95
M. Gas, J. Bujko, E. Chudobinska, M.W.A. Verstegen, R.E. Koopmanschap and V.V.A.M.
Schreurs
Effects of dietary protein level and feed intake on amino-acid gain and composition in
growing and fattening Iberian pigs 97
R. Nieto, R. Barea, I. Fernández-Fígares and J.F. Aguilera
Study, in model system, of the effect of oxidation on myofibrillar protein digestibility 99

L. Aubry, Ph. Gatellier, A. Vivion and V. Santé-Lhoutellier
Effects of maternal nutrition on birth weight and postnatal nutrient metabolism 101
J. Caton, K. Vonnahme, J. Reed, T. Neville, C. Effertz, C. Hammer, J. Luther, J. Taylor, D.
Redmer and L. Reynolds
Glucose metabolism and its relation to milk production in F2 offspring of Charolais ×

Holstein crosses 103
H.M. Hammon, P. Junghans, O. Bellmann, F. Schneider, R. Weikard and C. Kühn
Expression of beta-adrenergic receptors during lactation in Holstein dairy cattle 105

J.M. Sumner and J.P. McNamara
Investigations on the storage of ω-3 and ω-6 fatty acids in the dry period and their transfer
into milk fat in early lactation 107
H.-R. Wettstein, R.E. Hochstrasser, C. Elia, H. Leuenberger, M. Wanner and M. Kreuzer
Live weight, body size, fatness and carcass characteristics of young bulls
of fifteen European breeds 109
P. Albertí, B. Panea, C. Sañudo, J.L. Olleta, G. Ripoll, P. Ertbjerg, M. Christensen, S.
Gigli, S. Failla, A. Gaddini, J.F. Hocquette, R. Jailler, S. Rudel, G. Renand, G.R. Nute,
R.I. Richardson and J.L. Williams

Metabolic and contractile characteristics of Longissimus thoracis muscle of young bulls
from 15 European breeds in relationship with body composition 111
J.F. Hocquette, C. Jurie, B. Picard, P. Albertí, B. Panea, M. Christensen, S. Failla, S.
Gigli, H. Levéziel, J.L. Olleta, C. Sañudo, P. Ertbjerg, G.R. Nute and J.L. Williams
Deposition of protein, fat and energy in lambs of the breed German Merino Landsheep 113
G. Bellof and J. Pallauf
Impact of vitamin D3 and vitamin E by feeding on meat quality parameters of beef and pork 115
R. Lahucky, I. Bahelka, U. Küchenmeister, K. Vasickova, G. Nürnberg and K. Ender
Influence of various Iodine supply on growing-finishing performance and on Iodine status

of pigs 117
A. Berk, K. Franke, F. Schöne and G. Flachowsky
2A. Regulation of lipid deposition and intramuscular lipids
Intramyocellular lipid accumulation and insulin resistance development 121

C. Aguer, J. Mercier, C. Yong Wai Man, S. Bordenave, L. Metz and M. Kitzmann
Regional specifities in transcriptomic profiles of adipocytes isolated from skeletal muscle

or adipose tissues in growing pigs 123
I. Louveau, M. Damon and F. Gondret
Effect of a reduced protein diet on expression of lipogenic enzymes in relation to

intramuscular fat formation in the pig 125
O. Doran, F.M. Whittington, K.G. Hallett and J.D. Wood
Changes in liver metabolic pathways induced by dietary energy and selection for muscle
fat content in rainbow trout 127
C. Kolditz, M. Borthaire, F. Lefèvre, E. Quilletand F. Médale
Reduced intake of dietary amino acid promotes accumulation of intramuscular fat in the
Longissimus dorsi muscle of finishing pigs 129
M. Katsumata, M. Matsumoto, S. Ieiri and Y. Kaji
Triacylglycerol synthesis and secretion in Atlantic salmon hepatocytes: effect of dietary lipids 131
M.A. Kjær, A. Vegusdal, M. Todorčević, T. Gjøen, A.C. Rustan, B. Torstensenand B. Ruyter
Effect of fish and plant oils on lipid composition and fatty acid β‑oxidation in adipose
tissue of Atlantic salmon 133
M. Todorčević, A.Vegusdal, N. Djaković, M.A.Kjær, B. Torstensen and B. Ruyter
Performance, blood lipids and fatty acid composition of tissues of rabbits fed diets
supplemented with conjugated linoleic acid 135
M. Marounek, E. Skřivanováand A. Dokoupilová
Synthesis and desaturation of cis9, trans11 CLA in adipose tissue of Charolais steers and
cull cows 137
D. Gruffat, C. Rémond, D. Durand, O. Loreau and D. Bauchart

Inclusion of rapeseed oil in the diet to improve the performance of silage-fed fattening cattle 139
J. Jatkauskas and V. Vrotniakiene
2B. Regulation of milk production and composition
The regulation of amino acid utilisation by insulin in the lactating ewe mammary gland 143
B. Sinclair, P. Back, P. Harris, S. Davis, D. Mackenzie, M. Tavendale, W.C. McNabb, N.
Royand J. Lee
Does a dietary arginine deficiency limit milk protein yield? 145

T. Whyte, A. Hayirli, H. Lapierre and L. Doepel
Feeding measures to improve nitrogen efficiency in dairy cattle 147

S.M. van Zijderveld and W.M. van Straalen
Constraints in estimating N-excretion from the milk urea content in dairy cows 149
S. De Campeneere, D.L. De Brabander and J. Vanacker
Estimation of the intra-mammary metabolic fate of glucose and acetate in response to

longer milking intervals in dairy cows 151
J. Guinard-Flament, S. Lemosquet and E. Delamaire
Dietary and mammary components of milk fat depression: insights from a meta-analysis
of literature data 153
F. Glasser and Y. Chilliard
Effect of rumen-protected conjugated linoleic acid in combination with propylene glycol

or rumen protected fat on performance and metabolic parameters of early lactation dairy cows155
F.J. Schwarz, T. Liermann and A.M. Pfeiffer
Effect of dietary conjugated linoleic acid (CLA) on milk composition of dairy cows 157
U. Meyer, C. Brömmel, G. Flachowsky and G. Jahreis
Effect of flaxseed and rapeseed supplementation during the last trimester of pregnancy
and lactation in sows on fatty acid composition in their milk 159
B. Bałasińska, R. Zabielski, P. Ostaszewski and G. Kulasek
Variations in mammary extraction of nutrients under the effect of a 36-h milk

accumulation into the udder in dairy cows 161
J. Guinard-Flament, D. Causeur and E. Delamaire
Response to diet quality of two cattle genotypes at 200 and 3600 m altitude in milk yield
and composition and nitrogen and energy utilization 163
K. Bartl, C.A. Gomez, M. Kreuzer, H.D. Hess and H.-R. Wettstein
Milk production as a function of energy and protein source supplementation follows the
saturation kinetics typical of enzyme systems 165
R.P. Lana, D.C. Abreu, P.F.C. Castro and B. Zamperline

2C. Regulation of muscle protein deposition and loss
Development of low grade inflammation during aging impaired postprandial muscle

protein synthesis in rat skeletal muscle 169
I. Rieu, G. Mayot, C. Sornet, E. Pujos, M. Balage, I. Papet and D. Dardevet
Effect of immune system stimulation and dietary methionine plus cysteine intake on
protein deposition and digestibility in growing pigs 171
A. Rakhshandeh, M. Rademacher and C.F.M. de Lange
The ubiquitin and caspase systems are sequentially regulated in the rat gastrocnemius
muscle during casting immobilisation and recovery 173
E. Vazeille, A. Claustre, A. Codran, S. Ventadour, D. Taillandier, D. Béchet, D. Attaix, D.
Dardevet and L. Combaret
Relation between protein degradation and oxidative stress during aging in rat muscle 175
L. Mosoni, L. Combaret, C. Sornet and D. Dardevet
Changes in the expression of selected proteins elucidate skeletal muscle type-specific

resistance to glucocorticoid-induced muscle cachexia 177
P. Pawlikowska, M. Lokociejewska, J.F. Hocquette and A. Orzechowski
Amino acid signalling: methionine regulates the S6K1 pathway and protein synthesis in
avian QM7 myoblasts 179
S. Métayer Coustard, S. Crochet, E. Audouin, M. Derouet, Y. Mercier, P.A. Geraertand S.
Tesseraud
AMPK regulates the S6K1 pathway and protein synthesis in avian QM7 myoblasts 181
S. Métayer Coustard, A. Collin, E. Audouin, S. Crochet, J. Dupontand S. Tesseraud
Decreased nutritional responsiveness of S6K1 in the breast muscle of genetically fat chickens 183
S. Duchene, M. Abbas, I. Seiliez, E. Audouin, S. Crochet, S. Métayer Coustard and S.
Tesseraud
Leucine suppresses myofibrillar proteolysis by down-regulating ubiquitin-proteasome

pathway in chick skeletal muscles 185
K. Nakashima, A. Ishida, M. Yamazaki and H. Abe
Skeletal muscle response to an endotoxin injection followed by malnutrition is similar in

low-grade inflamed and non-inflamed old rats 187
G. Mayot, K. Vidal, L. Combaret, D. Breuillé, S. Blum, C. Obled and I. Papet
Estrogenic and isoflavonic actions on differentiation and protein metabolism in porcine

muscle satellite cell cultures 189
M. Mauand C. Rehfeldt
Protein deposition in the body, content of nucleic acids in the mld muscle of pigs as
affected by limitation of protein during growing period 191
G. Skiba, S. Raj, D. Weremko and H. Fandrejewski

2D. Perinatal metabolic programming
Temporary consumption of a soy-based diet induces persisting alterations in protein

metabolism and oxidative stress responsiveness in juvenile pigs 195
P. Junghans, M. Beyer, M. Derno, K.J. Petzke, U. Küchenmeister, U. Hennig, W. Jentsch
and M. Schwerin
Influence of metabolic imprinting during early growth period on muscle development and
intramuscular adipogenesis in Japanese Black steers 197
T. Gotoh, T. Fumita, T. Etoh, K. Hayashi, Y. Nakamura, J. Wegner and H. Iwamoto
Growth and differentiation of the chicken Pectoralis major muscle: effect of genotype and
early nutrition 199
C. Berri, E. Godet, T. Bordeau, N. Haj Hattab, S. Tesseraud and M.J. Duclos
Effect of in utero metabolic programming on postnatal growth of mink kits 201

C.F. Matthiesen, D. Blache and A.-H. Tauson
Glucose tolerance in pregnant sows and liver glycogen in neonatal piglets is influenced by
diet composition in gestation 203
P. Bikker, J. Fledderus, J. Kluess and M.J.H. Geelen
Heat production in chicken embryos 205

A. Chwalibog, A.-H. Tauson and G. Thorbek
Nutritional programming due to maternal high protein diet reduces offspring birth weight
and body weight and is genotype dependent in mice 207
M. Kucia, M. Langhammer, N. Dietrich, M. Derno, U. Renne, G. Nürnberg, U. Hennig
and C.C. Metges
2E. Novel techniques for novel results in energy expenditure and body composition
Comparing techniques to estimate energy expenditure on physical activity in individually

housed dairy cows 211
M.J.W. Heetkamp, W.J.J. Gerrits, A.T.M. van Knegsel and H. van den Brand
Quantitative partitioning of energy expenditure in pregnant ewes 213

A. Kiani, M.O. Nielsen and A. Chwalibog
A calorimetry system for metabolism trials 215

N.M. Rodríguez, W.E. Campos and M. Lachica
Inter-laboratory test for gas concentration measurements of the R3C network calorimetric
chambers 217
C. Montaurier, P. Even, C. Couet and S. Dubois
Determining cattle production efficiency without measuring the intake 219

A. Brosh, A. Shabtay, J. Miron, G. Adin and Y. Aharoni

Use of the heart rate method and GPS for direct field estimation of the energy cost of cow
grazing activity: effects of season and plot size 221
A. Brosh, Z. Henkin, E.D. Ungar, A. Dolev, A. Orlov, A. Shabtay and Y. Aharoni
Heart rate measurements as an index of energy expenditure and energy balance in

ruminants: a review 223
A. Brosh
Adaptation of night median heart rate in calorimetric chambers versus in free living
conditions: a critical evaluation 225
C. Montaurier, M. Vermorel, P. Ritz, A. Chamoux, Y. Boirieand B. Morio
Effect of cold exposure on natural abundance of 13C and heat production in Spanish goats
by the CO2 entry rate technique 227
M. Lachica, A.L. Goetsch and T. Sahlu
Characterisation of lipid distribution in rainbow trout by magnetic resonance, colour

vision and histology imaging techniques 229
A. Davenel, J. Bugeon, G. Collewet, E. Quilletand F. Médale
2F. Novel techniques for novel results in organ balance, in vitro tissue metabolism and
nutrient ‘tracing’ studies
Metabolic fate of carbohydrates from milk replacer in heavy milk-fed calves 233
J.J.G.C. van den Borne, G.E. Lobley, J.W. Blum and W.J.J. Gerrits
Measurement of fatty acid oxidation in swine using 13C labeled fatty acids 235
E. van Heugten, J.J.G.C. van den Borne, M.W.A. Verstegen, J. van Milgen and W.J.J.
Gerrits
Mathematical analysis of human [13CO2]-breath test results: post prandial fate of amino
acids is related to their dietary form 237
V.V.A.M. Schreurs, J.A. Nolles, K. Krawielitzki and J. Bujko
Effect of graded dietary tryptophan levels on [1-13C]leucine oxidation and nitrogen

deposition in growing pigs 239
U. Hennig, P. Junghans, J. Bartelt, C. Relandeau, A. Tuchschererand C.C. Metges
Endogenous nitrogen flows in the digestive tract of lactating dairy cows: comparison
between estimations using total 15N versus 15N-amino acid isotope dilution 241
D.R. Ouellet, R. Berthiaume, R. Martineau and H. Lapierre
A multi-isotope approach to studying arginine metabolism in neonatal piglets 243

K.L. Urschel, M. Rafii, P.B. Pencharz and R.O. Ball
Mutations in metabolic pathways, what role does genetic background play? 245
J. Marini, A. Erez and B. Lee
Gut function of pigs in relation to weaning 247

P.J.A. Wijtten, H. Bouritius, P.R.T. Bonekamp, G.J. Witte, J.J. Verstijnen and T.A.T.G.
van Kempen

Combination of two approaches to study the mammary use of glucose for lactose
synthesis in dairy cows: net mammary balance and RNA levels 249
M. Boutinaud, M.H. Ben Chedly, E. Delamaire and J. Guinard-Flament
Molecular markers for a delicate balance between protein and lipid metabolism in
subcutaneous fat tissue of beef cow 251
A. Shabtay, H. Eitam, A. Orlov, Y. Aharoni, I. Izhaki and A. Brosh
Part 3. ‘Omics’ in metabolism and nutrition studies
Lexic of the ‘Omics’ 255

J.F. Hocquette
Nutrigenomics: techniques and applications 259

A. Scalbert, D. Milenkovic, R. Llorach, C. Manach and C. Leroux
Hepatic gene expression of gluconeogenic and glycolytic enzymes as indicators of the

activity of carbohydrate metabolism in mink 277
C.F. Matthiesen, P.D. Thomsen and A.-H. Tauson
Dietary polyunsaturated fatty acids reduce expression of colonic inflammatory genes in

interleukin-10 knockout mice 279
B. Knoch, M.P.G. Barnett, W.C. McNabb, Y.E.M. Dommels, Z.T. Zhu, S.O. Knowles and
N.C. Roy
Skatole increases expression of detoxification genes in the ovine liver 281

M.H. Deighton, W.C. McNabb, D. Pacheco, M.H. Tavendale, Z.A. Park and N.C. Roy
Gene expression in adipose tissue of dairy cattle changes in early lactation or with
supplemental chromium, integrating expression into metabolic models 283
J.P. McNamara, J.M. Sumner, J. Vierck and A. Jourdan
Transcriptomic profile in mammary gland is modified by nutrition in lactating goats 285

C. Leroux, S. Ollier, S. Bes, M. Goutte, L. Bernard and Y. Chilliard
Effect of fasting on the thalamus/hypothalamus in cows - a proteomics analysis 287

B. Kuhla, S. Kuhla, P.E. Rudolph, D. Albrecht and C.C. Metges
Comparative transcriptomic and proteomic analyses of pig longissimus muscles differing

in intramuscular fat content 289
J. Liu, M. Damon, P. Ecolan, I. Guislde, N. Guitton, P. Cherel and F. Gondret
Bovine PRKAG3 gene expression and association with glycogen content 291
M. Roux, E. Ciani, D. Petit, A. Ouali, H. Levéziel and V. Amarger
Intestinal and ruminal epithelial and hepatic metabolism regulatory gene expression as
affected by forage to concentrate ratio in bulls 293
R.L. Baldwin VI, S.W. El-Kadi, K.R. McLeod, E.E. Connor and B.J. Bequette
Genomic tools to analyse bovine muscle and adipose tissues transcriptomes 295
A.K. Kadanga, C. Leroux, M. Bonnet, I. Cassar-Malek and J.F. Hocquette

Effect of inoculation with intestinal bacteria on intestinal gene expression in the
interleukin-10 knockout mouse 297
M.P.G. Barnett, S.-T. Zhu,A.L. Cookson, R. Broadhurst, B. Knoch, M. Davy, W.C. McNabb
and N.C Roy
Modification of gene expression involved in muscle growth and energy metabolism

according to growth capacity in young Charolais bulls 299
C. Bernard, I. Cassar-Malek, G. Renand and J.F. Hocquette
The myogenic influence of triiodothyronine nuclear and mitochondrial pathways explored

by transcriptome analysis 301
O. Baris, P. Seyer, M. Busson, S. Grandemange, C. Gouarné, L. Pessemesse, F. Casas, C.
Wrutniak-Cabello and G. Cabello
Functional genomics of rat skeletal muscle during aging 303

A. Listrat, I. Piec, J. Alliot, R.G. Taylor and D. Béchet
Intestinal mucosa proteome analysis in goat kids fed milk or soy protein based diets 305
S. Kuhla, P.E. Rudolph, D. Albrecht, U. Schönhusen, R. Zitnan, W. Tomek, K. Huber, J.
Voigt and C.C. Metges
Influence of catechin on urinary metabolic profiles and antioxidant status in hyperlipidic

diets-fed rats by using a LC-Qtof-based metabonomic approach 307
A. Fardet, R. Llorach, J.-F. Martin, C. Besson, E. Pujos, C. Obled and A. Scalbert
Impact of the ingestion of polyphenols and fruits rich in polyphenols on atherosclerosis

studied on apoE deficient mice by a transcriptomic approach 309
D. Milenkovic, S. Auclair, C. Besson, A. Mazur and A. Scalbert
AGENAE – GENANIMAL: the French research program in animal genomics 311

C. Chevalet, J.F. Hocquette, P. Sellier and P. Monget
FUGATO - Functional genome analysis in animal organisms 313

K. Sanders and G. Ostermann
Nutrigenomics New Zealand – tailoring New Zealand foods to match people’s genes; a
case study 315
N.C. Roy, M.P.G. Barnett and W.C. McNabb
Part 4. Coordination between tissues for the metabolic utilisation of nutrients
4. Splanchnic metabolism and interorgan exchanges of nutrients
Interorgan exchange of amino acids: what is the driving force? 319

Y.C. Luiking and N.E.P. Deutz
Intake of fermentable fiber stimulates mucin and mucosal protein synthesis in the pig colon 329
A.J. Libao-Mercado, C.L. Zhuand C.F.M. de Lange

Impact of a low dietary threonine supply on protein synthesis, amino acid deposition and
composition of the intestine and the carcass of piglets 331
A. Hamard, B. Sève, D. Melchior and N. Le Floc’h
Intestinal valine irreversible loss rate during an established Trichostrongylus colubriformis

infection in lambs 333
E.N. Bermingham, W.C. McNabb, B.R. Sinclair, M. Tavendale, B.P. Treloar and N.C. Roy
Contribution of the digestive tract and the liver to the whole body metabolism of
phenylalanine and leucine in growing lambs 335
I. Savary-Auzeloux, G. Kraft and I. Ortigues-Marty
Net flux of amino acids across splanchnic tissues of ewes during abomasal protein and
glucose infusion 337
H. Freetly, C. Ferrell and S. Archibeque
Effect of abomasal glucose infusion on splanchnic glucose metabolism in freshening dairy

cows 339
M. Larsen and N.B. Kristensen
Portal recovery of glucose infused into the abomasum of lactating dairy cows 341
Amino acid and energy metabolism by the portal-drained viscera of beef steers:
quantitative relationships with metabolizable energy intake 343
K.R. McLeod, S.W. El-Kadi, D.L. Harmon and E.S. Vanzant
Effect of polyethylene glycol on the net flux of amino acids in the mesenteric- and portal-
drained viscera in lactating ewes fed Sulla, a condensed tannin forage 345
D.L. Deighton, W.C. McNabb, B.R. Sinclair, B.P. Treloar and N.C. Roy
Splanchnic net release and body retention of nitrogen in growing lambs fed diets
unbalanced for energy and protein 347
G. Kraft, I. Ortigues-Marty and I. Savary-Auzeloux
Partitioning of nitrogen net fluxes across the portal-drained viscera in sheep: effect of
dietary protein rumen degradability 349
D. Rémond and C. Poncet
Can liver protein synthesis be affected by an imbalanced dietary supply of energy or

nitrogen in growing lambs? 351
A stimulation of protein metabolism in the whole intestine is the main cause of the
decreased nutritional value of a pectin supplemented diet 353
T. Pirman, M.C. Ribeyre, D. Rémond, L. Mosoni, C. Buffière, A. Lavrenčič, A. Pogačnik,
M. Vrecl, J. Salobir and P. Patureau Mirand
Ruminal fermentation, portal absorption and hepatic metabolism of glycerol infused into
the rumen of lactating dairy cows 355
N.B. Kristensen and B.M.L. Raun

Identification of a vacuolar H+-ATPase as a new energy consuming mechanism in rumen
epithelium of sheep and cattle 357
M. Schweigel, K.S. Heipertz, M. Kolisek, W. Jähme, E. Albrecht and R. Zitnan
4A. Regulations by nutrients, hormones and nervous system
Reduced insulin responses by asynchronous protein and lactose intake in veal calves
despite high plasma glucose levels 361
T. Vicari, J.J.G.C. van den Borne, W.J.J. Gerrits, Y. Zbinden and J.W. Blum
Modulation of adipose tissue metabolism in periparturient dairy cattle through pre partum
administration of thiazolidinediones 363
T.R. Overton and K.L. Smith
Glucagon-like peptide 2 inhibits intestinal lysosomal proteolysis and improves small

intestinal recovery in refed starved rats 365
A. Codran, S.E. Samuels, S. Ventadour, A. Claustre, M.-P. Roux, D. Taillandier, D. Béchet,
D. Attaix and L. Combaret
Postprandial blood hormone and metabolite responses influenced by feeding frequency

and feeding level in heavy veal calves 367
T. Vicari, J.J.G.C. van den Borne, W.J.J. Gerrits, Y. Zbinden and J.W. Blum
The role of feeding regimens in regulating metabolism of broiler breeders: hepatic lipid
metabolism, plasma hormones and metabolites 369
M. de Beer, R.W. Rosebrough, B.A. Russell, S.M. Poch, M.P. Richards, J.P. McMurtry,
D.M. Brocht and C.N. Coon
The relation between circulating ghrelin and vitamin A during the fattening period in
Japanese Black steers 371
M. Hayashi, K. Kido and K. Hodate
Effects of rumen protected choline on selected metabolites and liver constituents in dairy
goats within the first month of lactation 373
L. Pinotti, A. Campagnoli, F. D’Ambrosio, C. Pecorini, R. Rebucci, D. Magistrelli, V.
Dell’Orto and A. Baldi
Rare earth elements (REE) in piglets feeding – performance and thyroid hormone status 375
D. Förster, A. Berk, H.-O. Hoppen and G. Flachowsky
Role of the 5’AMP-activated protein kinase in reproduction: a possible involvement in the

central regulation 377
L. Tosca, G. Ferreira, E. Jeanpierre, S. Coyral-Castel, A. Caraty, D. Lomet, C. Chabrolle
and J. Dupont
Chicken liver glucokinase is activated by a glucokinase activator 379

N. Rideau, A. Picard and J. Grimsby
Effects of in ovo amino acids or glucose administration on hexokinase activity in hatching

muscle of broiler embryos 381
Y. Ohta, Y. Furuya, I. Yoshimura, H. Furuta and M. Sugawara

Effect of conjugated linoleic acid on gluconeogenesis, glycogen turnover and IGF-1
synthesis in primary culture of porcine hepatocytes 383
I. Fernández-Fígares, J.A. Conde-Aguilera, M. Lachica, R. Nieto and J.F. Aguilera
Serum profile of metabolites and hormones of growing Iberian gilts fed diets
supplemented with betaine, conjugated linoleic acid or both 385
I. Fernández-Fígares, M. Lachica, R. Nieto and J.F. Aguilera
4B. Health and metabolism
Association of plant extracts rich in polyphenols and vitamin E efficiently prevents

lipoperoxidation in plasma of sheep fed a n-3 PUFA rich diet 389
C. Gladine, E. Rock, C. Morand, D. Gruffat, D. Bauchartand D. Durand
Cardiac oxidative stress in high fat/high sucrose fed rats is mainly due to NADPH oxidase
overexpression. Protective effects of polyphenols 391
T. Sutra, C. Feillet-Coudray, G. Fouret, G. Cabello, J.-P. Cristol and C. Coudray
The effect of sodium ascorbate, trolox and 3-hydroxy-3-methylbutyrate on apoptosis

induced by oxidative stress in C2C12 cells without dystrophin 393
S.J. Berwid and P. Ostaszewski
Effects of inflammation in peripartum dairy cows on milk yield, energy balance and
efficiency 395
E. Trevisi, A. Gubbiotti and G. Bertoni
Dietary L-carnitine enhances the acute phase response in chickens 397

J. Buyse, Q. Swennen, T.A. Niewold, K.C. Klasing, G.P.J. Janssens, M. Baumgartner and
B.M. Goddeeris
Dietary β-hydroxy-β-methylbutyrate supplementation influences performance differently

after immunisation in broiler chickens 399
J. Buyse, Q. Swennen, B.M. Goddeeris, F. Vandemaele, K.C. Klasing, M. Baumgartner
and T.A. Niewold
The comparative study of effects of Immunowall® (prebiotics) and Avilamycin on

amounts of humoral immunity of broiler chickens 401
A. Zakeri, M. Fadaei, S. Charkhkar and S. Zakeri
Non-specific resistance state in rats fed a protein free diet 403

E. Sawosz, A. Chwalibog I. Kosieradzka, M. Grodzik, T. Niemiec and J. Skomiał
Feeding a lower-protein, amino-acid supplemented diet has no effect on growth

performance but reduces post-weaning diarrhoea in pigs 405
J.M. Heo, J.C. Kim, B.P. Mullan, D.J. Hampson, R.H. Wilson, J. Callesen, C.F. Hansen
and J.R. Pluske
Protein balance of lambs infected with Haemonchus contortus and fed tanniniferous
sainfoin (Onobrychis viciifolia) 407
A. Scharenberg, Y. Arrigo, F. Heckendorn, H. Hertzberg, A. Gutzwiller, H.D. Hess, M.
Kreuzer and F. Dohme

Venous blood gas in Holstein steers fed diets with different concentrate to alfalfa hay ratios 409
A.R. Vakili, M. Danesh Mesgaran, A.R. Heravi Mousavi and S. Danesh Mesgaran
Long-term physical activity does not influence the glycemic index in women 411
S. Mettler, P. Vaucher, P.M. Weingartner, C. Wenk and P.C. Colombani
4C. Nitrogen digestion and recycling
Source of nitrogen for pig gut microbes: effect of feeding fermentable fiber 415
A.J. Libao-Mercado, C.L. Zhu, J.P. Cant, H.N. Lapierre, J.N. Thibault, B. Sève, M.F.
Fuller and C.F.M. de Lange
Effects of metabolizable protein supply on nitrogen metabolism and recycling in lactating

dairy cows 417
D. Valkeners, H. Lapierre, J. Marini and D.R. Ouellet
Effect of ruminal degradable nitrogen deficit on nitrogen metabolism in growing double-

muscled Belgian Blue bulls fed beet pulp silage based diet 419
D. Valkeners, Y. Beckers, A. Lindebrings and A. Théwis
Methodological considerations for the determination of standardised ileal digestibilities of

amino acids in newly weaned pigs 421
M. Eklund, H.P. Piepho, M. Rademacher and R. Mosenthin
Effects of feeding duration and ruminal nitrogen and energy release rates on nitrogen
balance and microbial synthesis in sheep 423
T. Ichinohe and T. Fujihara
Timing of herbage and fasting allocation alters nutrient supply in grazing cattle 425
P. Gregorini, S.A. Gunter and P.A. Beck
Effect of water-soluble carbohydrate on rumen nitrogen kinetics of steers given perennial

ryegrass silage measured by 15N-tracer methodology 427
E.J. Kim, N.D. Scollan and J.V. Nolan
Effect of toasting organic field beans on starch and NDF digestibility and rumen microbial
protein synthesis in dairy cows 429
P. Lund, M.R. Weisbjerg, T. Hvelplund, M. Larsen and T. Kristensen
Pattern and rate of urea infusion into the rumen alter N balance and plasma NH4+ in wethers 431
M.I. Recavarren and G.D. Milano
Effects of dietary crude protein and ruminally-degradable protein on urea recycling and
microbial protein production in beef heifers 433
K. Baker, T. Mutsvangwa, J.J. McKinnon, G. Gozho and T.A. McAllister
Effect of potato pulp as a dietary carbohydrate source on nitrogen excretion and urea
metabolism in sheep 435
T. Obitsu, M. Tsunemine, K. Han, T. Sugino and K. Taniguchi

The effect of dietary pectin on concentration of free amino acids and urea in blood plasma
of young pigs 437
E. Święch, M. Taciak, A. Tuśnio and L. Buraczewska
Effect of different energy supply on microbial protein synthesis and renal urea handling in
Corriedale sheep consuming temperate fresh forages 439
I. Tebot, C. Echaides, A. Secchi and A. Cirio
The effect of potato protein and potato fibre on amino acid digestibility, small intestinal
structure and on N-balance and performance of young pigs 441
A. Tuśnio, B. Pastuszewska, E. Święch and L. Buraczewska
Part 5. From the parts to the whole of how to use detailed information to answer applied
questions
5. Protein-energy interactions
Protein-energy interactions: horizontal aspects 445

G.E. Lobley
Vertical integration from ‘omics’ to the whole organism 463

A. Cornish-Bowden and M.L. Cárdenas
Intravenous administration of lysine and threonine to a deficient diet results in low

nitrogen utilization in preruminant calves 473
S.J.J. Alferink, J.J.G.C. van den Borne, A. Habets,A.A. Jacobs and W.J.J. Gerrits
The use of glutamine and glutamate for gluconeogenesis and non-essential amino acid
synthesis in late term chicken embryos 475
N.E. Sunny, J. Adamany, S.L. Owens and B.J. Bequette
Metabolic flexibility of lactating mink (Mustela vison) is not reflected at transcriptional

level but by changes in functional liver mass 477
R. Fink, P.D. Thomsen and A.-H. Tauson
Heat production in broilers is not affected by dietary crude protein 479

J. Noblet, S. Dubois, J. van Milgen, M. Warpechowski and B. Carré
Diet-induced thermogenesis and feed intake regulation in the chicken: effect of diet and
genotype 481
Q. Swennen, A. Collin, G.P.J. Janssens, E. Le Bihan-Duval, A. Bordas, E. Decuypere and
J. Buyse
Protein intake but not feed intake may affect dietary net energy for finishing pigs 483
S. Moehn and R.O. Ball
Influence of dietary protein/energy ratio on growth, body composition, protein, lipid and
energy retentions as well as amino acid metabolism of Nile tilapia 485
J. Gaye-Siessegger, S.M. Mamun and U. Focken

Efficiency of energy and protein deposition in swine during compensatory growth
measured by dual energy X-ray absorptiometry (DXA) 487
A.D. Mitchell and A.M. Scholz
Dynamic integration of biological processes into models: contribution to prediction of

cattle growth and body composition 489
F. Garcia, R.D. Sainz, J. Agabriel and J.W. Oltjen
5A. Whole organism energy metabolism
The efficiency of utilisation of metabolisable energy of diets rich in saturated or

polyunsaturated fats in broiler chickens 493
G. Ferrini, M. Lachica, A.C. Barroeta, J.F. Aguilera and J. Gasa
Energy utilization and growth responses of broilers to supplementation of enzyme cocktails 495
O.A. Olukosi, A.J. Cowieson and O. Adeola
Phytase reverses negative effect of dietary phosphorus reduction on energy metabolism in

growing pigs fed restrictively 497
Y. Zhang, S. Moehn and R.O. Ball
Effects of feed intake in the rate of protein deposition in heavy Iberian pigs 499
R. García-Valverde, R. Barea, R. Nieto and J.F. Aguilera
Feeding frequency alters protein and energy metabolism of sows fed 1 × and 2 × the
energy requirement for maintenance 501
R.S. Samuel, S. Moehn, L.J. Wykes, P.B. Pencharz and R.O. Ball
Influences of feeding intensity on protein and energy deposition in calves 503

H. Janssen, U. Meyer and G. Flachowsky
Energy metabolism and energy requirement for maintenance of Brahman steers in tropical
conditions 505
A. Chaokaur, T. Nishida, I. Phaowphaisal, P. Pholsen, R. Chaithiang and K. Sommart
Effects of dairy cow genotype and plane of nutrition on energy partitioning between milk
and body tissue 507
T. Yan, R.E. Agnew and C.S. Mayne
Comparison of energy consumption and respiratory quotient in chicken embryos with

different growth rate 509
M. Sato, K. Noda, K. Kino, A. Nakamura and M. Furuse
Incubation circumstances affect energy metabolism in avian embryos 511

H. van den Brand, A. Lourens, R. Meijerhof, M.J.W. Heetkamp and B. Kemp
Heat production of two lineages of broilers fed diets of different physical form 513
L.J.C. Lara, W.E. Campos, N.C. Baião, N.M. Rodríguez, M.V. Triginelli and R.S. Leite
Energy metabolism of high productive laying hens in daily light and dark periods 515
A. Chudy

Studies on energy needs and nitrogen metabolism of cats during pregnancy and lactation 517
B. Wichert, L. Schade, B. Bucher, C. Wenk and M. Wanner
Changes in energy metabolism during gestation and lactation in sows 519

R.S. Samuel, S. Moehn, L.J. Wykes, P.B. Pencharz and R.O. Ball
Determination of the energy cost of physical activity in veal calves 521

E. Labussière, J. van Milgen, S. Dubois, G. Bertrand and J. Noblet
The relationships between fasting energy expenditure and intermediary metabolites in

growing lambs 523
A. Kiani, M.O. Nielsen and A. Chwalibog
Energy requirements of gestating Santa Inês ewes 525

W.E. Campos, G.L. Macedo Jr., M.I.C. Ferreira, I. Borges, N.M. Rodríguez and M.L.
Lachica
Adaptational response of energy metabolism to varying environmental conditions in

Hereford oxen 527
M. Derno, W. Jentsch, M. Schweigel, H.-D. Matthes and E. Mohr
5B. Whole organism protein metabolism
Changing dietary lysine level from a deficient to a sufficient level greatly enhances growth
rate of growing rats 531
A. Ishida, K. Nakashima and M. Katsumata
The effect of dietary methionine concentrations on the efficiency of energy utilisation in

broiler chickens 533
N. Priyankarage, S.P. Rose, S.S.P. Silva and V.R. Pirgozliev
Nitrogen metabolites and enzymatic activity during the weaning period in goat kids 535
D. Magistrelli, L. Pinottiand F. Rosi
Influence of dietary tryptophan concentration on performance and dietary selection by

starting pigs 537
T. Ettle, J. Bartelt, C. Relandeau and F.X. Roth
Histidine maintenance requirement and the efficiency of its utilisation for protein
accretion in pigs 539
P. Patráš, S. Nitrayová and J. Heger
Protein oxidation measured by breath test in mink fed bacterial protein meal 541
A.L.F. Hellwing, A.-H. Tauson and A. Skrede
Response of pigs in the weight ranges 35 to 60 kg and 80 to 100 kg to increasing ileal

digestible Threonine: ileal digestible Lysine ratios in the diet 543
M.K. O’Connell, C. Relandeau, M. Overend and P.B. Lynch
Lysine requirement for maintenance in growing pigs 545

J. Ringel and A. Susenbeth

Maintenance protein requirement and efficiency of utilization in poultry 547
N.K. Sakomura, J.B.K. Fernandes, R. Neme, C.B.V. Rabelo and F.A. Longo
Oral and intravenous phenylalanine kinetics in adult mixed hounds 549

A.K. Shoveller, G.M. Davenport, J.P. Cant, S. Robinson and J.L. Atkinson
Effect of feeding carefully dried and ensiled tanniniferous sainfoin (Onobrychis viciifolia)
on protein metabolism of lambs 551
A. Scharenberg, Y. Arrigo, A. Gutzwiller, H.D. Hess, U. Wyss, M. Kreuzer and F. Dohme
Intake of fermentable fibre and body protein deposition in pigs fed methionine or
tryptophan limiting diets 553
C.L. Zhu, M. Rademacher and C.F.M. de Lange
Influence of dietary benzoic acid on nitrogen metabolism in growing/finishing pigs 555

K. Bühler, S. Gebert and C. Wenk
Growth response of pigs to dietary threonine:lysine ratio is affected by the withdrawal of

anti microbial growth promoters 557
P. Bikker, J. Fledderus, L. le Bellego and M. Rovers
5C. Multicriteria evolution of nutritional recommendations
Empirical modelling by meta-analysis of digestive interactions and CH4 production in

ruminants 561
D. Sauvant and S. Giger-Reverdin
Evaluation of the German net energy system and estimation of the energy requirement of
cows on the basis of an extensive data set from feeding trials 563
L. Gruber, A. Susenbeth, F.J. Schwarz, B. Fischer, H. Spiekers, H. Steingass, U. Meyer, A.
Chassot, T. Jilg and A. Obermaier
Development of a simple nutrient based feed evaluation model: net energy versus
glycogenic nutrient supply in predicting milk output 565
H. van Laar, A. van Vugt, T. van de Broek, C. Soulet de Brugiere and J. Dijkstra
A new practical feed evaluation system for pigs 567

S. Boisen and P. Tybirk
Prediction of the metabolizable energy intake and energy balance of goats with the Small
Ruminant Nutrition System model 569
A. Cannas, L.O. Tedeschi and D.G. Fox
Inter- and intraindividual variation of feed intake and metabolic parameters of dairy cows
related to energy supply 571
U. Meyer, K. Horstmann, M. Kaske and G. Flachowsky

5D. Modelling and meta-analysis
Assessment of duodenal starch as a predictor of portal absorption of glucose in ruminants 575

C. Loncke, I. Ortigues-Marty, J. Vernet, H. Lapierre, D. Sauvant and P. Nozière
Energy and protein requirements of purebred and crossbred Nellore bulls, steers, and
heifers: a meta-analysis evaluation 577
M.L. Chizzotti, L.O. Tedeschi, S.C. Valadares Filho, P.V.R. Paulino and F.H.M. Chizzotti
Portal absorption of N: partition between amino acids and ammonia in relation with
nitrogen intake in ruminants 579
H. Lapierre, J. Vernet, R. Martineau, D. Sauvant, P. Nozière and I. Ortigues-Marty
Effects of protein supply on whole body glucose rate of appearance and mammary gland
metabolism of energy nutrients in ruminants 581
S. Lemosquet, G. Raggio, H. Lapierre, J. Guinard-Flament and H. Rulquin
Nitrogen transactions along the gastrointestinal tract in cattle: a meta-analytical approach 583
J. Marini, D.G. Fox and M. Murphy
Predicting in vivo production of volatile fatty acids in the rumen from dietary
characteristics by meta-analysis: description of available data 585
P. Nozière, F. Glasser, C. Martin, D. Sauvant
Relationship between intestinal supply of essential amino acids and their mammary
metabolism in the lactating dairy cow 587
H. Rulquin, G. Raggio, H. Lapierre and S. Lemosquet
Effect of species (ovine, bovine) and feeding level on portal blood flows and net volatile
fatty acid (VFA) fluxes: a meta-analysis 589
J. Vernet, P. Nozière, S. Léger, H. Lapierre, D. Sauvant and I. Ortigues-Marty
Bibliographical database applied to ruminant nutrition 591

J. Vernet, J.P. Brun and I. Ortigues-Marty
Alternative regression approaches when modelling energy components 593

M.S. Dhanoa, R. Sanderson, S. Lopez, J. Dijkstra, E. Kebreab and J. France
Comparison of mathematical models to evaluate various in situ ruminant feed crude

protein degradation kinetics 595
M. Danesh Mesgaran, T. Tashakkori, A.R. Vakili and A.R. Heravi Mousavi
Modelling energy partition in growing pigs 597

A. Strathe, A. Danfær, G. Thorbek and A. Chwalibog
Using meta-analysis to study residual feed intake and CVDS model predictions of feed
intake and efficiency in growing and finishing cattle 599
B.M. Bourg, L.O. Tedeschi, G.E. Carstens and P.A. Lancaster

Net partial efficiencies of metabolizable energy utilization for protein and fat gain in
Nellore cattle 601
P.V.R. Paulino, S.C. Valadares Filho, M.L. Chizzotti, E. Detmann, M.A. Fonseca, M.I.
Marcondes and R.D. Sainz
Residual feed intake, energy and protein metabolism in beef steers 603
R.D. Sainz, F.C.P. Castro Bulle, P.V.R. Paulino and J.F. Medrano
Body nutrient composition of two broiler chicken strains 605

N.K. Sakomura, S.M. Marcato, J.B.K. Fernandes and I.A.M.A. Teixeira
5E. Feed evaluation and methanogenesis
Rumen methanogenesis of dairy cows in response to increasing levels of dietary extruded

linseeds 609
C. Martin, A. Ferlay, Y. Chilliard and M. Doreau
Energy utilisation and methane conversion rate in Indonesian indigenous sheep fed Napier
grass supplemented with pollard 611
A. Purnomoadi, F.Y. Devi, R. Adiwinarti, E. Rianto, O. Enishi and M. Kurihara
Methane production in lactating dairy cows on fat or corn silage rich diets compared to
Intergovernmental Panel on Climate Change (IPCC) estimates 613
W.M. van Straalen, H. van Laar and H. van den Brand
Manipulation of rumen methanogenesis with saponin-containing plant extracts 615

J. Takahashi, B. Pen and R. Asa
Influence of tanniniferous shrubs (Calliandra calothyrsus and Flemingia macrophylla) in

tropical diets on energy metabolism and methane emission of lambs 617
T. Tiemann, H.-R. Wettstein, A.C. Mayer, M. Kreuzer, C.E. Lascano andH.D. Hess
Enteric methane emission of Japanese native goats 619

O. Enishi, N. Takusari, K. Higuchi, I. Nonaka, M. Kurihara and F. Terada
Energy value of wheat bran and dried beet pulp in finishing Italian heavy pigs 621
G.M. Crovetto, G. Galassi, L. Rapetti and S. Colombini
Energy value of crude glycerol in 11 and 110 kg pigs 623

P. Lammers, B. Kerr, T. Weber, W. Dozier, M. Kidd, K. Bregenhahl and M. Honeyman
Quantitative and qualitative analyses of seed storage proteins from toxic and non-toxic
varieties of Jatropha curcas L 625
N. Selje-Assmann, H.P.S. Makkar, E.M. Hoffmann, G. Francis and K. Becker
Potato protein concentrate – nutritional value and effects on gut morphology, ileal
digestibility, and caecal fermentation in rats 627
M. Taciak and B. Pastuszewska

Effect of mannan oligosaccharides on protein metabolism in broiler chickens depending
on the dietary fibre content of the feed 629
B. Prause, R. Messikommer, P. Spring and C. Wenk
Lowered feed consumption and utilisation of diets containing Acacia villosa leaves in
experimental female rats 631
E. Harlina, B.P. Priosoeryanto, B. Tangendjaja, L.K. Darusman and D. Sastradipradja
Estimating forage digestibility from faecal crude protein concentration in grazing sheep 633
C.J. Wang, B.M. Tas, T. Glindemann,G. Rave, L. Schmidt, F. Weißbachand A. Susenbeth
Effects of feeding different levels of lauric acid on ruminal protozoa kinetics and
fermentation pattern in dairy cows 635
A.P. Faciola, G.A. Broderick and A.N. Hristov
Effect of different energy supply on rumen fermentation of Corriedale sheep consuming

temperate fresh forage 637
I. Tebot, C. Cajarville, A. Pereira, J.L. Repetto, V. Elizondo, A.L. Falero and A. Cirio
Author index 639

Preface
In 2003, the International Symposia on Energy Metabolism in Animals and on Protein Metabolism
and Nutrition coincided. The first joint Symposium was held in Rostock-Warnemünde, Germany.
Considering the success of this shared symposium as well as the intricate relationships which exist
among all energetic and nitrogenous nutrients, the International Committee decided, in agreement
with the European Association for Animal Production (EAAP), to hold the 2nd Symposium on Energy
and Protein Metabolism and Nutrition in France in September 2007. There should be no further
question about keeping the two Symposia separate.
The Symposium is in profound evolution. It is oriented towards livestock science but also addresses
general aspects of protein and energy metabolism with applications to both Animal Production and
Biomedical Sciences.
The National Scientific Committee set out three objectives for the 2007
Symposium
The first objective was to develop a platform at which widely different (i.e. from fundamental to
integrative) approaches to and concepts of nutrition are discussed, which may eventually be integrated
into systems of nutritional recommendations. Indeed, while cutting edge research is developing
rapidly, it is important that more fundamental research is translated into practical outcomes through
active links with applied research and practical applications. The newest research techniques and
methods will thus be addressed and scientists/groups will demonstrate outcomes from their work
to achieve ‘integration’.
The second objective was to encourage the comparison of research strategies developed in different
species to address similar scientific issues. Indeed, over the last two decades the close interactions
between scientists working in the area of energy and protein metabolism and nutrition across species
(i.e. farm animals, humans and laboratory animals) has been of great benefit to the development of
new general nutritional and metabolic concepts and their applications.
The third objective was to organize an open and collegial Symposium based on a workshop spirit,
in order to stimulate formal and informal interactions between openly invited scientists actively
working on nutrient metabolism and nutrition. Attendance was limited to about 300 participants in
a semi-residential mode.
The symposium took place from Sunday 9 through Thursday 13 September 2007 in the Congress
Hall of Vichy, France.
The scientific programme

After an introductory session to highlight New and Prospective Visions on Energy and Protein
Metabolism Research in the Present Socio-Economical Context, the scientific programme was based
around five main topics organised in plenary sessions and workshops. Each main topic was introduced
(or concluded) by an invited lecture, followed by a plenary session, several parallel workshops to
deal with specific scientific or methodological questions and a general plenary discussion.
Each main topic was in the hands of a coordinator, assisted by the National and International
Scientific Committees and a general discussion leader. Each workshop was in the hands of a workshop
coordinator and workshop discussion leader

The first main topic dealt with Nutrition and Mitochondrial Functions. Mitochondria play a central
role in cell survival and are the main site of ATP generation. They are also involved in a crosstalk with
the nucleus which determines gene expression and thus cell development and metabolism. Finally,
they are the target of nutritional, oxidative stress and inflammatory signals, which are actually in
progress of being understood.
The second topic tackled the Regulation of Body Composition and/or Product Quality by Tissue
Metabolism. The control of body composition and tissue characteristics is crucial in farm animals
and humans whatever the endpoint (animal products or health preservation) with for example major
regulations of muscle protein deposition and loss. Glucose and lipid metabolism are also involved
in the control of intramuscular lipids with implications in meat quality and health in humans. Milk
composition, in terms of lipidic-, nitrogenous- and micro-constituents, is of growing interest. There
is recent evidence that growth, development and metabolism are influenced by pre- or early post-
natal events, this plasticity corresponds to the concept of perinatal metabolic programming. The
knowledge in these areas depends on rapid technical progresses in energy expenditure and body
composition measurements, as well as in organ balance, in vitro tissue metabolism and nutrient
tracing methodologies.
The third topic covered the ‘Omics’ in metabolism and nutrition studies. High throughput technologies
can allow great advances towards the comprehension of gene, protein, metabolite and flux regulation.
There is a need to define the relevant terminology, and to explain the bases of those techniques,
their methods of data acquisition and data integration as well as their potential benefits and limits to
the development of integrative biological approaches. The first challenge for this new research area
is to integrate and interpret this vast amount of data with the objective to develop new biological
concepts useful to the understanding of metabolism and nutrition.
The fourth topic dealt with the Coordination between Tissues for the Metabolic Utilisation of
Nutrients. So far, the metabolic fate of macronutrients has been extensively characterised, as well
as the quantitative importance of metabolism in the different organs and tissues. The new challenges
are to understand the metabolic interactions between macronutrients as well as between organs and
tissues. Competition between metabolic pathways may occur when specific functions of the organism
are solicited, such as for example in disease, inflammation and oxidative stress or in simultaneous
gestation and lactation. What makes a function or an organ a nutritional priority for the organism,
and how flexible are nutritional priorities? Coordination between pathways and between organs and
tissues depends on the availability of digestion end-products and is regulated by intricate nutrient-
hormone-nervous system interactions.
The fifth and last topic challenged the integration of knowledge From the Parts to the Whole or How
to Use Detailed Information to Answer Applied Questions. The energy and protein feeding systems
for animals and recommended nutritional allowances for humans are based on the same concepts
applied to the whole organism, and involve both energy and protein metabolism and physiology. So
far, feeding systems and nutritional recommendations have been focused either on the efficiency of
production for animals or on health for humans. However new nutritional challenges are appearing,
in particular dealing with concerns for environmental issues (such as methane emission), body
composition, tissue characteristics (e.g. product quality), health at different ages, longevity, welfare
and well-being. Consequently, multicriteria feed evaluation and nutritional recommendations need
to be developed. Such evolution requires integrating knowledge on nutrient metabolism and their
interactions, thanks to modelling and meta-analytical approaches.
The large number of proposals which has been submitted and the wide attendance, from about 40
countries of all continents, testify of a sustaining world-wide interest in nutrition and metabolism. The

willingness of all persons contacted to get actively involved in the scientific or practical organisation
of this symposium reflects the enthusiasm for international exchange.
The Scientific and Organising Committees dedicate this Symposium to all colleagues who preceded
us and who set the milestones for research on energy and protein metabolism and nutrition, and to
all present participants who have the responsibility to carry on, and to answer emerging questions
from society in a scientific way, using new technologies and scientific partnerships.
We thank everyone who contributed to make this Symposium an important scientific event.
For the National and International Scientific Committees as well as the Organising Committee,
Isabelle Ortigues-Marty

Part 1. Nutrition and mitochondrial functions
On-going and prospective research on nutrition and mitochondrial
functions
M.P. Mollica1§, L. Lionetti1§, A. Lombardi1, E. Silvestri2, F. Goglia2 and A. Barletta1
1Dipartimento Scienze Biologiche, Sezione Fisiologia, Università degli Studi di Napoli Federico II,
Via Mezzocannone 8, 80134 Napoli, Italy
2Dipartimento Scienze Biologiche ed Ambientali, Università del Sannio, Via Port’Arsa 11, 82100
Benevento, Italy
§These authors contributed equally to the work
Abstract
The review will focus on the effects of the nutritional status and dietary components on mitochondrial
functions. Mitochondria, the powerhouses of the cell, are able to adapt to the whole body/tissue
energy request modulating the activity of the respiratory chain thus meeting either a very high
demand for adenosine triphosphate (ATP) or a need for heat production. A number of factors,
among which the nutritional state of the animal as well as its diet, through both genomic and/or
nongenomic pathways may influence mitochondrial metabolism and oxidative phosphorylation
efficiency. As for the nutritional status the review is focused on fasting, caloric restriction and
refeeding, whereas as for dietary composition the focus is sharply on the amount and type of dietary
fat. Moreover, diet can affect the formation of mitochondrial free radicals, therefore the relationship
between nutrition, mitochondrial free radical production, oxidative damage and lifespan will also
be taken into consideration together with the influence of nutrition on both gene expression and the
mitochondrial apoptotic process.
Keywords: mitochondrial function, dietary composition, nutritional status, oxidative damage,

thyroid hormone
Introduction
The relationship between nutrition and mitochondrial function is a very interesting one to analyse
since mitochondria, the powerhouses of the cell, are where the different forms of chemical energy
derived from food and body stores are transduced into adenosine triphosphate (ATP), by processes
that involve the consumption of oxygen, called oxidative phosphorylation (OXPHOS). The system,
however, is not perfectly efficient and it is now accepted that the efficiency of the mitochondrial
machinery depends on the presence of a proton-leak pathway created by the intrinsic characteristics
of the internal membrane, such as its lipid composition, or by uncoupling proteins (UCP) (for reviews
see Lanni et al., 2003; Brand and Esteves, 2005; Cannon et al., 2006).
OXPHOS activity is controlled by a number of factors. Among these, the nutritional state of the
animal, as well as its diet, may influence mitochondrial metabolic pathways through genomic and
nongenomic effects on mitochondrial oxidative phosphorylation (Berdanier, 2001). This review of
nutrition and mitochondrial functions focusses on the effects of dietary components as well as on
the nutritional status on mitochondria. In the case of nutritional status, the focus is on fasting, caloric
restriction and refeeding, whereas in the case of dietary composition, the focus is sharply on the
amount and type of dietary fat (particularly because of the large amount of fat consumed in the so-
called ‘developed countries’ and its deleterious consequences). Since diet can affect the formation of
mitochondrial free radicals, this work also highlights the relationship between nutrition, mitochondrial
ROS (reactive oxygen species) production, oxidative damage and lifespan. Finally, the influence
of nutrition on both gene expression and the mitochondrial apoptotic process is discussed. We are

conscious that the literature in this field is so very rich that it cannot be exhaustively treated in a
single review article, so we apologise to the numerous very important contributors that we have not
cited in the present article, limited as it is to some particular aspects and with a didactic purpose.
Dietary components and mitochondrial functions

Lipid intake, age and mitochondrial functions
The current state of chronic overnutrition or positive energy balance (in developed countries) has
created a previously unknown metabolic conflict between carbohydrates and lipids. A metabolic
conflict develops under conditions in which chronic overnutrition results in simultaneous high
exposure to both carbohydrates and lipids. This is characterised by a facilitation of de novo lipogenesis,
decreased fatty acid oxidation and accumulation of toxic non-oxidised long-chain fatty acids, the
basis for a generalised lipotoxic state (Slawik and Vidal-Puig, 2006). Many studies have been
made on the effects of high-fat feeding on mitochondrial functions, and it seems that the effects are
dependent on the age at which the high-fat feeding starts (for review see Iossa and Liverini, 2000).
Also the capacity to counteract the development of diet-induced obesity is age-dependent (Iossa
and Liverini, 2000). In fact, it has been shown that when post-weaning rats, which are characterised
by an active phase of growing, are fed a high-fat diet for 2 weeks they exhibit an increased energy
intake with an associated increase in energy expenditure, the net result being to counteract obesity
development. These rats are able to utilise the excess lipid intake by a shift in lipid partitioning
between oxidation and storage. However, this resistance to obesity is lost in mature rats. In fact, in
mature rats the energy expenditure is not able to balance the excess energy intake, and the increase
in lipid oxidation only partly counteracts the excess lipid intake. The impairment in regulatory
mechanisms shown by mature rats may be the cause of the tendency to accumulate fat, a tendency
exhibited by both rats and humans, as they grow older (Iossa and Liverini, 2000). The mechanisms
underlying these effects involve changes in metabolic activity in single organs and tissues (Rolfe
and Brown, 1997) and changes in mitochondrial functions. Post-weaning rats fed a high-fat diet for
2 weeks exhibit a decrease in NADH oxidation in hepatic mitochondria, together with increased
respiration in liver cells, suggesting a fall in hepatic metabolic efficiency (Iossa et al., 1995; Lionetti
et al., 1996; Mollica et al., 1998). Similar studies on rat skeletal muscle have revealed an increase
in lipid oxidative capacity and an increase in fatty acid-induced uncoupling, which could help to
avoid the accumulation of fat (Iossa et al., 2002).
As cited above, a high-fat-diet may have different effects when administered to adults than when
administrated to immature individuals. In adult rats, a high-fat-diet did not elicit the regulatory
mechanisms at the mitochondrial level that are useful for counteracting obesity development. Such
an impairment in regulatory mechanisms in adult rats may be the cause of the tendency to accumulate
fat that is characteristic of both rats and humans as they grow older. Age-related changes also occur
in the involvement of particular biochemical mechanisms in the tissues and in the effects of the
dietary fatty acid composition.
Dietary fatty acid composition and mitochondrial functions
Dietary fats and fat-soluble vitamins (A, D, E, K) are sources of antioxidants and bioactive compounds.
Fatty acids can be subdivided into the following categories: saturated fatty acids (SFA, no double-
bonds between the carbons), monounsaturated fatty acids (MUFA, only one double-bond) and
polyunsaturated fatty acids (PUFA, more than one double-bond). SFA are predominantly present
in animal fat and animal derivates while MUFA and PUFA are predominantly vegetable in origin
(Table 1). The human body can manufacture all fatty acids except two: linoleic acid and alpha-
linolenic acid, which are widely distributed in plant and fish oils; they are called essential fatty acids
since they must be supplied in food. These two essential fatty acids are polyunsaturated and are the

Table 1. Presence of saturated (SFA), monounsaturated (MUFA) and polyunsaturated (PUFA) fatty
acids in some foods.
SFA MUFA PUFA
Fatty beef Olive oils Fatty fish (i.e. salmon)

Veal Peanut oils Sunflower
Lamb Canola Soybeans
Pork Many nuts
Lard etc.
Poultry fat
Butter
Cream
Milk
Cheese
Tropical oil:
coconut oil
palm oil
Cocoa butter
parent compounds of the omega-6 and omega-3 fatty acid series, respectively. Linoleic acid and
arachidonic acid are omega-6 fatty acids. Alpha-linolenic acid, eicosapentaenoic acid (EPA) and
docosahexaenoic acid (DHA), (component of the phospholipids in cell membranes, especially in
the brain and retina), present in marine fish oil, are examples of omega-3 fatty acids and are often
used as nutritional supplements.
Various studies have been carried out to investigate the effects of different dietary fat compositions
on mitochondria. Fatty acids are oxidised in mitochondria, which also produce ROS which, in turn,
lead to lipid peroxidation. This is a very dangerous event because of the capacity of ROS to initiate
a cascade of reactions leading to damage to macromolecules such as enzymes, DNA, etc.
The unsaturated fatty acids in the cellular membranes are the macromolecules most susceptible to
oxidative damage in cells, and this sensitivity increases as a function of their number of double-bonds.
Membranes rich in PUFA may be easily peroxidised, and this observation gave rise to the notion that
the relatively low double-bond content of the membranes of long-lived animals could have evolved
so as to protect them from oxidative damage (Herrero et al., 2001). In long-lived mammals, low
degrees of fatty acid unsaturation are shown by mitochondrial membranes and cell membranes in
tissues including the liver, heart, skeletal muscle and kidney, and this constitutively protects their
cellular membranes, proteins and DNA against lipid-peroxidation-derived damage (for review see
Pamploma et al., 2002; Hulbert, 2005).
A relationship between fatty acid unsaturation and oxidative damage is also observed in experimental
studies in vivo. The composition of the dietary fat components will affect free-radical generation,
diets rich in polyunsaturated fatty acids resulting in larger amounts of free radicals in selected tissues
(Berdanier, 2001). It has recently been observed that treating rats with dietary oils containing a small
number of double-bonds lowers the degree of fatty acid unsaturation in brain cellular membranes
and also lowers brain lipoxidation-derived protein modification (Pamplona et al., 2004). This makes
sense because lowering fatty acid unsaturation decreases the susceptibility of membranes to lipid
peroxidation.

The degree of saturation of the dietary fatty acids also influences various mitochondrial biochemical
pathways. The following summarises some interesting findings on this subject:
1. PUFA administration markedly decreased the tricarboxylate carrier (TCC) mRNA content and
TCC activity of rat liver mitochondria (as compared with saturated fatty acid administration)
(Giudetti et al., 2003). In addition, PUFA administration regulates the TCC gene at transcriptional
and post-transcriptional steps, whereas diets rich in saturated fatty acids or monounsaturated fatty
acids do not seem to affect either TCC activity or gene expression (Siculella et al., 2004).
2. Treatment with EPA, but not with DHA, lowered plasma triacylglycerol by increasing hepatic
mitochondrial fatty acid oxidation and carnitine palmitoyltransferase (CPT)-I activity (Madsen
et al., 1999). The 3-thia fatty acid, tetradecylthioacetic acid (TTA), and EPA each induced
mitochondrial growth in type I muscle fibres in both the diaphragm and soleus muscle (Totland
et al., 2000).
3. The ω-3 PUFA, DHA and EPA, may induce cell-cycle arrest and apoptosis by activating protein
phosphatases, which in turn lead to dephosphorylation of retinoblastoma protein (pRB) and
eventually to activation of the apoptotic enzyme caspase 3 through the protein Bcl2 and the release
of cytochrome c from mitochondria (see paragraph on apoptosis) (Siddiqui et al., 2004).
4. In cardiac mitochondrial membranes, a diet rich in ω-3 PUFA prevented the age-related
declines in ω-3 PUFA and in the inner mitochondrial membrane-specific phospholipid,
cardiolipin (diphosphatidylglycerol), as well as preventing the increases in ω-6 PUFA and in
phosphatidylcholine. In contrast, an ω-6 PUFA-rich diet augmented the age-related increases
in ω-6 PUFA and phosphatidylcholine and the declines in ω-3 PUFA and cardiolipin (Pepe,
2005).
Epidemiological evidence has established that ingestion of long-chain polyunsaturated omega-3 fatty
acids (omega-3 PUFA), abundant in fish oils, has profound effects on many human disorders and
diseases, including cardiovascular disease and cancer. There are also a number of products, including
bread and cereals, available to supplement the dietary intake of omega-3 fatty acids. Some of these
supplements are proposed to aid different pathological conditions. While the beneficial effects of
omega-3 fatty acids can no longer be doubted, their molecular mechanism of action remains elusive.
Without question, the action of omega-3 fatty acids is complex and involves a number of integrated
signalling pathways in which mitochondria play an important role (Siddiqui et al., 2004).
Nutritional status: fasting, caloric restriction, refeeding and

mitochondrial oxidative damage
Fasting and caloric restriction
Fasting results in marked changes in the metabolic activities of most organs. In skeletal muscle,
changes are directed toward the use of lipids and aminoacids as alternatives to glucose. This shift is
accompanied by changes in gene expression and mitochondrial activities (Gahill, 1976; Pilegaard
et al., 2003; De Lange et al., 2006). These events are time-dependent, species/organ-dependent and
transient. Examples include AMP-activated protein kinase (AMPK) and peroxisome proliferator-
activated receptor delta (PPARδ) (Munday et al., 1991; Witters et al., 1994; De Lange et al.,
2004; Luquet et al., 2004). During fasting, there is an increase in uncoupling protein 3 expression
(Himms-Hagen and Harper, 2001; Moreno et al., 2003) and in serum FA levels, paralleled by an
enhanced FA oxidation (Moreno et al., 2003; De Lange et al., 2006). Important changes are also
evident in caloric restriction and refeeding. It is well known that overeating shortens the lifespan
while caloric restriction extends it (Weindruch, 2003) in all mammalian species studied so far, and
this strongly suggests a role for energy metabolism in the aging process. Oxidative damage and
increased mitochondrial ROS production accompany aging in many tissues and are thought to lead
to a loss of efficiency in metabolic energetics (Bevilacqua et al., 2005). The only mechanism known
to inhibit this gradual age-associated dysfunction in bioenergetics is calorie restriction (CR), but it

remains unclear how such nutritional intervention accomplishes this end (for review see Hunt et al.,
2006). It is known that mitochondrial ROS (mtROS) production is decreased in tissues (including
skeletal muscle, kidney, liver, heart and brain) obtained from animals on calorie-restricted diet
(Lopez-Torres et al., 2002; Drew et al., 2003; Lambert and Merry, 2004; Gredilla and Barja, 2005).
The decrease in ROS generation in CR rats specifically occurs at complex I in all the organs studied
to date (heart, liver and brain) (Sanz et al., 2005). The CR–dependent decrease in ROS production
is accompanied by a dramatic decrease in mtDNA oxidative damage (Lopez-Torres et al., 2002) as
well as in mitochondrial lipid peroxidation (Pamplona et al., 2002; Bevilacqua et al., 2005) which in
turn, can prevent age-related disease. CR is inevitably associated with changes in certain circulating
nutrients and hormones (such as leptin, insulin, glucagon, thyroid hormones, etc.) able to control
gene expression by binding to their receptors. Among these, PPAR and CREBP (cAMP response
element-binding protein) play major roles in the cell- and tissue-response to CR. The lipid-activated
transcription factors, PPAR in particular, play an important role in obesity-related metabolic diseases
such as hyperlipidaemia, insulin resistance and coronary artery disease. They have subtype-specific
functions, indicating the therapeutic potential of these receptors. An important role in the modulation
of the transcriptional activities of such transcription factors has been ascribed to coactivators. In
particular, the PPARγ coactivator, PGC-1α, - originally described as a cold-inducible coactivator,
regulating adaptive thermogenesis by increasing the transcriptional activation of the UCP1 gene
via PPARγ and thyroid hormone receptor - interacts with several nuclear receptors and transcription
factors controlling the expression of genes involved in energy and nutrient homeostasis (Tiraby et
al., 2003), including those for nuclear-encoded mitochondrial proteins. In human adipose cells,
overexpression of PGC-1α results in the appearance of oxidative features and the expression of
genes typical of the brown adipocyte. A recent study investigating the effect of a low caloric diet on
human subcutaneous adipose tissue found a decrease in mRNA levels for genes involved in lipid
metabolism, especially PPARγ2, and an increase in PGC-1α mRNA levels (Viguerie et al., 2005).
Considerable evidence has accumulated to suggest that many effects of CR are mediated by insulin
and growth hormone(GH)-insulin-like growth factor I (GH/IGF-I) (Heilbronn and Ravussin, 2003),
the levels of which are significantly suppressed during CR (Sonntag et al., 1999). IGF-I-receptor
knockout mice live 26% longer than their wild-type littermates while maintaining normal energy
metabolism (Holzemberger et al., 2003). Moreover, it has become increasingly evident that CR-
associated changes in the circulating levels of insulin and IGF-I in mammals also dramatically affect
the expression and functions of genes such as Sirt1 and FOXO (Guarente and Picard, 2005; Mounier
and Posner, 2006) involved in chromatin silencing (Bordone and Guarente, 2005) and upregulation
of target genes functioning as life-extending factors (Greer and Brunet, 2005), respectively.
It has been proposed that the reduction in methionine intake during CR and protein restriction may
be the cause of the decrease in MtROS production and oxidative damage to mtDNA, and may be
responsible for around 50% of the increase in maximum life-span (MLSP) observed in caloric
restriction (Pamplona and Barja, 2006). Moreover, it has been found that methionine is the only amino
acid whose abundance in tissues strongly (and negatively) correlates with MLSP (i.e. the longer the
life-span, the lower the level of methionine in intracellular proteins). Methionine could induce damage
through a variety of possible mechanisms. In fact, the methionine residues in proteins are among the
amino acids most susceptible to oxidation by ROS, and the sensitivity of proteins to oxidative stress
increases as a function of their number of methionine residues. Oxidation of methionine residues
generates methionine sulphoxide in proteins, which deprives them of their function as methyl donors,
and may lead to loss of their biological activities (Ciorba et al., 1997). In any case, there is a strong
need for further experimental work on the ability of methionine restriction to reduce mitochondrial
oxidative stress and on the mechanisms by which it might lead to slower aging.

Effect of refeeding after caloric restriction on mitochondrial functions
In response to a diminished food supply (CR), humans and other mammals may undergo a reduction
in energy expenditure, which is partly attributable to an increased metabolic efficiency (Dulloo and
Girardier, 1993). This is viewed as an adaptive response to food scarcity because it slows the rate of
weight loss or reduces the energy cost of weight maintenance. Since suppressed thermogenesis also
persists for some time upon refeeding (Dulloo and Jacquet, 1998) and the energy spared is directed
specifically towards accelerating the recovery of body fat (or ‘catch-up fat’) (Dulloo and Girardier,
1990), it can also be viewed as the outcome of a control system that operates as a feedback loop
between depletion/repletion of the fat stores and suppressed thermogenesis (Dulloo and Jacquet,
2001). This energy-conservation mechanism, which probably evolved to optimise survival capacity
in a lifestyle characterised by periodic food shortage, is an important factor that nowadays contributes
to the relapse towards obesity after slimming, and hence to the poor efficacy of voluntary dietary
restriction in the management of obesity. It is also a factor contributing to the high rate of fat deposition
that characterises catch-up growth after earlier growth-retardation (Dulloo and Jacquet, 2001) and
has been implicated in the link between early malnutrition, catch-up growth and a subsequently
increased risk of type 2 diabetes and/or cardiovascular disease. This mechanism has been referred
to as ‘adipose-specific’ control of thermogenesis (i.e. an autoregulatory feedback system in which
signal(s) from the depleted adipose fat stores exert a suppressive effect on thermogenesis until the
fat stores are replenished) (Dulloo et al., 2002).
At present, skeletal muscle is thought to be the major effector site for this adipose-specific control
of thermogenesis (Dulloo et al., 2002). However, the underlying effector mechanisms remain
elusive. Early interest in the notion that such regulated thermogenesis in skeletal muscle might
involve mitochondrial uncoupling by UCP2 or UCP3 (two homologues of UCP1) has waned.
This is because it has been demonstrated that these uncoupling proteins are either up-regulated or
unaltered in response to starvation and refeeding (Harper et al., 2001; Crescenzo et al., 2003), a
pattern of expression inconsistent with their playing roles in the control of adaptive thermogenesis.
Recent studies suggest that both in caloric restriction and in refeeding, a diminished skeletal muscle
mitochondrial mass and function (specifically in the subsarcolemmal (SS) mitochondria) contribute to
the suppression of thermogenesis (Crescenzo et al., 2006). Moreover, refeeding reverses the beneficial
effects of caloric restriction on skeletal muscle mitochondrial oxidative damage. In fact, refed rats
show, in both SS and intermyofibrillar (IMF) muscle mitochondria, a lower aconitase activity, an
index of increased ROS. In addition, given the important role that SS mitochondria perform in
the bioenergetic support of signal transduction and substrate transport, and hence in the action of
insulin, the decreased SS mitochondrial mass and function may have particular relevance for the
mechanism by which suppressed thermogenesis in skeletal muscle leads to insulin-resistance during
the accumulation of catch-up fat. This may have implications for the physio(patho)logy of catch-up
growth, weight fluctuations and frequent ‘yo-yo’ dieting. In fact, SS mitochondria provide energy
for membrane-related processes, including signal transduction, ion exchange, substrate transport and
substrate activation (Hood, 2001), steps clearly relevant to the action of insulin. A role for reduced
SS mitochondrial energetics in the pathogenesis of skeletal muscle insulin-resistance in both obesity
and type 2 diabetes has recently been advanced. The basis for this is the lower SS mitochondrial
activity and lower SS electron transport chain activity found in skeletal muscle from obese or diabetic
patients than in that from nondiabetic lean volunteers (Ritov et al., 2005). Thus, because of the
potential importance of SS mitochondria for the bioenergetic support of insulin-signaling and insulin-
mediated glucose transport in skeletal muscle, the possibility arises that the decreased mitochondrial
energetics, specifically in the SS compartment (described here as occurring during refeeding), may
also play a role in the mechanism by which suppressed thermogenesis in skeletal muscle leads to
insulin-resistance during the accumulation of catch-up fat (Crescenzo et al., 2006).

Influence of nutrients and thyroid hormones on mitochondrial gene
expression
Dietary fats apart from being important macronutrients for growth, development and energy
metabolism, provide substrates for membranes and signalling molecules and also regulate gene
expression. Since the publication over a decade ago of the early evidence that dietary fats serve
as regulators of gene expression (for reviews see Jump, 2004; see also Pegorier et al., 2004; Jump
et al., 2005), many transcription factors have been identified as possible targets for fatty acid
regulation. These factors include the following: (i) the retinoid X receptor (RXR) (Ou et al., 2001);
(ii) hepatic nuclear factors (HNF- 4α and γ) (Wisely et al., 2002 and Dhe-Paganon et al., 2002);
(iii) peroxisome proliferator-activated receptors (PPARα, β and γ) (Xu et al., 1999) and (iv) sterol
regulatory element binding protein-1c (SREBP-1c) (Worgall et al., 1998). Most of the studies have
been carried out on the liver because of its key role in whole-body lipid metabolism. The key event
for the action of FA on gene expression involves FA entering the cell to affect the activity or level
of transcription factors. However, FA also affect the activity or level of G-protein-linked receptors
(Kostenis, 2004) but, neither tissue distribution nor ligand-specificity adequately explain the action
of fatty acid synthetase (FAS) on hepatic gene expression. Non-esterified fatty acids (NEFA) enter
the cell either through transporters (fatty acid transport protein (FATP) or fatty acid transporter CD36
(FAT)) or by diffusion. NEFA are converted to fatty acyl-CoA (FACoA) by FATP (Di Russo et al.,
2005) or fatty acyl-CoA synthetases (ACS) (Coleman et al., 2002). FACoA and NEFA are bound to
fatty acid binding protein FABP and acyl-CoA binding protein (ACBP) respectively, which carry FA
either into the cell compartments for metabolism (Hertzel and Bernlohr, 2000) or into the nucleus
where they affect transcription factors (for a schematic representation of all the above see Figure
1). FA also affect gene expression in other organs (e.g. skeletal muscle) and some of these aspects
will be mentioned in the next section.
It is now apparent that regulation can also occur at the level of the mitochondrial genome found in
the matrix of the mitochondrion that encodes 13 gene products that are important for the appropriate
functioning of OXPHOS as well as products essential for its transcription and translation. In contrast
NEFA
Fatty acid
Cell membrane
transporters Diffusion
ACS
FABP NEFA FA-CoA ACBP
TE
Nucleus
FABP NEFA
Oxidation
Fatty Acid
Transcription Synthesis
factor
Complex Lipid
Synthesis
Regulated
target gene
VLDL Membranes
Figure 1. Schematic representation of the pathways involved in non-esterified fatty acids (NEFA)
cell entry and their subsequent activity (for explanation see text).

to the nuclear genome, the mitochondrial genome is circular and consists of a light strand, a heavy
strand and a small fragment called the displacement loop or D-loop. Computational analysis of the
D-loop has suggested the presence of a number of response elements for proteins that bind vitamins
and hormones.
Among the nutrients that can affect mitochondrial transcription is retinoic acid, which seems to
stimulate mitochondrial gene expression (reviewed in Berdanier, 2006). Retinoic acid has been shown
to upregulate NADH dehydrogenase subunit 5 mRNA, as well as cytochrome c oxidase subunit I
and 16S rRNA (Everts et al., 2002). Everts and Berdanier reported that dietary vitamin A upregulates
ATPase 6 gene expression and optimizes OXPHOS in diabetes-prone BHE/Cdb rats (Everts and
Berdanier, 2002). Although other nutrients may also affect mitochondrial gene expression, some of
these have yet to be identified. Vitamin D, through the actions of its metabolite 1α,25‑dihydroxyvitamin
D, is known to affect the transcription of a wide range of nuclear genes. Chou et al. (1995) reported
that this nutrient affects both nuclear and mitochondrial gene expression in kidney and intestinal cells.
The effects of vitamin D are tissue-specific with respect to effects on mitochondrial transcription.
Indeed, no effect of vitamin D on mitochondrial OXPHOS or mitochondrial gene expression could
be found in hepatic tissue (Chou et al., 1995). Mitochondrial DNA is also affected by vitamin B12.
This vitamin is known to play an important role in nucleic acid synthesis, both DNA and RNA in
mitochondria being reduced in lymphocytes from vitamin B12-deficient humans. However, a lack
of this vitamin reportedly affects overall nucleic acid synthesis rather than having a direct effect on
the process of transcription or translation (Cantatore et al., 1998).
Another nutrient known to affect gene expression is zinc (Shay and Cousin, 1993). Primary hepatocytes
cultured with graded amounts of zinc displayed graded increases in mitochondrial transcripts and
mitochondrial gene products. Most likely, this was due to the role that zinc plays in the binding of
zinc-finger receptor proteins to DNA. Cells deficient in zinc would make fewer of these proteins, and
receptor proteins are integral players in the effects of vitamins and hormones on gene expression in
both the nucleus and the mitochondria. To our knowledge, in vivo studies are still lacking.
Iodine, found in seafood, iodised salt and some vegetables, is essential for thyroid-hormone synthesis.
An inadequate intake of dietary iodine can lead to an enlarged thyroid gland (goitre) and to other
iodine-deficiency disorders. Thyroid hormones (T4, T3) regulate the body’s metabolic rate and
promote growth and development throughout the body, including the brain. Mitochondria are
among the cellular targets of thyroid hormones. Very recently, evidence of the existence of a direct
T3 mitochondrial pathway was obtained. Numerous studies have reported short-term and delayed
T3 stimulations of mitochondrial oxygen consumption. Convincing data indicate that the early
influence occurs through an extra-nuclear mechanism that is insensitive to inhibitors of protein
synthesis. Although it has been proposed that diiodothyronines (Goglia et al., 1999 and 2002;
Goglia, 2005) could also be mediators of this short-term influence of T3, the detection of specific
T3-binding sites supports an additional, direct, influence by T3 itself. The more delayed influence
of thyroid hormone upon mitochondrial respiration probably results from mechanisms elicited
at the nuclear level, including changes in phospholipid turnover and stimulation of uncoupling
protein expression, leading to an increased inner-membrane proton leak. Both direct and indirect
pathways are obviously involved in the stimulation by T3 of mitochondrial genome transcription.
In particular, a 43 kDa c-erb A-alpha 1 protein located in the mitochondrial matrix (p43) induces
- by acting as a potent T3-dependent transcription factor for the mitochondrial genome - an early
stimulation of organelle transcription. In addition, T3 increases the expression of the mitochondrial
TFA, a mitochondrial transcription factor encoded by a nuclear gene. Similarly, the stimulation of
mitochondriogenesis by thyroid hormone probably involves both pathways. In particular, the c-erb
A-alpha gene simultaneously encodes a nuclear protein and p43, thus ensuring coordination of the
expression of the mitochondrial genome and that of nuclear genes encoding mitochondrial proteins
(for review Wrutniak-Cabello et al., 2000).

From the foregoing, it is apparent that mitochondrial gene expression is similar to nuclear expression
in several respects, transcription being responsive to some of the same nutrients that affect nuclear
transcription. Future research should add to our understanding of mitochondrial DNA and its
transcription.
Dietary bioactive agents and the mitochondria-mediated apoptotic

pathway
Apoptosis eliminates potentially deleterious mutated cells and so is one of the most potent defences
against cancer. Apoptosis occurs primarily through two well-recognised pathways in cells, including
the intrinsic, mitochondrially mediated, effector mechanism and the extrinsic, death-receptor mediated,
effector mechanism (Reed, 2004). Many diet-related genes are involved in carcinogenesis as well as
apoptosis, and thus are ultimately molecular targets for dietary chemoprevention (reviewed in Martin,
2006). Many dietary bioactive agents induce apoptosis through the intrinsic mitochondrially mediated
pathway (Chen and King, 2005) which is characterised by alterations in both mitochondrial membrane
potential (MMP) and the release of mitochondrial proteins, including cytochrome c and endonuclease
G, secondary mitochondria-derived activators of caspases. The release of cytochrome c then triggers
caspase activation and ultimately the execution of apoptosis. In the intrinsic mitochondrial pathway,
the Bcl-2 family of at least 18 pro- and anti-apoptotic proteins are pivotal regulators of apoptosis,
and all of them may be targets (Chen and King, 2005).
Dietary bioactive agents, such as the vanilloids curcumin (found in turmeric), capsaicin (found
in chilli peppers), flavonoid caicilin, and nordihydroguaiaretic acid (from chapparal) can induce
apoptosis by altering mitochondrial membrane function and dissipate the MMP via opening of the
mitochondrial permeability transition pore (MTP) (for review see Martin, 2006). Numerous examples
demonstrate that dietary bioactive agents (dietary ginger, including curcumin, flavonoid-rich grape-
seed extract, etc.) can induce a mitochondrial release of cytochrome c (reviewed in Martin, 2006).
Beta-carotene, a carotenoid found in carrots, induces a release of cytochrome c from mitochondria
and alters the mitochondrial membrane potential in various tumour-cell lines derived from leukaemia,
colon adenocarcinoma and melanoma cells (Palozza et al., 2003). Lycopene, a non-provitamin
A carotenoid found in tomatoes, delivered at physiological concentrations, can depolarise the
mitochondria of human prostate cells, induce cytochrome c release and ultimately induce apoptosis
(Hantz et al., 2005). This change is also induced by concentrations of lycopene equivalent to the
plasma level found in those consuming three to five daily servings of fruits and vegetables (Hantz
et al., 2005). Collectively, numerous diverse dietary bioactive agents (for example, beta-carotene,
the polyphenol stilbene resveratrol and curcumin) can induce apoptosis by modulating the Bcl-2
family of proteins, making them critical targets for bioactive agents (for review see Martin, 2006).
As mentioned above, the omega-3 PUFA, DHA and EPA, may induce cell-cycle arrest and apoptosis
(Siddiqui et al., 2004). Current strategies for cancer treatment, such as chemotherapy and ionising
radiation, induce apoptosis in cancer cells. Clearly, if apoptosis could be selectively induced in
cancer cells by dietary components (resveratrol, beta-carotene, etc.), diet could be employed as an
effective chemopreventive strategy.
Conclusion
This is a new era in research on the interaction of nutrients and genes. Although many nutrients
have been shown to influence the expression of a wide variety of nuclear genes, few studies have
been devoted to the study of nutrient effects on mitochondrial gene expression. However, we are
now entering the new age of mitochondrial medicine. Indeed, with a better understanding of how
mitochondrial activity is controlled and how it is related to some of the degenerative diseases (such
as diabetes and heart disease), we are gaining a new appreciation of the interactions that occur among
genetics, diet and mitochondrial function.

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Insulin modulates the decrease of oxidative phosphorylation induced by
ingested butyrate. NMR study on the isolated and perfused rat liver
M.-C. Beauvieux1, H. Roumes1, V. Rigalleau1,2, H. Gin1,2 and J.-L. Gallis1
1RMSB UMR5536 CNRS-Université Bordeaux2, 146 rue Léo Saignat, 33076 Bordeaux Cedex,
France
2Nutrition Diabétologie Hôpital Haut-Lévêque, Avenue de Magellan, 33604 Pessac, France
Introduction
We have demonstrated in the isolated and perfused liver of rat a positive and linear correlation, only
in the presence of insulin, between the net glucose-dependent fluxes of ATP and glycogen (Glg)
(Baillet-Blanco et al., 2005). On the contrary, the perfusion of fatty acids (FA) (butyrate, octanoate)
decreases the ATP content in this model (Beauvieux et al., 2001). This effect of FA could be partly
explained by the supply of FADH2 due to the β−oxidation and the subsequent decrease in the
stoechiometric ratio of NADH+H+ and FADH2, entering in the respiratory chain to be reoxidised.
That could lead to a decrease of the synthesis flux of mitochondrial ATP, disturbing thus the energy
supply necessary to glycogenosynthesis.
Since a decrease in the glycogen content, in the muscle but also in the liver, has been reported in
insulinoresistance (IR), mitochondrial ATP production could be a target of IR, in agreement with
the recent hypothesis proposed in the IR clinical context (Lowell and Shulman, 2005). Our aim was
to evaluate in the fasted rat the effects of a glucose+butyrate force-feeding on the parameters of
oxidative phosphorylation (OP) measured in the isolated liver, perfused or not with insulin.
Material and methods

Male Wistar rats (100 g) fasted from 48 h were force-fed with two isocaloric diets (7.28 kcal): (i)
1.82 g glucose (G) or (ii) 1.4 g G + 0.19 g butyrate (G+B). Between 0 to 10 h following the force-
feeding, the livers (n=3-7 for each time) were isolated and perfused (5 mL/min/g) with isotonic
Krebs-Henseleit buffer (pH 7.3; 37 °C; O2 95%, CO2 5%), containing 30 mM glucose, with or
without insulin (120 mUI/L). The changes of the ATP content were followed in the whole liver by
the measurement of the area of the resonance peak of ΑΤPβ by 31P Nuclear Magnetic Resonance
(Brucker DPX400, 9.4 T, dual probe 31P/13C). An external reference (methylene diphosphonic acid,
18.4 ppm) allowed accurate ATP quantitation.
At the ATP steady-state, 0.5 mM iodacetate (2 min) then 2.5 mM KCN (10 min) were added in the
perfusate (inhibiting glycolytic and mitochondrial ATP synthesis, respectively). Since at the onset of
KCN addition (t0), ATP synthesis rate = ATP consumption rate, the in situ rate (R) of ATP turn-over
(Beauvieux et al., 2002): ATP = Aexp-kt, R(t0) = -Ak was calculated.
Results
The 48-h fasting induced a dramatic decrease of ATP and Glg hepatic contents (-65 and -99%,
respectively). The force-feeding induced a progressive in vivo repletion of ATP and Glg contents.
The in vivo ATP and Glg contents, measured by NMR immediately after the liver excision, resulted
from both synthesis and consumption mechanisms. The perfusion sequence, described above,
allowed the specific measurement of OP parameters: time constant (k [min-1]) and rate of synthesis
(R [µmol/min×g]).

The addition of inhibitors in the perfusate pointed out the in vivo transient increase of k and R after
2-3 h following the force-feeding, duration corresponding to the gastric empty. Without insulin in
the perfusate, the ingested butyrate prevented these increases only at the 3-h post-ingestion delay,
this time corresponding to the post-prandial insulinemia peak: kG+B = 0.45 ± 0.07 (m ± SEM) vs. kG
= 1.55 ± 0.21, P = 0.01 and RG+B = 0.42 ± 0.03 vs. RG = 1.40 ± 0.23, P=0.04 (Figures 1a and 1b).
Whatever the delay was following the force-feeding with glucose alone, k and R were unchanged by
addition of insulin in the perfusate. In contrast, at 3-h following the force-feeding with both glucose
and butyrate, k (= 0.96 ± 0.19; P=0.04, G + B + I vs. G) and R (= 0.79 ± 0.18) increased when insulin
was perfused; this latter counteracted thus the effects of ingested butyrate.
Figure 1. Time constant (a) and rate of ATP synthesis (b). Fed (♦) or fasted (▲)control rats; G (■)
force-feeding [ff]; G+B [ff] (○); G+B [ff]+ perfusion with Ins (□).
Conclusion
The presence of butyrate in the diet transitory prevents the increase of ATP turn-over obtained with
glucose alone. Perfused insulin counteracts the effect of butyrate ingestion. The intrinsic catalytic
activity of the system seems concerned owing to the change of the time constant. These results
were in agreement with the improvement of the OP parameters reported in the presence of insulin
in hepatic mitochondria of diabetic rats (Huang et al., 2001).
References
Baillet-Blanco, L., M.-C. Beauvieux, V. Rigalleau, H. Gin and J.-L. Gallis, 2005. Insulin induces a positive relationship
between the rates of ATP and glycogen changes in isolated rat liver in presence of glucose; a 31P and 13C NMR
study. Nutr. Metab. 2, 32.
Beauvieux, M.-C., P. Tissier, H. Gin, P. Canioni and J.-L. Gallis, 2001. Butyrate impairs energy metabolism in isolated
perfused liver of fed rats. J. Nutr. 131, 1986-1992.
Beauvieux, M.-C., P. Tissier, P. Couzigou, H. Gin, P. Canioni and J.-L. Gallis, 2002. Ethanol perfusion increases the
yield of oxidative phosphorylation in isolated liver of fed rats. Biochim. Biophys. Acta 1570, 135-140.
Huang, Q., L. Shao, H. Jiang, Z.C. Miao, Q.D. Shi and S.S. Liu, 2001. Effect of insulin on oxygen free radicals and
oxidative phosphorylation in liver mitochondria of diabetic rats. Acta Pharmacol. Sin. 22, 455-458.
Lowell, B.B. and G.I. Shulman, 2005. Mitochondrial dysfunction and type 2 diabetes. Science 307, 384-387.

Effects of a diet enriched in trans fatty acids (trans MUFA) on muscle
mitochondrial functions and development of insulin resistance in rodents
A.-L. Tardy, P. Rousset, C. Giraudet, J.M. Chardigny and B. Morio
INRA, UMR1019, Clermont-Ferrand, F-63000, France
Introduction
Fatty acid composition of the diet could have an impact on the incidence of plurimetabolic syndrome
(Lovejoy et al., 2002). Epidemiologic data suggest that a chronic consumption of trans MUFA could
be noxious for insulin sensibility (Salmeron et al., 2001) but the mechanisms are still unknown.
Trans MUFA are present in partially hydrogenated vegetable oils of industrial origin and in meat
and dairy products of ruminants. In this last case, vaccenic acid (trans-11) is the major isomer, while
industrial hydrogenated fat contains mainly trans-9 (elaidic acid) and trans-10 isomers. The two
major sources of trans MUFA in food have a different isomeric profile and thus probably different
metabolic effects. Those effects may imply a disturbance in muscle mitochondrial oxidative and
phosphorylation (OXPHOS) functions known to be linked to insulino resistance development.
The objective of this work was to determine the impact of the trans fatty acids, elaidic and vaccenic
acids, on mitochondrial functions and insulin resistance in rodents.

Thirty-seven male (~400 g) Wistar rats were randomly divided into 3 groups and received during
8 wk either: oleic acid (C18:1-9 cis, OLE), elaidic acid (C18:1-9 trans, ELA) and vaccenic acid
(C18:1-11 trans, VAC) enriched diet. These iso-energy diets brought 96 Kcal/d and were balanced
in nutrients, vitamins, fibres and micronutriments. All diets had the same base and were enriched
at 4% of total energy intake with the fatty acid of interest. Insulin sensitivity was evaluated by the
glycemic and insulinemic responses to an intra-peritoneal injection of glucose (1 g/kg) (Chanseaume
et al., 2006). The mitochondrial OXPHOS capacities were assayed by oxygraphy (state 3 with
ADP 360µM) on isolated mitochondria from soleus and tibialis anterior with the substrates of the
respiratory chain glutamate, malate and succinate (5, 2.5 and 5 mM). ATP production was measured
by luminometry using the luciferase/luciferine reaction. P/O ratio which estimate OXPHOS coupling
was calculated. Respiratory control ratio (RCR) was calculated by dividing state 3 by state 4. The
mitochondrial superoxide anion radical (MSR) production was assayed using lucigenin (25 µM)
as chemilumigenic probe. Citrate synthase (CS) and cytochrome c oxidase (COX) activities were
determined spectrophotometrically.
Results
At slaughter, no differences in total body (588 ± 28 g) and tissue weight were observed between the
three groups. Insulin and glucose area under the curve (AUC) were similar in all groups (AUCI =
26.7 ± 7.8 nM and AUCG = 1457 ± 256 mM). Moreover, no group was insulin resistant. In soleus, no
differences in state 3 oxygen uptake rate was observed between the three groups, but ATP production
and the P/O ratio were higher in the OLE group than in the trans groups (P<0.01 vs. ELA and VAC).
Furthermore, in comparison with the trans groups, MSR production was increased in the presence
or not of rotenone in the OLE group (P<0.05 vs. VAC and ELA). CS activity was higher in the OLE
group than in the trans groups (P<0.05 vs. VAC and ELA) but COX activity was not modified (Table
1). In the tibialis anterior, state 3 oxygen uptake, ATP production, P/O and MSR production were
similar in the three groups. As in soleus, CS activity was higher in the OLE group than in the trans
groups (P<0.05 vs. VAC and ELA) but COX activity was not modified.

Table 1. Soleus mitochondrial function after 8 wk of feeding with the different diets (1,2,3).
OLE ELA VAC P value
OXPHOS (oxidative phosphorylation) activities

State 3 214.1±25.2 224.9±31.2 203.6±40.3 NS
State 4 54.7±7.4 50.6±9.0 50.4±9.3 NS
ATR 40.1±9.4 33.8±17.5 34.8±11.6 ND
RCR 3.95±0.53 4.52±0.71 4.08±0.43 NS
ATP 655.9±142.1a 479.2±126.9b 498.5±111.9b <0.01
P/O 3.061±0.608a 2.207±0.471b 2.658±0.558ab <0.01
MSR (mitochondrial superoxide anion radical) production

Rotenone – 65.7±21.6a 42.7±14.9b 37.4±14.0b <0.05
Rotenone + 73.6±21.1a 49.1±18.0b 42.2±16.1b <0.05
Mitochondrial enzyme activities

CS4 2.056±0.533a 1.610±0.287b 1.559±0.121b <0.05
COX5 3.271±1.173 3.045±0.683 3.162±0.414 NS
Values are means±SD. Means in line without a common letter differ, P<0.05; NS, non significant, P>0.05; ND, not
determined. The atractyloside test was used to validate the quality of mitochondrial preparation; CS: citrate synthase
and COX: cytochrome c oxidase.
Conclusion
These results suggest that the trans fatty acids of natural and industrial origin have similar effects
on mitochondrial oxidative functions and do not induce the development of insulino resistance.
On the contrary, in comparison with their cis counterparts they induce a decrease of mitochondrial
functions.
References
Chanseaume, E., C. Malpuech-Brugere, V. Patrac, G. Bielicki, P. Rousset, K. Couturier, J. Salles, J.P. Renou, Y. Boirie
and B. Morio, 2006. Diets high in sugar, fat, and energy induce muscle type-specific adaptations in mitochondrial
functions in rats. J. Nutr. 136, 2194-2200.
Lovejoy, J.C., S.R. Smith, C.M. Champagne, M.M. Most, M. Lefevre, J.P. DeLany, Y.M. Denkins, J.C. Rood, J. Veldhuis
and G.A. Bray, 2002. Effects of diets enriched in saturated (palmitic), monounsaturated (oleic), or trans (elaidic)
fatty acids on insulin sensitivity and substrate oxidation in healthy adults. Diabetes Care 25, 1283-1288.
Salmeron, J., F. Hu, J.E. Manson, M.J. Stampfer, G.A. Colditz, E.B. Rimm and W.C. Willett, 2001. Dietary fat intake
and risk of type 2 diabetes in women. Am. J. Clin. Nutr. 73, 1019-1026.

Up-regulation or activation of avian UCP attenuates mitochondrial ROS
production and oxidative damage in broiler chickens exposed to acute
heat stress
A. Mujahid, Y. Akiba, M. Kikusato and M. Toyomizu
Animal Nutrition & Biochemistry, Graduate School of Agricultural Science, Tohoku University, 1-1
Tsutsumidori-Amamiyamachi Sendai, 981-8555, Japan
Introduction
Our previous studies have shown that exposure to acute heat stress stimulates muscle mitochondrial
ROS production (Mujahid et al., 2006) and oxidative damage, possibly via down-regulation of
avUCP. Skulachev (1998) suggested that superoxide production may not only be enhanced through
substrate utilisation but also reduced by measures that decrease mitochondrial membrane potential.
In fact, Brand et al. (2004) demonstrated that superoxide production is sensitive to proton motive
force and can be decreased by mild uncoupling. Therefore, we hypothesised that up-regulation or
activation of avUCP can attenuate mitochondrial ROS production and oxidative damage in chickens
exposed to acute heat stress. Olive oil and CoQ10 were chosen to up-regulate avUCP and to activate
avUCP, respectively, in skeletal muscle mitochondria.

Forty 7-d-old broiler chickens (Cobb) were divided into 4 groups (n=10). One of three kinds of
diet was fed to each group for 8 d, i.e., a commercial diet was fed to 2 groups, one of which was a
normal control group (not exposed to heat stress) and the other was a heat-stressed control group.
An olive oil-containing diet and a CoQ10-containing diet were fed to two other heat-stressed
groups, respectively. Heat-stressed groups were exposed to 34 °C for 12 h while the control group
was maintained at 25 °C. The skeletal muscle pectoralis superficialis was used for the isolation of
total mitochondria by homogenisation, protein digestion and differential centrifugation. Muscle
mitochondria (0.35 mg/mL) were incubated in standard incubation buffer (120 mM KCl, 5 mM
KH2PO4, 3 mM HEPES, 1 mM EGTA and 0.3% (w/v) BSA, pH 7.2, 37 °C), containing 50 µg/mL
PHPA, 4 U/mL horseradish peroxidase, and 30 U/mL superoxide dismutase. After addition of 4 mM
succinate, the hydrogen peroxide generation rates were determined spectrofluorimetrically from the
change in fluorescence at excitation and emission wavelengths of 320 and 400 nm, respectively.
We also measured avUCP mRNA and protein expression (Real-time PCR and Western blotting)
and oxidative damage (MDA). The HNE-sensitive portion of CAT-upregulated mitochondrial ROS
production was calculated as the difference between CAT addition (5 µM) and CAT plus HNE (10
µM) treatments in order to estimate avUCP activity as shown in our previous paper (Abe et al.,
2006). Data were first analysed by a general linear model analysis of variance procedure and means
were compared using Duncan least significance multiple-range test. Differences were considered
significant for values of P<0.05.
Results and discussion

On exposure to heat stress, growth performance, feed consumption and feed efficiency were
significantly decreased in the control diet-fed chickens. This decrease in production performance on
exposure to heat stress was improved to some extent in olive oil- or CoQ10-fed chickens.
We confirmed that acute heat stress increased muscle mitochondrial ROS production (Figure 1a) and
MDA, and decreased avUCP mRNA transcript (Figure 1b) and protein content in skeletal muscle of
broiler chickens. Both the olive oil diet and the CoQ10 diet suppressed muscle mitochondrial ROS

a) H2O2 production b) avUCP mRNA c) avUCP activity
HNE-sensitive CAT-upregulated
H2 O2 as a ratio to basal H2 O2
Ratio of mRNA to 18s rRNA
0.4 2 0.6
a a
a
a
b
nmol/min/mg
c a
c b
0.2 1 0.3
b c
b
0 0 0
Olive Olive Olive
( ) CoQ oil ( ) CoQ oil ( ) CoQ
ol
ol
ol
oil
ntr
ntr
ntr
Heat stress Heat stress Heat stress
Co
Co
Co
Figure 1. Hydrogen peroxide production (a) avUCPmRNA and (b) HNE-sensitive portion of CAT-
Figure. Hydrogen peroxide production (a), avUCPmRNA (b) and HNE-sensitive portion of CAT-upregulated
upregulated hydrogen
hydrogen peroxide peroxide(C)
production production (c) inisolated
in mitochondria mitochondria isolated
from muscle fromand
of control muscle of control
nutritionally regulated,
andheat-stressed
nutritionallybroiler chickens. a-cp < 0.05 compared among groups for different treatments.
regulated, heat-stressed broiler chickens. a-c P<0.05 compared among groups for
different treatments.
production (Figure 1a) and MDA in heat-stressed broilers. The decreases in avUCP transcript due
to heat stress recovered to baseline levels in the olive oil-fed group, but not in CoQ10-fed groups
(Figure 1b). However, the heat stress-suppressed avUCP activity was increased by feeding either
olive-oil or CoQ10 (Figure 1c). In conclusion, the increased muscle mitochondrial ROS production
and oxidative damage in chickens exposed to acute heat stress can be decreased by up-regulation of
avUCP (olive oil feeding) or its activity (CoQ10 feeding).
References
Abe, T., A. Mujahid, K. Sato, Y. Akiba and M. Toyomizu, 2006. Possible role of avian uncoupling protein in down-
regulating mitochondrial superoxide production in skeletal muscle of fasted chickens. FEBS Lett. 580, 4815-
4822.
Brand, M.D., C. Affourtit, T.C. Esteves, K. Green, A.J. Lambert, S. Miwa, J.L. Pakay and N. Parker, 2004. Mitochondrial
superoxide: production, biological effects, and activation of uncoupling proteins. Free Radic. Biol. Med. 37,
755-767.
Mujahid, A., K. Sato, Y. Akiba and M. Toyomizu, 2006. Acute heat stress stimulates mitochondrial superoxide production
in broiler skeletal muscle, possibly via downregulation of uncoupling protein content. Poult. Sci. 85, 1259-265.
Skulachev, V.P., 1998. Uncoupling: new approaches to an old problem of bioenergetics. Biochim. Biophys. Acta
1363, 100-124.

Relationships between hepatic mitochondrial function and residual feed
intake in growing beef calves
P.A. Lancaster1, G.E. Carstens1, J. Michal2, K.M. Brennan2, K.A. Johnson2, L.J. Slay1, L.O. Tedeschi1
and M.E. Davis3
1Texas A&M University, College Station, TX, USA
2Washington State University, Pullman, WA, USA
3Ohio State University, Columbus, OH, USA
Introduction
Residual feed intake (RFI) is a feed efficiency trait that quantifies inter-animal variance in dry matter
intake (DMI) that is unexplained by variation related to body weight (BW) and growth rate–efficient
animals are those that consume less DMI than expected for a given BW and growth rate. Herd et al.
(2004) estimated that approximately one-third of the biological variation in RFI of growing calves
could be explained by inter-animal differences in digestion, heat increment, composition of growth
and activity, and posited that the remaining two-thirds was linked to inter-animal variation in energy
expenditure. The objective of this study was to determine if calves with divergent RFI phenotypes
differ in hepatic mitochondrial function.

Angus heifers (n=29; initial BW=256.0 kg) were individually fed a grain-based diet (ME=2.85
Mcal/kg DM) for 70 d using Calan-gate feeders (study 1). In study 2, Santa Gertrudis steers (n=119;
initial BW=308.4 kg) were individually fed a roughage-based diet (ME=2.2 Mcal/kg DM) for 70 d
using a GrowSafe feeding system. Within each study, RFI was calculated as the residual from linear
regression of DMI on ADG and mid-test BW0.75. In study 1, heifers with low and high RFI (n=7)
that were <0.5 SD and >0.5 SD from the mean RFI of 0.00 ± 0.64 kg/d, respectively, were selected,
while in study 2, steers with low (n=6) and high (n=8) RFI that were <1.0 SD and >1.0 SD from the
mean RFI of 0.00 ± 0.86 kg/d, respectively, were selected for mitochondrial measurements.
Mitochondria were isolated from liver biopsy samples, and proton leak kinetics were determined
according to Bevilacqua et al. (2004). In study 1, mitochondria were isolated, packaged on ice and
shipped overnight to Washington State University for respiration and proton leak measurements.
However, in study 2, mitochondria were isolated and respiration and proton leak measurements were
determined in the College Station laboratory. Mitochondrial respiration and proton leak data were
analyzed using the mixed procedure of SAS with RFI group (low vs. high RFI) as a fixed effect
and sampling d as a random effect. Least square means were computed for RFI group and mean
comparisons were performed using Tukey post hoc test.

Overall (± SD) final BW, ADG and DMI for the 29 heifers were 320 ± 25 kg, 1.30 ± 0.15, and 9.43
± 1.14 kg/d, respectively. Heifers with low RFI consumed less (P<0.01) DMI (8.94 vs. 10.32 ± 0.33
kg/d) than heifers with high RFI, even though ADG (1.31 and 1.33 ± 0.05 kg/d) and final BW (326
and 322 ± 9 kg) were similar. In study 2, overall (± SD) final BW, ADG and DMI for steers were
369 ± 27 kg, 0.83 ± 0.16 and 9.48 ± 1.00 kg/d, respectively. Steers with low RFI consumed less
(P<0.01) DMI (8.36 vs. 10.99 ± 0.32 kg/d) than steers with high RFI, even though ADG (0.92 and
0.78 ± 0.07 kg/d) and final BW (382 and 359 ± 10 kg) were similar.

In study 1, heifers with low RFI tended (P<0.10) to have greater respiration after addition of FCCP
(uncoupler) than heifers with high RFI (Table 1). In addition, heifers with low RFI had a greater
(P<0.05) acceptor control ratio (ACR; state 3 to state 2 respiration) than heifers with high RFI. In
study 2, steers with low RFI had a greater (P<0.05) ACR than steers with high RFI. Collectively,
these data suggest that calves with low RFI have greater control of oxidative phosphorylation by
ADP concentration than calves with high RFI. In both studies 1 and 2, the respiratory control ratio
(RCR) was similar between calves with low and high RFI. These data were similar to results of
Bottje et al. (2002) who reported greater ACR of liver mitochondria for broilers with improved feed
efficiency (feed:gain ratio), but similar RCR. However, Kolath et al. (2006) reported a greater RCR,
but similar ACR in skeletal muscle mitochondria from calves with low compared to high RFI. In both
studies, calves with low RFI had similar state 2 and state 4 respiration rates, but numerically greater
respiration rates during state 3. Kolath et al. (2006) reported statistically greater rates of respiration
during state 2 and 3 for calves with low RFI. Maximum proton leak-dependent oxygen consumption
was similar between calves with low and high RFI for both heifers and steers. These data suggest
that ADP may have greater metabolic control of respiration in calves with low RFI.
Table 1. Respiration rates and proton leak kinetics of liver mitochondria isolated from heifers and
steers with low and high RFI.
Study 1 Study 2
Low Hight SE Low Hight SE
State 2 respiration, nM O2/mg protein 52.5 54.5 3.4 28.8 28.2 8.9
FCCP, nM O2/mg protein 230.4a 191.2b 15.5 41.5 40.3 15.5
ACR 2.15x 1.79y 0.11 0.078x 3.36y 0.68
RCR 2.07 1.85 0.11 5.20 4.70 1.11
Membrane potential, mV 186.1 169.0 8.4 192.3 195.1 5.42
Proton leak O2, nM O2/mg protein 62.0 61.4 12.5 18.8 19.0 2.8
1Maximum proton leak-dependent O2 consumption during proton leak measurement.

xyMeans with different superscripts within study differ (P<0.05).
abMeans with different superscripts within study differ (P<0.10).
References
Bevilacqua, L., J.J. Ramsey, K. Hagopian, R. Weindruch and M.-E. Harper, 2004. Effects of short-and medium-term
calorie restriction on muscle mitochondria proton leak and reactive oxygen species production. Am. J. Physiol.
Endocrinol. Metab. 286, E852-E861.
Bottje, W., Z.X. Tang, M. Iqbal, D. Cawthorn, R. Okimoto, T. Wing and M. Cooper, 2002. Association of mitochondrial
function with feed efficiency within a single genetic line of male broilers. Poult. Sci. 81, 546-555.
Herd, R.M., V.H. Oddy and E. Richardson, 2004. Biological basis for variation in residual feed intake in beef cattle:
Review of potential mechanisms. Austr. J. Exp. Agric. 44, 423-430.
Kolath, W.H., M.S. Kerley, J.W. Golden and D.H. Keisler, 2006. The relationship between mitochondrial function and
residual feed intake in Angus steers. J. Anim. Sci. 84, 861-865.

Interaction between adipose tissue and skeletal muscle in the control of
muscle mitochondrial functions
E. Chanséaume1, P. Rousset1, C. Gryson1, S. Walrand1, Y. Boirie2 and B. Morio1
1INRA UMR1019, Human Nutrition Laboratory, BP 321, 58 Montalembert Street, 63009 Clermont-
Ferrand, France
2INRA UMR1019, Human Nutrition Laboratory, BP 321, 58 Montalembert Street, 63009 Clermont-
Ferrand, France; University Clermont 1, UFR Médecine; CHU Clermont-Ferrand, 63000 Clermont-
Ferrand, France
Introduction
The metabolic syndrome (MS) is characterised by a group of metabolic disorders which are associated
with increased risk of cardiovascular disease and type 2 diabetes. Among these risk factors, abdominal
obesity and insulin resistance are now recognised to be highly predictive of eventual development
of metabolic diseases. Furthermore, a sedentary lifestyle is an additional risk factor of the MS.
In skeletal muscle, a reduction in mitochondrial oxidative activity is frequently reported in insulin

resistant, obese or type 2 diabetic patients (Simoneau et al., 1999; Kelley et al., 2002; Petersen et
al., 2004). This observation has led to consider mitochondrial dysfunction as a key factor in the
aetiology of insulin resistance, obesity and associated metabolic disorders (Petersen and Shulman,
2006). Nowadays, the causes responsible for the decline in muscle mitochondrial functions remain
to be elucidated.
The aim of our work was to clarify the link between abdominal obesity, insulin resistance and
mitochondrial dysfunction within skeletal muscle of sedentary healthy men.

A total of 60 healthy male volunteers, aged 35 to 50 were included on the criterion of their abdominal
adiposity evaluated by waist circumference (from 75 to over 102 cm) and separated into 4 groups
based on this criterion (Table 1). A transverse scan of the whole body was performed by using DEXA
(Hologic QDR 4500 X-Ray bone densimeter, Hologic, Waltham, MA) for determination of total and
regional (arms, legs and trunk) body composition. After an overnight fast, volunteers were submitted
to a 3 h oral glucose tolerance test (OGTT, 75 g glucose), with measurement of blood insulin and
glucose at baseline and 15, 30, 60, 90, 120, 150 and 180 min after the glucose load. The subjects
were characterised by complete blood test (including plasma concentration of lipid metabolites), and
assessment of muscle mitochondrial functions from vastus lateralis biopsy. Mitochondrial respiration
rates (glutamate/malate/succinate 5/2/5 mM and ADP 1mM) and ATP production (bioluminescence)
were investigated on permeabilised muscle fibres and reactive oxygen species production (superoxyde
anion O2-), on isolated mitochondria (bioluminescence) (Figure 1).

Preliminary results show that mitochondrial functions according to waist circumference delineate
a bell curve, and not a linear decrease. Changes in the pattern of plasma lipid metabolites do not
explain this relationship. Moreover, a negative correlation exists between abdominal adiposity and
muscle mitochondrial production of reactive oxygen species (r=-0.357, P<0.05). Our data suggest
that the adaptations of mitochondrial functions within skeletal muscle result from a complex
interaction between adipose tissue mass and distribution and do not only depend on plasma lipid

Table 1. Subject characteristics (n=15 subjects per group).
Group 1 Group 2 Group 3 Group 4 ANOVA

<88 cm 88-94 cm 94-102 cm >102 cm
Age, y 40.9 ± 3.3 40.5 ± 3.0 42.3 ± 5.4 42.0 ± 4.9 ns

WC, cm 82.1 ± 4.1a 90.7 ± 1.3b 97.0 ± 2.6c 111.8 ± 9.0d <0.0001
Weight, kg 70.1 ± 9.1a 79.3 ± 3.6b 84.9 ± 5.3b 101.5 ± 11.5c <0.0001
BMI, kg/m2 23.0 ± 2.4a 25.8 ± 1.4b 26.8 ± 1.8b 33.0 ± 3.4c <0.0001
% Truncal fat mass 18.2 ± 4.6a 23.0 ± 3.5b 24.7 ± 3.4b 32.8 ± 5.4c <0.0001
Triglyceride, g/L 1.06 ± 0.68a 1.23 ± 0.47a 1.72 ± 0.52b 1.67 ± 0.53b <0.01
Glucose, mM 4.59 ± 0.34a 4.99 ± 0.49b 4.99 ± 0.52b 5.04 ± 0.48b <0.05
Belfiore ISI 1.19 ± 0.18a 1.16 ± 0.23a 1.04 ± 0.20ab 0.92 ± 0.23b <0.01
WC = Waist circumference, BMI = Body mass index, ISI = Insulin sensitivity index (Belfiore et al., 1998).
Values are means ± SD. Values significantly different have different letters.
16 * *
14
12
Mitochondrial respiration rate 10

(glutamate/malate/succinate 5/2/5 mM 8
and ADP 1mM) in nat/min/mg fibers
6
1 2 3 4
4
2
0
Figure 1.
Figure 1. Mitochondrial
Mitochondrial respiration
respirationrate evaluated
rate on on
evaluated permeabilized muscle
permeabilised fibersfibres (*P<0.01 vs.
muscle
group 1).
*p<0.01 vs group 1
pattern. The involvement of adipocytokines in the control of mitochondrial functions is in the course
of investigation.
References
Simoneau, J.A., J.H. Veerkamp, L.P. Turcotte and D.E. Kelley, 1999. Markers of capacity to utilize fatty acids in human
skeletal muscle: relation to insulin resistance and obesity and effects of weight loss. FASEB J. 13, 2051-2060.
Kelley, D., J. He, E. Menshikova and V. Ritov, 2002. Dysfunction of mitochondria in human skeletal muscle in type
2 diabetes. Diabetes 51, 2944-2950.
Petersen, K.F., S. Dufour, D. Befroy, R. Garcia and G.I. Shulman, 2004. Impaired mitochondrial activity in the insulin-
resistant offspring of patients with type 2 diabetes. N. Engl. J. Med. 350, 664-671.
Petersen, K.F. and G.I. Shulman, 2006. Etiology of insulin resistance. Am. J. Med. 119, S10-16.
Belfiore, F., S. Iannello and G. Volpicelli, 1998. Insulin sensitivity indices calculated from basal and OGTT-induced
insulin, glucose, and FFA levels. Mol. Genet. Metab. 63, 134-141.

Influence of protein-free diet on morphology of rat hepatocytes’
mitochondrium
E. Sawosz1, A. Chwalibog2, M. Grodzik1, T. Niemiec1, J. Skomiał1 and I. Kosieradzka1
1Department of Animal Nutrition and Feed Science, Warsaw Agricultural University, Ciszewskiego
8, 02-786 Warsaw, Poland
2Department of Basic Animal and Veterinary Sciences, University of Copenhagen, Groennegaardsvej
3, 1870 Frederiksberg C, Denmark
Introduction
Nutrition, environment, age and especially stress can influence morphological and/or functional
state of liver mitochondria. Changes in number, size and distribution of mitochondria in the cells are
probably a consequence of energy consumption. A low level of dietary energy decreases the number
of mitochondria, leading to a decrease of ATP synthesis as well as a decrease of mitochondrial radical
oxygen species (ROS) production. Possibilities to reduce ROS production via dietary procedures
may be a remedy for disease prevention and longevity. Although proteins are secondary sources
of energy, they play a vital role in transport across the mitochondrial membranes and stimulate
mitochondrial division. The aim of the experiment was to evaluate the influence of protein free diet
on morphology of liver mitochondria in rats.

Twenty-four Wistar male rats (200-250 g) were divided into two groups, fed ad libitum two semi-
synthetic, isoenergetic diets (14.9 MJ ME/kg diet); the control (192 g crude protein/kg diet) and
protein-free diet. The rats were kept in individual cages for 14 d at 22 °C, 60-70% relative humidity
and 12 h lighting. At the end of the experiment, the rats were fasted for 12 h and then sedated by
intramuscular ketamine. Immediately after killing, the slices of liver were fixed in glutaraldehyde,
embedded in EPON polymerised at 60 °C and stained in uranyl acetate. Sections were stained with
lead citrate. The samples were examined in the electron-transmission microscope Joel type JEM
1220.
Results
Mitochondria are usually found as snakelike tubules, widely distributed in the cytoplasm and often
interconnected into extended mitochondrial reticula. These mitochondrial networks are extremely
dynamic, with tubular processes undergoing frequent fragmentation, branching, and fusion, as well
as redistribution within the cytoplasm (Sogo and Yaffe, 1994).
In the present experiment, a protein-free diet changed the distribution of mitochondria in hepatocytes,
decreased the number by 75% and significantly (P<0.001) increased their size (width × length) from
1.1 × 1.4 to 1.8 × 2.7 µm (Figure 1). Visualisations of the mitochondrial structures have shown
substantial changes between the control and protein-free diet, concerning particularly ribosomes,
seen in the matrix as small dark bodies, suggesting an inhibition of polyribosome formation (Schieber
and O’Brien, 1985). The potential coupling of mitochondrial metabolism to mitochondrial protein
synthesis in the heart mitochondria was investigated by McKee et al. (1990) who observed that
mitochondrial protein synthesis required the presence of oxidiseable amino acids (as well as the
presence of ATP, however, ATP added above 0.5 mM became progressively inhibitory).
Our preliminary results suggest that dietary stress provoked by protein-free diet can influence a
dynamic equilibrium between different mitochondrial units. Lack of dietary oxidised compounds and

Figure 1. Hepatocytes of rats in 6000 × magnification: a - control; b - protein free diet and in
30 000× magnification; c,d - control; e,f - protein free diet.
relative overproduction of ATP (Crowley and Payne, 1998) can inhibit transport of electrons from
NADH to oxygen and consequently production of ROS. Sanz et al. (2004) carried out experiments
with protein restricted diet given to rats, and suggested that the decrease in aging rate induced by
energy restriction can be partly due to the reduced intake of proteins acting through decreases in
mitochondrial ROS release and oxidative DNA damage.
The present results demonstrate that dietary stress provoked by a short-term application of a
protein-free diet can decrease the number of mitochondria but increase their size as well as shape.
These morphologic changes can indicate shortening of inner mitochondrial membrane length and
consequently reduction of oxidative phosphorylation level and ROS production.
References
Crowley, K.S. and R.M. Payne, 1998. Ribosome Binding to Mitochondria Is Regulated by GTP and the Transit Peptide.
J. Biol. Chem. 273, 17278-17285.
McKee, E.E., B.L. Grier, G.S. Thompson, A.C. Leung and J.D. McCourt, 1990. Coupling of mitochondrial metabolism
and protein synthesis in heart mitochondria. Am. J. Physiol. 258, E503-E510.
Sanz, A., P. Caro and G. Barja, 2004. Protein restriction without strong caloric restriction decreases mitochondrial
oxygen radical production and oxidative DNA damage in rat liver. J. Bioenerg. Biomembr. 36, 545-552.
Schieber, G.L. and T.W. O’Brien, 1985. Site of Synthesis of the Proteins of Mammalian Mitochondrial Ribosomes:
Evidence from Cultured Bovine Cells. J. Biol. Chem. 260, 6367-6372.
Sogo, L.F. and M.P. Yaffe, 1994. Regulaton of mitochondrial morphology and inheritance by Mdm10p, a protein of
the mitochondrial outer membrane. J. Cell Biol. 126, 1361-1373.

Expression of uncoupling proteins and mitochondrial activity are
dependent on muscular fibre type in rabbits and chickens
R. Joubert1, A. Collin1, C. Berri1, L. Lefaucheur2, A. Vincent2, M. Fillaut2, P. Ecolan2, L. Mur1, E.
Godet1, S. Crochet1, T. Bordeau1, S. Skiba-Cassy3, S. Tesseraud1, P. Herpin4 and M. Damon2
1INRA, UR83 Recherches Avicoles, F-37380 Nouzilly, France
2INRA, UMR1079 Systèmes d’Elevage, Nutrition Animale et Humaine, F-35590 Saint-Gilles,
France
3INRA, UMR1067 Nutrition Aquaculture et Génomique, F-64310 Saint-Pée-sur-Nivelle, France
4INRA, Direction Scientifique Adjointe Animal et Produits Animaux, F-35590 Saint-Gilles,
France
Introduction
Uncoupling proteins (UCP) play a key role in the mitochondrial utilisation of energy substrates.
They could be involved in the mitochondrial proton leak (or uncoupling) that lowers the efficiency
of oxidative phosphorylation, in fatty acid or metabolic anion transports, and could also limit the
mitochondrial production of reactive oxygen species (ROS; Criscuolo et al., 2006). In mammals,
a higher expression of UCP3 has been reported in glycolytic muscles compared to oxidative ones
(Hesselink et al., 2001). In the chicken, the role of avUCP remains to be elucidated: this protein seems
to limit oxidative stress, but it could also be involved, as the mammalian UCP1, in thermogenesis
and fatty acid utilisation in muscles. The aim of this study was then to characterise the expression
of rabbit UCP3 and chicken avUCP according to muscle fibre type in order to relate their expression
to mitochondrial function.

In each species, UCP mRNA expression was analysed by real-time RT-PCR in either slow oxidative
(white part of the Adductor superficialis in the chicken and Semi-membranosus proprius in the rabbit)
or fast glycolytic (Pectoralis major in the chicken and Psoas major in the rabbit) muscle types. In the
chicken, expressions were also measured in other muscles characterised by intermediary metabolic
and contractile properties. The results are expressed as ratios between UCP mRNA and 18S rRNA.
Mitochondrial respiration rate and its regulation by a stimulator (hydroxynonenal HNE) and an
inhibitor (guanosine diphosphate GDP) of UCP activity were studied in isolated permeabilised
fibres from the glycolytic and oxidative muscles in the rabbit. Because of the fragility of chicken
isolated fibres, those parameters (as well as the stimulation of respiration by palmitate) were studied
in isolated muscle mitochondria in the chicken. The results were analysed by ANOVA and means
were compared using a Student test.

Both rabbit UCP3 and chicken avUCP expressions were higher in muscles containing fast glycolytic
fibres than in those containing slow oxidative ones (Figure 1). In the chicken, the fast oxido-glycolytic
Iliotibialis muscle also exhibited a high avUCP mRNA expression, while mixed types of muscles
presented intermediary expressions.
We next investigated if the higher expression of these UCP in fast muscles was associated with
increased stimulation of mitochondrial respiration by HNE or palmitate and greater inhibition by GDP
compared to slow oxidative muscles. In the rabbit, but not in the chicken, the higher expression of
UCP in glycolytic fibres was associated with a lower respiratory control ratio, indicative of a higher
uncoupling in glycolytic compared to oxidative muscle fibres. In both species, HNE- or palmitate-

Figure 1. Effect of muscle type on mRNA expression of rabbit UCP3 and chicken avUCP. In the
rabbit, SMP: Semi-membranosus proprius, PsM: Psoas major. In the chicken: ADSW and ADSR =
white and red parts of Adductor superficialis, respectively; ALD: Anterior latissimus dorsi, ADP:
Adductor profundus, SART: Sartorius, Ilio: Iliotibialis, PLD: Posterior latissimus dorsi, PeM:
Pectoralis major. a,b Within a species, means exhibiting different letters were significantly different
(P<0.05).
stimulated oxygen consumption was not higher in glycolytic fibres than in oxidative ones. In rabbit
fibres, HNE-stimulated oxygen consumption was not inhibited by GDP (2.5 mM) in the glycolytic
fibres and only slightly decreased in the oxidative ones. In chicken mitochondria, no inhibition of
respiration by GDP (2.5 mM) was shown after HNE or palmitate adjunction, while a higher GDP
concentration (5 mM) totally abolished the palmitate effect on respiration in both oxidative and
glycolytic muscle mitochondria.
Taken together, the present data do not support the hypothesis of a strong involvement of UCP3 and
avUCP in mitochondrial uncoupling of oxidative phosphorylation in rabbit and chicken muscles.
These proteins might rather be involved in the limitation of ROS production, which is reported to
be potentially higher in fast glycolytic fibres in mammals.
References
Criscuolo, F., J. Mozo, C. Hurtaud, T. Nubel and F. Bouillaud, 2006. UCP2, UCP3, avUCP, what do they do when
proton transport is not stimulated? Possible relevance to pyruvate and glutamine metabolism. Biochim. Biophys.
Acta 1757, 1284-1291.
Hesselink, M.K., H.A. Keizer, L.B. Borghouts, G. Schaart, C.F. Kornips, L.J. Slieker, K.W. Sloop, W.H. Saris and P.
Schrauwen, 2001. Protein expression of UCP3 differs between human type 1, type 2a, and type 2b fibers. FASEB
J. 15, 1071-1073.

Regulation of mitochondrial and tissue oxidations by thyroid hormones
in chicken muscle
A. Collin1, Q. Swennen2, S. Métayer Coustard1, S. Skiba-Cassy3, R. Joubert1, G. Briclot1, S. Crochet1,
E. Decuypere2, J. Buyse2 and S. Tesseraud1
2Laboratory for Livestock Physiology and Immunology, Department of Biosystems, Katholieke
Universiteit Leuven, Kasteelpark Arenberg 30, 3001 Leuven, Belgium
3INRA, UMR1067 Nutrition Aquaculture et Génomique, F-64310 Saint-Pée-sur-Nivelle, France
Introduction
Thyroid hormones are major hormones regulating development and energy expenditure in mammals
and in avian species (Decuypere et al., 1981; Silva, 1995). They modulate transcription of target
genes by interacting with intracellular thyroid receptors that bind to thyroid hormone response
elements (TRE) located on DNA. They can also act as direct stimulators of enzymes involved in
the respiratory machinery (Goglia et al., 1999). In mammals, thyroid hormones are also shown
to regulate fatty acid utilisation (Lanni et al., 2005). As in mammals, oxidative pathways might
be affected by thyroid status in the chicken. The aim of this study was therefore to investigate the
impact of thyroid hormones on the expression of genes involved in energy substrate utilisation and
on oxidative stress in the chicken Gastrocnemius muscle.

Eighteen chickens received, from one wk of age, either a standard diet (Control group), or a standard
diet supplemented with triiodothyronine T3 (T3 group; 1 mg/kg feed) or with the thyroid gland
inhibitor methimazole (MMI group; 1 g/kg feed), before being sacrificed at 28 d of age. MMI and T3
chickens were then clearly hypothyroid and hyperthyroid, respectively, with plasma T3 concentrations
of 0.24 pmol/mL and 9.30 pmol/mL, as compared to control chickens (3.17 pmol/mL; Collin et
al., 2003). To evaluate the effect of thyroid status on the regulation of muscular mitochondrial
oxidations, mRNA expression of genes involved in mitochondrial fatty acid transfers (muscle and
liver carnitine palmitoyltransferase 1: respectively M-CPT1 and L-CPT1; Skiba-Cassy et al., 2007),
fatty acid oxidation (hydroxyacyl dehydrogenase HAD) and respiratory chain activity (cytochrome
oxidase COX) were investigated by real-time Polymerase Chain Reaction. The protein expression
and activation of AMP-activated protein kinase (AMPK), a sensor of cellular energy status, was
determined by Western blot analysis. Oxidative stress in the Gastrocnemius muscle was evaluated by
measuring the thiobarbituric acid reactive substances (TBARS) and the ferric reducing/antioxidant
power (FRAP).

The results indicate highly significant overexpression of the messenger RNA studied, except for M-
CPT1, in T3-treated chickens as compared to control and MMI-treated chickens (Figure 1). Indeed,
the hepatic isoform of CPT1, previously shown to be regulated by nutritional status in chicken
muscles (Skiba-Cassy et al., 2007), and mRNA of the enzymes COX and HAD, were five to six-
fold increased in the T3 group as compared to MMI-treated and control chickens. This suggests a
stimulation of oxidative pathways by T3 in chicken muscle mitochondria.
Phosphorylation of the AMPKα1 catalytic subunit relative to AMPKα total protein was found to
be higher in the control and T3-treated groups than in the methimazole-treated one. Since AMPK is

Figure 1. Effect of thyroid status (MMI: hypothyroid chickens; T3: hyperthyroid chickens) on mRNA
expression of several genes involved in mitochondrial oxidations.
involved in the activation of pathways controlling glucose and fatty acid utilisation, this result could
reflect a higher stimulation of fatty acid oxidation in the control and hyperthyroid groups.
Finally, such an increase in muscular fatty acid oxidations in T3-treated chickens could favour
oxidative stress. Even though FRAP data were not different between treatments, TBARS were 4.7
and 10.4 fold higher in the T3-treated group than in the control and in the MMI groups, respectively.
These findings suggest an activation of muscle tissue peroxidation in the hyperthyroid state as
compared to the control or hypothyroid state.
In conclusion, this study highlights clear effects of thyroid hormones on fatty acid utilisation and
lipid peroxidation in the chicken muscle.
References
Collin, A., M. Taouis, J. Buyse, N.B. Ifuta, V.M. Darras, P. Van As, R.D. Malheiros, V.M.B. Moraes and E. Decuypere,
2003. Thyroid status, but not insulin status, affects expression of avian uncoupling protein mRNA in chicken. Am.
J. Physiol. Endocrinol. Metab. 284 (4), E771-E777.
Decuypere, E., S.C. Hermans, H. Michels, E.R. Kühn and J. Verheyen, 1981. Thermoregula-tory response and thyroid
hormone concentrations after cold exposure in young chicks treated with iopanoic acid and saline. Adv. Physiol.
Sci. 33, 291-298.
Goglia, F., M. Moreno and A. Lanni, 1999. Action of thyroid hormones at the cellular level: the mitochondrial target.
FEBS Lett. 452, 115-120.
Lanni, A., M. Moreno, A. Lombardi, P. de Lange, E. Silvestri, M. Ragni, P. Farina, G.C. Baccari, P. Fallahi, A. Antonelli
and F. Goglia, 2005. 3,5-diiodo-L-thyronine powerfully reduces adiposity in rats by increasing the burning of
fats. FASEB J. 19, 1552-1554.
Silva, J.E., 1995. Thyroid hormone control of thermogenesis and energy balance. Thyroid 5, 481-492.
Skiba-Cassy, S., A. Collin, P. Chartrin, F. Médale, J. Simon, M.J. Duclos and S. Tesseraud, 2007. Chicken liver and
muscle carnitine palmitoyltransferase 1: nutritional regulation of messengers. Comp. Biochem. Physiol. B, doi:
10.1016/j.cbpb.2007.01.007.

Variation in animal energy expenditure and mitochondrial function and
protein expression
J. Michal, K. Brennan, K. Ross and K.A. Johnson
Washington State University, Department of Animal Sciences, 99164, Pullman, USA
Introduction
Mitochondrial uncoupling is a potential source of variation in energy use, leading to the hypothesis
that animals with greater mitochondrial uncoupling are less efficient. Mitochondria are not completely
coupled (Rolfe and Brand, 1997) and proton leak may be 25% of basal metabolic rate (Brookes,
2005). The mechanism of proton leak and uncoupling is not well understood, but uncoupling
proteins (UCP) may play a role. Human genomic analysis found 3 markers in the UCP2 locus that
are linked to resting metabolic rate (Bouchard et al., 1997). UCP3 knockout mice have more coupled
mitochondria and abnormal skeletal muscle mitochondrial respiration and mice over expressing
UCP3 have reduced energy efficiency. Adenine nucleotide translocase (ANT1) facilitates transport
of ATP and ADP across the inner mitochondrial membrane (Sharer, 2005). Bovine factor B (FacB)
is a subunit of ATP synthase required for the activity of the mitochondrial ATP synthase complex
(Belogrudov, 2006). Expression of these important genes may help explain variation in energy
efficiency. The objective of this study was to examine the contribution of mitochondrial metabolism
and mRNA abundance to whole animal energy expenditure.

Heat production (HP), fasting heat production (FHP), and metabolizable energy for maintenance
(MEm) from Angus, Holstein and Wagyu heifers (n=8/breed; 10 mo) were measured using open-
circuit, indirect respiration calorimetry. Heifers were measured at low (L; 0.13 Mcal NEm/kg0.75),
and fasting (F) levels of intake. Assuming a semilog relationship between HP and ME intake (MEI),
MEm was determined by iteratively solving for the point where MEI equals HP. At the L level
of intake mitochondria were isolated from liver biopsies from the Angus and Wagyu heifers and
added to respiration medium for simultaneous measurement of oxygen consumption and membrane
potential at 37 °C. RNA was extracted from muscle biopsies of the Biceps femoris. Gene specific
primers for UCP2, UCP3, ANT1, and FacB were designed based on available bovine sequences
(Accession no: AF127029, AF092048, BC102994, AY780292, respectively). Relative abundances
were quantified by real-time PCR using β-Actin as a reference gene. The ratio of each target gene
in a sample was expressed versus a pooled control cDNA sample. SAS (PROC GLM) was used to
examine the effect of breed on transcript abundances for each target gene and PROC CORR was
used to identify relationships between mRNA gene expression and measures of energy metabolism
and proton leak.

Whole animal breed differences were found in HP, FHP, and MEm where Angus and Wagyu were
different than Holsteins (Table 1). Breed differences were also apparent in liver mitochondrial
efficiency measures including O2 consumption and membrane potential at the same leak rate.
For a given membrane potential Angus mitochondria had 2-2.5 fold higher proton leak rates than
Wagyu mitochondria (Figure 1). No breed differences were noted in UCP2, UCP3, ANT1, and
FacB expression. No significant correlations were identified between whole animal measures and
gene expression data. Gene expression patterns among three breeds of cattle were similar despite
differences in whole animal energy expenditure and mitochondrial energy metabolism.

Table 1. Whole animal and mitochondrial energy expenditure and relative abundance of mRNA for
several genes involved in mitochondrial energy metabolism.
Item Angus Holstein Wagyu SE
HP, kcal/kg0.75 145.3a 169.4b 145.3a 2.8

FHP, kcal/kg0.75 91.8a 109.6b 92.0a 2.6
MEm, kcal/kg0.75 113.5c 137.1d 105.2c 5.2
Hepatic mito O21 11.8e - 7.52f 1.4
Maximal membrane potential, mV 118.9c - 145.4d 5.1
UCP2 0.71 0.57 0.60 0.37
UCP3 2.17 1.8 1.17 0.37
ANT1 0.89 1.06 1.00 0.11
FacB 1.64 1.87 1.53 0.23
1nmoles O/min/mg protein; ab P<0.01; cd P<0.0001; ef P<0.05.
Figure 1. Proton leak kinetics in growing Angus and Wagyu heifers.
References
Belogrudov, G.I., 2006. Bovine factor B: Cloning, expression, and characterization. Arch. Biochem. Biophys. 451,
68-78.
Bouchard, C., L. Perusse, Y.C. Chagnon, C. Warden and D. Ricquier, 1997. Linkage between markers in the vicinity
of the UCP2 gene and resting metabolic rate in humans. Human Mol. Gen. 6, 1887-1889.
Brookes, P.S., 2005. Mitochondrial H(+) leak and ROS generation: an odd couple. Free Rad. Biol. Med. 38, 12-23.
Rolfe, D.F. and M.D. Brand, 1997. The physiological significance of mitochondrial proton leak in animal cells and
tissues. Biosci. Rep. 17, 9-16.
Sharer, J.D., 2005. The adenine nucleotide translocase type 1 (ANT1): a new factor in mitochondrial disease. Life
57, 607-614.

Cardiac muscle UCP-3 expression in relation to nutrient intake and beef
cattle metabolism
G.K. Murdoch1, W.T. Dixon2, J. Moibi2, B.T. Li2, M. Vinsky2 and R.J. Christopherson2
1University of Idaho, Department of Animal and Veterinary Science, College of Agricultural and
Life Sciences, Moscow, Idaho 83844-2330, USA
2University of Alberta, Department of Agricultural, Food and Nutritional Science, Edmonton,
Alberta, T6G 2P5 Canada
Introduction
Measurement of feed efficiency of cattle is important but it is essential that we develop the
accompanying understanding of the physiological, genetic and molecular basis of the observed
variability. This will allow utilization of gene markers for targeted selection programs, which is
a critical step for improving beef production efficiency in a sustainable manner. We reported an
association between uncoupling protein mRNA expression in adipose and skeletal muscle from acute
cold-stressed steers (Murdoch et al., 2005). Therefore, we tested the hypothesis that UCP-3 mRNA
expression in less frequently studied but metabolically important tissue; such as cardiac muscle,
may be associated with whole-body metabolism.

Thirty-six steers (3 breeds) individually fed either 1.2 times maintenance (1.2M) or higher (2.2M) of
a standard feedlot diet (90% w/w Barley conc., 10% w/w roughage, on average, ME 14.496 MJ/kg of
DM; CP 14.93% w/w DM) in a two-period cross-over design. Indirect calorimetry measures of HP
were taken during the last 10 d of each period. Average daily gain (ADG), energy retention (MER),
and maintenance requirement (MEm) were assessed. Post-mortem cardiac samples were collected
for RNA extraction. Rt-Polymerase Chain Reaction (PCR) with gene specific primers was used to
evaluate the UCP-3 mRNA content in each sample, relative to G3PDH.

Energy retention (MJ/d/kg0.75) was higher (P<0.05) in the 2.2M compared to the 1.2M, but there
was no effect of breed or interaction of breed and feeding (Table 1). The change in energy retention
(difference in ER at 2.2M and 1.2M intakes for each animal) per change in MEI was not affected
by breed type (P>0.05) (Table 1). Heat production (HP) (MJ/d/kg0.75) by the animals was increased
(P<0.05) at the 2.2M feeding when compared to the 1.2M. Our novel measures of UCP-3 mRNA
in bovine cardiac muscle did not correlate with measures of HP, MEI, ADG or MEm measured in
individual steers; UCP-3 mRNA in cardiac muscle was negatively correlated (P=0.04, r=-0.34)
with MER (Table 2B). No significant differences in UCP-3 mRNA relative to breed or feeding level
alone were noted (Table 2A).
As expected HP increases with the higher MEI, though there were not any significant breed-specific
differences in this increase. Large variability between individual animal’s UCP-3 mRNA, was typical
both within and between our breeds. Cardiac UCP-3 did not significantly correlate to measures of HP,
ADG nor MEm values for individual animals at either feeding level, which is perhaps not surprising
given this is but a single tissue. However, cardiac UCP-3 mRNA is inversely associated with MER,
which is novel and suggests that selection for high energy retention may inadvertently select against
cardiac UCP-3 content. Cardiac UCP-3 may be involved in fatty acid transport as well as reduction
in reactive oxygen species accumulation in scenarios of high metabolism (Bezaire et al., 2007)
outside of its possible role in mitochondrial energetic efficiency, any inadvertent selection against

Table 1. Metabolic measures for 3 breeds at 2 feeding levels.
Breed Angus Brangus Charolais
1.2M 2.2M SEM 1.2M 2.2M SEM 1.2M 2.2M SEM

Item
Weight gain, g/d 439.8b 954.5a 46.6 500.2b 1100.7a 61.8 491.2b 1030.9a 40.7
MEI, MJ/d/kg0.75 0.668b 1.195a 0.02 0.673b 1.168a 0.02 0.684b 1.144a 0.02
Heat production, 0.483b 0.70a 0.012 0.481b 0.711a 0.015 0.524b 0.696a 0.012
MJ/d/kg0.75
Energy retention, 0.186b 0.495a 0.023 0.195b 0.475a 0.031 0.160b 0.448a 0.022
MJ/d/kg0.75
a,b Statistically different (P<0.05), between different letters.
Table 2. (A) Cardiac muscle UCP-3 mRNA, relative to breed and feeding level, (B) UCP-3 correlations
with metabolic measures.
(A) UCP-3 mRNA (B) Correlation (r)
Feeding level 2.2M 587.6±25.0 Heat production -0.09

1.2M 603.1±40.5 ADG, g/kg0.75 -0.15
Breed Angus 598.9±34.3 MEI, MJ/kgbw0.75 -0.29

Brahman-Angus 596.3±34.3 MER -0.34
Charolais 591.6±34.3 ME maintenance 0.06
*Arbitrary densitometric units.
its presence in cardiac muscle may be deleterious. International use of ‘Residual feed intake’ (RFI)
for the selection of cattle with better feed-utilization attributes is becoming more common place
though the physiological causes of RFI variability have not been fully characterized, as such we
believe these are important preliminary results to present. Kolath et al. (2006) reported no significant
difference in longissmus lumborum UCP-2 or UCP-3 mRNA expression in relation to high or low
residual feed intake values, this single measure may not accurately reflect an individual animal’s
capacity to up-regulate UCP expression, nor represent expression of UCP3 outside of skeletal muscle.
It has yet to be determined whether the high RFI animals retain a greater capacity to increase their
respective UCP content in response to variable thermoregulatory or metabolite utilization demands
as compared with low RFI animals.
References
Bezaire, V., E.L. Seifert and M.-E. Harper, 2007. Uncoupling protein-3: clues in an ongoing mitochondrial mystery.
FASEB J. 21, 312-324.
Kolath, W.H., M.S. Keeley, J.W. Golden, S.A. Shahid and G.S. Johnson, 2006. The relationships among mitochondrial
uncoupling protein 2 and 3 expression, mitochondrial deoxyribonucleic acid single nucleotide polymorphisms,
and residual feed intake in Angus steers. J. Anim. Sci., 84, 1761-1766.
Murdoch, G.K., W.T. Dixon, E.K. Okine and R.J. Christopherson, 2005. Bovine tissue mRNA abundance related to
acute cold exposure and acute feeding restriction. Can. J. Anim. Sci. 85, 157-164.

Inter-muscular variability of metabolic properties of bovine muscle
fibres
M.P. Oury1, C. Jurie2, J.F. Hocquette2 and B. Picard2
1ENESAD, SEQAV, BP 87999, 21079 Dijon Cedex, France
2INRA, URH, C2M, Theix, 63122 Saint-Genès-Champanelle, France
Introduction
Studies on muscle physiology are of interest for medical and agronomical applications. The variability
of contractile and metabolic properties between different muscles is important (Hocquette et al.,
1996). Moreover, there also is variability in the contractile and metabolic properties of the same type
of fibres in different muscle. This variability has not been well analysed. So, the aim of this study
was to compare these properties in different muscle types located at different places on the carcass:
the rectus abdominis (RA), the longissimus thoracis (LT) and the triceps brachii (TB).

Samples were removed 24 h after slaughtering. The anaerobic glycolytic metabolism was assessed
by lactate dehydrogenase (LDH) activity (Ansay, 1974). The aerobic oxidative one was evaluated by
isocitrate dehydrogenase (ICDH; Briand et al., 1981) and cytochrome-c oxydase (COX; Piot et al.,
1998) activities. Muscle fibre size was determined by computerised image-analysis on 10-µm thick
sections cut with a cryotome at -25 °C. The delimitation of the major fibre types was based on the
combination of two histological classification schemes: myofibrillar ATPase (ATP; Guth and Samaha,
1972) and succinate dehydrogenase (SDH; Pearse, 1986) activities. Immunohistochemistry allowed
the identification of pure and hybrid fibres (Pons et al., 1986). The different types of myosin heavy
chain isoforms were determined on the basis of previously determined migration pattern (Picard
et al., 1995) on SDS polyacrylamide gel electrophoresis (Laemmli, 1970). The intramuscular lipid
content was determined by the Soxhlet standard method.

Comparison of rectus abdominis (RA) and triceps brachii (TB)
SO (slow oxidative), FOG (fast oxido-glycolytic) and FG (fast glycolytic) fibre areas were significantly
higher in the RA (from +18 to +129%). So, the mean area of fibres was also higher (+38%). The
RA also had a higher proportion of SO fibres (+35%) and a lower proportion of FOG fibres (-58%).
It is difficult to differentiate the fast FOG and FG fibres in the RA because the revelation of SDH
activity on histochemical sections is very low. Thus, we may have over/under-estimated the FOG/FG
fibres. Three reasons may explain the previous observation: i) the FOG fibres may be less oxidative
in the RA than in the TB, ii) the RA may have a smaller content in mitochondria than the TB, iii)
the enzymatic activity may be smaller in the RA than in the TB. We confirmed the third suggestion,
since the RA has enzymes with lower activities of ICDH (-49%), LDH (-19%) and COX (-27%)
than the TB. Despite this lower oxidative metabolism, the RA had more oxidative myosin isoforms
(MyHCI and IIa) than the TB and also had more lipids (+42%), as already observed (Brackebush
et al., 1991). In the RA, the FG fibres were the smallest ones, whereas the SO fibres were the
biggest ones (2868 vs. 3957 µm²). These observations contrast with those found on usually studied
muscles, where the SO fibres have the smallest area and FG the biggest ones (Picard et al., 2004).
The hypothesis of over-estimation of the FOG fibres was confirmed by the immuno-histochemistry
results. Indeed, they led to a higher proportion of I fibres (+57%) and a lower proportion of IIX
ones (-50%) in the RA.

Comparison rectus abdominis (RA) and longissimus thoracis (LT)
The mean area of fibres was significantly higher in the RA than in the LT (+28%). ICDH and COX
activities were higher (+8%; +60%), whereas LDH activity was lower (-45%) in the RA compared to
the LT. The RA seemed to have a more oxidative metabolism. That is associated with a higher content
in intramuscular fat (+14%). The proportions of the different myosin isoforms confirmed as well
these conclusions. Indeed, the RA appeared to have a higher proportion of MyHCI (+25%), balanced
by a lower proportion of MyHCIIa (-16%). No significant correlation was found between MyHCIIa
proportions and the oxidative enzyme activities in the RA, whereas the MyHCIIa proportions were
positively linked with ICDH (r=+0.31) and COX (r=+0.21) activities in the LT. Thus, it appeared
that the IIa isoforms have a more oxidative metabolism in the LT than in the RA. In the LT, previous
results indicated that IIA fibres, that contain IIa isoforms, might be separated into oxidative and
non-oxidative IIA fibres depending on their metabolic properties (Picard et al., 1998). In the RA,
the rather low SDH activity does not allow to distinguish these two types of IIA fibres (Picard et al.,
2004). So, it is not surprising to find that IIa isoforms are more oxidative in LT than in RA.
Conclusion
These two comparisons illustrated that the RA muscle has some particularities of fibre properties.
It appears that the FG fibres were the smallest, whereas the SO were the biggest. Indeed, it seems
that either the mitochondria number is lower in the RA or their metabolism has been exposed to a
modification, leading to a decrease of the SDH activity and muscular metabolism. This could explain
why the response of muscles to factors of variation such as nutritional factors could be different
according to the muscle studied.
References
Ansay, M., 1974. Individualité musculaire chez le bovin: étude de l’équipement enzymatique de quelques muscles.
Ann. Biol. Anim. Biochim. Biophys. 14, 471-486.
Brackebush, S.A., F.K. McKeith, T.R.Carr and D.G. McLaren, 1991. Relationship between longissimus composition
and the composition of the major muscles of the beef carcass. J. Anim. Sci. 69, 631-640.
Briand, M., A. Tamant, Y. Briand, G. Monin and B. Durand, 1981. Metabolic types of muscle in the sheep: I myosin
ATPase glycolytic and mitochondrial enzyme activities. Eur. J. Appl. Physiol. 46, 347-358.
Guth, H.L. and F.J. Samaha, 1972. Erroneous interpretations which may result from application of the myofibrillar
ATPase histochemical procedure to developing muscle. Exp. Neurol. 34, 465-475.
Hocquette, J.F., B. Picard and X. Fernandez, 1996. Le métabolisme énergétique musculaire au cours de la croissance
et après l’abattage de l’animal. Viandes et Produits Carnés 17, 217-230.
Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of the bacteriophage TA. Nature
227, 680-685.
Pearse, A.G.E., 1968. Histochemistry theorical and applied. J & A Churchill, London, vol. 2, 948-950.
Picard, B., J. Robelin and Y. Geay, 1995. Influence of castration and postnatal energy restriction on the contractile and
metabolic characteristics of bovine muscle. Ann. Zootech. 44, 347-357.
Picard, B., M.P. Duris and C. Jurie, 1998. Classification of bovine muscle fibres by different histochemical techniques.
Histochem. J. 30, 473-479.
Picard, B., M.P. Oury, C. Jurie, I. Cassar-Malek and J.F. Hocquette, 2004. Variabilité inter-musculaire des propriétés
contractiles et métaboliques de muscles de bovins. Cah. Nutrition Diététique, 39, 70.
Piot, C., J.H. Veerkamp, D. Bauchart and J.F. Hocquette, 1998. Contribution of mitochrondria and peroxisomes to
plamitate oxidation in rat and bovine tissues. Comp. Biochem. Physiol. 121, 69-78.
Pons, F., J. Leger, M. Chevallay, F. Tome, M. Fardeau and J. Leger, 1986. Immunocytochemical analysis of myosin
heavy chains in human fetal skeletal muscles. J. Neurol. Sci. 76, 151-163.

Part 2. Regulation of body composition and/or product quality
by tissue metabolism
2. Body composition and/or product quality

Regulation of marbling and body composition - Growth and
development, gene markers and nutritional biochemistry
D.W. Pethick1, W. Barendse2, J.F. Hocquette3, J.M. Thompson4 and Y.H. Wang2
1Co-operative Research Centre for Beef Genetic Technologies, Murdoch University, Perth, Western
Australia, 6150, Australia
2Co-operative Research Centre for Beef Genetic Technologies, CSIRO Livestock Industries,
Queensland Bioscience Precinct, Carmody Road, St Lucia, Queensland, 4067, Australia
3INRA, Herbivore Research Unit, 63122 Theix, France
4Co-operative Research Centre for Beef Genetic Technologies, School of Rural Science and
Agriculture, University of New England, Armidale, New South Wales 2351, Australia
Abstract
In this review we first discuss the role of intramuscular fat as a factor affecting consumer preferences
for meat. The development of intramuscular fat in pigs, sheep and cattle is then highlighted and the
importance of total carcass fatness and muscularity is discussed. Our current level of knowledge
relating to metabolic and genomic aspects of fat metabolism in intramuscular adipocytes is discussed
particularly with reference to glucose metabolism and regulatory genes. It is concluded that there is
a growing body of evidence that intramuscular adipocytes are metabolically different to other depots
(subcutaneous fat). Critical developmental time periods are proposed based on traditional growth
studies along with new evidence from gene expression experiments using micro array analysis. The
role of traditional quantitative genetics and gene marker assisted selection for intramuscular fat is
discussed in relation to changing fat distribution towards this site. Finally the possibility for nutritional
modification of intramuscular fat is discussed. Given our current knowledge, it is currently difficult to
preferentially stimulate the development of intramuscular fat in isolation to other depots by nutrition.
Hence it is suggested that further research on nutritional modulation be targeted to specific genotypes
within breeds of animals that have a bias toward intramuscular fat accretion.
Keywords: intramuscular fat, marbling, fatness, adipocytes, livestock
Introduction
In this review we first discuss the role of intramuscular fat as a factor affecting consumer preferences
for meat. The development of intramuscular fat in beef cattle is highlighted and were informative,
examples from other species are used to highlight the role of total carcass fatness and muscularity.
Our current level of knowledge relating to metabolic and genomic aspects of fat metabolism
in intramuscular adipocytes is discussed particularly with reference to glucose metabolism and
regulatory genes. The role of gene marker assisted selection for intramuscular fat is discussed in
relation to changing fat distribution toward intramuscular fat (that may benefit meat quality) at the
expense of other depots. Finally the influence of nutrition on the development of intramuscular fat is
discussed. The reader is guided to additional recent reviews of marbling and fat metabolism (Dunshea
and D’Souza, 2003; Harper and Pethick, 2004; Pethick et al., 2004, 2005a and b).
The role of intramuscular fat in meat palatability

Although marbling assessment is generally an integral part of any beef grading scheme the literature
suggests that it has only a minor association with palatability. Dikeman (1987) concluded that marbling
accounted for only 10 to 15% of the variance in palatability. The Meat Standards Australia (MSA)
research would agree and showed that the contribution of marbling to palatability was significant,

but importantly just one of several factors determining final palatability. However, Thompson (2004)
concluded that since variations in tenderness are controlled by schemes such as MSA, marbling will
become a more important determinant of palatability due to its specific contribution to juiciness and
flavour of grilled steaks for Australian consumers.
There is also a concern that very low levels of intramuscular fat will lead to meat that is perceived
as dry and less tasty. Such a situation has been found in young highly muscled lean cattle (double-
muscled cattle genotypes, young bulls from Belgian Blue or Blonde d’Aquitaine for instance) and
in many cuts from modern pig genotypes (Channon et al., 2001). The minimum requirement for
ether extractable fat in order to achieve acceptable consumer satisfaction for grilling ‘red meat’ cuts
(beef and lamb) is quoted at 3-4% by Savell and Cross (1986), and 5% for sheepmeat (Hopkins
et al., 2006). For pork, a figure of 2-2.5% is cited (Bejerholm and Barton-Gade, 1986) on a fresh
uncooked basis while a maximum level of 3.5% is thought to achieve optimal consumer acceptability
(Fernandez et al., 1999).
Development of intramuscular fat - Growth and development

Fat is deposited in specific depots which are similar for all mammals. The primary depots are
within the abdominal cavity (perirenal, mesenteric and omental), between muscles (intermuscular),
in subcutaneous and within muscles (intramuscular). However the proportions differ between the
species and are influenced by age and total body fatness. Thus the pig has more subcutaneous fat
(70% of total body fat) and less abdominal fat than sheep and beef cattle (Wood, 1984). In sheep
Thompson et al. (1987) measured chemical fat in mature Merinos and found the major depots
were abdominal (33% = 16% omental + 11% kidney + 6% mesenteric), subcutaneous (24%) and
intermuscular (20%). A common denominator to all species is that intramuscular fat is a small deport
(7% in Merino sheep).
Another common conclusion from animal developmental studies is that intramuscular fat is late
in developing relative to other fat depots (Vernon, 1981). Indeed the appearance of intramuscular
adipocytes is late when compared to other depots. For instance, adipocytes are identified in pig
muscles during the first month postnatally, whereas subcutaneous adipocytes develop during
gestation in this species (Hauser et al., 1997). However, because fat is deposited at a greater rate
than lean tissues later in life, the concentration of fat in muscle will inevitably increase later in an
animal’s life. Therefore the commercial trait, marbling, visible intramuscular fat or real percentage of
intramuscular fat is late maturing. This does not mean that the rate of fat accretion in intramuscular
adipocytes is also late maturing relative to other depots. The study of Johnson et al. (1972), showed
that the proportional distribution of fat between carcass pools is found to be constant over a wide
range of carcass fat contents in cattle indicating that the major fat depots grow in the same proportion
as animals fatten. The results of Pugh et al. (2005) are also consistent with this observation. Recent
developmental studies in fattening lambs and young sheep suggest that intramuscular fat is indeed
early maturing in this species. Thus in a serial slaughter experiment covering animals from 4 to 22
months of age and a diverse range of breeds/genotypes (see Hopkins et al., 2007a for description of
the experiment) there was a significant trend for the total fat content (g) of the m. longissimus thoracis
et lumborum to be higher relative to carcass fat at 4 months of age when the lambs were relatively
lean compared to when they were older and much fatter (Figure 1, Hopkins et al., 2007b).
Cellular studies in rabbits (Gondret et al., 1998) have shown that intramuscular fat develops due to
both an increased number and size of clustered adipocytes with associated increases in lipogenic
enzyme activity (Gondret et al., 1997). The available data in beef cattle would suggest that the
potential for cellular development of adipocytes is fixed relatively early in life and there after
changes in either size and/or number of cells occurs in proportion to the initial cell precursor numbers
and/or lipogenic proteins (Pethick et al., 2004). This would clearly indicate that a variety of ‘fat’

Figure 1. Relationship between carcass fatness (g) and the ratio of fat in the m. longissimus thoracis
et lumborum (loin): carcass fat (g/g) in lambs and young sheep.
measurements taken on muscle tissue in early life would hold great potential for predicting subsequent
intramuscular fat development. Examples would include intramuscular fat content (perhaps by non
invasive methods such as ultrasound), markers of adipocytes such as fatty acid carrier proteins and/or
functional lipogenic enzymes involved in fatty acid biosynthesis.
The development of intramuscular fat relative to carcass weight in beef cattle has been discussed
previously by the authors using evidence from the ‘fatter type’ beef breeds (Pethick et al., 2004).
The data suggests a period of minimal change of intramuscular fat content at young ages followed
by a linear increase between a carcass weight of about 200–400 kg at least for American Angus ×
Hereford (Duckett et al., 1993), Australian Angus (Pugh et al., 2005) or Japanese Black × Holstein
(Aoki et al., 2001) type cattle undergoing prolonged grain feeding. Based on this data we hypothesise
three drivers of intramuscular fat development (i) the potential for total carcass fat deposition (ii)
the potential for muscle growth and finally (iii) the extent of fat partitioning bias for intramuscular
fat versus other carcass depots.
The link between carcass fatness and intramuscular fat development is not always clear. In Figure
2 we have combined serial slaughter studies using modern pig genotypes (D’Souza et al., 2003),
Angus beef cattle (Pugh et al., 2005), Japanese Black × Holstein cross cattle (Aoki et al., 2001) and
Australian sheep breeds (Hopkins et al., 2007 plus personal communication) to show the influence
of carcass fatness on intramuscular fat development in the m. longissimus thoracis et lumborum.
Despite the heterogenetic nature of the experiments, variations in methodologies and that only
carcass fatness has been reported, there is a compelling case that intramuscular fat (%) is related to
total adiposity (and by difference muscularity). Excluding the Japanese Black cross cattle, it would
appear that intramuscular fat stays relatively constant as animals fatten until a carcass fatness of
about 30-35% is reached. At least for prime lambs and pigs, such levels of fatness are well beyond
that required for profitable production systems and also consumer expectations for leanness of retail
meat cuts. Premiums paid for marbled beef in some markets allow production systems with fatter
cattle to be profitable. Clearly the Japanese Black cross cattle stand out has being very different
since they develop intramuscular fat more strongly at lower levels of carcass fatness and this is the
only breed in the data set to have undergone prolonged genetic selection for increased intramuscular
fat. This result emphasizes the power of genetic manipulation for shifting the emphasis from one
depot to another.

Figure 2. Relationship between carcass fat (%) and intramuscular fat (%) of the m. longissimus
thoracis et lumborum (loin) in Angus and Japanese × Holstien (JB × Hol) beef cattle, lambs/sheep
and the modern pig (see text for sources).
Selection for high levels of muscularity is known to reduce both total carcass fatness and intramuscular
fat at a given carcass weight. Recent work in sheep has shown that lambs produced from sires with
a high estimated breeding value for post weaning eye muscle depth (using the Australian Lambplan
system) produce substantially leaner carcasses (Hegarty et al., 2006) with reduced intramuscular
fat and eating quality (Hopkins et al., 2005) when compared to lambs from sires with an elevated
estimated breeding value for post weaning growth rate. Mutations in the GDF8 gene of beef cattle,
which effects the development of the double muscle phenotype, affect muscle development to
generally cause increased muscle mass (McPherron et al., 1997). With respect to the development
of intramuscular fat, Wegner et al. (1998) demonstrated that the GDF8 mutant double muscled
animals have (i) fewer islands of fat cell development in the m. longissimus thoracis et lumborum
muscle, (ii) a lower rate of growth of these islands and (iii) smaller adipocytes in marbling islands
than conventional (i.e. non GDF8 mutant) cattle. Non genetic alterations in muscularity have similar
consequences. Thus low birth weight piglets have reduced muscle later in life (due to less muscle
fibers) and increased adipocity including increased levels of intramuscular fat compared to higher
birth weight controls (Gondret et al., 2006).
The intramuscular fat content of older livestock (culled cows and ewes) is typically higher than
in younger animals. Indeed the intramuscular fat content of Australian cast for age ewes (mutton)
can reach levels in the order of 7-12% and is dependant on carcass fatness and animal age (Pethick
et al., 2005c), the older the animal the higher the intramuscular fat. This effect of animal age is
not understood but is likely associated with changes in muscularity and/or different rates of fat
turnover in intramuscular versus other carcass fat depots. Thus reproducing ewes in the Australian
Mediterranean environment will undergo cyclical changes in total body fatness and protein. From a
fat metabolism perspective, the hypothesis is that all depots replete similarly during periods of high
nutrition (relative to metabolic demand) but that not all depots deplete at the same rate during weight
loss periods (such as during lactation and the dry summer period). From a protein perspective, it
might be that after periods of protein mobilisation associated with pregnancy and lactation, repletion
of muscle mass is incomplete and so intramuscular fat (%) increases.

Development of intramuscular fat - Metabolic understanding
Muscle type
A key feature associated with the development of intramuscular fat is the fibre type or metabolic
pattern of energy metabolism expressed by the muscle tissue. Within the one animal genotype the
more glycolytic muscle types (e.g. m. semitendinosus) generally have lower levels of intramuscular
fat (rabbits: Gondret et al., 1998; cattle: Hocquette et al., 2003; sheep: Gardner et al., 2006). Across
genotypes a similar response can be found. Thus in the study by Hocquette et al. (2003) where two
muscle types were contrasted across three breeds of cattle with disparate propensity to accumulate
intramuscular fat, there was also a strong correlation between intramuscular fat and the aerobic
markers cytochrome-c oxidase and isocitrate dehydrogenase as well as the adipose specific fatty
acid binding protein. Of course studies across genotypes, which positively correlate the extent of
anaerobic muscle metabolism to the level of intramuscular fat accumulation, are confounded by the
observation that highly muscled cattle are more glycolytic (Pethick et al., 2005b) and also have less
carcass fat. Moreover many game meats (venison, kangaroo) are dark red in colour (presumably due
to more aerobic metabolism) and at the same time have low levels of intramuscular fat, indicating
that cause and effect is not clear. However, these studies as well as others in rabbits (Gondret et al.,
2004) and sheep (Gardner et al., 2006) suggest that intramuscular fat content results from a balance
between catabolic and anabolic pathways rather than from the regulation of a specific biochemical
pathway. It has thus been speculated that a high fat turnover (which is a characteristic of oxidative
muscles) would favour fat deposition (Hocquette et al., 2003).
Additional biochemical work also underlines the contribution of the connective tissue to the
development of adipose cells, especially within intramuscular adipose tissues. For instance, it has
been hypothesised that type XII and XIV collagen contents may be associated with metabolism of
intramuscular lipids (Listrat et al., 2006), thus confirming previous in vitro studies (Tahara et al., 2004;
Ruehl et al., 2005). Type XII and XIV collagens are thus likely to both contribute to intramuscular
fat accumulation, but probably by different biological mechanisms since type XII collagen content
mainly differs between muscle types and type XIV collagen content mainly differs between breeds
(Listrat et al., 2006).
Metabolic differences between depots
There is clear evidence that different fat depots can have different metabolic activities. Hishikawa
et al. (2005) used RT-PCR to show evidence that genes were differentially expressed between
subcutaneous and visceral adipose tissues in Japanese Black cattle. Six genes were declared
differentially expressed between the two adipose tissue sites not only in cattle, but also in mice
and pigs but with some species specificities. In humans, Tchkonia et al. (2007) have studied the
expression profiles of undifferentiated preadipocytes and surprisingly differences were found between
omental versus subcutaneous and mesenteric. The cells of the two main visceral depots (omental and
mesenteric) are thus very distinct. It has been speculated that the microenvironment of fat depots
confers specific characteristics to cell progenitors during early development. These observations
have been recently extended to isolated porcine intramuscular adipocytes, that clearly exhibited low
lipogenic and lypolytic rates compared with adipocytes from subcutaneous and perirenal regions
(Gardan et al., 2006). In cattle there is evidence that marbling adipocytes show a preference for
glucose/lactate carbon, while subcutaneous adipose tissue uses mainly acetate as a source of acetyl
units for lipogenesis. This was initially proposed by Smith and Crouse (1984) and even inferred
by Whitehurst et al. (1981). More recently extensive collaboration between Australian and French
scientists have confirmed that an extensive range of enzymes, proteins and genes associated with
glucose metabolism and lipogenesis from glucose are more highly expressed in intramuscular fat
that had been purified by dissection (Hocquette et al., 2005; Bonnet et al., 2003; M. Bonnet et al.,

personal communication). Moreover the lipogenic pathway from glucose is highly responsive to
diet in ruminants (Smith et al., 1992; Pethick and Dunshea, 1996).
Gene expression pathways in muscle in relation to marbling
Further gene expression work has been undertaken by the Cooperative Research Centre for Beef
Genetic Technologies (Beef CRC) this time in collaboration with Japanese scientists (Wang et
al., 2005; Lenhert et al., 2006) and the results confirm that genetic changes in intramuscular fat
development could be identified quite early in life. Transcriptomes of whole m. longissimus thoracis
et lumborium tissue from Japanese Black cattle were compared to age matched Holstein animals.
The results showed that at 11 months, the genes associated with adipogenesis, mono-unsaturated
fatty acid synthesis and fatty acid accumulation were highly expressed in Japanese Black cattle
(Wang et al., 2005). This simply indicated that, by 11 months, Japanese Black cattle have developed
significantly more intramuscular fat than the Holstein cattle. In another breed comparison experiment,
gene expression was studied in m. longissimus tissue of progeny from Australian Hereford dams
joined to either Japanese Black or Piedmontese sires. The results indicated that the genes associated
to adipogenesis/lipogenesis are not differentially expressed between Japanese Black and Piedmontese
sires at both fetal and the new born stage (Lehnert et al., 2006). When the samples were compared
from young animals (3, 7 and 12 months), the expression of various fat related genes (adipocyte
fatty acid binding protein, adiponectin, C1Q and collagen containing domain and stearoyl-CoA
desaturase) were significantly up regulated in the Japanese Black sires only at 8 months of age (not
at 3 or 12 months, Wang unpublished data). This indicates that the intramuscular fat development, at
least in the Japanese Black cross animals, is very active between 3 and 8 months of age (before the
end of weaning). In the Japanese Black versus Holstein study, thyroid hormone responsive element
spot 14 (THRSP) was significantly more highly expressed in Japanese Black cattle. This expression
difference between two breeds was most pronounced at the youngest age of sampling (11 months)
such that THRSP expression was 25 fold higher in Japanese Black animals.
The gene expression studies clearly underpin that genetic potential for fat development is fixed
early in life and confirm the conclusions from our growth and developmental work cited above. The
challenge is now to understand the gene expression and metabolic switches that might influence
adipogenesis and lipogenesis. Some gene expression candidates have been highlighted in the
literature. Among them, the THRSP locus has shown some promise as a gene that might regulate
fat developmental pathways. It has been extensively studied in the liver and adipose tissue of the
rat and mouse and while all its functions are not fully understood, it is associated with lipogenesis.
Expression of THRSP is regulated by several nutritional and hormonal factors. Studies on THRSP
have indicated the gene isolated from both the human and rodent has a similar structure and response
to both thyroid hormone and glucose (Campbell et al., 2003). However, the THRSP response to
thyroid hormone and glucose differ in magnitude. The human THRSP promoter responds robustly
to thyroid hormone whereas rat THRSP robustly responds to glucose (Campbell et al., 2003). Given
that we have established a clear bias toward glucose metabolism in intramuscular fat in the bovine
more detailed nutritional and hormonal intervention work in young (pre-weaning) cattle would seem
justified. Moreover double muscle cattle, which show low levels of intramuscular fat development,
are also characterized by lower levels of triiodothyronine, insulin and glucose plasma concentrations
(Hocquette and Bauchart, 1999).
QTL and diagnostic tests for marbling in cattle

For a number of years several Australian cattle breeds have used the estimated breeding value for
various fat depots such as intramuscular fat, P8 rump fat and rib fat (Parnell, 2004; Angus Australia,
2006) as selection tools. This is based on using ultrasound scanning of live animals (bulls and progeny)
to estimate the various depots. The genetic relationships between fat depots or sites are known for

some well described cattle breeds such as the Angus. In this case the genetic correlations for two
different estimates of subcutaneous fat (rump versus rib sites) is high at +0.75. There is a lower
but still significant positive relationship between rib or rump fat and intramuscular fat % of +0.24
and +0.35 respectively (Angus Australia, 2006) which suggests considerable scope for differential
selection of different depots (i.e. subcutaneous versus intramuscular) – that is the genetic correlations
are well below 1. Similar genetic correlations between subcutaneous and intramuscular fat have
been widely reported in pigs.
To compliment the traditional genetic approach, the Beef CRC has undertaken to map genes affecting
meat quality. Some DNA markers have been discovered and commercialised from this mapping
activity. Several DNA tests for marbling have been identified (Barendse, 1997 and 2003; Barendse
et al., 2004), some of which are based on microsatellites (CSSM034 and ETH10) and some are
based on single nucleotide polymorphisms in the thyroglobulin (TG) and retinoic acid receptor-
related orphan receptor C (RORC) genes. One of these, based on the TG:g.-537C>T (TG5) single
nucleotide polymorphism, was licensed in June 2000 as the GeneSTAR® marbling test (Barendse et
al., 2004). Other scientific groups have published fat and marbling related polymorphisms based on
the leptin (LEP) (Buchanan et al., 2002) and growth hormone (GH1) (Sneyers et al., 1994; Chikuni
and Mitsuhashi, 2002) genes. The current status of many of these tests has recently been reviewed
(Kuhn et al., 2005; Jeon et al., 2006). However, not all the tests that are currently available have
been published or collected in reviews, and there is a trend towards non-disclosure by the testing
companies of the identity of the genetic variation underlying the test, especially since more genes
are added to the test panels for fatness traits.
The gene marker tests affecting marbling (and tenderness) have at least some biological relationship
to what is known about these traits. Thyroid hormone (Mears et al., 2001), retinoic acid levels
(Torii et al., 1996), and the impact of growth hormone (Dalke et al., 1992) and leptin (Wegner et
al., 2001) all show the importance of circulating factors in the development of marbling and other
fat depots. The RORC axis is likely involved in the maintenance of adipocytes and the ability to
process glucose (Barendse et al., 2007). This does not argue, however, that the only genes to affect
the fatness traits will be for metabolic pathways that are already known, or that further study of
these genes or traits will not reveal any new pathways or interactions. On the contrary, these are still
early days for the evaluation of genetic variation and their role in meat quality. An important aspect
of the study of these DNA tests is the validation of these tests in different countries and in different
breeds. So far, the LEP (leptin) and GH1 (growth hormone 1) tests for marbling have been tested in
Australia (Barendse et al., 2005 and 2006) and this is part of an international effort at validation. For
example the TG5 marker has been tested independently outside of Australia (Thaller et al., 2003;
Rincker et al., 2006), and the leptin test for marbling has been extensively studied (Nkrumah et
al., 2004 and 2005; Kononoff et al., 2005; Schenkel et al., 2005) see also http://www.ansci.cornell.
edu/nbcec/ucdavis/validation.htm.
These tests for marbling individually explain a small part of the total variation for marbling in the
slaughter cattle used to build the Beef CRC data base, with maximum differences between genotypes
being of the order of 0.2 to 0.5 of a phenotypic standard deviation. When the available tests for
marbling are added up, they account for approximately 0.7 of an AUSMEAT marbling score (about
2% units of intramuscular fat) in a 300 kg carcass weight beef animal, with TG5 accounting for
0.3 and GH1 accounting for 0.38 – the other loci (i.e. RORC) will combine to push this higher but
one can be less confident about the size of the effect of the other genes since they have either not
been evaluated in samples greater than 1000 or they are unpublished. The goal of the Beef CRC is
to account for at least half the genetic variation for a meat science trait firmly associated to gene
markers and this will require the use of more extensive genomic technologies which sample the
whole genome with single nucleotide polymorphisms at high resolution (whole genomic scans).

Nutritional modulation of intramuscular fat - Manipulating protein and
energy
Nutritional manipulation of intramuscular fat levels in pork via altering the dietary protein:energy
ratio has been investigated in a number of studies. Of course the basic premise is that by restricting
muscle development through a subtle protein deficiency, total carcass fatness will be increased
sufficiently to elevate intramuscular fat. The data in pigs shows very clearly that this approach can
lead to increases in intramuscular fat (Essen-Gustavsson et al., 1994; Cisneros et al., 1996; Eggert
et al., 1998; D’Souza et al., 2003; Doran et al., 2006). Using fine manipulation of protein deficiency
and dietary restriction, it seems possible to increase intramuscular fat without affecting carcass
fatness (Gondret and Lebret, 2002).
The results of manipulating the protein:energy ratio in beef cattle diets is less conclusive. The
conclusions from two studies (Oddy et al., 2000; Pethick et al., 2000) were that diets which contain
more or less protein than recommended amounts for feedlot animals do not lead to significant
differences in marbling or intramuscular fat. However, there was a trend for high protein diets to
produce less and low protein diets more marbling than control diets in both experiments. In the
case of Oddy et al. (2000) the low protein diets in combination with added dietary fat (to decrease
the protein:energy ratio) significantly decreased feed conversion ratio and cost of gain relative to
control and high protein diets. However in the case of Pethick et al. (2000) the low protein diets did
not change the feed conversion ratio perhaps because in this case they did not include dietary fat.
Certainly the data of Pethick et al. (2000) suggest that a simple diet based on barley and hay (with
no additional protein source in the form of grain legumes or urea) fed to Angus steers at a starting
live weight of 540 kg (P8 back fat = 12 mm) produced equal performance to more traditionally
formulated rations containing additional protein sources at an extra cost.
The literature reporting the effects of supplemental fat on marbling scores in beef cattle are mixed
and are well discussed by Andrae et al. (2001). These authors argue that marbling responses to dietary
fat have been more consistent when supplemental fat is added to diets based on grains that contain
less fat than corn (i.e. wheat, barley) and this is supported by their study where high oil maize (7%
fat in DM) was fed in comparison to traditional maize (4.7% fat in DM) to finishing cattle (final
HCW = approx. 330 kg). Simple measures of carcass fatness (fat thickness) and intramuscular fat
(visual marbling score) were similar when cattle were fed isoenergetic diets. However when the
high and low oil maize diets were formulated at the same inclusion level of maize the marbling
score was higher for the high oil maize based ration. This is predictable since at equal inclusion
in the ration along with equal dry matter intake, the high oil maize grain would supply more net
energy for fat synthesis.
Nutritional modulation of intramuscular fat - Vitamin A

Vitamin A deficiency is associated with an elevated intramuscular fat content (Harper and Pethick,
2004). Indeed recent gene marker studies in beef cattle (Barendse, 2004) have shown an association
between intronic and exonic alleles within the retinoid related orphan receptor C (gamma) gene and
marbling scores. It has been reported that a low intake of ß-carotene or vitamin A in young Wagyu
steers increases marbling (Oka et al., 1998). This was confirmed by Adachi et al. (1999) who showed
that vitamin A levels in cattle blood were negatively associated with marbling scores. More recent
work by the Beef CRC (Z.A. Kruk et al., unpublished results) showed a 35% increase in intramuscular
fat of the LT muscle (but not m. semitendinosus) in Australian Angus cattle fed a diet deficient in
Vitamin A for 300 days. This was also associated with increased seam fat thickness suggesting that
total carcass fatness had been influenced. D’Souza et al. (2003) also reported that feeding pigs a
grower and finisher diet deficient in vitamin A significantly improved the intramuscular fat levels in
the m. longissimus thoracis muscle from 1.3 to 2%. It has been proposed that the effect of Vitamin A

on intramuscular fat deposition is mediated by retinoic acid, a derivative of Vitamin A, which regulates
the adipogenic differentiation of fibroblasts, inhibiting the terminal differentiation of intramuscular
adipose tissue in cattle (Kuri-Harcuch, 1982). It has also been proposed that retinoic acid regulated
growth hormone gene expression (Bedo et al., 1989), which in turn decreases fat deposition and
intramuscular fat in steers (Dalke et al., 1992). Deficiencies in retinoic acid, therefore, may result
in lower growth hormone concentrations and increased fat deposition including intramuscular fat.
However in the case of the pig, D’Souza et al. (2003) found no change in carcass composition,
measured using dual X-ray absorptiometric analysis, suggesting that at least in the pig the effects of
Vitamin A deficiency might be more localised to intramuscular fat. However further work to assess
whole body fatness in the live animal would be needed to validate this suggestion.
Nutritional modulation of intramuscular fat - Fermentation pattern in

ruminants
Pethick et al. (1997 and 2004) postulated that diets which promote both (i) maximal fermentation
in the rumen to produce gluconeogenic precursors (propionate), and (ii) which maximise starch
digestion in the small intestine might increase intramuscular fat deposition. Such diets are usually
associated with high levels of processing which increase the accessibility of the dietary starch granule
to both microbial and animal amylases and so maximise the availability of glucose to the fattening
animal (Rowe et al., 1999). The logic behind this hypothesis was that (i) such diets would promote
increased levels of anabolic hormones (insulin) which are known to stimulate lipogenesis; (ii) the
logic parallels the observation in humans that diets with a high glycaemic index (i.e. diets that allow
rapid glucose absorption and concomitant high insulin levels) promote obesity (Ludwig, 2000); (iii)
such diets will also deliver increased levels of net energy for lipogenesis (the reason why grain feeding
promotes more intramuscular fat development compared with grass finishing, Pethick et al., 2004)
and (iv) there is evidence that marbling adipocytes show a preference for glucose/lactate carbon
while subcutaneous adipose tissue uses mainly acetate as a source of acetyl units for lipogenesis (see
above). The potential for this strategy to specifically stimulate intramuscular adipocyte development
is by no means clear, but as discussed above experiments targeted at early life interventions at this
axis would seem warranted in cattle where marbling genetics is well defined.
Nutritional modulation of intramuscular fat - n-6 polyunsaturated fatty

acids
In a stimulating review, Ailhaud et al. (2006) have postulated that the modern obesity epidemic in
humans affecting the developed world is partly related to the increased consumption of polyunsaturated
fatty acids (PUFAs) of the n-6 series during early life pre-weaning. Convincing evidence is put
forward to show that the n-6 fatty acids (in contrast to n-3 fatty acids) promote adipogenesis in vitro
and markedly favour adipose tissue development in rodents during the gestation/suckling period.
The suggested mechanism involves arachidonic acid (n-6 C20:4) stimulation via prostocyclin of
preadipocytes to form adipocytes. There is a well described increase in intramuscular fat levels in
beef cattle finished with cereal grain based diets compared to grass finished animals (Pethick et al.,
2004) which is typically attributed to differences in net energy consumption. However such diets
would also vary markedly in the ratio n-6/n-3 fatty acids with grass based diets lower and grain based
diets higher and it is tempting to speculate this may be related to the hypothesis developed by Ailhaud
et al. (2006). Again it would seem warranted to test this hypothesis using early life interventions at
this axis in livestock where the marbling genetics is well defined. The genetic propensity to favour
adipocyte development at the intramuscular site would be important since there is no suggestion
that the n-6 fatty acids affect one depot more than another.

Conclusion
Our knowledge of intramuscular fat is increasing. Largely this fat depot develops in parallel with
others, but its commercial expression (% fat in muscle or marbling) is also dependant on muscle
growth. It is proposed, based on data in three livestock species (cattle, pigs and sheep) that muscle
growth and fat growth within the muscle occur at a similar rate until a total carcass fatness of about
30-35% is reached. After this point muscle growth slows while fat growth is maintained resulting
in an increased expression of intramuscular fat (%). The exception is where a particular breed has
been selected to express intramuscular fat such that there is a bias or shift in fat distribution toward
the intramuscular site and expression can occur at lower and more efficient levels of total carcass
fatness. There is additional evidence that intramuscular adipocytes are metabolically different to
other depots (subcutaneous fat) and an understanding of this would appear important to underpin
strategies for genetic and non genetic manipulation of intramuscular fat. It is currently difficult to
preferentially stimulate the development of intramuscular fat in isolation to other depots by nutrition
as typically all depots are likely to respond. Hence it is suggested that further research on nutritional
modulation be targeted to specific genotypes within breeds of animals that have a bias toward
intramuscular fat accretion. Hence the importance of both quantitative and molecular genetics, if
increasing or maintaining the level of intramuscular fat, is important. Critical developmental time
periods (10 months of age or less for cattle) are proposed based on traditional growth studies along
with new evidence from gene expression experiments using micro array analysis. We suggest future
experiments be undertaken to further understand the differences in intramuscular fat from other
depots and that strategic nutritional intervention experiments be undertaken in genetically defined
animals early in life.
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Wood, J.D., 1984. Fat deposition and the quality of fat tissue in meat animals. In: Wiseman, J. (editor), Fats in animal
nutrition. Butterworths, London, 407-435.

Protein and selected mineral deposition in the body of pigs during
compensatory growth
D. Weremko1, G. Skiba, S. Raj and H. Fandrejewski
The Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences 05-110
Jabłonna, Poland
Introduction
The compensatory growth phenomenon is often used in modern systems of outdoor pig production.
The work on compensatory growth was focussed on metabolism of organic macro-nutrients e.g.
protein, fat usually (e.g. Bikker, 1994), but there is a lack of studies on mineral elements during this
kind of growth. Thus, the aim of this study was to determine protein, P, Ca, Mg and Zn deposition
in the pig’s body during compensatory growth.

Sixty crossbred pigs from 25 kg body weight were kept individually and fed ad libitum for 86 d. Two
diets - basal (B) and high fibre (F) were used. Diet B contained 13.1 MJ EM and 8.9 g digestible
lysine, 2.01 g digestible phosphorus, 6.7 g calcium (with Ca:P ratio of 1.3:1), and 119 g zinc.
Diet F was a mixture of diet B (80%) and grass meal (20%) and contained less energy, digestible
lysine, digestible phosphorus, Ca and Zn by 12, 17, 11, 7 and 8%, respectively. Control pigs (C)
were continuously fed the B diet; whereas, the F33 and F65 pigs were fed the F diet for 33 or 65 d,
respectively following diet B till d 86. Animals were slaughtered at the following: 25 kg (n=6),
after d 33 (6 each from groups B and F33), after d 65 (6 each from groups B, F33 and F65) and after
d 86 (12 from group C and 6 each from groups F33 and F65). The protein content was determined
according to standard methods (AOAC, 2004), and P spectrophotometrically using molybdovanadete
as the colour-forming reagent, however Ca, Zn and Mg from ash solutions by atomic absorption
spectrophotometry. Protein and mineral retention in the body were calculated from the difference
between their final and initial content at a particular growth stage. All calculations were performed
using analysis of variance (ANOVA) generated by Statistica 5.5 PL (StatSoft Inc., 1997).
Results
During feeding a grass meal diet, the F33 pigs deposited less protein and minerals compared to pigs
fed adequately (Table 1), but decreasing deposition of minerals was higher (mean 21%) than protein
(17%). When feeding the B diet was applied again (realimentation period), pigs of the F33 group
showed a compensatory response and consumed on average 4% more feed, deposited more protein
(by 28%) and minerals (by 21, 14, 25 and 7% for P, Ca, Mg and Zn, respectively) daily compared with
the C group, but only till d 65 of the study. During the following time, the compensatory response
disappeared and deposition of investigated components did not differ from the C pigs. However,
content of protein, Ca and P in the body of the F33 pigs at d 65 of the study did not differ from the C
animals. Therefore these pigs show complete compensation for protein, P and Ca already till d 65.
Final (at d 86) content of investigated body component of the F33 group did not differ from the
control group. It seems that in the case of Mg and Zn this recovery process proceeded slower and
complete compensation required a longer time. When feeding, a grass meal diet was prolonged till
d 65 (group F65) decreasing a deposition of protein and minerals was lower compared with a shorter
time of such feeding (group F33) due to the difference in voluntary feed intake, which unexpectedly
was greater in the F65 pigs. In the latter pigs also decreasing deposition of protein was lower (only
7%) than in minerals (mean 11%) (Table 2). The compensatory response of the F65 was poorer but
their deposition of protein, P and Zn were still higher than the control group (by 11, 12 and 4%,
respectively). Deposition of the other minerals did not differ between groups.

Table 1. Daily deposition of protein and mineral component in the C and F33 pigs.
Group Restriction Realimentation
for 33 d from 33 to 65 d from 65 to 86 d
Protein, g C 140a 132A 133

F33 116b 169B 133
P, g C 3.16a 4.20A 5.87
F33 2.53b 5.10B 6.07
Ca, g C 5.94 7.84A 10.64
F33 4.71 9.07B 10.83
Mg, g C 0.32A 0.24a 0.21
F33 0.23B 0.30b 0.24
Zn, g C 0.014a 0.019 0.019
F33 0.012b 0.020 0.022
a,b P<0.05; A,B P<0.01.
Table 2. Daily deposition of protein and mineral component in the C and F65 pigs.
Group Restriction Realimentation

for 65 d from 65 to 86 d
Protein, g C 136 133

F65 127 148
P, g C 3.73A 5.87a
F65 3.30B 6.56b
Ca, g C 6.98A 10.64
F65 5.99B 10.77
Mg, g C 0.28a 0.21
F65 0.25b 0.21
Zn, g C 0.017a 0.019
F65 0.015b 0.020
a,b P<0.05; A,B P<0.01.
In conclusion, the results of the study indicate that pigs responded stronger to restriction of mineral
components than protein, however it had no influence on animal health or physical activity. Moreover,
compensatory deposition of minerals differs between particular elements. Animals recovered
deficiency of P and Ca more easily, and protein as well, compared with the Mg and Zn, since recovery
of the latter two components required more time.
References
Association of Official Analytical Chemists, 2004. Official methods of analysis, 18th edition. AOAC, Arlington, VA,
USA.
Bikker, P., 1994. Protein and lipid accretion body components of growing pigs: effects of body weight and nutrient
intake. PhD Thesis, Wageningen University, 203 pp.
StatSoft Inc., 1997. Statistica 5.5 PL for Windows, Tulsa, USA.

Meal feeding regulates S6K1 phosphorylation but not that of 4E-BP1 in
rainbow trout (Oncorhynchus mykiss) muscle
I. Seiliez1, J.C. Gabillard2, S. Panserat1, S. Cassy1, J. Gutiérrez3, S. Kaushik1 and S. Tesseraud4
1INRA, UMR1067 Nutrition Aquaculture et Génomique, F-64310 St Pée-sur-Nivelle, France
2INRA, UR1037 Station Commune de Recherches en Ichtyophysiologie Biodiversité et Environnement,
Campus de Beaulieu, F-35000 Rennes, France
3Departament de Fisiologia, Facultat de Biologia, Universitat de Barcelona, Spain
Introduction
The synthesis of skeletal muscle proteins is rapidly stimulated after oral intake of nutrients, through
an acceleration of mRNA translation initiation (Yoshizawa et al., 1998). This activation involves
the phosphorylation of two key intracellular kinases, i.e. p70 S6 kinase (S6K1) and the translational
repressor eukaryotic initiation factor 4E binding protein (4E-BP1), regulated by the mammalian
target of rapamycin (mTOR) signalling pathway (Shah et al., 2000). The mechanisms by which
meal feeding modulates this cellular pathway are beginning to be elucidated and increasing evidence
points out a broad role for amino acids in this process (Kimball and Jefferson, 2006). However, the
molecular mechanisms implicated are complex and little understood.
The purpose of the present study was to characterise the effect of a single meal on the phosphorylation
and/or activity of several major kinases involved in the mTOR pathway in muscle of rainbow trout
(Oncorhynchus mykiss). Besides providing for the first time in a fish species, new insight into the
molecular mechanisms regulating the synthesis of skeletal muscle proteins by feeding, we show
in this study the nutritional regulation of the mTOR signalling pathway in a representative model
species known to require a high dietary amino acids supply.

Our results show that meal feeding induced an activation of the TOR pathway in rainbow trout muscle
by enhancing the phosphorylation and/or activity of TOR, the Protein kinase B (PKB) upstream of
TOR, and also S6K1 (Figure 1 and data not shown). The extent of phosphorylation of the former
kinases significantly increased approximately 30-60 min following meal feeding, whereas that of
S6K1 significantly increased only after 5 h. This was consistent with the long-term stimulation of
muscle protein synthesis (6 h after the meal) previously reported after refeeding in Rainbow trout
(McMillan and Houlihan, 1989) and may highlight a key function of S6K1 in controlling protein
synthesis in that species.
However, we observed no effect of feeding on the amount of 4E-BP1 in its γ form, the most highly
phosphorylated form known to be the only one involved in the regulation of mRNA translation
(Figure 1). This result contrasts with previous data in mammals showing a marked effect of meal
feeding on the phosphorylation of 4E-BP1 in muscle (Vary and Lynch, 2006) and suggests that the
phosphorylation of 4E-BP1 is independent of the feeding status and likely not limiting for protein
synthesis in trout muscle.
One possible explanation for the lack of meal feeding effect on the phosphorylation status of 4E-
BP1 would be an absence or a deficient action of the TOR protein on 4E-BP1. To investigate this
hypothesis, primary cultures of trout skeletal muscle cells were incubated with insulin or insulin plus
rapamycin (a specific inhibitor of TOR) and the phosphorylation status of 4E-BP1 was examined.
None of the treatments led to significant changes in the relative proportions of α, β and γ forms

Figure 1. Effect of feeding on phosphorylation of PKB, TOR, S6K1 and 4E-BP1. Equal amounts
of protein homogenates from white muscle of experimental animals were immunoblotted with an
antibody specific for the phosphorylated form of PKB (Ser473), TOR (Ser2448), S6K1 (Thr389) and
the three electrophoretic forms (termed α, β and γ) of 4E-BP1.
(data not shown). In contrast, the phosphorylation of S6K1 on residue Thr389 was increased by
insulin in trout cells (data not shown). Moreover, rapamycin treatment abolished insulin-induced
increase of S6K1 phosphorylation, suggesting the involvement of TOR in insulin action in fish as
in other species.
In conclusion, in this study we report for the first time in a fish species the existence and the nutritional
regulation of the major kinases involved in the TOR pathway. Our results showed that meal feeding
induced the activation of the TOR pathway in trout muscle by enhancing the phosphorylation and/
or activity of PKB, TOR and S6K1. However, we observed no effect of feeding status nor that of
insulin and/or rapamycin on the phosphorylation of 4E-BP1. The current research program aims at
elucidating the molecular basis of this lack of 4E-BP1 regulation by studying the respective effects of
insulin and amino acids and exploring in vitro the signal transduction pathways potentially involved
in 4E-BP1 activation.
References
Kimball, S.R. and L.S. Jefferson, 2006. Signaling pathways and molecular mechanisms through which branched-chain
amino acids mediate translational control of protein synthesis. J. Nutr. 136, 227S-231S.
McMillan, D.N. and D.F. Houlihan, 1989. Short-term responses of protein synthesis to re-feeding in rainbow trout.
Aquaculture 79, 37-46.
Shah, O.J., J.C. Anthony, S.R. Kimball and L.S. Jefferson, 2000. 4E-BP1 and S6K1: translational integration sites for
nutritional and hormonal information in muscle. Am. J. Physiol. Endocrinol. Metab. 279(4), E715-29.
Vary, T.C. and C.J. Lynch, 2006. Meal feeding enhances formation of eIF4F in skeletal muscle: role of increased eIF4E
availability and eIF4G phosphorylation. Am. J. Physiol. Endocrinol. Metab. 290, E631-642.
Yoshizawa, F., S.R. Kimball, T.C. Vary and L.S. Jefferson, 1998. Effect of dietary protein on translation initiation in
rat skeletal muscle and liver. Am. J. Physiol. 275, E814-820.

Dietary methionine supply affects the amino acid composition of body
proteins
J. van Milgen1, L. Brossard1, N. Le Floc’h1 and M. Rademacher2
1INRA, UMR1079 Systèmes d’Elevage, Nutrition Animale et Humaine, F-35590 Saint-Gilles,
France
2Degussa AG, Rodenbacher Chaussee 4, D-63457 Hanau, Germany
Introduction
The amino acid requirements for growing animals are often estimated using nitrogen balance studies.
In these studies, it is assumed that the amino composition of body protein is constant. However,
there are some doubts concerning the validity of this hypothesis (Gahl et al., 1994; Wei and Fuller,
2006). The objective of this study was to test the hypothesis that the amino acid composition of
body protein is affected by the methionine (Met) supply in piglets.

Four diets (based on wheat, barley, soybean meal, peas, and corn starch) were formulated in which
Met was the first-limiting amino acid for growth. The ileal digestible Met levels ranged from 0.183
to 0.257% and differences in Met supply were generated by inclusion of different levels of DL-Met.
Diets supplied 1.36% ileal digestible lysine, which exceeded requirements for growth. Also, other
amino acids were supplied in excess of requirements.
A total of 30 piglets (5 blocks of 6 piglets each) were selected at 35 d of age (7 d post-weaning). One
block of piglets received the diet containing the highest Met level for 8 d and piglets were slaughtered
thereafter (initial group). The other four blocks of piglets received one of four diets during 18 d.
The first 8 d were used to adapt the piglets to the diets, which were followed by a nitrogen balance
study for 10 d. The body weight of the piglets at the start of the balance trial averaged 14 kg. Feed
was offered slightly below ad libitum intake (560 g DM/d) and at identical levels within a block.
Following the balance study, piglets were slaughtered (without dehairing), the gastro-intestinal tract
and bladder were emptied and the chemical composition of the empty body (including viscera and
blood) was determined. The effect of age was tested by comparing the two groups of piglets that
received the diet with the highest level of Met (i.e. 8 d for the initial group or 18 d). For the balance
study, the effect of Met supply was assessed by linear regression.

As expected, body weight gain and nitrogen retention increased with increasing Met supply (Table 1).
The amino acid composition of the initial group was different from that of piglets receiving the
same diet for 18 d. For most amino acids, the content in body protein increased with age; only the
arginine content decreased with age.
An increased dietary Met supply increased the contents of most amino acid in body protein; only
the contents of arginine and histidine decreased. Also the sum of (analyzed) amino acids increased
with increasing Met supply. Due to the combined effects of age and Met supply, the amino acid
composition of protein gain was largely affected by the Met supply. The calculated Met content of
protein gain ranged from 1.3 to 2.5 g Met/16 g N.

Table 1. Performance, nitrogen retention, and body composition as a function of the methionine
supply in piglets.
Initial Final groups (dig. Met level, %) Probability1 RSD

group
0.183 0.208 0.232 0.257 Age Met
Final BW,kg 13.8 17.7 17.9 18.2 18.5

Daily gain, g/d n.a. 337 345 353 398 n.a. 0.02 40
N retention, g/d
balance study n.a. 9.49 10.18 10.54 11.74 n.a. <0.01 0.73
slaughter study n.a. 7.07 8.81 8.47 9.32 n.a. 0.02 1.28
Body composition2
Crude protein, % 15.71 15.08 15.84 15.66 15.52 0.59 0.33 0.57
Lysine 6.61 6.57 6.67 6.66 6.70 0.28 0.14 0.14
Methionine 1.67 1.61 1.68 1.80 1.79 <0.01 <0.01 0.05
Cystine 0.94 1.00 0.96 0.99 0.99 0.02 0.96 0.03
Threonine 3.35 3.34 3.37 3.59 3.59 <0.01 <0.01 0.09
Isoleucine 3.25 3.18 3.27 3.31 3.32 0.17 <0.01 0.08
Leucine 6.84 6.80 6.86 6.91 6.95 0.20 0.03 0.11
Valine 4.52 4.46 4.52 4.56 4.59 0.18 <0.01 0.07
Phenylalanine 3.65 3.61 3.63 3.64 3.66 0.65 0.18 0.06
Arginine 6.63 6.50 6.47 6.37 6.40 <0.01 0.09 0.11
Histidine 2.58 2.73 2.76 2.69 2.62 0.20 0.01 0.08
Glycine 8.86 8.65 8.46 8.68 8.72 0.55 0.49 0.31
Proline 6.67 6.69 6.49 6.33 6.53 0.26 0.19 0.23
Serine 3.50 3.47 3.44 3.71 3.70 <0.01 <0.01 0.12
Alanine 6.15 6.04 6.04 6.23 6.27 0.10 <0.01 0.13
Aspartate 7.52 7.41 7.49 7.90 7.94 <0.01 <0.01 0.16
Glutamate 12.06 11.76 11.94 12.46 12.55 0.01 <0.01 0.24
1The ‘Age’ effect corresponds to comparing pigs in the initial group with pigs in the final group receiving the highest
Met level. The ‘Met’ effect corresponds to a linear effect of Met for pigs in the final group.
2Amino acid contents are expressed as g/16 g N.
Different body proteins are known to have a very different amino acid composition. For example,
the Met content is 0.8% for collagen, 1.8% for the myosin heavy-chain, and 4.1% for actin (Pearson
and Young, 1989). If the animal can selectively modify the deposition of different body proteins,
this would constitute a great mechanism to cope with (and temporary store) an unbalanced supply
of amino acids. It remains to be confirmed if this mechanism also exists for other amino acids, for
older animals and whether it affects meat quality.
References
Gahl, M.J., T.D. Crenshaw and N.J. Benevenga, 1994. Diminishing returns in weight, nitrogen, and lysine gain of pigs
fed six levels of lysine from three supplemental sources. J. Anim. Sci. 72, 3177-3187.
Pearson, A.M. and R.B. Young, 1989. Muscle and meat biochemistry. Academic Press, London.
Wei, H.-W. and M. Fuller, 2006. Dietary deficiencies of single amino acids: whole body amino acid composition of
adult rats. Arch. Anim. Nutr. 60, 119-130.

Does a time interval for methionine supplementation favour weight gain
and body composition? A model study with growing rats
M. Gas1,2, J. Bujko2, E. Chudobinska2, M.W.A. Verstegen1, R.E. Koopmanschap1 and V.V.A.M.
Schreurs1
1Wageningen Institute of Animal Sciences (WIAS), Wageningen University, Marijkeweg 40, 6709
PG Wageningen, The Netherlands
2Department of Human Nutrition and Consumer Sciences, Warsaw Agricultural University,
Nowoursynowska 159C, 02-776 Warsaw, Poland
Introduction
Adequate supplementation of poor quality protein diets is critical when the dietary nitrogen intake
should remain marginal. The classical way of improving poor quality protein diets is just by mixing
the diet with a supplement of the missing amino acid(s) in free form. However, free amino acid (AA)
supplementation is not left without physiological consequences. In a previous study (Bujko et al.,
2003), postprandial oxidative losses of leucine were higher when a methionine (Met) deficient meal
was mixed with its Met supplement compared to the situation when the supplement was given after
a time interval of 1 h. The present study aimed at investigating the influence of 1 h time interval of
supplementation on weight gain and composition of the liver and the remaining carcass of growing
rats. The study was performed at a marginal (5%) and an adequate (13.8%) protein intake.

Twenty-four growing male Wistar rats were fed iso-energetic diets (ME=13.4 kJ/g) containing 13.8%
or 5% crude protein. The AA pattern of the dietary protein was 50% deficient in Met. Both protein
groups were divided into two sub-groups (six rats each), that received the free Met supplement with
a 0 h time interval (mixed with deficient meal) or with a 1 h time interval after the main meal. The
effects were studied on weight gain and the composition of the liver and the remaining carcass. The
latter were analysed separately for the dry matter (freeze-drying), crude protein (Kjeldahl method)
and fat content (Soxhlett-method) after 32 d. The MANOVA and post hock LSD test (Statgraphics
Plus, version 4.1) was used to determine statistically significant differences (P<0.05).

At d 0 the mean weight was 141 g (sd=7). The weight gain over 32 d was influenced by both the
protein level of the diet (P<0.0001) and the time interval of free Met supplementation (P=0.0242). An
interaction between both factors was observed (P=0.0002). The lowest weight gain was shown by the
group fed the 5% protein with 0 h time interval of supplementation (29 g, sd=4.6). Significantly higher
weight gain was shown by the group fed the 5% protein with 1 h time interval of supplementation
(35 g, sd=3.6). Animals fed the 13.8% protein diets showed a significantly higher weight gain
compared to the groups fed the 5% protein diet. However, adverse effects were observed for the
time interval of supplementation (74 g, sd=5.2 for 0 h vs. 62 g, sd=5.0 for 1 h). The composition of
the liver and the remaining carcass were clearly influenced by the protein level of the diet.
Liver and carcass composition. The body composition (Figure 1 left) showed a lower protein and
a higher fat content of the rats fed the 5% compared to the 13.8% protein diet. These observations
confirmed earlier findings of an increase in body fat content in rats fed low protein diets (Du et al.,
2000). Diets with reduced protein content were compensated for by increased carbohydrate content
in order to keep the diets iso-energetic. This could have caused a higher level of lipogenesis on the

Figure 1. Dry matter (DM) composition of bodies without the liver (left figure) and livers (right
figure) of rats after being 32 d on either a 13.8% or 5% protein diet deficient in methionine and
supplemented with free methionine at 0 or 1 h after the main meal was served. Significant differences
between groups (P<0.05) are indicated by different letters (small letters for fat and capital letters
for protein).
low dietary protein level. Additionally, 1 h interval between Met deficient meal and its supplement
increased fat and decreased protein content in the body compared to 0 h groups.
Fatty livers (20% of fat in DM) with lower protein content were observed in animals fed the 5%
protein diets (Figure 1 right). This suggests that release of the fat from the liver is impaired. In case
of a protein deficient diet and a higher level of lipogenesis, severe Met deficiency is expected. It is
possible that Met deficiency causes a reduced choline production. This might explain the accumulation
of triacylglycerols in the liver due to impaired release (Aoyama et al., 1998).
In summary, 1 h delay in free Met supplementation to Met deficient meal improved weight gain
only in 5% protein diets but at the same time, a higher fat and lower protein content was noticed
in the carcass.
References
Aoyama, Y., T. Inaba and A. Yoshida, 1998. Dietary cystine and liver triacylglycerols in rats: effects of dietary lysine
and threonine. Comp Biochem. Physiol. A Mol. Integr. Physiol. 119, 543-546.
Bujko, J., M. Gas, M. Krzyżanowska, R.E. Koopmanschap, J.A. Nolles and V.V.A.M. Schreurs, 2003. Optimal time
interval for AA supplementation as studied by AA oxidation during the postprandial phase. In: Souffrant, W.B.
(editor), Progress in research on energy and protein metabolism. Wageningen Academic Publishers, Wageningen,
EAAP publication No. 109, 681-684.
Du, F., D.A. Higginbotham and B.D. White, 2000. Food intake, energy balance and serum leptin concentrations in rats
fed low-protein diets. J. Nutr. 130, 514-521.

Effects of dietary protein level and feed intake on amino-acid gain and
composition in growing and fattening Iberian pigs
R. Nieto, R. Barea, I. Fernández-Fígares and J.F. Aguilera
Department of Animal Nutrition, Estación Experimental del Zaidín (CSIC), Camino del Jueves s/n,
18100 Armilla, Granada, Spain
Introduction
The concept of an ideal protein for growth in pigs relies on the assumption of a constant amino-acid
(AA) composition for protein gain; nevertheless, differences in relative proportions between organs
and carcass caused either by genotype or nutritional regimen may lead to dissimilar AA pattern, with
possible consequences on the optimum AA composition of dietary protein. To examine the effect of
dietary protein content (DPC), feeding level (FL) and body weight on AA gain and composition, a
study was performed with growing and fattening Iberian pigs, a slow growing obese porcine genotype
with a low potential for protein deposition (Nieto et al., 2002; Barea et al., 2007).

The study was performed with 24 purebred castrated male Iberian pigs growing from 15 to 50 kg
BW given three diets containing 175, 156 or 129 g CP/kg DM (14.4-15.4 MJ ME/kg DM), and with
24 fattening (50 to 100 kg BW) purebred castrated male Iberian pigs fed diets with 13.9 or 14.6 MJ
ME/kg DM and 145 or 95 g CP/kg DM, respectively. The pigs were fed at 80% or 95% of ad libitum
intake. Four and six animals per treatment combination were used for the growing and fattening
pigs, respectively. An additional group of eight pigs was slaughtered at 15 kg BW for initial body
composition. All experimental pigs were individually penned. They were weighed weekly and daily
food intake for the following wk was adjusted accordingly. The pigs were slaughtered at 50 ± 0.5
or 99 ± 0.5 kg BW. The gastrointestinal contents were removed and blood and organs collected and
weighed separately. Four components were obtained (carcass, head and feet, viscera, and blood)
which were kept at –20 °C until analysis. The right half of the carcass and the rest of the components
were separately ground, homogenised and sub-samples were taken for freeze-drying and subsequent
analysis. All analyses were performed in duplicate. Crude protein was determined in freeze-dried
samples of tissues by a Kjeldahl procedure and AA were analysed after protein hydrolysis (6 M
HCl plus 1% phenol) by HPLC as phenylisothiocianate derivates. The experimental protocol was
approved by the Bioethical Committee of the CSIC. The effects of DPC and FL within each body-
weight group were assessed by two-way ANOVA with four or six pigs per treatment combinations
for 15 to 50 kg and 50 to 100 kg pigs, respectively.

The average ME intake in the growing pigs was 23.8 and 20.5 MJ/d, depending on FL. Protein level
significantly affected weight gain (P<0.001); the highest value (516 g/d) was obtained with the
lowest DPC. For the fattening pigs, average ME intakes were 44.0 and 37.2 MJ/d and the highest
weight gain (772 g/d) was also obtained with the lowest DPC. At 50 kg the proportions of viscera
and carcass to empty body weight were affected significantly by the FL; viscera augmented (0.128
vs. 0.115, P<0.001) and carcass diminished (0.738 vs. 0.754) on raising the FL, but no effect of
DPC was detected. Nevertheless, at 100 kg BW viscera and carcass proportions had no variation
with the treatment factors (mean values, 0.103 and 0.782 respectively). The AA composition of the
protein from viscera was rather similar at 15, 50 and 100 kg BW. The AA gain (g/d) for viscera in
the growing pigs were positively influenced by FL (P<0.001; Table 1). In the fattening pig, a trend
was observed for gains in some essential (Arg, His, Leu, His) and nonessential AA on increasing

FL. Daily gains for most essential AA in viscera were from 30 to 50% higher in growing than in
fattening pigs. The AA composition of carcass protein remained fairly constant along the BW range
studied. Carcass protein showed a different AA pattern of the viscera protein. In general, no effect
of DPC or FL was observed for the AA composition of carcass protein (data not shown). During
the 15 to 50 kg period, higher carcass daily gain for both essential (Table 1) and nonessential AA
were obtained on increasing FL (P<0.001) and on decreasing DPC (P<0.01 to P<0.05). In the
heavier pigs a trend (P<0.10) was observed for higher daily gains of several AA on increasing FL
and on decreasing DPC. Carcass daily gain of most essential AA tended to be higher (10 to 20%) in
growing than in fattening pigs. These preliminary results suggest likely differences in the optimum
AA pattern for viscera and carcass growth. This fact deserves special attention in the Iberian pig
since greater viscera proportions (with high protein turnover rates) have been observed in this native
breed compared with conventional pig genotypes (Rivera-Ferre et al., 2005).
Table 1. Effect of dietary protein content (DPC g/kgDM) and feeding level (FL, 0.95×ad libitum
or 0.80×ad libitum) on amino acid gain (g/d) of viscera and carcass in growing (15-50 kg) and
fattening (50-100 kg) Iberian pigs1.
Viscera Carcass
His Leu Lys Thr Leu Lys Thr Val

Growing pigs
DPC, 175 0.23 0.53 0.60 0.28 3.1 a 4.0 a 1.8 a 2.1 a
156 0.24 0.55 0.62 0.29 3.4 ab 4.4 ab 2.1 ab 2.3 ab
129 0.23 0.52 0.59 0.28 3.7 b 4.8 b 2.2 b 2.5 b
P value ns ns ns ns ** ** ** **
FL, 0.95 × 0.28 0.64 0.72 0.34 3.8 a 4.9 a 2.2 a 2.6 a
0.80 × 0.19 0.43 0.48 0.23 3.1 b 4.0 b 1.8 b 2.1 b
P value *** *** *** *** *** *** *** ***
Fattening pigs2
Mean value3 0.15 0.37 0.26 0.18 2.7 3.8 1.6 1.6
1Means with different superscript letter differ (P<0.05).

2 DPC, 145 and 95 g/kg DM.
3 No significant effect of DPC or FL.
References
Barea, R., R. Nieto and J.F. Aguilera, 2007. Effects of the dietary protein content and the feeding level on protein and
energy metabolism in Iberian pigs growing from 50 to 100 kg body weight. Animal, in press.
Nieto, R., A. Miranda, M.A. García and J.F. Aguilera, 2002. The effect of dietary protein content and feeding level
on the rate of protein deposition and energy utilization in growing Iberian pigs from 15 to 50 kg body weight.
Br. J. Nutr. 88, 39-49.
Rivera-Ferre, M.G., J.F. Aguilera and R. Nieto, 2005. Muscle fractional protein synthesis is higher in Iberian than in
Landrace growing pigs fed adequate or lysine-deficient diets. J. Nutr. 135, 469-478.

Study, in model system, of the effect of oxidation on myofibrillar protein
digestibility
L. Aubry, Ph. Gatellier, A. Vivion and V. Santé-Lhoutellier
INRA, UR370 QuaPA, 63122 Saint Genès Champanelle, France
Introduction
Meat proteins serve as an important source of energy and essential amino acids for humans. To enter
the bloodstream, proteins must be broken down into aminoacids or small peptides by proteases during
the flow through the digestive tract. This process depends on the physicochemical state of proteins.
Meat processes (storage, mincing, cooking, salting) generate free oxygenated radicals which can
induce physical and chemical changes to the proteins resulting in alteration of their nutritional value.
Oxidation of amino groups into carbonyls, thiol oxidation and aromatic hydroxylation are the main
chemical modifications of aminoacids during oxidation (Davies et al., 1987). These reactions can lead
to protein aggregation and to changes in surface hydrophobicity. The interaction between proteolysis
and oxidation of proteins has been extensively studied in medical sciences with contrasting effects.
An increase of hydrophobicity can enhance protein degradation while the formation of aggregates
in highly oxidative conditions can make proteins less susceptible to proteolysis (Davies, 2001). In
meat, the link between protein oxidation and digestibility is poorly documented. However it has been
shown that myosin oxidation can affect its proteolytic degradation by enzymes of the digestive tract
(Liu and Xiong, 2000). The present study was designed to explore the effect of oxidative conditions
on in vitro digestibility of myofibrillar proteins from pig M. longissimus dorsi.

Myofibrils were incubated 3 h at 37 °C in a phosphate buffer (20 mM; pH 6) with FeSO4 and
H2O2 which generate the hydroxyl radical. The whole protein oxidation was estimated with 2.4-
dinitrophenyl hydrazine (DNPH) according to Oliver et al. (1987) and expressed in nmol of carbonyl
per mg of protein. Specific protein oxidation was evaluated by labeling protein carbonyls with
DNPH followed by immunoblotting (kit Chemicon) of proteins separated by 12.5% SDS-PAGE.
Proteolysis was performed with pepsin (20 U/mg protein) 1 h at 37 °C in glycine buffer (33 mM; pH
1.8) and its rate was measured by absorbance at 280 nm of the 15% TCA-soluble fraction. The non
soluble fraction was then hydrolysed 30 min at 37 °C in glycine buffer (33 mM; pH 8) by trypsin
and α-chymotrypsin (6.6 U and 0.33 U/mg protein).

Protein oxidation
Figure 1a shows the effect of increasing oxidising agent on whole protein oxidation. Carbonyls
increased linearly between 0 and 3 mM Fe2+/H2O2 to reach a maximum which was 12 folds the
initial level. Figure 2 confirms the formation of carbonylated proteins with increasing amounts of
oxidising agent. Among all myofibrillar proteins, only myosin and actin oxidation were detectable
and the level of oxidation was more pronounced in actin. From 1 mM of Fe2+/H2O2 formation of
smears indicated protein aggregation or fragmentation. In a previous study (Morzel et al., 2006) we
showed an important increase of disulfide and dityrosine bridges, implicated in protein aggregation,
at this level of oxidising agent.

18
a b c
Carbonyls (nM/mg prot.)
16 0,25
0,4
Proteolysis rate (OD/h)

14
Proteolysis rate (OD/h)

12 0,2
0,3
10 0,15
8
0,2
6 0,1
4 0,1 0,05
2
0 0 0
0
0,05
0,1
0,3
0,5
1
3
5
0
0,05
0,1
0,3
0,5
1
3
5
0
0,05
0,1
0,3
0,5
1
3
5
Fe2+ / H2O2 (mM) Fe2+ / H2O2 (mM ) Fe2+ / H2O2 (mM)
Figure 1. Effect of oxidising agent on (a) carbonyl content of myofibrillar proteins, (b) proteolysis
by pepsin, and (c) proteolysis by trypsin and α-chymotrypsin (n=4).
Fig 2
Myosin
Actin
0 0.05 0.1 0.3 0.5 1 3 5

Fe 2+ / H2O2 (mM)
Figure 2. Immunoblot analysis of oxidised proteins separated by 12.5% SDS Page.
In vitro digestibility
Figures 1b and 1c show the effect of a prior oxidative treatment on myofibrillar protein degradation
by digestive tract proteases. With pepsin, a protease of the stomach, a biphasic curve was observed.
Low levels of oxidising agent increased proteolysis rate while an important decreased rate was
observed in the highest oxidative conditions for which aggregation was described (Morzel et al.,
2006). Digestibility by pancreatic proteases was less affected by oxidation. A decrease in proteolysis
rate was observed only from 1 mM Fe2+/H2O2.
In conclusion, oxidative processes either increased or decreased digestibility of myofibrillar

proteins depending on the nature of the protease and the level of protein oxidation. The decrease of
proteolysis rate, observed for both proteases, at the highest oxidations could be attributed to protein
aggregation.
References
Davies, K.J.A., 2001. Degradation of proteins by the 20S proteasome. Biochimie 83, 301-310.
Davies, K.J.A., M.E. Delsignore and S.W. Lin, 1987. Protein damage and degradation by oxygen radicals. II.
Modification of amino acids. J. Biol. Chem. 262, 9902-9907.
Liu, G. and Xiong Y.L., 2000. Electrophoretic pattern, thermal denaturation, and in vitro digestibility of oxidized
myosin. J. Agr. Food Chem. 48, 624-630.
Morzel, M., Ph. Gatellier, T. Sayd, M. Renerre and E. Laville, 2006. Chemical oxidation decreases proteolytic
susceptibility of skeletal muscle proteins. Meat Sci. 73, 536-543.
Oliver, C.N., B.W. Alin, E.J. Moerman, S. Goldstein and E.R. Stadtman, 1987. Age-related changes in oxidized proteins.
J. Biol. Chem. 262, 5488-5491.

Effects of maternal nutrition on birth weight and postnatal nutrient
metabolism
J. Caton1, K. Vonnahme1, J. Reed1, T. Neville1, C. Effertz1, C. Hammer1, J. Luther1, J. Taylor2, D.
Redmer1 and L. Reynolds1
1Center for Nutrition and Pregnancy, Department of Animal and Range Sciences, North Dakota
State University, Fargo, ND 58015, USA
2USDA-ARS, U. S. Sheep Experiment Station, Dubois, ID 83423, USA
Introduction
Effective nutritional management during gestation has long been recognized as an important
component of livestock production practices. Recent observation in developmental programming
has elevated the concept of maternal onset of adult disease and lifelong performance to the forefront
of investigation in several laboratories. In addition, the 2007 Aspen Perinatal Biology Symposium
and the 2007 5th International Congress on Developmental Origins of Health and Disease, both
focus on developmental programming and have major sections that highlight relevance in animal
agriculture. Clearly, research in these directions with livestock species has and will likely continue
to produce relevant data for both livestock and biomedical interests. Nutrient restriction and excess
has been previously implicated in developmental programming and maternal selenium intakes may
be elevated in many parts of the world. Therefore, we hypothesize that maternal plane of nutrition
and dietary selenium level will result in measurable postnatal responses in sheep.

Pregnant Rambouillet ewe lambs (n=82) of equivalent BW (52.2 ± 0.8 kg) and condition score (BCS;
3.0 ± 0.05) were housed individually and allotted randomly to one of 6 treatments in a 3 × 2 factorial
design. The groups included plane of nutrition (60% [RES], 100% [CON], and 140% [HIGH] of
requirements for gestating ewes) and dietary levels of Se [adequate Se (7.4 µg/kg BW) vs. high Se
(85 µg/kg BW); from Se enriched yeast]. Selenium (Se) treatments were initiated at breeding and
nutritional treatments on d 40 of gestation. All diets were fed once daily in a complete pelleted form
(36.5% beet pulp, 22.3% alfalfa meal, 16.2% corn, 18% soybean hulls, and 7.0% soybean meal;
14.4% CP, 2.63 Mcal ME/kg; DM basis). At parturition, lambs were removed from the ewes before
nursing and provided artificial colostrum for the first 20 h, after which milk replacers were fed.
Lambs were weaned at 8 wk of age and managed similarly during the growing and finishing phases
of production. A subset of both ewe (n=30) and wether (n=29) lambs were penned individually
during two intensive intake and efficiency measurements (7 d each; occurring in mo 3 and 4 of age).
Following efficiency measurements, wether lambs were used in two 7-d N-balance and digestion
determinations in metabolism cages. Also following the first efficiency measurement, ewe lambs
(n=30; d=107 ± 0.71; 33.5 ± 0.76 kg) were used in a glucose challenge. Catheters were placed in
the jugular vein of each ewe lamb and blood samples were drawn 30 min after catheter placement.
Blood samples were drawn (t=-5, -2, 2, 5, 10, 15, 30, 45, 60, 90, 120, 150, 180, and 240 min). An
injection (0.5 g glucose/kg BW) was given at t=0. Glucose and insulin levels were determined by
colorimetric and Immulite, respectively. Area under the curve for each animal was determined
using Sigma Plot software. Lambs were necropsied at approximately 5 mo of age for organ mass
determinations. Data were analyzed using the GLM procedure of SAS (SAS Inst. Inc., Cary, NC)
for a 3 × 2 factorial with maternal nutrition and Se level being the main effects and fetal number was
used as a covariate. Means were separated with the method of least significant difference.

Results
At parturition, ewe BW and BCS were least (P<0.01) in RES, intermediate in CON, and greatest
in HIGH (53.9, 67.3, and 76.5 ± 1.4 kg and 1.60, 2.53, and 3.61 ± 0.10, respectively), as were ewe
average daily gain (ADG) and gain:feed ration (P<0.01). Ewe BW, BCS, ADG, gain efficiency, and
gestation length were not altered by Se supplementation. At birth, lamb BW, curved crown rump
length, and heart girth were lower (P=0.01) in RES and HIGH compared with CON. Weaning weight
8 wk post partum was less (P=0.01) in lambs from RES ewes compared with those from CON and
HIGH. Birth and weaning weight were unaffected by Se supplementation.
Maternal nutrition did not alter (P>0.10) dietary intake or digestion coefficients for dry matter (DM),
organic matter (OM), neutral detergent fiber (NDF), and acid detergent fiber (ADF) in wethers.
Likewise, maternal Se supplementation did not alter (P>0.10) intake in lambs; however, total tract
digestion of DM, OM, NDF, and ADF were reduced (P<0.05) by high maternal Se intakes. Maternal
dietary treatments did not alter (P>0.23) N retention, or N retention per unit of N consumed or
digested. Maternal plane of nutrition by Se level interactions were noted for ADG and gain:feed ratios.
In lambs from HIGH ewes, supplementing Se increased (P<0.01) ADG and gain:feed ratios.
There was no effect of nutrition or Se on the amount of circulating glucose. However, there was a
nutrition by Se interaction (P<0.05) with ewe lambs from RES ewes fed adequate dietary Se and
having reduced insulin release compared with ewe lambs from other treatments.
Live BW, hot carcass weight, and dressing percent of lambs at necropsy were not affected (P>0.26)
by maternal dietary treatment. Lamb pancreatic mass (g) was reduced (P=0.06) in RES and HIGH
compared with CON, as was blood mass (g and g/kg of empty BW; P<0.08). Mass of the reticulum
(g) was also reduced (P=0.10) in lambs from the RES and HIGH compared with CON ewes. In the
lambs, maternal Se supplementation increased (P<0.01) mass (g and g/kg empty BW) of the ovaries,
decreased (P<0.09) mass of the heart and kidney (g/kg empty BW), and increased (P=0.04) total
visceral adiposity compared with adequate Se. Maternal nutrition by Se interactions were observed
(P<0.06) for the omasum, small intestine, ileum, and lungs.
Conclusions
We suggest that maternal plane of nutrition but not supplemental Se affects the BW, BCS, and
gestation length of ewes and birth and weaning weights of lambs. Maternal Se supplementation
results in reduced postnatal digestion coefficients, increased ADG and feed:gain in lambs from
HIGH fed ewes, altered internal organ masses, and increased visceral adiposity in lambs at necropsy.
Maternal RES and HIGH diets reduced pancreatic mass in lambs and Se supplementation in RES
ewes resulted in reduced insulin release postnatally.
These data provide strong evidence for developmental programming in offspring from pregnant
ewes receiving various nutritional levels and supplemental Se.

Glucose metabolism and its relation to milk production in F2 offspring of
Charolais × Holstein crosses
H.M. Hammon, P. Junghans, O. Bellmann, F. Schneider, R. Weikard and C. Kühn
Research Institute for the Biology of Farm Animals, Dummerstorf (FBN), Wilhelm-Stahl-Allee 2,
18196 Dummerstorf, Germany
Introduction
Nutrient transformation and endocrine regulation of nutrient flow distinctly differ between Charolais
(beef breed; accretion type) and German Holstein (dairy breed; secretion type) (Bellmann et al.,
2004). Furthermore, the marked differences in milk production between Charolais and Holstein
cows are related to changes in insulin-dependent glucose metabolism (Pareek et al., 2007). Breeding
segregating F2 cows of crosses from Charolais and German Holstein provides an animal model that
combines dairy and beef genetic background and allows to study the regulation of nutrient flow with
respect to milk production (Kühn et al., 2002). Interestingly, milk production during first lactation
markedly differed in two F2 families with exchanged paternal and maternal grandsires (Hammon
et al., 2006). Therefore, we hypothesised that glucose metabolism during second lactation differs
between cows of these two F2 families and is related to differences in milk production.

The crossbreed cows included in our study have been generated by mating Charolais bulls (named
A and B) to German Holstein cows and a following intercross of the F1 individuals using embryo
transfer to German Holstein recipients to establish full sib and half sib F2 offspring. Eighteen F2
cows (n=9 for family Ab and Ba, A or a and B or b denoting two different Charolais sires, capital
letters indicating the respective paternal grandsire) were investigated during second lactation.
Cows received intravenous glucose infusions (1 g/kg BW0.75) 10 d before and 30 and 93 d after
parturition. Blood samples were taken before and 7, 14, 21, and 28 min after glucose challenge.
Glucose and insulin plasma concentrations were measured. Glucose half-time (T½gluc) as well as
areas under the concentration curve for glucose (AUCgluc) and insulin (AUCins) were calculated.
On d 94 a U-13C6-D-glucose bolus (3 mg/kg BW0.75) was given intravenously and blood samples
were taken before and 5, 10, 20, 30, 60, 90, 120, 150, 180 min after injection for determination
of glucose production (GP) and plasma concentrations of glucose, NEFA, β-OH-butyrate, insulin,
and glucagon. Cows were 12 h without food before glucose challenge tests and GP measurements,
respectively. Cows were slaughtered on d 100 after parturition and liver samples were snap-frozen
in liquid nitrogen and stored at –80 °C until analysed. Total RNA was extracted and gene expression
for pyruvate carboxylase (PC, EC 6.4.1.1), cytosolic and mitochondrial phosphoenolpyruvate
carboxykinase (PEPCK; EC 4.1.1.32), glucose 6-phosphatase (G6-Pase; EC 3.1.3.9) and facilitative
glucose transporter 2 (GLUT2) were quantified by real-time RT-PCR relative to a housekeeping
gene (GAPDH). Data for milk yield, glucose challenge tests, and plasma concentrations on d 94
were analysed using the Mixed Model of SAS with family and time as fixed effects and individual
cows as random effects. Differences were identified by the Tukey t-test. Data for GP and hepatic
gene expression were analysed by general linear model of SAS with family as the fixed effect. The
PROC CORR procedure of SAS was used to calculate correlations between milk yield, plasma
concentrations, AUC, GP and hepatic gene expression.

Milk yield was generally on a low level, ranging from 0.5 to 25 kg milk/d on the first 93 d in milk,
and was higher (P<0.05) in the Ba than in the Ab family. Because sire A was mated to paternal

half sisters sired by B and vice versa, this difference in production might indicate possible sex
chromosomal or imprinting effects. On d 93 AUCins was lower (P<0.05) and T½gluc was higher in
Ba than in Ab. AUCins did not differ between families on d -10 and 30 relative to parturition. On d 94
plasma glucose and insulin were lower (P<0.05) and plasma β-OH-butyrate and the glucagon/insulin
ratio were higher (P<0.05) in Ba than in Ab. An inverse relationship between insulin status and milk
production is also seen when comparing dairy and beef cows (Bines and Hart, 1978; Pareek et al.,
2007). In liver tissue, mRNA levels of PC, G6-Pase and GLUT2 were higher (P<0.05) and levels of
cytosolic PEPCK tended to be higher (P<0.1) in Ba than in Ab. GP was positively related (r=0.78;
P<0.05) to milk production, although not significantly different between families. Milk yield was
negatively associated with basal plasma concentrations of glucose and insulin and AUCins, but was
positively associated with T½gluc, and mRNA levels of PC and G6-Pase (r=-0.44, -0.57, -0.33, 0.52,
0.54 and 0.69; P<0.05, respectively). GP was positively associated with mRNA levels of PC and
G6-Pase (r=0.38 and 0.41; P=0.1, respectively).
Data presented herein indicate that a higher level of milk production in F2 cows from crosses of
Charolais and German Holstein was associated with a lower insulin status, greater glucose production
rates and an elevated expression of enzymes involved in gluconeogenesis and glucose transport.
These results support the importance of glucose supply for milking performance (Rigout et al., 2002),
and that insulin status affects nutrient partitioning by favouring glucose uptake in tissues besides the
mammary gland (Kronfeld et al., 1963).
References
Bellmann, O., J. Wegner, C. Rehfeldt, F. Teuscher, F. Schneider, J. Voigt, M. Derno, H. Sauerwein, J. Weingärtner
and K. Ender, 2004. Beef versus dairy cattle: a comparison of metabolically relevant hormones, enzymes, and
metabolites. Livest. Prod. Sci. 89, 41-54.
Bines, J. A. and I. C. Hart, 1978. Hormonal regulation of the parition of energy between milk and body tissue in adult
cattle. Proc. Nutr. Soc. 37, 281-287.
Hammon, H.M., O. Bellmann, J. Voigt, F. Schneider and C. Kühn, 2007. Glucose-dependent insulin response and milk
production in heifers within a segregating resource family population. J. Dairy Sci., in press.
Kronfeld, D.S., G.P. Mayer, J.M. Robertson and F. Raggi, 1963. Depression of milk secretion during insulin
administration. J. Dairy Sci. 46, 559-563.
Kühn, C., O. Bellmann, J. Voigt, J. Wegner, V. Guiard and K. Ender, 2002. An experimental approach for studying the
genetic and physiological background of nutrient transformation in cattle with respect to nutrient secretion and
accretion type. Arch. Tierz. 45, 317-330.
Pareek, N., J. Voigt, O. Bellmann, F. Schneider and H.M. Hammon, 2007. Energy and nitrogen metabolism and insulin
response to glucose challenge in lactating German Holstein and Charolais heifers. Livest. Sci., doi:10.1016/
j.livsci.2007.02.001.
Rigout, S., S. Lemosquet, J.E. van Eys, J.W. Blum and H. Rulquin, 2002. Duodenal glucose increases glucose fluxes
and lactose synthesis in grass silage-fed dairy cows. J. Dairy Sci. 85, 595-606.

Expression of beta-adrenergic receptors during lactation in Holstein
dairy cattle
J.M. Sumner and J.P. McNamara
Department of Animal Sciences, Washington State University, Pullman 99164-6351, USA
Introduction
There is great variation among dairy cattle in body condition loss during early lactation, which affects
all aspects of production, health and longevity. As a result of previous research on body condition
score, metabolism of fat and regulation of lipolysis in dairy cattle, we are now able to examine the
control of lipolysis and thus body fat use at a genetic level.
The objective was to determine the expression of beta-adrenergic receptors in adipose tissue of
Holstein dairy cattle during pregnancy and lactation. Activity of hormone sensitive lipase and
responsiveness to beta-adrenergic stimulation increases during early lactation; and the adaptation
varies among cattle with different milk production ability. However, expression of beta-adrenergic
receptor subtypes and their potential role in the control of lipolysis is unknown for dairy cattle.

Twenty Holstein dairy cattle were grouped by lactation number (1, 2, and 3 or more) and subcutaneous
adipose tissue was sampled via biopsy to measure in vitro lipolytic rates and gene expression. Gene
expression was measured using real-time RT-PCR and primers were designed using Primer express
software for the three beta-adrenergic receptor subtypes, hormone sensitive lipase, and perilipin and
two reference genes, beta actin and ribosomal S2 protein.
Results
Production of 305 d adjusted lactation for 1st, 2nd and higher parity cows were 14936, 12791,
and 11326 (SE 931). Fat averaged 3.55 and protein 3.3%. Body weight and body condition score,
measured at -30, 30, 90 and 270 d around calving averaged 682, 598, 634 and 638 kg and 3.3, 2.3,
2.5, and 2.7 body condition score units.
Lipolysis was measured in in vitro incubations at 6 doses of isoproteranol to determine basal rates
and response to adrenergic stimulation, to relate to gene expression information. Basal lipolysis
increased 10% following parturition, stimulated lipolysis peaked at 60% above pre partum at 90 d
post partum. This is consistent with previous work showing such an increase.
The beta-1 receptor was expressed at all time points and the relative expression increased at all post
partum sample points. The mean increase relative to pre partum was 170%, 72% and 112% at 30,
90, and 270 d in milk (DIM). The beta-2 receptor was expressed at all time points and the expression
increased at all post partum sample points (P=0.06). The mean increase was 75%, 121% and 100%
at 30, 90, and 270 DIM compared to 30 d pre partum. The beta-3 receptor was expressed and the
expression increased post partum. The mean increase was 111%, 125% and 69% at 30, 90, and 270
DIM. This is the first demonstration of an up-regulation of gene expression of lipolytic proteins in
the adipose tissue of dairy cattle during lactation and is consistent with the increased rates of basal
and stimulated lipolysis.
Hormone sensitive lipase was expressed at all time points measured and the expression increased
(P=0.09) post partum and was the highest in primiparous animals (P=0.002). The mean increase

was 180%, 359% and 47% at 30, 90, and 270 d of lactation compared to pre partum. Perilipin was
expressed at all time points and the expression increased (P=0.04) post partum. The mean increase
was 227%, 1847% and 126% at 30, 90, and 270 DIM. This is the first time perilipin has been measured
in the adipose tissue of dairy cattle.
Discussion
This work has demonstrated that during lactation the expression of key genes involved in the catabolic
pathways in adipose tissue are increased. Prior to this research, it had been shown that the sensitivity
of adipose tissue to lipolytic stimulus was increased during lactation (McNamara, 1994; and several
references therein). This was demonstrated by showing increased sensitivity to lipolytic stimuli in
lactation and further, increased sensitivity in higher producing animals (McNamara and Hillers,
1989). We now know that this increase is mediated at least in part by increases in the expression
of the beta-adrenergic receptor subtypes within the cell membrane and the expression of hormone
sensitive lipase and perilipin, its cofactor, within the cell.
The animals used in this study had a calculated negative energy balance in early lactation and
were losing approximately 1 kg of body weight per d at 30 DIM. At 90 DIM, they were gaining
approximately 1 kg of body weight per d at the same time that they had the highest levels of lipolytic
gene regulation and the highest rates of stimulated lipolysis. This provides support for the hypothesis
of simultaneously high rates of both lipogenesis and lipolysis.
Further research in this area will result in improvements both in animal health and to the models
of nutrient use developed from the previous work (McNamara, 1994). These models can be used
to develop and test specific hypotheses and lead to improved management and profitability for the
dairy industry.
References
McNamara, J.P. and J.K. Hillers, 1989. Regulation of bovine adipose tissue metabolism during lactation 5. Relationships
of lipid synthesis and lipolysis with energy intake and utilization. J. Dairy Sci. 72, 407-418.
McNamara, J.P., 1994. Lipid metabolism in adipose tissue during lactation: A model of a metabolic control system.
J. Nutr. 124, 1383S-1391S.

Investigations on the storage of ω-3 and ω-6 fatty acids in the dry period
and their transfer into milk fat in early lactation
H.-R. Wettstein1, R.E. Hochstrasser1,2, C. Elia3, H. Leuenberger4, M. Wanner2 and M. Kreuzer1
1ETH Zurich, Institute of Animal Sciences, Universitaetstr. 2, 8092 Zurich, Switzerland
2University of Zurich, Vetsuisse Faculty, Winterthurerstr. 270, 8057 Zurich, Switzerland
3University of Padova, Dept. of Animal Sci., V.le dell’Università, 16, 35020 Legnro, Padova, Italy
4ETH Zurich, ETH Research Stations, Chamau, 6331 Hünenberg, Switzerland
Introduction
Recently, the fatty acid composition of milk, and especially the content of ω-3 fatty acids has gained
increasing attention due to the associated human health aspects. Several studies showed that fatty
acid profile can be favourably influenced directly by the diet offered (e.g. Collomb et al., 2004).
However, there might also be indirect influences, especially of feeding history, in times when body
fat is intensively mobilised. This was shown in cows grazing high altitude pastures (Leiber et al.,
2005). Soppela and Nieminen (2002) described that ω-3 fatty acids, previously deposed in adipose
tissue, seem to be mobilised preferentially in energy deficiency. This hypothesis should be tested by
strategic feeding of dairy cows in late pregnancy and early lactation, when deposition and mobilisation
of adipose fat is prevalent.

Two concentrates either characterised by crushed linseed (L) or sunflower seed (S) were fed to 4×6
dairy cows in different order from 6 wk before calving to 6 wk after calving. In the first treatment
(LL/LL) the cows got 2 kg/d of L and in the second (SS/SS) 2 kg/d of S before and after calving.
In the two other treatments, cows either received the L concentrate at a level of 2 kg/d or a mixture
of the L- and the S concentrate (1 kg/d each), but this only until 5 d before expected calving date,
from when on these cows received 2 kg/d of S until the end of the experiment (treatments LL/SS and
LS/SS, respectively). Additionally, the cows were fed a roughage mixture and, after calving, low-fat
energy and protein concentrates according to requirements. The roughage mixture consisted of grass
silage and straw before calving and of grass silage, corn silage and hay after calving. Before and
after calving, feed intake was recorded and feed samples and biopsies of the subcutaneous adipose
tissue from the pelvic region were taken. After calving, milk samples were taken and milk yield
was recorded. The main milk constituents were analysed by using infrared technique. Fatty acid
methyl esters (FAME), generated by the cold trans-esterification method (Suter et al., 1997), were
analysed chromatographically using a Supelcovax® column. The results were subjected to statistical
analysis by using the mixed procedure of SAS regarding treatment, sampling time and the respective
interaction with the animal as subject for repeated measurement.
Results
Milk yield and milk protein content were not affected by treatments (P>0.05) and, on average,
accounted for 35.7 kg milk/d and 3.24% protein for all four treatments across the first 40 d of
lactation. In milk fat content a trend toward the highest value with LL/SS (4.66%) was found
(P=0.05) compared to 4.06, 4.46 and 3.82% found with LL/LL, SS/SS and LS/SS respectively. In
colostrum, the α-linolenic acid (ALA) proportion was the highest (P<0.001) in the milk fat of cows
exposed to the LL/LL treatment (Figure 1). The values for the cows receiving linseed concentrate
before calving and sunflower concentrate after calving were intermediate, and the values were the
lowest in the cows always receiving sunflower concentrate. After the colostrum period, a trend for
a declining ALA proportion in milk fat occurred in all treatments. In the cows where the linseed

Figure 1. Proportions of α-linolenic acid and of linoleic acid in milk fat.
concentrate was replaced by the sunflower concentrate shortly before calving, the proportion of
ALA approached the values of the SS/SS cows within the first 30 d. The proportion of linoleic acid
(LA), however, was always the lowest with the LL/LL treatment and the highest with LL/SS, but
was only intermediate in SS/SS and in LS/SS.
The ALA proportions in adipose tissue before calving were 0.57, 0.45, 0.55 and 0.53 g/100 g FAME
for LL/LL, SS/SS, LS/SS and LL/SS (P>0.1) and decreased in all four treatments during the first
6 wk of lactation (P<0.001). The highest decrease was found with LL/SS (‑0.21 g/100 g FAME,
P<0.05) followed by SS/SS (‑0.11 g/100 g FAME), whereas the decrease was quite similar with
LL/LL and LS/SS (‑0.05 and ‑0.04 g/100 g FAME). On the contrary to ALA, the proportion of LA
in adipose tissue stayed similar or increased slightly.
The results indicate a certain influence of feeding history on milk fat ALA proportion at the very
beginning of the lactation and, therefore, a proportionately higher mobilisation of ALA compared
to other fatty acids whereas for LA this does not seem to be the case. Overall, intake of these fatty
acids during the dry period was of only minor importance for milk fat composition.
References
Collomb, M., H. Sollberger, U. Büttikofer, R. Sieber, W. Stoll and W. Schaeren, 2004. Impact of a basal diet of hay
and fodder beet supplemented with rapeseed, linseed and sunflower seed on the fatty acid composition of milk.
Int. Dairy J. 14, 549-559.
Leiber, F., M. Kreuzer, D. Nigg, H.-R. Wettstein and M.R.L. Scheeder, 2005. A study on the causes for the elevated
n-3 fatty acids in cows’ milk of alpine origin. Lipids 40, 191-202.
Soppela, P. and M. Nieminen, 2002. Effect of moderate wintertime undernutrition on fatty acid composition of adipose
tissues of reindeer (Rangifer tarandus tarandus L.). Comp. Biochem. Physiol. A 132, 403-409.
Suter B., K. Grob and B. Pacciarelli, 1997. Determination of fat content and fatty acid composition through 1-min
transesterification in the food sample; principles. Z. Lebensm. Unters. Forsch. A 204, 252-258.

Live weight, body size, fatness and carcass characteristics of young bulls
of fifteen European breeds
P. Albertí1, B. Panea1, C. Sañudo2, J.L. Olleta2, G. Ripoll1, P. Ertbjerg3, M. Christensen3, S. Gigli4, S.
Failla4, A. Gaddini4, J.F. Hocquette5, R. Jailler5, S. Rudel5, G. Renand6, G.R. Nute7, R.I. Richardson7
and J.L. Williams8
1CITA de Aragón, 50080, Zaragoza, Spain
2Departamento de Producción Animal y Ciencia de los Alimentos, Universidad de Zaragoza, 50013
Zaragoza, Spain
3Department of Food Science, University of Copenhagen, DK-1958 Frederiksberg C, Denmark
4CRA, Istituto Sperimentale per la Zootecnia, 00016 Monterotondo, Italy
5INRA, UR1213, 63122 Theix, France
6INRA, UR337, 78352 Jouy-en-Josas cedex, France
7Division of Farm Animal Science, University of Bristol, BS40 5DU, United Kingdom
8Parco Tecnologico Padano, Via Einstein, Polo Universitario, Lodi, 26900, Italy
Introduction
This work is part of the European Project GemQual (Genetics of Meat Quality), which involved
fifteen cattle breeds from five different countries. Its objectives were to compare animal performances,
body fatness and carcass characteristics, meat quality across breeds, and identify genetic markers of
beef quality. The objective of this paper was thus to describe the relationships between performances,
body size characteristics and carcass traits of young bulls from fifteen Western European cattle pure
breeds, fed with concentrates and slaughtered at 15 mo of age.

A total of 436 pure-breed bulls were reared on five experimental research centres in the United
Kingdom, Jersey (JER), South Devon (SD), Aberdeen Angus (AA) and Highland (HIG); Denmark
Holstein (HOL), Danish Red Cattle (RED) and Simmental (SIM)]; Spain Asturiana de los Valles
(ASV), Casina (CAS), Avileña (AVI) and Pirenaica (PIR); Italy Piemontese (PIE) and Marchigiana
(MAR), and France Limousin (LIM) and Charolais (CHA). The standardised diet consisted mainly
of a concentrate compounded from barley flakes (80 to 84%). The energy density ratio and protein
content of the diet were similar for all breeds. The weight, body length, height at withers and
pelvis width, of the animals were recorded at 9, 12 and 15 mo of age. After slaughter, 15 carcass
variables were recorded, including carcass weight, conformation and fatness scores, morphological
measurements and dissection data. With these variables, analysis of variance and principal component
analysis were performed to evidence relationships between the studied traits.

The use of ADG rate and live weight has not been consistent to sort breeds by type (dairy, beef, or
local) since for a short interval of weights, it was possible to find dairy breeds (Holstein and Danish
Red) that grew slowly and fattened later than expected from their target weights in comparison
with other breeds, and beef breeds that grow more quickly than expected by its final live weights as
Aberdeen Angus and South Devon.
However, the body size measurements and the carcass traits were more useful tools to discriminate
cattle breeds, as meat specialised (PIE, ASV, PIR, LIM, SD, CHA and AA), local and dairy breeds
(JER, CAS, HIG, HOL and RED) and intermediate breeds (AVI, MAR and SIM). Muscle percentage
measured from the dissection of the 6th rib dissection ranged from 58.9% in Holstein to 79.9% in

Piemontese, whereas fat percentage varied from 3.2% in Piemontese to 21.7% in Aberdeen Angus.
Dairy and local breeds had the highest values for fat and the lowest values for muscle percentages,
as would be expected.
On Figure 1, the first factor of the principal component analysis explained 48.8% of variability,
was related to the blockiness index, carcass weight, loin area, minimum diameter of the loin,
dressing percentage and conformation score; the second factor explained 24.6%, was related to the
fat percentage measured at dissection, fatness score and kidney knob and channel fat weight, and
negatively related to muscle percentage at dissection. Breeds that are grouped as specialised-beef
breeds (PIE, ASV, PIR, LIM, SD, CHA and AA), have high muscle score but they differ clearly in
fatness. Local and dairy breeds are placed on the left side of the plot (JER, CAS, HIG, HOL and
Figure 1. Breeds plotted by carcass traits on principal component analysis.
RED), they were badly shaped and the dairy highly fattened. The intermediate group (AVI, MAR and
SIM) have intermediate features: medium conformation, medium fatness level, they have undergone
some selection and have improved conformation characteristics.
2.5
badly shaped well shaped
greatly fattened 2 greatly fattened
Factor 2: 24.6%; fatness
1.5
AA
HOL RED
1
HIG CHA
0.5 SD
LIM
0 SIM
-2.5 -1.5 CAS -0.5 0.5 1.5 2.5
PIR
AVI -0.5
ASV
JER MAR
-1
-1.5
PIE well shaped

badly shaped poorly fattened
-2
poorly fattened
Factor 1: 48.8%: conformation
-2.5
Figure 1. Breeds plotted by carcass traits on principal component analysis.
In conclusion, this study verifies that body dimensions and carcass characteristics are objective traits
that differ among breed, and in this study it was useful to sort group cattle breeds on homogeneous
types. Furthermore, this study shows that no consistent relationship exists between carcass weight
or conformation and fatness of bovine breeds.

Metabolic and contractile characteristics of Longissimus thoracis muscle
of young bulls from 15 European breeds in relationship with body
composition
J.F. Hocquette1, C. Jurie1, B. Picard1, P. Albertí3, B. Panea3, M. Christensen4, S. Failla5, S. Gigli5,
H. Levéziel2, J.L. Olleta6, C. Sañudo6, P. Ertbjerg4, G.R. Nute7 and J.L. Williams8
1INRA, UR 1213, 63122 Theix, France
2INRA-Université de Limoges, UMR 1061, 87060 Limoges, France
3Centro de Investigación y Tecnología Agroalimentaria, Zaragoza, 50080, Gobierno de Aragón,
Spain
4Department of Food Science, University of Copenhagen, DK-1958 Frederiksberg C, Denmark
5Istituto Sperimentale di Zootecnia, Monterotondo, 00016 Italy
6Departamento de Producción Animal y Ciencia de los Alimentos, 50013, Universidad de Zaragoza,
Spain
7Division of Farm Animal Science, BS40 5DU, University of Bristol, United Kingdom
8Roslin Institute EH25 9 PS, United Kingdom
Introduction
This work is part of a European Project, GemQual (Genetics of Meat Quality), which involved nine
partners from five different countries. Its objective was to identify genetic markers of beef quality.
However, the experimental design also allows the comparison of animal performance, carcass
characteristics (companion paper by Alberti et al., 2007. Int. Symp. on Energy and Protein Metabol.
and Nutr., Vichy, France) and beef quality across breeds according to their body composition. This
paper focusses on the metabolic and contractile properties of muscle fibres of the fifteen studied
bovine breeds.

A total of 436 pure-breed bulls were reared in 5 experimental research centres in the UK [Jersey
(JER), South Devon (SD), Aberdeen Angus (AA) and Highland (HIG)], Denmark [Holstein (HOL),
Danish Red Cattle (RED) and Simmental (SIM)], Spain [Asturiana de los Valles (ASV), Casina
(CAS), Avileña (AVI) and Pirenaica (PIR)], Italy [Piemontese (PIE) and Marchigiana (MAR)]
and France [Limousin (LIM) and Charolais (CHA)]. The standardised diet consisted mainly of a
concentrate compounded from barley flakes (80 to 84%). The energy density ratio and protein content
of the diet were similar for all breeds. Animals were slaughtered at 15 mo of age (carcasses were not
electrically stimulated) and samples of longissimus thoracis were taken within 1 h from slaughter for
the measurement of metabolic activity characteristics of glycolytic (lactate dehydrogenase [LDH]),
oxidative muscle metabolism (isocitrate dehydrogenase [ICDH], citrate synthase [CS], cytochrome-c
oxydase [COX]) and for the determination of the proportions of the different myosin heavy chain
(MyHC) isoforms (type I: slow oxidative; type IIa: fast oxido-glycolytic, type IIx: fast glycolytic).
Relationships between variables were studied using principal component analysis.
Results
As presented in a companion paper by Alberti et al., AA is the fattest breed followed by HIG, HOL
and RED whereas PIE is the leanest breed (Table 1).
Based on COX and CS activities, the two Spanish hardy breeds (CAS, AVI) had the most oxidative
muscles. Based on ICDH, the JER (a small breed) had the most oxidative muscles. The proportions
of oxidative MyHC (types I and IIa) were the highest for JER and HIG (a fat breed). The lowest

proportions of type IIx MyHC were observed in HIG and JER, followed by AA (which is also a fat
breed). Based on LDH, the most glycolytic fibres were for RED, followed by HOL, ASV and PIE.
The muscles of PIE (a lean breed) had the highest proportion of type IIx MyHC and the lowest CS
and COX activities.
Table 1. Body composition of the studied breeds.
Breed AA ASV AVI CAS CHA HIG HOL JER
Carcass weight, kg 336de 349cd 325ef 245h 387a 245h 320efg 190h
Fat, % 21.7a 7.8g 12.6ef 14.7cd 15.4c 19.6b 19.3b 13.0def
Muscle, % 61.4h 75.0b 69.1de 66.3g 67.7efg 62.8h 58.9i 66.5fg
Breed LIM MAR PIE PIR RED SD SIM
Carcass weight, kg 360bc 307g 336de 372ab 319fg 347cd 345cd

Fat, % 13.2def 8.9g 3.2h 9.7g 19.5b 14.2cde 11.7f
Muscle, % 71.9c 70.0d 79.9a 72.9c 61.1h 68.5def 67.8efg
a-g Means significantly differ at P<0.05 (SNK test).
In Figure 1, the first factor of the principal component analysis was mainly explained by the MyHC
isoforms (oxidative type vs. fast-glycolytic type). The beef breeds were scattered along this axis
with fat breeds (HIG, AA) as opposed to lean breeds (MAR, PIE, PIR, CHA, SD, LIM). The second
factor was explained by CS and COX activities. Hardy (CAS, AVI), dairy (JER, RED, HOL) or dual
purpose (SIM) breeds had the highest CS and COX activities. Furthermore, breeds from the same
geographical area (circles) are grouped together.
In conclusion, differences in muscle characteristics across breeds can be associated with their
biological type (hardy, beef or dairy) or their geographical origin. The hardy or dairy breeds tend
to have more oxidative muscles. The muscles from Spanish or British breeds also tend to be more
oxidative unlike the Italian breeds (which are very lean). One important observation is that the
fattest breeds (AA, HIG) are sometimes, but not always (HOL, RED), characterised by the highest
muscle oxidative metabolism.
3,0
Fast
2,5 B
Oxidative fibres Glycolytic
Asturiana fibres
2,0 COX de los valles
(ASV) LDH
1,5 Avileña (AVI)
Red Danish (RED)
1,0 CS Casina (CAS) Holstein (HOL)
Fact. 2 : 15,78%
0,5 Simmental (SIM) Limousin (LIM) MyHC IIx

0,0 Pirenaica (PIR)
MyHC I South Devon (SD)
-0,5 ICDH Charolais (CHA)
Jersey (JER)
-1,0 MyHC IIa
Aberdeen Angus (AA) Piemontese (PIE)
-1,5 Oxidative
fast or slow Marchigiana (MAR)
-2,0 fibres Highland (HIG)
-2,5
-8 -6 -4 -2 0 2 4 6 8
Fact. 1 : 58,75%
Figure 1. Principal component analysis to discriminate breeds.

Deposition of protein, fat and energy in lambs of the breed German
Merino Landsheep
G. Bellof1 and J. Pallauf2
1Weihenstephan University of Applied Sciences, Section Animal Nutrition, D-85350 Freising,
Germany
2Justus Liebig University Giessen, Institute of Animal Nutrition and Nutrition Physiology, D-35392
Giessen, Germany
Introduction
An understanding of nutrient and energy deposition in animals is crucial to the management of feeding
intensity for optimal animal growth as well as for minimal excretion of nutrients. The purpose of
this study was to assess the deposition of fat, protein and energy during the growth period of lambs
of the most important German sheep breed, the German Merino Landsheep.

Fifty-four male and 54 female lambs were fed at three different feeding levels from 18 kg body
weight (BW) onwards and slaughtered at various body weights (BW): 18, 30, 45, and 55 kg. Varying
amounts of concentrate (main ingredients grain and soybean meal, pelleted) and hay in the daily
feeding rations were used. The feeding intensity ‘low’ corresponded to a daily ration consisting of
60% concentrate in the feedstuffs, whereas the feeding intensity level ‘high’ had a daily feeding ration
consisting of 90% concentrate in the feedstuffs. Within each feeding level there was no difference in
rations of the feed according to the gender of the animals. The feeding of the animals on demand-
feeding stations made it possible to record the daily feeding intake of both feedstuffs for each animal
and at the same time that of the group (separated according to gender).
Based on the method of the comparative slaughter technique the total body of each animal was
analysed. From the data of empty-body gain, the fat, protein and energy deposition in the different
fattening periods was calculated. The statistical analysis of the individual performance data was
conducted using the statistics program SAS®. In each case a 3-factorial analysis of variance was
conducted using the GLM-procedure. The factors ‘Feeding intensity’, ‘Gender’, and ‘Final weight’
represented fixed effects in the equations.
Results and conclusion

For concentrate 12.4 MJ ME/kg DM and for hay 9.2 MJ ME/kg DM were derived. The ME
concentration in the dietary dry matter of the ration varied between 11.5 (feeding intensity ‘low’) to
12.1 MJ (‘high’). Increasing feeding intensity (low, medium, high) led to a daily increase in weight
gain (208, 253, 305 g). At the same time there was a lowered energy intake per kg body gain (50.9;
47.9; 45.9 MJ ME/kg). At a comparative feeding intensity and body weight level (e.g. ‘high’, 45
kg), the male animals showed a higher daily increase in body weight and a lower energy intake per
kg body weight gain in comparison to the female animals (338 g vs. 262 g; 41.9 MJ ME/kg vs. 52.8
MJ ME/kg BW gain respectively). Increasing body weight (30, 45, 55 kg) led to a decreased energy
efficiency ratio (41.2; 50.0; 53.5 MJ ME/kg BW).
The male lambs showed at all body weights tested and at all feeding levels a lower daily fat deposition
and a higher daily protein deposition compared to the female lambs. The deposition of fat increased
in both genders with increasing body weight. The amount of increase differed between the three
feeding levels. The male lambs showed at all body weights and at all feeding levels a higher daily

gain but a lower daily deposition of energy compared to the female lambs. However, except for one
group, the gender difference did not reach statistical significance. Parallel to the daily deposition of
fat the daily deposition of energy increased in both genders with increasing body weight.
Male animals had an average energy content of 8.5 MJ/kg of empty body weight (30 kg BW), while
female animals had distinctly higher values of 10.0 MJ/kg.
Based on the deposition of fat and protein during the growth period of the lambs of all three feeding
levels the composition of the empty body was estimated according to the ARC (1980) by allometric
equations (Figure 1).
300
280
260
240 Fat females

Protein or fat (g/kg)
220
200 Fat males
180
160 Protein
females
140
Protein
120 males
100
80
60
10 15 20 25 30 35 40 45 50 55
Empty body weight (kg)
Figure 1. Composition of the empty body of lambs (log10 y = a + b log10 x; Fat females, all feeding
levels: b=1.9599, a=-2.1744, R2=0.963; Fat males, all feeding levels: b=1.8324, a=-2.1375,
R2=0.968; Protein females, all feeding levels: b=0.9329, a=-0.6219, R2=0.988; Protein males, all
feeding levels: b=0.9794, a=-0.6744, R2=0.990.
The equations show a consistent pattern of protein concentration decreasing slightly and fat
concentration rising considerably with increasing empty body weight.
The fat concentration in empty body gain rose with increasing empty body weight. The ARC (1980)
estimated for non-Merino female sheep at 20 and 45 kg empty body weight a content of fat of 291
and 642 g/kg. For Merino females of these weight classes in the present study 208 and 433 g/kg
were predicted.
In conclusion, a higher protein and lower fat concentration in empty body gain was found as compared
to the literature on the genotype investigated.
References
Agricultural Research Council, 1980. The Nutrient Requirements of Ruminant Livestock. Technical Review by an
Agricultural Research Council Working Party. CAB, Farnham Royal, UK.

Impact of vitamin D3 and vitamin E by feeding on meat quality
parameters of beef and pork
R. Lahucky1, I. Bahelka1, U. Küchenmeister2, K. Vasickova1, G. Nürnberg2 and K. Ender2
1Slovak Agriculture Research Centre, 94992 Nitra, Slovak Republik
2Research Institute for the Biology of Farm Animals, 18196 Dummerstorf, Germany
Introduction
The results from a number of studies showed that feeding of supra/nutritional levels of some
nutrients may improve the antioxidative capacity and meat quality parameters. Thus nutrients include
magnesium, vitamin (vit.) E and others (Buckley et al., 1995; Swigert et al., 2004). Montgomery et
al. (2000) have reported that feeding very high concentrations of vit. D3 to cattle results in increased
levels of plasma and muscle calcium and improved tenderness of beef. Vit. D3 supplementation
did not affect quality characteristics or tenderness of pork (Swigert et al., 2004) but changes in L*
and a* values were positively influenced (Wiegan et al., 2002). The objective of this research was
to characterise the effects of dietary supplementation with vit. D3 individually, and in combination
with vit. E on meat quality characteristics of beef and pork.

Thirty bulls (Spotted breed) were fed standard feed (control group, group C, n=10) and experimental
groups (each n=10) were fed feed supplemented with vit. D3 (7.5×106/animal/d, for 7 d, D group) and
with vit. D3 (7.5×106/animal/d, for 7 d) and vit. E (2.500 α–tocopherylacetate/animal/d, 6 wk, DE
group) before slaughter. In total 36 pigs (Slovak White Meaty) were genotyped (RYR1, DNA based
test). The control group (group C, n=12) received a standard diet. The experimental groups (each
n=12) received a supplemental level of vit. D3 (500 000 IU/kg of feed intake for 5 d, D group) and a
supplemental level of vit. D3 (500 000 IU/kg of feed intake for 5 d) and vit. E (ROVIMIX® E-50 SD,
500 mg α-tocopherol/kg of feed for 30 d, DE group) before slaughter. The animals (bulls at average
weight of 550 kg, pigs 110 kg) were slaughtered according to standard commercial procedures in
an Institute abattoir. Blood was collected and calcium was determined (atomic absorption). The pH
value (45 min) of the carcass, colour (24 h and 5 d) by Miniscan and drip loss (24 h) were measured.
The longissimus muscle (LD) was removed for further analyses. Vit. E (α-tocopherol) level was
measured by the HPLC method. Lipid oxidation and antioxidant capacity (muscle homogenate
stimulated by Fe2+/ascorbate) were assessed by the 2-thiobarbituric acid method (TBARS, expressed
as malondialdehyde – MDA). Shear force was determined in cooked samples (internal temperature
75 °C) with a Warner-Bratzler (W-B) apparatus. Data were analysed by a three-way ANOVA
(software package SAS® version 9.1). All pairwise comparisons were done by a Turkey-test. The
data are expressed as Least-Squares Means (LSM) ± standard error (SE).

Supplementation of vit. D3 exhibited plasma calcium concentration in bulls (2.60 vs. 2.78 mmol/L)
and greater in pigs (2.71 vs. 3.90 mmol/L) as was shown also by Wiegan et al. (2002). Feeding
supplemental vit. E increased (P<0.05) the level of α-tocopherol in beef (2.81 vs. 4.18 mg/kg) and
in pork (2.01 vs. 3.21 mg/kg). The results were in agreement with those published earlier (Buckley
et al., 1995). Colour data (a* value) were higher (P=0.07) for LD muscle (5 d) from vit. D3 and vit.
D3 and vit. E treated pigs (Table 1). Similar results were introduced also by Wiegan et al. (2002).
Shear force (W-B) values of longissimus dorsi muscle were not influenced in pigs (Table 1) but
positively (P<0.05) influenced in bulls (14 d W-B 5.9 kg at control vs. 4.3 and 4.1 kg at group E+D3
and group D3, respectively). The results from this study support other studies (Montgomery et al.,
2000; Wiegan et al., 2002).

Table 1. Meat quality (longissimus dorsi) of pigs.
Trait Control, LSM Group D, LSM Group D+E, LSM SE
pH1 6.28 6.38 6.30 0.0749

Drip loss, % 3.26 3.09 3.28 0.2437
Colour L* 48.62 46.53 46.20 1.0018
Colour a* 2.14 2.33 2.10 0.2662
Colour L* - 5 d 49.14 49.17 48.24 0.9697
Colour a* - 5 d 2.79 3.71 3.54 0.2931
Shear force (W-B), kg 4.82 4.71 4.99 0.2892
Antioxidant capacity measured as MDA (nM/mg protein) after muscle homogenate incubation with
Fe2+/ascorbate was improved (at 120 min, P<0.05) by vit. E and partially by vit. D3 in beef (Figure
1a) and pork (Figure 1b) when compared to control animals.
a b
MDA, nmol/mg protein
MDA, nmol/mg protein

0.5
0.6
0.4 0.5
0.3 0.4
0.2 0.3
0.2
0.1 0.1
0 0.0
0 30 60 120 0 30 60 90 120
Time of incubation with Time of incubation with
Fe2+/ascorbate (min) Fe2+/ascorbate (min)
Control vitD vitED
Figure 1. Antioxydant capacity of longissimus dorsi muscle in (a) bulls and (b) pigs.
In conclusion, supplementation of vit. D3 increases blood calcium concentration and improves

tenderness of beef but not of pork. Colour (a* value) can be improved in pork.
Supplementation of vit. E increases α-tocopherol in beef and pork. Antioxidant capacity was improved
in beef and pork by vit. E and partially by vit. D3 supplementation.
References
Buckley, D.J., P.A. Morrissey and J.I. Gray, 1995. Influence of dietary vitamin E on the oxidative stability and quality
of pig meat. J. Anim. Sci. 73, 3122-3131.
Montgomery, J.L., F.C. Jr Parish, D.C. Beitz, R.L. Horst, E.J. Huff-Lonergan and A.H. Trenkle, 2000. The use vitamin
D3 to improve beef tenderness. J. Anim. Sci. 78, 2615-2621.
Swigert, K.S., F.K. McKeith, T.C. Carr, M.S. Brewer and N. Culbertson, 2004. Effects of dietary vitamin D3, vitamin
E, and magnesium supplementation on pork quality. Meat Sci. 67, 81-86.
Wiegan, B.R., J.C. Sparks, D.C. Beitz, F.C. Jr. Parish, R.L. Horst, A.H. Trenkle and R.C. Ewan, 2002. Short-term of
vitamin D3 improves color but does not change tenderness of pork loin chops. J. Anim. Sci. 80, 2116-2112.

Influence of various Iodine supply on growing-finishing performance
and on Iodine status of pigs
A. Berk1, K. Franke1, F. Schöne2 and G. Flachowsky1
1Institute of Animal Nutrition, Federal Agricultural Research Centre (FAL), Bundesallee 50, D-
38116 Braunschweig, Germany
2Thüringer Landesanstalt für Landwirtschaft (TLL), Naumburger Straße 98, 07743 Jena,
Germany
Introduction
Iodine is essential for man and animals. In the prevention of iodine deficiency, there are efforts
to concentrate the trace element in milk, eggs and meat by iodine addition to the feed (beside the
salt iodination regarding food). To improve the content of iodine in meat by feeding there are very
different results of experiments reported in the literature (He et al., 2002; Schöne et al., 2002). For
example, there is a very small range between human requirements (circa 200 µg/d) and the ‘upper
level’ (500-600 µg/d) of iodine. Therefore, the EFSA (2005) asked for more dose-response studies
with food producing animals to assess the contribution of various sources of iodine intake in men.

The experiment was carried out with 70 BHZP-hybrids (35 females, 35 castrated males) in the
live weight period from 27 to 115 kg in five groups with 14 animals each. The feed of group 1 was
a cereals-soybean meal based diet, which served as the control and contained 0.17 mg/kg iodine
of native origin. This diet was supplemented with 0.5; 1; 2 and 5 mg/kg (groups 2 to 5) iodine.
In each group, four pigs were slaughtered after fattening and cut into the fractions innards/blood,
bones and muscle/fat, which followed the removal of the thyroid. Iodine was analysed by ICP-MS
(Leiterer et al., 2001). Mean comparisons were conducted by the Tukey test with the SAS (2002)
GLM procedure.

The results of the iodine analyses in feed are given in Table 1. The data reflect the supply relatively
good. The iodine content in the feed of group 1, which was of native origin, nearly meets the
requirements of pigs (GfE, 2006; National Research Council, 1998).
Table 1. Iodine contents in the feed of the various groups (mg/kg feed, 88% DM).
group 1 group 2 group 3 group 4 group 5
Iodine supply, planned 0 0.5 1.0 2.0 5.0

Mean of two analyses 0.17 0.41 0.99 2.20 4.38
The extremely different iodine supply did not affect the feed intake, the growth intensity (weight
gain) and the feed to gain ratio (Table 2).
The thyroid represented by far the highest iodine concentration compared to the further parts of the
pig body (Table 3). Looking at the extrathyreoidal fractions of the whole body analyses, there were
the highest iodine concentrations in the innards/blood whereas the muscle/fat and the bones contained
very low amounts of the trace element. Up to 2 mg/kg diet, the feed iodine dosages increased the

thyroid iodine content; the higher iodine supplement resulted in a constant level, i.e. plateau. With
the exception of the innards/blood the iodine concentration of all investigated fractions was elevated
by the feed iodine supplementation. In comparison to the control group (without added iodine) the
highest iodine addition (5 mg/kg diet) caused a 3.6 fold empty body iodine concentration (calculated
from the content of the investigated fractions). The distribution of the iodine in the body remained
widely unaffected by the iodine supplementation. On average amongst the groups, the thyroid
contained 80% of the body iodine, innards/blood 14%, muscle/fat 5% and the bones 1%.
Table 2. Animal performance in dependence on iodine supplementation.
Groups 1 2 3 4 5
Iodine, mg/kg - 0.5 1.0 2.0 5.0
Daily weight gain, g 837 ± 70 819 ± 99 811 ± 93 851 ± 84 867 ± 63

Feed intake, kg/d 2.26 ± 0.10 2.24 ± 0.13 2.21 ± 0.10 2.24 ± 0.13 2.26 ± 0.10
Feed/gain ratio, kg/kg 2.72 ± 0.21 2.77 ± 0.29 2.74 ± 0.28 2.65 ± 0.18 2.62 ± 0.16
a, b Different letters in one line show significant differences (P<0.05).
Table 3. Iodine concentration of the investigated fractions of the slaughtered pigs.
Groups 1 2 3 4 5
Iodine, mg/kg - 0.5 1.0 2.0 5.0
Thyroid, µg/g 620c ± 71 1054b ± 280 1154b ± 191 1699a ± 184 1645a ± 159
Innards/blood, µg/kg 94 ± 61 63 ± 40 138 ± 73 230 ± 145 126 ± 38
Bones, µg/kg 15b ± 5 19b ± 10 17b ± 4 18b ± 2 37a ± 4
Muscle/fat, µg/kg 3.9c ± 0.6 6.0c ± 1.9 8.5b ± 1.9 10.8b ± 1.2 17.1a ± 1.5
a, b, c Different letters in one line show significant differences (P<0.05).
References
EFSA, 2005. Opinion of the Scientific Panel on Additives and Products or Substances used in Animal Feed on the
request from the Commission on the use of Iodine in feedingstuffs (Question nr. EFSA-Q-2003-058). www.efsa.
eu.int/science/feedap/feedap-opinions/808/iodine1.pdf, (14.09.2005), 49 pp.
GfE, 2006. Empfehlungen zur Energie- und Nährstoffversorgung von Schweinen. DLG-Verlag Frankfurt/M.,
Germany.
He, M.L., W. Hollwich and W.A. Rambeck, 2002. Supplementation of algae to the diet of pigs is a new possibility to
improve the iodine content in the meat. J. Anim. Physiol. Anim. Nutr. 86, 97-104.
Leiterer, M., D. Truckenbrodt and K. Franke, 2001. Determination of iodine species in milk using ion chromatographic
separation and ICP-MS detection. Eur. Food Res. Technol. 213, 150-153.
National Research Council, 1998. Nutrient Requirements of Swine. 10th rev. ed. National Academy of Sciences,
Washington D.C., USA.
Schöne, F., M. Leiterer, U. Kirchheim, K. Franke and G. Richter, 2002. Jodkonzentration in Schweine-, Rind- und
Schaffleisch und ihre Beeinflussung. In: Breves, G. (editor), Proceedings of the Society of Nutrition Physiology.
DLG-Verlag Frankfurt/M., Germany, Series 11, 58.

2A. Regulation of lipid deposition and intramuscular lipids

Intramyocellular lipid accumulation and insulin resistance development
C. Aguer, J. Mercier, C. Yong Wai Man, S. Bordenave, L. Metz and M. Kitzmann
INSERM ERI 25 ″Muscle et Pathologies″, CHU A. de Villeneuve, 371 avenue Doyen G. Giraud,
Bât. Crastes de Paulet, Montpellier, F-34295 Cedex 5, France
Introduction
Muscle insulin resistance (IR) is a major metabolic abnormality observed in type 2 diabetes (T2D).
A strong relationship between intramuscular lipid (IMCL) accumulation and degree of IR exists
(Petersen et al., 2004). Mechanisms that result in excess accumulation of IMCL in IR states may
result from abnormal increases in fatty acid (FA) transport through plasma membrane and/or
diminished FA oxidation. Myotubes established from T2D subjects present the same defects as in
muscle biopsies, including IR (Gaster et al., 2002). The aim of this study was to use cell culture
of primary human myotubes isolated from T2D subjects in order to study mechanims involved in
IMCL accumulation in relation to IR.

Subjects and material
Primary human myotubes were purified from muscular biopsies of 3 insulin sensitive overweight
controls (BMI: 25-30 kg/m²) (C), 5 overweight and 4 obese (BMI>30 kg/m²) T2D subjects and
cultured as described (Kitzmann et al., 2006).
IMCL content
Myotubes were stimulated with insulin (1 μM) and/or palmitate (0.6 mM) during 16 h with or without
inhibition of FA transporters by phloretin (400 μM). IMCL content was quantified after oil red O
staining (Gaster and Beck-Nielsen, 2006).
Western-blots
Western-blots were performed using antibodies against P-IRS1Ser307, P-AktSer473 and Tubulin,
with or without insulin stimulation (10 min).

Myotubes from overweight C and T2D show similar IMCL accumulation whereas myotubes from
obese T2D show significant greater IMCL accumulation than C myotubes (Figure 2). C myotubes
are sensitive to insulin (increase in the phosphorylation of IRS-1 and Akt after insulin stimulation)
(Figure 1, left panel). In contrast to myotubes from overweight and obese TD2 subjects showing
constitutive phosphorylation of IRS-1 and no response after insulin stimulation (Figure 1, right
panel).
Inhibition of active FA transport (phloretin) decreased IMCL content in myotubes from obese T2D
to reach the same value as C myotubes (Figure 2). Thus, the increase in IMCL content in obese T2D
myotubes is related to an increase in FA transporter activity.
Mitochondrial activity inhibition by antimycin increased the content of IMCL in myotubes from C
and overweight T2D but not in myotubes from obese T2D (data not shown). This result suggests

Figure 1. Western blot for Ser307 P-IRS-1 and Ser473 P-Akt in myotubes from C, T2D subjects
(overweight and obese) with or without insulin stimulation. Tubulin was used as a loading charge
control.
Figure 2. IMCL accumulation in myotubes established from C (white bars), overweight T2D
(grey bars) and obese T2D (black bars) subjects exposed to palmitate, after insulin and phloretin
stimulation. (* P<0.05 vs. C and obese T2D myotubes; # P<0.05 vs. obese T2D myotubes stimulated
by palmitate).
that IMCL accumulation in myotubes from obese T2D could be linked to defective mitochondrial
lipid oxidation capabilities.
Conclusion
From the results presented herein, it seems that the accumulation of IMCL (due to an increased activity
of FA transporters and a decreased mitochondrial oxidation ability) was related to obesity rather than
IR (C and overweight T2D have similar IMCL content and none lipid metabolism abnormalities).
References
Gaster, M., I. Petersen, K. Hojlund, P. Poulsen and H. Beck-Nielsen, 2002. The diabetic phenotype is conserved in
myotubes established from diabetic subjects: evidence for primary defects in glucose transport and glycogen
synthase activity. Diabetes 51, 921-927.
Gaster, M. and H. Beck-Nielsen, 2006. Triacylglycerol accumulation is not primarily affected in myotubes established
from type 2 diabetic subjects. Biochim. Biophys. Acta 1761, 100-110.
Kitzmann, M., A. Bonnieu, C. Duret, B. Vernus, M. Barro, D. Laoudj-Chenivesse, J.M. Verdi and G. Carnac, 2006.
Inhibition of Notch signaling induces myotube hypertrophy by recruiting a subpopulation of reserve cells. J. Cell.
Physiol. 208, 538-48.
Petersen, K., S. Dufour, D. Befroy, R. Garcia and G. Shulman, 2004. Impaired mitochondrial activity in the insulin-
resistant offspring of patients with type 2 diabetes. N. Engl. J. Med. 350, 664-671.

Regional specifities in transcriptomic profiles of adipocytes isolated from
skeletal muscle or adipose tissues in growing pigs
I. Louveau, M. Damon and F. Gondret
INRA, UMR1079, Systèmes d’Elevage Nutrition Animale et Humaine, 35590 Saint Gilles, France
Introduction
Body fat distribution is of special interest for both animal production and human physiopathology.
Excessive deposition of fat is detrimental for the commercial value of animal carcasses. However,
intramuscular lipid content which is correlated with the number and size of adipocytes clustered along
myofiber fasciculi, positively influences sensory quality traits and consumer’s acceptability of the
fresh meat (Fernandez et al., 1999). In humans, excessive lipid accumulation in muscle is associated
with insulin resistance and related disorders (Goodpaster and Wolf, 2004). Triglycerides represent
the most variable fraction of tissue lipids, and are mainly stored in the adipocytes. Differences in
metabolic activity or gene expression between subcutaneous and visceral adipocytes have been
previously reported in both humans and animal models (Giorgino et al., 2005). In a recent study, we
showed that adipocytes isolated from pig skeletal muscle exhibit much lower lipogenic and lipolytic
capacities than subcutaneous and perirenal adipocytes (Gardan et al., 2006). The present study was
undertaken to further examine the functional specificity of intramuscular adipocytes using a high
through-output micro-array technology.

Eighty-d-old and 210-d-old female pigs (30 and 150 kg body weight, n=5-6/group) were killed
after an overnight fast. Adipocytes were isolated by collagenase treatment from skeletal muscle
(trapezius) or from dorsal subcutaneous, perirenal or intermuscular (below trapezius) adipose
tissues. An aliquot of adipocyte suspension was digitised and diameters of isolated cells were
measured by image analysis. Total RNA was then extracted from adipocytes using the guanidium
isothiocyanate-phenol-chloroform extraction. RNA integrity was controlled with the Agilent 2100
bioanalyzer. Porcine generic micro-arrays (9 k) were used. They were developed by INRA as a
part of the AGENAE program and they were supplied by the biological resource center GADIE
(INRA, Jouy-en-Josas, France). Each product was spotted on nylon membrane using a Microgrid
II robot (Biorobotics). After hybridisation with an oligonucleotide 33P-probe, membranes were then
hybridised with complex cDNA 33P-target. The membranes were then exposed to phosphor screens
and images were acquired using the FUJIFILM BAS 5000. Hybridisation signals were quantified
and were then normalised.

Whatever the age, diameters of adipocytes were the smallest in muscle, whereas they did not differ
between the 3 adipose tissues (e.g. 24 vs. 33, 38 and 42 µm for muscle vs. intermuscular, perirenal
and subcutaneous adipocytes in 80-d-old pigs). Interestingly, the diameter of muscle adipocytes in
210-d-old pigs (39 µm) largely resembled that of adipocytes isolated from 80-d-old pig adipose
tissues. A first analysis of transcriptome data indicates that there were large differences between
intramuscular adipocytes and adipocytes isolated from the other depots whatever the age considered.
Among the differentially expressed clones, 25% were up-regulated whereas 75% were down-regulated
in intramuscular adipocytes compared with other adipocytes. Fewer differences were detected
between subcutaneous, intermuscular and perirenal adipocytes.

Conclusion
The expression pattern of genes in intramuscular adipocytes compared with non muscular adipocytes
further supports the view that there are regional differences in adipocyte functions. The identification
of the genes that were highly expressed in intramuscular adipocytes compared to other fat locations
should allow us to get a better understanding of the specific function of these adipocytes. Furthermore,
the comparison of gene transcript profiles in the different adipocytes should provide new insights
into the control of body fat depots. It should also give us new markers that could be further used
in animal selection.
Acknowledgements
We wish to thank F. Pontrucher and C. Tréfeu for their expert technical assistance in adipocyte
isolation. We also wish to thank A. Le Cam and J. Montfort (INRA, SCRIBE, Plateau Technique
Transcriptome Beaulieu IFR GFAS 140, F-35042 Rennes cedex) for their expertise in microarray
hybridization. The biological resource center ‘GADIE’ (INRA, UMR INRA-CEA, LREG, Domaine
de Vilvert, F-78352 Jouy-en-Josas) is also acknowledged for the supply of microarrays.
References
Fernandez, X., G. Monin, A. Talmant, J. Mourot and B. Lebret, 1999. Influence of intramuscular fat content on the
quality of pig meat - 1. Composition of the lipid fraction and sensory characteristics of m. longissimus lumborum.
Meat Sci. 53, 59-65.
Gardan, D., F. Gondret and I. Louveau, 2006. Lipid metabolism and secretory function of porcine intramuscular
adipocytes in comparison with subcutaneous and perirenal adipocytes. Am. J. Physiol. Endocrinol. Metab. 291,
E372-E380.
Giorgino, F., L. Laviola and J.W. Eriksson, 2005. Regional differences of insulin action in adipose tissue: insights from
in vivo and in vitro studies. Acta Physiol. Scand. 183, 3-30.
Goodpaster, B.H. and D. Wolf, 2004. Skeletal muscle lipid accumulation in obesity, insulin resistance, and type 2
diabetes. Pediatr. Diabetes 5, 219-226.

Effect of a reduced protein diet on expression of lipogenic enzymes in
relation to intramuscular fat formation in the pig
O. Doran, F.M. Whittington, K.G. Hallett and J.D. Wood
Division of Farm Animal Science, School of Clinical Veterinary Science, University of Bristol,
Langford, Bristol, BS40 5 DU, United Kingdom
Introduction
One focus of the pork industry is the production of lean meat. However, reduction in the level of
subcutaneous fat has also resulted in a reduction of intramuscular fat (IMF) to values which are below
those for optimum eating quality (Eikelenboom and Hoving-Bolink, 1994). Therefore strategies
which would allow an increase in the amount of IMF without increasing subcutaneous fat content
would be of benefit to the meat industry.
The amount of IMF can be increased by feeding a reduced protein diet (RPD) to pigs in the finishing
period of growth (Adeola and Young, 1989; Wood et al., 2004). The mechanism of the dietary
manipulation of IMF formation has not yet been understood. One of the possible explanations for
increasing IMF by a RPD could be activation of expression of lipogenic enzymes in pig muscles,
and hence increasing the rate of fatty acid biosynthesis.
The aims of the present study were (i) to investigate the effect of a reduced protein diet on expression
of major lipogenic enzymes in muscle and subcutaneous adipose tissue of pigs and (ii) to investigate
the relationship between expression of the lipogenic enzymes and IMF content. The enzymes
investigated in this study were: acetyl CoA carboxylase (ACC), fatty acid synthase (FAS) and
stearoyl-CoA-desaturase (SCD).The level of individual fatty acids, formation of which is catalysed
by the above-mentioned enzymes has also been investigated.

The study was performed on 10 entire male pigs (0.5 Duroc × 0.25 Large White × 0.25 Landrace)
fed different diets between 40 and 100 kg live weight. The diets were formulated to contain 14
MJ digestible energy per kg, 5% of soyabean oil and either 21% or 18% crude protein. Samples
of longissimus thoracis and lumborum muscle and subcutaneous adipose tissue were obtained
immediately after slaughter. The expression of microsomal protein SCD and the cytosolic proteins
ACC and FAS was estimated by Western blotting. Microsomes from one particular pig were present
at all the blots (reference sample), and the intensity of the signal for this sample was taken as 100
arbitrary units. The intensity of signals for all other samples on the blot was expressed as a fraction
of the reference sample. Fatty acid composition was analysed by HRGC.
Results
Reduction of protein in the diet from 21% to 18% resulted in a significant increase of total and
individual fatty acids in muscle (intramuscular fat) (Table 1). In contrast to IMF, reduction of the
dietary protein did not have a significant effect on the total fatty acid content of subcutaneous fat
and only a small effect on individual fatty acids in this tissue (Table 1).
The increase in total and individual fatty acids in muscles under the diet with a reduced protein level
was accompanied by an increase in the expression of one of the major lipogenic enzymes, SCD,
but not ACC or FAS (Table 2). There was a significant positive relationship between SCD protein
expression and the level of intramuscular fat (r=0.73, P<0.001).

Table 1. Effect of dietary protein on fatty acid composition.
Fatty acids, Muscle Subcutaneous adipose tissue

mg/100g tissue
Control RPD1 Control RPD
Total fatty acids 1580±35 2850±130* 70247±156 74258±346

16:0 367±19 719±138* 14850±426 17059±1185
18:0 179±10 339±72 7535±207 9084±500*
16:1 53±7 110±22* 1502±123 1592±137
18:1n-9 478±31 1169±245* 21649±573 26070±1192*
1RPD=Reduced protein diet, *P<0.05.
Table 2. Effect of dietary protein on expression of lipogenic enzymes in muscle.
Enzyme expression, arbitrary units
ACC FAS SCD
Control RPD Control RPD Control RPD
108.1±4.4 118.4±7.1 54.9±14.1 124.4±32.9 60.3±11 119.8±7.3*
*P<0.05.
Conclusions
We found the following: (i) the reduction of protein in the diet increased the level of intramuscular
but not subcutaneous fat in pigs; (ii) the increase in intramuscular fat was accompanied by activation
of expression of the lipogenic enzyme SCD but not FAS and ACC; (iii) a strong positive relationship
was established between intramuscular fat content and the SCD level in muscles. SCD is a rate-
limited enzyme catalysing the synthesis of monounsaturated fatty acids, mainly 16:1 and 18:1, which
are major components of tissue lipids. It is suggested that SCD might be one of the physiological
candidate genes controlling intramuscular fat formation in pigs. These results are a part of a peer-
reviewed paper, which has been recently published in ‘British Journal of Nutrition’ (2006, 95,
609-617).
References
Adeola, O. and L.G. Young, 1989. Dietary protein-induced changes in porcine muscle respiration, protein synthesis
and adipose tissue metabolism. J. Anim. Sci. 67, 664-673.
Eikelenboom, G. and A.H. Hoving-Bolink, 1994. The effect of intramuscular fat on eating quality of pork. Proceeding
of 40th International Congress of Meat Science and Technology, The Hague, The Netherlands.
Wood, J.D., G.R. Nute, R.I. Richardson, F.M.Whittington, O. Southwood, G. Plastow, Mansbridge, N. Da Costam
and K.C. Chang, 2004. Effect of breed, diet and muscle on fat deposition and eating quality in pigs. Meat Sci.
67, 651-667.

Changes in liver metabolic pathways induced by dietary energy and
selection for muscle fat content in rainbow trout
C. Kolditz1, M. Borthaire1, F. Lefèvre2, E. Quillet3 and F. Médale1
1INRA, UMR NuAGe, 64310 Saint-Pée-sur-Nivelle, France
2INRA, UR SCRIBE, Campus de Beaulieu, 35042 Rennes Cedex, France
3INRA, Laboratoire de Génétique des Poissons, 78350 Jouy-en-Josas, France
Introduction
Levels of fat storage and allocation within fish body compartments strongly affect product quality
(Johansson et al. 2000; Morkore et al., 2001). On the contrary to other farmed animals, there is no
agreement on the ideal muscle fat content in fish, since a moderate level is preferred for fresh products
and a higher level for smoked fillet. Genetic selection and dietary treatment are currently the main
tools to manage muscle fat content, but their respective effects on metabolic pathways responsible
for muscle fattening is still poorly understood. Fattening results from the balance between the supply
by both dietary fat and de novo synthesis of fatty acid from carbohydrates, and fatty acid oxidation
for energy production. In fish, lipogenesis mainly occurs in the liver, the centre of intermediary
metabolism. The objective of the present study was to identify hepatic metabolic pathways that
contribute to muscle fat deposition under both genetic selection and dietary manipulation.

Experimental animals and diets
Two lines of rainbow trout, designed as L (lean muscle line) and F (fatty muscle line), were obtained
after three generations of divergent selection for high or low muscle fat content evaluated using
a non-destructive method in live fish (Quillet et al., 2005). Triplicate groups of fish of both lines
were fed diets containing either 100 (LE diet) or 230 (HE diet) g lipid/kg dry matter, from the first
feeding, for 6 mo. At the end of the trial, livers were sampled from 9 fish per condition, 24 h after
the meal.
Gene expression analysis
Variations in liver gene expression among lines and dietary treatments were studied both through
measurements of expression of selected key genes involved in energy metabolism using qRT-
PCR and through liver transcriptome analysis. The candidate genes were either related to lipid
metabolism (fatty acid synthase as a marker of de novo synthesis, NADP-isocitrate dehydrogenase
that generates NADP required for lipogenesis, carnitine palmitoyl transferase 1, acyl-CoA oxidase
and hydroxyacyl-CoA dehydrogenase as markers of fatty acid β oxidation pathway, and PPAR α and
β that are known as key regulators of lipid metabolism in mammals) or to energy production from
glycolysis (hexokinase 1 and pyruvate kinase) and amino acid oxidation (glutamate dehydrogenase).
Overall oxidative capacities were also examined by measuring citrate synthase and cytochrome-c
oxidase (COX complex IV). Most of them were also assayed for enzyme activity.
Liver transcriptome expression was analysed after hybridisation of RNA samples on Nylon micro-
arrays produced by the INRA-GADIE biological resources centre with 9152 rainbow trout cDNA
from a normalised pooled-tissues library. Data from micro-array hybridisation were treated using
BASE (BioArray Software Environment) software (bioinformatic platform Sigenae). Statistical
analyses were done using TMEV (TIGR Multiple Experiment Viewer) software and variations in

gene expression were considered as significantly different between experimental conditions when
P value was <0.01.

The experimental design resulted in a relevant model to study genetic and nutritional determinism
of muscle fat deposition. At the end of the feeding trial, trout were distinguished by different muscle
fat content: the lowest values (4.2% wet weight) were observed in the L line fed the LE diet, and the
highest (10% wet weight) in the F line fed the HE diet. Interestingly, the two other groups (L-HE
and F-LE) had similar muscle fat content (6.4% wet weight), suggesting equivalent effects of genetic
selection and dietary treatment on this trait. Studies of selected metabolic key factors showed that
an increase in dietary energy (HE diet) enhances expression of liver genes involved in fatty acid
catabolism and confirmed that it decreases activity of enzymes involved in de novo lipid synthesis
(Dias et al., 1998). Micro-array analysis revealed that 186 clones were differentially expressed
between the two dietary treatments. Biological functions were attributed to 108 of them, and 64
were related to metabolism. The main dietary-induced changes were observed for lipid transport
and energy metabolism. For example, gene expression of apolipoprotein A-I, (ApoA-I), the major
protein component of high density lipoproteins (HDL), was increased with the HE diet.
Differences in metabolic pathways between lines were characterised for the first time. Catabolism
of glucose and amino acid, as well as energy metabolism, were enhanced in the liver of the F line
whereas genes involved in fatty acid β-oxidation were down regulated. From micro-array analysis,
176 clones were differentially expressed between the two lines and a biological function was assigned
to 99 of them. Fifty were involved in metabolic pathways. In contrast with dietary effects, mRNA
level of acetyl-CoA binding protein and hormone sensitive lipase were increased in the liver of the
F line, whereas no changes were found in H-FABP or ApoA-I mRNA levels.
It thus appears that the dietary treatments and the genetic selection procedure used to modify muscle
lipid content affected different metabolic pathways in the rainbow trout liver.
References
Dias, J., M.J. Alvarez, A. Diez, J. Arzel, G. Corraze, J.M. Bautista and S.J Kaushik, 1998. Regulation of hepatic
lipogenesis by dietary protein/energy in juvenile European sea bass (Dicentrarchus labrax). Aquaculture 161,
169-186.
Johansson, L., A. Kiessling, K.H. Kiessling and L. Berglund, 2000. Effects of altered ration levels on sensory
characteristics, lipid content and fatty acid composition of rainbow trout (Oncorhynchus mykiss). Food Qual.
Pref. 11: 247-254.
Morkore, T., J.L. Vallet, M. Cardinal, M.C. Gomez-Guillen, P. Montero, O.J. Torrissen, R. Nortvedt, S. Sigurgisaldottir
and M.S. Thomassen, 2001. Fat content and fillet shape of Atlantic salmon: relevance for processing yield and
quality of raw and smoked product. J. Food. Sci. 66, 1348-1354.
Quillet, E., S. Le Guillou, J. Aubin and B. Fauconneau, 2005. Two-way selection for muscle lipid content in pan-size
rainbow trout (Oncorhynchus mykiss). Aquaculture 245, 49– 61.

Reduced intake of dietary amino acid promotes accumulation of
intramuscular fat in the Longissimus dorsi muscle of finishing pigs
M. Katsumata1, M. Matsumoto2, S. Ieiri3 and Y. Kaji2
1NILGS, Tsukuba, Ibaraki 305-0901, Japan
2KONARC, Koh-shi, Kumamoto 861-1192, Japan
3Kumamoto Agricultural Research Center, Koh-shi, Kumamoto 861-113, Japan
Introduction
Reduced intake of dietary lysine up-regulated GLUT4 expression and activated citrate synthase,
an indicator of oxidative capacity of the cell, in porcine muscle (Katsumata et al., 2003). Lipids
were stored mainly in type I oxidative fibers in porcine muscle (Essén-Gustavsson et al., 1994)
and activities of oxidative enzymes highly correlated with triacylglycerol content of bovine muscle
(Hocquette et al., 2003). We hypothesised from those findings that reduced intake of dietary lysine
might promote intramuscular fat (IMF) accumulation in porcine muscle. To test this hypothesis,
we conducted a feeding trial (Investigation 1); fattening gilts were fed a diet with 70% of the
recommended lysine content (NRC 1998). Current movements towards developing recycling-based
societies and recent increase in the price of corn due to promotion of using bioethanol for fuel
encourage us to substitute food co-products for a certain amount of corn as a feed ingredient. The
pig industry in Japan has conducted several attempts to use food co-products for feeds of pigs and
it is now well recognised that feeding breadcrumbs (BS) to finishing pigs enhances IMF content.
The industry competes for BS because the enhancement of IMF induced by BS feeding has been
thought to be a specific response. Consequently, price of BS has kept rising and even shortage of BS
occurs. In order to prove the enhancement of IMF was not a specific response to BS and to ease the
competition, it was necessary to elucidate underlying mechanisms of this response. We hypothesised
that enhancement in IMF content induced by BS feeding could be attributed to a shortage of amino
acids in the diets. Thus, we conducted investigation 2 to test the hypothesis that a shortage of amino
acids in BS may affect IMF.

In investigation 1, eleven gilts aged 110 d were used. The average initial body weight of the pigs was
62 kg. Six pigs were assigned to the low lysine diet (LL, lysine content 0.40%) and five pigs were
assigned to the control diet (lysine content 0.68%). The diets were iso-energetic and iso-protein,
and contained all essential amino acids (apart from lysine) in the recommended amounts. The pigs
were fed these diets until their body weights reached 110 kg.
In investigation 2, we prepared three diets as follows; a control diet, a diet including 30% BS (BS
diet, BS content 30%), and a diet including BS supplemented with amino acids, lysine, methionine,
threonine, and tryptophan, to meet their requirements (BS+AA diet). We used twenty-four pigs in
this investigation, and the average initial body weight of the pigs was 40 kg. Four barrows and four
gilts were assigned to one of the three diets. The pigs were fed these diets until their body weights
reached 110 kg.

Investigation 1
IMF content in Longissimus dorsi (L. dorsi) muscle of the LL group was twice as high as that of
the control pigs (P<0.01, Table 1). Thus, this investigation clearly demonstrates that reduced intake

of a single amino acid, lysine, promotes the accumulation of IMF in the L. dorsi of finishing pigs.
Although neither back fat depth nor L. dorsi area was affected by dietary lysine levels (data not
shown), live weight gain and feed efficiency tended to be lower in the LL group (P=0.118 and
P=0.052, respectively, Table 1). Pigs from the LL group took 5 d longer to reach 110 kg (P<0.01).
Thus, there was a trade-off between enhancement of IMF content and growth performance. In order
to minimise this disadvantage, further studies are required to obtain more detailed relations between
dietary lysine levels and IMF content in L. dorsi.
Table 1. Effects of dietary lysine levels on growth performance and IMF content in L. dorsi1.
Control Low lysine P value
Feed intake, g/d 2494±146 2374±136 NS

Live weight gain, g/d 784±39 715±36 0.118
Feed efficiency 0.32±0.01 0.30±0.01 0.052
Age at slaughter, d 173±1 178±1 P<0.01
IMF in L. dorsi, % 3.5±0.7 6.7±0.7 P<0.01
1 Values are expressed as least square mean ± standard error.
Investigation 2
Daily live weight gain and feed efficiency of the pigs did not differ among the groups (data not
shown). IMF content in the L. dorsi of pigs fed on the BS diet was higher than those of the other two
groups (3.52% for the BS groups, 2.29% for the control, and 1.98% for the BS+AA, respectively;
BS vs. control P=0.0515 and BS vs. BS+AA P<0.05, respectively). Thus, the enhanced IMF level
induced by BS feeding returned to the control level by supplementation of amino acids to the diet.
Although we were not able to identify which amino acid was limiting in this response from this
result, it supported our hypothesis that response of IMF to BS feeding was due to a shortage of amino
acids in BS and that the enhancement was not a specific response to BS. We infer that feeding other
food co-products from cereals such as co-products of noodle or cooked rice enhances IMF because
their amino acid contents are similar to that of BS.
References
Katsumata, M., M. Matsumoto and Y. Kaji, 2003. Effects of a low lysine diet on glucose metabolism in skeletal muscle of
growing pigs. In: Souffrant, W.B. and C.C. Metges (editors), Progress in research on energy and protein metabolism.
Wageningen Academic Publishers, Wageningen, the Netherlands, EAAP series 109, 187-190.
Essén-Gustavsson, B., A. Karlsson, K. Lundstrom and A.-C. Enfalt, 1994. Intramuscular fat and muscle fibre lipid
contents in halothane-gene-free pigs fed high or low protein diets and its relation to meat quality. Meat Sci. 38,
269-277.
Hocquette, J.F., C. Jurie, Y. Ueda, P. Boulesteix, D. Bauchart and D.W. Pethick, 2003. The relationship between muscle
metabolic pathways and marbling of beef. In: Souffrant, W.B. and C.C. Metges (editors), Progress in research on
energy and protein metabolism. Wageningen Academic Publishers, Wageningen, the Netherlands, EAAP series
109, 513-516.
NRC, 1998. Nutrient requirements of swine. Tenth revised edition. National Academy Press, Washington DC, USA,
111 pp.

Triacylglycerol synthesis and secretion in Atlantic salmon hepatocytes:
effect of dietary lipids
M.A. Kjær1, A. Vegusdal1, M. Todorčević1, T. Gjøen2, A.C. Rustan3, B. Torstensen4 and B. Ruyter1
1AKVAFORSK, Institute of Aquaculture Research, Box 5010, 1432 Ås, Norway
2Department of Microbiology, Institute of Pharmacy, University of Oslo, Box 1072 Blindern, 0316
Oslo, Norway
3Department of Pharmaceutical Biosciences, Institute of Pharmacy, University of Oslo, Box 1072
Blindern, 0316 Oslo, Norway
4NIFES, National Institute of Nutrition and Seafood Research, Box 2029 Nordnes, 5817 Bergen,
Norway
Introduction
Marine fish oils (FO), rich in highly unsaturated fatty acids (FA), have been used as the main lipid
constituent in fish feed. However, FO is a limited source and prices vary a lot. This has led to an
interest in developing alternative sources for use in fish diets. Plant oils is one candidate, but how
these plant oils affect lipid secretion and fish health is far from clear. There is considerable evidence
that the consumption of FO reduces hepatic secretion and serum triacylglycerol (TAG) levels in
humans (Harris, 1989; Nestel, 1990). It has also been found that eicosapentanoic acid (EPA) may
have a reducing effect on secretion of TAG rich lipoproteins in Atlantic salmon (Vegusdal et al.,
2005). The mechanism by which n-3 FA in the diet reduces TAG secretion is not fully known. The
goal of this study was therefore to investigate the effects of rapeseed oil (RO) and n-3 rich diets on
the accumulation and secretion of 3H-glycerolipids by salmon hepatocytes in culture.

Salmon were fed for 17 wk on four different diets supplemented with either 13.5% FO, RO, EPA
or docosahexaenoic acid (DHA) enriched oils. Hepatocytes were isolated from all dietary groups
and incubated with 3H-glycerol. Lipids were extracted from both cells and their media, and the lipid
classes and lipid composition were analysed. In addition we analysed the recovery of radiolabelled
lipids.
Results
Our results show that the dietary FA composition markedly influenced the endogenous FA content
and composition of the hepatocytes. Lipid accumulated to a greater extent in liver from fish fed
RO than it did in liver from fish fed the n-3 rich diets. The percentage of the longer chain n-3 FA
in the total hepatocyte lipids was approximately 1.6 times greater in the FO, EPA and DHA groups
than in the RO group. On the contrary, the percentage of n-6 FA in the total hepatocyte lipids was
approximately twice as high in the RO group as in the EPA and DHA groups, and 2.8 times as high
as in the FO group. This change in endogenous FA profile further affected the rate of secretion of
radioactive glycerolipids which was much lower from hepatocytes isolated from fish fed n-3 rich
diets than in hepatocytes from fish fed the RO diet (Table1).

Table 1. Intracellular, secreted and total level of 3H-glycerolipids in hepatocytes from fish fed FO,
RO or EPA and DHA enriched oils.
Total nmol recovered in FO RO EPA DHA

radiolabelled lipids1
Intracellular
TAG 5.52±0.45a 11.69±0.82b 2.50±0.49c 3.89±0.58ac
MDG 0.48±0.05 0.89±0.06 0.51±0.03 0.81±0.14
PL 2.55±0.41 3.24±0.25 1.76±0.21 1.86±0.22
Secreted
TAG 8.33±2.14a 9.57±1.39a 2.68±0.65b 3.19±0.38b
MDG 0.19±0.04 0.29±0.05 0.10±0.01 0.07±0.01
PL 0.34±0.05 0.53±0.05 0.26±0.03 0.22±0.03
Total
TAG 13.85±6.51a 21.26±5.84b 5.18±2.81c 7.08±2.92c
MDG 0.66±0.24 1.18±0.30 0.61±0.14 0.89±0.46
PL 2.88±1.13 3.76±0.75 2.02±0.59 2.08±0.71
1 The total nmol were in each sample measured in cells and media from one cell flask, with approximately
1×107 cells. The total average protein intracellular concentration was 3.4 mg/cell flask, with no differences between
the groups. Data are means ± SEM (n=10, except for FO and EPA, where n=8). Different letters indicate significant
differences (P≤0.05).
Discussion
The marked difference in FA composition due to the diets had a pronounced effect on the secretion of
glycerolipids. Our results agree with mammalian studies, showing that an endogenous FA composition
with high n-6/n-3 ratio promotes lipid secretion to a level that is higher than that obtained with an
endogenous FA composition with a lower n-6/n-3 ratio. Several vegetable oils rich in 18:1n-9 have
already become attractive substitutes for FO in salmon diets. However, the stimulating effect on lipid
secretion that we observed, and the negative effects of some vegetable oils on fish heart histology
that have been reported (Bell et al., 1991 and 1993) should be considered. These effects may give
rise to problems that are similar to problems related to coronary heart diseases in humans.
References
Bell, J.G., A.H. McVicar, M.T. Park and J.R. Sargent, 1991. High dietary linoleic acid affects the fatty acid compositions
of individual phospholipids from tissues of Atlantic salmon (Salmo salar): association with stress susceptibility
and cardiac lesion. J. Nutr. 121, 1163-1172.
Bell, J.G., J.R. Dick, A.H. McVicar, J.R. Sargent and K.D. Thompson, 1993. Dietary sunflower, linseed and fish oils
affect phospholipid fatty acid composition, development of cardiac lesions, phospholipase activity and eicosanoid
production in Atlantic salmon (Salmo salar). Prostaglandins Leukot. Essent. Fatty Acids 49, 665-673.
Harris, W. S., 1989. Fish oils and plasma lipid and lipoprotein metabolism in humans: a critical review. J. Lipid. Res.
30 (6), 785-807.
Nestel, P.J., 1990. Effects of n-3 fatty acids on lipid metabolism. Annu. Rev. Nutr. 10, 149-167.
Vegusdal, A., T. Gjøen, R.K. Berge, M.S. Thomassen and B. Ruyter, 2005. Effect of 18:1n-9, 20:5n-3, and 22:6n-3 on
lipid accumulation and secretion by Atlantic salmon hepatocytes. Lipids 40, 477-486.

Effect of fish and plant oils on lipid composition and fatty acid
β‑oxidation in adipose tissue of Atlantic salmon
M. Todorčević1, A. Vegusdal1, N. Djaković1, M.A. Kjær1, B. Torstensen2 and B. Ruyter1
1AKVAFORSK, Institute of Aquaculture Research, Box 5010, 1432 Ås, Norway
2NIFES, National Institute of Nutritional and Seafood Research, Box2029 Nordnes, 5817 Bergen,
Norway
Introduction
Visceral adipose tissue and myosepta surrounding the muscle are both primary sites of fat storage in
Atlantic salmon. A high amount of lipid (28 to 40%) is currently used in commercial salmon diets.
It is widely reported that high fat intake generally leads to increased fat deposits both in the fillet
and in visceral adipose tissue. In order to avoid the unwanted increase in the amount of visceral
adipose tissue, it is important that the fatty acids (FA) of the dietary oil is easily oxidised instead of
being primarily deposited.
Fish oil (FO) has traditionally been used as the dominating lipid component in fish feed, but the
limited supply and variable price of FO have increased the use of plant oils in fish diets. It is, however,
not known how these plant oils affect lipid utilisation of fatty acids in adipose tissue. The objective
of the present study was to evaluate the effects of fish oils rich in n-3 polyunsaturated fatty acids
(PUFA) and a plant oil rich in 18 carbons FA on lipid utilisation in adipose tissue.

Four groups of salmon were fed for 17 wk in fresh water tanks on four diets supplemented with either
13.5% FO, rapeseed oil (RO), eicosapentanoic (EPA) or docosahexaenoic (DHA)-acids enriched oils.
At the end of the feeding trial, fatty acid compositions of adipose tissue and an isolated fraction of
mitochondria and peroxisomes were determined as well as fatty acid β-oxidation activity in isolated
mitochondrial and peroxisomal fractions.
Results
The FA composition of the diets markedly affected the FA composition of adipose tissue and its
subcellular organelles. Fish fed the RO had increased levels of 18:1n-9 and decreased levels of EPA
and DHA than compared to fish fed the n-3 rich diets. The total FA fraction of adipose tissue and
its organelles were further separated into polar and neutral lipid fractions. FA compositions of polar
and neutral lipids are presented in Tables 1 and 2. The mitochondrial β-oxidation activity was the
highest in the FO group, while it was significantly lower in the RO group. In comparison, increased
percentages of 20:5n-3 and 22:6n-3 (EPA and DHA enriched dietary groups) had no detectable
mitochondrial β-oxidation activity. The peroxisomal β-oxidation activity was higher in the DHA
enriched group than in any other dietary groups.

Table 1. FA composition of adipose tissue polar and neutral lipids.
Fatty acids Phospholipids (PL) Triacylglycerol (TAG)
FO RO EPA DHA FO RO EPA DHA
18:1n-9 11.7±0.38ª 21.9±0.44b 10.4±0.74ª 11.8±4.02ª 13.9±0.08b 34.6±0.44c 9.7±0.99ª 11.5±0.51ª

20:5n-3 9.8±0.62ª 8.1±0.39ª 17.5±2.43b 7.7±0.27ª 4.1±0.01ª 2.3±0.15a 23.9±2.01b 5.2±0.13ª
22:6n-3 18.7±0.11ª 16.6±0.69ª 15.0±2.29ª 27.5±0.72b 12.9±0.44b 7.6±0.05a 14.28±0.54b 31.6±0.54c
Table 2. FA composition of crude mitochondrial and peroxisomal fraction.
Fatty Phospholipids (PL) Triacylglycerol (TAG)

acids
FO RO EPA DHA FO RO EPA DHA
18:1n-9 6.5±0.18ª 19.4±3.11b 8.2±2.37ª 6.5±0.48ª 12.3±1.09ª 33.8±3.04b 7.6±0.59ª 8.5±0.39ª

20:5n-3 4.02±0.29ª 1.9±0.64ª 11.7±1.26b 4.8±0.96ª 1.6±0.25ª 1.5±0.11ª 23.4±0.05c 5.6±0.67b
22:6n-3 12.8±1.17ªb 8.8±0.99ª 12.3±1.34ªb 19.9±4.37b 5.4±1.15ª 5.2±0.14ª 13.8±0.44b 32.2±2.13c
1,2 The quantity of each fatty acid is given in percentage of total fatty acids. Data are means ± SEM (n=3). Different
letters indicate significant differences (P≤0.05).
Discussion
Until now, little was known about how different dietary fatty acids affect the fatty acid composition
of salmon adipose tissue. The dietary lipids influenced the FA composition of adipose tissue in a
similar way as previously shown for other tissues like muscle and liver (Thomasen and Røsojo,
1989; Bell et al., 2001; Torstensen et al., 2004). Mitochondrial β-oxidation activity was not detected
in the EPA and DHA dietary group, indicating the possibility that the β-oxidation activity was
inhibited by the high levels of 20:5n-3 and 22:6n-3 in these dietary groups. One explanation may
be that the high levels of EPA and DHA found in the mitochondrial PL fraction caused membrane
lipid peroxidation. Our results show that dietary DHA increases peroxisomal β-oxidation activity
in salmon adipose tissue, probably due to the fact that 22:6n-3 is known to be a good substrate for
peroxisomal β-oxidation.
References
Bell, J., J. McEvoy, D.R. Tocher, F. McGhee, P.J. Campbell and J.R. Sargent, 2001. Replacement of fish oil with
rapeseed oil in diets of Atlantic salmon (Salmo salar) affects tissue lipid compositions and hepatocyte fatty acid
metabolism. J. Nutr. 131, 1535-1543.
Thomassen, M.S. and C. Røsjø, 1989. Different fats in feed for salmon: Influence on sensory parameters, growth rate
and fatty acids in muscle and heart. Aquaculture 79, 129–135.
Torstensen, B.E., L. Frøyl and Ø. Lie, 2004. Replacing dietary fish oil with increasing levels of rapeseed oil and olive
oil - Effects on Atlantic salmon (Salmo salar) tissue and lipoprotein composition and lipogenic enzyme activities.
Aquacult. Nutr. 10, 175-192.

Performance, blood lipids and fatty acid composition of tissues of rabbits
fed diets supplemented with conjugated linoleic acid
M. Marounek1,2, E. Skřivanová1 and A. Dokoupilová1
1Institute of Animal Science, Přátelství 815, CZ-104 00 Prague, Czech Republic
2Institute of Animal Physiology and Genetics, Vídeňská 1083, CZ-142 20, Czech Republic
Introduction
Over the past decades multitude health benefits have been attributed to conjugated linoleic acid
(CLA) in animal experiments (Belury, 2002). Although short-time health-promoting effects of CLA
are not fully conclusive for humans, the interest in animal trials with CLA persists. The knowledge
of the effects of dietary CLA in rabbits is not complete. Three studies by Corino et al. (2002, 2004
and 2007) deal with the effect of CLA on growth, meat quality and lipid metabolism of New Zealand
White rabbits. The authors concluded that the response of rabbits to CLA depends on age, level of
supplementation and length of CLA feeding. The aim of this study was to establish the extent to which
performance, fatty acid (FA) pattern and blood lipids of rabbits are influenced by feeding CLA.

Fourty Hyplus rabbits, weaned at 5 wk of age were housed individually. Rabbits of the experimental
groups were fed diets supplemented with Luta-CLA® 60 from BASF (Germany) at 5 and 10g/kg, for
the whole fattening period (6 wk) or 3 wk before slaughter. The fat composition of loin muscle, hepatic
tissue, perirenal fat was determined by GLC, after extraction of lipids and alkaline transmethylation.
CLA isomers were determined employing an HPLC instrument equipped with 3 Ag-impregnated
columns. Serum triacylglycerols, total cholesterol and its fractions were determined enzymatically.
One-way ANOVA, followed by the Tukey test, was used to assess the significance of differences
between dietary groups.

Average daily gains of rabbits ranged from 41 to 44 g. No performance improvement in CLA-fed
rabbits was observed. CLA significantly increased serum triacylglycerols, total and VLDL cholesterol
(Table 1). Dietary CLA significantly increased the proportion of saturated fatty acids (SFA) at the
expense of monounsaturated fatty acids (MUFA) in muscle and liver lipids. In perirenal fat, CLA
Table 1. Blood lipids in rabbits fed a control diet and diets supplemented with CLA-oil (mean values
and residual mean square errors).
CLA-oil 0 5 g/kg 10 g/kg RMSE
Wk of treatment: - 1-6 4-6 3-6
Cholesterol, µmol/mL
Total 1.60a 2.14b 2.31b 2.48b 0.35
LDL 0.38 0.52 0.58 0.55 0.24
HDL 0.73 0.66 0.77 0.64 0.17
VLDL 0.49a 0.96b 0.96b 1.29b 0.23
Triacylglycerols, µmol/mL 1.21a 2.11ab 2.10ab 2.84b 1.07
a,b Values in the same row with unlike superscript differ (P<0.05).

decreased the proportion of MUFA and increased polyunsaturated fatty acids (PUFA, Table 2). CLA
supplementation increased CLA concentration in meat, liver and fat. CLA concentration in tissues was
influenced by its content in the diet rather than by the length of CLA feeding (3 vs. 6 wk). Adipose
and hepatic tissue incorporated the greatest and the lowest amount of CLA, respectively. Isomers of
CLA did not accumulate in tissue lipids proportionally to their concentration in the CLA-enriched
diet. In all tissues, the relative proportion of cis-9, trans-11 CLA was lower than in the diet. Minor
CLA isomers (other than cis-9, trans-11 and trans-10, cis-12), which were almost absent in CLA-oil
appeared in all tissues. Thus, CLA modified FA composition of tissue lipids and tissue metabolism
modulated isomeric composition of deposited CLA. In this study, there was no difference in serum
lipid content between 3 vs. 6 wk of CLA feeding. Lee et al. (1994) observed that CLA at first increased
serum lipids in rabbits, but its effect was reversed after 8 wk of feeding.
Table 2. Fatty acid profile of tissue lipids (mg per 1 g FA determined) in rabbits fed a control diet
and diets supplemented with CLA-oil (mean values and residual mean square errors).
CLA-oil: 0 5 g/kg 10 g/kg RMSE
Wk of treatment: - 1-6 4-6 3-6
Loin
SFA 349a 363ab 380ab 390b 19
MUFA 326a 273b 274b 282b 16
CLA 0.9a 22b 19b 30c 4
PUFA (including CLA) 325a 364b 346ab 328ab 27
Liver
SFA 387a 420ab 449b 481c 40
MUFA 251a 173b 171b 167b 36
CLA 0.6a 12bc 10b 15c 3
PUFA (including CLA) 362 407 380 352 58
Perirenal fat
SFA 325 321 325 335 29
MUFA 367a 327b 332b 328b 15
CLA 0.5a 30b 23c 44d 5
PUFA (including CLA) 308a 352b 343b 337ab 24
a-d Values in the same row with unlike superscript differ (P<0.05).
References
Belury, M.A., 2002. Dietary conjugated linoleic acid in health: physiological effects and mechanisms of action. Annu.
Rev. Nutr. 22, 505-531.
Corino, C., J. Mourot, S. Magni, G. Pastorelli and F. Rosi, 2002. Influence of dietary conjugated linoleic acid on
growth, meat quality, lipogenesis, plasma leptin and physiological variables of lipid metabolism in rabbits. J.
Anim. Sci. 80, 1020-1028.
Corino, C., F. Filetti, M. Gambacorta, A. Manchisi, S. Magni, G. Pastorelli, R. Rossi and G. Maiorano, 2004. Influence
of dietary conjugated linoleic acids (CLA) and age at slaughtering on meat quality and intramuscular collagen in
rabbits. Meat Sci. 66, 97-103.
Corino, C., D.P. Lo Fiego, P. Macchioni, G. Pastorelli, A. Di Giancamillo, C. Domeneghini and R. Rossi, 2007.
Influence of dietary conjugated linoleic acids and vitamin E on meat quality, and adipose tissue in rabbits. Meat
Sci. 76, 19-28.
Lee, K.N., D. Kritchevsky and M.W. Pariza, 1994. Conjugated linoleic acid and atherosclerosis in rabbits. Atherosclerosis
108, 19-25.

Synthesis and desaturation of cis9, trans11 CLA in adipose tissue of
Charolais steers and cull cows
D. Gruffat1, C. Rémond1, D. Durand1, O. Loreau2 and D. Bauchart1
1National Institut of the Agronomic Research, Research Unit on Herbivores, Nutrients and Metabolisms
group, Research Centre of Clermont-Ferrand/Theix, 63122 Saint Genès-Champanelle, France
2CEA, Saclay, 91191 Gif/Yvette Cedex, France
Introduction
Bovine meat is, with dairy products, the major dietary source of conjugated linoleic acid (CLA) for
humans which is hypothesised to have several health benefits. The presence of CLA in ruminant
products results, on the one hand, from the bacterial biohydrogenation and trans isomerisation of
dietary polyunsaturated fatty acids (PUFA) in the rumen and, on the other hand, from an endogenous
synthesis in tissues by ∆9 desaturation of vaccenic acid (VA, trans11-18:1), another intermediate of
bacterial biohydrogenation of PUFA. In lactating cows, it has been clearly demonstrated that VA is
desaturated into CLA in the mammary gland (Griinari et al., 2000). However, in growing or finishing
ruminants, only one study was interested in the tissue synthesis of CLA and showed that the liver is
not involved in the endogenous synthesis of CLA (Gruffat et al., 2005). Moreover, CLA metabolism
is poorly documented. Among the tissues potentially involved in CLA synthesis and metabolism,
the subcutaneous adipose tissue (SAT) is known to play a central role in CLA deposition as part of
triglycerides in ruminants (Bauchart et al., 2002).

In this context, the capacity of SAT to synthesise CLA from VA and the different pathways of its
metabolism were evaluated by using the method of incubated slices of SAT from Charolais cull
cows (n=2) and steers (n=6). SAT slices were incubated until 16 h in the presence of [1-14C]-VA or
[1-14C]-cis9,trans11 CLA added to the medium. The in vitro functional viability of SAT explants
was determined by measuring the glucose-6-phosphate deshydrogenase activity and glucose uptake
by adipocytes (data not shown). The extent of cellular uptake of VA and CLA and their subsequent
esterification into neutral and polar lipids were determined by liquid adsorption chromatography on
SiO2 aminopropyl cartridges. Moreover, the fatty acid bioconversions by desaturation and elongation
reactions were evaluated by gas-liquid chromatography coupled with a radiodetector (GC-RAM).

The intensity of uptake of VA and cis9,trans11 CLA by SAT was 1.66 and 1.80 fold higher
respectively, in cull cows than in steers (Table 1). This result suggests a higher metabolic activity
in SAT of cull cows compared to steers. This was in agreement with the preferential fat storage of
cull cows during the finition period at the difference of steers that use fats as energetic substrates
for protein accretion (Robelin, 1978). VA and cis9,trans11 CLA taken up by SAT were around 10%
more esterified in cull cows than in steers (Table 1), confirming a preferential lipogenesis in cows.
However, esterification of VA and cis9,trans11 CLA concerned mainly the neutral lipid synthesis
pathway in both types of animals.
Bioconversions of VA and cis9,trans11 CLA into more unsaturated FA in SAT differed in intensity
between cull cows and steers (Table 1). Indeed, VA was more desaturated into cis9,trans11 CLA
(+31.8%) in SAT of cull cows whereas the desaturation of cis9,trans11 CLA into cis6,cis9,trans11
18:3, undetectable in cull cows, amounted to 14.5% of CLA taken up by SAT in steers. Differences
in VA desaturation have already been demonstrated in rat by Kraft et al. (2006) showing a higher

intensity of desaturation of VA in females than in males suggesting a possible effect of sexual
hormones on synthesis or activity of desaturases. This hypothesis would imply the higher activity
of ∆9 desaturase for bioconversion of VA and a lower ∆6 desaturase activity for cis9,trans11 CLA
bioconversion in cull cows compared to steers.
Table 1. Intensity of uptake, esterification into neutral and polar lipids and bioconversion of VA and
cis9,trans11 CLA in SAT of Charolais cull cows and steers (ND = not detectable).
Charolais cull cows Charolais steers
VA c9,t11 CLA VA c9,t11 CLA
FA uptake, % of FA in medium 66.0±0.65 77.2±1.8 39.8±3.6 42.8±3.7

FA esterification, % of FA taken up by cells 95.6±2.0 97.7±0.4 83.7±2.6 88.2±2.3
into neutral lipids, % of esterified FA 92.2±3.2 91.2±6.2 92.6±1.9 94.1±1.2
into polar lipids, % of esterified FA 7.8±3.3 8.8±6.3 7.4±1.9 5.9±1.2
FA bioconversion, % of bioconverted FA 6.5±3.2 ND 4.4±0.5 14.5±1.2
Conclusion
We demonstrated, for the first time, that SAT is well involved in tissue synthesis of CLA from
VA in both steers and cull cows. In contrast, desaturation of CLA into conjugated PUFA, the
cis6,cis9,trans11 18:3, seems to occur exclusively in adipose tissue of steers although this result
must be confirmed on a more significant number of animals.
References
Bauchart, D., A. De La Torre, D. Durand, D. Gruffat and A.Peyron, 2002. L’apport de graines de lin riches en acide
linolénique favorise le dépôt de CLA principalement dans les triglycérides du muscle chez le bouvillon. Viandes
et Produits Carnés 9, 73-74
Griinari, J.M., B.A. Corl, S.H. Lacy, P.Y. Chouinard, K.V.V. Nurmela and D.E. Bauman, 2000. Conjugated linoleic acid
is synthesized endogenously in lactating dairy cows by Delta(9)-desaturase. J. Nutr. 130, 2285-2291.
Gruffat, D., A. De La Torre, J.M. Chardigny, D. Durand, O. Loreau and D. Bauchart, 2005 Vaccenic acid metabolism
in the liver of rat and bovine. Lipids 40, 295-301.
Kraft, J., L. Hanske, P. Mockel, S. Zimmermann, A. Hartl, J.K. Kramer and G. Jahreis, 2006. The conversion efficiency
of trans-11 and trans-12 18:1 by Delta9-desaturation differs in rats. J. Nutr. 136, 1209-1214.
Robelin, J., 1978. Répartition des dépôts adipeux chez les bovins selon l’état d’engraissement, le sexe et la race. Bull.
Technol. CRZV, Theix-INRA 34, 31-34.

Inclusion of rapeseed oil in the diet to improve the performance of
silage-fed fattening cattle
J. Jatkauskas and V. Vrotniakiene
Department of Animal Nutrition and Feeds, Institute of Animal Science of Lithuanian Veterinary
Academy, Baisogala, LT-82317 R. Zebenkos 12, Radviliskis distr., Lithuania
Introduction
Lean beef has an intramuscular fat content of around 5% or less with approximately 47, 42 and
4% of total fatty acids as saturated fatty acids (SFA), monounsaturated fatty acids (MUFA) and
polyunsaturated fatty acids (PUFA), respectively (Moloney et al., 2001). Enhancing the PUFA and
decreasing the SFA content of beef could improve the healthful characteristics of the fat produced by
ruminants (Scollan et al., 2003). Increasing the dietary supply of PUFA is one of the most important
strategies allowing modification of beef fat composition. Hence, feeding diet rich in unsaturated acids
(18:1, 18:2, 18:3) can increase the levels of these MUFA and PUFA in the meat (Felton and Kerley,
2004). Rapeseed oil may be an available fat source that contains large quantities of unsaturated fatty
acids (FA). Thus, the objective of this study was to investigate the effects of feeding rapeseed oil in
the diet of fattening bulls on animal performance, carcass characteristics and the FA composition of
skeletal and cardiac muscles and adipose tissue.

Fourteen fattening young bulls of the Lithuanian Black-and-White breed with an initial mean live
weight of 373 kg were randomly allocated to one of two dietary treatments, each consisting of seven
animals. All animals were offered a first cut legume-grass silage ad libitum plus one of the two
concentrates: RAP0 - barley (83%), soybean meal (15%), mineral-vitamin mixture (2%), or RAP1-
barley (72%), soybean meal (15%), mineral-vitamin mixture (2%), rapeseed (canola) oil (11%).
Canola oil has high content of unsaturated FA 18:1, 18:2 and 18:3 (59.5, 24.3 and 8.6% of total fat,
respectively).The experiment was carried out according to the analogous design, consisting of 21
d of pre-experimental period and 168 d of the experimental period. Three animals from each group
were slaughtered at the end of the experiment and blood and heart muscle samples were taken. The
carcasses were held in chill for 48-h before butchering and sampling. Complete cross-sections of
the longissimus thoracis muscle at the 10-12th rib level and depot fat (adipose tissue) samples from
the left side of each carcass were taken and chemical composition was analysed. The composition
of FA methyl esters were measured using gas-liquid chromatography. Samples of ruminal contents
were taken. All the data were subjected to general one-way ANOVA (Genstat 5) with diets as the
main factor.
Results
The average daily feed intake was 18.5 and 18.1 g DM/kg liveweight with a forage: concentrate
ratio of 75:25 (DM basis), the average daily gain(ADG) was 1.1 and 1.2 kg/d and the average final
weight 557 and 571 kg in RAP0 and RAP1 groups, respectively.
Most individual saturated FA were not influenced by the rapeseed oil diet. Generally, differences
between the two treatments in FA composition were greater in blood serum and heart muscle than
in longissimus thoracis muscle (Table 1). Proportions of 18:1n-9 and 18:2n-6 were greater (P<0.05)
or tended to be greater in blood serum, heart muscle and longissimus thoracis muscle from cattle
fed rapeseed oil compared with the diet without oil. FA 20:4n-6 decreased significantly in blood
serum but tended to increase in longissimus thoracis muscle and heart muscle. Contrasting with the

higher 18:2n-6 acid content of the longissimus thoracis muscle and heart muscle due to rapeseed
oil application, this effect was not observed for blood serum 18:2n-6 content. Oils containing high
concentrations of C18 unsaturated FA have consistently decreased protozoal counts or activity
(Oldick and Firkins, 2000). Our present data showed no significant difference in analysed rumen
fluid infusoria count, however microbial protein synthesis significantly (P<0.05) decreased in the
rapeseed oil diet.
Table 1. Effect of rapeseed supplementation on FA composition of tissues in fattening bulls fed silage
diet (% Total FA).
Individual Diets
fatty acids
RAP0 RAP1
LTM BS DF HM LTM BS DF HM
18:1n-9 46.95 8.73 31.89 24.37 48.07 18.52* 37.66 23.06

18:2n-6 0.98 34.22 0.44 17.29 1.92* 31,16 0.42 24.43*
18:3n-3 0.52 9.88 0.30 1.92 0.58 10.70 0.36 2.82
20:4n-6 0.42 1.19 5.16 0.50 0.9 5.50
SFA 45.97 36.07 63.66 45.27 44.08 31.54 58.63 40.20
MUFA 51.65 17.62 34.48 28.47 52.44 25.00** 39.70 25.20
PUFA 2.38 46.31 1.86 26.26 3.49 43.46 1.67 34.60*
LTM - longissimus thoracis muscle; BS - blood serum; DF - depot fat; HM - heart muscle; SFA - saturated FA
(C14:0 + C16:0 + C18:0); MUFA - monounsaturated FA (C16:1 + C18:1); PUFA - polyunsaturated FA (C18:2n-6 +
C18:3n-3 + C20:4-n-6 + C20:5n-3 + C22:4n-6 + C225-n-3 + C22:6n-3).
In the present experiment feeding canola oil to fattening bulls during the finishing period resulted
in a small decrease in SFA content and in a higher (P<0.05) level of 18:2 n6 and PUFA in both
beef and heart intramuscular fat. However, microbial protein synthesis decreased (P<0.05) in the
rapeseed oil diet.
References
Felton, E.E. and M.S. Kerley, 2004. Performance and carcass quality of steers fed different sources of dietary fat. J.
Anim. Sci. 82, 1794-1805.
Moloney, A.P., M.T. Mooneu, J.P. Kerry and D.J. Troy, 2001. Producing tender and flavoursome beef with enhanced
nutritional characteristics. Proc. Nutr. Soc. 60, 221-229.
Oldick, B.S. and J.L. Firkins, 2000. Effects of degree of fat saturation on fiber digestion and microbial protein synthesis
when diets are fed twelve times daily. J. Anim. Sci. 78, 2412-2420.
Scollan, N.D., M. Enser, S.K. Gulati, I. Richardson and J.D. Wood, 2003. Effects of including a ruminally protected
lipid supplement in the diet on the fatty acid composition of beef muscle. Br. J. Nutr. 90, 709-716.

2B. Regulation of milk production and composition

The regulation of amino acid utilisation by insulin in the lactating ewe
mammary gland
B. Sinclair1, P. Back1,2,3, P. Harris1, S. Davis1, D. Mackenzie2, M. Tavendale1, W.C. McNabb1, N.
Roy1 and J. Lee1
1Food, Metabolism & Microbiology, Food & Health Group, AgResearch Grasslands, Palmerston
North, New Zealand
2Institute of Food, Nutrition and Human Health, Massey University, Palmerston North, New
Zealand
3Current address: Dexcel Limited, Private Bag 3221, Hamilton, New Zealand
Introduction
Insulin plays an important role in regulating the partitioning of nutrients to the mammary gland
(Bauman, 2000). Bequette et al. (2002) showed in concentrate fed goats that, despite a fall in
circulating amino acid (AA) concentrations observed during a hyperinsulinaemic euglycemic clamp
(HEC), mammary blood flow and transport activity of several essential AA (EAA) were increased,
with the branched-chain AA being the most affected, and with mammary leucine oxidation reduced
by 38%. These data suggest that insulin might exert differential effects on AA metabolism in the
mammary gland. There is evidence that the nutritional status of the animals might also affect the
animal’s response to insulin (Bequette et al., 2002). This is largely untested in lactating ruminants fed
fresh forage. In this study, we hypothesise that insulin increases milk protein production by increasing
mammary AA uptake and protein synthesis and reducing mammary AA oxidation. An HEC was
used to assess the effects of insulin on leucine kinetics and net AA flux in the mammary gland and
to relate these effects to whole body leucine kinetics in early lactating ewes fed fresh forage.

Twelve ewes were housed indoors individually and offered perennial ryegrass (Lolium perenne)/white
clover (Trifolium repens) fresh forage (cut daily; intake: 288 g crude protein/d; 16.2 MJ total ME/d)
every four h for the experimental period. The ewes were fitted with a permanent aortic catheter and
a blood flow probe around the pudic artery on about d 70 of gestation and a temporary catheter was
placed into the caudal superfical epigastric (mammary) vein prior to sampling. Ten d after lambing,
the ewes were concurrently infused with a fixed rate of bovine insulin (HEC ewes; about 1 µg/kg
body weight0.75/h; in sterile filtered 5 g/L bovine serum albumin) or bovine serum albumin solution
(control ewes) and a variable rate of glucose into the jugular vein of HEC ewes for 4 d. On d 4, [1‑13C]-
leucine (1.9 mg/min) was infused into the jugular vein for 7.5 h, with 1.5 h blood samples taken at
4.5, 6 and 7.5 h and mammary blood flow estimated simultaneously. Blood was harvested from the
aorta and mammary vein to measure plasma AA concentration by HPLC analysis and leucine, keto
isocaproate and CO2 isotopic enrichment by gas chromatography or isotope ratio mass spectrometry
(Back, 2002). The data were analysed as a randomised block design. Since the ewes were blocked
according to lambing date, the analyses included HEC as a fixed effect with block as a random effect
and time as a repeated factor. The analyses used the GLM procedure from SAS (1988).

Feed intake was similar between the HEC and control ewes and averaged 1.4 kg dry matter/d. There
was no change (P>0.10) in milk protein output (79.9 ± 3.8 (SEM) g/d) and mammary blood flow
(713 ± 167 (SEM) mL/min) during the HEC. The HEC decreased (15-32%; P<0.05) arterial plasma
concentration of EAA except that of histidine and tyrosine (data not shown). Whole body leucine
irreversible loss rate tended to be lower (P<0.10) in HEC ewes (10.46 vs. 11.21 ± 0.2 (SEM) mmol/h).

The mammary uptake of isoleucine, leucine, methionine and phenylalanine was reduced (P<0.05)
by the HEC (Table 1). The ratio of mammary leucine uptake to leucine output in milk (0.83 ± 0.04
(SEM)) was not affected by the HEC. Mammary protein synthesis was reduced (P<0.05) by the
HEC but mammary leucine oxidation was not affected (P>0.10; Table 1). Reduced mammary protein
synthesis is likely to be related to decreased plasma EAA and mammary EAA uptakes (Tesseraud
et al., 1992). These changes in AA fluxes did not translate to increased milk protein production in
HEC ewes in early lactation fed fresh forage. The results show that the ewe mammary gland can
adapt to changed AA supply to maintain milk protein production in early lactation.
Table 1. Mammary essential amino acid (EAA) uptake and leucine kinetics (mmole/h; ± SED) in
early lactation ewes with or without a hyperinsulinaemic euglycaemic clamp (HEC).
Mammary parameters HEC Control P value
Arginine uptake 1.78 (± 0.13) 1.93 (± 0.10) >0.10

Histidine uptake 0.57 (± 0.22) 0.55 (± 0.17) >0.10
Isoleucine uptake1 1.71 (± 0.12) 2.05 (± 0.09) 0.05
Leucine uptake1 2.91 (± 0.16) 3.62 (± 0.13) 0.01
Lysine uptake1 1.82 (± 0.11) 1.89 (± 0.08) >0.10
Methionine uptake1 0.67 (± 0.03) 0.84 (± 0.02) 0.001
Phenylalanine uptake1 0.84 (± 0.04) 1.09 (± 0.04) 0.001
Threonine uptake1 1.45 (± 0.18) 1.73 (± 0.14) >0.10
Tyrosine uptake1 0.89 (± 0.22) 1.31 (± 0.17) >0.10
Valine uptake1 2.67 (± 0.19) 3.13 (± 0.15) 0.10
Mammary leucine oxidation2 1.48 (± 0.43) 1.54 (± 0.35) >0.10
Mammary leucine for protein synthesis2 4.11 (± 0.19) 4.69 (± 0.15) 0.05
1Arterio-venous concentration difference of these AA reduced (P<0.10) by the HEC.

2Precursor pool: mammary vein keto isocaproate.
References
Back, P.J., 2002. The role of insulin in the regulation of milk protein synthesis in pasture-fed lactating ruminants. PhD
Thesis, Massey University, New Zealand.
Bauman, D.E., 2000 Regulation of nutrient partitioning during lactation: Homeostasis and homeorhesis revisited. In:
Cronje, P.B. (editor), Ruminant Physiology: Digestion, Metabolism, Growth and Reproduction. CAB International,
Wallingford, UK, 311-328.
Bequette, B.J., C.E. Kyle, L.A. Crompton, S.E. Anderson and M.D. Hannigan, M.D., 2002. Protein metabolism in
lactating goats subjected to the insulin clamp. J. Dairy Sci. 85, 1546-1555.
SAS, 1988. SAS System for mixed models. SAS Institute Inc., Cary, NC.
Tesseraud, S., J. Grizard, B. Makarski, E. Debras, G. Bayle and C. Champredon, 1992. Effect of insulin in conjunction
with glucose, amino acids and potassium on net metabolism of glucose and amino acids in the goat mammary
gland. J. Dairy Res. 59, 135-140.

Does a dietary arginine deficiency limit milk protein yield?
T. Whyte1, A. Hayirli1, H. Lapierre2 and L. Doepel1
1University of Alberta, 410 Agriculture-Forestry Centre, T6G 2P5, Edmonton, AB, Canada
2Agriculture and Agri-Food Canada, Lennoxville Stn, J1M 1Z3, Sherbrooke, QC, Canada
Introduction
Milk proteins are built from individual amino acids (AA), classified as non-essential (NEAA) if
they can be synthesized by the animal or essential (EAA) if solely provided from the diet and rumen
microbial protein synthesis. Arginine (Arg) is an equivocal AA in terms of its essentiality; despite
the fact that ruminants can synthesize Arg from other N precursors, it is usually classified as an
EAA because de novo synthesis is insufficient to cover the requirements of a high-yielding dairy
cow (NRC, 2001). Arginine also has the peculiarity of being the only AA for which net uptake from
the blood by the mammary gland exceeds by two to three times the amount that appears in milk
protein (Mepham, 1982). The Arg taken up in excess is potentially used to synthesize NEAA, whose
mammary uptake is less than milk protein output. To determine whether milk protein synthesis could
be limited by insufficient Arg supply, an experiment was carried out to study how milk protein yield
and mammary metabolism of AA and energy substrates change when Arg supply is deficient.

Six Holstein cows (199 ± 5 DIM) were used in a replicated 3 × 3 Latin square with 14-d periods.
Cows were fed a diet formulated to supply 100% of the net energy requirements and 75% of the
metabolizable protein (MP) requirements (NRC, 2001). The treatments were continuous abomasal
infusions of (i) water (Ctl); (ii) all EAA, including Arg at 5.5 mmol/h, in amounts sufficient to meet
100% of MP requirements (Arg+); and (iii) as treatment 2 but excluding Arg (Arg-). The AA infusions,
based on casein profile, were delivered in 8 L of water. Blood from the coccygeal vessel (arterial)
and mammary vein (venous) was collected on d 14 every other h between the twice daily milkings
(n=6). Samples were analyzed for AA, glucose, and β-hydroxybutyrate. Mammary plasma flow was
estimated using the Fick principle (using Phe and Tyr as the AA markers). Mammary arterio-venous
difference, uptake and extraction rate of nutrients were analyzed using the mixed procedure of SAS
(1999). Preplanned contrasts compared Ctl vs. the AA infusions, and Arg+ vs. Arg-.
Results
Amino acid infusions increased milk yield despite a decreased mammary plasma flow (Table 1). In
relation with the increment in milk protein yield, AA infusions increased mammary uptake of all
EAA but only the uptake:output ratio of the BCAA and Lys. Deletion of Arg from the EAA mixture
did not impair the milk response to AA infusion, despite a decrease in Arg concentration (P=0.002;
85.3, 104.7 and 85.2 ± 2.7 μM for Ctl, Arg+ and Arg-, respectively). A low supply of Arg did not
alter mammary plasma flow and despite a numerical increase in extraction efficiency (30.1, 31.9,
and 35.3% ± 2.5 for Ctl, Arg+ and Arg-, respectively), mammary uptake of Arg was lower with
Arg- than with Arg+.
Deletion of Arg from the AA mixture, however, did not affect mammary uptake of other AA, glucose
(409, 345 and 370 ± 37 mmol/h), and β-hydroxybutyrate (115, 125 and 122 ± 19 mmol/h) for Ctl,
Arg+ and Arg-, respectively.

Table 1. Amino acid mammary gland uptake (MG: mmol/h) and the ratio uptake:milk output.
AA Parameter Treatment SEM P-value
Ctl Arg+ Arg- Ctl vs. AA Arg+ vs. Arg-
Milk yield, kg/d 24.4 25.9 25.8 0.5 0.04 0.88

Protein yield, g/d 756 831 836 16.1 <0.01 0.83
Fat yield, g/d 624 599 603 16.1 0.27 0.86
Plasma flow, L/h 579 512 480 22.0 0.02 0.34
Arg MG1 14.0 16.8 14.2 0.64 0.10 0.03
U:O2 2.14 2.35 2.04 0.09 0.67 0.06
3
BCAA MG 60.6 78.0 73.7 4.51 0.03 0.53
U:O 1.05 1.23 1.18 0.06 0.07 0.59
His MG 5.9 6.6 6.6 0.20 0.04 0.79
U:O 1.04 1.05 1.09 0.04 0.49 0.54
Lys MG 20.1 27.9 26.8 1.12 <0.01 0.53
U:O 1.08 1.35 1.33 0.05 <0.01 0.75
Met MG 6.3 7.2 6.9 0.31 0.09 0.57
U:O 1.01 1.05 1.04 0.02 0.21 0.68
Thr MG 12.8 14.8 14.0 0.50 0.05 0.29
U:O 1.08 1.13 1.09 0.02 0.25 0.31
Ala MG 1.39 5.09 6.90 5.19 0.50 0.81
U:O 0.09 0.25 0.47 0.37 0.58 0.69
Gln MG 38.8 38.3 34.6 2.06 0.38 0.26
U:O 1.80 1.62 1.50 0.06 0.02 0.22
Glu MG 16.8 14.4 14.3 0.66 0.02 0.91
U:O 0.63 0.48 0.48 0.02 <0.01 0.97
1Calculated
as (arterial- venous concentration) × mammary plasma flow.
2MG uptake to milk output ratio; milk AA output = milk protein yield × 0.965 × AA composition.
3BCAA (branched-chain AA) = Ile + Leu + Val.
Conclusion
Overall, the mammary gland was able to face a deficiency in Arg supply and a decreased mammary
uptake of Arg without compromising milk yield. Uptake of the other measured AA used to synthesize
milk protein, however, did not increase when Arg was deficient. This suggests that increased uptake of
AA other than those used for protein synthesis, such as those involved in the Arg cycle, like Orn and
Cit, may be required to supply additional N needed for NEAA synthesis during an Arg deficiency.
References
Mepham, T.B, 1982. Amino acid utilization by lactating mammary gland. J. Dairy Sci. 65, 287-298.
National Research Council, 2001. Nutritional requirements of dairy cattle. 7th revised edition. National Academy
Press, Washington, DC.

Feeding measures to improve nitrogen efficiency in dairy cattle
S.M. van Zijderveld and W.M. van Straalen
Schothorst Feed Research B.V., Meerkoetenweg 26, P.O. Box 533, 8200AM, Lelystad, The
Netherlands
Introduction
Nitrogen (N) excretion by dairy cows in the Netherlands is one of the major sources of environmental
N-pollution. In order to reduce this pollution, dairy farmers are only allowed to produce a limited
amount of N per hectare of available land. If any surplus is produced, manure has to be removed
from the farm, introducing additional costs for the dairy farmer. The N-excretion from dairy cows
in the Netherlands is calculated from the annual milk production per cow and the annual average
of milk urea (MU) contents in bulk milk. Earlier research has shown a strong, positive correlation
between MU-levels and nitrogen excretion (i.e. Jonker et al., 1998).
This study was set up to explore feeding measures that can be employed to reduce MU-levels
and thus improve nitrogen efficiency in dairy cattle, without negatively affecting the production
parameters of the animals.

The experiment was designed as a randomised block design with 5 treatments including 14 animals per
treatment. The experimental animals were all Holstein-Friesians in midlactation (average production
30 kg milk/d, 141 d in milk). During a 3-wk adaptation period all animals were fed the same diet.
Data from this period were used as covariables in the statistical analysis. After the 3-wk preperiod,
animals were switched to one of five treatments for a period of 7 wk (main period).
During the entire trial, all animals received the same roughage mixture which consisted of (on a dry
matter basis) 70% grass silage, 25% maize silage and 5% dried alfalfa. The differences between
treatments were established by feeding different concentrate types. The treatments are described
in Table 1.
Table 1. Description of experimental treatments.
Treatment Abbreviation Description
1 CON Control treatment, resembling normal Dutch feeding conditions

(160 g crude protein (CP)/ kg DM ration).
2 LCP Treatment with low CP (150 g CP/ kg DM ration) through feeding
low CP-concentrates.
3 BP Treatment with high inclusion of beet pulp to improve nitrogen
capture in the rumen and large intestine.
4 EO Treatment with added essential oils to reduce protein degradation in
the rumen.
5 COMBI Treatment in which all measures were combined (low CP, inclusion
of beet pulp and essential oils).

The results from the trial are summarised in Table 2.
Table 2. Feed intake, milk production and composition during the main period of the experiment.
Treatment lsd**
CON LCP BP EO COMBI
Roughage intake, kg DM/d 15.6c* 14.4a 15.0b 15.3bc 14.6ab 0.53

Concentrate intake, kg DM/d 7.2ab 7.3b 7.3abc 7.2a 7.3c 0.07
Milk production, kg/d 31.9c 29.9ab 30.8b 31.8c 29.6a 0.95
Fat content, % 4.17ab 4.24b 4.12a 4.18ab 4.24b 0.10
Protein content, % 3.49ab 3.50ab 3.47a 3.46a 3.53b 0.05
Milk urea content, mg/dL 23.9c 21.0b 23.3c 23.9c 19.9a 1.02
N-efficiency, % 30.9a 32.7b 30.3a 31.1a 32.2b 1.14
* Values with a different superscript within a row are significantly different (P<0.05); ** lsd=least significant
difference (P<0.05).
Reducing CP-contents of the diet resulted in an improved conversion of feed N to milk N (treatments
LCP and COMBI). This was also reflected in the lower MU-levels on these treatments. Reducing
CP-contents of dairy cow diets has been shown to improve N-efficiency before (Frank and Swensson,
2002; Nouisianen et al., 2004). Despite the improvement in utilisation of N for milk protein
production, cows on treatments in which CP-contents were lowered, displayed a lower roughage
intake. As a result of this, energy intake was lower on these treatments, resulting in lower milk
production.
Calculation of revenues for the farmer for all treatments (data not shown) revealed that reduced income
from the lower milk production outweighed the decrease in costs for removal of surplus manure
under these circumstances. For Dutch dairy farmers it appears financially unattractive to reduce CP-
contents of the diets further than 160 g/kg DM to improve N-efficiency of their animals.
In conclusion, reducing the CP-content of dairy cow diets leads to an improved conversion of feed
N to milk N, which is reflected in the lower milk urea contents of the milk. However, in this trial,
roughage intake declined as a result of the lower CP-content of the diet. This resulted in a lower
milk production on these treatments. Reducing CP-contents below 160 g/kg DM will result in an
improved N-efficiency in dairy cows, but will decrease revenues for the dairy farmer.
References
Frank B. and C. Swensson, 2002. Relationship between content of crude protein in rations for dairy cows and milk
yield, concentration of urea in milk and ammonia emissions. J. Dairy Sci. 85, 1829-1838
Jonker J.S., R.A. Kohn and R.A. Erdman, 1998. Using milk urea nitrogen to predict nitrogen excretion and utilization
efficiency in lactating dairy cows. J. Dairy Sci. 81, 2681-2692
Nouisianen J., K.J. Shingfield and P. Huhtanen, 2004. Evaluation of milk urea nitrogen as a diagnostic of protein
feeding. J. Dairy Sci. 87, 386-398

Constraints in estimating N-excretion from the milk urea content in
dairy cows
S. De Campeneere, D.L. De Brabander and J. Vanacker
ILVO, Animal Science Unit, Scheldeweg 68, 9090 Melle, Belgium
Introduction
Several models (Jonker et al., 1998, Nousiainen, 2003) have been developed to predict nitrogen
(N) excretion of dairy cows from milk urea content (MUC) and some additional parameters. The
accuracy of these models is thought only to be affected by nutritional factors, like feed protein intake
and the protein/energy ratio in the diet. However, De Campeneere et al. (2006) compared 100%
maize silage (100MS) and 100% prewilted grass silage (100PGS) based diets in a feeding trial and
found MUC values of 100MS (230 mg/L) to be significantly different from that of 100PGS (171
mg/L). After correction for differences in energy and protein supply (based on De Brabander et al.,
1999), MUC of the 100MS was 71 mg/L higher than that of 100PGS. Additional N-balance trials
confirmed that although total N-excretion was almost identical for 100MS and 100PGS: 392 and
389 g/d, the MUC content (after correction for differences in energy and protein supply) for 100MS
was 84 mg/L higher than for 100PGS.
These results suggest that MUC is also influenced by factors other than protein and energy in the
diet. The current trial was performed to test if the differences in MUC between 100MS and 100PGS
found by De Campeneere et al. (2006) were due to differences in K and Na content.

A trial was set up with 18 Holstein cows fed a 100%MS diet for all 3 treatments. To obtain comparable
K- and Na-contents as in PGS-diets the following treatments were applied: 1) control; 2) control +
380 g of KCl/d and 3) control + 510 g of NaCl/d. Preliminary to the trial, during 1 reference wk,
all cows were fed the control diet with MS ad libitum and supplemented with soybean meal and
balanced concentrate to achieve 105% of the energy and protein requirements. Thereafter, during the
whole trial (6 wk) group 1 remained on the control diet, while groups 2 and 3 formed a cross-over
with treatments 2 and 3 during the first and second period of 3 wk.
Milk production was recorded daily and milk composition (fat, protein, urea) was recorded during
the last 4 milkings of the reference wk and of each of the last 2 wk of both experimental periods.
Statistical analysis was done as a GLM procedure with period, treatment and period × treatment
as fixed factors and with the observed value of the respective parameter during the reference wk
as covariable. The difference between the control and treatment group was analysed with contrast
procedure.

DM-intake of KCl and NaCl treatments tended to be higher than that of the control (P-value of
contrast with control P=0.09 and P=0.11 respectively). The results indicate that KCl and NaCL
decreased MUC with 10% and 22%, respectively. However, these differences were partly caused
by differences in energy and protein intake. Net energy supply was lower for the control than KCl
and NaCl treatments (107 vs. 111 and 109% of requirements, respectively), while protein supply and
rumen degradable protein balance were higher (100 vs. 95 and 95% of requirements, and 148 vs.
103 and 106 g/d, respectively). When MUC values were corrected for these differences (based on
De Brabander et al., 1999) and covariated for the MUC value observed during the reference wk, the

differences between treatments were reduced (Table 1). The influence of KCl on MUC disappeared,
while the effect of NaCl on MUC remained important (P<0.05; 14%). Adding the salts increased
milk production and milk protein content, while the fat content remained unchanged in our trial.
In contrast to our results, Sehested and Lund (2006) found a significant decrease in the MUC from
4.4 mM to 2.9 mM/L when increasing the dietary K content (with KCl) from 12 to 35 g/kg DM.
Within a high K content, a further increase in Na in the diet (from 1 to 10 g/kg DM; with NaCl) did
not influence the milk urea content any further. These authors also found a positive effect of KCl
on milk yield and protein content, but no further effect of NaCl (within a high KCl content) on milk
yield or milk protein.
Table 1. Least square means of MUC, milk yield and milk fat and protein content for the three
treatments.
Control KCl NaCl SEM P-value
MUC, mg/L 228 235 197* 8 0.008

Milk, kg 27.9 29.4* 30.0** 1.5 0.003
Fat, % 3.90 3.83 3.92 0.11 ns
Protein, % 3.10 3.25*** 3.21* 0.04 0.002
DM-intake, kg/d 18.9 19.9† 19.8 0.44 0.17
1Sign. level of the contrast of each treatment group with the control † P<0.10; * P<0.05; **P<0.01; ***P<0.001.
These results indicate that K could not explain the difference found by De Campeneere et al.
(2006) between 100MS and 100PGS while Na could only explain part of it. Probably also other
parameters are involved, which should be quantified before MUC can be used as a reliable N-
excretion management tool.
References
De Brabander, D.L., S.M. Botterman, J. Vanacker and Ch.V. Boucqué, 1999. The milk urea concentration as indicator for
nutrition and N-excretion. (in Dutch). Report nr 1109. Dep. Animal Nutrition and Husbandry, Centre Agricultural
Research Ghent, Belgium, 8 pp.
De Campeneere, S., D.L. De Brabander and J.M. Vanacker, 2006. Milk urea concentration as affected by the roughage
type offered to dairy cattle. Livest. Sci. 103, 30-39.
Jonker, J.S., Kohn, R.A. and R.A. Erdman, 1998. Using milk urea nitrogen to predict nitrogen excretion and utilization
efficiency in lactating dairy cows. J. Dairy Sci. 81, 2681-2692.
Nousiainen, J., K.J. Shingfield. and P. Huhtanen, 2003. Evaluation of milk urea nitrogen as a diagnostic of protein
feeding. J. Dairy Sci. 87, 386-398.
Sehested, J. and P. Lund, 2006. NaCl and KCl intake affects milk composition and freezing point in dairy cows. In:
Van der Honing et al. (editors), Book of the abstract of the 57th Annual Meeting of the EAAP. EAAP, Antalya,
Turkey, 17-20 September, 295.

Estimation of the intra-mammary metabolic fate of glucose and acetate
in response to longer milking intervals in dairy cows
J. Guinard-Flament, S. Lemosquet and E. Delamaire
INRA, Agrocampus Rennes, UMR1080, Production du Lait, F-35590 St-Gilles
Introduction
A reduction in milking frequency, i.e. once daily milking, reduces milk production and increases
milk fat content in dairy cows. Previous results suggest that the decline in milk yield is associated
with a reduced amount of nutrients taken up by the udder (Delamaire and Guinard-Flament, 2006).
However, little is known about the intra-mammary metabolic adaptations concerning glucose and
acetate, even though both are essential for milk fat synthesis and ATP furniture, and glucose is
essential for milk volume determination through lactose synthesis. This study was aimed at gaining
a better understanding of the metabolic fate of these two substrates in response to longer milking
intervals from mammary balance data and metabolite levels in milk.

As described previously by Delamaire and Guinard-Flament (2006), the same three dairy cows in mid
lactation were milked at 8-, 12-, 16- or 24-h intervals during the last 7 d of 2 wk periods according
to a Youden square with 5 periods. The cows were fitted with an ultrasonic flow probe implanted
around the left external pudic artery and two permanent catheters inserted into the left carotid and
left subcutaneous abdominal vein to determine the quantities of nutrients taken up from or discharged
into blood plasma by the udder (glucose, acetate, BHBA, lactate, total glycerol, and CO2). The milk
was analysed to determine contents of fat, protein, fatty acids, lactose, and metabolites of lactose
synthesis (glucose, glucose-6-P and glucose-1-P, Rigout et al., 2002). The mammary efficiency to
use nutrients for milk synthesis was estimated using milk output/uptake ratio (in %) assuming that
lactose and synthesised glycerol in milk and venous plasma lactate were provided from glucose,
and that milk C4 to C14 and 50% of C16 fatty acids were provided from BHBA and acetate. The
remaining glucose and acetate were estimated to be oxidised.

In this study, the milk yield decreased from 38.1 to 31.4 kg/d (P<0.05) and the milk fat content tended
to increase from 3.98 to 4.47% as milking intervals increased (P<0.10). Lactose is synthesised from
glucose and galactose, the latter coming from glucose converted into glucose-6-P and then converted
into glucose-1-P by a phosphoglucomutase. The levels of glucose and glucose-1-P decreased in
milk by 14 and 69% between the 8- and 24-h milking intervals, respectively (P<0.06; Table 1).
The glucose-6-P/glucose ratio did not vary but the glucose-1-P/glucose-6-P ratio decreased by 0.50
points. This result suggests an inhibition of glucose-6-P conversion into glucose-1-P.
Whereas the mammary uptake of glucose and acetate decreased in response to longer milking
intervals (Delamaire and Guinard-Flament, 2006), the proportion of glucose taken up by the udder
and converted into lactose, lactate, and CO2 remained unchanged (Table 2). Only the relative part
of glucose converted into glycerol tended to increase by 2.3 points (P<0.11). In contrast, the relative
part of acetate converted into milk short- and medium-chain fatty acids increased significantly from
38 to 63% in response to longer milking intervals, provoking a decline of its oxidation.

Table 1. Milk metabolites of lactose synthesis according to milking intervals in dairy cows.
Milking interval RMSE Effects
8h 12 h 16 h 24 h Linear Quadratic Cubic
Glucose, mM/L 0.70 0.75 0.62 0.60 0.055 0.057 0.130 0.171
Glucose-6-P, mM/L 0.116 0.116 0.120 0.107 0.0154 0.658 0.510 0.638
Glucose-1-P, mM/L 0.078 0.061 0.045 0.024 0.0153 0.013 0.386 0.968
Glucose-6-P/Glucose 0.16 0.16 0.19 0.18 0.027 0.351 0.847 0.258
Glucose-1-P/Glucose-6-P 0.72 0.54 0.37 0.22 0.159 0.029 0.659 0.865
Table 2. Metabolic fate of glucose and acetate in the udder according to milking intervals in dairy
cows (in % of glucose or acetate taken up by the udder).
% Milking interval RMSE Effects
8h 12 h 16 h 24 h Linear Quadratic Cubic
Glucose → Lactose 83 82 96 87 11.6 0.435 0.841 0.297

Glucose → Glycerol 6.5 6.3 8.5 8.8 1.58 0.105 0.441 0.400
Glucose → Lactate 0.07 0.04 0.88 1.62 2.000 0.388 0.609 0.884
Glucose → CO2 11 12 -5 3 13.5 0.317 0.990 0.311
Acetate → C4–C14+C16/2 38 28 63 63 10.0 0.020 0.094 0.052
Acetate → CO2 62 72 37 37 10.0 0.020 0.084 0.052
The present results indicate that the udder produced less milk and lactose in response to longer
milking intervals by regulating its glucose uptake to its needs. No major changes occurred in glucose
partition between intra-mammary metabolic pathways. The lactose synthesis pathway could be
affected in its first steps, i.e. the galactose synthesis (present study), and its last step, i.e. the activity
of galactosyltransferase (Wilde and Knight, 1990).
The improved conversion of acetate towards milk fatty acid synthesis and of glucose towards glycerol
synthesis was consistent with the enhanced milk fat level. The modifications in acetate partition
between fatty acid synthesis and oxidation could occur to adapt mammary cell energy balance in
response to reduced metabolic activity due to longer milking intervals.
References
Delamaire, E. and J. Guinard-Flament, 2006. Increasing milking intervals decreases the mammary blood flow and
mammary uptake of nutrients in dairy cows. J. Dairy Sci. 89, 3439-3446.
Rigout, S., S. Lemosquet, J.E. van Eys, J.W. Blum and H. Rulquin, 2002. Duodenal glucose increases glucose fluxes
and lactose synthesis in grass silage-fed dairy cows. J. Dairy Sci. 85, 595-606.
Wilde, C.J. and C.H. Knight, 1990. Milk yield and mammary function in goats during and after once-daily milking.
J. Dairy Res. 57, 441-447.

Dietary and mammary components of milk fat depression: insights from
a meta-analysis of literature data
F. Glasser and Y. Chilliard
INRA, UR1213 Herbivores, Site de Theix, F-63122 Saint-Genès-Champanelle, France
Introduction
Reduction in milk fat yield, known as milk fat depression (MFD) has been described and studied for
several decades, and is observed under various nutritional conditions. Milk fatty acids (FA) come
from both mammary uptake of plasma FA (mainly C18 FA) and mammary de novo synthesis (C4-
C16 FA), which are regulated by distinct effectors. For a better understanding of MFD processes,
in order to know whether MFD is a unique process or may result from distinct metabolic controls,
it is necessary to study to which extent each of these two metabolic pathways are affected in the
various conditions that induce MFD.

From a database of published experiments reporting milk FA composition, we selected the experiments
inducing MFD (based on a minimum 15%-decrease in milk fat yield). The 74 selected experiments
(184 treatments) included four main types of protocols: unsaturated plant lipid supplementation
(n=31 experiments), fish oil supplementation (n=20), t10,c12-18:2 (hereafter t10c12) post-ruminal
infusions (n=11), variation in forage:concentrate (F:C) ratio (n=12). We studied the daily milk yields
of the sums of C4-C16 FA and C18 FA. Indeed, even if part of milk C16 originates in mammary
uptake from plasma, milk C16 yield was highly correlated (R²=0.85) to C4-C14 yield. Data were
analysed separately for each type of protocol, using GLM models (Minitab ®), with a fixed effect
of the experiment.

Concerning the mean variations in daily milk FA yields, plant lipid supplementations (-261 g/d of
C4-C16, +23 g/d of C18) differed from the three other protocols, which did not significantly differ
(-165 g/d of C4-C16 and -89 g/d of C18).
For plant lipid supplementation, milk C4-C16 and C18 yields were quadratic functions of supplemental
C18 (Figure 1, r²=0.94 and 0.98). The decrease in C4-C16 yield was partly compensated for by an
increased incorporation of C18 in milk lipids. There was also a linear decrease in DMI with increasing
supplemental C18 (slope 2.2 g/g) and confusion between the two variables did not enable to study
separately the effect of each one. Both the increase in C18 and decrease in DMI (i.e. in precursors
of FA synthesis) could be responsible for the observed decrease in de novo synthesis, but the data
available do not allow distinguishing these effects.
For fish oil experiments, both C4-C16 and C18 yields were quadratic decreasing functions of fish
oil intake (Figure 1, r²=0.94 and 0.92). There were not enough experiments in the database with
simultaneous variations in C18 and fish oil (substitution of plant lipids by fish oil or simultaneous
supply of fish oil+plant lipids) to study the potential interactions between both lipid sources. Milk
yield of C20-C22 FA increased linearly with the C20-C22 supplied by the supplement, with a slope
of 0.13 (r²=0.83). The increase in C20-C22 FA did not by far compensate the decrease in C18 FA.
In post-ruminal t10c12 infusions, there was a quadratic decrease in both C4-C16 and C18 yields
with increasing daily infusion of t10c12 (Figure 1, r²=0.88 and 0.96).

Figure 1. Adjusted models of C4-C16 and C18 yields (g/d) in the different types of MFD experiments
(r² of the models are in the text). X-axis varies between 0 and 1, representing the maximum value of
the explicative variable in each case: 1000 g/d of supplemental C18 for plant lipid supplementation,
500 g/d of fish oil intake for fish oil supplementation, 40 g/d of t10c12 for t10c12 infusions and 100%
forage in DMI for variations in F:C ratio.
In experiments of F:C variation, the behaviour of C4-C16 (r²=0.91) and C18 (r²=0.93) yields was
similar: the yields were maximal for a F:C ratio around 50:50. For both higher and lower F:C ratios,
decreases in both C4-C16 and C18 yields were observed. The DMI followed a similar feature. A
decrease in FA intake was also observed with high-forage diets.
For both t10c12 infusions and plant lipid supplementation, there was a sharp decrease in C4-C16 yield,
and either a smaller decrease (t10c12 infusions) or a limited increase (plant lipid supplementation)
in C18 yield. C18 proportion in milk lipids was thus increased. De novo synthesis was probably
inhibited by t10c12 and/or other trans unsaturated C18 isomers, and we could hypothesise that the
shortage in C4-C16 could limit the incorporation of C18 in milk lipids, through constraints on FA
esterification. Indeed, supplementation of more saturated lipids or protected lipids, preventing ruminal
production of trans isomers, prevent the decrease in C4-C16 yield and increase milk fat secretion
through higher incorporation of supplemental C18 (linear increase of milk C18 yield, with a mean
slope of 0.35, obtained from 4 other experiments).
Fish oil supplementation and variations in F:C ratio induced proportional decreases in C4-C16 and
C18 yields, and the proportions of both FA pools in milk fat were not significantly altered. The
processes involved in these cases are less clear, since few detailed and recent data are available,
especially for F:C experiments. Fish oil highly increases the trans-18:1 isomers produced in the
rumen, at the expense of 18:0. A regulation through milk fat melting point and/or ruminal production
of trans isomers inhibiting de novo synthesis could be involved. For F:C ratio, fat secretion decreased
both below and above medium ratios. The processes implied could be numerous and probably differ
according to the F:C range: for low-forage diets, trans isomers produced in the rumen could inhibit
de novo synthesis. For high-forage diets, intake of C18 and/or precursors of de novo synthesis could
limit milk fat secretion.
In conclusion, a decrease in C4-C16 yield was observed in all situations, and the contribution of
trans isomers produced in the rumen is a likely hypothesis in all cases except high-forage diets.
Regulations at the esterification step, where both C4-C16 and C18 are involved, and/or melting
point constraints could explain, at least in part, the low incorporation or decrease of milk C18 yield
in plant lipid supplementations, t10c12 infusions and fish oil experiments.

Effect of rumen-protected conjugated linoleic acid in combination with
propylene glycol or rumen protected fat on performance and metabolic
parameters of early lactation dairy cows
F.J. Schwarz1, T. Liermann1 and A.M. Pfeiffer2
1Section of Animal Nutrition, Department of Animal Science, Technical University of Munich,
Hochfeldweg 6, 85350 Freising-Weihenstephan, Germany
2BASF-AG, Nutrition Research Station, Offenbach/Queich, Germany
Introduction
Conjugated linoleic acid (CLA) used as a fat supplement can reduce the content of milk fat and,
therefore, has the potential beneficial effect of reducing energy requirements of the early lactating
dairy cow (Griinari and Bauman, 2006). Propylene glycol (PG) as a glucose precursor may increase
glucose supply, rumen-protected fat (RF) may help to increase energy supply. Therefore, feeding
rumen-protected CLA (CLA) alone or in combination with PG or RF is of interest with regards to
energy partitioning and the effect of an additional energy source of the diet on performance and
metabolic responses of dairy cows.

Two experiments (EI, EII) with Red Holstein × Fleckvieh cows were conducted. In EI and EII, 50
(31 multiparous, 19 primiparous cows), and 53 (40 multiparous, 13 primiparous cows) dairy cows
were respectively used from wk 1 until wk 14 of lactation. Multiparous cows were blocked based
on their previous lactation performance. The experimental schedule was designed as follows: EI:
1) control, 2) 40 g/d of CLA (CLA-1) and 3) 40 g/d of CLA + 200 g/d of PG (CLA-PG). EII: 1)
control, 2) 40 g/d of CLA (CLA-2) and 3) 40 g/d of CLA + 700 g/d of RF (CLA-RF). All cows were
fed ad libitum once a d a partial mixed ration (PMR) (50.0% corn silage, 28.5% grass silage, 15.5%
concentrate (AKF), 6.0% hay, dry matter basis). Additional concentrate (LKF) was offered when daily
milk yield was above 21 kg. The CLA supplement was lipid encapsulated (containing 10% trans-10,
cis-12 CLA; Lutrell, BASF) and was provided with the LKF. At the same time PG was sprayed
on to the LKF with a liquid dispenser prior to feeding in EI and RF (Dunafett 100, EURODUNA)
was added additionally in EII. Intake of PMR, LKF, CLA and PG was recorded daily for each cow
as well as milk yield. Milk samples and blood samples were taken once per wk. Aliquots of blood
samples were stored at –20 °C until analysis of plasma glucose, NEFA and BHBA. Body condition
score (BCS) and body weight (BW) were determined weekly. The energy balance of each cow was
calculated from daily feed intake, maintenance requirement and milk energy output. Analysis of
variance was conducted using the GLM procedure of SAS (1998).
Results
Overall performance of cows in EI and EII is shown in Table 1. Daily feed intake (DMI) was
unchanged for all treatments in both experiments. In EI milk yield tended to be higher (P<0.10) for
CLA-1 (7%) and CLA-PG (5%) in contrast to the control. In EII increased milk yield for CLA-2 and
CLA-RF was apparent from wk 9 to wk 14 with a final increase of 2.4 kg (CLA-2) and 3.9 kg (CLA-
RF) in wk 14 in comparison to the control. Milk fat content was reduced by 17% in both EI and EII,
whereas milk protein was unaffected by CLA alone. However, CLA-RF (EII) tended to reduce milk
protein percentage. At the start of the experiment all cows were in severe negative energy balance
(–39 MJ NEL/d). CLA treated cows reached positive energy balance in wk 6-7 (EI) or in wk 5-6 (EII)
which was sooner compared to the control (wk 9-10 in EI and EII). BW loss of cows was similar in
all treatments with –30 kg on average until wk 6, further on the BW was stable until wk 14.

Table 1. Effect of treatments on performance and energy balance during lactation period wk 1 to
14 in EI and EII.
Experiment 1 (EI) Experiment 2 (EII)
Control CLA-1 CLA-PG SEM Control CLA-2 CLA-RF SEM
DM intake, kg/d 17.3 18.0 17.7 0.13 19.0 19.0 18.8 0.13
Milk, kg/d 32.1 34.2 33.7 0.24 34.8 35.9 35.8 0.26
Fat, % 3.82a 3.09b 3.20b 0.03 3.66a 3.00b 3.04b 0.02
g/d 1210a 1050b 1060b 0.01 1280a 1070b 1080b 0.01
Protein, % 3.07 3.02 2.99 0.01 3.16 3.10 2.93 0.01
g/d 980 1020 1010 0.01 1090 1110 1050 0.01
Energy intake 123.3 126.5 126.2 0.94 133.3 132.8 136.4 0.89
Energy balance, MJ NEL/d -10.6a -4.3b -4.8b 0.87 -9.5a -2.0b 1.8b 0.88
a,bMeans within a row with different superscripts differ (P<0.05).
Overall, for EI no significant treatment differences in plasma parameters were observed. However,
plasma glucose showed a steadily increasing level with time after parturition for all treatments with
the lowest point at wk 2 and a further increase up to wk 14 whereas CLA-PG seems to have the
highest course after wk 2. NEFA values were short-time increased for CLA-1 and CLA-PG in wk
2 and 3 in comparison to the control; further on, the treatments did not differ. BHBA continuously
decreased over the entire experimental period with a numerically lower ranking for CLA-PG. In
EII, metabolic parameters for CLA-RF treatment neither showed a positive effect on plasma glucose
levels (it was lower), nor on NEFA, with a tendency for higher values during the period. BHBA
levels were numerically higher and showed a small and late increase in wk 3 to 5 in comparison to
control and CLA-2.
Conclusion
Diet supplementation with CLA had a tendency to increase milk yield. CLA decreased milk fat
in a consistent manner and subsequently cows reached positive energy balance sooner. This was
consistent with previous studies supplementing rumen protected CLA in early lactation (Griinari
and Baumann, 2006). CLA-PG tended to have a beneficial effect on production and metabolic
parameters when applied via the feed. CLA-RF tended to have beneficial effect on production but
no further positive effect on metabolic parameters. Finally it shall be emphasised that these two
studies showed that cows supplied with CLA and CLA-energy supplemented-diets came back earlier
to a positive energy balance.
References
Griinari, J.M. and D.E. Bauman, 2006. Milk fat depression: Concepts, mechanisms and management applications.
In: Sejrsen, K., T. Hvelplund and M.O. Nielsen (editors), Ruminant Physiology: Digestion, Metabolism, and
Impact of Nutrition on Gene Expression, Immunology and Stress. Wageningen Acad. Publ., Wageningen, The
Netherlands, 389–417.

Effect of dietary conjugated linoleic acid (CLA) on milk composition of
dairy cows
U. Meyer1, C. Brömmel1, G. Flachowsky1 and G. Jahreis2
1Federal Agricultural Research Centre (FAL), Institute of Animal Nutrition, Bundesallee 50, D38116
Braunschweig, Germany
2Friedrich Schiller University, Institute of Nutrition, Dornburger Str. 24, D07743 Jena, Germany
Introduction
Influencing the milk fat content and the fatty acid pattern by diet composition and feed supplements
has been an aim of animal nutritionists for a long time. Apart from diet variation and supplementation
of unsaturated fatty acids (Flachowsky et al., 2006), some authors showed that dietary supplementation
of conjugated linoleic acids (CLA) inhibits milk fat content and alters fatty acid composition (e.g.
Kraft et al., 2000; Bauman and Griinari, 2003). Mainly the trans-10, cis-12 C18-isomers are recognised
to be responsible for the reduction of milk fat concentration (Lock et al., 2006). A decrease in milk
fat yield could potentially reduce the energy demand in order to minimise the post partum negative
energy balance of high-yielding dairy cows. On the grounds that CLA are claimed for health
promoting effects in human, it is occasionally recommended to increase the CLA concentration of
milk. However, the discussion about the effects of CLA on human nutrition remains controversial.
The preliminary study was conducted to assess the effects of increasing amounts of dietary CLA on
feed intake and milk composition of cows in early lactation.

Five multiparous Holstein cows averaging 584 kg initial live weight were individually fed a TMR
consisting of 30% corn silage, 20% grass silage and 50% concentrate on dry matter basis for ad
libitum consumption. The concentrate contained 25% wheat, 25% corn, 17% soybean meal, 15%
peas, 15% sugar beet pulp as well as 3% mineral and vitamin premix. Drinking water was freely
available.
To detect the effects of CLA in the diet, the experiment was divided into four consecutive periods.
Seven d without supplementation (Period I) were followed by 14 d of supplementation (25, 50
and 100 g/d CLA supplement) during Periods II, III and IV. After each of these periods the cows
received an unsupplemented TMR for seven d. The CLA supplement contained 17.5% trans-10,
cis-12 CLA and 17.5% cis-9, trans-11 CLA in a rumen-protected form to prevent hydrogenation
by rumen microbes.
Cows were milked twice a d. Feed intake and milk yield were recorded daily. Milk samples for
the determination of fat, protein and fatty acid contents were collected at each milking. Milk fat
and protein were determined by Fourier transform spectroscopy (Milkoscan FT 6000, Foss A/S,
Denmark) and milk fatty acid composition was analysed by gas chromatography (flame ionisation
detector). The statistical analysis was performed with the SAS procedure Paired-Samples T-Test
(SAS Institute Inc., Cary, NC, USA).
Results
The effect of CLA supplementation on feed intake and milk production is shown in Table 1. Dry
matter intake (DMI), milk yield and milk protein percentage averaged 18.2 kg/d, 32.8 kg/d and 2.9%
during the trial. Milk yield was not significantly influenced by treatment. The milk fat depression
occurs during the whole experiment.

Table 1. Effect of dietary CLA supplementation on dry matter intake (DMI), milk yield and milk
composition.
Period I (n=5) P1 II (n=5) P2 III (n=5) P3 IV (n=4)
CLA supplement g/d 0 25 50 100

DMI kg/d 18.3 0.041 19.1 0.082 18.7 0.017 16.6
Milk yield kg/d 33.1 0.229 33.7 0.604 34.0 0.070 30.4
Milk fat % 3.68 0.008 3.19 <0.0001 2.64 0.010 2.22
Milk protein % 2.92 0.974 2.92 0.004 2.84 0.015 3.03
1Period I compared with II.

2 Period II compared with III.
3 Period III compared with IV.
Milk fat percentage was reduced by 0.5, 0.6 and 0.4%-points between Periods I and II, II and III as
well as III and IV. The milk fat yield was just as well reduced considerably and the results show,
furthermore, that the milk fat depressing effect persisted when the CLA supplementation was finished.
Total trans fatty acids and total CLA in milk increased dramatically (from 2.35 to 4.81% and from
0.56 to 1.12% of total fatty acids in Periods I or IV, respectively).
Conclusion
The supplementation of the dairy diet with CLA leads to a distinct milk fat depression and an increase
of CLA in milk, respectively. Long-term experiments are necessary to evaluate the effects of dietary
CLA on animal metabolism and animal health.
References
Bauman, D.E. and J.M. Griinari, 2003. Nutritional regulation of milk fat synthesis. Ann. Rev. Nutr. 23, 203-227.
Flachowsky, G., K. Erdmann, L. Hüther, G. Jahreis, P. Möckel and P. Lebzien, 2006. Influence of roughage/concentrate
ratio and linseed oil on the concentration of trans-fatty acids and conjugated linoleic acid in duodenal chyme and
milk fat of late lactating cows. Arch. Anim. Nutr. 60, 501-511.
Kraft, J., P. Lebzien, G. Flachowsky, P. Möckel and G. Jahreis, 2000. Duodenal infusion of conjugated linoleic acid
mixture influences milk fat synthesis and milk CLA content in dairy cow. Occ. Publ. Brit. Soc. Anim. Sci. 25,
143-147.
Lock, A.L., B.M. Teles, J.W. Perfield II, D.E. Bauman and L.A. Sinclair, 2006. A conjugated linoleic acid supplement
containing trans-10, cis-12 reduces milk fat synthesis in lactating sheep. J. Dairy Sci. 89, 1525-1532.

Effect of flaxseed and rapeseed supplementation during the last
trimester of pregnancy and lactation in sows on fatty acid composition in
their milk
B. Bałasińska, R. Zabielski, P. Ostaszewski and G. Kulasek
Department of Physiological Science, Warsaw Agricultural University, 02-787 Warszawa ul.
Nowoursynowska 159, Poland
Introduction
Newborn piglets possess low levels of fatty tissue and therefore are exposed to various stress factors
which in turn may decrease their viability. Proper composition of a diet for pregnant sows may
elevate the viability of piglets and as a result, increase indices of swine productivity. Milk is the
first nutrient consumed by the infant. This natural fodder contains not only nutritional compounds,
but also many other bioactive components, among them hormones, peptides, n-3 and n-6 PUFA
(polyunsaturated fatty acids). A precursor for the n-3 family is α-linolenic acid – ALA (C18:3n-3),
and for the n-6 family it is linoleic acid – LA (C18:2n-6). The composition of milk fatty acids is
affected by the diet for sows. The addition of ALA to the diet increased ALA content in milk (Bazinet
et al., 2003). Fish oil administration increased the level of long-chain n-3 fatty acids (Arbuckle and
Innis, 1993). During late pregnancy growth, CNC in the foetus results in a fast increase of DHA
(docosahexaenoic acid – C22:6n-3) and AA (arachidonic acid – C20:4n-6) in brain tissue. A proper
ratio of LA/ALA in the diet for piglets stimulates an increase in DHA content in tissues (Blanc et
al., 2002). However it is unknown whether diet for sows enriched with LA and ALA influence the
content of AA and DHA in their milk.
The aim of our study was to evaluate whether diet for pregnant sows enriched with flax and rape
seeds administrated at the end of pregnancy and during lactation effects the composition of fatty
acids in colostrum and milk.

The experiment was performed on 13 sows divided into 2 groups: control – C (n=6) receiving a
standard diet and experimental – D (n=7) receiving a diet enriched with supplements (flaxseed -
4%, rapeseed - 4%). The experimental diet was administered from the 80th d of pregnancy until the
28th d of lactation. Colostrum and milk were collected immediately following oxitocin injection.
Polyunsaturated fatty acids were determined by gas chromatography.
Results
Protein and fat content in sow milk did not differ significantly during lactation between groups C
and D (Table 1). In turn, fat content increased significantly during lactation. In group C this rise
amounted to 10% in colostrum and 15% in milk on the 28th d of lactation and in group D, 9% and
17%, respectively (P≤0.05, Table 1). Protein content decreased slightly, however there was no
significant difference between the given d of lactation. Milk from sows in group D had higher ALA
content than milk from sows in group C. In contrast, in group C there was significantly higher AA
content in milk than in group D.
The content of each fatty acid in milk changed during lactation (Table 2). High LA content was
observed in colostrum regardless of the group, whereas in milk the lower LA content was noted
in the 28th d of lactation in all groups. The diet of sows influenced the ALA content in their milk
though ALA was present in trace amounts in the control group, however the animals from that group

Table 1. Protein and fat content in colostrum and milk during lactation (g/100 mL milk.)
Colostrum 7d 28 d
C D C D C D
Fat 10.00±1.50 9.25±0.65 12.4±2.63 9.00±0.79 14.95±1.95 16.98±1.37

Protein 9.33±1.19 9.72±1.46 7.10±0.45 6.15±0.22 7.37±0.22 6.78±0.35
Table 2. Fatty acid concentration (% of total fatty acids) in colostrum and milk in 7 and 28 d of
lactation.
Colostrum 7 d 28 d Average P≤
SEM
Linoleic acid – LA, C18:2n-6 C 11.60x 10.01xa 6.48y 1.03 0.016

D 13.72x 9.64ya 8.63y 1.03 0.003
α-linolenic acid – ALA, C18:3n-3 C 0.76xa 0.64xya 0.41ya 0.07 0.025
D 5.60xb 4.45xyb 2.85yb 0.8 0.09
Arachidonic acid – AA, C20:4n-6 C 0.92xa 0.66ya 0.48ya 0.08 0.006
D 0.55xb 0.34yb 0.27yb 0.06 0.009
Σ n-3 C 0.98 0.73 0.5
D 5.75 4.45 2.85
Σ n-6 C 11.83 10.77 7.21
D 13.94 10.09 9.11
Σ n-6/Σ n-3 C 12 15 14
D 2 2 3
a, b Significantly differences between groups – C and D.

x, y Significantly differences in group in consecutive day of lifetimes.
were not supplemented with that acid. The content of this acid in sow’s milk in group D was about
7 fold higher than that in sow’s milk in group C regardless of the lactation d. The content of AA was
significantly higher in milk in group C than in group D. There was no DHA detected in any group.
The addition of rape and flax seeds to the diet for sows decreased n-6/n-3 ratio in milk. n-6/n-3 ratio in
sow’s milk from the control group amounted to 12, and in the experimental group amounted to 2.
In conclusion, rape and flax seed added to the sow’s diet has an impact on the composition of
polyunsaturated fatty acids in milk and colostrum; fatty acid composition in milk and colostrum
changed with time whereas the n-6/ n-3 ratio remained the same during the whole experiment within
each group. Therefore diet enriched with rape and flax seed with lower n-6/n-3 ratios (2 vs. 12)
seems to be better for piglet breeding.
References
Arbuckle, L.D. and A.M. Innis, 1993. Docosahexanoic acid is transferred through maternal diet to milk and to tissues
of natural milk-fed piglets. J. Nutr. 123, 1668-1675.
Bazinet R., E.G. McMillan and S.C. Cunnane, 2003. Dietary alpha-linolenic acid increases the n-3 PUFA content of
sow’s milk and the tissues of the suckling piglet. Lipids 38, 1045-1049.
Blanc C., M.A. Neumann, M. Makrides and R.A. Gibson, 2002. Optimizing DHA in piglets by lowering the linoleic
acid to α-linolenic acid ratio. J. Lipid Res. 43, 1537-1543.

Variations in mammary extraction of nutrients under the effect of a 36-h
milk accumulation into the udder in dairy cows
J. Guinard-Flament1, D. Causeur2 and E. Delamaire1
1INRA, Agrocampus Rennes, UMR1080, Dairy Production, F-35590 St-Gilles
2CNRS IRMAR, Agrocampus Rennes, UMR 6625, CS 84215, F-35042 Rennes
Introduction
To synthesise milk components, the udder extracts nutrients from the blood compartment. Because
of variations in arterial concentrations and mammary blood flow (MBF), the arterial flow of nutrients
to the udder varies differently throughout the day. As a result, the udder has to adapt its extraction
efficiency to secure the amount of nutrients taken up or to modify the metabolic fate of nutrients.
Since little is known about uptake relationships between nutrients, a trial was carried out to provide
more details about correlation between MBF, arterial concentrations, and mammary ability to
extract nutrients. The trial consisted in measuring the mammary use of nutrients throughout milk
accumulation into the udder over an extended period of 36-h length in order to compare the first
and the last 12-h periods of mammary activity that are known to be different with regards to the
milk secretion rate.

Three multiparous dairy cows in mid lactation and yielding 34 kg of milk/d were fitted with an
ultrasonic flow probe implanted around the left external pubic artery to measure MBF and with
two catheters inserted into the left carotid and subcutaneous milk vein to determine the nutrient
arteriovenous or venoarterio differences of concentrations (AV or VA, respectively). Before the
trial, cows were fed and milked twice daily at 06.30 and 18.30 h, with free access to feed for 8 h.
During the experiment, they were milked at 06.30 h (d 1) and at 18.30 h (d 2). During this 36-h
accumulating period, blood samples were collected at 07.00, 08.00, 09.30, 11.00, 14.00 and 18.00 h
on both d. They were used to determine the levels of glucose, acetate, BHBA, NEFA, total glycerol,
O2 and CO2. Trial was repeated 1 wk later on 2 cows.
The arterial availability of nutrients was estimated via MBF, arterial concentration, and arterial flow
(MBF × arterial concentration). The mammary extraction ability was estimated via AV (or VA) and
extraction rates (AV/arterial concentrations × 100). A principal component analysis was performed
on within-cow centred data to explore the correlation between nutrients and the effects of milk
accumulation. Only variables correlated with factor map were kept (|r| > 0.5).

Milk accumulation over 36 h induced a decrease in milk yield and milk fat and protein yields by 46,
18 and 46% respectively, compared to a 36-h milk production of cows milked twice daily. The first
and second factorial dimensions explained about 49% of the total variance in centred data (Figure 1).
The dimension 1 represented the variables varying throughout time within 12-h period. Dimension 2
represented the variables varying between the two 12-h periods. High values of acetate, BHBA, O2
and CO2 were positively correlated to dimension 1. High values of MBF and low values of arterial
concentration of glucose were positively correlated to dimension 2 (Figure 1a).
The changes throughout kinetics were similar between 12-h periods but the mean levels between
12-h periods were different (Figure 1b). O2 and CO2 variables that illustrate the mammary oxidative

1.0
(a)
MBF
AF-NEFA
4
17
AF-O2 ER-Acetate (b)
24
2518.00
12 4
18 6 13
5 1
30 7.00 2
ER-Glucose 29 10 11.00
7 3 8
0.5
16
14.00
2
2611 28 8.00
AF-Glucose 19 21
23 9
AF-Acetate 43 22 1st 12-h period27 14
Dimension 2
55
Dimension 2 (18.3%)
47 46 9.30
31 7.00
AV-Acetate 49
0
48 36 15
VA-CO2 20
ER-O2
0.0
AV-BHBA 33 44
AV-O2 60
34 37
18.00 3rd 12-h59period
50
A-Acetate 53 8.00 38
-2
14.0042 41
57
54 56 45
A-BHBA 32 51
11.00 9.30
35
-0.5
A-Glucose 39
-4
52 40
-1.0
-6
-1.0 -0.5 0.0 0.5 1.0 -5 0 5
Dimension 1 (30.6%) Dimension 1
Figure 1. Principal component analysis showing uptake relationships between nutrients under a
36-h milk accumulation period in dairy cows. (a) variable factorial map: A, arterial concentrations;
AV (or VA), arteriovenous (venoarterio) differences; ER, extraction rates; AF, arterial flow. (b)
individual factor map: □ time of blood sampling.
metabolism appeared to be highly correlated to acetate and BHBA. AV and extraction rates of O2 and
VA of CO2 were positively correlated to the arterial concentrations and AV of acetate and BHBA.
The results from this study suggest that mammary oxidative metabolism was increased by higher
arterial concentrations of acetate and BHBA, resulting from feeding. This phenomenon could be
due to the increased ATP needs to sustain the stimulated synthesis of milk short and medium chain
fatty acids from acetate and BHBA.
Milk accumulation into the udder induced a decline in MBF and an increase in the arterial
concentrations of glucose during the last 12-h period. Such results were previously reported in dairy
cows milked once daily (Delamaire and Guinard-Flament, 2006a and b). The reduction in MBF leads
to depressed arterial availability of nutrients in response to the milk accumulation into the udder
except for glucose: its arterial availability is less affected due to its higher arterial concentration that
probably results from the depressed mammary use of glucose.
References
Delamaire, E. and J. Guinard-Flament, 2006a. Longer milking intervals alter mammary epithelial permeability and
the udder’s ability to extract nutrients. J. Dairy Sci. 89, 2007-2016.
Delamaire, E. and J. Guinard-Flament, 2006b. Increasing milking intervals decreases the mammary blood flow and
mammary uptake of nutrients in dairy cows. J. Dairy Sci. 89, 3439-3446.

Response to diet quality of two cattle genotypes at 200 and 3600 m
altitude in milk yield and composition and nitrogen and energy
utilization
K. Bartl1,2, C.A. Gomez 2, M. Kreuzer1, H.D. Hess3 and H.-R. Wettstein1
1ETH Zurich, Institute of Animal Sciences, Universitaetstr. 2/LFW, 8092, Zurich, Switzerland
2Universidad Nacional Agraria La Molina, Department of Nutrition, Lima, Peru
3Agroscope Liebefeld-Posieux (ALP), Rte de la Tioleyre 4, 1725, Posieux, Switzerland
Introduction
Milk production in the Andes, although very common, faces problems of both high altitude (low
oxygen partial pressure; Leiber et al., 2005) and extended dry season periods with scarcity of forage
with sufficient quality. In the Andes, dairy cows are kept on pastures at altitudes exceeding 4000 m
a.s.l. Our own unpublished results of a preliminary study carried out in the central Peruvian Andes
demonstrate that both milk yield and milk quality decreased during the dry season. About 80% of
annual precipitation occurs during October to March whereas, during the rest of the yr, lack of water
reduces growth and quality of non-irrigated forage resources. In particular, contents of crude protein
decrease significantly (-29%) from the rainy to the dry season (Flores et al., 2005). Another factor
affecting the productivity of dairy cattle in the highlands is the genotype of the cows. The dominant
cow type used by smallholders at high altitudes is the indigenous ‘Criollo’ cattle. Among the European
breeds, the use of the Brown Swiss is the most common. The aim of the present study was to test
whether there are major differences in the response of indigenous (Criollo) and Brown Swiss cattle
to nutrient supply at different altitudes. Jenet et al. (2006) demonstrated an almost complete lack of
response in milk yield of the indigenous Boran breed (Bos indicus), but indigenous Bos taurus might
have different strategies to cope with scarcity of high quality feed. The diets tested were designed
to cover the variation in feed quality caused by season.

Twelve Brown Swiss and 12 Criollo cows, adapted to high altitude, were assigned to three different
dietary treatments in a repeated 3 × 3 Latin square design (total n=72) at the Peruvian coast and in
the highlands at altitudes of 200 and 3600 m a.s.l., respectively. The experimental diets represented
the quality of highland dry-season forage (D; oat straw:maize stover; 0.54:0.46 on dry matter (DM)
basis) and highland rainy-season forage (R; oat hay:alfalfa hay:maize stover, 0.54:0.32:0.14), and
a diet optimised to meet the cows’ requirements (O; forage: alfalfa hay:maize stover, 0.81:0.19;
concentrate: maize:wheat bran, 0.58:0.42). Diets D, R and O contained, per kg DM, 41, 80 and
131 g of crude protein and 3.9, 5.0 and 4.9 MJ of net energy for lactation (NEL), respectively. The
cows had ad libitum access to roughages. Cows on diet O received concentrate in a fixed amount in
proportion to their metabolic body weight. Feed intake, milk yield and composition were recorded
daily. Blood plasma samples were analysed for various hormones and metabolites. Data of wk 3
of feeding the respective treatment diet were analysed using two different models and the MIXED
procedure of SAS. Model 1 included cow type (C), feeding (F), period (P) and altitude (A) as fixed
factors and all possible double and triple interactions between F, C and A. Model 2 included C, F
and P as fixed factors and the C × F interaction. Animal within genotype and site was defined as
subject for a random effect in both models.
Results
Intake of DM (kg/d) of Criollo and Brown Swiss cows with diets D, R and O was 4.80 and 6.30,
8.92 and 13.08, and 9.89 and 15.28, respectively. The intake of protein (kg/d) increased (P<0.05)

from diet D to O from 0.19 to 1.27 for Criollos and from 0.25 to 1.98 for Brown Swiss. Both cow
types increased energy-corrected milk yield (ECM, kg/head/d) by a factor of two when switching
from diet D to O (Brown Swiss from 4.28 to 8.88; Criollos from 2.46 to 4.77). The major part of this
increase was, however, already found when replacing diet D by R. At both sites, the relative increase
of ECM of Criollos (+103%) from diet D to R was higher than that of Brown Swiss (+74%; diet ×
genotype interaction, P<0.001). The milk fat content was higher (P<0.01) in Criollos than in Brown
Swiss and was higher (P<0.05) by 8% in the highlands than in the lowlands. In the highlands, milk
protein content was 4% smaller with diets R and O and 2.5% higher with diet D than in the lowlands
(overall diet × altitude interaction, P<0.01). With diet D, NEL intake was smaller than assumed NEL
requirements (both MJ/d) for both cow types and at both sites, while intake exceeded requirements
in all other cases. For the Criollos, NEL deficit with diet D was less pronounced (-12.1 and -8.8 in the
high- and lowlands, respectively) than for Brown Swiss (-18.7 and -13.4) and surpluses with the other
diets were smaller (diet × cow type interaction, P<0.001). This was consistent with correspondingly
lower (P<0.001) levels of plasma glucose, T3 and insulin and higher (P<0.01) levels of non-esterified
fatty acids with diet D than with the other diets. The apparent utilisation of dietary N for milk N
excretion was much higher (P<0.001) with the low-protein diet D (0.54 and 0.40 at high and low
altitude, respectively) than with diets R and O (0.19; F × A interaction, P<0.001).
In conclusion, the response of the two cow types to diet quality varied, depending on altitude.
Differences in milk yield between Brown Swiss and Criollos diminished with decreasing diet quality
at both sites. Diet responses were the smallest when switching from diet R to O. Both breeds heavily
relied on body reserves when fed dry-season feeds but energy shortage of Criollos with diet D was
smaller compared to Brown Swiss. This indicates that Criollo cows are better adapted to altitude and
diets low in energy and protein and that they have a higher capacity than Brown Swiss to overcome
temporary lack of protein and energy by mobilisation of body reserves. Nevertheless, they are able
to respond in milk yield to dietary improvements. The latter is clearly different from the reaction to
feed improvement by Bos indicus cattle (Jenet et al., 2006).
References
Flores, E.R., J. Cruz and J. Ñaupari, 2005. Utilización de praderas cultivadas en secano y praderas naturales para la
producción lechera. Boletín Técnico CICCA-FDA-INCAGRO, Lima, Peru.
Jenet, A., S. Fernandez-Rivera, A. Tegegne, H-R. Wettstein, M. Senn, M. Saurer, W. Langhans and M. Kreuzer, 2006.
Evidence for different nutrient partitioning in Boran (Bos indicus) and Boran × Holstein cows when re-allocated
from low to high or high to low feeding level. J. Vet. Med. A 53, 383-393.
Leiber, F., M. Kreuzer, B. Jörg, H. Leuenberger and H-R. Wettstein, 2004. Contribution of altitude and alpine origin
of forage to the influence of alpine sojourn of cows on intake, nitrogen conversion, metabolic stress and milk
synthesis. Anim. Sci. 78, 451-466.

Milk production as a function of energy and protein source
supplementation follows the saturation kinetics typical of enzyme
systems
R.P. Lana1,2, D.C. Abreu1, P.F.C. Castro1 and B. Zamperline1
1Departamento de Zootecnia-UFV, 36.571-000, Viçosa, MG, Brazil
2Sponsored by CNPq - Brasília, DF, Brazil
Introduction
Marginal response in milk production to increasing amounts of concentrate has been described
as curvilinear, i.e. the marginal increase in milk per kg of concentrate decreases as the amount of
concentrate increases. Substitution rate, or the reduction in pasture dry matter intake per kilogram
of concentrate, is a factor that could explain the variation in milk response to supplementation
(Bargo et al., 2003).
However, a more broad theory to explain this effect can be found in the work of Lana et al. (2005),
in which the curvilinear responses to nutrients (energy, protein, or minerals) observed in animals
and plants are similar to that described by Michaelis-Menten for enzymatic systems and by Monod
(1949) for microorganisms when substrate concentration is increased.
The objective of this study was to demonstrate the use of saturation kinetics models to explain milk
response to energy and protein source supplementation in grazing cows.

Three experiments were aimed at evaluating the effects of energy and protein supplementation on
milk production by cows at tropical pasture of Elephant-grass (Pennisetum purpureum, Schum),
which presents 6% CP and 50% TDN (or 2 Mcal ME/kg DM) during the dry season.
Twelve-crossbred Holstein-Zebu cows (520 kg) were allotted into three-4×4 Latin squares, in four
periods of seven d. The cows were milked twice daily, and the calf had access to the cow for a short
period of time in order to stimulate milk ejection. The milk was measured on the last d of each
period, in which the milk ingested by the calf was not recorded.
The treatments consisted of increased levels of wheat middlings-WM (0.5, 1.0, 2.0 and 4.0 kg/cow/d),
soybean meal-SM (0.0, 0.65, 1.3 and 2.6 kg/cow/d) and mixture of corn meal:soybean meal 57:43-
CSM (0.0, 1.5, 3.0 and 6.0 kg/cow/d). The concentrate supplements were fed twice a d, at milking
time, and mineral salt was offered free choice.
The experiments were analyzed as Latin square design including effects of treatments, animal and
period. Linear regressions of the reciprocal of milk production as a function of the reciprocal of
supplement intake were developed according to Lana et al. (2005).
The marginal increase in milk production was calculated, dividing the accretion in milk production
(Y2 - Y1) by the increase in supplement intake (X2 - X1), from a specified level of supplement to
the previous one.

There were treatment effects (P<0.05) on milk production for SM and CSM. The responses to
supplementation were curvilinear, even for WM, following a Michaelis-Menten relationship of
enzyme systems, and being explained by the following equations of Lineweaver-Burk:
1/Milk = 0.0061 × (1/WM) + 0.0917; r2=0.86; SE=0.001123; n=4 (1)

1/Milk = 0.0226 × (1/SM) + 0.0825; r2=0.95; SE=0.001928; n=4 (2)
1/Milk = 0.0394 × (1/CSM) + 0.0866; r2=0.97; SE=0.001374; n=4 (3)
The observed marginal increase in milk production was reduced with increasing supplementation
(1.79, 0.28 and -0.11 kg of milk/kg of WM; 1.57, 1.67 and 1.19 kg of milk/kg of SM; and 0.94, 0.60
and 0.36 kg of milk/kg of CSM). The decrease in marginal efficiency was more pronounced on WM,
followed by CSM and SM. This result can be explained by the energy/CP ratio of the supplements,
in which the TDN/CP ratios were 4.0, 3.4 and 1.9, respectively. Probably the limiting nutrient in
the low quality tropical grass pasture was protein and not energy and then, the animals were more
responsive to protein supply.
The estimated marginal increase in milk production, determined using the Equations 1, 2 and 3,
reduced with increasing supplementation, and the calculated amounts of supplements to reach
marginal cost-benefit zero were 0.9, 1.25 and 1.7 kg for WM, SM and CSM, respectively.
The NRC (2001) and the Cornell System - version 5.0 (Fox et al., 2003) consider linear response of
2.2 to 2.6 kg of milk/kg of concentrate for both energy (net and metabolizable energy, respectively)
and protein (metabolizable protein) supplementation. The observed efficiency values were much
smaller than the NRC and CNCPS model predictions, and these differences cannot be explained by
the unaccounted calf milk ingestion, considering low difference in milk production and then similar
milk ingestion among concentrate levels. Then, models of saturation kinetics are more appropriate
to explain the animal responses to nutrients and to make nutrient recommendations, based on the
cost-benefit ratio of supplementation.
Conclusion
The milk response to energy and protein source supplementation in grazing cows is curvilinear and
follows a Michaelis-Menten relationship of enzyme systems.
References
Bargo, F., L.D. Muller, E.S. Kolver and J.E. Delahoy, 2003. Invited review: Production and digestion of supplemented
dairy cows on pasture. J. Dairy Sci. 86, 1-42.
Fox, D.G., T.P. Tylutki, L.O. Tedeschi, M.E. Van Amburgh, L.E. Chase, A.N. Pell, T.R. Overton and J.B. Russell, 2003.
The net carbohydrate and protein system for evaluating herd nutrition and nutrient excretion, CNCPS version 5.0.
Department of Animal Science, Cornell University, Ithaca, NY, USA.
Lana, R.P., R.H.T.B. Goes, L.M. Moreira, A.B. Mâncio, D.M. Fonseca and L.O. Tedeschi, 2005. Application of
Lineweaver-Burk data transformation to explain animal and plant performance as a function of nutrient supply.
Livest. Prod. Sci. 98, 219-224.
Monod, J., 1949. The growth of bacterial cultures. Ann. Rev. Microbiol. 3, 371-394.
NRC, 2001. Nutrient Requirements of Dairy Cattle. 7th edition. Natl. Acad. Press, Washington, DC, USA.

2C. Regulation of muscle protein deposition and loss

Development of low grade inflammation during aging impaired
postprandial muscle protein synthesis in rat skeletal muscle
I. Rieu, G. Mayot, C. Sornet, E. Pujos, M. Balage, I. Papet and D. Dardevet
INRA, Theix, UMR 1019 Unité de Nutrition Humaine, 63122 St-Genès-Champanelle, France
Introduction
During aging, a decline in skeletal muscle mass occurs in both humans and rodents. This atrophy is
associated with a loss of muscle strength, which directly affects the mobility and health of elderly
people. The mechanisms leading to sarcopenia are still unclear but result from an imbalance between
rates of protein synthesis and degradation. This imbalance is not obvious when basal rates of protein
turnover are measured, (Mosoni et al., 1993; Dardevet et al., 1994; Volpi et al., 2001) but is detected
in the postprandial state. An apparent defect in the stimulation of muscle protein synthesis has been
shown in old rats (Mosoni et al., 1995) and elderly humans (Arnal et al., 1999) after the ingestion
of a normal protein meal. Studies agreed to demonstrate that aged muscle is less sensitive to the
stimulatory effects of amino acids at physiologic concentrations (Volpi et al., 1999; Arnal et al.,
1999; Dardevet et al., 2000 and 2002; Paddon-Jones et al., 2004). Aging is also characterised by
the development of a low grade inflammation with an increase of cytokines and acute phase protein
levels (fibrinogen, C-reactive protein and α2-macroglobulin (α2-m); Papet et al.; 2003; Visser et al.,
2002). We hypothesised that the resistance of muscle protein synthesis to amino acids during aging
may be a consequence of the development of this low grade inflammatory status.

Old rats (18-20 mo-old) were studied for 4 mo. They had free access to water and a standard pellet
chow. Animals were weighted each wk and food intake was recorded as well. Once a mo, a blood
sample was withdrawn to assess plasma fibrinogen and α2-m levels. Animals were then separated
into two groups. A group of rats showing no increase of both fibrinogen and α2-m levels (Control
(C)) and a group in which fibrinogen and α2-m levels increased over the 4 mo period of time (Low
Grade Inflammation (LGI). Rats showing excessive α2-m (>600 mg/L) and fibrinogen levels (>6
g/L) were considered to present a pathological inflammation and not included to any of the groups.
In each group, rats were studied at the post absorptive or after food intake (90-120 min after the last
meal intake (postprandial state)). Muscle protein synthesis was assessed for 45 min with the flooding
dose method using L-[1-13C]-Phe as a tracer. Protein synthesis (% per d) was calculated as follows:
(Eb×100/Ea)×1440/t where Eb, Ea are the enrichment at time t of the protein-bound Phe and the
enrichment of the Phe precursor pool between time 0 and time t. The Ea of the tissue fluid will be
used as the Ea of the precursor pool. Values presented are means ± SEM. Statistical evaluation of
the data was performed by 2-way ANOVA to analyse the inflammation status and nutritional effects.
When a significant overall effect was detected, differences among individual means were assessed
with fisher test to determine significant differences.
Results and conclusions

Over the 4 mo period of time, LGI rats lost significantly more body weight than the C rats (79 ±
10 vs. 128 ± 12 g) despite a similar food intake between the two groups. Fibrinogen levels did not
increase significantly in both groups. α2-m increased slightly but not significantly in C rats (58 ± 12
to 87 ± 13 mg/L) whereas it increased by 4.9 times in LGI rats over the 4 mo period (143 ± 26 to 508
± 106 mg/L). Moreover, albumin levels were significantly decreased in the LGI group compared to
the C group by 23%. Muscle protein synthesis was unchanged at the post absorptive state between
the 2 groups (4.06 ± 0.16%/d vs. 4.17 ± 0.2%/d in C and LGI rats, respectively) (Figure 1). Food

Figure1. Effect of food intake on muscle protein synthesis in control and LGI rats.
intake increased significantly muscle protein synthesis by 20% in C rats whereas it had no effect
in LGI rats (Figure 1). This difference is not due to a difference of the last meal intake since both
groups ate 10.14 ± 0.69 and 9.30 ± 0.86 g for C and LGI rats respectively.
In conclusion, the development of low-grade inflammation during aging impaired muscle protein
synthesis stimulation by food intake and may be responsible for the enlargement of muscle loss during
aging. Future experiments will assess the effect of anti-inflammatory drugs (NSAID) or nutrients
(vitamin E, polyphenols as rutin) during aging on the response of muscle protein metabolism to
food intake.
References
Arnal, M.-A., et al., 1999. Protein pulse feeding improves protein retention in elderly women. Am. J. Clin. Nutr. 69,
1202-1208.
Dardevet, D., C. Sornet, D. Taillandier, I. Savary, D. Attaix and J. Grizard, 1995.Sensitivity and protein turnover
response to glucocorticoids are different in skeletal muscle from adult and old rats. Lack of regulation of the
ubiquitin-proteasome proteolytic pathway in aging. J. Clin. Invest. 96, 2113-2119.
Dardevet, D., et al., 2000. Stimulation of in vitro rat muscle protein synthesis by leucine decreases with age. J. Nutr.
130, 2630-2635.
Dardevet, D., et al., 2002. Postprandial stimulation of muscle protein synthesis in old rats can be restored by a leucine-
supplemented meal. J. Nutr. 132, 95-100.
Mosoni L., M.C. Valluy, B. Serrurier, J. Prugnaud, C. Obled, C.Y. Guezennec and P.P. Mirand, 1995. Altered response
of protein synthesis to nutritional state and endurance training in old rats. Am. J. Physiol. 268, E328-E335.
Mosoni, L., P.P. Mirand, M.L. Houlier and M. Arnal, 1993. Age-related changes in protein synthesis measured in vivo
in rat liver and gastrocnemius muscle. Mech. Ageing Dev. 68, 209-220.
Paddon-Jones, D., et al., 2004. Amino acid ingestion improves muscle protein synthesis in the young and elderly. Am.
J. Physiol. 286, E321-E328.
Papet, I., et al., 2003. Acute phase protein levels and thymus, spleen and plasma protein synthesis rates differ in adult
and old rats. J. Nutr. 133, 215-219.
Visser, M., et al., 2002. Relationship of interleukin-6 and tumor necrosis factor-alpha with muscle mass and muscle
strength in elderly men and women: The health ABC study. J. Gerontol. A Biol. Sci. Med. Sci. 57, M326-M332.
Volpi, E., et al., 2001. Basal muscle amino acid kinetics and protein synthesis in healthy young and older men. JAMA
286, 1206-1212.
Volpi, E., et al., 1999. Oral amino acids stimulate muscle protein anabolism in the elderly despite higher first-pass
splanchnic extraction. Am. J. Physiol. 277, E513-E520.

Effect of immune system stimulation and dietary methionine plus
cysteine intake on protein deposition and digestibility in growing pigs
A. Rakhshandeh1, M. Rademacher2 and C.F.M. de Lange1
1Department of Animal and Poultry Science, University of Guelph, Guelph, ON, Canada; 2Degussa
AG, Hanau, Germany
Introduction
Immune system stimulation (ISS) can result in substantial reductions in animal productivity. In
growing animals, reductions in whole body protein deposition (PD) that are induced by chronic ISS can
be attributed largely to reductions in protein gain in muscle, as a result of increased protein degradation
and decreased protein synthesis in this tissue (Breuille et al., 1997). Amino acids (AA) released from
muscle are redirected towards organs involved in the animal’s immune response. Previous reports
have suggested additional requirements for the sulphur containing AA (SAA), methionine or cysteine,
during ISS for synthesis of metabolites that have important roles in the animal’s immune response, such
as glutathione, homocysteine, taurine, polyamines, choline and carnitine (Malmezat et al., 2000; Yu et
al., 1993). An increased need for SAA may be linked directly to increased muscle protein degradation
during ISS. Also, ISS can cause morphological and physiological changes in the gastrointestinal
tract such as edema, change in gut motility, permeability, and microflora (Yamada, 1995). However,
the impact of ISS on protein and AA digestibility has not been evaluated. Our objectives were to
determine the impact of ISS on PD, protein and AA digestibility, and to explore whether additional
dietary SAA supply can reduce the negative impact of ISS on PD in growing pigs.

Twenty-four Yorkshire barrows (23 ± 0.3 kg body weight) were fed restricted (850 g/d), and exposed
to either low (3.6 g/d) or medium (4.7 g/d) dietary intakes of SAA. Half of dietary SAA was supplied
from methionine and diets were formulated to be first limiting in SAA. Pigs were acclimatised to the
diet and experimental environment for 9 d. Following a 5-d N-balance period (pre-challenge), half
of the pigs at each dietary SAA levels were injected intramuscularly with either saline or increasing
amounts of Escherichia coli lipopolysaccharide (LPS, serotype 55:B5) every 48 h and for at least
5 d. A second 3-d N-balance (during ISS) was conducted after the start of LPS or saline injections.
Titanium dioxide was included in the diet (0.1%) and used as indigestible marker for measuring
apparent ileal and fecal nutrient digestibility. At the end of the second N-balance period, pigs were
weighed and euthanised. The abdomen was opened, weight of the liver determined, and ileal digesta
was collected for measuring apparent digestibility. The repeated measurements procedure (proc.
Mixed in SAS), was used for statistical data analysis.

Repeated injection with LPS increased liver weight (P<0.01; Table 1) and body temperature (40.2
versus 39.8 °C; SE 0.07; P<0.05), confirming that the pigs’ immune system was stimulated (Breuille
et al., 1997). Apparent ileal and fecal digestibility of crude protein (CP) were not affected by ISS
(P>0.10; Table 1). Values for fecal digestibility were higher than those for ileal digestibility of CP,
reflecting protein fermentation in the pigs’ hindgut (Moughan and Donkoh, 1991). Pre-challenge
PD (P<0.01), but not PD during ISS (P>0.10), was increased with increasing intake of SAA.
Administration of LPS reduced PD at both SAA intake levels (P<0.01). No interactive effects between
ISS and SAA intake on PD were found (P>0.10). However, the reduction in PD due to ISS was
smaller at the medium SAA intake level than at the low SAA intake level (16.4 ± 1.9 versus 10.1 ±
1.6 g/d; P<0.03), indicating that additional SAA intake reduces the negative effects of ISS on PD.

Table 1. Impact of immune system stimulation (ISS) and intake of methionine plus cysteine (SAA)
on apparent digestibility of crude protein (CP) and body protein deposition (PD).
SAA intake Low (3.6 g/d) Medium (4.7 g/d) SEM P values
ISS - + - + ISS SAA ISS × SAA
Liver weight, % of BW
During ISS1 2.3 2.7 2.4 2.7 0.05 0.01 0.67 0.82
Fecal CP digestibility, %
Pre ISS1 88.0 87.5 0.43 NA2 0.3 NA
During ISS1 88.6 87.8 88.8 88.7 0.72 0.5 0.5 0.62
Ileal CP digestibility, %
During ISS 78.7 78.0 74.0 69.4 3.5 0.44 0.02 0.60
PD2, g/d
Pre ISS 83.2 95.9 1.75 NA 0.003 NA
During ISS 89.4 73.0 95.2 85.1 1.37 0.001 0.27 0.50
1Before(Pre ISS) or during ISS (during ISS) with a repeated and increasing dose of LPS.
2Notapplicable.
3Body protein deposition derived from N-balance data (N retention × 6.25).
In conclusion, immune system stimulation per se does not affect apparent ileal and fecal digestibility
of crude protein in growing pigs. This study has provided evidence that additional dietary intake of
methionine plus cysteine can reduce the adverse effects of immune system stimulation on whole body
protein deposition in growing pigs, and probably accelerates the recovery. In our laboratory further
studies are in progress to more closely assess interactive effects of immune system stimulation and
multiple methionine plus cysteine intake levels on whole body protein deposition in growing pigs.
The underlying mechanisms need to be explored further as well.
References
Breuille, D., M. Arnal, F. Rambourdin, G. Bayle and D. Levieux, 1997. Sustained modification of protein metabolism
in various tissue in a rat model of long lasting sepsis. Clin. Sci. 94, 413-423.
Malmezat, T., D. Breuille, P. Captain, P. Minard and C. Obled, 2000. Glutathione turnover is increased during acute
phase of sepsis in rats. J. Nutr. 130, 1239- 1246.
Moughan, P. J. and A. Donkoh, 1991. Amino acid digestibility in non-ruminants: a review. Recent Adv. Anim. Nutr.
Australia, 172–184.
Yamada, T., 1995. Textbook of gastroenterology. Second edition. Lippincott, Philadelphia.
Yu, Y., J.F. Bruke and V.R. Young, 1993. A kinetic study of L-2H3-methyl-1-13C-methionine in patients with severe
burn injury. J. Trauma. 35, 1-7.

The ubiquitin and caspase systems are sequentially regulated in the rat
gastrocnemius muscle during casting immobilisation and recovery
E. Vazeille, A. Claustre, A. Codran, S. Ventadour, D. Taillandier, D. Béchet, D. Attaix, D. Dardevet
and L. Combaret
Human Nutrition Research Center, Human Nutrition Unit, National Institute for Agricultural
Research, 63122 Ceyrat, France
Introduction
Muscle wasting in disuse conditions results from an imbalance between protein synthesis and
degradation, between apoptosis and differentiation/regeneration processes, but also from a slow-to-
fast fibre type transition. Altogether these adaptations result in muscle remodelling. Immobilisation
produces remarkable morphological, physiological, and biochemical alterations in skeletal muscle
fibres leading to their degeneration and atrophy. Uncontrolled and sustained muscle wasting impairs
human movement, leading to difficulties in performing daily activities and to increased morbidity.
Therefore a better understanding of the mechanisms underlying muscle protein wasting and recovery
following immobilisation is required to develop new strategies for limiting muscle wasting and
improving subsequent recovery.
Muscle wasting during immobilisation results in part from the activation of ubiquitin-proteasome-
dependent proteolysis (Gomes et al., 2001). Immobilisation is also characterised by the activation
of apoptosis (Degens and Always, 2006). However, the potential link between the activation
of proteolysis and pro-apoptotic processes remains to be elucidated in immobilisation-induced
muscle wasting. This work was designed to study the regulation of ubiquitin-proteasome-dependent
proteolysis and apoptosis during cast-induced muscle atrophy and regeneration in the rat.

Adult rats were subjected to unilateral hindlimb immobilisation to induce maximal atrophy of
the gastrocnemius muscle, the controlateral non-casted leg being the control. Immobilisation was
imposed for 4 (I4), 6 (I6) or 8 (I8) d. In gastrocnemius muscle regeneration studies, casts were
removed after 8 d of immobilisation and animals were allowed to recover for 10 (R10), 15 (R15),
20 (R20), and 30 (R30) d.
Caspase-3 and -9 activities were measured using fluorogenic substrates in cytosolic muscle protein
extracts.
Ubiquitin-dependent proteolysis was assessed by measuring the accumulation of ubiquitin-protein

conjugates in the myofibrillar protein fraction by Western blotting using the FK1 antibody, which
recognises polyubiquitin chains. Rates of ubiquitination were determined by incubating soluble
proteins with biotinylated-ubiquitin at 37 °C and with the deubiquitinating enzyme inhibitor,
ubiquitin aldehyde.
Statistical analyses were performed using the paired Student T test (casted leg vs. controlateral non-
casted leg). The significance level was defined at 5%.

Results
Immobilisation period
Animals reduced their food intake by ~30% during immobilisation as soon as I4, so that their body
weight decreased by 12% at I8. The gastrocnemius muscle atrophied progressively from I4 (-8%,
P<0.05) to I8 (-23%, P<0.0001).
The caspase-3 and caspase-9 activities increased by 75% and 45% respectively in immobilised
gastrocnemius muscles at I8 (P<0.05), without any modification at early time points.
By contrast, ubiquitin protein conjugates increased in the gastrocnemius from I4 (+40%, P<0.05)
and remained elevated until the end of the immobilisation period (+64%, P<0.05). Similarly, rates
of ubiquitination increased at I6 (+56%, P<0.05) and I8 (+50%, P<0.05).
Recovery period
The immobilised gastrocnemius was still atrophied by 15% (P<0.05) at R30. The caspase-3 activity
remained elevated at R10 (+35%, P<0.005), was down-regulated below basal levels at R15 (-20%,
P<0.05), and normalised at R20. The increased caspase-9 activity was suppressed at R10, not
significantly down-regulated below basal levels (-20%, P=0.11) at R15, and normalised at R20.
The increased rate of ubiquitination and the accumulation of polyubiquitin conjugates were suppressed
at R10, down-regulated below basal levels by ~15% at R15 (P<0.05), and normalised at R20.
Conclusions
Our data showed that both ubiquitin- and caspase-dependent proteolytic systems are up-regulated
during immobilisation. Interestingly, a sequential activation and normalisation of these two systems
seems to prevail. Altogether our data suggest a two-stage process in which ubiquitin-dependent
proteolysis is rapidly up- and down- regulated when muscle atrophies and recovers, respectively,
whereas apoptotic processes may be involved in the late stages of atrophy and recovery.
However, the caspase-dependent cascade and the degradation of polyubiquitin conjugates by the
proteasome both involved several proteases, enzymes and cofactors (Attaix et al., 2005). Thus, further
investigations are clearly required to determine the physiological significance of this sequential
regulation during muscle wasting and recovery.
References
Attaix, D., S. Ventadour, A. Codran, D. Bechet, D. Taillandier and L. Combaret, 2005. The ubiquitin-proteasome system
and skeletal muscle wasting. Essays Biochem. 41, 173-186.
Degens, H. and S.E. Always, 2006. Control of muscle size during disuse, disease, and aging. Int. J. Sports Med. 27,
94-99.
Gomes, M.D., S.H. Lecker, R.T Jagoe, A. Navon and AL. Goldberg, 2001. Atrogin-1, a muscle-specific F-box protein
highly expressed during muscle atrophy. Proc. Natl. Acad. Sci. USA 98, 14440-14445.

Relation between protein degradation and oxidative stress during aging
in rat muscle
L. Mosoni, L. Combaret, C. Sornet and D. Dardevet
INRA, NSP-UNH, Centre de Clermont-Ferrand – Theix, 63122 Theix, France
Introduction
Muscle loss observed during aging can limit the autonomy of elderly individuals and also limit their
capacity to recover from stress. It is important to understand the mechanisms involved in order to
develop strategies to limit this process.
Oxidative stress could be involved in the loss of muscle proteins during aging. Oxidation of key
metabolic enzymes and alterations of mitochondria function could lead to apoptosis or necrosis and
to a loss of muscle fibers. Oxidation of long-lived proteins like myofibrillar proteins could accelerate
their proteolysis and decrease muscle protein mass.
Although several studies have analysed age-related changes in oxidation level of soluble muscle
proteins, detecting most of the time an increase with age, few studies have been interested in the
oxidation level of myofibrillar proteins. We showed that the carbonyl content of a non collagenic
fraction of muscle proteins (soluble + myofibrillar proteins) was remarkably stable during aging in
rat skeletal muscle: it did not increase with age, and even decreased in the soleus muscle in the oldest
rats (Mosoni et al., 2004). These results suggested that degradation of oxidised proteins is rapid and
efficient, which was unexpected in a tissue which has a relatively slow turnover. To confirm these
results, our aim was to inhibit muscle protein degradation in the absence of oxidative stress and to
analyse the consequences on myofibrillar protein carbonyl content.

We used male Wistar rats aged 12 or 24 mo (n=10 per age). Left and right epitrochlearis muscles
were excised under anaesthesia and incubated 2 h in conditions currently used in our laboratory to
measure in vitro muscle protein synthesis and degradation rates (37 °C, Krebs-Henselheit buffer).
Protein synthesis was inhibited by 0.5 mM cycloheximide. In addition, in right muscles, proteolysis
was inhibited by addition of insulin (0.1 u/mL) and branched chain amino acids (0.2 mM valine,
0.17 mM leucine, 0.1 mM isoleucine), protease inhibitors (10 mM methylamine, 50 µM leupeptine,
40 µM MG132) and by excluding CaCl2 from incubation buffer. At the end of the incubation,
epitrochlearis muscles were weighed and frozen in liquid nitrogen. Tyrosine released during
incubation was measured by fluorimetry in incubation buffer. Myofibrillar proteins were purified
and their protein carbonyl content was measured using DNPH according to Fagan et al. (1999).
Statistical differences were analysed by paired Student t test and by two-way variance analysis (age
and inhibition effects).
Results
We showed that there was no difference in myofibrillar carbonyl content in a 2 h incubated muscle
and in a non incubated muscle. In addition, antioxidant capacities of control buffer and inhibiting
buffer were not different.
At both ages, proteolysis was inhibited in supplemented buffer and the difference was highly
significant (Figure 1A, left panel). Variance analysis also detected a significant increase in proteolysis
(Figure 1B, right panel), which increased significantly when proteolysis was inhibited. The effect

Figure 1. Proteolysis rates (A) and myofibrillar carbonyl content (B) in epitrochlearis muscles from
12 and 24 mo-old rats incubated in control buffer or inhibiting buffer.
tended to be more marked in old rats (significant difference between control and inhibited conditions
only in old rats) but the interaction between age and inhibition effects was not significant (P=0.15).
Carbonyl content was not different between 12 and 24 mo-old rats.
Conclusion
Although carbonyl content is not increased with age in basal conditions, as soon as protein degradation
is inhibited, even in the absence of oxidative stress, carbonyl content increases, and it tends to increase
more in old rats. Thus, protein degradation seems to be the major determinant of protein carbonyl
content in skeletal muscle of rats. In healthy rats, proteolysis is likely to prevent any accumulation
of oxidised myofibrillar proteins.
References
Fagan, J.M., B.G. Sleczka and I. Sohar, 1999. Quantitation of oxidative damage to tissue proteins. Int. J. Biochem.
Cell. Biol. 31, 751-757.
Mosoni L., D. Breuille, C. Buffiere, C. Obled and P. Patureau Mirand, 2004. Age-related changes in glutathione
availability and skeletal muscle carbonyl content in healthy rats. Exp. Gerontol. 2004 39, 203-210.

Changes in the expression of selected proteins elucidate skeletal muscle
type-specific resistance to glucocorticoid-induced muscle cachexia
P. Pawlikowska1, M. Lokociejewska1, J.F. Hocquette2 and A. Orzechowski1
1Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw Agricultural
University, 02-776 Warsaw, Poland
2INRA, Unité de Recherches sur les Herbivores, Theix, 63122 Saint-Genès Champanelle, France
Introduction
Metabolic type of muscle fibres (oxidative vs. glycolytic) establishes considerable differences in
skeletal muscle susceptibility to glucocorticoid-dependent (GD) muscle cachexia (MC). Excess of
glucocorticoids evokes secondary diabetes mellitus (DM) associated with marked body weight loss.
Thus, upon glucocorticoid overdose (DEX-treated rats received 1 mL of dexamethasone disodium
phosphate (Sigma, St. Louis, MO, USA) dissolved in saline (0.85% w/v NaCl) by intragastric
tube in a daily dose of 2 mg/kg BW/d during 5 consecutive d. oral treatment with 2 mg/kg BW/d)
developing hyperglycemia is accompanied by oxidative stress (OS). It is believed that antioxidants
might correct the consequences of OS. Furthermore, upon glucocorticoid-induced diabetic state the
somatic index [(organ wet weight×100/body weight), SI in percentage] of the gastrocnemius muscle
(glycolytic-oxidative) is significantly reduced, whereas that of the soleus muscle (oxidative) is either
at least unchanged or elevated. The aim of this study was to find out whether any remarkable changes
in the expression of proteins that are crucial for insulin signalling and energy formation occur in the
gastrocnemius (GM) vs. soleus muscle (SM) isolated from rats treated with dexamethasone (DEX),
sodium ascorbate (ASC) or ascorbic acid phosphate (ASC-P) alone or combined together.

Wistar male rats at the age of six wk were subjected to an experiment as described in detail by
Lokociejewska et al. (2006). Western-blot analyses were performed as previously described.
After transfer, membranes were immunoblotted with Akt (protein kinase B), phosphoSer473-Akt,
NCOIV subunit of cytochrome-c oxidase antiserum. Next, they were stripped and re-probed with
β-actin to show that equal amounts of protein were loaded in each lane. Anaerobic metabolism
was characterised by phosphofructokinase (PFK) and lactate dehydrogenase (LDH) activities. The
aerobic metabolism was studied by measurements of isocitrate dehydrogenase (ICDH) activity.
The results were statistically evaluated using one way ANOVA and the Tukey multiple comparison
test by GraphPad PrismTM version 4.0 software and are expressed as mean ± SEM and a value of
P<0.05 was determined to be significant, P<0.01 as highly significant and P<0.001 as very highly
significant.

In contrast to the glycolytic-oxidative muscle (GM), the somatic index of SM increased in
dexamethasone-treated (DEX) growing rats (Łokociejewska et al., 2006). In turn, the somatic
index of GM increased by co-treatment with sodium ascorbate (ASC) or ascorbic acid phosphate
(ASC-P) indicating that antioxidants might directly counteract catabolic action of glucocorticoids or
that they indirectly improve GM sensitivity to insulin. No changes were observed in the expression
of Akt, phospho-Ser473-Akt, subunit IV of mitochondrial cytochrome-c oxidase in soleus muscle.
The activity of LDH was not affected, either. Interestingly, PFK activity in SM was significantly
elevated in experimentally treated animals (P<0.001) and the highest was observed after ASC-P.
In turn, ICDH activity peaked in SM after dexamethasone treatment (P<0.05) and dropped during
co-treatment with ascorbate (DEX/ASC) (Figure 1). In contrast, the average expression of NCOIV

ICDH activity LDH activity PFK activity
120 3500 250
3000
c a
umol /min/g tissue

200
umol/min/g tissue
umol/min/g tissue
100
ac a ac 2500
2000 150
80
b 1500 100
1000 b ab b B
60 ab a
50
500
40 0 0
C
-P
-P
RL
SC
-P
-P
RL
SC
-P
-P
RL
X
C
DE
DE
DE
AS
AS
AS
AS
SC
SC
SC
C
C
CT
CT
CT
A
A
AS
AS
AS
X/
X/
X/
A
A
DE
DE
DE
X/
X/
X/
DE
DE
DE
Figure 1. Box and whisker charts illustrating differences in the activity of enzymes of aero- and
anaerobic metabolism in SM. Boxes represent average values ± SD/SEM obtained from post mortem
measurements after treatment. Bars marked with different letters differ at least highly significantly
(capital letter, P<0.01) or significantly (lower case letters, P<0.05).
was significantly higher in GM than in SM, although it decreased considerably after DEX treatment
or co-treatment. DEX was also shown to significantly reduce the expression of phospho-Ser473-Akt
in GM. The results of immunoblotting indicate that dexamethasone does not affect insulin sensitivity
in SM. However, the rise in the activity of the step-limiting enzyme of carbohydrate catabolism
(PFK) suggests that SM started to utilise excess glucose during DEX-induced hyperglycemia. A
glucose tolerance test revealed that both ASC and ASC-P significantly increased sensitivity to insulin.
In contrast to SM, however, the activity of PFK in GM was significantly lower after treatment or
co-treatment with antioxidants as compared to the average control value from GM. Taken together,
these results pointed to the metabolic profile of the skeletal muscle that determines the resistance to
glucocorticoid-dependent muscle cachexia. It also indicates, that some of the disturbances related
to secondary diabetes could be partially corrected by antioxidants.
References
Łokociejewska, M., J. Wagner, J. Zarzyńska, M. Jank, P. Ostaszewski, A. Burdzińska, T. Sadkowski, J. Olczak, A.
Mrówczyńska and A. Orzechowski, 2006. Sodium ascorbate (ASC) and ascorbic acid phosphate (ASC-P) differently
modulate glucocorticoid-dependent metabolic effects in growing rats. Arch. Tierz. (Dummerstorf) 49, 41-51.

Amino acid signalling: methionine regulates the S6K1 pathway and
protein synthesis in avian QM7 myoblasts
S. Métayer Coustard1, S. Crochet1, E. Audouin1, M. Derouet1, Y. Mercier2, P.A. Geraert2 and S.
Tesseraud1
1UR83 Recherches Avicoles, INRA Tours, 37380 Nouzilly, France
2Adisseo France SAS, 42 Avenue Aristide Briand, 92160 Antony, France
Introduction
Amino acids have recently been shown to act as regulators of metabolic pathways. Amino acids
regulate protein synthesis and breakdown (i.e. protein turnover) and consequently protein deposition,
which corresponds to the balance between the two processes. Elucidating the mechanisms involved
in such regulation is important from fundamental and applied points of view, since it can provide
a basis to optimize amino acid requirements and controlling protein mass, body composition, etc.
Protein synthesis occurs when all amino acids are available. It is affected when the level of an
essential amino acid, i.e. one not synthesised in the body, is insufficient. However, amino acids, that
have long been considered simply as precursors of protein synthesis, are now recognised to exert
other significant influences, i.e. they are precursors of essential molecules, act as mediators or signal
molecules and affect numerous functions. For example, amino acids, particularly the branched-chain
amino acid leucine, control mRNA translation by affecting intracellular protein kinases involved in
such regulation. Although studies are sparse for methionine, this sulphur amino acid may also exert
a ‘signal’ function through the activation of the p70 S6 kinase (S6K1), a key enzyme controlling
protein synthesis (Shigemitsu et al., 1999; Tesseraud et al., 2003).
The aim of the present study was therefore to explore the signalling cascade involved in the action
of sulphur amino acids (i.e. methionine and its analogues and/or metabolites) and the consequences
on the regulation of protein synthesis in quail muscle (QM7) myoblasts.

QM7, a stable myogenic cell line (Antin and Ordahl, 1991), was used in this study. QM7 myoblast
cells were grown in McCoy medium supplemented with 10% fetal calf serum and 1% chicken
serum to 90-100% confluence, fasted 16 h in serum-free medium, washed once with complete
RPMI medium or RPMI medium without methionine, and incubated in the same medium for 2 h.
The cells were then incubated in a fresh RPMI medium added with different isomers of methionine
or its analogues (0.10 mM).
At the end of incubation, QM7 myoblasts were prepared as described by Tesseraud et al. (2003).
QM7 lysates (40 µg of protein) were subjected to SDS-PAGE gel electrophoresis and western blotting
using the appropriate phospho-specific antibody. After washing, membranes were incubated with
an Alexa Fluor secondary antibody (Molecular Probes, Interchim, Montluçon, France). Bands were
visualised by Infrared Fluorescence by the Odyssey® Imaging System and quantified by Odyssey
infrared imaging system software.
Protein synthesis was measured by incorporation of L-[U-14C]phenylalanine (0.25 µCi/mL) into

proteins. Protein synthesis was expressed as nanomoles of phenylalanine incorporated per mg
protein/h.

We first showed that the availability of either methionine or leucine regulated the S6K1 pathway
(data not shown), indicating that both methionine and leucine exert a signal effect. Methionine
deprivation (0) caused a decrease in p70S6K phosphorylation on Thr389 and Thr421/Ser424
compared to control cells (control) (Figure 1A). The supply of the lacking methionine as L- and
DL-methionine isomers (L-Met and DL-Met) restored S6K1 phosphorylation to the levels observed
in control cells. Methionine also activated S6K1 downstream targets, the ribosomal protein S6 and
the eukaryotic elongation factor 2 (eEF2), and increased protein synthesis (Figure 1B). The supply
of the lacking methionine as D-methionine (D-Met) or its analogue (DL-HMTBA) has no effect on
S6K1 activation and protein synthesis. The activation of the S6K1 pathway was also induced by a
methionine precursor, the keto analogue of methionine (2-keto-4-methylthiobutyric acid) but not
by cysteine (data not shown). The methionine-related activation of the S6K1 pathway was obtained
from very low concentrations of the amino acid since only 5 µM of methionine was sufficient to
phosphorylate S6K1.
Figure 1. Effects of methionine (Met) on the S6K1 pathway and protein synthesis in QM7 myoblasts.
After a 2h-preincubation in a Met-free medium, the cells were incubated in media without Met
(0), or added with Met (L-, DL- or D-Met isomers) or its analogue (DL-HMTBA); the cells were
alternatively maintained in a complete medium as the control. (A) Phosphorylation of several kinases
involved in the S6K1 pathway; Representative Western blots. (B) Protein synthesis measured by
phenylalanine incorporation.
In conclusion, our results suggest that methionine as leucine can act as a ‘nutrient signal’ to stimulate
protein synthesis in QM7 cells. Further studies more specifically focussed on sulphur amino acids
are likely to have new nutritional applications in the future.
References
Antin P.B. and C.P. Ordahl, 1991. Isolation and characterization of an avian myogenic cell line. Dev. Biol. 143, 111-
121.
Shigemitsu K, Y. Tsujishita, H. Miyake, S. Hidayat, N. Tanaka, K. Hara and K. Yonezawa, 1999. Structural requirement
of leucine for activation of p70 S6 kinase. FEBS Lett. 447, 303-306.
Tesseraud S., K. Bigot and M. Taouis, 2003. Amino acid availability regulates S6K1 and protein synthesis in avian
insulin-insensitive QM7 myoblasts. FEBS Lett. 540, 176-180.

AMPK regulates the S6K1 pathway and protein synthesis in avian QM7
myoblasts
S. Métayer Coustard1, A. Collin1, E. Audouin1, S. Crochet1, J. Dupont2 and S. Tesseraud1
2Unité de Physiologie de la Reproduction et des Comportements, INRA Tours, 37380 Nouzilly,
France
Introduction
The AMP-activated protein kinase (AMPK) is an evolutionarily conserved sensor of cellular energy
status and a major cellular regulator of lipid and glucose metabolism (Corton et al., 1994; Hardie
and Carling, 1997).
AMPK corresponds to a heterotrimetric complex comprising a catalytic alpha subunit and two
regulatory beta and gamma subunits; there are several isoforms for each subunit (α1, α2, β1, β2, γ1,
γ2, γ3). Binding of AMP to the γ subunit of AMPK triggers phosphorylation on threonine 172 of the
catalytic alpha subunit by the upstream AMPK kinase called LKB1, resulting in increased enzyme
activity. AMPK plays a key role in controlling cell energy metabolism, including the regulation of
glucose utilisation, fatty acid oxidation and glycogen metabolism (Carling, 2004). In some models,
AMPK can also inhibit protein synthesis via the target of rapamycin (TOR)/p70 S6 Kinase (S6K1)
pathway (Hardie, 2005) even if some intriguing observations have recently shown heightened insulin
responses of S6K1 following AMPK activation (Longnus et al., 2005).
The aim of the present study was therefore to investigate the regulation and the role of AMPK in the
molecular mechanisms involved in the control of protein synthesis in QM7 myoblasts.

QM7 cells are a stable and permanent myogenic cell line developed by Antin and Ordahl (1991).
QM7 myoblast cells were grown in McCoy medium supplemented with 10% fetal calf serum and
1% chicken serum to 90-100% confluence, fasted 16 h in a serum-free medium, washed once with
RPMI medium, depleted in the same medium for 2 h and then stimulated during 1 h with various
doses of AICAR, largely used as a pharmaceutical activator of the AMPK system.
QM7 myoblasts were then lysed as described by Tesseraud et al. (2003) and then subjected to
SDS-PAGE gel electrophoresis and western blotting using the appropriate antibody. Membranes
were incubated with an Alexa Fluor secondary antibody (Molecular Probes, Interchim, Montluçon,
France). Bands were visualised by Infrared Fluorescence by the Odyssey® Imaging System and
quantified by Odyssey infrared imaging system software.
To measure protein synthesis in QM7 myoblasts, L-[U-14C]phenylalanine (0.25 µCi/mL) was added
to the medium during the stimulation period. At the end of incubation (60 min), cells were washed
once in ice-cold PBS, and then in ice-cold 10% trichloroacetic acid (TCA). TCA-insoluble material
was washed three times with 10% TCA and solubilised in 0.5M NaOH at 37 °C for determination
of protein and radioactivity incorporated into QM7 protein.

Firstly, we characterised AMPK in the quail muscle (QM7) cell line. cDNA corresponding to
fragments of the AMPK catalytic α1 and α2 subunits, the AMPK regulatory β1 and β2 subunits,

and the AMPK regulatory γ1 and γ2 subunits were expressed in quail QM7 myoblasts (data not
shown). We also showed that the AMPKα subunit was phosphorylated on Thr172 in a dose- and
time-dependent manner in the presence of AICAR (data not shown). Moreover, the addition of
AICAR led to a significant decrease in the phosphorylation level (on Thr421/Ser424 and Thr389
residues) and activity of S6K1 (Figure 1). Similarly, it altered the phosphorylation of two S6K1
downstream targets, the ribosomal protein S6 and the eukaryotic elongation factor 2, consequently
inhibiting protein synthesis.
Figure 1. Effects of Aicar on the S6K1 pathway and protein synthesis in QM7 myoblasts. In A,
nitrocellulose membranes were incubated with specific antibodies raised against phospho-T389
S6K1, phospho-T421/S427 S6K1, phospho-S235/236 S6, phospho-T56 eEF2 and α-tubulin. In B,
protein synthesis was determined as described in the material and methods.
Our results suggest that AMPK can regulate the S6K1 pathway and protein synthesis in avian
myoblasts. Since amino acids are known to act on the S6K1 pathway, further studies are under
investigation to analyse the role of AMPK in the amino acid-induced S6K1 activation and to determine
the effect of amino acids on AMPK regulation.
References
Antin P.B. and C.P. Ordahl, 1991. Isolation and characterization of an avian myogenic cell line. Dev. Biol. 143, 111-
121.
Carling D., 2004. AMPK. Curr. Biol. 14, R220.
Corton J.M., J.G. Gillespie and D.G. Hardie, 1994. Role of the AMP-activated protein kinase in the cellular stress
response. Curr. Biol. 4, 315-324.
Hardie D.G., 2005. New roles for the LKB1-->AMPK pathway. Curr. Opin. Cell Biol. 17, 167-173. Review.
Hardie D.G. and D. Carling, 1997. The AMP-activated protein kinase-fuel gauge of the mammalian cell? Eur. J.
Biochem. 246, 259-273. Review.
Longnus S.L., C. Segalen, J. Giudicelli, M.P. Sajan, R.V. Farese and E. Van Obberghen, 2005. Insulin signalling
downstream of protein kinase B is potentiated by 5’AMP-activated protein kinase in rat hearts in vivo. Diabetologia
48, 2591-2601.
Tesseraud S., K. Bigot and M. Taouis, 2003. Amino acid availability regulates S6K1 and protein synthesis in avian
insulin-insensitive QM7 myoblasts. FEBS Lett. 540, 176-180.

Decreased nutritional responsiveness of S6K1 in the breast muscle of
genetically fat chickens
S. Duchene1, M. Abbas1, I. Seiliez2, E. Audouin1, S. Crochet1, S. Métayer Coustard1 and S.
Tesseraud1
2INRA, UMR1067 Nutrition, Aquaculture et Génomique, F-64310 St Pée-sur-Nivelle, France
Introduction
S6K1 is a major effector of cell growth. For example, mice deficient for this kinase are smaller than
controls and have reduced muscle mass (Ohanna et al., 2005). In mammals, S6K1 is phosphorylated
in response to insulin via a signal transduction pathway involving phosphatidylinositol 3’kinase
(PI3K), protein kinase B (PKB also called AKT) and the mammalian target of rapamycin (mTOR).
Surprisingly, S6K1 is activated by refeeding and insulin in chicken muscle despite a relative insulin
resistance in the early steps of insulin receptor signalling in this tissue (PI3K pathway) (Tesseraud
et al., 2006; Duchene et al., 2007a). The Mitogen-activated protein kinase (MAPK) / Extracellular
signal-regulated protein kinase (ERK) 1/2 pathway may participate in the insulin-dependent regulation
of S6K1 in chicken muscle as shown in avian cells (Duchene et al., 2007b).
The aim of the present study was therefore to further investigate the regulation and activation of
S6K1 in the chicken muscle. S6K1 signalling was studied in two experimental lines of chickens
that exhibit great differences in body composition and muscle development. These lines have been
divergently selected on fattening and there is about 12% increase of the weight of breast muscle in
lean chickens vs. fat chickens at 6 wk of age (Pym et al., 2004).

Six-wk-old genetically lean and fat chickens were assigned to the following nutritional states: food-
deprived for 16 h, refed for 30 min or fed. Pectoralis major muscles were sampled in six chickens
per genotype and per treatment, quickly frozen in liquid nitrogen and stored at -80 °C until analyses.
The expression and/or the phosphorylation of S6K1 and two kinases potentially involved in its
activation, i.e. AKT and ERK1/2 were measured by Western-blot on muscle lysates. S6K1, AKT
and ERK1/2 activities were determined after immunoprecipitation of each kinase in the presence of
ATP and an artificial substrate. Statistical analysis was performed using ANOVA.

S6K1 content was higher in the Pectoralis major muscle of lean chickens compared to their counterparts
(Figure 1A) (P<0.01; ERK used as a loading control). We showed differences in the phosphorylation
of S6K1 according to the genotype: there were lower S6K1 phosphorylation on T389 (P<0.05) and
conversely higher S6K1 phosphorylation on T421/S424 (P<0.001) in the lean line than in the fat
one. Interestingly, muscle S6K1 activity assayed by the ability of the enzyme to phosphorylate its
substrate was regulated by the nutritional state (fed and refed states vs. fasted state) in lean chickens
but not in fat chickens (data not shown). To explore the underlying mechanisms, we investigated
the regulation of two kinases involved in the control of S6K1 in avian cells (Duchene et al., 2007b).
AKT was regulated by the nutritional state, but there was no line-related difference concerning its
phosphorylation level, i.e. activation (Figure 1B), and/or activity (data not shown). Conversely,
we observed clear differences between the two genotypes concerning the activation and activity of
ERK1/2, with a regulation by the nutritional state recorded only in lean chickens. These findings
indicate that ERK1/2 is strongly involved in the control of S6K1 in muscle of avian species.

Figure 1. Regulation and activation of S6K1 in lean line (LL) and fat line (FL) chickens food-deprived
for 16 h (S), fed (F) or refed for 30 min (R30). (A) S6K1 phosphorylation on T389 and T421-S424
and S6K1 expression. (B) Phosphorylation and expression of ERK and AKT. Representative Western
blots.
In conclusion, our results suggest differences in S6K1 activation and regulation between genetically
lean and fat chickens. The underlying mechanisms (e.g. in vivo regulation of the S6K1 pathway
by insulin and/or amino acids) and the physiological consequences of these findings remain to be
explored.
References
Duchene, S., S. Metayer, E. Audouin, K. Bigot, J. Dupont and S. Tesseraud, 2007a. Refeeding and insulin activate the
AKT/p70S6 kinase pathway without affecting IRS1 tyrosine phosphorylation in chicken muscle. Domest. Anim.
Endocrinol. Epub 2006 Sep 25.
Duchene, S., E. Audouin, S. Crochet, M.J. Duclos, J. Dupont and S. Tesseraud, 2007b. Involvement of the ERK1/2
MAPK pathway in insulin-induced S6K1 activation in avian cells. Domest. Anim. Endocrinol. Epub 2006 Nov
29.
Pym, R.A.E., B. Leclercq, F.M. Tomas and S. Tesseraud, 2004. Protein utilisation and turnover in lines of chickens
selected for different aspects of body composition. Br. Poult. Sci. 45, 775-786.
Ohanna, M., A.K. Sobering, T. Lapointe, L. Lorenzo, C. Praud, E. Petroulakis, N. Sonenberg, P.A. Kelly, A. Sotiropoulos
and M. Pende, 2005. Atrophy of S6K1(-/-) skeletal muscle cells reveals distinct mTOR effectors for cell cycle and
size control. Nat. Cell Biol. 7, 286-294.
Tesseraud, S., M. Abbas, S. Duchene, K. Bigot, P. Vaudin and J. Dupont, 2006. Mechanisms involved in the nutritional
regulation of mRNA translation: features of avian model. Nutr. Res. Rev. 19,104-116.

Leucine suppresses myofibrillar proteolysis by down-regulating
ubiquitin-proteasome pathway in chick skeletal muscles
K. Nakashima, A. Ishida, M. Yamazaki and H. Abe
National Institute of Livestock and Grassland Science, Tsukuba 305-0901, Japan
Introduction
Amino acids, in combination with hormone, are known to be primary regulators of body protein
turnover (Kadowaki et al., 2003). Branched chain amino acids (valine, leucine, and isoleucine, and
BCAA) are essential amino acids and the major nitrogen source in skeletal muscle. Among the
BCAA, particularly leucine stimulates protein synthesis but suppresses protein degradation in skeletal
muscles. The exact mechanism of the suppression of protein degradation by leucine is still unclear.
BCAA have a regulatory effect on proteolysis in skeletal muscle, but their effect on the muscle
proteolytic pathway has not yet been reported. To clarify these issues, the present study investigated
the effects of leucine on myofibrillar proteolysis in cultured chick myotubes and chicks.

Cell culture
The cells were isolated from the thigh muscles of 13-d-old chick embryos (Nakashima et al., 2004).
The myotubes were incubated with valine (Val, 1 mM), leucine (Leu, 0.2, 0.4 and/or 1 mM) or
isoleucine (Ile, 1 mM) for 2 and 6 h in a Krebs-Henseleit-Hepes buffer supplemented with 0.1%
BSA, 10 mM Hepes, 2 mM pyruvate, and 5 mM glucose. After 6 h incubation, the medium was
collected, and the Nτ-methylhistidine concentration, as an index of myofibrillar proteolysis, was
measured by the HPLC method after derivatisation of fluorescamine with a treatment of perchloric
acid and heating.
Animal preparation and experimental protocol
On d 7, twenty-four birds of similar body weight were selected and housed in wire-bottomed
aluminum cages, and six replications were made per treatment. At the start of the experiment,
14-d-old chicks weighing 140 ± 3 g were divided into groups and caged separately. Chicks were
deprived of food for 24 h and then randomly assigned to continue as food-deprived (Control) or
to receive one of three dietary treatments by oral gavage as follows: L-valine (Val), L-leucine
(Leu) or L-isoleucine (Ile). The amount of each amino acid administered was 225 mg/100 g body
weight, prepared as 3.8 g/50 mL in distilled water. The amount of each amino acid administered
was equivalent to the amount of leucine consumed by chicks of this age and strain during 24 h of
free access to a diet. The chicks were killed at 2 h after administration, and gastrocnemius muscles
and blood samples were obtained.

The effects of leucine on the degradation of myofibrillar protein using Nτ-methylhistidine released into
the medium and the mRNA level of proteolytic-related genes in chick myotubes were investigated.
Leucine (0.2, 0.4 and 1 mM) suppressed Nτ-methylhistidine release into medium in a dose-dependent
manner in chick myotubes. Ubiquitin and proteasome C2 subunit mRNA expressions were also
decreased by leucine in a dose-dependent manner, and m-calpain large subunit mRNA expression
was decreased by leucine (1 mM) in chick myotubes. However, cathepsin B mRNA expression
was not decreased by leucine. This result indicates that leucine reduces myofibrillar proteolysis

and inhibits ubiquitin-proteasome and Ca2+-calpain proteolytic pathways in chick myotubes. The
effects of BCAA (valine, leucine and isoleucine) on the degradation of myofibrillar protein and the
expression of ubiquitin, and proteasome C2 subunit mRNA expression in chick myotubes were
studied. Leucine and isoleucine but not valine suppressed Nτ-methylhistidine release (Figure1A)
and ubiquitin and proteasome C2 subunit mRNA expressions (Figure 1B) in chick myotubes in vitro.
This result indicates that leucine and isoleucine, as BCAA, reduce myofibrillar proteolysis and the
ubiquitin-proteasome pathway in chick myotubes.
Figure 1. Effect of BCAA on myofibrillar proteolysis, ubiquitin and proteasome C2 subunit mRNA
expression in chick myotubes and chick skeletal muscles.
The effects of BCAA (valine, leucine and isoleucine) on myofibrillar proteolysis and ubiquitin and
proteasome C2 subunit mRNA expression in chicks in vivo were examined. Plasma Nτ-methylhistidine
concentration, as an index of myofibrillar proteolysis, was decreased by oral administration of
leucine and isoleucine but not valine in chicks (Figure 1C). Ubiquitin mRNA expression was not
inhibited by BCAA in chick gastrocnemius muscles. However, leucine and isoleucine but not
valine inhibited proteasome C2 subunit mRNA expression in chick skeletal muscles (Figure 1D).
This result indicates that leucine and isoleucine suppress myofibrillar proteolysis by inhibiting the
proteasome pathway in chick skeletal muscles. The present study is the first study to demonstrate
that an important component of muscle proteolysis inhibition by leucine has the ability to suppress
ubiquitin-proteasome proteolytic pathway, and degradation of myofibrillar protein.
References
Kadowaki, M. and T. Kanazawa, T, 2003. Amino acids as regulators of proteolysis. J. Nutr. 133, 2052S-2056S.
Nakashima, K., I. Nonaka and S. Masaki, 2004. Myofibrillar proteolysis in chick myotubes during oxidative stress. J.
Nutr. Sci. Vitaminol. (Tokyo) 50, 45-49.

Skeletal muscle response to an endotoxin injection followed by
malnutrition is similar in low-grade inflamed and non-inflamed old rats
G. Mayot1, K. Vidal2, L. Combaret1, D. Breuillé2, S. Blum2, C. Obled1 and I. Papet1
1INRA, UMR1019 Unité de Nutrition Humaine, 63122 Saint-Genès-Champanelle, France
2Nutrition and Health, Nestlé Research Center, 1000 Lausanne 26, Switzerland
Introduction
Skeletal muscle mass is gradually lost during ageing, a process referred to as sarcopenia (Greenlund
and Nair, 2003). Ageing is associated with chronic inflammation, whose intensity is very variable
within old individuals (Ferrucci et al., 2002; Mayot et al., 2007). The age-associated low-grade
inflammation has been suggested to contribute to sarcopenia (Roubenoff, 2003). We have recently
shown that sarcopenia is not enhanced in low-grade inflamed old rats compared to age-matched non-
inflamed rats, when they are maintained in their breeding conditions (Mayot et al., 2006). However,
low-grade inflammation could be a factor associated with an increased risk of muscle mass loss in
case of any additional stress in old individuals.
The aim of the study was to analyse the response of skeletal muscle mass and protein turnover to
a stress applied to old rats being or not low-grade inflamed. Inflammation was assessed by plasma
α2-macroglobulin and fibrinogen levels (Mayot et al., 2007). The stress was obtained by an endotoxin
injection followed by protein energy malnutrition. It mimics a transient febrile event followed by a
period of poor appetite in elderly people.

Wistar male rats, 22 mo old and apparently healthy, were acclimatised in individual cages and
received an 18% casein diet for 3 wk. On d 0 (D0), they were stratified into 2 classes with or without
initial low-grade inflammation by means of the hierarchical clustering using Ward distance on α2-
macroglobulin and fibrinogen measured at d 7. Each class was then divided into 2 groups receiving
either the control or the stress treatment. For the control treatment, the pre-experimental conditions
were maintained for 5 non-inflamed rats (NC group) and 5 inflamed rats (IC group). For the stress,
a single intra-peritoneal injection of lipopolysaccharide from Escherichia coli 0127:B8 (0.4 mg/kg)
was performed and followed by a 23-d period of malnutrition i.e. a 4% casein diet was distributed
in quantities limited to 50% of spontaneous food intake. The stress was applied to 5 non-inflamed
rats (NS group) and 7 inflamed rats (IS group). At d 23, protein turnover was measured in vitro
in the epitrochlearis muscle and posterior leg skeletal muscles were dissected and weighed. The
results are expressed as means ± SE. Data were analysed by two-way (initial inflammatory status
and treatment) ANOVA.
Results
As expected, stress decreased the body weight and gastrocnemius and tibialis anterior masses
(Table 1). Surprisingly, none of the parameters were affected by the initial inflammatory status.
However, the relative body weight loss induced by the stress (NS and IS groups) was significantly
correlated with initial concentrations of either α2-macroglobulin or fibrinogen (not shown).

Table 1. Effect of stress in low-grade inflamed and non-inflamed old rats.
Group NC NS IC IS ANOVA1
Body weight d 0, g 663 ± 59 603 ± 22 576 ± 17 648 ± 35 ns

Body weight d 23, g 647 ± 54 476 ± 20 563 ± 21 493 ± 29 T
Gastrocnemuis, g 2.31 ± 0.06 2.00 ± 0.09 2.29 ± 0.10 2.03 ± 0.11 T
Tibialis anterior, mg 844 ± 40 719 ± 35 786 ± 23 762 ± 38 T (0.06)
Extensor digitorum 189 ± 018 179 ± 8 196 ± 9 190 ± 12 ns
longus, mg
Soleus, mg 156 ± 017 163 ± 17 172 ± 6 188 ± 6 ns
Protein synthesis, 0.16 ± 0.02 0.14 ± 0.01 0.15 ± 0.01 0.15 ± 0.01 ns
nmol tyr/mg prot/h
Protein degradation, 1.83 ± 0.08 1.78 ± 0.04 1.98 ± 0.08 1.80 ± 0.08 ns
nmol tyr/mg prot/h
1Two-way (initial inflammatory status and treatment) ANOVA, ns: none significant effect, T: significant effect of
treatment P<0.05, P value for tendency is into brackets.
Discussion
No effect of initial inflammatory status and no interaction between treatment and initial inflammatory
status for muscle masses and protein turnover were observed. So, despite the fact that the relative
body weight loss induced by the endotoxin injection followed by protein energy malnutrition was
slightly higher in low-grade inflamed rats compared to non-inflamed controls, the response of
skeletal muscle was not worsened by the low-grade inflammation. It is not excluded that skeletal
muscle protein turnover might have been differentially affected earlier after the induction of the
stress. Nevertheless, skeletal muscle masses were not differentially affected thereafter. Our results
extend our previous findings and confirm that age-associated low-grade inflammation does not
enhance sarcopenia.
References
Ferrucci, L., B.W. Penninx, S. Volpato, T.B. Harris, K. Bandeen-Roche, J. Balfour, S.G. Leveille, L.P. Fried and J.M.
Guralnik, 2002. Change in muscle strength explains accelerated decline of physical function in older women with
high interleukin-6 serum levels. J. Am. Geriatr. Soc. 50, 1947-1954.
Greenlund, L.J.S. and K.S. Nair, 2003. Sarcopenia-consequences, mechanisms; and potential therapies. Mech. Ageing
Dev. 124, 287-299.
Mayot, G., D. Breuillé, C. Obled and I. Papet, 2006. Muscle mass and protein synthesis are not modified by low grade
inflammation in old rats. 28th ESPEN Congress, P0333.
Mayot, G., K. Vidal, J.F. Martin, D. Breuillé, S. Blum, C. Obled and I. Papet, 2007. Prognostic values of [alpha]2-
macroglobulin, fibrinogen and albumin in regards to mortality and frailty in old rats. Exp. Gerontol., in press.
Roubenoff, R., 2003. Catabolism of aging: is it an inflammatory process? Curr. Opin. Clin. Nutr. Metab. Care 6,
295-299.

Estrogenic and isoflavonic actions on differentiation and protein
metabolism in porcine muscle satellite cell cultures
M. Mau and C. Rehfeldt
Research Institute for the Biology of Farm Animals (FBN), Muscle Biology and Growth Research
Unit, Wilhelm-Stahl-Allee 2, D-18196 Dummerstorf, Germany
Introduction
The role of estrogens and estrogen-like compounds, such as isoflavonic phytoestrogens, in pig skeletal
muscle growth is largely unknown. Estrogens are not considered to be effective growth enhancers in
pigs (Roche and Quirke, 1986), whereas, as constituents of soy, isoflavones have been discussed to
promote growth performance (Ren et al., 2001). However, the isoflavonic phytoestrogens genistein
and daidzein (≥10 µM) were demonstrated to act as toxins and inhibitors of porcine myoblast growth,
whereas 17β-estradiol and estrone were shown to decrease DNA synthesis rate at supra-physiological
concentrations (Mau et al., 2006). Moreover, there are indications from in vitro experiments with
a rat muscle cell line (L8) that genistein inhibits myotube formation in a dose-dependent manner
(Ji et al., 1999). The impact of estrogens and isoflavones on the differentiation of porcine skeletal
muscle has received no attention so far. Therefore, the aim of this study was to investigate the effects
of various concentrations of estrogens and genistein and daidzein on the in vitro growth of porcine
muscle satellite cell cultures during differentiation.

3
Cells were seeded in six matrigel-coated 24-well plates at about 5×10 cells per well and grown
for 96 h in DMEM plus 10% fetal bovine serum (FBS) and 10% horse serum (HS), until the cells
reached 80% confluence. After growing in DMEM plus 10% FBS and 1 µM insulin for 24 h, confluent
cultures were differentiated during 72 h in serum-free differentiation medium (MEMα:MCDB 110
(4:1) plus 1 µM cytosine arabinoside) supplemented according to Doumit et al. (1996). During the
last 26 h of each experiment, the cells were treated with different concentrations of 17β-estradiol
(E2), genistein or daidzein. Protein synthesis and degradation were measured by the incorporation or
release of L-[2,6-³H]-phenylalanine in separate experiments after adding 148 kBq (4 µCi) per well
according to Rehfeldt et al. (2002). In another experiment, creatine kinase (CK) activity was measured
as a marker of differentiation. Data were subjected to analyses of variance (SAS). Significance of
differences between least squares means was tested by the Student t-test (P<0.05).
Results
Protein synthesis rate was not affected by 17β-estradiol. At a concentration of 100 µM, both genistein
and daidzein slightly reduced protein synthesis by 13% and 5%, respectively. Furthermore, 17β-
estradiol (0.1, 1 nM), genistein (0.1, 1, 10 µM) and daidzein (0.1, 1, 10, 100 µM) reduced protein
degradation to 96-98% (Figure 1). At concentrations of 20 and 100 µM, genistein reduced the CK
activity per well to 90-92% of the control. However, the specific CK activity (IU/protein) remained
unchanged with genistein, and likewise with 17β-estradiol, and daidzein at any concentration tested.
Total protein was exclusively affected by 20 and 100 µM genistein and 100 µM daidzein inducing
significant reductions by ca. 5%.

Figure 1. Exposure to E2 (0.1, 1 nM), daidzein (0.1 - 100 µM) and genistein (0.1 – 10 µM) for 26 h
lowered protein degradation rate in differentiating porcine satellite cells. Bars represent the relative
changes compared to control (=100%) and are based on the least squares means of L-[2,6-3H]-
phenylalanine release (% dpm acid soluble) (n=24 per treatment; *P<0.05).
Conclusions
The results suggest that estrogens as well as genistein (0.1 to 10 µM) and daidzein (0.1 to 100 µM)
are able to selectively inhibit protein degradation in differentiating porcine muscle cell cultures, while
the degree of differentiation does not seem to be influenced. High isoflavone concentrations (20 - 100
µM), however, decrease protein synthesis and/or appear to be toxic. Conclusively, 17β-estradiol and
low concentrations of genistein and daidzein may have the potential to enhance net protein accretion
in porcine skeletal muscle by inhibiting protein degradation, whereas higher concentrations may
have detrimental effects.
References
Doumit, M.E., D.R. Cook and R.A. Merkel, 1996. Testosterone up-regulates androgen receptors and decreases
differentiation of porcine myogenic satellite cells in vitro. Endocrinology 137, 1385-1394.
Ji, S., R.L. Losinski, S.G. Cornelius, G.R. Frank, G.M. Willis, D.E. Gerrard, F.F.S. Depreux and M.E. Spurlock, 1998.
Myostatin expression in porcine tissues: tissue specificity and developmental and postnatal regulation. J. Am.
Physiol. Regul. Integr. Comp. Physiol. 275, 1265-1273.
Mau, M., T. Viergutz and C. Rehfeldt, 2006. Influence of estrogens and isoflavones on porcine muscle satellite cell
growth. Arch. Anim. Breed. 49 (Special Issue), 81-85.
Rehfeldt, C., K. Walther, E. Albrecht, G. Nürnberg, U. Renne and L. Bünger, 2002. Intrinsic properties of muscle
satellite cells are changed in response to long-term selection of mice for different growth traits. Cell Tiss. Res.
310, 339-348.
Ren, M. Q., G. Kuhn, J. Wegner and J. Chen, 2001. Isoflavones, substances with multi-biological and clinical properties.
Eur. J. Nutr. 40, 135-146.
Roche, J. F. and J. F. Quirke, 1986. The effects of steroid hormones and xenobiotics on growth of farm animals. In:
Buttery, P.J., D.B. Lindsay and N.B. Haynes (editors) Control and manipulation of animal growth. Butterworths,
London, 39.

Protein deposition in the body, content of nucleic acids in the mld muscle
of pigs as affected by limitation of protein during growing period
G. Skiba, S. Raj, D. Weremko and H. Fandrejewski
The Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences 05-110
Jabłonna, Poland
Introduction
The aim of this study was to determine the changes of protein deposition in the body, and the content
of the nucleic acids in the mld muscle in animals consuming lower amounts of protein during early
stages of their growth. It was assumed that protein restriction in an early age of pigs would influence
a rate of protein deposition in the body during the following growth period, and the content of the
nucleic acids in the muscle as well.

Two groups of gilts, 12 animals each, during the growing period (25 -50 kg BW) were fed in another
way. The C pigs were offered a grower diet (13.2 MJ ME, 8.3 g digestible lysine per kg diet), but
the E animals consumed a diet with lower energy and lysine content (12.1 MJ ME, 5.8 g digestible
lysine per kg diet). Daily allowances for the E pig was fixed to what the C animals consumed
respectively, with 35 and 15% less lysine and metabolisable energy as compared with the C pigs.
Subsequently all pigs were fed the grower diet till 80 kg BW following a finisher diet (12.8 MJ
ME, 6.8 g digestible lysine/kg diet) till 105 kg BW. A comparative slaughter technique was used
to determine protein deposition in the body. Animals were slaughtered at 50, 80 and 105 kg BW.
Moreover, musculus longisimus dorsi (mld) was separated, weighed and samples (last rib region)
were taken and analysed for chemical composition (Association of official Analytical Chemists,
2004) and nucleic acid content (Oksbjerg et al., 2000).
Results
During the growing period, the pigs of the E group compared with the pigs of the C group grew slower
(572 vs. 792 g/d, respectively, P<0.01) and deposited daily less protein in the body (96 vs. 133 g,
respectively, P<0.01). Protein content in the body and mld muscle did not differ between treatments
(mean 167g/kg EBW and 203.6 mg/g, respectively). After return to feeding with a grower diet the
E pigs grew faster compared with the C animals (mean 896 vs. 864 g/d, respectively, P<0.01) and
deposited daily more protein (on average 145 vs. 117 g, respectively, P<0.01), and finally their body
contained more protein (166 vs. 154 g/kg, P<0.05). Mass and protein content of the mld muscle, at
the end of the growing period, did not differ between the C and E animals (Table 1). Restriction of
protein supply for the E pigs lowered the total amount of DNA (by 15%) and RNA (by 9%), but the
RNA:DNA ratio was increased (by 8%) compared to the C animals. Oksbjerg et al. (2002) showed a
similar observation in the semitendinosus muscle of pigs fed restrictively. These data suggest that the
rate of protein turnover of these pigs was lower, however an increased ratio of RNA:DNA indicate
that the potential of restricted pigs for protein synthesis was unchanged or even slightly higher. Final
mass of the mld muscle (Table 1) and its protein content (mean 215.6 mg/g) did not differ between
the groups of pigs. Total content of the DNA in the muscle of the E pigs was higher at both 80 (by
15%) and 105 kg BW (by 4%). However, the total content of the RNA was slightly lower (at 80 kg
BW) or unchanged (at 105 kg BW). In the study by Kristensen et al. (2002) total content of both
the DNA and RNA of pigs fed restrictively in the growing period was slightly lower. Analysis a
content of the nucleic acids using allometric equation (Table 2) indicate that the rate of gain of the
total amount of the both nucleic acids in mld muscle of the E pigs was higher, since the growth

Table 1. Characteristic of the mld muscle at 50, 80 and 105 kg BW.
Item 50 kg BW 80 kg BW 105 kg BW
C E S.E. C E S.E. C E S.E.

(n=4) (n=4) (n=4) (n=4) (n=4) (n=4)
Mass, g 1281 1247 78.4 2195 2224 127.7 2895 2826 156.4
Total DNA content, g 0.570 0.482 0.04 0.745 0.781 0.04 0.800 0.830 0.05
Total RNA content, g 3.061 2.775 0.24 4.444 4.233 0.20 5.050 5.111 0.35
Ratio of RNA:DNA 5.36 5.76 0.17 6.00 5.43 0.19 6.35 6.14 0.18
Table 2. Content of DNA and RNA in the mld muscle using the relationship Y = aXb (Y: component,
a: intercept, X: body weight, b: slope ratio) in the pigs grown from 25 to 105 kg.
Group Y a X b r2 S.E.
C Total DNA 0.021 Body weight 0.46 0.768 0.070

E Total DNA 0.004 Body weight 0.65 0.894 0.060
C Total RNA 0.045 Body weight 0.59 0.931 0.264

E Total RNA 0.012 Body weight 0.76 0.919 0.343
coefficient (b) in these animals was higher by 40% (for DNA) and 29% (for RNA) as compared
with the C pigs. A higher rate of gain of the total amount of DNA in the mld muscle of the E pigs
could indicate that the higher rate of satellite cell proliferation could contribute to greater protein
deposition in the body of these pigs, because satellite cells are only a source of the new nuclei for
protein synthesis during postnatal growth (Allen et al., 1979). Moreover, higher rate of gain of the
total content of RNA can suggest that rate of protein synthesis in the muscle of these pigs could be
greater, compared with the ‘normally’ fed animals, since the amount of RNA is positively related
to the rate of protein synthesis in many situations (Millward et al., 1975).
References
Allen, R.E., R.A. Merkel and R.B. Young, 1979. Cellular aspect of muscle growth: myogenic cell proliferation. J.
Anim. Sci. 49, 115-127.
Association of official Analytical Chemists, 2004. Official methods of analysis. 18th edition. AOAC, Arlington, VA,
USA.
Kristensen, L., M. Therkildsen, B. Riis, M.T. Sorensen, N. Oksbjerg, P.P. Purslow and P. Ertbjerg, 2002. Dietary-
induced changes of muscle growth rate in pigs: effects on in vivo and postmortem muscle proteolysis and meat
quality. J. Anim. Sci. 80, 2862-2871.
Millward, D.J., P.J. Garlik, J.C. Steward, D.O. Nnanyelugo and J.C. Waterlow, 1975. Skeletal muscle growth and
protein turnover. Biochem. J. 150, 235-243.
Oksbjerg, N., J.S. Petersen, I.L. Sorensen, P. Henckel, M. Vestergaard and P. Ertbjerg, 2000. Long-term changes
in performance and meat quality of Danish Landrace pigs: a study on a current compared with an unimproved
genotype. Anim. Sci. 71, 81-92.
Oksbjerg, N., M.T. Sorensen and M. Vestergaard, 2002. Compensatory growth and ist effect on muscularity and
technological meat quality in growing pigs. Acta Agric. Scand. A Anim. Sci. 52, 85-90.

2D. Perinatal metabolic programming

Temporary consumption of a soy-based diet induces persisting alterations
in protein metabolism and oxidative stress responsiveness in juvenile pigs
P. Junghans1, M. Beyer1, M. Derno1, K.J. Petzke2, U. Küchenmeister3, U. Hennig1, W. Jentsch1
and M. Schwerin4
1Research Institute for the Biology of Farm Animals (FBN), Research Unit Nutritional Physiology
’Oskar Kellner‘, 18196 Dummersdorf, Germany
2German Institute of Human Nutrition (DIFE), Nuthetal, Germany
3FBN, Research Unit Muscle Biology and Growth, Dummerstorf, Germany
4FBN, Research Unit Molecular Biology, Dummerstorf, Germany
Introduction
Studies in growing pigs fed highly purified soy protein isolate (SPI) as compared to casein (CAS)
diet have shown that the amino acid imbalance in SPI leads to decreased protein reten-tion and
growth retardation as well as modified hepatic gene and protein expressions associa-ted with
protein biosynthesis and oxidative stress responsiveness (Schwerin et al., 2002; Junghans et al.,
2004). Exposure of infants to soy protein based diet is reported to have long-term effects due to
methionine deficiency (Mendez et al., 2002). Therefore, the purpose of the present work was to
examine whether in juvenile pigs an SPI diet can induce persistent alterations of protein and energy
metabolism, nutrient oxidation and redox homeostasis.

Animals and diets
Male, castrated pigs (German Landrace) were weaned in experiment 1 (Exp. 1) at 28 d or at 21 d

of life (Exp. 2) and reared on a starter diet. In Exp. 1, 18 pigs (16 kg BM) consumed a CAS based
control diet for 24 d. Nine pigs were switched to a SPI based test diet which was fed for another 31
d while the other nine pigs continued on the CAS diet as the control group. Exp. 2 was designed to
investigate potential persisting dietary effects of the SPI diet. In this experiment 36 pigs (11 kg BM)
were fed a CAS diet in period 1 for 24 d. In period 2, 18 pigs (15 kg BM) were randomly assigned
to either the CAS or the SPI diet which were fed for the next 31 d. In the third 31-d period 18 pigs
in each group were either continued on the CAS diet (control group) or switched back from SPI to
the CAS diet (test group). The isoenergetic and isonitrogenous diets contained (% of DM) wheat
starch (41), CAS or SPI as sole protein source (9), fat (15), sucrose (20), cellulose (7), minerals and
vitamins (8). ME of the rations amounted to 1600 kJ/kg BM0.62/d.
Methods
Whole body protein synthesis (WBPS) was calculated using the [15N]glycine end-product method
(Golden and Waterlow, 1977). Heat production, carbohydrate and fat oxidation were determined
by gas exchange measurements. Hepatic thiobarbituric acid reactive substances (TBARS) were
measured to evaluate the hepatic lipid peroxidation (Buege and Aust, 1978). Reduced thiol (GSH,
cysteine) concentrations in liver tissue samples were measured using monobromobimane (Petzke
et al., 2000). Quantitative RT-PCR was carried out as described by Schwerin et al. (2002). All
measurements were performed at the end of period 2 (Exp. 1) or period 3 (Exp. 2). Age of slaughter
was 110 or 130 d, respectively.

Results
WBPS was different between the test and control group in Exp. 1 (Table 1). In Exp. 2, WBPS tended
to be different. In the test group GSH was decreased in Exp. 1 and increased in Exp. 2. TBARS was
increased in the test group in Exp. 1. In Exp. 2, TBARS was decreased in the test group. In Exp. 1 and
2, hepatic mRNA abundances were different between test and control groups, except endopeptidase
24.16 (Exp. 2). The SPI diet did not affect heat production and nutrient oxidation.
Table 1. Parameters of protein metabolism and redox homeostasis in pigs fed SPI or CAS diets
(Means ± SEM).
Experiment 1 Experiment 2
Sequence of feeding Control Test P≤ Control Test P≤

CAS-CAS CAS-SPI CAS-CAS-CAS CAS-SPI-CAS
WBPS g protein/kg/d 11.2 ± 1.9 5.9 ± 0.8 0.01 16.0 ± 1.5 12.1 ± 1.3 0.06
GSH µmol/g liver fresh 4.57 ± 0.10 0.87 ± 0.15 0.001 6.82 ± 0.19 7.61 ± 0.19 0.05
TBARS µmol/g homogenate 0.22 ± 0.05 1.24 ± 0.19 0.001 0.45 ± 0.06 0.25 ± 0.03 0.05
protein
GT ×10-3 1.38 ± 0.32 4.39 ± 1.16 0.001 1.09 ± 0.07 1.43 ± 0.15 0.05
PMSR ×10-3 Copy number of 2.88 ± 0.27 4.36 ± 0.48 0.001 2.34 ± 0.22 2.99 ± 0.24 0.05
transcripts/10
LAP ×10-4 µg RNA 2.76 ± 0.22 3.73 ± 0.34 0.001 1.79 ± 0.29 3.17 ± 0.58 0.05
EP ×10-4 3.11 ± 0.21 6.21 ± 0.62 0.001 1.95 ± 0.22 2.92 ± 0.50 0.08
GT, Glutathione-S-transferase α; PMSR, Peptide methionine sulfoxide reductase; LAP, Leucine aminopeptidase; EP,
Endopeptidase 24.16.
Conclusion
In summary, our results suggest that SPI-induced changes in protein metabolism and redox homeostasis
in growing pigs can persist 4 wk after the cessation of SPI feeding. Results on persisting dietary effects
suggest links to findings on the phenomenon of nutritional programming. Persisting effects could be
triggered by methionine as methyl donor which is depleted in the SPI diet. It is believed that methionine
affects gene expression by methylation of DNA known as epigenetic effects (Waterland and Jirtle, 2004).
Additionally, methionine is involved in the initiation of protein biosynthesis and synthesis of GSH.
References
Buege, J.A. and S.D. Aust, 1978. Microsomal lipid peroxidation. Methods Enzymol. 52, 302-310.
Golden, M.H. and J.C. Waterlow, 1977. Total protein synthesis in elderly people, a comp-arison of results with
[15N]glycine and [14C]leucine. Clin. Sci. Mol. Med. 53, 277-288.
Junghans, P., M. Derno, W. Jentsch, S. Kuhla and M. Beyer, 2004. Effect of a soy protein diet on protein and energy
metabolism and organ development in protein-restricted growing pigs. Arch. Anim. Nutr. 58, 453-461.
Mendez, M.A., M.S. Anthony and L. Arab, 2002. Soy-based formulae and infant growth and development, a review.
J. Nutr. 132, 2127-2130.
Petzke, K.J., A. Elsner, J. Proll, F. Thielecke and C.C. Metges, 2000. Long-term high protein intake does not increase
oxidative stress in rats. J. Nutr. 130, 2889-2896.
Schwerin, M., U. Dorroch, M. Beyer, H. Swalve, C.C. Metges and P. Junghans, 2002. Dietary protein modifies hepatic
gene expression associated with oxidative stress responsiveness in growing pigs. FASEB J. 16, 1322-1324.
Waterland, R.A. and R.L. Jirtle, 2004. Early nutrition, epigenetic changes at transposons and imprinted genes, and
enhanced susceptibility to adult chronic diseases. Nutrition 20, 63-68.

Influence of metabolic imprinting during early growth period on muscle
development and intramuscular adipogenesis in Japanese Black steers
T. Gotoh1, T. Fumita1, T. Etoh1, K. Hayashi1, Y. Nakamura1, J. Wegner2 and H. Iwamoto1
1Kuju Agricultural Research Center, Faculty of Agriculture, Kyushu University 8780201, Kuju,
Japan
2Research Institute for Biology of Farm Animals, 18196 Dummerstorf, Germany
Introduction
Wagyu (Japanese Black cattle) are known for their excellent marbled beef which is achieved by
feeding them with a considerable amount of concentrate (4000 to 5000 kg altogether, until slaughter
at 28-30 mo of age). More than 90% of this feed has been imported from the USA and other countries,
which imposes a heavy dependence on imported feed on the Japanese economy. Cattle are ruminants,
which means they have an important ecological niche that capitalises on the symbiotic relationship
between fibre fermenting ruminal microbes and mammalian demand for usable nutrients. We aim
to produce a high-quality safe beef product while maximising the use of domestic grass resources.
It has been shown, however, that alterations in foetal and early postnatal nutrition and endocrine
status may result in developmental adaptations that permanently change the structure, physiology,
and metabolism in adult life in rats, domestic species and mice and humans (Levin et al., 2000).
This phenomenon is referred to as ‘metabolic imprinting’. However, there are very few reports of
metabolic imprinting in cattle.
In this study, in order to study the effects of metabolic imprinting on beef production, a fattening
experiment was conducted. In this experiment, we feed mainly roughage and investigated how
metabolic imprinting affects the expression of meat quality or quantity and related genes in Japanese
Black steers.

In this study, Japanese Black steers were nursed artificially until 2 mo of age when they were divided
into two groups: groups R (n=7) and C (n=7). In group R, the calves were fed only roughage. In
group C, they were fed a considerable amount of concentrate (over 2.5% of their body weight)
and given ad libitum access to Italian ryegrass hay (roughage). After 10 and until 26 mo of age
both groups were fattened with only Italian ryegrass hay. Muscle samples were taken by biopsy
from longissimus muscles (LM) at 2, 10, 17 and 22 mo of age. Total RNA was isolated from these
tissues with ISOGEN. Semi-quantitative analysis of realtime reverse transcription-polymerase chain
reaction (RT-PCR) was used to measure the mRNA expression of meat quality-related (PPARγ2,
C/EBPα, β, and δ, Leptin, G6P, SCD, FASN, ADRP) and quantity-related genes (myostatin, IGF-I,
IGF-I receptor, MyoD, myogenin, MRF4, Myf5, and PGC-1α). G3PDH was used as a standard for
each PCR reaction. Volume percentages of intramuscular fat were measured by the Soxhlet method.
Adipose cell size diameter was observed using Oil-red O staining methods. ANOVA was used for
statistical analyses

The average live weight was 2.2-fold higher in group C (265 ± 18 kg) than in group R (117 ± 12
kg) just after 10 mo of age (P<0.001). At slaughter, group C was just 1.3-fold heavier (472 ± 42 kg)
than group R (357 ± 25 kg) (P<0.001).

Many factors regulating the determination of adipocytes and the process of differentiation have
been reported (Lazar, 2002; Mandrup and Lanes, 1997). The expression of PPARγ2 mRNA in LM
of group C was significantly higher than in group R only at 22 mo of age (P<0.05). The expression
of C/EBPα, β and δ, Leptin, G6P, ADRP, SCD and FASN in LM was significantly higher in group
C than in group R at 5 or 10, and 22 mo of age. The diameter of intramuscular adipose cells in
LM was significantly larger in group C than in group R at 10 and 22 mo of age. The percentage
of intramuscular fat content in LM at 26 mo of age was significantly higher in group C (10.3%)
than in group R (6.2%) (P<0.05). In both groups, subcutaneous fat thickness was quite thin (group
C: 0.8 mm, group R: 0.2 mm) at 26 mo of age. These results indicate that the effect of metabolic
imprinting, or fattening regimen during the early growth period affected the expression of adipocyte
differentiation factors and the accumulation of intramuscular adipose tissue. On the contrary, with
this feeding system, waste fat such as subcutaneous did not greatly accumulate in group C.
The mRNA expression of MyoD, IGF-I in LM was significantly higher in group C than in group R
at 5 mo of age (respectively, P<0.05, P<0.01). The mRNA expression of myogenin and MRF4 and
myostatin in LM was significantly higher in Group C than in group R at 10 mo of age (respectively,
P<0.01, P<0.001). For, myostatin, the mRNA expression in LM was significantly higher in group R
than in group C at 22 mo of age (P<0.05). On the contrary, the mRNA expression of IGF-I receptor
in LM was siginificantly higher in group R than in group C at 5, 10 and 22 mo of age (P<0.05). In
Myf5, there were no significant differences in mRNA expression in LM between two groups from 2
to 22 mo of age. The MRF family plays an important role as transcription factors during myogenesis
(Berkes and Tapscott, 2005).
Myogenesis occurs in two steps; ‘determination’ and ‘differentiation’. In vitro, precursor cells are
determined to be myoblasts, which subsequently proliferate to become myofibers. MyoD and Myf5
are expressed mainly in ‘determination’ at an early stage during myogenesis. On the contrary, after
determination, myogenin and MRF4 are expressed mainly in differentiation from the myoblast to
myotube, and maintain and mature myotube. In vivo, satellite cells may be on an equality level
with precursor or stem cells in vitro. In this study, the high expression of MyoD at 5 mo of age and
myogenin and MRF4 at 10 mo in Group C might suggest that ‘determination’ and ‘differentiation’
occurred and that myoblasts differentiated from satellite cells, which may influence the hypertrophy
and hyperplasia of muscle fibre.
Conclusion
The feeding level during the early growth stage influenced mRNA expression in skeletal muscle.
The growth size, meat quantity and quality were markedly different between groups. This may
be caused by the effect of metabolic imprinting induced by a high feeding level during the early
growth stage.
References
Berkes C. A. and S.J. Tapscott, 2005. MyoD and the transcriptional control of myogenesis. Semin. Cell. Dev. Biol.
16, 585-595.
Levin, B.E., 2000. The obesity epidemic: metabolic imprinting on genetically susceptible neural circuits. Obes. Res.
8, 342-347.
Lazer, M.A., 2002. Becoming fat. Genes Dev. 16, 1-5.
Mandrup, S. and M.D. Lanes, 1997. Regulating adipogenesis. J. Biol. Chem. 272, 5367-5370.

Growth and differentiation of the chicken Pectoralis major muscle: effect
of genotype and early nutrition
C. Berri, E. Godet, T. Bordeau, N. Haj Hattab, S. Tesseraud and M.J. Duclos
INRA, UR83 Recherches Avicoles, 37380 Nouzilly, France
Introduction
Selection for growth and improved breast muscle yield in chickens is associated with muscle fibre
hypertrophy (Guernec et al., 2003) and metabolic changes with consequences in terms of meat
quality (Berri et al., 2001). This points out to a need for better understanding of the mechanisms
underlying muscle growth, differentiation and metabolism. The present study was conducted to
test whether variations in growth rate, induced by nutrition or genotype, could alter the expression
of genes involved in muscle cell growth and differentiation, as well as the number and activity of
satellite cells.

In a first experiment, chicks from two experimental lines divergently selected for high (HG) or
low growth (LG) rate were compared. Chicks of both lines were either fed (F) as of hatch or 2
d later (DF for delayed feeding). The Pectoralis major (P. major) muscle were sampled on d 15
and 18 in ovo, at hatch and on d 2, 4, 7 and 43 post-hatch. The differentiation of the muscle fibres
were assessed by the measure of the three developmental isoforms of fast MyHC (Myosin Heavy
Chain), embryonic 3, neonatal and adult. PCNA and PAX7 were chosen as markers of proliferation
and satellite cell number, respectively. The corresponding mRNA was quantified by qRTPCR.
Absolute mRNA levels of genes under investigation were corrected for 18S rRNA levels to give a
relative mRNA level. In addition, for the ages of 2, 4, 7 d post-hatch, the number of satellite cells
was estimated in situ on frozen muscle cross-section using a monoclonal antibody against PAX7
(DSHB, Iowa, USA), which specifically labels satellite cells, and the number of muscle nuclei by
Hoechst 33258 staining. In a second experiment, the P. major muscle of chicks injected with BrdU
1 h before sacrifice (100 µg BrdU/g) was sampled. First, 3 d-old LG and HG chicks fed as of hatch
were compared. Second, HG chicks at hatch and on d 3 post-hatch were compared. In this case, the
3 d-old chicks were either fed as of hatch or unfed. For this experiment, the number of satellite cells
and nuclei were estimated in situ as in the first experiment and the proliferative activity of cells by
using a monoclonal antibody against BrdU (Biomeda). The effects of nutrition, genotype or age
were analysed by ANOVA. Comparisons of means for each significant effect of ANOVA (P<0.05)
were performed by the Student Newman Keuls test.
Results
Whatever the stage of development and the regimen, the HG chicks exhibited higher body and P.
major muscle weight than the LG chicks. In HG chickens, delayed feeding (DF) induced a delay
in growth and muscle development. In LG chickens, DF did not alter overall body growth, but
decreased P. major muscle weight and yield at 2 and 4 d post-hatch. At d7 post-hatch, PAX7 and
PCNA expression was lower in F than in DF birds. At d7, the expression of PAX7 was also lower in
HG than in LG. The transition between the different fast MyHC isoforms varied with the genotype
and the nutritional status. From d7 after hatch, there was a trend for higher levels of the adult isoform
mRNA in HG birds, and at d43 they expressed lower levels of the neonatal MyHC than LG birds.
Delayed feeding led to a lower expression of the neonatal MyHC isoform and higher embryonic to
neonatal MyHC ratio at d2.

The PAX7/nucleus ratio decreased with age during the first wk. This decrease occurred between 2
and 4 d in DF chicks but only after 4 d in F chicks. At 3 d post-hatch, a slightly lower PAX7/nucleus
ratio was detected in HG than in LG birds. By contrast, the BrdU/nucleus or BrdU/PAX7 ratio was
much lower in LG than in HG chicks, suggesting a higher proliferative activity in the latter genotype
(Figure 1). In the same way, feeding induced a significant increase in the BrdU/nucleus (P<0.001)
and BrdU/PAX7 (P<0.0001) ratios between hatch and 3 d in fed birds suggesting increased cell
proliferation. Feeding also induced a slight decrease in the PAX7/nucleus ratio between hatch and
3 d (P<0.05). At 3 d, F birds exhibited slightly lower PAX7/nucleus ratio and much greater BrdU/
nucleus or BrdU/PAX7 ratio (Figure 1). These data suggest that cell proliferation is delayed in LG
compared to HG, and in DF compared to F.
Figure 1. Effect of genotype (left) and feeding (right) on the number of PAX7 positive cells and
proliferative activity estimated through BrdU incorporation in 3-d old chicks.
In conclusion, our data show that variations in growth rate alter the expression of markers of muscle
development, and therefore the cellular processes of muscle growth and differentiation. The measure
of fast MyHC isoform transitions shows that delayed growth is linked with delayed contractile
differentiation of the muscle fibres. Assuming that PAX7 expression measures satellite cell number
and PCNA or BrdU incorporation proliferation, our data also show that variations in growth rate,
induced by nutrition or genotype, are related to variations in satellite cell proliferative activity rather
than in satellite cell number.
References
Berri, C., N. Wacrenier, N. Millet and E. Le Bihan-Duval, 2001. Effect of selection for improved body composition on
muscle and meat characteristics of broilers from experimental and commercial lines. Poult. Sci. 80, 833-838.
Guernec, A., C. Berri, B. Chevalier, N. Wacrenier-Ceré, E. Le Bihan-Duval and M.J. Duclos, 2003. Muscle development,
insulin-like growth factor-I and myostatin mRNA levels in chickens selected for increased breast muscle yield.
Growth Horm. IGF Res. 13, 8-18.

Effect of in utero metabolic programming on postnatal growth of mink kits
C.F. Matthiesen1, D. Blache2 and A.-H. Tauson1
1Department of Basic Animal and Veterinary Sciences, Faculty of Life Sciences, University of
Copenhagen, Grønnegaardsvej 3, DK- 1870 Frederiksberg C, Denmark
2School of Animal Biology, Faculty of Natural and Agricultural Sciences, The University of Western
Australia, 35 Stirling Highway, Crawley WA 6009, Australia
Introduction
The mink is a strict carnivore and a seasonal breeder with one annual breading season. The mating
season starts in early March in the Northern hemisphere. It is increasingly recognised that intra-
uterine malnutrition can cause long-term metabolic programming in the offspring. The outcome
of an intra-uterine protein malnutrition resulting in a metabolic programming or imprinting in the
offspring may depend on when the malnutrition is imposed and whether it is imposed during certain
sensitive time periods in gestation (Lucas, 1991). It appears that an inadequate nutrient supply in
each of these critical periods may cause a different metabolic programming response in the offspring
(Tauson et al., 2006). The objective of the present study was to investigate how in utero nutritional
metabolic programming affects growth and metabolism of mink kits during the first two mo of life,
a period of rapid growth.

Thirty-two male mink kits were used in the present study. Amongst these, 16 (P) were exposed to
in utero metabolic programming by feeding their dams a low protein gestation diet for three wk
in late gestation, whereas the dams of the remaining 16 kits (C) had been adequately fed during
gestation. All animals were fed ad libitum and feed intake was recorded. The kits were divided into
two feeding groups, each comprising 8 C and 8 P. One group was given a level of protein that met
the requirements (A; 30% of metabolisable energy (ME) from protein) and the other was given an
insufficiently low level of protein (L; 14% of ME from protein) for a three wk period, starting at
weaning when the kits were 7 wk old. Respiration and balance experiments were performed by
means of indirect calorimetry in an open-air circulation system with two kits in each cage. The
following metabolic parameters were calculated: respiratory quotient (RQ), heat production (HE),
(ME) intake, retained energy (RE) and retained nitrogen (RN). The animals were killed at the end
of the experiment for collection of blood and organ material. Plasma samples were analysed for
the following hormones: IGF-1, cortisol, GH, leptin by radioimmunoassays (RIA). The statistical
analyses of data were carried out by mean of the MIXED procedure in SAS, according to the
following model: Yij = µ + αi + εij, where µ is the general mean, αi is the fixed effect of treatment
combination (CA, CL, PA, PL) and εij is the residual ~ N(0,σi2). All results are presented in relation
to metabolic body size (kg0.75).

The body (LW) and liver weights of kits fed the A diet postnatally were significantly higher (P<0.001)
than those of kits fed the L protein diet, irrespective of in utero nutrition. These results indicate that
possible effects of in utero protein restriction on body weights and liver size were alleviated by an
adequate protein supply during postnatal growth. This was consistent with findings on body weights
for rats where an intra-uterine protein restriction also was alleviated by an accelerated growth
postnatally when animals were adequately fed (Hales and Baker, 2001). The results of the quantitative
metabolism data also support that the intra-uterine protein restriction was alleviated and that the
results in the present experiment were more influenced by postnatal dietary treatment (Table 1).

Plasma concentrations of the measured hormones showed that the concentration of GH and leptin
were significantly lower for kits fed the A diet postnatally irrespectively of intra-uterine treatment,
the results for GH being consistent with the higher ME intake on the A diet and those for leptin
possibly reflecting a relatively lower fat retention for A than for L kits (Table 2).
In conclusion, these results suggest that in utero nutrition effects were not evident in 10 wk old kits,
but strong effects of postnatal dietary treatment were documented.
Table 1. Animal live weights, liver weights and quantitative metabolism data.
Variable Treatment RR1 P-value2
CA CL PA PL
LW, g 1033a 769b 985a 719b 98.96 <0.001

Liver, g/kg0.75 37.41a 30.38b 36.74a 30.09b 2.44 <0.001
Liver, % of LW 3.71a 3.25b 3.69a 3.28b 0.24 <0.001
RN, g/kg0.75/d 2.06a 0.72b 2.45a 0.72b 0.42 <0.001
ME, kJ/kg0.75 1438a 970b 1538a 959b 156.30 <0.001
HE, kJ/kg0.75 797 611 822 737 223.45 NS
RE, kJ/kg0.75 642ab 359ac 717 b 223c 222.98 <0.05
RQ 0.83 0.89 0.83 0.92 0.06 NS
1 Rootof residual.
2Effectof treatment.
Values with different superscripts differ significantly.
Table 2. Hormone concentrations in plasma.
Variable Treatment RR1 P-value2
CA CL PA PL
IGF1, ng/mL/kg0.75 344.03 363.72 392.85 428.24 97.95 NS

GH, g/mL/kg0.75 22.71a 38.67b 24.10a 40.99b 14.72 <0.05
Cortisol, g/mL/kg0.75 19.65 11.36 8.92 7.40 10.87 NS
Leptin, ng/mL/kg0.75 0.71a 0.99b 0.73a 1.05b 0.17 <0.001
1Root of residual.
2Effectof treatment.
Values with different superscripts differ significantly.
References
Hales, C.N. and D.J.P. Barker, 2001.The thrifty phenotype hypothesis. Br. Med. Bull. 60, 5-20.
Lucas, A., 1991. Programming by early nutrition in man. In: Bock, G.R. and J. Whelan (editors), The childhood
environment and adult disease. Wiley, Chichester, UK, Ciba Foundation Symposium 156, 38-55.
Tauson, A-H., P. Harris and M. Coenen, 2006. Intrauterine nutrition – effect on subsequent health. In: Miraglia, N.
and W. Martin-Rosset (editors), Nutrition and feeding of the broodmare. Wageningen Academic Publishers, The
Netherlands, EAAP Publication 120, 367-386.

Glucose tolerance in pregnant sows and liver glycogen in neonatal piglets
is influenced by diet composition in gestation
P. Bikker1, J. Fledderus1, J. Kluess1,2 and M.J.H. Geelen3
1Schothorst Feed Research, PO Box 533, 8200 AM Lelystad, The Netherlands
2Present address: Adisseo France SAS, CERN, route de Chamblet, 03600 Commentry, France
3Dept. of Nutrition, Fac. of Vet. Medicine, Utrecht University, Utrecht, The Netherlands
Introduction
In sows, as in many other species, glucose tolerance and insulin sensitivity decrease in late gestation
to meet the increasing demand for glucose by the foetuses (Père et al., 2007). Glucose is the main
energetic substrate utilized by the foetus and is stored in the form of glycogen in the liver and muscle.
Adequate glycogen stores are necessary to ensure survival of the neonatal piglets when colostrum
intake is below requirements. However, in sows little information is available about the relationship
between insulin resistance, development of glycogen stores and viability in neonatal piglets. Also
the influence of diet composition during gestation is scarcely studied. Van der Peet-Schwering et al.
(2004) found a reduction in glucose tolerance in sows receiving an additional amount of fat in late
gestation. Corson et al. (2003) suggested an effect of timing and composition of added dietary fat
in the gestation diet on glucose tolerance in pregnant sows. The effects on piglet development were
not reported. Therefore, the objective of this study was to determine the effect of energy source in
gestation diets on the development of glucose tolerance and insulin resistance in pregnant sows,
liver glycogen in newborn piglets and viability of suckling piglets.

This study was conducted with two treatments (T1 and T2) and 50 sows per treatment in one cycle,
from mating until weaning. Sows of T1 received a diet high in unsaturated fats from 3% linseed
oil and 3% soybean oil from mating until farrowing. Sows of T2 received a diet high in saturated
fats from 6% palm oil from mating until d 85 of gestation and subsequently a low fat diet high in
maize starch (18%) until farrowing. Sows of both treatments received an equal amount of energy
and amino acids per d. The feeding scheme was 23.8 MJ NE/d from d 1-80 of gestation and 28.2
MJ NE/d from d 80 until farrowing. During lactation all sows received the same lactation diet with
9.6 MJ NE/kg. Forty sows per treatment were used to determine reproductive performance of sows,
and survival and growth of their offspring. In 10 sows per treatment glucose tolerance and insulin
resistance were determined on d 7, 84 and 112 of gestation after intra-venous infusion of a 2.5 M
glucose solution (0.25 mL/kg of body weight). Blood samples were taken 1, 3, 5, 7, 9, 12, 15, 18, 21,
25, 30, 35, 40, 45, 50 and 60 min after infusion. In addition, from these sows two neonatal piglets
per litter were sacrificed for analysis of liver glycogen.

T1 sows gradually developed a decreased glucose tolerance and increased insulin resistance during
gestation as illustrated for glucose in Figure 1a. Glucose half life in the glucose tolerance test (GTT)
increased from 13.3 min to 29.8 min on d 7 and 112 of gestation, respectively. In T2 sow glucose
tolerance and insulin sensitivity decreased until d 84, without any further decrease thereafter.
Consequently, in the GTT on d 112 of gestation we found lower blood glucose and insulin levels
in sows of T2 compared to T1 as illustrated for glucose in Figure 1b. The rapid clearance of blood
glucose in T2 sows may result in lower glucose availability for the foetuses. Indeed we found lower
liver glycogen contents in piglets of T2 sows compared to piglets of T1 sows, 134 vs. 151 mg/g liver
(P<0.05). These differences can probably be explained by the lower insulin resistance and higher

b Plasma glucose level, day 112
a Plasma glucose level, treatment 1
16,0 16,0
T1 T2
14,0 14,0
12,0 Day 7 12,0

Glucose (mmol/L)
Glucose (mmol/L)
*
10,0 Day 84 10,0 **
8,0
Day 112
8,0 * *
* *
6,0 6,0
* * *
4,0 4,0
2,0 2,0
0,0 0,0
0 10 20 30 40 50 60 0 10 20 30 40 50 60
Time after glucose infusion (min) Time after glucose infusion (min)
Figure 1. Plasma glucose levels in a glucose tolerance test (GTT) on d 7, 84 and 112 of gestation
in sows fed a high fat diet (a) and on d 112 of gestation in sows fed a high fat (T1) or high starch
diet (T2) in late gestation (b).
glucose tolerance in T2 sows at the end of gestation. However, viability in piglets of T2 sows did
not seem to be compromised. Mortality of piglets in the suckling period was numerically lower for
T2 compared to T1, 6.2 versus 7.8%. Moreover, the influence of diet composition on the mortality
of piglets increased with litter size. In a subset of 45 sows with more than 12 piglets per litter (mean
total born 14.5; birth weight 1340 g), mortality of suckling piglets was 5.5 and 10.6% for T2 and
T1 (P=0.1) respectively. This suggests a higher viability of piglets of T2 sows, despite the lower
insulin resistance in sows and the lower liver glycogen in offspring.
These results indicate that diet composition can influence glucose tolerance and insulin resistance in
pregnant sows and hence glycogen storage in foetal piglets. Diets inducing a lower insulin resistance
in sows may improve piglet viability, despite lower glycogen storage in piglets at birth. These results
need further validation and clarification, before practical application to improve viability of piglets
is recommended.
References
Corson, A.M., J.C. Litten, K.S. Perkins, J. Lawes and L. Clarke, 2003. The effect of maternal nutrition during the first
or second half of gestation on glucose tolerance of the sow. Endocrine Abstracts 6, OC20. 194th Soc. of Endocr.
Meeting, London, UK, 3-5 Nov. 2003.
Père, M.C. and J.Y. Dourmad, 2007. Insulin sensitivity during pregnancy, lactation, and postweaning in primiparous
gilts. J. Anim. Sci. 85, 101-110.
Van der Peet-Schwering, C.M.C., B. Kemp., G.P. Binnendijk, L.A. den Hartog, P.F.G. Vereijken and M.W.A. Verstegen,
2004. Effects of additional starch or fat in late-gestating high nonstarch polysaccharide diets on litter performance
and glucose tolerance in sows. J. Anim. Sci. 82, 2964-2971.
Supported by EU ‘Feed for Pig Health’ FOOD-CT-2004-506144 and ‘EARNEST’ FOOD-CT-

2005-007036

Heat production in chicken embryos
A. Chwalibog, A.-H. Tauson and G. Thorbek
Department of Basic Animal and Veterinary Sciences. Faculty of Life Sciences, Copenhagen
University, Groennegaardsvej 3, 1870 Frederiksberg C, Denmark
Introduction
Different growth performance between different lines of broilers or even between individuals may be
expressed during embryonic life. Considering that 1/3 of a broiler’s life takes place during the prenatal
phase, quantitative determination of heat production during incubation may be a crucial parameter
predicting metabolic rate and consequently, growth performance of chickens post-hatching.
The aim of this investigation was to measure gas exchange and to calculate heat production in
embryos from a fast and slow growing line of chickens. Using the indirect calorimetry technique,
it was also possible to evaluate the amount of oxidised fat during embryonic development and to
compare daily fat oxidation with changes in the fat content of the eggs.

Fort-eight embryos from a fast growing line, Ross 308 (RO) and 48 from a slow growing line,
Labresse (LA) of White Plymouth Rock were used. Gas exchange was measured in an open-air-
circuit respiration unit (Micro-Oxymax from Columbus Instruments, Columbus, Ohio, USA). The
unit contained four small chambers with a volume of 2000 cm3, in which the temperature was kept
constant at 37.8 °C as in the incubator. The eggs were kept in the incubator during the experimental
time, while the gas exchange was measured in the respiration chambers for three h after 10, 13,
16 and 19 d in the incubator. Heat production (HE) and oxidation of fat (OXF) was calculated in
accordance with Chwalibog (2002).
In addition to the respiration experiment, chemical analyses were carried out on 120 fertilised eggs,
equally divided between the two lines. The eggs were placed in the incubator, and 20 eggs from
each line were removed after 7, 13 and 19 d of incubation, boiled, peeled and analysed in groups of
4-5 eggs for dry matter, nitrogen, fat and gross energy.
Statistical analyses were done using the GLM procedure in SAS (SAS Institute Inc., 1990). Parameters
analysed in the model were the fixed effects of embryonic age, genetic line and their interaction
effects. In order to compare eggs with different weight, egg weight was included in the model as
a covariate.
Results
Gas exchange was below 10 mL/h for RO and LA at 10 and 13 d of age, increasing steeply until d
16 and slowing down between 16 to 19 d. The pattern of the curves for gas exchange was identical
for RO and LA, but on a lower level for LA. The energy expenditure followed a similar pattern with
a mean value around 50 J/h on d 10, increasing to 528 (RO) and 402 (LA) J/h on d 19 (Figure 1).
The main source of HE was OXF contributing up to nearly 100% to the total HE. OXF from d 10
to 19 corresponded to 1.63 g (RO) and 1.92 g (LA). The differences between the two lines were
highly significant (P<0.001).
All chemical compounds of fertilised eggs were significantly different between the lines; RO had lower
water, protein and fat contents and consequently, lower energy content than LA (Table 1). During the

Figure 1. Mean values of (A) O2 consumption ○ Ross, ∆ Labresse and CO2 production ● Ross, ▲
Labresse; (B) Heat production ○ Ross, ∆ Labresse, during the incubation period.
Table 1. Egg weight (EW), content of water, protein, fat and gross energy (GE) in eggs from Ross
(RO) and Labresse (LA) after incubation for 7, 13 and 19 d. Least squares means and residual mean
square error (RE).
Day 7 13 19 RE P-value
Line RO LA RO LA RO LA Line Age
EW,g 53.4 56.1 51.6 56.5 49.7 54.5 2.68 0.004 0.147
Water, g 35.7 37.1 34.4 37.3 33.9 36.7 0.33 0.002 <0.001
Protein, g 6.86 6.89 6.93 7.05 6.96 7.18 0.15 0.028 0.002
Fat, g 4.89 5.68 4.29 5.38 2.92 4.13 0.19 <0.001 <0.001
GE, kJ 355 392 339 392 282 334 7.24 <0.001 <0.001
incubation time, the amount of water and protein was constant for each line, while the fat content
decreased. The total decrease in fat from d 7 to 19 was 1.97 g (RO) and 1.55 g (LA), corresponding
fairly well to the results from the respiration experiment being measured from d 10 to 19.
The results from both experiments demonstrate that embryos from the fast growing line (RO) had a
higher metabolic rate and oxidised more fat than the embryos from the slow growing line (LA). It
is remarkable that the differences between the two lines, as demonstrated in chickens (Chwalibog
et al., 2004) were already evidenced during embryonic development.
References
Chwalibog, A, 2002. Substrate oxidation and retention in pigs and poultry. Anim. Sci. J. 73, 95-104.
Chwalibog, A., A-H. Tauson, C. Matthiesen, A. Ali, K. Thorhauge, E. Sawosz and G. Thorbek, 2004. Oxidation of
carbohydrates and fat in newly hatched chickens. J. Anim. Feed Sci. 13 (Suppl. 2), 3-6.
SAS, 1990. SAS® Procedure Guide. Version 6, Third Edition. SAS Institute Inc., Cary, NC.

Nutritional programming due to maternal high protein diet reduces
offspring birth weight and body weight and is genotype dependent in mice
M. Kucia, M. Langhammer, N. Dietrich, M. Derno, U. Renne, G. Nürnberg, U. Hennig and C.C.
Metges
Research Institute for the Biology of Farm Animals (FBN), 18196 Dummerstorf, Germany
Introduction
In many populations worldwide epidemiological evidence relates low birth weight to increased risk
for syndrome X, coronary heart disease, and high blood pressure in adult age. On the basis of these
observations it was suggested that low birth weight is causally related to foetal under- or malnutrition
and subsequent foetal growth retardation or disproportionate growth which permanently affects
adult health. An analysis of human data on dietary protein supplementation during pregnancy on the
outcome of pregnancy came to the conclusion that high-protein or balanced protein supplementation
alone is not beneficial and may be harmful to the infant (cited in Metges, 2005). We were the first
to demonstrate in a rat model that maternal dietary high protein (HP) intake throughout pregnancy
results in low birth weight, decreased energy expenditure and increased body fatness in the offspring
(Daenzer et al., 2002). Recently, others have shown that maternal high protein diet during pregnancy
and lactation programs blood pressure, food efficiency, and body weight (Thone-Reineke et al., 2006).
We were now interested to know whether the observed effects of a maternal HP diet throughout
pregnancy and/or lactation can be reproduced in mice and whether effects of maternal HP diet on
offspring growth are genotype dependent.

Virgin female mice (n=50) long-term selected for either high body weight (HBW) or long distance
running (LDR) and an unselected control (CON) were housed individually and maintained at 22 °C
on a 12 h light-dark cycle. They were mated and d 1 of gestation was taken when vaginal plug was
first visible. Mice were fed isoenergetic HP (40% protein) or control (C) diets (20%) from mating
to end of lactation (21 d). Protein content was increased at the expense of starch. Pups were cross-
fostered and litters were standardised to 10 pups at birth. Offspring was tested in 3 combinations
of pre- and postnatal dietary exposure: HP-C, C-HP, and C-C (n=10 litters each). Offspring growth
was monitored until age 6 mo.

CON and LDR offspring showed reduced birth weight when their mothers received HP diet
throughout pregnancy (CON 1.40 vs. 1.46 g; LDR 1.58 vs. 1.62 g; P<0.05), whereas in HBW no
difference between HP and C diet was found (2.17 vs. 2.12 g) (Figure 1). Litter size at birth was lower
in CON dams fed the HP diet as compared to the C diet (10.96 vs. 12.03 pups in C; P<0.05). Body
mass gain in pregnant mothers from conception to parturition was lower in CON and HBW dams.
Until weaning, losses were the highest among C-HP pups (CON 23% and HBW 16% of all litters).
In all strains body mass gain per litter between birth and age 21 d were lowest in C-HP (CON 43.9;
LDR 51.8; HBW 88.1 g), as compared to HP-C (95.1; 71.7; 199.9 g) and C-C (95.7; 78.7; 197.3 g)
offspring (lactation diets HP vs. C; P<0.05). Growth development in offspring born to mothers fed
the C diet during pregnancy and reared by foster dams having received the HP diet during pregnancy
and lactation was reduced until age 6 mo (P<0.05). Reduced birth weight as observed in offspring
of mothers receiving the HP diet during pregnancy was compensated until weaning.

Figure 1. Birth weight in mouse offspring of dams fed a HP diet during pregnancy is decreased in
a genotype dependent manner (*P< 0.05).
Conclusion
Lactating dams fed the HP diet were less able to support normal growth development in pups until
weaning possibly due to a limitation in milk production. The LDR strain was less susceptible to early
postnatal exposure to maternal HP diet. In the HBW strain foetal growth in offspring of HP dams is
maintained presumably due to more intense body reserve mobilisation of their mothers.
References
Daenzer, M., S. Ortmann, S. Klaus and C. C. Metges, 2002. Prenatal high protein exposure decreased energy expenditure
and increased adiposity in young rats. J. Nutr. 132, 142-144.
Metges, C.C., 2005. Longterm effects of pre- and postnatal exposure to low and high dietary protein levels. Evidence
from epidemiological studies and controlled animal experiments. Adv. Exp. Med. Biol. 569, 64-68.
Thone-Reineke, C., P. Kalk, M. Dorn, S. Klaus, K. Simon, T. Pfab, M. Godes, P. Persson, T. Unger and B. Hocher, 2006.
High-protein nutrition during pregnancy and lactation programs blood pressure, food efficiency, and body weight
of the offspring in a sex-dependent manner. Am. J. Physiol. Regul. Integr. Comp. Physiol. 291, R1025-R1030.
This study was supported by supported by EC (EARNEST, Food-CT-2005-007036).

2E. Novel techniques for novel results in energy expenditure

and body composition
Comparing techniques to estimate energy expenditure on physical
activity in individually housed dairy cows
M.J.W. Heetkamp1, W.J.J. Gerrits2, A.T.M. van Knegsel1,2 and H. van den Brand1
1Adaptation Physiology Group, Wageningen Institute of Animal Sciences, Wageningen University,
P.O. Box 338, 6700 AH Wageningen, the Netherlands
2Animal Nutrition Group, Wageningen Institute of Animal Sciences, Wageningen University, P.O.
Box 338, 6700 AH Wageningen, the Netherlands
Introduction
Measurements of physical activity are required to separate activity related from non-activity related
energy expenditure. In our calorimetry facilities, we use a radar device for this purpose. Changes
in frequency of radar waves, reflected by a moving subject (=Doppler effect) are monitored and,
when exceeding a threshold value (≈sensitivity), counted. Theoretically, continuous standing or
lying could result in the same number of activity counts, whereas the energetic implications are quite
different. Furthermore, small changes in the sensitivity of the sensors can have large implications for
the estimates of activity related energy expenditure. Our objective was to compare the radar method
with activity data computed from short term weight variation.

We conducted an experiment (van Knegsel et al., 2007), with eight pairs of two lactating dairy cows,
housed per pair, in a tie-stall set-up, in a climate respiration chamber for 9 wk. Cows were fed one of
two different diets. Physical activity was measured continuously per cow by each of two methods:
(i) radar sensors; (ii) counting short term weight changes measured with a built-in weighing device
in each cow box. Cow weights were recorded once every 0.85 s. Successive weight changes were
counted when exceeding a predefined threshold. This threshold was step-wise varied between 100
and 1000 g with 100 g increments. The activity counts obtained by both methods were cumulated
over 9 min. intervals, during which heat production (Q) was measured using indirect calorimetry. The
energy costs for each unit of activity was then computed for both methods of activity measurements
by linear multiple regression: Q = β1 × Act1 + β2 × Act2, where β1 and β2 are the regression
coefficients of activity measurements, and, Act1 and Act2 are the activity measurements of the two
cows in one chamber, either based on the radar or on the weight measurements. Activity related heat
production (Qact) was calculated as Qact = β1 × Act1 + β2 × Act2.

When increasing the threshold value in the weight method the relationship between Qact,radar and
Qact,weight (Qact,weight = a × Qact,radar + b) changed dramatically. While the R2 of this relationship
remained constant between 0.71 and 0.74, the estimated intercept (b) increased from -330 to 0 kJ/
kg0.75/d and the regression coefficient (a) decreased from 8.4 to 0.5 when the threshold increased
from 100 to 1000 g. For a variety of reasons, a threshold of 500 g (a = 0.996 and b=35.2) was
considered optimal. A threshold below 300 g caused estimates of Qact,weight to exceed 50% of Q,
which is not realistic. Threshold values above 800 appeared too insensitive because too much zeros
were found. At threshold level 500, remarkably, the intercept of 35 kJ/kg0.75/d indicates that the
radar technique is more sensitive in measuring small movement like for example breathing and
ruminating. On average Qact,weight was 173 kJ/kg0.75/d and Qact,radar was 208 kJ/kg0.75/d. No diet
effect (P>0.1) was present.

Furthermore, an effort was made to distinguish standing from lying, using the weight data counts.
The posture of each cow was derived from analysis of the frequency distribution of 1-min counts of
weight changes using the 500 g threshold data, according to the method of Schrama et al. (1993).
Radar activity data could not be used for this purpose because counts of the sensor above one cow in
response to movements of the other cow within the chamber could not be excluded. For the weight
activity data, as Schrama (1993), with increasing number of activity counts per min, we observed
an initial decrease in the frequency, after which a peak was observed, as illustrated in Figure 1.
Throughout the experiment, 20 to 48 counts per min seemed to be the distinction count between
standing and lying depending on cow and weight system. To all 1-min data records then a variable
‘posture’ (0=lying, 1=standing) was added. It was assumed that lying and standing periods at least
had a length of 3 min. A repeated-measurements model was used with diet and trial included as fixed
effects, and observations on the same pair of cows taken as repeated measures. No effect of diet
(P>0.1), was found on posture variables. On average, cows spent 61% of the total time budget standing
(range 16-78) with a frequency of 16 (range 4-27) times a day during 55 (range 15-260) min.
The energy costs of standing were estimated from the 9 min heat production data, ignoring the time
during which only one of two cows was standing. The energy cost of standing (ECst), calculated
as the difference between Q during standing and lying, were 104 kJ/kg0.75/d. We assumed that
heat production unrelated to physical activity was not different between postures. The average
daily amount of energy expenditure due to standing, 61% (time spent standing) of ECst, was 63
kJ/kg0.75/d. No effect of diet was found on ECst (P>0.1) nor on daily energy expenditure due to
standing (P>0.1).
Figure 1. An example of a frequency distribution of a cow over a 1-wk period.
In conclusion, (i) both radar and weight technique generated more counts when cows were standing.
This illustrates that the radar technique may adequately account for the energy costs related to
standing. Positioning of the radar sensors just above each cow may be crucial. (ii) Tuning the
sensitivity of any system quantifying physical activity is crucial for obtaining reliable estimates of
energy expenditure on physical activity. Video analysis may help to further explore the required
sensitivity of different activity measuring systems. (iii) Energy expenditure of standing vs. lying
was estimated to be close to 100 kJ/kg0.75/d.
References
Schrama, J.W., 1993. Energy metabolism of young, unadapted calves. Thesis, Wageningen Agricultural University,
The Netherlands.
Van Knegsel A.T.M., et al., 2007. Dietary energy source in dairy cows in early lactation: energy partitioning and milk
composition. J. Dairy Sci. 90, 1467-1476.

Quantitative partitioning of energy expenditure in pregnant ewes
A. Kiani1,2, M.O. Nielsen and A. Chwalibog1
1Department of Basic Animal and Veterinary Sciences, Faculty of Life Sciences, Copenhagen
University, Groennegaardsvej 7, DK-1870 Frederiksberg C, Denmark
2Animal Sciences Group, Faculty of Agricultural Sciences, Lorestan University, Iran
Introduction
An increase in whole body energy expenditure in late gestation is termed energy expenditure of
gestation (EEgest). Quantitative partitioning of late gestation energy expenditure is problematic due
to (i) different metabolic rates in maternal and foetal tissues and (ii) the homeorhetic metabolic
adaptations which occur simultaneously in the maternal organs of the pregnant animal.
Theoretically whole body energy expenditure in pregnant animals originates mainly from (a) basal
metabolism of the maternal tissues (EEbm); (b) homeorhetic metabolic adaptations (EEhomeorhetic),
(c) energy expenditure of maternal weight gain or loss (EEweightchange) and (d) energy expenditure
of conceptus development (EEconceptus). (b) and (a) are the energy expenditure of non-gravid tissues
(EEnon-gravid tissues) and (b) and (d) are the contributors of the EEgest. Comparative slaughter technique
(Rattray et al., 1974) and measuring the O2 consumption of gravid tissues (Reynolds et al., 1986;
Bell et al., 1987) have traditionally been used to estimate the EEconceptus, however, these techniques
do not provide any quantitative information regarding EEhomeorhetic. In the present method, the
whole body energy expenditure is measured by indirect calorimetry and then EEgest is calculated
and partitioned into EEconceptus and EEhomeorhetic using a regression approach.

Twenty multiparous twin-bearing ewes were fed with relatively wide ranges of metabolisable energy
(ME) 220-440, 350-700, 350-900 (kJ/W0.75) at wk seven, five, two pre partum respectively. At
different wk, whole body energy expenditure was determined from respiratory gaseous (O2, CO2
and CH4) and urinary nitrogen according to Brouwer (1965). The EEbm was estimated form the
linear regression of retained energy on ME intake. The EEweightchange was calculated using energy
concentration values of 23.85 MJ/kg and 20.0 MJ/kg for weight gain and weight loss, and 0.64 and
0.84 values for the efficiencies of the energy use for gain and body reserve utilisation (AFRC, 1993),
respectively. Conceptus weight at different wk pre partum was estimated according to Robinson et
al. (1977). EEconceptus was calculated by subtracting EEbm and EEhomeorhetic from the whole body
energy expenditure after adjustments to zero EEweightchange. Furthermore, EEconceptus was linearly
regressed on conceptus weight (kg).
Results
For the twin-bearing ewes in the present study, basal metabolism energy expenditure was about 6.9
MJ/d. The EEhomeorhetic and EEconceptus were 274 and 234 kJ/kg conceptus respectively (Figure 1).

12000 EEnon-gravid tissues
EEweightchange
11000
EEconceptus
10000
9000
n=56
8000 0 .7 7 ,
7 R2 =
g) + 6 90
7000 W (k
274 C
u e s ( kJ ) =
s
kJ.d-1
is
6000 v id t n=56
n - g ra 0 .7 9 ,
EEno R2 =
g)
5000 W (k
234 C
tu s (k J ) =
4000 n cep
EEco
3000
2000
1000
0
0 2 4 6 8 10 12 14 16 18
Conceptus weight (kg)
Figure 1. Quantitative partitioning of energy expenditure in twin-bearing ewes.
Conclusions
The present value of EEconceptus corresponds well with values obtained by other techniques (Reynolds
et al., 1986; Bell et al., 1987), indicating the validity of the method. The results indicate that about
50% of the late gestation energy expenditure in the ewe is associated with metabolism of maternal
tissues and the rest is attributed to the conceptus development.
References
AFRC, 1993. Energy and protein requirements of ruminant. An advisory manual prepared by the AFRC Technical
Committee on Response to Nutrient. CAB International, Wallingford.
Bell, A.W., F.C. Battaglia. and G. Meschia, 1987. Relation between metabolic rate and body size in the ovine fetus.
J. Nutr. 117, 1181-1186.
Brouwer, E., 1965. Report of sub-committee on constants and factors. In: Blaxter, K.L. (editor), Energy Metabolism
of Farm Animals. Academic Press, London, 441-443.
Rattray, P.V., W.N. Garrett., N.E. East and N. Hinman, 1974. Efficiency of utilization of metabolizable energy during
pregnancy and the energy requirement for pregnancy in sheep. J. Anim. Sci. 38, 383-393.
Reynolds, L.P., C.L. Ferrell., D.A. Robertson. and S.P. Ford, 1986. Metabolism of the gravid uterus, fetus and utero-
placenta at several stages of gestation in cows. J. Agr. Sci. 106, 437-444.
Robinson, J.J., I.A. MacDonald., C. Fraser and R.M.J. Crofts, 1977. Studies on reproduction in profile ewes. 1. growth
of the products of conceptus. J. Agr. Sci. 88, 539-552.

A calorimetry system for metabolism trials
N.M. Rodríguez1, W.E. Campos1 and M. Lachica2
1Escola de Veterinária da Universidade Federal de Minas Gerais (EV-UFMG), Brasil
2Animal Nutrition Department, Estación Experimental del Zaidın, Granada, Spain
Introduction
A respiration chamber that can be used as an indirect open or closed system was built at the Veterinary
School of Universidade Federal de Minas Gerais (Brazil). The volume of 5237 L allows its use as
a confinement system for small animals, such as fowls, for up to 24 h, or larger animals like swine
or lambs for shorter periods of measurement. As far as we know, this is the first chamber of this
type in Brazil.
Chamber description and analyzer calibration

The chamber was built with 6 mm transparent acrylic resin plates with external dimensions of 1.2
m (wide) × 2.0 m (height) × 2.1 m (length) fixed by an external aluminum frame. On the top, there
is a steel bar holding the whole chamber which can be raised and lowered up and down with an
electric device. Since the chamber has no floor, it is placed on a steel base which has a canal (5 cm
wide × 5 cm depth) filled with water that surrounds the lower edges when the chamber is placed on
the base, voiding the entry or exit of air. The chamber was frequently tested for leaks by altering the
atmospheric pressure inside and then observing changes in water level in a glass manometer. The
chamber was found to be air tight. Chamber air is suctioned continuously through a 3.6 cm (internal
diameter) PVC tubing at a controlled flow rate that can vary from 10 to 100 L/min.
When the chamber is operating, samples of the inside air are continuously sent to the analysis
equipment. For every sample analyzed from the chamber there is a simultaneous determination of the
outside air composition, assuring a reliable base line. The analyzers are mounted in serial connection
and the results are automatically recorded by a specific software every three to 6 min.
Since O2 paramagnetic analyzers are influenced by the CO2 concentration, a correction factor should
be established. For such purpose a variant of the system followed by Aguilera and Prieto (1985)
was used. Standardized O2:CO2:N2 mixtures were used at the following concentrations: 21:0:79;
21:0.5:78.5; 21:1:78 and 20.5:1:78.5. These gases were alternatively injected in the analyzers and
the average of recorded values used to calculate the correction factor. This was established according
to the following equation: [O2]c = [O2] – (0.0053X2 + 0.00117X) where [O2]c is the corrected value,
[O2] is the recorded value and X is the percentage of CO2 related to O2 (r2=0.999). This shows the
need of working with correction factors due to the influence of CO2 on O2 determination by analyzers
that use the paramagnetic principle. Otherwise O2 may be overestimated.
Besides the analyzers, it is necessary to check the whole system efficiency for gas detection simulating
the gas exchange produced by an animal inside the chamber. This evaluation was made injecting
pure CO2, CH4 and N2 gases. Gases were injected for 6 h and the total measurement period was
21 h which was sufficient to resemble an experimental run, once sufficient gas was released for an
acceptable accuracy.
The calibration was performed with a flow rate of 70 L/min which is adequate for experiments
using adult sheep. Calibration factors were established comparing the volume of gases injected and
detected by the system. The values found were 1.0012 for oxygen, 1.0616 for carbon dioxide and
1.1782 for methane.

Metabolism trial
Six castrated male sheep (39 kg) housed in metabolic cages and fed Tanzania grass silage (Panicum
maximun – Tanzânia) ad libitum were used on a digestibility trial. The respirometric data was obtained
during 20 h for each animal and inside chamber air samples were analyzed every 5 min.
The average daily heat production of the sheep was 383.4 kJ/kg0.75 (Table 1) and metabolizable
energy (ME) intake was 390.9 kJ/kg0.75 which resulted in a slightly positive energy balance (116.3
kJ/d). This value represents 1.9% of total daily heat production. If basal metabolism was considered
to be 293 kJ/kg0.75 (NRC 2006) then the heat increment would be 90.4 kJ/kg0.75.
Table 1. Sheep calorimetric data (mean values).
kJ/kg0.75 kJ/kg LW kJ/d
Heat production 383.4 153.4 5981

ME intake 390.9 156.4 6097
Basal metabolism 293.0 1173 4570
Energy balance 7.5 3.0 116.3
Heat increment 90.4 36.2 1410.1
When efficiency of use of ME for maintenance was calculated, a value of 76.4% was obtained which
is close to the 80% assumed by NRC (2006). Also, the RQ was equal to 1.0 which is an expected
value for animals fed ad libitum.
The high efficiency of detection of injected gases shows that the respirometric system built is suitable
for heat production determinations in medium size animals. Tanzania grass silage was adequate to
supply energy for maintenance of castrated male sheep.
References
Aguilera, J.F. and C. Prieto, 1985. An open respiration unit for calorimetric studies with small animals. Arch. Tierernähr.
35, 825-83.
National Research Council, 2006. Nutrient Requirements of Small Ruminants. NRC, Washigton, DC, USA.

Inter-laboratory test for gas concentration measurements of the R3C
network calorimetric chambers
C. Montaurier1, P. Even2, C. Couet3 and S. Dubois4
1INRA UMR 1019 UNH-MLE, BP 321, 63009 Clermont Ferrand Cedex 1, France
2INRA/INAP-PG UMR 914, 16 rue Claude Bernard, 75005 Paris, France
3Hôpital Bretonneau, Médecine Interne A, 2 bd Tonnelle, 37044 Tours Cedex, France
4INRA UMR SENAH, 35590 Saint Gilles, France
Introduction
Four sites (Clermont Ferrand, Paris, Rennes, and Tours) involved in the R3C (Réseau de Compétence
en Chambre Calorimétrique) network own a setup to determine energy expenditure (EE) by indirect
calorimetry. EE is computed from the measurements of the O2 and CO2 concentration changes in
the calorimetric chamber (CC) during the stay of a volunteer or an animal (depending on the site).
Because we want reliable measured values, the gas analysers have to be calibrated with standard
gases. But the precision we need for these gases is not available in the gas trade. Thus each site has
built its own standard gas and controls it with its previous standard. Then the values of the standard
gases may drift step by step for each site. Moreover such a drift could impact on EE calculation.
The objective was to involve the sites of the R3C network in order to organise an inter-laboratory test,
that is, to verify if the values of concentrations measured by each site using the same gas mixtures
are in accordance. This may help us to correct the installations that present the farthest values from
the mean values. Thus we want to ensure that during studies involving many sites, EE determinations
are equivalent from one site to another.
Design of the study

Each site has a CC for humans or animals (mouse, rat, pig, mini-pig, calf, etc.), each fitted out with a
couple of analysers (O2 and CO2). Two cylinders (C1 and C2) were filled with two different mixtures
of gas to obtain concentrations about 20.200% and 20.600% (O2), and 0.800% and 0.400% (CO2),
respectively for C1 and C2. Both cylinders were moved from one site to another. For each set of
analysers available on each site, concentrations were measured five non-consecutive times. From
measurements of all the sites, we obtained the real mean values for O2 and CO2 concentrations of
C1, and the same for O2 and CO2 concentrations of C2.
Results
O2 and CO2 concentrations measured by each site are detailed in Table 1.
For C1 and relative to the mean value of O2 concentration (20.189 ± 0.024%), the differences
of measured values on the different sites are from 0.2 to 4.4% of the mean value with a standard
deviation of about 3.2%.
For C2, O2 concentration’s mean value is about 20.608 ± 0.006%, and relative differences to it are
from 0.2 to 2.7% with a standard deviation of about 2.0%.
Concerning CO2, concentration’s mean value are about 0.763 ± 0.017% and 0.367 ± 0.010%
respectively for C1 and C2. The relative differences to the mean values are respectively from 0.6
to 2.6% and from 0.1 to 3.9%, with a standard deviation of about 2.2 and 2.8% respectively for C1
and C2.

Table 1. Mean ± sd of O2 and CO2 concentrations measured for C1 and C2 by each site, and mean
± sd of O2 and CO2 concentrations for all RT3 sites involved (Total).
Cylinder 1 (C1) Cylinder 2 (C2)
O2, % CO2, % O2, % CO2, %
Site 1 20.213 ± 0.002 0.758 ± 0.005 20.617 ± 0.004 0.360 ± 0.006

Site 2 20.199 ± 0.006 0.768 ± 0.003 20.607 ± 0.018 0.381 ± 0.003
Site 3 20.187 ± 0.007 0.743 ± 0.007 20.606 ± 0.004 0.360 ± 0.005
Site 4 20.157 ± 0.001 0.783 ± 0.009 20.601 ± 0.005 0.367 ± 0.005
Total 20.189 ± 0.024 0.763 ± 0.017 20.608 ± 0.006 0.367 ± 0.010
The measured O2 concentration of site 1 is statistically different from the others for C1. The
differences are non significant between the three other sites for C1, whereas they are significant for
all the sites for C2. Mean CO2 concentrations are statistically different for C1 except between sites
1 and 3, and the same for C2 except between site 4 and the other sites.
This shows us that measured gas concentration differences do exist between the four sites involved
in this test. Nevertheless, these results have to be temperate by considering respiratory exchange
in our calorimetric chamber context. Even if statistics show significant measured concentration
differences between the four sites, considering the respiratory quotient (RQ), the differences between
the sites are only about 0.016 ± 0.040 for C1 and 0.018 ± 0.051 for C2. Moreover, for example
and information only, a difference of concentrations (O2 and CO2) about 2% may influence EE
calculation by about 1.6%.
To be rigorous, we have to take these differences into account to adjust the values. Thus we could
combine data of EE from many sites of the R3C network when performing multi-site studies. From
this work, we now have to establish methods to manage these corrections efficiently.
We set up this inter-laboratory test as a ‘Quality’ action to ensure a good comparison of our results
of EE estimation.
Conclusions
The results underscore that differences of measurement exist between the four sites of the R3C
network involved in this test. From this first inter-laboratory test, we can quantify differences of O2
and CO2 concentration measurements between the sites. We can take these differences into account,
even if they are low, in order to manage multi-sites and epidemiologic studies providing comparable
results of EE, after correction if necessary.
The setting of correcting methods and the writing of a rigorous inter-laboratory test procedure are
the next steps of this work.

Determining cattle production efficiency without measuring the intake
A. Brosh1, A. Shabtay1, J. Miron2, G. Adin3 and Y. Aharoni1
1ARO, Newe Yaar Research Center, P.O. Box 1021 Ramat Yishay 30095, Israel
2ARO, Department of Cattle and Genetics, P.O.Box 6, Bet Dagan, 50250, Israel
3Extention service ministry of agriculture P.O. Box 6, Bet Dagan, 50250, Israel
Introduction
Selection for low residual feed intake (RFI), the measured minus expected feed intake, is becoming
the common value for identifying production efficiency. The expense of measuring feed intake has
limited the implementation of selection programs that target efficiency. Most of the metabolisable
energy (ME) consumed (MEI) by cattle is lost as energy expended (EE) as heat. The energy balance
of non-draft animals is defined as MEI = EE + Retained Energy (RE), where RE includes both
secretions in milk and body energy deposition. Knowledge of any two of the above variables allows
the solution of the equation and calculation of energy efficiency. Consequently, theoretically it is
possible to define a new parameter, residual EE (REE) that is similar to RFI by its nature (normal
distribution and linear negative correlation with efficiency) and will define efficiency by calculating
the measured EE minus expected EE. Using the REE value instead of RFI was limited in the past by
the difficulty to measure EE of free-range animals. The advance in measuring EE reliably by the heart
rate method has apparently created an opportunity to use it for determining animal efficiency without
intake measurement (Aharoni et al., 2006). Moreover, we hypothesise that lower EE in relation to the
production rate is the main reason for the variations among animal production efficiency. The objective
of the paper is to present preliminary studies where REE was used to characterise cattle efficiency.

The EE (kJ/kgBW0.75/d) was measured by means of the heart rate (HR) method, i.e. HR that was
measured throughout 3-4 d was multiplied by the oxygen consumption (VO2) per heartbeat, measured
throughout 10-15 min, and by 20.47 kJ per liter VO2. In Experiment 1, we used 19 Holstein dairy
cows. Their EE and the energy in their secreted milk were measured four times during four mo. The
predicted EE of these cows was calculated from their energy in milk and their BW, assuming 0.57
MJ/kgBW0.75 for maintenance and efficiency of 64% of ME for milk production, and was subtracted
from their measured EE to calculate their residual EE (REE). Thus, an efficient cow has a negative
REE (predicted EE higher than measured EE). In experiment 2 we used nine Holstein dairy cows.
Their EE, energy in milk and calculated REE were carried out as in experiment 1. The individual
intake of the cows was measured during one wk, diet ME was calculated from diet content and table
values for individual MEI calculations. The RFI was calculated and EE was measured in the same
wk. The correlation between RFI and REE was tested.

The repeatability of the variation between cow REE in experiment 1 (Figure 1) was significant
(P<0.001), and the deviation in relation to the average value ranged from –17% to +13% (range of
30% of total EE for cows at the same level of production). In experiment 2 (Figure 2), the residual EE
was positively correlated (P<0.01, R2 = 0.70) with RFI. Two cows with the lowest RFI (-6 and -4 kg
DM/d) were also the same two cows with the lowest residual EE (-16 and -24 MJ/d). Total RE (153
and 139 MJ/d) and milk energy (155 and 136 MJ/d) of these two cows were higher than the group
averages; total group RE was 121 ± 17.4 MJ/d, and milk energy was 119 ± 18.0 MJ/d, respectively.
The presented preliminary results suggest that short-term measurements of REE can be used as a
viable biomarker of energetic efficiency of lactating cows, without the need to measure feed intake.

180 EE 30
Expected EE
160
Observed and Expected EE (MJ/day)

REE
20
140
120 10
REE (MJ/day)
100
0
80
60 -10
40
-20
20
0 -30
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Figure 1. The EE of 19 dairy Holstein cows that was measure three to four times for each cow
throughout the milking period. EE of individual cow was a covariate corrected according to the
cow energy in milk production.
15
High yielding Dairy cows
REE to RFI
10
REE (MJ/d)
5
Lineair (REE (MJ/d))
0
-7 -6 -5 -4 -3 -2 -1 0 1 2 3 4
RFI (kgDM/d) -5
-10
REE (MJ/d)
REE to RFI -15
y = 3.4806x - 1.7167 -20

2
R = 0.7004
-25
-30
Figure 2. Correlation between residual feed intake (RFI, abscissa) and residual energy expenditure
(REE, ordinate), measured during one wk on nine dairy Holstein cows.
References
Aharoni, Y., A. Brosh and E. Kafchuk, 2006. The efficiency of utilization of metabolizable energy for milk production:
a comparison of Holstein with F1 Montbeliarde × Holstein cows. Anim. Sci. 82, 101–109.

Use of the heart rate method and GPS for direct field estimation of the
energy cost of cow grazing activity: effects of season and plot size
A. Brosh1, Z. Henkin1, E.D. Ungar2, A. Dolev3, A. Orlov1, A. Shabtay1 and Y. Aharoni1
1ARO, Newe Yaar Research Center, P.O. Box 1021 Ramat Yishay 30095, Israel
2ARO, Dept. of Natural Resources, P.O. Box 6, Bet Dagan 50250, Israel
3MIGAL - Galilee Tech Center, Qiryat Shemona, P.O. Box 90000, Rosh Pinna 12100, Israel
Introduction
Time spent, distance traveled and energy cost of the activities of cows grazed on plots of 28 ha
throughout the entire day were first measured by Brosh et al. (2006). Plot size may affect animal
behavior because in larger plots each individual cow has a larger area to explore for herbage selection,
and this may affect the distance traveled and the time, and energy spent on activities; consequently
it may affect the total daily EE (TEE). The objective of the present study was to compare the energy
cost of activities and TEE of cows grazed on large plots (LP) to previous data received in smaller
plots (SP).

The data required to calculate EE were obtained by the heart-rate method and Global Positioning
System (GPS) collars equipped with motion sensors. The cows were observed on a Mediterranean
foothill rangeland covered with herbaceous vegetation. Measurements were taken in 3 seasons:
late winter (LW), early summer (ES) and late summer (LS) throughout two yr; in the first year on
a plot of a smaller size (SP) of 28 ha and in the second year on larger plots (LP) of 78 and 146 ha.
In each season the herbage biomass and nutritional value were similar in the SP and in the LP. The
energy cost of a specific activity was calculated as the cost of the activity above the EE of lying
down. Three activities were defined: standing, walking idle and grazing; they are presented in units
of (kJ/kgBW0.75/d), which is the cost of engaging in a specific activity for 24 h. In addition, the cost
of horizontal (HOR) and vertical (VER) locomotion was defined in units of (J/kgBW0.75/m), i.e. the
cost of moving 1 meter of horizontal or vertical distance. Fourteen and three statistical models were
evaluated as to their suitability to estimate activity costs in the first and second study respectively.
The daily cost of each activity was calculated as the sum of activity cost X daily time of engaging
in the activity + daily locomotion distances (HOR and VER) X the locomotion costs.

Across seasons and treatments TEE (kJ/kgBW0.75/d) ranged from 679 (LP) and 477 (SP) in non-
lactating cows in ES up to 1 014 (LP) and 954 (SP) in lactating cows in LW. The energy cost of
activities and of horizontal and vertical movement (models’ average) for the cows grazed on SP
(Brosh et al., 2006) and LP is presented in Table 1.
The estimations of the costs of activity and of locomotion were highly significant. The estimated cost
of walking idle and of vertical positive locomotion in the LP was greater than in the SP (P<0.05) by
1.2 and 15 times respectively; all other estimated costs were similar in the LP and SP. The sum of
grazing costs for cows on LP was similar to that on SP when cows grazed on high quality, green, lush
herbage in LW, but higher by 43% and 33% on LP than on SP in early and late summer, respectively.
We assume that this difference was the result of greater selection for higher quality herbage in LP
vs. SP plots. The sum of walking costs for cows on LP were 67% and 71% of the cost on SP in LW
and ES respectively, but 3.55 times the cost on SP in LS. The difference in the length of time spent
walking idle (2.9 times longer in LS on the LP than on SP) explains most of the difference.

Table 1. Model average estimations, for small (SP) and large (LP) plots, of coefficients of energy
expenditure of grazing cows in relation to lying down attributed to their activities and distances
traveled (horizontal and vertical positive locomotion).
Activity above lying down1 Movement2
Model R2 ST WA GR SEM P< HOR SEM P< VEP SEM P<

SP 0.60 43.6 72.6 95.9 4.8 0.001 2.806 0.129 0.001 1.63 0.50 0.001
LP 0.58 44.0 87.7 94.5 4.3 0.001 2.843 0.138 0.001 23.85 1.38 0.001
1Activity states (kJ/kgBW0.75/d): ST = standing, WA = walking idle (without grazing), GR = grazing; 2Movement
(J/kgBW0.75/d for m locomotion): HOR = horizontal, VRP = vertical positive locomotion; R2 defines the fraction of
total variance that was accounted for by the entire model.
The sum of all activity energy costs (Sum EAC) was mainly affected by the cost of grazing and least
affected by the cost of walking idle. The daily cost of vertical locomotion was very low in proportion
to Sum EAC, thus errors in estimating its contribution to Sum EAC in the SP are negligible in
relation to the contribution of the grazing cost. Sum EAC was maximal, and there was no difference
between LP and SP in late winter. Sum EAC was similar in LP vs. SP in the early summer in spite
of the longer grazing periods and greater grazing cost in LP vs. SP; this happened because the cost
of standing in LP was smaller than in SP. The biggest difference between Sum EAC in LP vs. SP
(25% greater in LP) was measured in late summer. It was the result of longer grazing periods and
consequently greater energy cost of grazing and walking in LP vs. SP. Similar to the finding in SP
(Brosh et al., 2006), the relationship between TEE and Sum EAC was positive, but when Sum EAC
was expressed as a fraction of TEE, this fraction was smaller when TEE was greater.
In conclusion, in the summer, when the dry biomass is sufficient, larger plots probably offer cows
a wider range of herbage selection; consequently cows graze longer and expend more energy, but
select herbage of better quality and larger quantity than in small plots, as was expressed by the greater
daily energy expenditure in LP vs. SP in early and late summer.
References
Brosh, A., Z. Henkin, E.D. Ungar, A. Dolev, A. Orlov, Y. Yehuda and Y. Aharoni, 2006. Energy cost of cows’ grazing
activity: the use of heart rate GPS methods for direct field estimation. J. Anim. Sci. 84, 1951-1967.

Heart rate measurements as an index of energy expenditure and energy
balance in ruminants: a review
A. Brosh
ARO, Newe Yaar Research Center, P.O. Box 1021 Ramat Yishay 30095, Israel
Introduction
A major part of the ME consumed by ruminants (MEI) is dissipated as heat; this is the energy
expenditure (EE), measured mostly as oxygen consumption (VO2). Measurement of EE in controlled-
condition chambers does not reflect the complexity of the natural, environmental and social conditions
of free-ranging animals. In mammals most of the measured VO2 is transferred to the tissues through
the heart; therefore, calibration of heart rate (HR) against VO2 can potentially be used for estimating
the EE of free-ranging. This paper discusses the potential of HR calibrated to VO2 for estimating
EE and energy balance (EB) of ruminants.

The data presented and discussed in this article were taken from published articles and unpublished
studies which are cited and presented by Brosh (2007). The animals used were domestic ruminants,
mostly cows (lactating and non-lactating beef and dairy cows in different reproduction states; goats
and sheep were also used). The data were obtained when animals were fed and grazed on a wide
range of diet qualities under normal, low and high thermal conditions. Daily EE was measured by
multiplying the long (several d) measurement of HR by the VO2 to HR ratio received for short
periods, 10 to 30 min.

EE estimation: When an animal does not perform significant exercise a constant value of VO2 per beat
(O2 pulse, O2P) measured over a short period of time (10 to 15 min) is used (the O2P-HR method);
during exercise O2P increases, and the regression equation of VO2 against HR can be used. The most
accepted way for estimating EE from HR for sedentary and exercise conditions together is the Flex
method, which is based on two equations that represent the VO2:HR relationship during exercise
and in a sedentary condition respectively. Intensive exercise for a significant daily period of time is
a very rare event even for grazing cows; therefore, the simple O2P-HR method is preferable because
it enables measuring a large number of animals without using special devices and trained animals.
Under extreme heat load, HR increases to improve heat dissipation, and O2P decreases; therefore,
the effect of heat load on O2P needs to be taken into account. Cold stress that doubles EE does not
affect O2P. Under defined conditions of diet ME, cow management, reproduction state (RepSt) and
production level, an average VO2 and consequently the EE of a group of cattle can be estimated by
multiplying the group average daily HR measurements by the published measured O2P. A list of
O2P values (μLO2/kgBW0.75/d) for cows (Brosh, 2007) is presented in Table 1.
Energy balance of non-draft animals is defined as MEI = EE + RE. When the dependency of EE on
MEI is linear (Brosh, 2007), the regression equation is: EE = b × MEI + C, therefore, the following
variables could be calculated: fasting EE (regression intercept, C), heat increment (regression
slope, b), energy efficiency (1 - regression slope) and maintenance energy requirement, which
is mathematically calculated as (fasting EE)/(1 - heat increment). The coefficients of the linear
regressions of the dependencies of cows’ EE on their MEI, which were calculated from several
studies (Brosh, 2007), are presented in Table 2.

Table 1. Average O2 Pulse of lactating Holstein cows and beef cows under grazing and confined
conditions in three reproductive states.
Animal Holstein Beef
Lactating Non-lac1 Preg2 Lactating Non-pregnant Preg2
Grazing - - 374c 337ab 294ab

Confined 450d 328ab 287ab 273a 268a
1Non-lactating, 2Pregnant
from 181 d up to calving; a,b,c,d Means within rows and columns of the same variable that
do not have a common superscript differ significantly (P<0.05).
Table 2. Five linear regressions of energy expenditure (EE, kJ/kgBW0.75/d) on ME intake (MEI,
kJ/kgBW0.75/d), regression coefficients and calculated EE in maintenance (MEm). Data of Grazed
(GR) and Confined (Con) beef cows on different diets ME (MJ/kgDM).
Condition R2 Slope Intercept MEm
1. GR on herbage (14 periods), ME (6.7 to 11.8) 0.792 0.375 328 525

2. GR on woodland (3 periods), ME (6.2 to 8.8) 0.794 0.282 342 476
3. Con on 9 diets (9 periods), ME (4.6 to 8.1) 0.616 0.219 340 435
The dependency of EE on MEI was found to be high and significant (P<0.001) in all studies. When
changes in MEI were induced by changes in dietary ME (trials 4 and 5) the R2 values were greater
than 0.9; when other variables and factors affected MEI and EE (i.e., the biomass in the grazed area
and the reproductive state), the R2 values were smaller.
It can be concluded that O2P-HR method is a practicable means of monitoring the EE of large numbers
of domestic ruminants; changes in their EE and HR level present reliable indications of changes
in their energy status. In the near future, when automatic HR monitoring of domestic ruminants is
available at a reasonable price, monitoring HR might provide producers with a sensitive tool for
identifying changes in their animals’ energy status. This will also significantly help to shorten the
time needed to identify health problems of individual animals.
References
Brosh, A., 2007. Heart rate measurements as an index of energy expenditure and energy balance in ruminants: A review.
J. Anim. Sci. 85, 1213-1227. doi:10.2527/jas.2006-298.

Adaptation of night median heart rate in calorimetric chambers versus
in free living conditions: a critical evaluation
C. Montaurier1, M. Vermorel1, P. Ritz2, A. Chamoux3, Y. Boirie1 and B. Morio1
1INRA UMR 1019 UNH-MLE, BP 321 63009 Clermont Ferrand Cedex 1, France
2Unit of Clinical Gerontology, University Hospital, Angers, France
3Work medical service, University Hospital, Auvergne University, Clermont-Ferrand, France
Introduction
Heart rate (HR) is an interesting variable for the assessment of energy expenditure in humans because
it is strongly related to oxygen consumption and therefore to energy expenditure (EE) across a wide
range of values (Ainslie et al., 2003). Twenty-four h-HR recording using a cardiofrequencemeter
(CFM) is frequently used in cohorts of free living subjects for the calculation of daily energy
expenditure (Lazzer et al., 2003) as for the estimation of the cardiac cost of physical activities
(Chatterjee et al., 2002), after a calibration period in a calorimetric chamber (CC) using ECG (Treuth
et al., 1998). These energy metabolism measurements are often used to establish nutritional energy
intake recommendations. The aim of the study was (i) to determine if a stress does exist due to HR
and EE recording methods, (ii) to compare and to establish a conformity between both methods of
HR recording in order to optimise future evaluation of EE at home from CC measurements.
Methods
Since night median heart rate (HRnight) is a good indicator of stress, we studied its variation during
two consecutive nights spent in CC in 120 volunteers, and in free living conditions in 69 volunteers.
HRnight corresponded to the 50th percentile of data recorded during the 6 lower consecutive h of
the night. We also compared simultaneous HR recordings using both appliances (ECG and CFM)
in CC in 20 men, and the HRnight and the daily median heart rate (HRday) determined in CC and
at home in 62 volunteers. HRday corresponded to the 50th percentile of the active period during
the d, i.e. from 08.00 h to 21.00 h.
Results
The HRnight for the second night spent in CC was significantly lower than the first one (–0.8 ± 2.5
bpm; P<0.01) (Figure 1a), whereas no significant difference was found using CFM at home. These
results suggest that a minor but significant adaptation period existed in CC and that the volunteers
required a period of adaptation before the measurements start, whereas there was no adaptation period
to the CFM in free-living conditions. Simultaneous recording using ECG and CFM showed significant
difference (0.9 ± 0.6 bpm; P<0.0001). This bias may lead to an average underestimation of daily EE
in free living conditions of 2.5 ± 0.8%. After correction of this difference between the two methods
of recording, HRnight (Figure 1b) and HRday determined in CC were significantly lower than those
determined at home (-2.5 ± 3.7 bpm; P<0.0001 and -5.9 ± 7.8 bpm; P<0.0001, respectively). This
suggests that a higher level of activities in free-living conditions may have a persistent effect on the
HRnight and may alter the comparison between HRnight determined at home or in CC.

Figure 1. Individual differences between the night median heart rates determined (a) during the first
and the second nights in the calorimetric chambers in 120 subjects (b) in the calorimetric chambers
and in free living conditions..
Conclusions
In conclusion, when HR-EE equations are used to calculate daily EE, HR kinetics have to be corrected
for the bias observed between the appliances used for HR recordings. Moreover, the intensity of the
activities performed during the day may have a significant influence on HRnight. This may explain
in particular why HRnight determined in the calorimetric chambers is lower than that determined in
free living conditions, and why it appears to be a significant adaptation effect on HRnight between
the first and the second night in the calorimetric chambers. From the present study, it appears that
HRnight recording is a robust method for the determination of energy expenditure and cardiac
cost, once bias introduced by use of different appliances is taken into account. Considering these
points is a basis to establishing good relationships between HR and EE and accurately evaluating
the activity level.
References
Ainslie P., T. Reilly and K. Westerterp, 2003. Estimating human energy expenditure: a review of techniques with
particular reference to doubly labelled water. Sports Med. 33, 683-698.
Chatterjee S., J. Sen and P. Chatterjee, 2002. Recovery cardiac cost following short duration high intensity exercise in
prepubertal, just pubertal, and postpubertal girls and its relationship with physical and physiological parameters.
J. Hum. Ergol. (Tokyo) 31, 53-58.
Lazzer S, Y. Boirie, A. Bitar, C. Montaurier, J. Vernet, M. Meyer and M. Vermorel, 2003. Assessment of energy
expenditure associated with physical activities in free-living obese and nonobese adolescents. Am. J. Clin. Nutr.
78, 471-479.
Treuth M.S., A.L. Adolph and N.F. Butte, 1998. Energy expenditure in children predicted from heart rate and activity
calibrated against respiration calorimetry. Am. J. Physiol. 275, E12-18.

Effect of cold exposure on natural abundance of 13C and heat production
in Spanish goats by the CO2 entry rate technique
M. Lachica1, A.L. Goetsch2 and T. Sahlu2
1Dept. of Animal Nutrition. Estación Experimental del Zaidín (CSIC). Camino del Jueves, s/n. 18100
Armilla, Granada, Spain
2American Institute for Goat Research. Langston University. Langston (OK 73050), USA
Introduction
The isotopic composition of the whole animal body is similar to that of its diet; however, fat tissue
is more depleted of 13C than the diet (DeNiro and Epstein, 1978). A change in enrichment can result
from a change in the substrates oxidized for energy (Prieto et al., 2001). Animals maintain body
temperature by increasing heat production (HP) through the muscular activity of shivering and also
through increased metabolic rate. This increased HP originates from fat catabolism (Graham et al.,
1958). Production of CO2 and, therefore HP, can be measured by isotopic dilution using NaH13CO3
(Corbett et al., 1971). The aim of this work was to study the effect of cold exposure on natural
abundance of 13C and estimate HP by CO2 entry rate (CER) technique.

Eight dry adult Spanish female goats (37 kg BW on average) were used, with two treatments, four
unshorn (U) and four shorn (S), fitted with a parotid gland catheter for saliva sampling and four
of them also with a jugular one for NaH13CO3 infusion, and kept in metabolic cages inside a barn.
Two diets, at maintenance level, were offered to the goats: first, one depleted of 13C (barley grain:
lucerne hay, 75:25; diet C3) to all of them; later, a second one enriched in 13C (corn grain:lucerne
hay, 75:25; diet C4) only to the not 13C infused. Both diets provided similar amounts of nutrients
and energy. Previously, all goats were fed with the diet C3 ad libitum for eight wk. Daily saliva
production was collected continuously over the length of the experiment. The first part involved
thirteen consecutive d. Then, the group split up: one with two U and S goats and diet switched from
C3 to C4 for thirteen consecutive d; and the other with two U and S goats, kept under diet C3, for
isotope infusion. Goats were continuously infused for at least 8 h prior to initiating saliva collection
and for four consecutive d to obtain the enrichment of 13C. Proper recovery values were applied (0.723
for U and 0.779 for S goats) to calculate CO2 production (Prieto et al., 2001). The ratio 13C:12C in
saliva samples was determined by isotope ratio mass spectrometry. During the experiment minimum
and maximum temperature averaged from 5.0 to 13.8 °C, respectively. ANOVA-I were applied to
data to compare differences between U and S treatments within diet C3 and C4, respectively; and
between diet C3 and C4 within treatments U and S (with the same U and S goats before and after
the diet switch), respectively.

Data are shown in Table 1. Cold exposure (shearing) and diet significantly affects the natural
abundance of 13C except for S goats switched from C3 to C4 diet, confirming the use of lipid reserves
for thermogenesis and that shivering involves the slow twitch oxidative fibers (Hinds et al., 1993).
Cold exposure significantly affects the HP (Graham et al., 1958; Symonds et al., 1986; Schaeffer et
al., 2001). The U goats showed a HP value in agreement with the MEm; this fact can be considered
as an index of the validity of the method. The S goats showed a value of 1.98 × MEm.

Table 1. Natural abundance of 13C (at %13C), CER values (CO2, mL/kg BW/h) and HP (kJ/kg0.75/d)
for unshorn and shorn goats with the diet C3 and C4 (mean ± se).
Diet C3 Diet C4
Unshorn Shorn Unshorn Shorn
At %13C 1.08960±4.2·10-4 a 1.08779±4.2·10-4 b 1.09518±6.4·10-4 a 1.08880±8.2·10-4 b

(n=52) (n=49) (n=26) (n=26)
CER 415.3±27.4 a 794.0±68.8 b
(n=8) (n=8)
HP* 437.9±28.4 a 868.0±73.7 b
(n=8) (n=8)
Unshorn Shorn
Diet C3 Diet C4 Diet C3 Diet C4
1.08951±5.5·10-4 a 1.09518±6.4·10-4 b 1.08706±5.6·10-4 a 1.08880±8.2·10-4 a

(n=26) (n=26) (n=25) (n=26)
a,b Withinthe same diet (or treatment) and row, values bearing different superscripts are significantly different
(P<0.05); *Calculated from: HP=24.07 CO2r; CO2r=apparent recovery of 13CO2×CER value (Prieto et al., 2001).
In conclusion, cold exposure produced a significant change in natural abundance of 13C associated
with a shift in relative rates of oxidation of metabolic substrates for thermogenesis; CER using
NaH13CO3 is a suitable technique to determine HP; in cold weather goats should be in good body
condition for shearing since it drastically increases the energy requirements for maintenance.
References
Corbett, J.L., D.J. Farrell, R.A. Leng, G.L. McClymont and B.A. Young, 1971. Determination of the energy expenditure
of penned and grazing sheep from estimates of carbon dioxide entry rate. Brit. J. Nutr. 26, 277-291.
DeNiro, M. and S. Epstein, 1978. Influence of diet on the distribution of carbon isotopes in animals. Geochim.
Cosmochim. Acta 42, 495-506.
Graham, N.McC., F.W. Waimnan, K.L. Blaxter and D.G. Armstrong, 1958. Environmental temperature, energy
metabolism and heat regulation in sheep. I Energy metabolism in closely clipped sheep. J. Agr. Sci. 52, 13-24.
Hinds, D.S., R.V. Baudinette, R.E. Macmillen and E.A. Halpern, 1993. Maximum metabolism and the aerobic factorial
scope of endotherms. J. Exp. Biol. 182, 41-56.
Prieto, C., M. Lachica, R. Nieto and J.F. Aguilera, 2001. The 13C-bicarbonate method: its suitability for estimating the
energy expenditure in grazing goats. Livest. Prod. Sci. 69, 207-215.
Schaeffer, P.J., J.F. Hokanson, D.J. Wells and S.L. Lindstedt, 2001. Cold exposure increases running VO2max and cost
of transport in goats. Am. J. Physiol. Regulatory Integrative Comp. Physiol. 280, 42-47.
Symonds, M.E., M.J. Bryant and M.A. Lomax, 1986. The effect of shearing on the energy metabolism of the pregnant
ewe. Br. J. Nutr. 56, 635-643.

Characterisation of lipid distribution in rainbow trout by magnetic
resonance, colour vision and histology imaging techniques
A. Davenel1, J. Bugeon2, G. Collewet1, E. Quillet3 and F. Médale4
1CEMAGREF, Rennes cedex, F-35044 France
2INRA, UR1037 SCRIBE, IFR140, Ouest-Genopole, F-35000 Rennes, France
3INRA, UR544 Laboratoire de Génétique des Poissons, F-78350 Jouy-en-Josas, France
4INRA, UMR1067 NuAGe, F-64310 Saint-Pée-sur-Nivelle, France
Introduction
Lipids are often studied as global contents without considering their anatomic distribution. Depending
on species, lipids are deposited either in non specialised organs and tissues (liver, muscle) or in
adipose tissues (Fauconneau et al., 1997). Because of the links between the location of fat stores
and product quality, precise data on distribution in different body compartments and within some
tissues (skeletal muscle) are needed. This paper describes data from three imaging techniques used
as tools to characterise the fat content and distribution in two lines of rainbow trout issued from the
third generation of a two-way selection for muscle lipid content (Quillet et al., 2005).

Two triploid pure lines (Lean line LL3 and Fat line FF3) were produced to obtain large size sterile
fish 19 mo old (about 2 kg). Eighteen fish were sampled by line. To check the anteroposterior lipid
distribution, 13 transverse MRI images were scanned using a Siemens Open 0.2T imager (0.7×0.7×4
mm3 voxel). The lipid distribution was analysed between the 13 sections and between six anatomic
zones within the same section (Figure 1) (Toussaint et al., 2005). Images of two cutlets sampled in
front of the dorsal at section 4 and anal fin at section 8 were analysed as described by Marty-Mahé et
al. (2004) using a colour image segmentation method to quantify quality traits (area of fat and muscle
tissues). Muscle samples including the skin and subcutaneous fat were processed for histological
measurements as described by Bugeon et al. (2003); the area of subcutaneous fat was measured by
image analysis with an automatic segmentation using Visilog for Windows.
Figure 1. Automatic segmentation of MRI section in 6 anatomic zones.

Results
MRI showed a decreasing anteroposterior gradient of lipid content, particularly in zones 3 and 6
(Figure 2) and a total lipid content of 9.7 and 12.7% for LL3 and FF3 lines respectively. Lipid content
determination by NMR relaxometry (Toussaint et al., 2002) performed in cutlets at sections 4 and
8 confirmed that lipid content was higher in the Fat line than in the Lean one.
zone 1
20 zone 2
lipid content in the flesh
zone 3
15 zone 4
by MRI (%)
zone 5
10 zone 6
0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
darne
Figure 2. Anteroposterior lipid content distribution in anatomic zones by MRI.
The higher percentage of peripheral adipose tissue in the FF3 line quantified by MRI was also
verified with the colour vision system on the cutlets. Histological measurements confirmed that
the subcutaneous fat in section 4 was thicker (P<0.001) in the Fat line than in the Lean line (1.37
versus 0.87 mm). Moreover MRI measured a higher lipid content in the peripheral of the Fat line
than in that of the Lean line.
These results confirm that the Fat line presented higher lipid content in muscle (Quillet et al., 2005)
but also higher lipid content in peripheral adipose tissue (mainly subcutaneous fat). Among the
different techniques used in this experiment, MRI allowed to quantify tissues in terms of relative
areas but also of fat content of a specific tissue like muscle or peripheral fat tissue. These techniques
bring new data on the impact of selection on lipid distribution.
References
Bugeon, J., F. Lefevre and B. Fauconneau, 2004. Correlated changes in skeletal muscle connective tissue and flesh
texture during starvation and re-feeding in brown trout (Salmo trutta) reared in seawater. J. Sci. Food. Agric. 84
(11),1433-1441.
Fauconneau, B., S. Andre, J. Chmaitilly, F. Krieg and S.J. Kaushik, 1997. Control of cellularity of muscle and adipose
tissues in rainbow trout. J. Fish Biol. 50, 296– 314.
Marty-Mahé, P., P. Loisel, B. Fauconneau, P. Haffray, D. Brossard and A. Davenel, 2004. Quality traits of brown trouts
(Salmo trutta) cutlets described by automated color image analysis. Aquaculture 232 (1-4), 225-240.
Quillet, E., S. Le Guillou, J. Aubin and B. Fauconneau, 2005. Two-way selection for muscle lipid content in pan-size
rainbow trout. Aquaculture 245 (1-4), 49-61.
Toussaint, C., F. Médale, A. Davenel, B. Fauconneau, P. Haffray and S. Akoka, 2002. Determination of the lipid
content in fish muscle by a NMR relaxometry method: comparison with classical chemical extraction methods.
J. Sci. Food Agric. 82 (2), 173-178.
Toussaint C., B. Fauconneau, F. Medale, G. Collewet, S. Akoka, P. Haffray and A. Davenel, 2005. Description of the
heterogeneity of lipid distribution in the flesh of brown trout (Salmo trutta) by MR imaging. Aquaculture 243,
255-267.

2F. Novel techniques for novel results in organ balance, in

vitro tissue metabolism and nutrient ‘tracing’ studies
Metabolic fate of carbohydrates from milk replacer in heavy milk-fed calves
J.J.G.C. van den Borne1, G.E. Lobley2, J.W. Blum3 and W.J.J. Gerrits1
1Animal Nutrition Group, Wageningen University, P.O. Box 338, 6700 AH, Wageningen, The
Netherlands
2Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen, AB21 9SB, United Kingdom
3Veterinary Physiology, Vetsuisse Faculty, University of Berne, CH-3012, Berne, Switzerland
Introduction
Short chain fatty acids supply the majority of the digestible energy (DE) intake in ruminating calves
and acetate is the main precursor for body fat deposition. In contrast, milk-fed calves ingest large
amounts of lactose (~37% DE) and fat (~37% DE) from milk replacer. In most species with a high
glucose absorption, such as milk-fed calves, temporary storage of glucose (as glycogen) is limited.
Therefore, glucose is either oxidised or deposited as fat.
Increasing the feeding level (FL) has been associated with increased rates of fat deposition in milk-fed
calves (Van den Borne et al., 2006) and with increased rates of de novo fatty acid synthesis in other
species. In milk-fed calves the proportion of daily lactose intake, used for de novo fatty acid synthesis
is unknown. It could be low considering that the intake of fat through the diet greatly exceeds the
daily rate of body fat deposition. In addition, heavy milk-fed calves may develop insulin resistance
(Blum and Hammon, 1999) with subsequent losses of glucose in the urine. In this study, we aimed
to quantify the effects of FL on glucose excretion in urine, substrate oxidation and de novo fatty
acid synthesis from glucose in milk-fed calves.

Eighteen male Holstein Friesian calves of 136 ± 3 kg body weight were measured for two 10 d
experimental periods, each preceded by an adaptation period of four weeks, to study the effect of
FL (1.5 and 2.5 times maintenance in period 1 and 2 respectively) on substrate oxidation. Calves
were also assigned to one of three feeding frequencies (1, 2 or 4 meals daily), i.e. 6 calves were
measured at each feeding frequency. Protein, fat and lactose contents of the milk replacer were 191,
196 and 470 g/kg dry matter, respectively. Only effects of FL are presented because interactions with
feeding frequency were non-significant. Excretion of glucose in urine was quantified. Heat production
was measured by conventional indirect calorimetry. Average daily oxidation rates of protein, fat
and carbohydrates were estimated from urinary nitrogen excretion and gas exchange. Oral bolus
doses of [U-13C]glucose and [2-13C]glucose were used to assess the pattern of dietary carbohydrate
oxidation and to estimate de novo fatty acid synthesis from dietary glucose. Sequestration of labelled
bicarbonate was measured by intravenous infusion of [13C]sodium bicarbonate. Excretion of 13CO2
in the breath was measured by non-dispersive infrared spectrometry.

Urinary glucose excretion of individual calves varied between 0 and 91 g/d and increased with higher
FL (P<0.01). This corresponded with a lower insulin sensitivity, as indicated by a higher ratio between
postprandial plasma insulin and glucose concentrations (Vicari et al., 2007) in the same calves.

Table 1. Influence of feeding level (high vs. low) on urinary glucose excretion, substrate oxidation
and oxidation of orally provided [U-13C]glucose and [2-13C]glucose in heavy milk-fed calves.
Results were pooled over feeding frequencies (1, 2 or 4 meals/d).
Feeding level P-value1
Low High
Dry matter intake, g/d 1353 ± 25 2943 ± 52 -

Urinary glucose excretion, g/d 11.7 ± 3.0 48.5 ± 9.1 **
Substrate oxidation, % of intake
Protein 45.7 ± 0.9 43.9 ± 1.1 NS
Carbohydrates 94.2 ± 0.9 91.4 ± 0.9 NS
Fat 76.8 ± 2.6 30.6 ± 2.1 ***
Isotope recovery, % of dose
[U-13C]glucose 78.5 ± 0.6 80.0 ± 0.9 NS
[2-13C]glucose 76.3 ± 0.7 81.6 ± 1.3 NS
1*P<0.05; **P<0.01; ***P<0.001; NS: not significant. Isotope recovery from [U-13C]glucose and [2-13C]glucose
were not different (P>0.10) and interactions (FF×FL) were not significant for all parameters.
Dietary glucose was predominantly oxidised (approx. 80% based on [13C]glucose kinetics and 94%
from indirect calorimetry), regardless of the FL (Table 1). De novo fatty acid synthesis was considered
negligible in milk-fed calves, based on the similar recoveries of CO2 from [U-13C]glucose and [2-
13C]glucose and the low estimates from indirect calorimetry (5.8-8.6% of carbohydrate intake). At
the high FL more dietary glucose (1264 vs. 599 g/d) was catabolised with a corresponding decrease
in the oxidation rates of fatty acids (both fractional and absolute). Instead, most dietary lipid was
transferred into body fat stores, explaining the greater deposition at the higher FL (Van den Borne
et al., 2006). In conclusion, the metabolic fate of dietary carbohydrates in heavy milk-fed calves
is predominantly either oxidation (91-95%) or loss in urine (0-8%). It is speculated that the low
retention of glucose is due to the genetic background or to the high fat intake in ontogenetically
ruminant, but milk-fed, calves.
References
Blum, J.W. and H.M. Hammon, 1999. Endocrine and metabolic aspects in milk-fed calves. Domest. Anim. Endocrinol.
17, 219-230.
Chwalibog, A., K. Jakobsen, S. Henckel and G. Thorbek, 1992. Estimation of quantitative oxidation and fat retention
from carbohydrate, protein and fat in growing pigs. J. Anim. Physiol. Anim. Nutr. 68, 123-135.
Van den Borne, J.J.G.C., M.W.A. Verstegen, S.J.J. Alferink, R.M.M. Giebels and W.J.J. Gerrits, 2006. Effects of feeding
frequency and feeding level on nutrient utilization in heavy preruminant calves. J. Dairy Sci. 89, 3578-3586.
Vicari, T., J.J.G.C. Van den Borne, W.J.J. Gerrits, Y. Zbinden and J.W. Blum, 2007. Postprandial blood hormone and
metabolite concentrations are differentially influenced by feeding frequency and feeding level in veal calves.
Domest. Anim. Endocrinol., in press.

Measurement of fatty acid oxidation in swine using 13C labeled fatty acids
E. van Heugten1, J.J.G.C. van den Borne2, M.W.A. Verstegen2, J. van Milgen3 and W.J.J. Gerrits2
1Department of Animal Science and Interdepartmental Nutrition Program, North Carolina State
University, Box 7621, Raleigh, NC 27695, USA
2Animal Nutrition Group, Wageningen University, PO Box 338, 6700 AH, Wageningen, The
Netherlands
3INRA, UMR1079, Systèmes d’Elevage, Nutrition Animale et Humaine 35590 Saint Gilles,
France
Introduction
Fat is an attractive energy source for pigs because of its high energy density and its low heat
increment compared to starch. In addition, fat composition of the carcass can be manipulated by
supplemental fat to satisfy consumer demands. Halas (2004) indicated that unsaturated dietary
fatty acids are primarily oxidized, whereas saturated fatty acids tend to be deposited. Kloareg et
al. (2005) suggested that 70% of digested n-6 and 50% of digested n-3 fatty acids were retained
in the body. These slaughter balance studies provide valuable information on the net deposition of
nutrients, but do not demonstrate the specific fate of individual nutrients. The aim of the present
study was to measure the fate of 13C labeled glucose and fatty acids in pigs offered diets differing
in fat content and composition.

Six barrows (initial body weight: 31.5 ± 0.60 kg) were individually housed and adjusted to the
experimental diets for 7 d. Diets consisted of a low fat diet (2.0% soy oil), a high saturated fat diet
or a high unsaturated fat diet in which 20% starch was replaced with 9.7 and 9.1% of animal fat or
soy oil, respectively (on a DE basis). The experiment consisted of three trials. Within each trial, 2
pigs were randomly assigned to one of three experimental diets (overall total of 2 observations per
treatment) and housed in 1 of 2 identical respiration chambers for 7 d. Experimental diets were fed
twice daily at a rate of 1200 kJ DE/kg BW0.75/d. Bolus doses of [U-13C] labeled α-linoleic acid
(C18:2; 318 mg) and stearic acid (C18:0; 316 mg) were administered with the feed on d 1 and 3,
respectively, and [U-13C] glucose (53 mg) was administered 1 h after feeding on d 5. Whole body
heat production was measured by indirect calorimetry and 13C enrichment in CO2 by non-dispersive
infrared absorption.

Total heat production was greater (P<0.05) for the low fat diet compared to the high fat diets 6 and
7 h after the afternoon meal and 3 h after the morning meal (Figure 1). The respiratory quotient
(RQ) was greater (P<0.05) for pigs fed the low fat diet compared to those fed the high fat diets
following feeding (Figure 1). Recovery of 13C from glucose (48.2%) did not differ between diets
(data not shown). Enrichment of CO2 with 13C (Figure 2) and recovery of 13C was greater (P=0.02)
for C18:2 (11.3%) than for C18:0 (6.3%). Numerically, recoveries (pooled SEM=2.32) were greater
for the high fat diets compared to the low fat diet for both 18:0 (3.2, 8.9, and 6.9% for low fat, high
animal fat and high vegetable fat, respectively) and 18:2 (7.8, 13.6, and 12.7% for low fat, high
animal fat and high vegetable fat, respectively). Peak 13C enrichment (Figure 2) was reached 15 h
after administration of both C18:0 and C18:2 and returned to base levels at 24 to 26 h after feeding.
For glucose, the peak was already reached at 1 h after feeding and returned to the base level 11 h
after feeding. Thus, in spite of relatively low recoveries, continued oxidation of labeled fatty acids
could be detected past multiple feedings. The 13CO2 enrichment measured in this study reflects the

composite effect of digestion, absorption, and complete oxidation of nutrients. In conclusion, the
pattern of oxidation of dietary fatty acids differs substantially from that of glucose and depends on
the dietary fat content and fat composition. The combined use of 13C labeled fatty acids and indirect
calorimetry therefore provides new and interesting biological information on the fate of dietary
glucose and fatty acids in pigs.
Figure 1. Heat production and respiratory quotient in pigs fed diets low or high in fat (n=2 per
diet). Arrows indicate feeding times. Contrast comparisons (P<0.05) for Starch vs. Animal fat (a),
Starch vs. Vegetable fat (b), and Animal fat vs. Vegetable fat (c) are indicated.
Figure 2. Enrichment with 13C in expired CO2 in pigs (n=6) given an oral bolus of [U-13C] α-linoleic
acid (C18:2) or stearic acid (C18:0) with the first meal (at 16.00 h) or [U-13C] glucose 1 h after
the meal. Isotope was given at time 0 (arrow). Contrast comparisons (P<0.05) for C18:0 vs. C18:2
(a), C18:0 vs. glucose (b), and C18:2 vs. glucose (c) are indicated.
References
Halas, V., 2004. Dietary influences on nutrient partitioning and anatomical body composition of growing pigs. PhD
Thesis, Wageningen University, the Netherlands, 217 pp.
Kloareg, M., J. Noblet and J. Van Milgen, 2005. Deposition of dietary fatty acids, de novo synthesis and anatomical
partitioning of fatty acids in finishing pigs. Br. J. Nutr. 93, 803-811.

Mathematical analysis of human [13CO2]-breath test results: post
prandial fate of amino acids is related to their dietary form
V.V.A.M. Schreurs1, J.A. Nolles1, K. Krawielitzki2 and J. Bujko3
1Human and Animal Physiology Group, Wageningen University, Marijkeweg 40, 6709 PG
Wageningen, The Netherland
2Oskar Kellner Institute, Wilhelm Stahl Allee 2, 18196 Dummerstorf-Rostock, Germany
3Department of Human Nutrition and Consumer Sciences, Warsaw Agricultural University,
Nowoursynowska 159C, 02-776 Warsaw, Poland
Introduction
Dietary free amino acids can replace dietary proteins but both forms of dietary amino acids can
not always be exchanged without physiological consequences. This pilot study with humans used
a [13CO2]-breath test approach to study kinetics of [1-13C]-leucine catabolism during the post
prandial phase of a meal. Bujko et al. (2007) discussed that information obtained in the post prandial
phase can be complementary to information obtained by steady state methods during the fed state.
A mathematical analysis of the breath test response curves was used to quantify nutrition-relevant
differences when free or protein-derived amino acids are present in the diet.

Five students participated in this pilot study, they maintained their personal lifestyle and were tested
without a period of adaptation to the composition of the test meals.
The breath tests were started, after an overnight fast, by ingestion (<15 min) of a test meal (268
kJ amino acids or protein, 860 kJ carbohydrates, 668 kJ fat). The test meals differed only in
composition of their amino acid part. The amino acid part of the meals contained free amino acids
or egg white protein (‘100’ meals) or a 1:1 mixture (‘50’ meals). All meals were labelled with [1-
13C] - leucine. In the EW-meals (EW100, EW50) the label was bound in egg white protein. In the
F-meals (F100, F50) the label was present in free form. So, the EW50 and F50 meals had the same
nutritional composition but differed in the site of labelling. When all egg white in the EW100 meal
was intrinsically labelled the meal contained 36.5 mg [1-13C] - leucine. In the F100 meal 60 mg of
the leucine present was exchanged for the tracer. All subjects received each of the four breath test
meals twice, in random order.
Breath samples were collected just before the meal (blank) and thereafter in 15 or 30 min intervals
during 6 h. The time length of this collection period was arbitrarily chosen but was supposed to
be sufficiently long to monitor post prandial differences. Breath samples were analysed for [13C]-
enrichment (Finnigan Delta C Isotope Ratio Mass Spectrometer). CO2 production of subjects was
derived from basal metabolic rate as based on their metabolic body weight (W0.75). Recovery of
ingested label was expressed as % dose/h.
As previously described (Schreurs and Krawielitzki, 2003) parameters for absorption and oxidation
were derived from the breath test response curves by making use of an iterative mathematical
principle. The ascending (absorption) and descending (oxidation) part of the response curves were
described by exponential functions. These functions were used to calculate values for the half-life
of absorption and oxidation and the percentile oxidation of the tracer dose. The programme provides
Sr-values (rest deviation) to establish statistical differences between the curves.

Expired air became enriched within 15 min (Figure 1). All response curves showed a transient
increase in [13C]-enrichment and reached nearly blank values again within 6 h. For the F-tracer
meals the response till 2 h was significantly higher compared to the EW- tracer meals. The recovery
of tracer was not influenced by the presence of a 1:1 mixture of both dietary forms of amino acids.
The more rapid absorption of free amino acids is associated with a higher oxidative post prandial
loss compared to protein derived amino acids. Mathematical parameters characterising the [13CO2]-
expiration curves (Table 1) can facilitate the physiological interpretation of the breath test results.
In the F-form the tracer had a shorter half-life for absorption and oxidation and a higher level of
oxidation compared to tracer in the EW-form. Higher values for oxidation probably suggest lower
values for post prandial protein synthesis. For the mixed meals all calculated values tended to be
lower values. These lower values are probably due to a lower competition for the tracer at the sites
of absorption and oxidation.
F 100 F 50 EW 50 EW 100
10
recovery (% of dose/hour)
0
0 1 2 3 4 5 6
time (hour)
Figure 1. Recovery of [13CO2] in the breath expressed as percentage of the dose ingested with
different meals containing [1-13C]-leucine in different dietary forms (see text).
Table 1. Mathematical parameters characterising [13CO2]-expiration curves.
EW 50 EW 100 F 50 F 100
t½ absorption, h 0.76 0.88 0.24 0.35

t½ oxidation, h 1.59 1.69 1.38 1.49
Oxidation, % dose 14.2 16.0 18.1 20.1
Sr, % dose/h 0.04 0.04 0.15 0.10
References
Bujko, J., V.V.A.M. Schreurs, J.A. Nolles, A.M. Verreijen, R.E. Koopmanschap and M.W.A. Verstegen, 2007.
Application of a [13CO2]-breath test to study short term amino acid catabolism during the post prandial phase of
a meal. Br. J. Nutr. 97, 891-897.
Schreurs, V.V.A.M. and K. Krawielitzki, 2003. Mathematical analysis of [13CO2]-expiration curves from human
breath tests using [1-13C]-amino acids as oral substrate. In: Souffrant, W.B. and C.C. Metges (edtors), Progress
in research on energy and protein metabolism. Wageningen Academic Publishers, The Netherlands, EAAP publ.
109, 239-242.

Effect of graded dietary tryptophan levels on [1-13C]leucine oxidation
and nitrogen deposition in growing pigs
U. Hennig1, P. Junghans1, J. Bartelt2, C. Relandeau3, A. Tuchscherer1and C.C. Metges1
1Research Institute for the Biology of Farm Animals, Wilhelm-Stahl-Allee 2, 18196 Dummerstorf,
Germany
2Lohmann Animal Health GmbH & Co KG, Heinz-Lohmann-Str. 4, 27472 Cuxhaven, Germany
3Ajinomoto Eurolysine S.A.S., rue de Courcelles 153, 75817 Paris, Cedex 17, France
Introduction
Tryptophan (Trp) is an indispensable amino acid (AA) incorporated in body proteins as muscle,
but also in functional proteins like serotonin. Low dietary levels can limit animal performance
and metabolism. Dietary Trp is known to be a limiting factor of serotonin synthesis. Therefore,
we aimed to estimate the animal requirement of Trp based on the three different methodologies in
a pilot study: serum serotonin, nitrogen (N) balance, and Indicator AA Oxidation (IAAO). Since
IAAO and N deposition are inversely correlated to the dietary concentration of a limiting AA, the
oxidation of [1-13C]leucine as an indicator of the protein synthesis level and the N deposition were
comparably measured in pigs.

Six groups of pigs were fed six equivalent diets based on wheat, corn and soy bean meal with graded
concentrations of standardised ileal digestible (SID) Trp below and above the recent recommendations
(1.33, 1.51, 1.67, 1.82, 2.00, 2.16 g/kg feed, 88% DM) and 10.31 g/kg feed SID lysine for a period
of three wk beginning at 54 d of age (DOA). The designed SID Trp:Lys ratios ranged from 0.13 up
to 0.22. The daily feed intake was increased up to 85 g DM/kg0.75 BW and was kept constant during
the last 10 d of the experiment. The mean intercalated BW ± SD of total 41 castrated male German
Landrace pigs was 24.8 ± 2.8 to 26.8 ± 3.1 kg (unequal class allocation; Table 1).
In the tracer study 22 pigs were continuously fed at 12.00 h half-hourly small meals within 5 h
(planned were 24 randomly selected pigs; two were failed). On 67, 68 and 69 DOA at 8.00 h a 10
h primed continuous i.v. infusion of [1-13C]leucine in 0.9% NaCl solution was started (prime: 5.25
µmol/kg BW, infusion rate: 3.5 µmol/kg BW×h) via the jugular catheter. Plasma was sampled at
15.00 h during the fed steady state to determine α-[1-13C]ketoisocaproic acid enrichment which is
considered to represent the enrichment of the intracellular leucine pool (Matthews et al., 1982). At
71 DOA the serotonin concentration was analysed in fasted serum. The N balance was conducted
between 70 and 76 DOA.
Table 1. Numbers of evaluated animals in the total experiment.
Diets
1 2 3 4 5 6
Tryptophan analysed, g/kg DM 1.84 2.05 2.23 2.40 2.61 2.79
Tracer study 4 4 3 3 4 4
Serum serotonin 7 6 6 5 7 8
N balance study 7 6 7 5 8 8

Results
Serum serotonin levels in the fasted state linearly increased from 600 to 1000 µg/L parallel to increase
of Trp intake per kg0.75BW/d but obviously no plateau was achieved as shown in Figure 1a. The N
deposition linearly increased from 1200 to 1550 mg/kg 0.75BW (y = 2.72 x + 887; slope p=0.0002).
Using a two-phase linear regression crossover analysis according to Robbins et al. (1979) the N
deposition intersected at 211 mg Trp/kg0.75BW intake and ≈ 1480 mg deposited N/kg0.75BW (Figure
1b). This intersection point corresponded to a daily intake of 2.13 g SID Trp and to a concentration
of 2.16 g SID Trp/kg feed, and resulted in a Trp:Lys ratio of 0.21. The 13C-leucine oxidation rate
linearly decreased from 3 to 2 mmol/h (r2=0.116, slope p=0.120) but no intersection point could be
calculated (Figure 2). All data suggest that the Trp requirement of these animals here could be even
equal or higher than the highest Trp concentration tested.
1700
1600
BW/day
1600
y = 3,14 x + 264 1500
2
r = 0.114
0.75
1200 1400
Serotonin, µg/L
N deposition, mg/kg
1300
800 1200
1100
400 1000
900
0 800
100 120 140 160 180 200 220 240 100 120 140 160 180 200 220 240
0.75
Trp intake, mg/kg BW/day Trp intake, mg/kg
0.75
BW/day
Figure
Figure1.1: (a) Serum
Serum serotonin
serotonin in fed
in pigs pigs and (b) N-deposition in pigs fed graded levels of
tryptophan.graded levels of tryptophan Figure 2: N-deposition in pigs fed
graded levels of tryptophan
C-Leucine oxidation, mmol/h
5,0
4,0
3,0
2,0
y = -1,0872x + 5,1531
1,0
0,0
13
1,7 1,9 2,1 2,3 2,5 2,7 2,9

Trp concentration, g/kg DM
13
Figure
Figure 2. 13C-leucine oxidation 3: C-leucine
in pigs oxidation
fed graded levels ofinTryptophan.
pigs fed
graded levels of tryptophan
In conclusion, all three parameters show a high variability typically for population measurements.
However, these results indicate that the minimal tryptophan need of 20 to 30 kg BW pigs tested here
corresponded to a SID Trp:Lys ratio of 0.21. Additional Trp concentration levels higher than 2.45 g
SID Trp/kg DM should be tested to allow reliable intersection point calculations.
References
Robbins, K.R., H.W. Norton and D. H. Baker, 1979. Estimation of nutrient requirements from growth data. J. Nutr.
109, 1710-1714.
Matthews, D.E., H.P. Schwarz, R.D. Yang, K.J. Motil, V.R. Young and D.M. Bier, 1982. Relationship of plasma leucine
and alpha-ketoisocaproate during a L-[1-C-13]-labeled leucine infusion in man. A method for measuring human
intracellular leucine tracer enrichment. Metab. Clin. Exp. 31, 1105-1112.

Endogenous nitrogen flows in the digestive tract of lactating dairy cows:
comparison between estimations using total 15N versus 15N-amino acid
isotope dilution
D.R. Ouellet, R. Berthiaume, R. Martineau and H. Lapierre
Agriculture and Agri-Food Canada, STN Lennoxville, J1M 1Z3, Sherbrooke, QC, Canada
Introduction
One isotope dilution method used to estimate endogenous nitrogen (EN) flows in the intestinal
tract is to infuse 15N-Leu several d to label EN. The EN flows are estimated through the dilution
of the isotopic enrichment (IE) of total-N in the digesta relative to the IE of EN. The challenge is
to adequately determine the IE of EN: EN originate from saliva, gastric juices, bile, pancreatic
secretions, sloughed epithelial cells, and mucin and therefore is not a homogenous entity. First
studies have used the IE of total-N of the trichloro-acetic acid (TCA)-soluble fraction of plasma as
representative of the IE of EN. But its relevance is questionable, even more in ruminants, due to
the important absorption of ammonia and subsequent production of unlabelled urea, present in this
fraction. Therefore, other fractions have been suggested to better represent the IE of EN than the
plasma TCA-soluble fraction, such as the mucosa of the gut and even milk casein, an ‘export’ protein
(Ouellet et al., 2002). In addition, as a result of transamination and return of labeled urea to the gut,
15N is not solely present in Leu but also in other amino acids (AA) and N-containing molecules
and this offers the opportunity to estimate EN using isotope dilution of total-N, Leu or other AA.
Estimations of EN flows obtained with single AA vs. total-N dilution using plasma TCA-soluble
fraction as representative of the IE of EN, however, showed difference in pigs (Lien et al., 1997).
The EN flows estimated using isotope dilution of total-N or single AA needed to be compared, in
cattle, with an appropriate representation of the IE of EN such as gut mucosa.

Four lactating cows (620 ± 31 kg BW and 20.0 ± 6.0 kg/d of milk) equipped with ruminal and
duodenal cannula were fed 14 d to adapt to a silage-based diet before a 6-d total collection of faeces
and urine was performed. On d 19, a jugular infusion of 15N2urea (0.5 mmol/h) was started for 72
h to determine incorporation of urea into bacteria. To label the EN, starting on d 27, an infusion of
15N-Leu (0.45 mmol/h) was performed for 200 h. Feed, duodenal digesta, faeces, intestinal and rumen
mucosa biopsies were sampled on d 34 and 35. The IE of 15N-total was analyzed by combustion-
isotope ratio mass spectrometer (C-IRMS) whereas the IE of 15N-AA was obtained, after protein
hydrolysis, by gas-chromatography-C-IRMS. The EN flows were estimated as previously described
(Ouellet et al., 2002). The IE of the rumen and duodenal mucosa were used as representative of
the IE of EN for pre-duodenal and intestinal EN flows, respectively. To allow comparison between
the different isotope dilution calculations, AA flows were converted as flows of crude protein (CP),
using the composition of abomasal isolate (36, 69, 94, 35, 49 g AA/kg CP for Leu, Asx, Glx, Ile, Ser,
respectively; Ørskov et al., 1986) and of endogenous CP at the terminal ileum of pigs (42.0, 66.2,
80.6, 27.3, 48.2 g AA/kg CP for Leu, Asx, Glx, Ile, Ser, respectively; Jansman et al., 2002). Data
were analysed using the MIXED procedure of SAS (SAS Inst. Inc.), with the method (total-N and
AA-N) and cow as main factors, using the Tukey-Kramer test to separate the means.

Total CP flows at the duodenum and in the faeces averaged 2523 ± 647 and 840 ± 198 g CP/d,
respectively. In contrast to what have been observed in pigs using the plasma TCA-soluble fraction
(Lien et al., 1997), estimations of EN using mucosa as representative of the IE of EN were not different

if estimated with isotope dilution of total-N and Leu, both at duodenal and fecal levels. However,
calculations using the isotope dilution of other AA generally yielded values higher than total-N and
Leu. Contributions of EN to N duodenal flow averaged 0.25 and 0.28 for total-N and Leu. Free EN
at the duodenum represented between 0.34 and 0.64 of total EN, depending on the 15N used. The
proportion of fecal EN originating from undigested EN present at the duodenum was similar among
the isotope dilution methods and was the major contributor to total EN in the faeces.
Table 1. Endogenous duodenal and fecal nitrogen (EN) flows estimated using isotope dilution of
total 15N versus 15N-amino acid and mucosa of the gut as representative of the EN.
Endogenous flow, g CP/d Based on the isotope dilution of 15N

15N-total 15N-Leu 15N-Asx 15N-Glx 15N-Ile 15N-Ser SEM
Duodenal
Total 614a 752ab 1193c 1095bc 1042bc 1084bc 82
In bacteria 367a 376a 785c 400a 463ab 654bc 47
Free 247a 375a 408ab 696b 579ab 429ab 78
Fecal
Total 262ab 215a 383c 349c 363c 321bc 14
Undig. duod.1 186a 173a 246a 214a 253a 211a 22
From intestine 77a 42a 137a 135a 110a 110a 29
1 From undigested duodenal EN; Values in the same row with different letters differ at P<0.05.
Conclusions
These results show that, using an appropriate source to estimate the IE of EN, the mucosa of the gut
in this case, the isotope dilution methods using total-N and Leu yield similar duodenal and fecal EN
estimations. The duodenal and fecal EN estimations calculated from 15N-Asx, 15N-Glx, 15N-Ile or
15N-Ser dilution, however, gave higher values than 15N-total and 15N-Leu.
References
Jansman, A.J.M, W. Smink, P. van Leeuwen and M. Rademacher, 2002. Evaluation through the literature data of the
amount and amino acid composition of basal endogenous crude protein at the terminal ileum of pigs. Anim. Feed
Sci. Tech. 98, 49-60.
Lien, K.A., W.C. Sauer and M.E. Dugan, 1997. Evaluation of the 15N-isotope dilution technique for determining the
recovery of endogenous protein in ileal digesta of pigs: effect of the pattern of blood sampling, precursor pools,
and isotope dilution technique. J. Anim. Sci. 75, 159-169.
Ørskov, E.R., N.A. MacLeod and D.J. Kyle, 1986. Flow of nitrogen from the rumen and abomasum in cattle and sheep
given protein-free nutrients by intragastric infusion. Br. J. Nutr. 56, 241-248.
Ouellet, D.R., M. Demers, G. Zuur, G.E. Lobley, J.R. Seoane, J.V. Nolan and H. Lapierre, 2002. Effect of dietary fiber
on endogenous nitrogen flows in lactating dairy cows. J. Dairy Sci. 85, 3013-3025.

A multi-isotope approach to studying arginine metabolism in neonatal
piglets
K.L. Urschel1, M. Rafii2, P.B. Pencharz1,2 and R.O. Ball1,2
1Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB,
T6G 2P5 Canada
2The Research Institute, Hospital for Sick Children, Toronto, ON, M5G 1X8 Canada
Introduction
In wk-old piglets, the estimated daily metabolic use of arginine for its various metabolic functions,
including protein synthesis, urea cycle function and the synthesis of nitric oxide and creatine is 1.08
g/kg/d, which is higher than the estimated intake of 0.43 g/kg/d from sow’s milk (Wu et al., 2000).
Endogenous arginine synthesis is then critical for meeting the daily metabolic arginine requirement
in piglets. Arginine supplementation improved piglet growth (Kim et al., 2004), providing evidence
that the combination of intake from milk and endogenous synthesis is insufficient for optimal piglet
performance. Proline has been identified as a major arginine precursor, in vivo (R.O. Ball et al.,
2007, unpublished results); however, other precursors may also be important. The objectives of the
present research were to determine the effect of arginine intake on arginine synthesis and the rates
of conversion among proline, ornithine, citrulline and arginine.

On d 0, 10 male piglets (1-2 d old) underwent surgery to implant gastric catheters for diet and isotope
infusion and femoral vein catheters for blood sampling (Urschel et al., 2005). Piglets received a
complete diet until the morning of d 3 and were then allocated to receive either a deficient (-Arg;
0.20 g/kg/d; n=5) or generous (+Arg; 1.80 g/kg/d; n=5) intake of arginine for the remainder of
the trial (Urschel et al., 2005). On d 7, all piglets received a 6-h primed (in µmol/kg), constant (in
µmol/kg/h) intragastric infusion of [guanido-15N2]arginine (prime: 12, constant: 20), [ureido-13C;
5,5-2H2]citrulline (prime: 5, constant: 5), [U-13C5]ornithine (prime: 13, constant: 20) and [15N; U-
13C ]proline (prime: 40, constant: 20). Whole-body fluxes and the rates of conversion of precursor
5
to product amino acid (Qprecursor to product) were calculated using previously described formulas
(Castillo et al., 1993).
Results
The effect of arginine intake on the whole-body metabolism of arginine and its precursors is shown
in Table 1. Of particular interest, is that the rate of total arginine synthesis (Qcitrulline to arginine), was
two times greater in piglets receiving the –Arg diet than in those receiving the +Arg diet. Regardless
of diet, the Qproline to arginine was ~60% of the Qcitrulline to arginine, showing that proline was the primary
arginine precursor. The rate of nitric oxide synthesis (equivalent to the rate of Qarginine to citrulline) was
twice as great in piglets receiving the +Arg versus –Arg diet.
Discussion and implications

The sum of the maximum rate of arginine synthesis (0.51 g/kg/d), and the estimated arginine intake
from sow’s milk (0.43 g/kg/d) was not enough to meet the estimated daily requirements
(Wu et al., 2000), which could explain why arginine supplementation was previously shown to
improve milk replacer-fed piglet growth (Kim et al., 2004). When dietary arginine was plentiful,
there was a greater rate of arginine metabolism to nitric oxide and there appeared to be a lower

rate of net arginine synthesis (Qcitrulline to arginine – Qcitrulline to ornithine) due to a greater hydrolysis of
endogenously synthesised arginine, than when dietary arginine was deficient, where there was a
greater rate of conversion of the precursors to arginine. Arginine is an indispensable amino acid for
piglets and the metabolism of arginine and its precursors is dependent on arginine intake.
Table 1. Whole-body amino acid fluxes and conversion rates1.
+Arg (n=5) -Arg (n=5) Pooled SE
Arginine flux 813 353* 39

Citrulline flux 111 136 12
Ornithine flux 452 191* 34
Proline flux 606 483 49
Qarginine to citrulline (nitric oxide synthesis) 105 46* 10
Qcitrulline to arginine (total arginine synthesis) 67 120* 15
Qcitrulline to ornithine 71 79 10
Qornithine to citrulline 159 142 16
Qornithine to arginine 119 114 14
Qornithine to proline 196 60* 24
Qproline to ornithine 142 112 17
Qproline to citrulline 39 67* 6
Qproline to arginine 42 74* 5
1All values are least squares means in µmol/kg/h. *Denotes an effect of diet (P<0.05).
References
Castillo, L., T.E. Chapman, M. Sanchez, Y.M. Yu, J.F. Burke, A.M. Ajami, J. Vogt and V.R. Young, 1993. Plasma
arginine and citrulline kinetics in adults given adequate and arginine-free diets. Proc. Natl. Acad. Sci. USA 90,
7749-7753.
Kim, S.W., R.L. McPherson and G. Wu, 2004. Dietary arginine supplementation enhances the growth of milk-fed
young pigs. J. Nutr. 134, 625-630.
Urschel, K.L., A.K. Shoveller, P.B. Pencharz and R.O. Ball, 2005. Arginine synthesis does not occur during first-pass
hepatic metabolism in the neonatal piglet. Am. J. Physiol. Endocrinol. Metab. 288, E1244-E1251.
Wu, G., C.J. Meininger, D.A. Knabe, F.W. Bazer and J.M. Rhoads, 2000. Arginine nutrition in development, health
and disease. Curr. Opin. Clin. Nutr. Metab. Care 3, 59-66.

Mutations in metabolic pathways, what role does genetic background play?
J. Marini1, A. Erez2 and B. Lee2
1Baylor College of Medicine, 1100 Bates Street, Houston, TX 77030, USA
2Baylor College of Medicine, 635E One Baylor Plaza, Houston, TX 77030, USA
Introduction
Ornithine transcarbamylase (OTC) is a key enzyme for the synthesis of urea and the endogenous
synthesis of arginine. OTC is present in hepatocytes and enterocytes and catalyzes the synthesis of
citrulline. Although the spf-ash mutation results in a reduction in enzyme abundance, ureagenesis
is maintained if urea cycle intermediates (UCI) are provided (Marini et al., 2006a and b). However,
preliminary work has shown that UCI were not necessary to maintain ureagenesis in a new line of spf-
ash mutant mice. The objective of the present work was to determine the effect of genetic background
in the manifestation of the spf-ash mutation regarding urea, UCI, and nitric oxide production.

The OTCspf-ash mutation, originally present in B6EiC3Sn (B6) mice, was bred into an outbred
mouse line (ICR). After 10 generations of backcrossing, mice were studied utilizing a multiple
tracer protocol during an unbalanced nitrogen load (glycine-alanine, 6.1 mmol/kg/h). A primed
continuous infusion of 13C18O urea, 15N15N arginine and 13C2H4 citrulline was conducted for 4 h to
determine the entry rate of these compounds. Additionally, the conversion of arginine to citrulline
(a measurement of NO production) and citrulline to arginine were calculated. Western analysis to
determine the abundance of OTC protein was performed in hepatic and intestinal tissue; α-tubulin
was utilized to normalize the OTC signal.

A reduction in hepatic and intestinal OTC abundance was observed as a consequence of the spf-ash
mutation (Table 1). Although no difference in the expression of OTC was found in mutant mice of
the two genetic backgrounds, B6 control animals had a higher expression of the enzyme in the liver,
but a lower expression in the intestine when compared to ICR controls. The reduction in intestinal
OTC activity resulted in a reduced citrulline flux in mutant mice from both genetic backgrounds.
Arginine flux, however, was only reduced in B6 spf-ash mice (Table 1). Nitric oxide (arginine to
citrulline flux) production was higher in ICR mice than in animals of the B6 genetic background;
furthermore, a trend (P<0.10) for higher nitric oxide production in ICR mutant mice was observed
when compared to B6 controls, despite the reduction in OTC enzyme activity which was thought
to limit arginine availability for nitric oxide synthesis. ICR mice were able to convert a greater
proportion of the citrulline flux into arginine than B6 mice (72 vs. 63%, P<0.001), which might
explain these observations. The reduction in hepatic OTC abundance and activity seen in spf-ash mice,
resulted in a reduced urea flux only in mice of the B6 background (Table 1). The inability to sustain
ureagenesis under the unbalanced nitrogen load resulted in the development of hyperammonemia
in B6 mutant animals.

Table 1. Ornithine transcarbamylase abundance in liver and intestine, and fluxes of related metabolites
in mice harboring the spf-ash mutation of two different genetic backgrounds.
B61 ICR SEM B3 M B×M
WT spf-ash2 WT spf-ash
Western analysis, abitrary units

Liver OTC 156a 45c 99b 35c 8.6 0.004 0.001 0.027
Intestine OTC 162b 32 c 207a 27c 8.0 0.038 0.001 0.015
Metabolite fluxes, µmol/kg/h

Arginine 477a 376b 515a 513a 23.2 0.001 0.031 0.038
Citrulline 163b 61d 190a 103c 7.1 0.001 0.001 0.308
Urea 5707a 3418b 5717a 5525a 249.4 0.001 0.001 0.001
Arg → Cit 5.3b 4.6b 14.a 7.0b 1.1 0.001 0.001 0.006
Cit → Arg 103b 39d 145a 71c 6.6 0.001 0.001 0.485
Plasma ammonia, µmol/L 85b 671a 52b 146b 61.2 0.001 0.001 0.001
1B6 and ICR: B6EiC3Sn and ICR genetic backgrounds, respectively.

2spf-ash=Ornithine transcarbamylase deficient mice.
3B, M, and B×M, main effects (Background and Mutation) and interaction (Background×Mutation).
Conclusions
Although genetic background did not affect the abundance of OTC enzyme in mutant mice, the fluxes
through this enzyme were very different. Mice of the B6 background produced less citrulline and
urea, which translated into a reduced arginine flux and hyperammonemia. The choice of appropriate
genetic background for the expression of mutations in metabolic pathways is crucial for the study
of these complex processes.
References
Marini, J.C., B. Lee and P.J. Garlick, 2006a. Reduced ornithine transcarbamylase activity does not impair ureagenesis
in Otcspf-ash mice. J. Nutr. 136, 1017-1020.
Marini, J.C., B. Lee and P.J. Garlick, 2006b. Ornithine restores ureagenesis capacity and mitigates hyperammonemia
in Otcspf-ash mice. J. Nutr. 136, 1834-1838.

Gut function of pigs in relation to weaning
P.J.A. Wijtten1, H. Bouritius2, P.R.T. Bonekamp1, G.J. Witte2, J.J. Verstijnen1 and T.A.T.G.
van Kempen3
1Provimi B.V., P.O. Box 5063, 3008 AB, Rotterdam, The Netherlands
2Numico Research B.V., P.O. Box 7005, 6700 CA, Wageningen, The Netherlands
3Provimi RTC, Lenneke Marelaan 2, 1932, Sint-Stevens-Woluwe, Belgium
Introduction
Immediately post-weaning the gut of the pig is subjected to a degenerative phase of 3 to 5 d followed
by a regenerative phase with rapid growth and maturation of enterocytes. Gut development post-
weaning has been studied using villous height and crypt depth as important parameters. Ussing
chambers have been used to study permeability of gut mucosa in vitro. These parameters can only
be obtained in terminal experiments and are labour intensive. In human medicine dual-sugar tests are
used to study the ratio between disaccharides (para-cellular transport) and monosaccharides (mainly
trans-cellular transport) in vivo (Uil et al., 1997). The ratio of these sugars detected in the urine gives
information of changes in gut permeability. Plasma citrulline was recently identified as a quantitative
marker for enterocyte mass (Crenn et al., 2003). The present study was conducted to see whether
citrulline and the dual-sugar test are helpful markers to characterise gut function post-weaning.

Twenty-four barrows were weaned at a mean age of 20 d and individually housed. All pigs had free
access to water and a pelleted nursery diet. For the dual-sugar test and blood sampling, the pigs were
divided into 3 equal groups of 8 pigs each. This minimises the effect of pig handling and residuals
of sugars in the urine of previous test d (each pig is only handled once per 3 d). The dual sugar test
was performed on d 0, 3, 6, 9, and 12 post-weaning for group A, on d 1, 4, 7, 10, and 13 for group B
and on d 2, 5, 8, 11, and 14 for group C. On test d, the pigs were deprived of feed from 2 h before till
2 h after sugar administration. Each pig was intragastrically dosed with a sugar solution containing
about 0.6 g/kg BW0.75 lactulose (disaccharide) and 0.04 g/kg BW0.75 rhamnose (monosaccharide).
Urine was collected until at least 9 h post sugar administration. Blood samples were taken from the
jugular vein of all pigs at 1 d before weaning and at 1 h post sugar administration. The urine and
blood plasma were stored at -20 °C. Sugar contents in the urine were analysed with high-pressure
anion-exchange chromatography and citrulline in blood plasma with HPLC.

At weaning the average weight of the pigs was 6.8 kg and average weight gain was 25 g/d from 0-7
d and 219 g/d from 7-14 d post-weaning. For all groups the L/R ratio was significantly (P<0.05)
higher on 2 or 3 test d during the second wk post-weaning compared with the first test d of each
group (Figure1). This might indicate an increased para-cellular permeability especially in the second
wk post-weaning. Permeability tests with Ussing chambers described in the literature have mainly
focussed on the first wk post-weaning and give conflicting results (Lallès et al., 2004). Permeability
problems in the present study were a lot later in time than what was expected based on the literature.
This discrepancy may be explained by the hypothesis that permeability is a function of recovery rather
than damage. The increased L/R ratio in our experiment might be related to the increase of mitotic
activity towards the end of the first wk post-weaning as has been described by Van Dijk (2001).
It can be hypothesised that the higher mitotic activity results in more immature enterocytes in which
tight junctions are not fully developed and para-cellular permeability might be increased. This is in

line with observations that diarrhoea problems and oedema disease generally occur in the second
wk post-weaning. For all groups, plasma citrulline levels were lower (P<0.05) on all d post-weaning
compared to one d before weaning (Figure 2) and numerically increased again towards the end of
the second wk post-weaning. In conclusion, L/R ratios and citrulline might be useful parameters to
study gut function post-weaning.
3,0 Group A 3,0 Group B a 3,0 Group C a

a
L/R (mol/mol)
a ab
2,0 b 2,0 2,0 abc
bc
1,0 bc 1,0 b 1,0 b
b c
c
0,0 0,0 0,0
0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14
Days post weaning Days post weaning Days post weaning
Figure 1. L/R ratios in the urine of pigs during the first two wk post-weaning.
140 a 140 140

a a
120 120
Citrulline (µmol/L)
Group A Group B 120 Group C

100 b 100 100
c b
bc b b bc bc
80 c c c 80 80
bc c c c
60 60 60
40 40 40
-2 0 2 4 6 8 10 12 14 -2 0 2 4 6 8 10 12 14 -2 0 2 4 6 8 10 12 14
Days post weaning Days post weaning Days post weaning
Figure 2. Citrulline concentrations in blood plasma from 1 d before until 14 d post-weaning.
References
Crenn, P., K. Vahedi, A. Lavergne-Slove, L. Cynober, C. Matuchansky and B. Messing, 2003. Plasma citrulline: a
marker of enterocyte mass in villous atrophy-associated small bowel disease. Gastroenterology 124, 1210-1219.
Lallès, J.-P., G. Boundry, C. Favier, N. Le Floc’H, I. Luron, L. Montagne, I.P. Oswald, S. Pié, C. Piel and B. Sève,
2004. Gut function and dysfunction in young pigs: physiology. Anim. Res. 53, 301-316.
Uil, J.J., R.M. Van Elburg, F.M. Van Overbeek, C.J.J. Mulder, G.P. Vanberghe-Henegouwen and H.S.A. Heymans, 1997.
Clinical implication of the sugar absorption test: intestinal permeability test to assess mucosal barrier function.
Scand. J. Gastroenterol. 223, 70-78.
Van Dijk, A.J., 2001. Spray-dried animal plasma in the diet of weanling piglets: influence on growth performance and
underlying mechanisms. PhD Thesis, Utrecht University, The Netherlands.

Combination of two approaches to study the mammary use of glucose for
lactose synthesis in dairy cows: net mammary balance and RNA levels
M. Boutinaud, M.H. Ben Chedly, E. Delamaire and J. Guinard-Flament
INRA, Agrocampus Rennes, UMR1080, Dairy Production, F-35590 St-Gilles, France
Introduction
The net mammary balance is a quantitative approach that produces comprehensive results about the
mammary uptake of nutrients. However, the study of the intramammary fate of nutrients remains
difficult in animals equipped for this approach. A non-invasive method for cell collection was
developed previously to analyse mammary gene expression in goat milk (Boutinaud et al., 2002).
This method has now been improved so that purified mammary epithelial cells (MEC) can be prepared
from cow milk. This paper constitutes the first study that combines the use of mammary epithelial
cells isolated from milk and the net mammary balance approach in order to better understand the
mammary use of plasma glucose for lactose synthesis. Since lactose is the major osmotic agent of
milk, its synthesis can regulate the milk volume. The aim of this study was thus to determine the
mechanisms underlying milk yield reductions in response to feed restriction and once daily milking
(ODM).

Five multiparous Prim’Holstein cows in their 5-6 th lactation mo were subjected to a double Latin
square under which the cows were milked once or twice daily while being fed a diet providing either
98% or 70% of their needs as determined the wk before the trial. Cows were fitted with an ultrasonic
flow probe implanted around the left external pudic artery and two permanent catheters were inserted
into the left carotid and the left subcutaneous abdominal vein, to determine the quantities of glucose
taken up from blood plasma (Guinard et al., 1994). On d 7 of each experimental wk, milk and lactose
yields and the net mammary balance for glucose were measured. Cells were isolated from fresh milk
by centrifugation (Boutinaud et al., 2002) and MEC were purified using magnetic beads (Alcorn et al.,
2002) associated with an anti Cytokeratin 8 antibody. Total RNA were extracted from purified milk
MEC using the Trizol Reagent. Real-time RT-PCR was performed to determine variations in mRNA
levels of genes involved in two critical steps for lactose synthesis, glucose transmembrane transport
(GLUT-1 and SGLT-1) and the last enzymatic reaction which is catalysed by galactosyltransferase
(GAT-(1,4) and its co-factor α−Lactalbumin (α-LA). The mRNA levels were expressed as a ratio
of cyclophilin mRNA level. The data were analysed using the SAS mixed procedure, considering
the effects of cows, periods, feeding levels, sub-periods, milking frequencies and the interaction
between feeding levels and milking frequencies.

The results are given in Table 1. Feed restriction and ODM provoked significant reductions in milk
yield (-3.4 and -5.0 kg/d, respectively) associated with a decrease in lactose yield (-181 and -274 g/d,
respectively) as had been expected (Guinard et al., 2006). The mammary uptake of glucose decreased
under both treatments (on average -22% under both treatments) as a result of depressed mammary
blood flow (-16 and -14%, respectively, for feed restriction and ODM) and different modifications
of mRNA level according to the treatments. The decrease in glucose mammary uptake under feed
restriction was associated with a drop in mRNA levels of glucose transmembrane transporters
(significant -53% reduction for GLUT-1 and a tendency for -50% reduction for SGLT-1). Feed
restriction had no effect on either α-LA or GAT-(1,4) mRNA levels. Thus under feed restriction the
glucose availability for lactose synthesis may have been a limiting factor for milk production. The

decrease in glucose mammary uptake during ODM was associated with a decrease in AVD (-9%)
due to a decrease in glucose extraction rate (-12%). During ODM, α-LA mRNA level decreased
(-73%) with no change in GAT-(1,4), SGLT-1 and GLUT-1 mRNA levels. ODM appeared to act
on activity of the lactose synthase enzyme, as reported by Wilde and Knight (1990) regarding the
regulation of the α-LA co-enzyme at a transcriptional level. During ODM, the further metabolic use
of glucose may have limited glucose uptake. The two approaches, net mammary balance and mRNA
levels in purified MEC, produced consistent results. They suggested that with the two treatments, the
glucose uptake and lactose production by the mammary gland were reduced under one similar and
two different mechanisms: a common reduction in mammary blood flow, transcription regulations of
glucose transporters under feed restriction and of α-LA under ODM. Thus the analysis of mammary
epithelial cells obtained from milk is a convenient method to combine studies on mammary balance
and gene expression, producing interesting and comprehensive results which can be used to examine
the effect of breeding practices on the regulation of mammary nutrient use.
Table 1. Effect of milking frequency (MF) and feeding level (FL, 98% vs. 70%) on milk and lactose
yields, mammary blood flow and glucose mammary balance, and on mRNA levels of genes involved
in lactose synthesis in purified Mammary Epithelial Cells on d 7 of treatments.
98% 70% SEM Effect
TDM1 ODM1 TDM1 ODM1 MF FL MF×FL
Milk yield, kg/d 27.0 21.5 23.1 18.7 0.62 *** * NS

Milk lactose yield, g/d 1316 1027 1120 861 28.0 *** * NS
Mammary blood flow2, 11.9 10.3 10.0 8.6 0.45 * * NS
L/min
Glu. AVD, mmol/L 0.86 0.79 0.83 0.75 0.038 * NS NS
Glu. Extraction rate, % 21.5 18.8 21.0 18.5 0.72 ** NS NS
Glu. Mammary uptake 2, 7.46 5.84 6.00 4.50 0.244 *** ** NS
mmol/min
α-LA mRNA 63.3 24.5 82.3 14.7 12.11 ** NS NS
GAT-(1,4) mRNA 94.6 104.8 75.5 95.1 15.96 NS NS NS
GLUT-1 mRNA 568 362 186 225 56.5 NS * NS
SGLT-1 mRNA 13.4 5.0 3.9 4.1 1.89 NS † †
1Twice Daily Milking (TDM), Once Daily Milking (ODM). 2Estimated from measured performed in the right half-
udder; †P≤0.10, *P≤0.05, **P≤0.01, ***P≤0.001.
References
Alcorn J., X. Lu, J.A. Moscow and P.J. Mcnamara, 2002. Transporter Gene Expression in Lactating Human Mammary
Epithelial Cells Transcription-Polymerase Chain Reaction. J. Pharmacol. Exp. Ther. 303, 487-496.
Boutinaud, M., H. Rulquin, D.H. Keisler, J. Djiane and H. Jammes, 2002. Use of somatic cells from goat milk for
dynamic studies of gene expression in the mammary gland. J. Anim. Sci. 80, 1258-1269.
Guinard, J., H. Rulquin and R. Vérité, 1994. Effect of graded levels of duodenal infusions of casein on mammary
uptake in lactating cows. 1 major nutrient. J. Dairy Sci. 77, 2221-2231.
Guinard-Flament, J., E. Delamaire, S. Lemosquet, M. Boutinaud and Y. David, 2006. Changes in mammary uptake
and metabolic fate of glucose with once-daily milking and feed restriction in dairy cows. Reprod. Nutr. Dev. 5,
589–598.
Wilde, C.J. and C.H. Knight, 1990. Milk yield and mammary function in goats during and after once-daily milking.
J. Dairy Res. 57, 441-447.

Molecular markers for a delicate balance between protein and lipid
metabolism in subcutaneous fat tissue of beef cow
A. Shabtay1, H. Eitam1, A. Orlov1, Y. Aharoni1, I. Izhaki2 and A. Brosh1
1Department of Cattle and Genetic Sciences, Institute of Animal Science, ARO, Newe Ya’ar Research
Center, P. O. Box 1021, Ramat Yishay 30095, Israel
2Department of Biology, University of Haifa at Oranim, 36006 Tivon, Israel
Introduction
Various types of stresses may account for the decreased weaning rates of beef cattle herds in Israel.
Among these stresses, reduced protein levels in the diet, especially during the hot and dry seasons
may be of a high probability to occur. Low-protein intake limits the utilisation of high roughage diet
as a source of energy, due to low supply of nitrogen to the rumen microbes. Under protein deficient
diets, dry matter intake and digestibility in turn fall below requirements for maintenance, leading to
detrimental effects on energy balance, performance, milk production and weight gains. Heat shock
proteins (HSP) are a subset of molecular chaperones that are involved in ‘house keeping’ functions
in the cell (protein folding, protein assembly and translocation between compartments). In addition,
they work in accordance with the proteasome machinery to degrade misfolded or aggregated proteins.
Fatty acid binding proteins (FABP; P15) are small, abundantly expressed, cytoplasmic proteins, which
among other roles mediate lipolysis and regulate the release of fatty acids from adipose tissue. Since
no indications are available for the continuous decrease in weaning rate, the present study was an
attempt to develop specific molecular markers for low-protein intake, a highly probable challenge
of free grazing cattle.

We studied the expression profile of HSP, C2 proteasome subunit and FABP in subcutaneous
fat tissue of gestated beef cows fed limited protein concentration (6% instead of 11%) in an iso-
energetic, ad libitum, high-fibre diet. Subcutaneous fat biopsies were sampled (and directly frozen
in liquid nitrogen) from the para lumbar fosa, after a local, epinephrine free anesthesia. Cows were
placed individually, to allow measurements of individual feed intake. We used the heart rate-oxygen
consumption (HR-VO2) method to measure energy expenditure (EE) and to estimate energy balance
of 6 individuals. EE was calculated from 4 d heart rate measurements multiplied by the oxygen
consumption per heart beat and by 20.47 kJ per liter VO2. The expression pattern of HSP and C2
proteasome subunit were obtained by Western blot analysis, using mouse monoclonal and rabbit
polyclonal antibodies, respectively. The identification of FABP was achieved after a separation
of subcutaneous fat protein extraction on 1D SDS-PAGE. The 15 kDa band was cut from the gel,
digested by trypsin, analysed by LC-MS/MS on LTQ Orbitrap and identified by Pep-Miner and
Sequest software against human, mouse, rat, bovine and rabbit databases.

After 3 wk on low-protein diet, dry matter intake and heart rate decreased significantly, and most
cows reached a negative energy balance, which was also reflected in their body condition score.
After 3 mo on low-protein diet, dry matter intake decreased further (Figure 1), but energy balance
went slightly higher as was also reflected in body condition score. At this time-point the expression
of HSP70, HSP90 and C2 proteasome subunit decreased markedly, while FABP levels increased
significantly (Figure 2).

30,00
25,00
Food Intake (g DM/kgBW*d)

20,00
15,00
10,00
5,00
0,00
control diet low protein diet-3 weeks low protein diet-3 months
Figure 1. Food intake of gestating beef cows (n=6) fed control (11%) or low-protein (6%) diet. The
results are presented as means ± std.
Figure 2. Representative protein expression pattern of HSP, C2 proteasome subunit and FABP in
subcutaneous fat of gestating control (C) and experiment (Exp) beef cows fed 3 mo normal (11%)
or low-protein (6%) diet.
While being involved in protein synthesis and protein degradation, HSP70, HSP90 and the proteasome
machinery promote protein turnover in the cell. HSP also serve as stress protein, being up-regulated
in response to various stimuli. Theoretically, a stress caused by protein demand, as presented in
this study, could have evoked a typical over-expression of HSP. However, such response would be
metabolically ‘expensive’, and it seems, at the level of subcutaneous fat tissue, that cows deal with
low protein intake by reducing protein turnover. On the contrary, low-protein intake adversely affects
energy balance, and increases the need for energy from internal sources. This, in turn promotes the
expression of FABP that participate in lipid degradation. Our results provide sensitive molecular
markers and indicate a new set-point for a delicate balance between protein and lipid metabolism
in subcutaneous fat of protein-deprived beef cattle.

Part 3. ‘Omics’ in metabolism and nutrition studies
Lexic of the ‘Omics’
J.F. Hocquette
INRA, UR1213, Unité de Recherches sur les Herbivores, Theix 63122 Saint-Genès Champanelle,
France
Genomics has brought with it a true biological revolution and can be applied to all areas of the
life sciences. Genomics is, however, sometimes misunderstood as well as its present and potential
applications for instance in metabolism and nutrition which is the subject of this symposium. The
aim of this short introduction of the session entitled ‘Omics in metabolism and nutrition studies’ is
to define basic words or expression, which are associated with the advent of ‘omics’ disciplines. The
definitions indicated in italic types have been proposed on the following website: http://en.wikipedia.
org/wiki/Main_Page. The others are taken from some key review papers (Greenbaum et al., 2001;
Hocquette, 2005).
Before genomics, molecular biology aimed at investigating single genes, their structure, functions and
roles. In other words, molecular biology is the study of biology at a molecular level. It is today very
common in biological research but cannot be said to be genomics. A weakness of many molecular
biology approaches is to study genes in isolation from the context of others.
Genomics is the study of an organism’s entire genome. The definition of genomics thus refers to
that of the genome. In biology the genome of an organism is its whole hereditary information and is
encoded in the DNA (or, for some viruses, RNA). The genome thus includes both the genes and the
non-coding sequences of the DNA. Whereas the term ‘genome’ appeared in the literature in 1920-
1930, the term ‘genomics’ appeared in the 1980s, and took off in the 1990s with the initiation and
development of genome projects for several biological species. The advent of genomics is thus linked
to the development of high-throughput techniques that allows studying the genome of organisms
as a whole. In other words, genomics provide scientists with methods to quickly analyse genes and
their products. Others define genomics as the identification of organisms’ genes and understanding
how the genes work using new biotechnological approaches.
One major consequence of the advent of genomics is that, today, scientists have the opportunity to
analyse at the genome level interactions between genes (and between their products) and therefore
to understand on a large-scale basis the interactions between the various systems of a cell. These
include the interrelationship of its DNA, RNA and synthesised proteins and learning how these
interactions are regulated.
Another consequence of the development of genomics comes from the increasing amount of
information derived from high-throughput studies. Genomic data need to be analysed and this has
favoured the development of bioinformatics, a term that refers to the new science of understanding
and organising large amounts of complex biological information.
As indicated before, genomics was mainly concerned historically with sequencing the genomes
of various organisms. DNA sequencing is the process of determining the nucleotide order of a
given DNA fragment. The sequence of DNA encodes the necessary information for living things
to survive and reproduce. The number of sequenced genomes is increasing rapidly (Figure 1). It
originally concerned mainly viruses and to a lesser extent bacterial species, but more recently also
some eukaryote organisms. A rough draft of the human genome was completed by the Human
Genome Project in early 2001 (http://www.hgsc.bcm.tmc.edu/projects/human/), and a draft of the
bovine genome is today available (http://www.hgsc.bcm.tmc.edu/projects/bovine). Comparison of
genomes (also called comparative genomics) has resulted in many progress (discovery of genes and of

500
450
400
350
300
250
200
150
100
50
0
1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006
Figure 1. Evolution of the number of completely sequenced genomes from 1995 to 2006 (from http://
genomesonline.org/gold_statistics.htm and from Cahiers Agriculture 16 (3), 163-169).
regulatory DNA sequences for instance). Having sequenced the genomes of several different species,
genomics
1400 techniques now allow us to identify polymorphisms and to study gene structure. It is the
characterisation of the physical nature and of the structure of whole genomes. This is the reason why
this discipline
1200 used to be called by some authors ‘structural genomics’. This definition is, however,
a matter of controversy because structural genomics consists nowadays in the determination of the
1000 Transcriptom*
three dimensional structure of all proteins of a given organism.
800 Metabolom*
After the sequencing of a great number of genomes, genomics is now shifting to the study of gene
expression and function. This is called by some authors the ‘post-genomic area’. Others called this
600
new discipline ‘functional genomics’. This is in fact a new field that attempts to make use of the vast
400
wealth of data produced by genomic projects (such as genome sequencing projects) to describe gene
(and protein)
200 functions and interactions. In other words, functional genomics allows the detection
of genes that are turned on or turned off at any given time and in any physiological or nutritional
situation.0 Thus, unlike basic genomics, functional genomics focus on the dynamic aspects such as
1995 1996 1997
gene transcription, translation, and1998 1999 2000
gene interactions, 2001
as opposed 2002 2003
to the static 2004
aspects of2005 2006
the genomic
information such as DNA sequence or structures.
One key target of functional genomics is the transcriptome, which is the set of all messenger RNA
(mRNA) molecules, or ‘transcripts’, produced in one or a population of cells. The term ‘transcriptome’
can be applied to the total set of transcripts in a particular cell type, in a whole tissue or in a given
organism. Unlike the genome, which is roughly fixed for one individual, the transcriptome can
vary with external factors. In other words, the transcriptome reflects the genes that are expressed
at any given time in any specific condition. The study of the transcriptome is possible thanks to
the development of high-throughput techniques for gene expression studies based for instance
on DNA microarray technology. The Science which aims to study the transcriptome is called
transcriptomics.
Similarly, proteomics is the large-scale study of proteins, particularly their structures and functions.
The term ‘proteomics’ was coined to make an analogy with genomics, the study of the genes.
Whether proteomics belongs or not to functional genomics is a matter of debate, but it seems that
it does not from the major part of the literature. The word ‘proteome’ derives from ‘proteins’ with
the suffix -ome which means ‘as a whole’. Since the genome of an organism is its set of genes, and
the transcriptome its set of transcripts, so its proteome is its set of proteins (produced by these gene
transcription and traduction). In this context, ‘functional proteomics’ aims at comparing proteomes to
understand how they change in response to changes at the genomic level or in respect to a treatment.
Indeed, whereas one given organism has only one genome, it has infinity of transcriptomes and

of proteomes because the expression of transcripts and proteins from a given set of genes varies
with time, age, physiological conditions and other environmental factors. Some scientists are more
interested in proteomics than in genomics because it can be argued that this gives a much better
understanding of an organism than genomics. Indeed, transcripts (mRNA) may be degraded rapidly or
translated inefficiently, resulting in a poor level of synthesised proteins. In addition, post-translational
modifications of proteins may profoundly affect their activities. Additional methods associated
with proteomics (for instance phosphoproteomics which study phosphorylation of all proteins and
glycoproteomics which study their glycolyslation on a large scale) are used to better understand
protein modifications and activities. It is also possible that one transcript may give rise to more than
one protein, and different proteins may have to combine together in an enzymatic complex to exert a
biological activity. Another complexity of proteomics is that an organism may contain only 20 000
to 25 000 genes, but these can transcribe upwards of 1 million proteins, thus making proteomics
more ambitious than genomics.
In line with the previous definitions, the metabolome represents the collection of all metabolites
in a biological organism, which are the end products of its gene expression. Metabolomics is the
comprehensive analysis of the whole metabolome under a given set of conditions. Metabonomics is
the quantitative measurement of time-related multiparametric metabolic responses of multicellular
systems to pathophysiological stimuli or genetic modifications. In other words, unlike metabolomics,
metabonomics examines the changes of metabolites in a tissue or a biofluid. This subtle difference is,
however, not shared by all experts. There seems to be no doubt about the strength of the metabolomics/
metabonomics approach in plant and microbial systems where metabolites are more easily sampled
compared to mammals for which blood/plasma or urine are the most studied biofluids.
Generally speaking, users of the suffix ‘-ome’ frequently take it as referring to totality of some sort.
Some Omics are well established while others are speculative. The scientific community keeps or
rejects each new term depending upon their continued usage or avoidance in the scientific literature.
For instance, the wider view of physiological mechanisms is called the study of the ‘Physiome’, a
word which comes from ‘physio’ (which means life) and ‘ome’ (as a whole) by analogy with genome.
The Physiome is by definition the ‘quantitative description of the physiological dynamics or functions
of the whole organism’ (http://www.physiome.org/). The most common terms found in the literature
with the suffix ‘-ome’ are genome, transcriptome, proteome, metabolome (these are words which
were cited in PubMed for the first time between 1995 and 1998) and also interactome (which is the
set of protein interactions or a list of interactions between all macromolecules in a cell). The use of
these terms is rapidly increasing in the scientific literature demonstrating the scientific revolution
that is occurring thanks to the development of the ‘omic’ disciplines (Figure 2).
The last discipline to be defined is ‘systems biology’ which aims to integrate proteomic, transcriptomic,
and metabolomic information to give a more complete picture of living organisms. Generally
speaking, with the increasing desire to find biologically meaningful patterns by genomic approaches,
bioinformatics is crucial not only for the analysis of genomic data but also for data integration
from genomes to phenotypes. This new discipline is clearly in development but bears with it great
potential for the future.
In conclusion, there is no doubt that the ‘omics’ approaches have already and will continue to provide
an explosion in our understanding of Biology. This is no more so than in the area of nutrition. Here,
arguably, the application of ‘omics’ technologies has started to bring nutrition (which hitherto has been
mainly considered a ‘Soft’ or ‘Cinderella’ science) into the mainstream of the Life Sciences. Thus,
the ability to examine at the molecular level how nutrition (or specific nutrients) can affect metabolic
pathways, hormonal signals and the whole balance of homeostasis should eventually provide means
of distinguishing sustainable health from the early steps in the development of disease, not to mention
the ability to identify from an individual’s genetic make-up whether he/she is at risk of developing

1400
1200
1000 Transcriptom*
800 Metabolom*
600
400
200
0
1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006
Figure 2. Annual number of scientific publications in the data base ISI Web of Science with the term
‘transcriptom*’ (including transcriptome, transcriptomics, etc.) or the term ‘métabolom*’ (including
metabolome, metabolomics, etc.) in the title, the keywords or the abstract between 1995 and 2006.
From Cahiers Agriculture 16 (3), 163-169..
particular diseases. This approach, which has been termed nutrigenomics, has been defined as the
application of the sciences of genomics, transcriptomics, proteomics and metabolomics to nutrition,
especially to the relationship between nutrition and health in human beings. At the biomedical level,
this has started to identify how nutrients can act like pharmaceuticals in the early stages of disease
development and has started to spin-out concepts such as ‘functional foods’ to describe those dietary
ingredients that provide important health-sustaining nutrition.
References
Greenbaum, D., N.M. Luscombe, R. Jansen, J. Qian and M. Gerstein, 2001. Interrelating different types of genomic
data, from proteome to secretome: ‘Oming in on function. Genome Res. 11, 1463-1468.
Hocquette, J.F., 2005. Where are we in genomics? J. Physiol. Pharmacol. 56 (Suppl. 3), 37-70.

Nutrigenomics: techniques and applications
A. Scalbert1, D. Milenkovic1, R. Llorach#1, C. Manach1 and C. Leroux2
1Unité de Nutrition Humaine, INRA, Centre de Recherche de Clermont-Theix, 63122 Saint-Genès-
Champanelle, France
2Unité de Recherches sur les Herbivores, INRA, UR1213 Centre de Recherche de Clermont-Theix,
63122 Saint-Genès-Champanelle, France
Abstract
The characterisation of phenotypes in nutrition research has so far largely relied on the estimation of
a limited number of markers. The techniques available today allow the simultaneous estimation of the
expression of the whole genome or the concentrations of several hundreds of proteins or metabolites
in a given sample. These techniques open considerable perspective to describe subtle metabolic
variations related to health and diseases or induced by the consumption of nutrients, foods or diets.
The techniques of transcriptomics are mature whereas those used for proteomics and metabolomics
are still evolving rapidly. They are briefly reviewed and examples of nutrigenomic applications
illustrate how they can contribute to the characterisation of phenotypes and to the elucidation of the
mechanisms of action of nutrients in human health and disease and livestock management.
Keywords: nutrigenomics, transcriptomics, metabolomics, nutrition, health
Introduction
The scope of nutrition research has very much evolved during the last 20 years from the satisfaction
of basic metabolic needs for building and maintaining the body functions over life span, to the
understanding of the regulation of the metabolism by nutrients and the diet and its consequence on
human health. Health is largely influenced by the diet and a proper diet helps prevent various diseases,
such as cardiovascular diseases, non-insulino-dependant diabetes, or osteoporosis. These diseases
result from chronic metabolic imbalances and it is therefore essential to understand how metabolism
is affected by diet and dietary factors and how these metabolic changes influence health.
Nutrients may not only affect metabolism through allosteric regulation of enzymes or modulation of
hormone secretion but also at the level of gene expression (Kaput and Rodriguez, 2004). Progress
in molecular biology has led to the identification of key signalling pathways modulated by macro-
or micronutrients. Nutrients can bind to nuclear receptors that act as sensors for various dietary
ligands. They can also inhibit the phosphorylation of different kinases involved in cell signalling
pathways. They may also act in an indirect way. As substrates, some nutrients also modify the
relative concentrations of endogenous metabolites, and particularly of some metabolites involved
in cell signalling. Activation or inhibition of these cell-signalling pathways modulate in turn the
expression of a large diversity of genes, eventually translated into metabolic or regulatory proteins
with various metabolic consequences. Metabolic homeostasis can thus be influenced both on a short
and long term basis.
Embracing the complexity of these metabolic effects in a global way has been a long-lasting
challenge. The sequencing of the whole human genome (Venter et al., 2001), more recently the
chicken and bovine genomes and very soon the porcine genome has made possible the study of
#RafaelLlorach was firstly holder by a grant from Ministerio de Educación y Ciencia (Spain) (EX2005-0310) and
secondly holder by a contract from Instituto Carlos III, programma F.I.S. (CD06/00161).

gene structure and function on a whole genome scale. These studies belong to genomics. The
suffix -ome- refers to a totality. The transcriptome, proteome and metabolome are respectively the
totality of transcripts, proteins and metabolites in a given cell or tissue at a given time. Genomics
encompasses both the determination of the sequence of the genome and their comparison between
individuals or species. Beyond the structure of the genome, genomics is also concerned with gene
function: functional genomics covers gene transcription into mRNA (transcriptomics), translation
into proteins (proteomics) and protein-protein interactions. Structural genomics consists in the
determination of the 3-dimensional structure of all proteins in a species on a genome-wide scale.
Modern analytical techniques also allow to simultaneously analyse thousands of metabolites in
metabolomics studies. All these studies, when applied to nutrition research, are parts of nutrigenomics
(Müller and Kersten, 2003).
Systems biology aims at integrating these different omics dimensions to characterise the function
of whole biological systems, taking into account all interactions between the components of the
system (Ideker et al., 2001a).
Genomics appears particularly adapted for exploring the complex links between diet and health.
Firstly the large amount of data generated by such approaches allows characterising a metabolic
state with a much greater accuracy. Early metabolic imbalances can be detected before a disease
is diagnosed better than with commonly used surrogate markers. Such an assessment has been
based so far on a limited number of biomarkers. For example, blood cholesterol correlates to the
risk of atherosclerosis and its level is influenced by the diet. A reduction of blood cholesterol in
populations has become a public health objective to reduce the incidence of cardiovascular diseases.
However, other independent risk factors are also known for cardiovascular diseases and it appears
today unrealistic to rely on a single or too limited number of biomarkers for evaluating risks of such
multifactorial diseases.
Secondly, Genomics may allow the identification of how the diet influences these imbalances to reach
a more healthy metabolic state (German et al., 2003). Known biomarkers are clearly insufficient
to evaluate the influence of the diet or nutrients on disease risk. Nutrients differ from drugs by the
diversity and often low magnitude of their metabolic effects. A specific biomarker may or may not
respond depending on genetic, physiological or environmental factors. Thirdly, assessing the role of
the diet in disease prevention must be global to determine its effects on any pathways that could lead
to disease. The considerable information generated by omics approaches already allows characterising
metabolic states with far more precision than the classical approaches. Sets of biomarkers (transcripts,
proteins or metabolites) can be extracted from metabolic fingerprints (also called metabolic signatures)
and used routinely for metabolic assessment (Fiehn, 2002; Müller and Kersten, 2003).
In livestock species, nutrigenomics seeks to describe the complex biological processes involved
in assimilation and processing of nutrients by various tissues and organs, and to quantify nutrient
movement (flux) through those processes. Applied nutrition is the selection and proportioning of
feedstuffs and ingredients to supply the correct amounts and balance of nutrients required for optimal
productive and reproductive performance.
In this review, we will describe the main techniques developed today to characterise disease and
health and the effects of the diet on human health or on animal productive function. Various examples
of applications of nutrigenomics studies will be given to illustrate how they can contribute to better
characterise phenotypes and the effects of the diet or nutrients on these phenotypes. Moreover, some
examples on livestock species will be discussed to present the advances in animal nutrigenomics.
We do not pretend covering the whole field of nutrigenomics in this short review but rather aim at
presenting the main techniques and their limits and at illustrating their potential value to unravel the
complex links between diet and health or production.

Omics technologies for nutrition and health studies
Transcriptomics
High throughout gene expression technologies emerged in the 1990s enabling the simultaneous
measurement of thousands of gene transcripts in a biological sample. A microarray analysis is a
multiple-step procedure that starts with a well-defined biological question, experimental design,
target RNA preparation, microarray hybridisation, image acquisition and statistical analyses of data,
and biological interpretation of identified differentially expressed genes.
Principle of microarray technology
The microarray technique is based on hybridisation of two complementary single strand DNA
molecules. DNA is strongly immobilised on a solid support (nylon membrane or glass) where it is
available for hybridisation with complementary DNA (cDNA). The immobilised DNA referred to as
‘probes’, are nucleic acid sequences each corresponding to a particular gene. Fifty thousand probes
are commonly spotted in a grid-like format on a microscope slide with dimensions of 25 mm × 75
mm (Barrett and Kawasaki, 2003). Each spot on such microarray is used to measure the expression
of one gene in a cell or tissue sample. Two fluorescent dyes (typically cyanin 3 and cyanin 5) are
used to label cDNA synthesised from messenger RNA in both control and experimental samples.
The labelled cDNA are then hybridised to the same microarray. The intensity of the two fluorescent
dyes is measured with a scanner and compared for each spot to deduce whether the expression of
a particular gene is upregulated, downregulated, unchanged or absent in the experimental sample
compared to the control (Schulze and Downward, 2001; White and Salamonsen, 2005). The sensitivity
of the assay is high; microarrays have been reported to detect the presence of one mRNA per cell,
that is, a concentration of one mRNA per ≥100 000 molecules (Barrett and Kawasaki, 2003).
Different microarray systems
There are different types of microarrays depending upon the kind of probes and approach used to
position the probes on the surface. The probes can either be cDNA (long sequences that can be few
hundred base pairs long) or oligonucleotide (short sequences of about 50 to 70 base pairs (bp)).
Before the availability of complete or near-complete genome sequences, the genes expressed were
identified through sequence analysis of cDNA banks. The inserts of each plasmid were PCR amplified
and spotted and fixed onto surface-modified glass slides.
Long oligonucleotides are more largely used today. They are synthesised from requested sequences,
using several algorithms. Basically, the oligonucleotides should have very similar melting temperatures
and/or guanosine–cytosine content, have very little homology with other oligonucleotides, be entirely
contained within an exon, and have no repetitive or hairpin sequences. Oligonucleotides from several
commercial sources are now available. Sets of ready-made oligonucleotides are available for different
species, mainly humans, mice, rats, and Arabidopsis. For other species with fewer genomic sequences
available, cross-species hybridisation can be efficiently used (Ollier et al., 2007).
Oligonucleotides can also be synthesised in situ on the plate. The first large-scale manufacturing of
such microarrays was developed by Affymetrix using photolithographic methods (Lockhart et al.,
1996). In brief, synthetic linkers with photolabile protecting groups are attached to a glass substrate,
and a mask is used to direct light to predetermined areas on the substrate to remove the exposed
groups. These de-protected groups are then available for reaction with bi-functional deoxynucleosides,
resulting in chemical coupling. A new mask is used to direct coupling at other sites, and the step is
repeated until the desired sequence and length of the oligonucleotide is synthesised. The present
format is a 1.28 × 1.28 cm chip containing up to 500 000 different oligonucleotide sequences of 25

bases in length (Lipshutz et al., 1999). The second and more recent approach for in situ synthesis
of oligonucleotides is the ink jet technology. In this method, ink-jet pumps, similar to those used in
printers, are used to dispense 100-picoliter reagent droplets in as many successive reaction cycles
as required to reach the desired oligonucleotide length. The advantages of the in situ inkjet method
are that no masks are required, synthesis is faster because each cycle attaches one base (four cycles
per base are required with photolithography). This versatile system can routinely produce arrays
with >25 000 elements (Hughes et al., 2001).
The completion of numerous genomic sequences and increased efficiency of production of

oligonucleotides have combined to make the use of long oligonucleotides (50–80 bases) for gene
expression studies a highly attractive alternative to short oligonucleotide or cDNA arrays initially
predominant (Woo et al., 2004). Their longer length enables more specificity in hybridisation than
shorter lengths and their similitude in design (length, melting temperature and G–C content) enables
more consistent hybridisation conditions for every gene on the array. Compared with cDNA arrays,
it is much easier to lay the same concentration of DNA per spot with oligonucleotides. Reduced
cost of synthesis of long oligonucleotides diminished the cost of microarray production making it
more attractive, especially for academic laboratories.
Besides pangenomic microarrays, focused ones containing few hundred targeted genes are available.
These microarrays can be used to study the expression of genes associated with a disease, selected
pathways or for diagnosing chemotherapeutic drug resistance in a cancer cell (El-Bayoumy et al.,
2003). A large set of focussed microarrays are commercially available.
Microarray data analyses
Once hybridised and scanned, the use of proper methods for data analysis is critical. Different
softwares are available to align the grid and quantify the signal intensity at each location. Data
regarding poor-quality spots are removed from further study. Data from dual colour fluorescence
microarrays are normalised to account for differences between microarrays, print-tip groups and
fluorescent dye channels (Smyth and Speed, 2003). There is no universally accepted method of
microarray data normalisation, however intensity-dependent normalisation methods, such as printtip
loess (local weighted regression) are most commonly used (Leung and Cavalieri, 2003).
The aim of a microarray experiment is usually to identify differentially expressed genes, with a
measure of statistical significance (Cui and Churchill, 2003; Allison et al., 2006). Most microarray
experiments are designed with only one categorical factor (e.g. treatment or genotype) and in
consequence, it is common practice to consider a univariate testing problem for each gene and
calculate t-statistics. However, all statistical inferences have a probability of being incorrect. To
set the significance for one statistical test, an acceptable α e rror must be chosen in advance (e.g.
the familiar P-value α 0.05 criterion). This means that applying a t-test to a list of 10 000 genes
will produce approximately 500 genes that appear to be differentially expressed even if they are in
fact random. The errors can be of two types: false positives (type I errors) are incorrect inferences
of differential expression and false negatives (type II errors) are failures to detect true differential
expressions (Nadon and Shoemaker, 2002). Different statistical tests have been developed in order
to reduce type I and II errors. The multiple hypothesis-testing problem is conventionally tackled by
conservative approaches that control the family-wise error rate (FWER), the probability of having
at least one false positive among all testing hypotheses. A classical example is the Bonferroni
correction. Controlling the FWER can be too stringent and limits the power to identify significantly
differentially expressed genes. It is often acceptable to have few false positives if the majority of
true positives are chosen. Therefore it might be more practical to control the false discovery rate
(FDR), the expected proportion of false positives among the number of rejected hypotheses (Cui and
Churchill, 2003; Leung and Cavalieri, 2003). There are many different software packages available

for performing normalisation and statistical analysis, with the most popular being SAM (Statistical
Analysis of Microarray).
DNA microarrays provide an exceptional capacity for whole genome profiling as indicated above.
However, the quality of gene expression obtained from microarrays can vary according to platform
and procedures used. Hybridisation errors may occur due to cross-hybridisation between transcripts of
high homology. Statistical tests used for microarray data analysis are yet to be standardised. Therefore,
the results obtained with microarray require further validation. Quantitative real-time RT-PCR is
commonly used as a validation tool for confirming gene expression results obtained from microarray
analysis since it has higher sensitivity and specificity and has lower RNA quantity requirement than
Northern blot (White and Salamonsen, 2005; Morey et al., 2006). Owing to the cost of independent
verifications, expression levels are measured for only a limited number of transcripts, typically less
than 20 genes (Draghici, 2003). Overall, significant correlation was observed between microarray
and qPCR results. Filtering of microarray data for measures of quality (fold-change and ρ-value)
proves to be the most critical factor, with significant correlations of ρ>0.80 consistently observed
when quality scores are applied (Morey et al., 2006).
Proteomics
Biological function cannot be predicted from the genome sequence and gene expression alone.
Translation into proteins varies in magnitude according to transcripts, protein are eventually secreted
and compartmentalised often resulting in poor correlations between transcripts and the corresponding
proteins (Gygi et al., 1999b; Ideker et al., 2001b). Furthermore, biological activity is also often
linked to post-translational modifications such as phosphorylation, glycosylation, ubiquitination,
methylation and oxidation. This is why, beyond transcriptomics, much interest is being paid to the
characterisation of the proteome.
Techniques for proteomics still evolve rapidly and must address several challenges due to considerable
variations of concentrations according to proteins (some proteins like albumin in plasma are
largely dominant), limited overlap in the nature of the proteins between different cells or tissues, or
large differences in protein properties with in particular hydrophobic proteins not easily analysed
(Anderson and Anderson, 2002; de Hoog and Mann, 2004). The technique initially developed to
characterise the proteome associates a separation step by 2D-electrophoresis to mass spectrometry
analysis. Several hundreds of proteins can be separated. Protein spots are excised, digested with a
sequence-specific protease such as trypsin, and the peptides analysed by LC-MS. Proteins can also
be labelled with fluorophores similarly to transcripts, to reveal differences between two samples.
Using 2D-electrophoresis, more than 250 proteins corresponding to 1000-1500 post-translationally
modified protein spots have been identified in human plasma (Anderson and Anderson, 2002). A
limit of the technique is its relatively low sensitivity.
Proteins can also be digested before separation, and the peptide mixture analysed by LC-MS.
Peptides are characterised by tandem (MS-MS) mass spectrometry and identified by comparison
with published structures. Due to the different propensities for ionisation and the lack of standards
for absolute protein/peptide quantification, only relative concentrations are usually determined. The
two samples to be compared are tagged with a reagent, either isotopically labelled or not (isotope-
coded affinity tag; ICAT) (Gygi et al., 1999a). The two samples are then mixed and the difference in
concentrations of a given protein is determined by the ratio of the two tagged peptides, as estimated by
mass spectrometry. The most commonly used reagent is biotin, labelled with zero or eight deuterium
atoms, reacting with cysteine in the proteins. A limit of this technique is the large amount of sample
required. Alternatively, proteins can be metabolically labelled by growing the cells in the presence
of a labelled precursor.

Sets of a priori known proteins can also be quantified in a targeted approach using protein arrays
(Schweitzer et al., 2003). Antibody arrays can be used for example to estimate cytokines with a
much greater sensitivity than the techniques described above.
Metabolomics
A eukaryotic organism might contain up to 20 000 metabolites which represent real end-points of
physiological regulatory processes (Fernie et al., 2004). The considerable variety of their chemical
structures and physico-chemical properties makes the analysis of the metabolome far more difficult
than that of the transcriptome or proteome, both based on the assembly of a limited number of
nucleic acids or amino-acids. There are today no universal platforms able to quantify all metabolites
present in a given sample. Two different approaches should be distinguished: metabolic profiling
and metabolic fingerprinting (Dettmer et al., 2007). Metabolic profiling is a targeted approach:
metabolites belonging to a specific category and sharing common physico-chemical properties are
simultaneously analysed. Improvement of the sensitivity and resolution of the analytical methods has
made possible the analysis of a much larger number of metabolites of a given class in a single analysis,
in comparison to former analytical methods focused on a more limited number of metabolites.
This led to the emergence of disciplines such as lipidomics and peptidomics (Watkins et al., 2002;
Baggerman et al., 2004). Metabolic profiling is not a truly omic approach in the sense that they are
developed to analyse metabolites known a priori. Only the integration of various metabolic profiling
methods could pretend to characterise the whole metabolome.
In metabolic fingerprinting, metabolites are analysed in a truly global manner, using more universal
analytical methods such as nuclear magnetic resonance (NMR) or mass spectrometry (MS), with
no a priori hypothesis on the nature of the metabolites of interest. Metabolic patterns of a series
of samples originating from different cells, animals of human subjects, are compared and samples
classified using multivariate statistic tools. Proton NMR has been used for over 20 years for such
applications. Any molecule containing one or more protons gives a signal with a chemical shift
characteristic of its chemical environment in the molecule. Chemical shifts are therefore characteristic
of a given metabolite and can be used for identification of a priori unknown markers. NMR analysis
offers several advantages: the intensity of each signal is proportional to the concentration of the
proton-containing molecule with a wide dynamic range. It is robust and fairly reproducible even
when different NMR equipments are used (Keun et al., 2002). Spectrum acquisition is fast and
simple and several hundred samples can be analysed in a day. The main limit of NMR is its lack of
sensitivity. Only metabolites present in millimolar concentration are usually detected. This explains
the limited variety of metabolites generally considered in NMR metabolomic studies, currently 20-
40 in tissue samples and 100-200 in urine samples (Griffin and Shockcor, 2004). An NMR-based
metabolomic approach applied to the characterisation of metabolic effects of about 100 different
toxins led to the identification of no more than 30 associated biomarkers in either urine, plasma or
bile. This limited sensitivity explains why only limited new biological knowledge has so far been
generated using NMR metabolomics.
MS is far more sensitive with detection limits in the micromolar range. Most organic metabolites can
be ionised and ions can be separated according to their mass/charge (m/z) value. To limit the problems
of ion suppression and to separate isobaric metabolites, metabolites are most often separated by gas
or liquid chromatography before mass analysis (Dettmer et al., 2007). Gas chromatography (GC)
can be applied to volatile compounds or to polar metabolites after derivatisation with trimethylsilyl,
methoxime or methyl groups. In liquid chromatography (LC), analytes are usually separated on
reverse phase columns with particule size of 3-5 µm. Ultraperformance liquid chromatography
(UPLC) on columns with a particule size of 1.4-1.7 µm is also increasingly used to reduce run times
and increase resolution of the chromatograms (Nordström et al., 2006). Both reverse and direct
phases are used to analyse respectively polar and apolar metabolites. Several thousand variables

can be measured within 30 min or less for a given biological samples, each characterised by its m/z
value, retention time and intensity.
Biomarkers of interest, as revealed by multivariate data analysis, can then be identified by comparison
of the corresponding mass information to that stored in libraries or database. In GC-MS analysis,
metabolite spectra can be reconstructed by peak deconvolution and compared to those of spectra
libraries (Tikunov et al., 2005). The most largely used equipments in LC-MS metabolomics are high-
resolution mass spectrometers such as time-of-flight mass spectrometers (Tof-MS). Accurate mass of
each ion are obtained and this allows calculating corresponding empirical formula (Kind and Fiehn,
2006). A search in publicly available databases such as KEGG, MetaCyc or Human Metabolome
Project provides tentative annotation of the different ions. However, the pseudomolecular ion is not
always observed due to the formation of adducts or fragments in the electrospray source. In practice,
it is still very difficult to annotate the markers due to the lack of comprehensive databases.
MSn experiments with Q-Tof, ion-trap or FT-ICR MS instruments are needed for structure elucidation
of unknown biomarkers (Dettmer et al., 2007). Unknowns can also be classically isolated by
preparative chromatography and their structure determined by MS and NMR techniques (Hodson
et al., 2007).
These difficulties met in the identification of the nature of the markers have encouraged some groups
to develop platforms to analyse the main metabolites of interest in a given field of research. For
example, a capillary electrophoresis-Tof-MS method was developed to analyse 569 metabolites
expected to be present in mouse tissues (Soga et al., 2006). However many markers identified by
the fingerprint approach fell out of this list of expected metabolites emphasising the limits of these
approaches. No more than 132 metabolites out of 1859 detected peaks could be identified in the mouse
tissue extracts. Identification of new and unexpected markers and related mechanisms of action of
drugs, toxins or nutrients will depend on our capability to identify these markers in the future. This
will require a large effort and big investments to further develop the metabolite databases.
Characterising and understanding health and diseases

Using these different omic approaches, a phenotype can be defined by a set of values describing the
expression of genes, and concentrations of proteins and metabolites. An individual can be positioned
in a metabolic hyperspace made of as many dimensions as variables. Subtle metabolic differences
between individuals or for a same individual between environmental conditions can be identified
using proper multivariate statistical tools. Different vectors characteristic of factors as diverse as
gender, age, oestrus cycle, body mass index, disease, drug treatment, toxin exposure or diet can
be defined within this space (Nicholson and Wilson, 2003). The more variables, the highest is the
chance to differentiate such subtle differences characterising each of these phenotypes. For example,
113 unknown metabolites detected by HPLC in urine samples were found to better discriminate
patients with liver cancer from those with hepatitis or hepatocirrhosis, as compared to 15 known
urinary nucleosides which supposedly accumulate in cancer cells due to a high turn over of tRNA
(Yang et al., 2004).
Such high-throughput approaches open promising perspectives for the diagnosis and prognosis of
various diseases. Disease states can be differentiated from a healthy state by transcriptomic, proteomic
or metabolomic approaches. A large meta-analysis of 3762 DNA microarrays from 40 publications led
to the identification of genes differentially expressed in cancer (Rhodes et al., 2004). A metasignature
made of 67 genes was found to be a significant predictor of cancer, independently of the cancer
type. Different sets of genes were characterised to recognise differentiated from undifferentiated
cancers, cancers according to their outcome, metastatic from primary cancers or oestrogen receptor
positive from negative cancers.

Metabolites as the endpoint of physiological regulatory processes may also be good predictors of
disease states. Comparison of metabolic profiles in heart extracts by 1H-NMR allowed differentiating
four genetic mouse models of cardiac diseases (Jones et al., 2005). However, the different strain
genetic backgrounds also affected the metabolic profiles and particularly some metabolisms related
to vessel function, therefore reducing the capacity of the model to recognise the diseases. Similar
NMR metabolomic analyses were applied to sera collected from patients with various degrees of
vascular stenosis. The status of the patients could be better predicted using metabolomics than by
measuring conventional risk factors. Lipid signals contributed most to the prediction (Brindle et
al., 2002). More recently, other authors showed that this approach only weakly predicts the disease
due to a confounding effect of treatments with lipid-lowering drugs such as statins (Kirschenlohr
et al., 2006).
A transcriptomic analysis of the aorta of mice fed a high fat diet and of apoE deficient mice, a widely
used model of atherosclerosis, showed that the expression of over 700 genes was affected by the
disease (Tabibiazar et al., 2005). These genes were differentially expressed over time, along the
development of the disease. A set of 38 genes accurately classified five stages of the disease. The
genes affected at the earlier stage of the disease, before the formation of detectable vessel lesions,
may be particularly important as diagnostic markers.
Omic approaches also allow exploring the mechanisms underlying pathological processes. Genes
identified by these approaches may have been previously linked to the disease but novel genes are
also often identified, throwing new lights on the mechanisms characterising or leading to disease. For
example, many of the genes over-expressed in the aorta of apoE deficient mice were inflammatory
genes, some of them newly associated to atherosclerosis (Tabibiazar et al., 2005). Functional
annotation of these genes through gene ontology confirmed the contribution of known pathways
such as wound healing, apoptosis or nitric oxide mediated signal transduction or cell adhesion and
migration, but also revealed new biological processes associated to the development of atherosclerotic
lesions, such as carbohydrate metabolism, complement activation, calcium ion homeostasis or
collagen catabolism. A proteomic approach showed a modification of the concentrations of 79 protein
species in the aorta of these same apoE deficient mice, with some post-translational modifications
of redox-sensitive proteins and an increase in the abundance of malic enzyme, a key enzyme in the
maintenance of a reducing intracellular environment (Mayr et al., 2005).
Similarly, the general cancer meta-signature described above made of 67 genes, includes many
genes previously associated with different cancers (Rhodes et al., 2004). These genes are likely key
transcriptional factors in the neoplastic transformation. Some of them encode for enzymes such as
topoisomerase II or for members of the proteasome complex already known to participate in the
neoplastic transformation, and established targets of chemotherapeutic drugs. However, other genes
in this signature were not previously known and might become novel targets for drugs.
Genomics can also be applied to characterise common physiological processes. For example, aging
was explored in mice by comparing the gene expression profile of the muscle of young and aged
mice (Lee et al., 1999). Out of the 6347 genes surveyed, 113 showed a more than two fold increase
or decrease in expression over aging. Functional analysis of these genes suggested an increase in
stress responses and neuronal growth, and a decrease in energy metabolism and in the biosynthesis
of some lipids and proteins.
An interesting question is to know to what extent some markers, either genes, proteins or metabolites,
identified in experimental animals are also relevant for human studies. Transcriptional signatures of
atherosclerotic lesions in the aorta of apoE deficient mice were compared to those of coronary artery
samples dissected from explanted hearts of patients undergoing heart transplantation (Tabibiazar et

al., 2005). Out of the 667 genes over expressed in mice atherosclerotic aorta, over 100 genes were
over expressed in atherosclerotic lesions of human patients.
An NMR-based metabonomic study was developed to identify urinary markers of osteoarthritis

(Lamers et al., 2005). About 40 out of the 125 signals able to discriminate osteoarthritic patients
were also present in the guinea pig profile. These results clearly show that metabolic profiling in
well-controlled animal studies can be useful to identify disease specific markers in human subjects.
These markers could be less easily discovered in human studies due to various confounding effects
such as drug treatment or not easily controlled diet.
Understanding the interactions between diet and health

Diets and nutrients affect human metabolism and health. Nutrition research aims at understanding
these links in order to better prevent diseases. Effects on common disease factors are often only
revealed after long experimental periods. High-throughput nutrigenomics methods offer new
perspectives for unravelling at an early stage, the metabolic changes of low magnitude which most
often characterise the consumption of different diets and nutrients. Applications of genomics tools to
nutrition research have been discussed in some excellent recent reviews (Afman and Muller, 2006;
Rezzi et al., 2007). Some examples are given here to illustrate their potential and limits.
Transcriptomic tools have been used to characterise in well-controlled animal experiments the
effects of a deprivation or supplementation of particular nutrients. Caloric restriction was shown to
reverse the changes in the expression of several genes associated with aging in the skeletal muscle
of rats (Lee et al., 1999). More particularly, the activity of genes involved in fatty acid and protein
biosynthesis and in energy metabolism was restored. Genes involved in the repair of macromolecule
damage were also over expressed. High-fat diets, when compared to standard chow diets in mice,
induced major changes in the liver transcriptomic profiles, mainly related to lipid metabolism, defence
response and detoxification (Kreeft et al., 2005). The same effects were observed independently of
the mouse strain considered, apoE3Leiden or C57BL/6J. Many of the genes affected were under the
control of nuclear receptors, ligands of biliary acids, fatty acids and cholesterol.
Phenotypes associated to vitamin deficiency and their normalisation by vitamin supplementation

have been characterised by proteomics and metabolomics in animal models (Griffin et al., 2002;
Chanson et al., 2005) and human subjects (Weissinger et al., 2006). These approaches helped to
determine the role of vitamin deficiency in disease syndromes.
Phytochemicals present in foods are often characterised by a wide array of metabolic effects
and genomics appears particularly adapted to the characterisation of their effects. Genistein, a
phytoestrogen present in soy foods, is thought to participate in the prevention of cardiovascular
diseases. Endothelial cells challenged by homocysteine, a risk factor for cardiovascular diseases, were
exposed to genistein (Fuchs et al., 2005). Several metabolic pathways related to atherosclerosis and
influenced by genistein could be identified by protein fingerprinting. Genistein blocked the alterations
induced by homocysteine on 17 proteins out of 700 quantified. These proteins were involved in
metabolism, gene regulation, protein folding, detoxification and apoptosis.
Genistein supplemented to the diet of mice was also found to fully reverse the expression of 80
genes out of the 97 genes differentially expressed (>2 fold) in the liver upon a high-fat diet (Kim
et al., 2005). These genes encoded for enzymes involved in lipid and carbohydrate metabolism or
were related to detoxification, inflammation, apoptosis and transcription regulation. These changes
in gene expression were linked to a reduction of body weight and an improvement of various lipid
parameters.

The first application of metabolomics in nutrition was also related to soy phytoestrogens. NMR
analyses of plasma and urine after consumption of various isoflavone-containing soy products
by women also suggested some effects of isoflavones on energy metabolism and an increase in
anaerobic metabolism (Solanky et al., 2003 and 2005). Similar NMR-based metabolomic studies
were carried out on beverages rich in phenolic compounds such as tea and chamomile (Wang et al.,
2005; Van Dorsten et al., 2006). A decrease in blood glucose and an increase in the urinary excretion
of several intermediates in the tricarboxylic acid cycle after tea consumption suggested some effects
on energy metabolism.
Mechanisms of action of micronutrients have been explored using genomics approaches. For example,
the role of some cell signalling pathways and the different functions depending on this pathway
can be studied in mice or cultured cells where the pathway of interest is invalidated. Comparison
of the genes regulated by epigallocatechin gallate (the main phenolic antioxidant in tea) in Nrf2 -/-
or Nfr2 +/+ mice showed that the response was largely controlled by the Nrf2 signalling pathway
(Shen et al., 2005). A similar study carried out on Nrf2 -/- and Nfr2 +/+ mouse astrocytes exposed
to t-butylhydroquinone, a synthetic antioxidant and food additive, led to similar conclusions and
allowed the authors to propose a global picture of the mechanisms of action of this compound (Lee
et al., 2003).
The considerable amount of information collected with omic tools could also be used to compare
the biological activities of nutrients or foods. Transcriptomic or metabolomic approaches have been
developed to compare the toxicity of known drugs and to predict the toxicity of unknown chemicals
(Hamadeh et al., 2002; Shockcor and Holmes, 2002). Similar work could be conducted on different
structurally or functionally related nutrients. Omic approaches have already been used to compare
related foods, such as refined and wholemeal flour, or black and green tea (Van Dorsten et al., 2006;
Fardet et al., 2007). These methods may soon become essential to assess the health properties of the
food products developed by the industry.
Applications of nutrigenomics in human intervention studies

Applications of nutrigenomics in human studies impose specific constraints due to the large variability
in metabolism between subjects. Comparison of gene expression profiles in cells of fasting blood
samples showed that the expression of most genes was relatively constant for a given individual
(87% of the genes showed less than 50% variation in expression) (van Erk et al., 2006). However,
a larger inter-individual variability was observed for 2330 other genes, most of them involved in
immune response. Such a large inter-individual variability was also observed for plasma proteins
(Anderson and Anderson, 2002). Various factors, genetic, physiological or environmental, contribute
to explain this inter-individual variability. Factors like gender, age, period in oestrus cycle, fasting or
non fasting state, gut microbiota, smoking, physical exercise, habitual diet and nutritional status are
all known to influence transcriptomic, proteomic or metabolomic profiles (Anderson and Anderson,
2002; Nicholson and Wilson, 2003; Whitney et al., 2003). These sources of variability should be
tightly controlled as much as possible and the individuals used as their own reference in cross-
over clinical trials. These conditions being satisfied, genomics data can be more easily exploited
to unravel metabolic changes induced by a diet or nutrient. A first study on the effects of a dietary
intervention on gene expression profiles in blood cells was published recently (van Erk et al., 2006).
Two different breakfasts, rich in carbohydrate or protein, were consumed by eight volunteers in a
randomised cross-over study. Changes in gene expression measured two hours after consumption of
the breakfast meal were quite small (fold changes generally below 1.7). Most of the genes affected
by the consumption of the breakfast were involved in immune response and signal transduction.
The main limit of such transcriptomic approaches in human intervention studies is that metabolic
effects occurring in other tissues may not be seen at the level of blood cells. Collecting biopsies

from other tissues in healthy human subjects for gene expression studies is largely limited to muscle
and subcutaneous adipose tissues. The plasma metabolome may better reflect metabolic changes at
the level of the whole organism. As illustrated above, several metabolomic studies on the effect of
the consumption of foods or beverages have already been published. However, the present limits of
such studies are the low sensitivity for NMR-based metabolomics or the lack of annotated databases
for MS-based metabolomics.
Applications of metabolomics in cohort studies is further complicated by large variations in the

metabolome resulting from the presence in the plasma and excretion in urine of drug metabolites
(xenometabolome) or metabolites formed from the non-nutrient phytochemicals abundant in
foods of plant origin (food metabolome) (Gibney et al., 2005). These exogenous compounds often
account for a major part of the variations in the metabolome particularly at the urine level (Lenz
et al., 2004; Dumas et al., 2006; Stella et al., 2006; Walsh et al., 2006). Analysing the endogenous
metabolome to compare metabolic phenotypes in different populations is still today difficult due to
the overlap with this exogenous metabolome. Progress in this field still awaits the construction of
comprehensive metabolite databases for a straightforward identification of the endogenous markers
of interest. Before that, the simultaneous analysis of the largely annotated transcriptome and the
poorly annotated metabolome may help identifying the markers of interest in the metabolome
fingerprints (Ippolito et al., 2005).
Nutrigenomics in livestock studies

Until the last decade, the nutritional regulation of molecular mechanisms underlying physiological
or productive functions in livestock species has largely been carried out through candidate gene
approaches. However, the recent sequencing of livestock species genomes (especially chicken and
bovine) and the advent of functional omics tools in the domestic animal has allowed to extend the
studies for evaluating the factors influencing the animal performance and health and improving the
quality of their products (Hocquette, 2005; Drackley et al., 2006; Tuggle et al., 2007). Nutrigenomics
applications in domestic animal species are still few. The first studies reported concern the effect
of extreme nutritional status such as undernutrition, on animal gene expression profiles in different
tissues such as the muscle, liver and mammary gland. Alteration of biological processes taking place
during nutritional restriction and their regulation are of interest in the context of managing livestock
for optimal production efficiency and product quality.
Much progress has been made in our understanding of the biological processes that contribute to
the delivery of consistent quality meat (Bendixen et al., 2005; Mullen et al., 2006; Hocquette et
al., 2007). Through the application of genomics and proteomics we are gaining a deeper insight
into these processes and their interaction with nutritional factors. For example, a dedicated skeletal
muscle cDNA-microarray approach was used in porcine to detect 46 and 126 genes differentially
expressed depending on the diet level, respectively in white and red muscles (da Costa et al.,
2004). Diet restriction resulted in the increased expression of genes involved in metabolism such as
glycolysis, fatty acid oxidation or in translation and post-translational transport as well as an increase
of potential for growth modulation or differentiation. More recently, Dal Monego et al. (2007) have
used a human cDNA microarray to determine the impact of energy restriction and identified 20 and
22 genes respectively up- and down-regulated in muscle. Most of the genes up-regulated by this
restriction are involved in the glycolytic and oxidative metabolism and in the contractile apparatus.
Genes down-regulated encode for muscle proteins associated with slow fibre type, and for signal
transduction.
In bovine, the first study using cDNA microarrays, concerned the response of bovine skeletal
muscle transcriptome to short-term dietary restriction (Byrne et al., 2005). This study highlighted
29 and 28 unique genes, which were respectively up- and down-regulated. Differentially expressed

genes are involved in protein turnover. Down-regulation of extracellular matrix transcription and
energy metabolism and up-regulation of structural protein transcription and genes involved in
metabolic detoxification were observed. Lehnert et al. (2006) also showed that after a long period
of undernutrition, body weight loss is accompanied by a down-regulation of genes encoding muscle
structural proteins, muscle metabolic enzymes, and extracellular matrix, when compared with
animals on a rapid growth diet. Among the up-regulated genes, one is involved in the prevention
of oxidative damage.
The impact of fasting on the pig liver transcriptome has also been studied using cDNA microarray
(Cheon et al., 2005). The central role of PPARα in the adaptation to nutritional restriction was
emphasised. A restricted intake of moderated-energy diets was also shown to affect the expression
of 85 genes in bovine liver around parturition (Cheon et al., 2005). Restricted energy intake pre
partum resulted in more pronounced up-regulation of genes with key functions in hepatic fatty acid
oxidation, gluconeogenesis, and cholesterol synthesis.
Mammary mechanisms underlying milk secretion and composition have been studied during
lactation under food-deprivation. The first comparison of gene expression in the mammary gland
using a microarray containing 500 oligonucleotides has pointed out 44 genes differentially expressed
(Ollier et al., 2005). The 22 up-regulated genes included 10 genes involved in cellular machinery,
cell adhesion or the cytoskeleton. The 22 down-regulated genes included genes involved in milk
component synthesis. More recently, using a larger bovine microarray containing 8379 genes the
expression of 161 genes was altered by food-deprivation (Ollier et al., 2007). As observed during
stress responses, most genes altered by food-deprivation (88%) are down regulated. In particular, the
decrease in expression of genes involved in milk protein, lactose and lipid metabolism is in agreement
with the drop of milk protein, lactose and fat secretion. In addition, this study highlighted modification
of the expression of at least 14 genes which could be responsible for a slowdown in mammary cell
proliferation and differentiation and/or an increase in programmed cell death in response to 48-h
food-deprivation in goats. Moreover, a proteomics approach was used to characterise biochemical
and cellular mechanisms governing effects of peripubertal feeding on heifer mammary development
(Daniels et al., 2006). These authors showed that dietary treatment influenced the expression of 131
protein spots and suggested possible roles of some proteins in mammary development.
Such nutrigenomic studies applied to livestock should allow to improve animal health and well being,
as well as meat and dairy production. They should contribute to generate better models to predict
nutritional requirements and better define rations to meet those requirements.
Integrating omics data in systems biology

Most nutrigenomics studies published so far explore the effects of the diet or of some nutrient at a
single level, either transcriptomic, proteomic or metabolomic. Combining these different approaches
should offer a more comprehensive view of the phenomenon observed. The correspondence between
gene expression and protein and metabolite concentrations cannot usually be analysed for a single
mRNA transcript which would code for a single protein involved in a given metabolic pathway.
Interpretations are rather made at the pathway levels to generate a coherent picture of the effects
of a given intervention. Systems biology seeks to explain biological phenomenon through the net
interaction of all cellular and biochemical components with a cell or organism (Liu, 2005). Such an
integration of the data should allow to predict biological outcomes, given the list of the components
involved.
A consistent qualitative picture can be obtained by combining different omic approaches. For
example, combining transcriptomics or proteomics with metabolomic approaches allows to better

characterise the key pathways involved in physio-pathological processes (Ippolito et al., 2005;
Mayr et al., 2005).
Beyond this qualitative description, pathways can be explored by modelisation to predict their
behaviour in a biological system. So far, this has been more particularly applied to single cells and
applications to multicellular organisms remains difficult, in particular due to cross-talk between
tissues (Nicholson et al., 2004). Models can be constructed at a high or low level (Ideker and
Lauffenburger, 2003). At the high level, correlations between different variables under different
perturbations of the system are characterised to identify inter-relationships between the parts of the
model (genes, proteins or metabolites). These parts of the model can then be modelled at a lower
level. The models so far developed in the field of nutrition are largely statistical models (Clish et
al., 2004). Few low-level models that integrate physicochemical parameters have been developed.
Among low-level models, a good example corresponds to a model of folate homeostasis which
may allow to better understand the links between deficiency and diseases (Nijhout et al., 2004).
Application of systems biology in the field of nutrition is still in its infancy. Further development
will require major progress in computational sciences to analyse massive amounts of data, and to
make their interpretation by biologists possible.
Conclusions
The wealth of information collected in genomics experiments offers considerable promise to
characterise the complex links between diet and health. Major advances have already been achieved
in disease diagnostic and drug development (Butcher et al., 2004; Perez-Diez et al., 2007) but
progress in nutrition science has been relatively slow despite large expectations (German et al., 2005).
Progress is still hampered by high costs and technological hurdles (Afman and Muller, 2006). The
high cost of transcriptomics limits the number of samples analysed in a given experiment. Techniques
for proteomics and metabolomics still require substantial developments. It is not possible today to
measure the whole proteome and metabolome on a unique analytical platform, and the identification
of the markers of interest from their mass spectra is still difficult. Big investments are being made
to fully annotate the proteome and metabolome in comprehensive databases (see for example the
Human Metabolome Project Database, www.hmdb.ca) to facilitate the identification of these markers
and the interpretation of the data. A few more years will still be needed before these databases are
completed. Joint efforts from researchers of various disciplines such as nutrition, medicine, molecular
biology, chemistry, mathematics or bioinformatics will be needed to further develop nutrigenomics.
Scientific societies and networks have recently been organised. Good examples are the European
Nutrigenomics Organization (www.nugo.org), the Human Proteome Organization (www.hupo.org)
and the Metabolomics Society (www.metabolomicssociety.org). Similarly, in livestock species,
sequencing of the whole chicken, bovine and very soon porcine genomes have made the development
of genomics studies in these species possible. Animal scientific society organisations are based on
French (Genanimal programs: www.inra.fr/agenae), European (e.g. Fugato: www.fugato-forschung.
de/projects) or international (e.g. Milk genomics and Human Health: milkgenomics.fil-idf-pr.com)
programmes leading to the recent advances in animal genomics. These initiatives should contribute
to make nutrigenomics fully operative in the future and the derived data biologically meaningful in
human and livestock species.
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Hepatic gene expression of gluconeogenic and glycolytic enzymes as
indicators of the activity of carbohydrate metabolism in mink
C.F. Matthiesen, P.D. Thomsen and A.-H. Tauson
Department of Basic Animal and Veterinary Sciences, Faculty of Life Sciences, University of
Copenhagen, Grønnegaardsvej 3, DK- 1870 Frederiksberg C, Denmark
Introduction
The mink is a strict carnivore and a seasonal breeder exhibiting induced ovulation and delayed
implantation. After a true gestation of 30 ± 3 d it gives birth to altricial young (see Tauson and
Valtonen, 1992). Mink diets usually have a high protein and low carbohydrate content, suggesting that
glucose homeostasis is supported to a large extent by gluconeogenesis. However, glycolytic capacity
is high (Fink et al., 2002a and b). Another strict carnivore, the cat, seems to lack ability to adapt
activity of gluconeogenic enzymes to protein supply (Rogers et al., 1977). For the mink it remains
to be elucidated if it has the capacity to adapt the activity of key hepatic enzymes in gluconeogenesis
and glycolysis to different nutrient supply. Methods that allow changes in carbohydrate metabolism
to be estimated are therefore crucial for answering these questions.
The activity and some kinetic parameters of the key enzymes of glycolysis, gluconeogenesis and
amino acid catabolism have previously been reported (Sørensen et al., 1995) but it has not been
clarified whether the activity of these enzymes is reflected at the transcriptional level. The objective
of this study was therefore to estimate relative abundances of the mRNA of glucose-6-phosphatase
(G-6-Pase), phosphoenolpyruvate carboxykinase (PEPCK), fructose-1,6-biphosphatase (Fru 1,6-
P2ase) and pyruvate kinase (PK) by quantitative RT-PCR in livers from mink dams and their foetuses.
Because the foetus obtains glucose from the mother through the placenta the working hypothesis
was that foetal values for PEPCK, Fru 1,6-P2ase and G-6-Pase could be expected to be lower than
those of the mother, and that this would be reflected in the mRNA expression.

Three dams were euthanised 30-34 d after last mating, when they were estimated to be in mid to late
true gestation. Foetal and maternal livers were quickly excised and flash frozen in liquid nitrogen.
Crown to rump length of foetuses ranged from 23-27 mm (2 litters) and 35 – 40 mm (1 litter).
RNA was isolated from dam liver and from 4, 4 and 1 foetal livers per litter using RNeasy Mini Kit
(Quiagen). It was translated into cDNA using random hexamer priming. PCR primer oligonucleotides
were designed from canine mRNA sequences for cytosolic PEPCK, Fru 1,6-P2ase, PK and G-6-
Pase and tested on mink liver cDNA samples and on canine liver cDNA and canine genomic DNA
samples as the control. A fragment of the mink 18S rRNA was amplified and used as a measure of
RNA in the PCR. The primers chosen for this study produced amplification of the expected band on
canine cDNA samples and of similar size on mink DNA. Quantifying of enzyme cDNA levels was
subsequently done by real time PCR using SYBR Green I detection and the LightCycler System
(Roche Diagnostics).

Data from these three dams suggest that the abundance of mRNA for G-6-Pase relative to the
reference gene 18S was much greater among dams which showed a clear expression than among
foetuses, which had undetectable or very low expression (data not shown). This was consistent
with findings in the mouse where virtually no expression was detected during foetal life (Pan et al.,
1998). Relative abundance of mRNA (mean ± STD) for PK was 4 to 10 times higher in dams than

in foetuses (Figure 1a). Relative PEPCK levels were low and no difference was detectable between
mothers and foetuses. For Fru 1, 6-P2ase, mRNA levels were consistently 3 to more than 10 times
higher in foetuses mean ± STD than in dams (Figure 1b).
The mink dams, but not the foetuses, could be expected to have a high gluconeogenic activity, likely
to be reflected in high abundances of mRNA for G-6-Pase, PEPCK and Fru 1, 6-P2ase. Our findings
of a high abundance of mRNA for G-6-Pase in dams compared to foetuses hence suggest that G-
6-Pase level may be used as an indicator of gluconeogenic activity. The high PK mRNA levels in
dams is most likely a reflection of a negative energy balance demanding glycolytic activity, but may
also reflect catabolism of mobilised glycerol.
Figure 1. mRNA abundance for (a) PK (left panel) and (b) Fru 1, 6-P2ase (right panel).
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Dietary polyunsaturated fatty acids reduce expression of colonic
inflammatory genes in interleukin-10 knockout mice
B. Knoch1,2, M.P.G. Barnett1, W.C. McNabb1, Y.E.M. Dommels3, Z.T. Zhu4, S.O. Knowles1 and
N.C. Roy1
1Food, Metabolism & Microbiology, Food & Health Group, AgResearch Grasslands, Tennent Drive,
Palmerston North 4474, New Zealand
2Institute of Food, Nutrition & Human Health, Massey University, 4474, New Zealand
3Crop and Food Research, Palmerston North, 4474, NZ and current address: Unilever Food &
Health Research Institute, Vlaardingen, the Netherlands
4Faculty of Medical and Health Sciences, The University of Auckland, Park Rd, Grafton, Auckland
1023, New Zealand
Introduction
Dietary lipids, especially fatty acids, modulate the course of Inflammatory Bowel Diseases (IBD;
Deckelbaum et al., 2006). n-3 long chain polyunsaturated fatty acids (PUFA) such as eicosapentaenoic
acid (EPA) have shown anti-inflammatory and immunoregulatory effects but also fail to prevent
IBD and favoured remission (Bassaganya-Riera and Hontecillas, 2006). The n-6 PUFA arachidonic
acid (AA) is a precursor for pro-inflammatory eicosanoids and can induce intestinal inflammation
(James et al., 2000). This study investigates the molecular effects of dietary EPA and AA on intestinal
inflammation in a bacterially inoculated interleukin-10 knockout (IL10-/-) mice that develops IBD-
like colitis (Roy et al., 2007).

Twenty-four male IL10-/- mice (C57BL/6J background) and twenty-four male C57BL/6J (control)
mice were inoculated at 5 wk of age with Enterococcus faecalis/faecium strains and complex intestinal
flora to obtain consistent and reproducible intestinal inflammation (Roy et al., 2007). The mice
were fed a powdered diet based on AIN-76A for six wk according to a 2 × 4 factorial design. Diets
provided 5% total fat from (1) corn oil; (2) esters of oleic acid (OA); (3) esters of AA; (4) esters of
EPA. The diets containing fatty acid ethyl esters (<98% purity) included 1% corn oil plus linoleic
acid and alpha-linolenic acid to meet dietary requirements. At 11 wk of age, duodenum, jejunum,
ileum and colon were harvested for histological and transcriptomic analyses. Statistical analyses
of histological injury scores (HIS) were performed in GenStat (8th edition, 2005). Cyanine3- and
cyanine5-labelled cRNA was synthesised from purified RNA extracted from the colon and then
hybridised onto mouse 44 k oligonucleotide arrays according to a reference design. Microarray spot
quantification was performed with GenePix 6.0 software. Gene list comparisons were generated in
limma (Bioconductor framework; Smyth, 2005) using moderated probability value for t-statistics,
false discovery rate control (alpha=0.05) and a differential expression cut-off of 1.5-fold or greater.
In this paper, we describe differentially expressed genes clustered in the ‘immune response’ (75
genes) and ‘inflammatory disease’ (38 genes) pathways using Ingenuity Pathway Analysis.

Total intestinal HIS in IL10-/- mice was higher than for C57 mice (9.3 ± 1.5 (SD) vs. 1.3 ± 0.4,
P<0.01) with the colon being the most inflamed intestinal tissue. IL10-/- mice on the EPA and AA
diets had 40% lower colonic HIS scores (P<0.05) than IL10-/- mice on control (AIN-76A or OA)
diets. Colonic inflammation in IL10-/- mice was associated with an up-regulation of immune-mediated
response genes known to be up-regulated in IBD (e.g. chemokines (CXCL3, Table 1) and cytokines
(TNF, IL1ß, IL18R1, Ltß; data not shown). There was also an up-regulation of genes involved in

tissue remodelling and leukocyte extravasation (MMP13, TIMP1), neutrophil infiltration (S100A8,
S100A9) and epithelial metabolism and biosynthesis (PTGS2 or COX-2) in IL10-/- mice. When
feeding the AA-enriched diet to IL10-/- mice, the expression of some of these genes was significantly
down-regulated (e.g. CXCL3, F3, MMP13, PTGS2, S100A8 and TNF; Table 1), whereas their
expression remained unchanged (P>0.05) in IL10-/- mice fed an EPA-enriched diet (data not shown).
Our data indicate that dietary AA supplementation had a beneficial effect on colonic inflammation
and down-regulated ‘immune’ and ‘inflammatory response’ genes.
Table 1. ‘Immune response’ and ‘inflammatory disease’ genes differentially expressed in inflamed
colon of IL10-/- mice fed AIN-76A diet (1) compared to C57 mice fed AIN-76A, and (2) compared
to IL10-/- mice fed AA diet.
Gene name Symbol Refseq ID Fold change (P<0.05)
(1) (2)
IL10-/- vs. AA vs. AIN-
C57 (AIN- 76 (IL10-/-)
76A)
Chemokine ligand 3 CXCL3 NM_009140 +3.0 -2.8

Coagulation factor III F3 NM_010171 +3.3 -2.5
Matrix metallopeptidase 13 MMP13 NM_008607 +7.6 -3.2
Prostaglandin-endoperoxidase PTGS2 NM_011198 +3.7 -3.1
synthase 2
S100 calcium binding protein A8 S100A8 NM_013650 +22 -2.8
Tumour necrosis factor TNF NM_013693 +5.8 -1.5
References
Bassaganya-Riera, J. and R. Hontecillas, 2006. CLA and n-3 PUFA differentially modulate clinical activity and colonic
PPAR-responsive gene expression in a pig model of experimental IBD. Clin. Nutr. 25, 454-465.
Deckelbaum, R.J., T.S. Worgall and T. Seo, 2006. n-3 Fatty acids and gene expression. Am. J. Clin. Nutr. 83, 1520S-
1525S.
James, M.J., R.A. Gibson and L.G. Cleland, 2000. Dietary polyunsaturated fatty acids and inflammatory mediator
production. Am. J. Clin. Nutr. 71, 343S-348S.
Roy, N.C., M.P.G. Barnett, B. Knoch, Y.E.M. Dommels and W.C. McNabb, 2007. Nutrigenomics applied in animal
model of Inflammatory Bowel Disease: transcriptomic analysis of the effects of EPA and AA enriched diets.
Mutat. Res., in press.
Smyth, G.K., 2005. Limma: linear models for microarray data. Bioinformatics and Computational Biology Solutions
using R and Bioconductor. In: Gentleman, R., V. Carey, S. Dudoit, R. Irizarry and W. Huber (editors), Springer,
New York, 397-420.
Nutrigenomics New Zealand is a collaboration between AgResearch Limited, Crop & Food Research,
HortResearch and The University of Auckland and is largely funded by the Foundation of Research,
Science and Technology (FRST). M.P.G. Barnett is funded by a FRST Postdoctoral Fellowship. B.
Knoch PhD Fellowship is funded by Nutrigenomics New Zealand.

Skatole increases expression of detoxification genes in the ovine liver
M.H. Deighton, W.C. McNabb, D. Pacheco, M.H. Tavendale, Z.A. Park and N.C. Roy
Food, Metabolism & Microbiology Section, Food & Health Group, AgResearch Grasslands, Tennent
Drive, 4442, Palmerston North, New Zealand
Introduction
Skatole (3-methylindole) is a toxic xenobiotic produced during rumen bacterial degradation of
tryptophan. Skatole contributes to the unique flavour palate known as pastoral flavour (characterised
by ‘sheepy’ or ‘grassy’ odours) that differentiates meat and milk products of pasture-fed ruminants
from those finished under grain-based production systems. Accumulation in milk and meat fat occurs
when skatole absorbed from the reticulorumen exceeds the detoxification capacity of the liver.
These conditions arise when ruminants are fed fresh forages with high rumen degradable protein
(RDP) content. We hypothesise that under conditions of minimal endogenous skatole production
(low dietary RDP) a continuous rumen infusion of skatole will elevate absorption and modify liver
metabolism of skatole within 72 h. This hypothesis is supported by a 50% reduction in peripheral
plasma skatole concentration between 16 and 72 h of continuous skatole infusion (Deighton et al.,
2006). Therefore, cDNA microarray analysis was used to identify differential gene expression in the
ovine liver in response to a 72 h skatole exposure administered via rumen infusion.

Twelve, ten mo old castrate males from a single sire were prepared with rumen cannula and exposed to
a contrasting level of skatole for a 72 h period. Endogenous skatole production was minimised using
a non-forage diet with low RDP content (ME: 13.4 MJ/kg DM; CP: 130 g/kg DM; estimated RDP:
40 g/kg DM). Sheep received a constant rumen infusion of propane-1,2-diol (5 mL/h) with or without
skatole (28 mg/mL) and were euthanised 72 h after commencement of infusion. Samples of liver
were frozen in liquid nitrogen and stored at -85 °C. Total hepatic RNA was isolated using TRIzol and
purified using a Qiagen RNeasy kit. RNA was quantified with a Nanodrop ND-100 spectrophotometer
and integrity was assessed using the Agilent 2100 Bioanalyser. Only total RNA with an OD 260/280
ratio >2.0 and a Bioanalyser 28s/18s peak ratio >1.4 was used for microarray hybridisation. cDNA
was prepared for microarray screening using the Invitrogen SuperScript Indirect cDNA Labelling
System incorporating Cy3 and Cy5 mono-reactive cyanine dye. Preparation, hybridisation and
scanning of the 20 736 expressed sequence tag (EST) bovine microarrays were performed following
the methods of Diez-Tascón et al. (2005). The EST were selected from AgResearch’s proprietary
database that represents approximately 80% of the bovine genome. The prospect for successful
analysis of ovine tissue using bovine EST is high since the estimated homology of protein coding
sequence between sheep and cattle is >97% (Kijas et al., 2005). The experiment consisted of an 18
array augmented two loop design and was balanced for dye bias. Microarray data was analysed using
the limma package in Bioconductor (Smyth, 2005). For each EST a modified t-test was performed
and the probability values were then adjusted for multiple testing using the Benjamini and Hochberg
correction (Benjamini and Hochberg, 1995), resulting in a False Discovery Rate (FDR). The FDR
is the probability that a gene declared to be significant is in fact not significant. EST with FDR less
than 0.01 were considered to be of interest.

This study shows that the expression of genes encoding enzymes with xenobiotic detoxification
activity was up-regulated in the hepatic tissue of sheep receiving a significant hepatic exposure of
skatole via rumen infusion. EST with significant (FDR<0.01) differential expression in either direction

represented about 14% of those assessed. Amongst these, five had a fold change >2 in response
to skatole treatment (Table 1). All five of these genes encode enzymes involved in detoxification
processes. Since the site of first pass metabolism of skatole absorbed from the gastrointestinal tract
the capacity of liver enzymes to metabolise skatole, through phase I and II detoxification processes,
there was a direct impact upon the amount of skatole entering the peripheral (post hepatic) circulation.
Thus, the concentration of skatole appearing in the lipidic components of meat and milk products
of forage fed ruminants is influenced by the animal’s hepatic detoxification capacity. This research
represents the first use of microarray technology to identify differential hepatic gene expression during
controlled skatole administration in ruminants. Further investigation is now required to establish the
specific role of these enzymes during the hepatic metabolism of skatole in sheep.
Table 1. Genes with differential skatole- induced expression in ovine hepatic tissue.
Gene name Symbol Accession number Fold change FDR1
Aldehyde dehydrogenase 1 ALDH1A1 OAU12761 +2.94 6.3E-10

family member A1
NAD(P)H dehydrogenase NQO1 DY484462 +2.56 7.6E-8
quinone 1
Leukotriene B4 12- LTB4DH DY507800 +2.51 9.1E-11
hydroxydehydrogenase
Glutathione S-transferase A1 GSTA1 AF140223 +2.08 1.2E-8
Stearoyl-CoA desaturase SCD NM_001009254 +2.05 2.5E-10
1 False Discovery Rate.
References
Benjamini, Y. and Y. Hochberg, 1995. Controlling the false discovery rate: a practical and powerful approach to multiple
testing. J. Roy. Stat. Soc. B 57, 289-300.
Deighton, M.H., D. Pacheco, M.H. Tavendale, N.M. Schreurs, J.S. Peters, W.C. McNabb and N.C. Roy, 2006. Increased
hepatic skatole exposure via rumen infusion increases skatole concentration in peripheral circulation and inter-
muscular fat in sheep. Proc. NZ Soc. Anim. Prod. 66, 295-299.
Diez-Tascón, C., O.M. Keane, T. Wilson, A. Zadissa, D.L. Hyndman, D.B. Baird, J.C. McEwan and A.M. Crawford,
2005. Microarray analysis of selection lines from outbred populations to identify genes involved with nematode
parasite resistance in sheep. Physiol. Genomics 21, 59-69.
Kijas, J.W., M. Menzies and A. Ingham, 2005. Sequence diversity and rates of molecular evolution between sheep and
cattle genes. Anim. Genet. 37, 171-174.
Smyth, G.K., 2005. Limma: linear models for microarray data. In: Gentleman, R., V. Carey, S. Dudoit, R. Irizarry and
W. Huber (editors), Bioinformatics and Computational Biology Solutions using R and Bioconductor. Springer,
New York, 397-420.

Gene expression in adipose tissue of dairy cattle changes in early
lactation or with supplemental chromium, integrating expression into
metabolic models
J.P. McNamara1, J.M. Sumner1, J. Vierck1 and A. Jourdan2
1Washington State University, 233 Clark Hall, Pullman, WA, 99164-6351, USA
2Kemin Industries, Inc., 2100 Maury Street, Des Moines, IA, 50306-0070, USA
Introduction
The role of adipose tissue in reproduction has been a matter of study for decades. The pinnacle of
adipose tissue function is reached in pregnancy and lactation, in which hormonal and neural signals
combine to first increase adipose stores and then use them during later pregnancy or lactation, followed
by restoration for the next reproductive cycle. In fact, several hormones that function in control of
mammalian pregnancy and lactation, such as estrogen, progesterone, prolactin, somatotropin and
placental lactogen, have important direct effects on adipose tissue metabolism and gene expression.
In our laboratory we have studied intensively the role of adipose tissue in the energetics of pregnancy
and lactation in cattle, pigs and laboratory animals. Adipose tissue, in addition to being a major
energy storage organ, is also a source of regulatory and health proteins including IGFI, leptin,
and immune function proteins. We conducted an analysis of gene expression in adipose tissue of
dairy cattle in late pregnancy and early lactation. One objective was to determine changes in gene
expression between 30 d pre partum and 30 d post partum; another objective was to determine if
supplemental Chromium Propionate (as KemTRACE ®brand Chromium Propionate Cr; CrP) altered
intake, production, adipose metabolism or gene expression. The long-term objective is to begin to
integrate gene expression maps into models of energy and nutrient metabolism.

Adipose tissue was biopsied from the tailhead region from 20 high-producing Holstein dairy cattle at
30 d pre and post partum (milk production was 43.1 ± 1.4 kg/d). We extracted complete mRNA from
samples from 3 cows at each time point (same cows, repeated measures) for gene array analysis. The
samples were prepared, amplified and analyzed on the Affymetrix Bovine Gene Chip at the WSU
Bioinformatics Core Laboratory. The determination of expression of genes in bovine adipose tissue
in lactating dairy cattle using gene array procedures was demonstrated, for the first time.
In a separate study (McNamara and Valdez, 2005), we fed 10 mg/d of chromium propionate (CrP)
from 21 d pre partum through 35 d in milk (DIM) to 12 cows in each group. The dry period diet
was 24% alfalfa silage, 60% grass hay and 16% concentrate. Lactation diets were alfalfa and alfalfa
silage based. Amongst these animals, 3 in each group were used for the gene expression chip assay.
Using the software analysis tools Gene Chip Operating Software (GCOS) and Genespring, a two-way
analysis of variance (diet, d and interaction), as well as the JMP procedure of SAS, we investigated
the patterns of gene expression. Although we used the traditional P value of 0.05, because of the
exploratory nature of this work and the lack of quantitative knowledge on gene expression in cattle
adipose tissue, we also report some changes with a P value of 0.10 or less.

Among those genes increasing (P<0.05) in expression from 30 d pre partum to 30 d post partum
were those signaling for myosin heavy chain, several immunoglobulins and receptors, and neutrophil
beta-defensin 5. Among those decreasing (P<0.05) in expression from 50 to over 75% included acetyl
CoA Carboxylase (-79%), several glycolytic enzymes; ATP citrate lyase (-75%), insulin receptor

induced protein (-77%), IGFI (-30 to – 50%), and IGFBP3 (-55%). Leptin expression (Genbank:
NM_173928.1) was reduced 57%). Whilst such changes in expression of metabolic control genes
was not surprising, it is still the first definitive demonstration that gene expression is a key element
of metabolic response to lactation in adipose tissue. The physiological role of potential changes in
immune proteins is as yet a mystery. In the chromium propionate study, CrP increased DMI after
calving by 3.1 kg/d; milk yield was 2.6 kg/d greater due to CrP from d 1 to d 90. For d 57 to 90 this
was 4.6 kg/d greater. Serum NEFA were decreased (P<0.05) about 20% by Cr supplementation.
Addition of CrP increased adipose lipogenesis (P<0.05) at 14, 28 and 56 d DIM. There was a lag of
milk yield response to CrP, the feed intake effect was obvious pre partum, but the milk yield effect
increased over time (0.8 kg/d for the first 35 d, increasing to 4.6 kg/d for d 57 to 90). Consistent
with the mode of action of chromium to increase insulin sensitive glucose uptake and thus increase
lipogenesis and decrease lipolysis, an effect of CrP may be to decrease lipolysis, allowing a greater
increase in feed intake, allowing more substrates for the mammary gland. This is similar to the
well-known ‘fat cow syndrome’ in which cows with less fat actually eat more because of less fatty
acid inhibition of feed intake.
There were 223 genes that were increased 2-fold or more at d -7 by CrP versus controls, and 1150
were decreased 50% or more compared to control (all at P<0.10 for effect of treatment or time).
At 28 DIM, 175 genes were up-regulated in CrP supplemented cows, 3517 genes were lower in
CrP supplemented cows in which at least one of the signals had an expression of 50 or more on the
Affymetrix chip, standardized to a signal strength of 125. There were 24 genes that increased and 347
that decreased in CrP supplemented animals at both d. The primary functional cluster of genes that
were up regulated in KT fed animals included those functioning in cell synthesis and in the immune
system. These included myosin heavy chain and immunoglobulin A receptor. Genes decreased
common to both times and CrP supplementation were NADH dehydrogenase, t-cell receptor, and
prostaglandin PGH2. At d 28, additional genes up-regulated by CrP included immunoglobulin heavy
and light chains, bovine retinoic acid binding protein, Bos taurus complement component C4 Leptin
expression was decreased by KT at d 28, as was growth hormone receptor mRNA. There were
several other genes that were down regulated or up regulated, with no consistent pattern related to
gene function emerging as yet.
These gene expression array results are obviously preliminary in nature, but many genes that control
metabolic pathways were changed in a direction consistent with the known changes in metabolic
rates (catabolic path enzymes increased, analbolic path enzymes increased). The biological meaning
of changes in expression of other genes remains to be determined. Data are being integrated into a
second-generation metabolic map, built on the mechanistic model of dairy cattle metabolism from
Baldwin and colleagues (Baldwin et al., 1987). We will present an integrated map of expression of key
genes that regulate adipose tissue lipogenesis, esterification, carbohydrate oxidation and lipolysis.
References
Baldwin, R.L., J. France and M. Gill, 1987. Metabolism of the lactating cow. I. Animal elements of a mechanistic
model. J. Dairy Res. 54, 74-105.
McNamara, J.P. and F. Valdez, 2005. Effects of dietary chromium propionate and calcium propionate on adipose tissue
metabolism and milk production of dairy cattle in the transition period. J. Dairy Res. 88, 2498-2507.

Transcriptomic profile in mammary gland is modified by nutrition in
lactating goats
C. Leroux, S. Ollier, S. Bes, M. Goutte, L. Bernard and Y. Chilliard
URH - UR1213, Equipe TALL, INRA, F-63122 Saint Genès-Champanelle, France
Introduction
During lactation, mammary epithelial cells synthesise and secrete large quantities of milk components
including proteins, lipids and lactose. Milk production and composition are affected by genetic,
nutritional and physiological factors. In particular, animal nutrition considerably affects the
composition of milk lipids which influences its nutritional quality (Chilliard and Ferlay, 2004). To
fulfill its primary function which is to synthesise milk components, the mammary gland requires
efficient transcriptional, translational and secretory machineries involving multiple genes whose
nutritional regulation remains poorly known. Until now, the studies of the effect of nutritional factors
on mammary lipogenesis were focused on few genes encoding the key lipogenic enzymes in the
cow and goat (Bernard et al., 2007). However, recently, high-throughput gene expression profiling
using array technologies is becoming a powerful way to simultaneously analyse the expression
pattern of hundreds or thousands of genes in a variety of ruminant biological samples (Leroux
et al., 2003; Suchyta et al., 2004; Bernard et al., 2005). Thus, to better understand the mammary
mechanisms underlying milk secretion and composition in response to dietary factors, the objective
of the present study was to identify genes whose expression is regulated by feeding level, using
microarray methodology.

Twelve lactating goats were assigned to two groups based on their feeding level (control diet ad
libitum vs. 48-h food-deprivation, FD). Milk and mammary tissue were collected to analyse their
composition and their transcriptome, respectively. Transcriptomic analyses were performed using a
bovine microarray (8379 oligonucleotides). Data have been deposited in NCBI’s Gene Expression
Omnibus and are accessible through GEO (accession number GSE6380). Six comparisons were
performed and for each comparison, data from the four replicated spots per oligonucleotide (2
intra- and 2 inter-slides) were normalised, averaged and statistical analyses were performed, after
filtering, with a standard Student t test to detect differentially expressed genes between the two groups.
Probability values were adjusted using the Bonferroni correction (at 0.01%) for multiple testing to
eliminate false positives. RT-PCR validations were performed using the LightCycler FastStart DNA
Master SYBR Green I kit (Roche Applied Science) according to the manufacturer’s instructions,
with 10 pmol of specific primers designed from area close to that of the microarray probe either
from caprine sequences, when available, or from bovine sequences.

Milk and plasma analyses
As expected, after 48 h of treatment compared to control, milk yield (2.57 vs. 0.4 kg/d), milk
lactose (119.0 vs. 12.6 g/d), protein (81.6 vs. 20.4 g/d), and fat (87.3 vs. 39.2 g/d) yields as well
as plasma insulin (98.8 vs. 37.9 pmol/L) and glucose (3.32 vs. 2.71 mmol/L) concentrations were
significantly lower in FD than in control goats whereas plasma 3-hydroxybutyrate (0.495 vs. 0.985
mmol/L) and nonesterified fatty acid (0.368 vs. 2.23 mmol/L) concentrations were significantly
higher. Secretion of C4:0 to C16:0 saturated FA was significantly lower in FD than in control
goats (53.89 vs. 12.99 g/d).

Mammary transcriptome analysis
Hybridisation of the RNA studied on the bovine oligochip showed that 7140 genes of the total 8379
different probes spotted were expressed in caprine lactating mammary tissue. Statistical analyses
allowed 161 differentially expressed genes in mammary tissue to be identified between the two
feeding levels studied. Changes in expression of 10 genes among the 161 identified were confirmed
by real-time RT-PCR. Most of these genes (141) were downregulated in FD compared with control
goats, whereas only 20 genes were upregulated. These genes were classified into 11 functional
categories according to Gene ontology annotation (Figure 1).
Among them, we identified genes involved in the drop of milk production and milk component
secretion. Moreover, our results highlighted genes that could be responsible for a slowdown in
mammary cell proliferation and differentiation in response to FD and for an orientation of mammary
cells toward program cell death that could correspond to an early step in mammary gland involution.
In conclusion, to our knowledge, this is the first transcriptomic analysis studying the impact of
nutrition on ruminant mammary gene expression, which demonstrates that FD alters mammary
transcriptome simultaneously to milk production and composition.
Cell cycle, proliferation, differentiation & death

Cell adhesion & Cytoskeleton
Signal transduction
Regulation of transcription & RNA metabolism
Cellular protein metabolism and transport
Cellular lipid metabolism and transport
Metabolism (other)
Transport (other)
Other
Biological process unknown
Expressed Sequence Tags
40 30 20 10 0 10
Figure 1. Distribution into functional categories of the 161 genes altered (downregulated: and
upregulated:Figure; P<0.0001)
1 : Distributionby
intofood-deprivation
functional categoriesin caprine
of the mammary
161 genes tissue.
altered (downregulated:
and upregulated: ; p<0.0001) by food-deprivation in caprine mammary tissue.
References
Bernard, C., S. Degrelle, S. Ollier, E. Campion, I. Cassar-Malek, G. Charpigny, S. Dhorne-Pollet, I. Hue, J.F. Hocquette,
F. Le Provost, C. Leroux, F. Piump, G. Rolland, S. Uzbekova, E. Zalachas and P. Martin, 2005. A cDNA macro-
array resource for gene expression profiling in ruminant tissues involved in reproduction and production (milk
and beef) traits. J. Physiol. Pharmacol. 56, 215-224.
Bernard, L., C. Leroux and Y. Chilliard, 2007. Expression and nutritional regulation of lipogenic genes in the ruminant
lactating mammary gland. In: Bernard L., C. Leroux and Y. Chilliard (editors), Advances in experimental medicine
and biology: bioactive components of milk, Springer, in press.
Chilliard, Y. and A. Ferlay, 2004. Dietary lipids and forages interactions on cow and goat milk fatty acid composition
and sensory properties. Reprod. Nutr. Dev. 44, 467–492.
Leroux, C., F. Le Provost, E. Petit, L. Bernard, Y. Chilliard and P. Martin, 2003. Realtime RT-PCR and cDNA macroarray
to study the impact of the genetic polymorphism at the alphas1-casein locus on the expression of genes in the goat
mammary gland during lactation. Reprod. Nutr. Dev. 43, 459-469.
Suchyta, S.P., S. Sipkovsky, R.G. Halgren, R. Kruska, M. Elftman, M. Weber-Nielsen, M.J. Vandehaar, L. Xiao, R.J.
Tempelman and P.M. Coussens, 2003. Bovine mammary gene expression profiling using a cDNA microarray
enhanced for mammary-specific transcripts. Physiol. Genomics 16, 8-18.
Supported by the LIPGENE EU-FP6 project (contract FOOD-CT-2003-505944, coordinated. by

M. Gibney, Irland)

Effect of fasting on the thalamus/hypothalamus in cows - a proteomics
analysis
B. Kuhla1, S. Kuhla1, P.E. Rudolph2, D. Albrecht3 and C.C. Metges1
1Research Units Nutritional Physiology ‘Oskar Kellner’, Research Institute for the Biology of Farm
Animals, 18196 Dummerstorf, Germany
2Genetics and Biometry, Research Institute for the Biology of Farm Animals, 18196 Dummerstorf,
Germany
3Institute of Microbiology, Ernst-Moritz-Arndt-University, 17487 Greifswald, Germany
Introduction
The physiological regulation of feed intake is influenced by a number of humoral metabolites,
hormones, stress and environmental parameters, which have to be balanced to maintain energy
homeostasis and body weight in animals and humans. A disturbance of this sensitive regulatory
system for example initiated by permanent or periodic fasting may result not only in temporary weight
loss but also in pathophysiological appearances such as anorexia or bulimia in humans. Similarly,
animals have to adapt to reduced food amounts during seasons and even more dramatically when
dairy cows are confronted with drastic reduction in nutrient supply during the dry-off period or with
an inadequate feed intake around the parturient period.
As the main neural control and regulation centre, the hypothalamus receives and integrates an array of
changing humoral and neural signals, among them circulating nutritional metabolites, hormones, and
neuronal afferents. Under the influence of overfeeding or food restriction, changing concentrations
of these signals are detected by ‘nutrient sensors’. One of the most important ‘nutrient sensor’ is the
5’-AMP-activated protein kinase (AMPK), which is sensitive to the intracellular AMP:ATP ratio
(Xue and Kahn, 2006). Also, mitochondrial reactive oxygen species (ROS) have been identified to
act as a redox regulator of food intake (Benani et al., 2007). Both regulators trigger a number of
responsive mechanisms including aberrant gene expressions directed to modulate feed intake, glucose
production, fatty acid oxidation, mitochondrial biogenesis, and energy expenditure. These findings
link food energy intake and regulation of energy homeostasis with the level of cellular ATP and ROS.
However, biochemical links are poorly understood and a more systemic approach to identify regulated
hypothalamic proteins would complement the understanding of the complex mechanism involved
in food intake regulation and diet-related diseases. Therefore, our aim was to elucidate aberrantly
expressed hypothalamic proteins after dietary energy restriction using proteome analysis.

In our study, ten lactating German Holstein cows, which were comparable in age, body weight
and milk yield, were fed a total mixed ration (TMR) or straw ad libitum for 2.5 d. NEL intake was
reduced approx. 6 fold in the straw-fed group. Animals had free access to water, were milked twice
daily, and were killed by exsanguination.
Thalamic/Hypothalamic tissues were subjected to 2-dimensional gel electrophoresis and proteins

were stained with Coomassie. After tryptic digestion of the excised gel spots and MALDI TOF/TOF
analysing, proteins were identified on the basis of peptide mass fingerprinting using the NCBInr
data base.

A map composed of 189 identified spots with 129 different proteins was obtained. Proteins from this
map were grouped according to their main cellular location (A) and function (B) and the composition
is illustrated in Figure 1.
Eight proteins were significantly (P≤0.05, t-test) up-regulated after straw feeding. Two of these
originated from mitochondria and six from the cytoplasm, although two of the latter may also occur
in the nucleus. According to their function, five enzymes are involved in nucleotide and energy
metabolism, among them brain creatine kinase, AICAR/IMPCH and tER-ATPase, underlying the
particular meaning of ATP in the regulation of energy homeostasis. Furthermore, one protein acts
in cell signalling (DRP-2) and two play a role in cellular defense against oxidative stress (STI-1,
CuZnSOD), suggesting that the latter two might be involved in redox regulation of food intake.
While all aberrantly expressed proteins were 1.2 – 2.0 fold up-regulated, no protein identified was
significantly down-regulated.
Endoplasmic
Reticulum
3%
Membrane Lysosome Carbohydrate Lipid Metabolism
5% 1% Metabolism 3%
8%
Nucleus Protein
Nucleotide/
5% Metabolism
Energy Metabolism
8%
25%
Extracellular
5%
Cytoskeleton
9%
Cytoplasm
Mitochondria 60%
21% Protein Folding &
Cell signaling
13% Cellular defense
20%
Transport
14%
A B
Figure 1. Main cellular location (A) and function (B) of 129 different hypothalamic proteins
identified.
In conclusion, the identified proteins describe new candidate molecules that are potentially involved
in maintaining energy homeostasis but need to be studied in more detail in future investigations.
References
Benani, A., S. Troy, M.C. Carmona, X. Fioramonti, A. Lorsignol, C. Leloup, L. Casteilla and L. Penicaud, 2007. Role
for mitochondrial reactive oxygen species in brain lipid sensing: redox regulation of food intake. Diabetes 56,
152-160.
Xue, B. and B.B. Kahn, 2006. AMPK integrates nutrient and hormonal signals to regulate food intake and energy
balance through effects in the hypothalamus and peripheral tissues. J. Physiol. 574, 73-83.

Comparative transcriptomic and proteomic analyses of pig longissimus
muscles differing in intramuscular fat content
J. Liu1, M. Damon1, P. Ecolan1, I. Guislde2, N. Guitton3, P. Cherel4 and F. Gondret1
1INRA, UMR1079 Systèmes d’Elevage Nutrition Animale et Humaine (SENAH), 35590 Saint Gilles,
France
2INSERM U533, 44000 Nantes, France
3Plate-forme protéomique Ouest-genopole, Campus de Beaulieu, 35042 Rennes, France
4France Hybrides, 45808 St Jean de Braye Cedex, France
Introduction
Variability in pork quality has to be better understood. Intramuscular (i.m.) fat content is known as a
key determinant of sensory traits and the consumer’s acceptability of meat and meat-derived products
(Fernandez et al., 1999). Interestingly, muscle fat content at a cellular level represents the lipids
stored inside the myocytes as well as in the adipocytes clustered along myofiber fasciculi. Therefore,
many metabolic pathways in both adipocytes and myofibers, as cell compartments involved in fatty
acids storage or utilisation, respectively, probably account for the variability of muscle fat content.
However, most studies have been focussed on only few indicators involved in muscle metabolism
(Gondret and Hocquette, 2006 for a review). Therefore, the present high-throughput study was aimed
to better depict and analyse i.m. fat variability by comparison of both proteomic and transcriptomic
profiles of pig longissimus muscles differing in i.m. fat content.

Sixteen longissimus muscle (LM) samples were chosen among a larger set of 1000 pigs originating
from commercial F2 crossing between Piétrain and a synthetic line (France-Hybrides) slaughtered
at nearly 110 kg bodyweight, to have either a high (4.58 ± 0.30%, n=8, [HF]) or a low (1.36 ± 0.21,
n=8, [LF]) i.m. fat content. The cytosolic proteins (n=6 in each group) were extracted and separated
by 2D gel electrophoresis [3-10 pH NL range; 12.5% SDS-PAGE]. The gels made in duplicate
per sample were visualised by sensitive silver staining and analysed by ImageMaster Platinum
software. The protein spots of interest were further identified by Maldi-Tof mass spectrometry. The
transcriptomic analysis was performed using a human myochip containing 6681 muscle specific
oligonucleotides (MyochipsOuestGenopole). Total RNA from homogenate muscles were labelled
according to the Amino Allyl MessageAmp™ II aRNA Amplification Kit protocol (Ambion). Four
and two hybridisations for each sample in HF or LF groups, respectively, (n=8 in each group) were
performed, using a mix of the 8 LF samples as a reference. GenePix software was used for image
analysis and differential expression analysis was made with Genespring software. ANOVA statistical
analyses were performed with the main effects of group, replicate, and the interaction between both
factors, in order to generate lists of differentially expressed proteins or genes. Real-time quantitative
RT-PCR analyses were also performed for some of these genes and transcription factors known to be
involved in lipid metabolism. Molecular network investigation was then conducted using Ingenuity
Pathway Analysis (IPA) software.
Results
Proteomic analyses resulted in 19 proteins showing a significant difference in intensity level
(ratio≥|1.3|, P<0.05) between the two groups. Two proteins also tended (P<0.10) to be expressed
differently between the groups. Proteins involved in glucose anaerobic degradation (enolase-1;
glyceraldehyde-3-phosphate dehydrogenase) were up-regulated in the HF group, whereas enzymes
participating in the generation of NAPDH (isocitrate dehydrogenase) and glycerol-3 phosphate

(glycerol-3-phosphate dehydrogenase) needed for fatty acid synthesis and esterification, respectively,
were down-regulated in HF pigs compared to the LF group. Interestingly, esterase protein content,
as a key enzyme in fatty acid lipolysis, was lower in HF pigs compared to LF pigs. Others may be
worthy to further investigate, such as proteins involved in misfolding damage reparation (DJ-1),
detoxification of impaired proteins (peroxiredoxin 6), or dynamics of myofilaments (cofilin, myosin
light chain 2). Gene expression analysis led to 29 genes with significant differential expression
(P<0.05), and 29 additional genes only tended to discriminate the two pig groups (P<0.10).
Preliminary IPA analysis failed to identify any significant network related to lipid metabolism,
but highlighted two networks for carcinogenesis. This may be due either to the use of software
devoted to human health and disease, or to the low specificity of hybridisation between pig RNA
and human oligonucleotides. However, among the 33 genes up-regulated in HF pigs compared
to LF pigs, 3 genes were suggested to be involved in adipogenesis, adiposity or intramuscular fat
content (claudin, caveolin 3, carbonic anhydrase). The 25 genes that were up-regulated in the LF
pigs were not clearly related to lipid metabolism, and further investigations are needed. Only one
gene (the redox-sensitive protein glutathione S-transferase P1, [GSTP1]) was up-regulated in HF
pigs at both mRNA and protein levels.
Conclusion
Altogether, our results show that differences in i.m. fat content within pigs of the same breed
slaughtered at the same weight and age did not involve major differences in muscle lipid metabolism.
We suggest that differences in the number of adipocytes clustered along myofiber fasciculi in pigs
might be rather involved in i.m. fat variability.
References
Fernandez, X., G. Monin, A. Talmant, J. Mourot and B. Lebret, 1999. Influence of intramuscular fat content on the quality
of pig meat. 1. Composition of the lipid fraction and sensory characteristics of muscle longissimus lumborum. 2.
Consumer acceptability of muscle longissimus lumborum. Meat Sci. 53, 59-72.
Gondret, F. and J.F. Hocquette, 2006. La teneur en lipides de la viande: une balance métabolique complexe. INRA
Prod. Anim. 19, 327-338.

Bovine PRKAG3 gene expression and association with glycogen content
M. Roux1, E. Ciani2, D. Petit1, A. Ouali3, H. Levéziel1 and V. Amarger1,4
1Animal Molecular Genetics Unit, INRA, University of Limoges, France
2Departement of Animal Production, University of Pisa, Italy
3Livestock Products Quality (QuaPA) Unit, INRA, Clermont Ferrand-Theix, France
4Nutritional Adaptations Physiology (PhAN) Unit, INRA, Nantes, France
Introduction
Skeletal muscle metabolism is a very complex parameter that has a major influence on meat quality
because it influences both the structure and the biochemical characteristics of the muscle. It is
closely linked to energy intake and use and thus to some environmental factors that can be more or
less controlled. However, muscle metabolism has a strong genetic determinism that is still poorly
understood because of its complexity in both the number of genes involved and the close interaction
between them. The protein kinase, AMP-activated, γ3 non-catalytic subunit (PRKAG3) gene is a
member of the 5’-AMP-activated protein kinase (AMPK) gene family. AMPK is a heterotrimer
consisting of an α catalytic subunit, and non-catalytic β and γ subunits. AMPK is an important
energy-sensing enzyme that monitors cellular energy status (Carling, 2005). Mutations affecting the
genes encoding the regulatory γ subunits have been shown to influence AMPK activity. In the pig,
mutations on the PRKAG3 gene affect meat quality by influencing muscle glycogen content (Milan
et al., 2000). Glycogen content in the muscle is correlated to meat quality in livestock because it
influences the post-mortem maturation process and ultimate pH. Multiple bovine PRKAG3 variations
(46 SNP polymorphisms and two alternative splicing sites) have been identified that might encode
a set of sub-isoforms of the γ3 subunit (Roux et al., 2006). Preliminary work has been conducted in
order to find possible associations between specific polymorphisms and phenotypic traits.

A total of 100 Holstein calves, raised in different herds with a diet which could be different according
to the herd, were slaughtered, in the same conditions, in three groups, at approximately 4 mo of
age. For each animal, different phenotypic traits including meat characteristics (pH value, glycogen
content, drip loss) were measured in the Longissimus thoracis muscle. A TaqMan® 5’ allelic
discrimination assay (Applied Biosystems) and a sequencing approach using Big Dye Terminator
chemistry on a ABI PRISM 3130 cycle sequencer were used to obtain genotypic data. Haplotypes
were reconstructed from unphased genotypes using the computer program PHASE v. 2.1 (Stephens
et al., 2001). The association between meat quality traits and haplotypes was tested by ANOVA
using the software JMP v. 5.0 (2002). Expression of the PRKAG3 gene was measured by a semi-
quantitative RT-PCR approach on another set of animals, belonging to two common French beef
breeds (Charolais and Limousin) and raised in the same experimental herd.
Results
In this study, 100 Holstein animals were genotyped for 14 markers and three major haplotypes were
identified (66%, 12% and 11.5%). We found a significant association (P<0.05) between glycogen
content and two polymorphisms present on the PRKAG3 gene (Figure 1), located in exon 4 (SNP
2343) and intron 4 (SNP 2643). Association is independent of the herd of origin. The higher glycogen
content is found in heterozygous animals, glycogen content is significantly different between the
three different genotype populations.

Figure 1. The association between glycogen content and PRKAG3 gene. Variance analysis evidences a
significant association (P<0.05) between glycogen content and SNP 2343 (a) and SNP 2643 (b).
We also identified, in another set of animals genotyped for the same 14 markers that one of them
influences the expression level of the gene in the Longissimus thoracis muscle (Figure 2). Variation of
PRKAG3 mRNA levels was associated with the 2339 G/C missense mutation. Heterozygous animals
have an expression level 2 to 5 times higher than that of animals homozygous for the G allele.
Figure 1.These
The three
association between
polymorphisms glycogen
are located content
in a short and
region of 300PRKAG3 gene.
bp that seems to be Variance
an importantanalysis
evidencesregulating region. association
a significant These preliminary results may
(P<0.05) representglycogen
between significant clues for assessing
content and SNP the impact
2343 (a) and
of the PRKAG3 gene on meat quality in cattle. This work will be completed by a highly quantitative
SNP 2643method
(b). of allele discrimination, to compare the relative expression of the PRKAG3 alleles from
individuals heterozygous for the interesting SNP.
Figure 2. Variation of PRKAG3 mRNA levels in bovine skeletal muscle. Genotype at the 2339 SNP
position is indicated below each sample.
References
Carling, D., 2005. AMP-activated protein kinase: balancing the scales. Biochimie 87, 87-91.
Milan, D., J.T. Jeon, C. Looft, V. Amarger, A. Robic, M. Thelander, C. Rogel-Gaillard, S. Paul, N. Iannuccelli, L. Rask,
Figure 2. Variation
H. Ronne,ofK.PRKAG3
Lundstrom, N.mRNA
Reinsch, J.levels
Gellin, E.in bovine
Kalm, skeletal
P.L. Roy, P. Chardonmuscle. Genotype
and L. Andersson, at the
2000. A mutation 2339
SNP position is indicated below each sample.
in PRKAG3 associated with excess glycogen content in pig skeletal muscle. Science 288, 1248-1251.
Roux, M., A. Nizou, L. Forestier, A. Ouali, H. Levéziel and V. Amarger, 2006, Characterization of the bovine PRKAG3
gene: structure, polymorphism, and alternative transcripts. Mamm. Genome 17, 83-92.
Stephens, M., N.J. Smith and P. Donnelly, 2001. A new statistical method for haplotype reconstruction from population
data. Am. J. Hum. Genet. 68, 978-989.

Intestinal and ruminal epithelial and hepatic metabolism regulatory
gene expression as affected by forage to concentrate ratio in bulls
R.L. Baldwin VI1, S.W. El-Kadi2, K.R. McLeod2, E.E. Connor1 and B.J. Bequette3
1Agricultural Research Service, USDA, Beltsville, MD, 20705, USA
2University of Kentucky, Lexington, KY 40546, USA
3University of Maryland, College Park, MD 20742, USA
Introduction
In ruminants, the gastrointestinal tract (GIT) represents <10% of empty body weight (McLeod and
Baldwin, 2000), yet its metabolic activity accounts for 25 to 35% of whole body energy consumption
(Burrin et al., 1989). Extent and type of energetic substrates used by the GIT varies with energy
intake and diet energy density (e.g. forage:concentrate ratio). When compared, GIT from ruminants
fed high forage-low concentrate (HF) rations use more energy (McLeod and Baldwin, 2000) and
amino acids (AA; MacRae et al., 1995) than those fed low forage-high concentrate (HC) rations.
In consequence, a reduction in post-absorptive accretion of AA by carcass is observed (50 vs. 59%;
MacRae et al., 1995). Mechanisms underlying this selective use of substrates by the GIT are largely
unknown. Therefore, we sought to establish if regulation of substrate use by the GIT and liver occurs
at gene transcription by evaluating expression of several known committed enzyme steps in substrate
catabolism. Comparisons were made between bulls fed a HC vs. a HF ration.

Angus bulls (n=12; 391 ± 34 kg) were randomly assigned to two rations comprised of orchard grass
silage and grain-concentrate in a 75:25 (HF) or 25:75 (HC) ratio. Rations were offered at equal
dry matter intake (DMI)/kg body weight and refusals were recorded. Feed offered was adjusted
weekly based on body weight. After a 28 d feeding period, bulls were slaughtered and intestinal
and rumen epithelia, and liver, were harvested for determination of gene expression (qRT-PCR) of
several key enzymes involved in substrate metabolism: sodium glucose co-transporter I (SGLT-1),
monocarboxylic acid transporter-1 (MCT-1), acetyl/propionyl-CoA synthetase 2 (ACAS2L), butyryl-
CoA synthetase 1 (BUCS1), branched chain aminotransferase 2 (BCAT2), branched chain keto acid
dehydrogenase (BCKDHA), glutaminase (GLS), glutamate dehydrogenase (GLUD1), glutamate-
oxaloacetic transaminase 2 (GOT2), and glucose-6-phosphate dehydrogenase (G6PDH). Amplified
products were verified by sequencing. Total RNA was obtained using RNeasy isolation kits with
on-column DNase digestion and quality and concentration were determined. Starting quantities were
quantified using a standard curve (1×102 to 1×108 copies). Assay correlation coefficients ranged
from 0.995 to 0.999 and PCR efficiencies ranged from 87-114%. Negative control reactions detected
no amplification of the product.

By design, DMI was similar for the two rations resulting in 33% greater metabolizable energy intake
for the HC vs. HF bulls and average daily gains of 1.6 kg/d and 1.56 kg/d for HC and HF, respectively.
Thus, differences in gene expression reflected changes due to substrate and/or ME supply and not
DMI. Abundance of BCAT2 transcripts were at or below detection and abundance of MCT-1, SGLT-1,
ACAS2L and GLS, while present, were unaffected by treatment. Abundance of BCKDHA transcripts
(Table 1) increased in rumen (P<0.01) of HF bulls but was unaffected in intestinal epithelium and
liver. Abundance of BUCS1 transcripts tended to decrease in rumen (P=0.1) and increased (P<0.03)
in the jejunum and liver in HF bulls. Expression of G6PDH was greater (P<0.05) in the rumen and
liver of HF bulls, but abundance in the intestinal segments was unaffected by treatment. Expression

of GOT2 was greater in the rumen (P=0.0001) and jejunum (P<0.1) of HF bulls whereas expression
in the duodenum and ileum was not affected by ration. In vivo portal drained viscera removal of
branched chain amino acids been demonstrated to increase with increased dietary forage (Seal et
al., 1992) and here we report elevated rumen BCKDHA expression with increased forage. While
the activation step for butyrate metabolism is up regulated (BUCS1), there was not a concomitant
increase in the CoA activation enzyme (ACAS2L) for propionate and acetate. These data demonstrate
in cattle differential expression of regulatory genes involved in substrate catabolism by the GIT in
response to altered nutrient supply using complex rations.
Table 1. Gene expression (transcripts/µgram RNA) of bulls fed either concentrate (HC) or forage(HF)
based diets.
Tissue
Transcript Diet Rumen Duodenum Jejunum Ileum Liver
BCKDHA1 HC 50957** 9082 7682 9211 9373

HF 70551 10881 9576 9616 11482
SEM 5090 784 966 978 1218
BUCS11 HC 17976* 1059 162*** 1096 1120293**
HF 1103 14323 519 280 1523446
SEM 7048 5981 68 344 123991
ACAS2L1 HC 3711 52110 96657 161481 5353
HF 4622 51614 92205 152006 5312
SEM 393 3788 10269 21274 554
GOT21 HC 128525*** 105551 72926* 96011 ND2
HF 226387 85179 89448 118887 ND
SEM 14969 9942 6750 13722
*,†,‡
Means of HF and HC are significantly different. *P≤0.1,**P≤0.05,***P≤0.001.
1 Transcript
identifications are presented in Material and methods.
2 ND = not determined.
References
Burrin, D.G., C.L. Ferrell, J.H. Eisemann, R.A. Britton and J.A. Nienaber, 1989. Effect of level of nutrition on splanchnic
blood flow and oxygen consumption in sheep. Br. J. Nutr. 62, 23-34.
MacRae, J.C., L.A. Bruce and D.S. Brown, 1995. Efficiency of utilization of absorbed amino-acids in growing lambs
given forage and forage-barley diets. Anim. Sci. 61, 277-284.
McLeod, K.R. and R.L. Baldwin, 2000. Effects of diet forage: concentrate ratio and metabolizable energy intake on
visceral organ growth and in vitro oxidative capacity of gut tissues in sheep. J. Anim. Sci. 78, 760-770.
Seal, C.J., D.S. Parker and D.P.J. Avery, 1992. The effect of forage and forage concentrate diets on rumen fermentation
and metabolism of nutrients by the mesenteric-drained and portal-drained viscera in growing steers. Br. J. Nutr.
67, 355-370.

Genomic tools to analyse bovine muscle and adipose tissues
transcriptomes
A.K. Kadanga, C. Leroux, M. Bonnet, I. Cassar-Malek and J.F. Hocquette
INRA, Herbivore Research Unit, Theix, 63122 Saint-Genès-Champanelle, France
Introduction
Microarray technology allows us to explore a major subset or almost all genes for an organism.
Experimental biases in gene expression profiling occur due to a varied total amount of hybridised
mRNA, differing label incorporation rate, spot quantification methodology for image analysis, or
bleaching effects of the dye (Ahmed et al., 2004). The accuracy and reproducibility of data generated
using microarray technology would be enhanced by the use of a common set of standards as recently
proposed (Shi et al., 2006). Although normalization methods have been developed to assess spot
quality in an extended examination of spot size, signal-to-noise ratios, background uniformity and the
saturation status, the variability introduced by image analysis methods needs to be assessed. There
is no consensus about what is the best way to analyse microarray data. The aim of this work was
to assess different microarray procedures, based on different image analysis and statistic methods,
by looking at the variability of the results. We have analysed two different data sets from the same
experiment by two normalisation methods and two statistical approaches. Utilisation of GenePix/
Madscan (GP/M) or ImaGene/Genesight (IG/G) software influenced the outcome of differentially
expressed genes regardless of the statistical method.

Total RNA was extracted from subcutaneous (SC) adipose tissue and from Longissimus thoracis (LT)
muscle. RNA was purified with RNeasy® Mini kit (Qiagen). RNA integrity was checked with the Lab
Chip Agilent technology. The reference was made of total RNA from SC, LT and mammary gland.
cDNA labelling with cyanine 5 or 3 (Amersham) was performed with ‘Pronto Plus Direct Systems’
(Corning-Promega) following recommendations of the supplier. The concentration and frequency of
incorporation were determined by spectrophotometry. Hybridisation was performed on a microarray
(Corning Ultra GAP, Toulouse) containing 8400 bovine oligonucleotides (Operon) with 40 pmoles
of labelled cDNA, at 42 °C for 16 h followed by washes and scan on 428 MWG Array Scanner
(MWG Biotech). We compared two experimental designs, triplicate and dye swap. Two forms of
software, GenePix Pro 6.0 (Axon Instruments) or ImaGene 6.0 (BioDiscovery) were used for spot
definition. The normalisation of data was done using Madscan 6.0 (http://cardioserve.nantes.inserm.
fr/mad/) or Genesight 4.1.6 (BioDiscovery). The data were then submitted to significance analysis
of microarray (SAM) (P=5%) or Student t-test with multi-test correction (Bonferroni 1%).

We compared two preprocessing methods (GP/M and IG/G) under two different statistical methods
(SAM and Student t-test) in two experimental designs (triplicate and dye swap). Our results show
that for a high fold change (FC>2; not shown), both image approaches were quite similar in the
number of differentially expressed genes. For a low fold change (FC>1.4), the number of genes
that exhibited significant changes in expression was higher for the GP/M combination than for the
IG/G in triplicate using SAM (Table 1). Moreover, the presence of six positive control genes was
indeed considered as a test of reliability in this study. Within the triplicate, only one gene with GP/
M compared to six with IG/G was declared differentially expressed. In the dye swap experimental
design, all six compared to five were outlined with the two procedures, respectively.

Table 1. Number of differentially expressed genes after treatment by GenePix (GP), Madscan (M),
ImaGene (IG), Genesight (G), SAM and multi-test.
Sample/Treatment GP/M/SAM IG/G/SAM IG/G/Multi-test
Triplicate
LT FC>1.4 352 140 207
Control genes 1 6 6
SC FC>1.4 384 216 138
Control genes 1 5 5
Dye-swap
SC FC>1.4 197 - 116
Control genes 6 - 5
The combination of image analysis and data normalisation is an important aspect of microarray
experiments; one that can have a potentially large impact on downstream analyses such as the
identification of differentially expressed genes. There was no significant difference related to
statistical treatment in the number of genes even though designing procedures to control the False
Discovery Rate (criterion to identify differentially expressed genes) is challenging (Verducci et al.,
2006). Although Dobbin et al. (2005) reported a minimal impact of dye biases, in our experiment,
dye swap sorted more control genes than the triplicate design. We demonstrated the high impact of
extracting raw data, before running the statistical analyses. From the above discussion, it is obvious
why there can be a great deal of discordance in results obtained from different or within the same
microarray platforms. By presenting the experimental design and performance advantages of both
procedures, we provide insight and guidance for properly selecting the best approach to meet research
needs. For future work, we chose to use IG/G approach in a dye-swap manner, which was the more
accurate regarding cost and experimental design consideration.
References
Ahmed, A.A., M.Vias, N.G. Iyer, C. Caldas and J.D. Brenton, 2004. Microarray segmentation methods significantly
influence data precision. Nucleic Acids Res. 32, e50.
Dobbin, K.K., E.S. Kawasaki, D.W. Petersen and R.M. Simon, 2005. Characterizing dye bias in microarray experiments.
Bioinformatics 21, 2430-2437.
Shi, L., L.H. Reid, W.D. Jones, R. Shippy, J.A. Warrington, S.C. Baker, P.J. Collins, F. de Longueville et al., 2006. The
MicroArray Quality Control (MAQC) project shows inter- and intraplatform reproducibility of gene expression
measurements (MAQC Consortium). Nat. Biotechnol. 24, 1151-1161.
Verducci, J.S., V.F. Melfi, S. Lin, Z. Wang, S. Roy and C.K. Sen, 2006. Microarray analysis of gene expression:
considerations in data mining and statistical treatment. Physiol. Genomics 25, 355-363.
This study was funded by a national grant from the ‘Agence Nationale de la Recherche’ and from
APIS-GENE (GENANIMAL call, national programme AGENAE) for the project entitled ‘MUGENE’
(GENEs of the MUscle tissue).

Effect of inoculation with intestinal bacteria on intestinal gene
expression in the interleukin-10 knockout mouse
M.P.G. Barnett1, S.-T. Zhu2, A.L. Cookson1, R. Broadhurst3, B. Knoch1,4, M. Davy5, W.C. McNabb1
and N.C Roy1
1Food, Metabolism & Microbiology, Food & Health Group, AgResearch Grasslands, Tennent Drive,
2Faculty of Medical and Health Sciences, The University of Auckland, Park Rd, Grafton, Auckland
1023, New Zealand
3National Resources Group, AgResearch Ruakura, East Street, Hamilton 3214, New Zealand
4Institute of Food, Nutrition & Human Health, Massey University, Palmerston North 4474, New
Zealand
5HortResearch, 120 Mt Albert Road, Sandringham, Auckland 1025, New Zealand
Introduction
The complex genetic nature of Inflammatory Bowel Diseases (IBD: Crohn Disease and Ulcerative
Colitis), and the interaction between food, host genetic background and resident intestinal bacteria,
means that animal models of IBD are essential to study these interactions. The interleukin-10
knockout (IL10-/-) mouse has been reported to spontaneously develop Crohn Disease-like intestinal
enterocolitis, caused in part by an inappropriate response to normal intestinal bacteria, when raised
under conventional conditions, and has been used extensively as a model of IBD (Jurjus et al., 2004).
In a previous study, we found that intestinal inflammation in IL10-/- mice (C57BL/6J background) was
not significantly higher than in C57BL/6 (control) mice due to a high variability of response between
IL10-/- mice (Barnett et al., unpublished data). Our aim was to test whether an oral inoculation of
IL10-/- mice with normal intestinal bacteria would result in a more consistent intestinal inflammation
and to characterise gene expression changes in intestinal cells resulting from this inoculation.

At 5 wk of age, five C57BL/6J and five IL10-/- (C57BL/6J background) mice were orally inoculated
with 12 Enterococcus faecalis and faecium strains (EF; sourced from cattle and poultry) and/or
complex intestinal flora (CIF) collected from healthy, age-matched C57BL/6 mice raised under
conventional conditions. At 12 wk of age, intact intestinal sections were assigned a histological
injury score based on inflammatory cell infiltrates, tissue destruction and tissue repair (Matsumotu
et al., 1998). RNA extracted from intact colon was labelled with Cy3 dye and hybridised on
Agilent 44k arrays with a Cy5-labelled reference RNA sample. Microarray spot quantification was
performed with GenePix 6.0 software. Gene list comparisons were generated in limma (Bioconductor
framework, Smith 2005) using moderated probability values for t-statistics, false discovery rate
control (alpha=0.05) and a differential expression cut-off of 1.5-fold or greater. Genes were clustered
using GeneSpring GX and Ingenuity Pathway Analysis software.

Colon histological injury scores of IL10-/- mice inoculated with the EF.CIF combination were higher
(P<0.05) than control mice inoculated with EF. CIF combination and non-inoculated IL10-/- mice
(Barnett et al., 2006). Gene expression in inflammatory pathways was consistently up-regulated, while
detoxification pathways (e.g. cytochrome P450 family) showed down‑regulation in EF.CIF-inoculated
IL10-/- mice (Table 1). Expression of 4185 genes was altered in IL10-/- mice due to inoculation with
EF.CIF; only 57 genes changed in control mice due to this inoculation. These results show that
IL10-/- mice inoculated with EF.CIF have characteristics of chronic inflammatory diseases such as

a dysregulated immune response, making them an improved model of intestinal inflammation with
which to identify food components of potential benefit to people with IBD.
Table 1. Differentially expressed inflammation and detoxification genes in colonic epithelium tissue
of IL-10-/- mice in response to EF.CIF bacterial inoculation.
Gene name Symbol Refseq ID IL10-/- vs. C57

(all EF.CIF)
Inflammation
CD74 molecule, MHC Class II invariant chain CD74 NM_010545 +5.7
MHC Class II, DM alpha HLA-DMA NM_010386 +2.9
Suppressor of cytokine signaling 3 SOCS3 NM_007707 +4.8
Interferon, gamma IFNG NM_008337 +2.5
Detoxification
ATP-binding cassette, sub-family B (MDR/ ABCB1 NM_011076 -2.8
TAP), member 1
Sulfotransferase family, cytosolic, 1A, phenol- SULT1A1 NM_133670 -4.5
preferring, member 1
Cytochrome P450, family 2, subfamily C, CYP2C40 NM_010004 -7.4
polypeptide 40
Cytochrome P450, family 4, subfamily B, CYP4B1 NM_007823 -15.1
polypeptide 1
References
Barnett, M.P.G., S.T. Zhu, R. Broadhurst, W.C. McNabb and N.C. Roy, 2006. Effect of Enteroccocus inoculation on
intestinal gene expression in inflamed colon of interleukin-10 knockout mouse. Reprod. Nutr. Dev. 46, S67.
Jurjus, A.R., N.N. Khoury and J.-M. Reimund, 2004. Animal models of inflammatory bowel disease. J. Pharmacol.
Toxicol. Methods 50, 81-92.
Matsumoto, S., N. Watanabe, A. Imaoka and Y. Okabe, 2001. Preventive effects of Bifidobacterium- and Lactobacillus-
fermented milk on the development of inflammatory bowel disease in senescence-accelerated mouse P1/Yit strain
mice. Digestion 64, 92-99.
Smyth, G.K., 2005. Limma: linear models for microarray data. In: Gentleman, R., V. Carey, S. Dudoit, R. Irizarry and
W. Huber (editors), Bioinformatics and computational biology solutions using R and Bioconductor. Springer,
New York, 397-420.
Nutrigenomics New Zealand is a collaboration between AgResearch Limited, Crop & Food
Research, HortResearch and The University of Auckland and is largely funded by the Foundation
of Research, Science and Technology (FRST). MPG Barnett is funded by FRST Postdoctoral
Fellowship AGRX0504. B Knoch is a PhD student whose Fellowship is funded by Nutrigenomics
New Zealand.

Modification of gene expression involved in muscle growth and energy
metabolism according to growth capacity in young Charolais bulls
C. Bernard1, I. Cassar-Malek1, G. Renand2 and J.F. Hocquette1
1INRA, UR1213, Unité de Recherches sur les Herbivores, Equipe Croissance et Métabolisme du
Muscle, Theix, 63122, Saint-Genès-Champenelle, France
2INRA, UR337, Station de Génétique Quantitative et Appliquée, 78352, Jouy-en-Josas, France
Introduction
In cattle, genetic selection in France has been directed in favour of high muscle development at the
expense of fat in order to produce leaner carcass and to quantitatively increase meat production.
However, it has influenced muscle characteristics, and therefore can have a significant impact on
meat quality. Indeed, some studies performed on two lines of Charolais young bulls, divergently
selected on muscle growth capacity, showed that the muscle modifications driven by selection may be
favourable to tenderness thanks to the strengthening of the fast-glycolytic muscle properties (which
favour meat ageing) but detrimental to flavour due to a reduction in intramuscular fat deposition
(Renand et al., 1994, Sudre et al., 2005).
The aim of this study was to identify differentially expressed genes according to muscle growth capacity
in order to get a better understanding of the effect of this selection on the muscle characteristics, with
the ultimate objective of controlling meat quality variability. To this end, our strategy consisted in
analysing and comparing the transcriptome of Longissimus thoracis (LT) muscle of 15- and 19-mo-old
Charolais young bulls divergently selected for their high (H) or low (L) muscle growth capacity.

This study was conducted with 25 young Charolais bull calves from an INRA experimental herd.
Animals were characterised in terms of growth performances (breeding values for muscle mass
and fat content) and muscle properties (enzyme activities; lipid content; collagen characteristics)
as described by Cassar et al. (2004) and Sudre et al. (2005). Total RNA was extracted from LT
muscle, using TRIZOL® Reagent (Life Technologies). RNA quality was assessed using an Agilent
2100 bioanalyser (Agilent Technologies). Microarray experiments were performed as described in
Cassar-Malek et al. (2007). We searched for differentially expressed genes between groups with high
or low muscle growth capacity at 15 and 19 mo of age (4 animals per extreme group). For carcass
composition and biochemical data, analysis of variance was performed using the GLM Procedure
(Statistical Analysis System Institute, 1996). In order to calculate percentage of muscle growth
variability explained by muscle characteristics and gene expression levels, a correlation study was
performed using the Statistica software (StatSoft, France).

The impact of genetic selection on animal characteristics was as high as that of age (19 vs. 15 mo).
Animals divergently selected for muscle growth capacity were characterized by highly significant
differences in muscle mass (+17%; P<0.05) and fat content in the carcass (-17%; P<0.05). By
contrast, the cytochrome-c oxidase (COX) activity in LT muscle was influenced by age (+41.6%;
P<0.05) but not by the genetic type, whereas intramuscular lipid and triglyceride contents were only
significantly affected by genetic type (-35.0% and –47.3%; P<0.05 respectively in H animals). The
other muscle characteristics were not affected either by the genetic type or by the age.

In order to identify gene expression differences according to muscle growth capacity, SAM analyses
(FDR≤1%) of the data were performed for each age group. They enabled identifying 262 differential
genes according to muscle growth potential whatever the age of the animals. Among them, 173 had
a similar variation at both ages of which 88 were up-regulated and 85 were down-regulated in H
animals whereas the 89 remaining genes underwent different expression profiles according to animal
age. About one third of these 173 genes were not classified in any Gene Ontology (GO) biological
process. The others were mostly involved in signal transduction (18.5%), protein metabolism and
its modification (16.8%), notably proteolysis and protein phosphorylation, immunity and defence
(10.4%), nucleic acid metabolism (9.2%) and more particularly mRNA transcription, developmental
processes (8.7%), and transport (8.1%).
Transcriptomic data revealed the up-regulation of about two thirds of the genes involved in the
glycolytic pathway in H animals at both 15 and 19 mo of age. Expression of genes involved in the
oxidative metabolism (citrate cycle) was also found to be down-regulated at 15 mo and up-regulated
at 19 mo of age. Lastly, expression of some genes was associated with muscle mass in the carcass
but was independent of fat deposition and meat quality. For instance, genes (FGF6, PLD2) involved
in muscle growth pathways (mTOR signalling) were found to be differentially expressed between
H and L animals.
Conclusion
Genetic selection in favour of muscle growth capacity induced changes in muscle gene expression.
These were associated with an increase in glycolytic metabolism and a reduction in oxidative
metabolism, especially at 15 mo of age. The differential expression of some genes was likely involved
in muscle mass development independently of fat deposition. However, none of these genes was
associated with meat sensory quality suggesting that selection upon muscle growth would not modify
meat quality to a great extent.
References
Cassar-Malek, I., J.F. Hocquette, C. Jurie, A. Listrat, R. Jailler, D. Bauchart, Y. Briand and B. Picard, 2004. Muscle-
specific metabolic, histochemical and biochemical responses to a nutritionally induced discontinuous growth
path. Anim. Sci. 79, 49-59.
Cassar-Malek, I., F. Passelaigue, C. Bernard, J. Léger and J.F. Hocquette, 2007. Target genes of myostatin loss-of-
function in muscles of late bovine fetuses. BMC Genomics 8, 63-74.
Renand, G., P. Berge, B. Picard, J. Robelin, Y. Geay, D. Krauss and F. Menissier, 1994. Genetic parameters of young
Charolais bulls progeny of divergently selected sires. Proc 5th World Congress Genet. Appl. Livest. Prod., Guelph,
Canada, 19, 446-449.
Statistical Analysis System Institute, 1996. SAS/STAT guide for personal computers, SAS Institute Inc., Cary, NC.
Sudre, K., I. Cassar-Malek, A. Listrat, Y. Ueda, C. Leroux, C. Jurie, C. Auffray, G. Renand, P. Martin and J.F. Hocquette,
2005. Biochemical and transcriptomic analyses of two bovine skeletal muscles in Charolais bulls divergently
selected for muscle growth. Meat Sci. 70, 267-277.
This study was funded by a national grant from the ‘Agence Nationale de la Recherche’ and from
APIS-GENE (GENANIMAL call, national programme AGENAE) for the project entitled ‘MUGENE’
(GENEs of the MUscle tissue).

The myogenic influence of triiodothyronine nuclear and mitochondrial
pathways explored by transcriptome analysis
O. Baris, P. Seyer, M. Busson, S. Grandemange, C. Gouarné, L. Pessemesse, F. Casas, C. Wrutniak-
Cabello and G. Cabello
UMR866, Différenciation Cellulaire et Croissance, INRA, 2 pl. Viala, 34060, Montpellier, France
Introduction
In our previous works, we showed that triiodothyronine (T3) is a major regulator of myoblast
differentiation. Triiodothyronine not only stimulates myoblast cell-cycle exit, a key event of cellular
differentiation, but also enhances their terminal differentiation. This hormonal myogenic influence
is mediated by two pathways: first, a nuclear pathway, through the use of nuclear receptors acting
as T3-dependent transcription factors. The binding sites of c-ErbA α1 (TR), the main receptor in
myoblasts, differ during proliferation and differentiation, thus implying different target genes (Busson
et al., 2006). The second is a mitochondrial pathway, identified in our laboratory, through the use
of a truncated form of TR, p43, located in the mitochondrial matrix (Wrutniak et al., 1995). When
bound to T3, p43 acts as a transcriptional factor of mitochondrial DNA (Casas et al., 1999) and
influences mitochondrial activity and transcription of nuclear genes such as c-myc and myogenin
(Seyer et al., 2006). Although the physiological relevance of these two pathways is well established,
the molecular mechanisms involved are not fully elucidated.
Results
Our aim was to identify as exhaustively as possible the transcriptional targets of T3 nuclear and
mitochondrial pathways during myoblast differentiation, in order to better understand the molecular
mechanisms by which the hormone exerts its myogenic influence. Transcriptome analysis experiments
using glass slide oligonucleotide arrays were performed on the following: (1) C2C12 myoblasts treated
or not with T3 (1nM); five independent experiments; (2) C2C12 myoblasts stably overexpressing p43
and control cells treated or not with chloramphenicol (antagonist of the T3 mitochondrial pathway);
five independent experiments. After normalisation of raw microarray data, we performed a supervised
analysis using SAM with a FDR≤1% to identify the differentially expressed genes.
T3 global transcriptional effects during myoblast differentiation
T3 treatment led to the up-regulation of 764 genes and the down-regulation of 37 genes during
myoblast differentiation. Interestingly, among these two lists are genes already known to be regulated
by the hormone (Acta1, Atp2a1, Atp2a2, Bteb1, Camk4, Myod, Myog, Ryr, Strbp, Ucp2…) thus
validating our experimental approach. Induced genes were mainly involved in muscle structure
and function, cell signalling, cell junction and cell to matrix communication. Repressed genes were
involved in cell adhesion and cell to matrix communication.
We confirmed the differential expression of 10 genes chosen for their potential involvement in
myogenic differentiation (Tead4, p21, Bin1, Rb1cc1, Itga7, Vamp5, Mef2d, P2rx5, Nov, Trim54) and
myogenin, as a positive control, by quantitative PCR experiments, using the Rps9 gene as a reference
to normalise data. For 3 of these genes (p21, Rb1cc1, Nov) involved in cell proliferation and/or cell
cycle withdrawal, we identified putative Thyroid Response Elements in their promoters, suggesting
their direct regulation by the hormone. Time course experiments are currently being performed to
check this hypothesis. Furthermore, these sequences have been cloned into a reporter gene plasmid
that will be transfected into C2C12 cells, in order to test their physiological relevance.

Mitochondrial activity transcriptional targets during myoblast differentiation
This part of our project is currently under analysis, but we already identified genes potentially
regulated by mitochondrial activity, mainly involved in metabolism, cell signalling cell cycle and
cell structure (Adfp, p21, Col1a1, Fabp3, Gstp2, Igfbp5, Itgb1bp2, Ly6a, Mfap5, Ndrg4, Ndufb7,
Ndufb8, Scya2, Tnfrsf12a, Vim). Several genes were found to be similarly regulated by T3 treatment
and p43 overexpression, including myogenin, as previously observed, and genes coding for various
isoforms of troponins and tropomyosins. Interestingly, Ndufb7 and Ndufb8, over-expressed in C2C12
cells stably expressing p43, code for 2 subunits of the mitochondrial respiratory chain complex I,
in agreement with the p43 influence on mitochondrial biogenesis. Moreover, Ly6a, repressed in
these same cells has been shown to negatively regulate myoblast proliferation and differentiation
(Mitchell et al., 2005).
Conclusion
Our results should lead to a better knowledge of the mechanisms by which T3 influences myogenic
differentiation and suggest that this hormone plays a wider role in this process than previously
envisaged. Indeed, apart from its action on cell-cycle exit and terminal differentiation, T3 may act
at several levels in the cell, influencing cell adhesion and cell to matrix communication and inducing
changes in the intracellular signalling such as calcium signalling.
References
Busson, M., L. Daury, P. Seyer, S. Grandemange, L. Pessemesse, F. Casas, C. Wrutniak-Cabello and G. Cabello, 2006.
Avian MyoD and c-Jun coordinately induce transcriptional activity of the 3,5,3’-triiodothyronine nuclear receptor
c-ErbAalpha1 in proliferating myoblasts. Endocrinology 147, 3408-3418.
Casas, F., P. Rochard, A. Rodier, I. Cassar-Malek, S. Marchal-Victorion, R.J. Wiesner, G. Cabello and C. Wrutniak,
1999. A variant form of the nuclear triiodothyronine receptor c-ErbAalpha1 plays a direct role in regulation of
mitochondrial RNA synthesis. Mol. Cell. Biol. 19, 7913-7924.
Mitchell, P.O., T. Mills, R.S. O’Connor, E.R. Kline, T. Graubert, E. Dzierzak and G.K. Pavlath, 2005. Sca-1 negatively
regulates proliferation and differentiation of muscle cells. Dev. Biol. 283, 240-252.
Seyer, P., S. Grandemange, M. Busson, A. Carazo, F. Gamaleri, L. Pessemesse, F. Casas, G. Cabello and C. Wrutniak-
Cabello, 2006. Mitochondrial activity regulates myoblast differentiation by control of c-Myc expression. J. Cell.
Physiol. 207, 75-86.
Wrutniak, C., I. Cassar-Malek, S. Marchal, A. Rascle, S. Heusser, J.M. Keller, J. Flechon, M. Dauca, J. Samarut, J.
Ghysdael and G. Cabello, 1995. A 43-kDa protein related to c-Erb A alpha 1 is located in the mitochondrial matrix
of rat liver. J. Biol. Chem. 270, 16347-16354.

Functional genomics of rat skeletal muscle during aging
A. Listrat1, I. Piec2, J. Alliot3, R.G. Taylor4 and D. Béchet2
1URH, INRA-Theix, 63122 Saint-Genès-Champanelle, France
2UNH, INRA-Theix, 63122 Saint-Genès-Champanelle, France
4QuAPA, INRA-Theix, 63122 Saint-Genès-Champanelle, France
3Université de Clermont-II, 63177 Aubière, France
Introduction
Skeletal muscle accounts for 40% of mammalian body weight, and the age-related reduction in
skeletal muscle mass ranges from 20 to 30% between ages 20 to 80 in humans, even in healthy
individuals (Carmeli et al., 2002). The progressive decline in skeletal muscle mass and function due
to aging (sarcopenia) therefore contributes significantly to loss of functional autonomy, increased
prevalence for falls, and greater morbidity. Age-related degenerative changes are reflected in
alterations in muscle morphology, function, and biochemical properties. Sarcopenia is thus associated
with muscle fibre atrophy (Jubrias et al., 1997), loss of satellite cell regenerative capacity Renault et
al. (2002), and remodelling of pre- and postsynaptic neuronal structures (Kamel, 2003). A change
in the composition of muscles from fast-twitch (type II) to slow-twitch (type I) fibres (Lexell et
al., 1988) and reduced synthesis rates of proteins such as myosin heavy chain are also observed
(Balagopal et al., 1997). To identify the mechanisms underlying healthy aging of skeletal muscle,
we have undertaken an analysis by proteomic, transcriptomic and histology in young adult, mature
adult, and old LOU/c/jall rats. This study is one of the first to describe in a systematic way aging-
induced changes in skeletal muscle proteins.

Fifteen male LOU/c/jall rats (7, 18, and 30 mo) were obtained from the Neurobiology of Aging
laboratory (Clermont-Ferrand II University, France), and 5 animals were used for each age group.
The rats had free access to water and food and were subjected to standard conditions of humidity,
temperature (21 ± 1 °C), and 12 h light cycle. Rats were killed by decapitation, and gastrocnemius
muscles from each animal were immediately dissected, weighed, and frozen in liquid nitrogen.
Muscles were stored at –80 °C until processed. Two dimensional gel electrophoresis were performed
according to Piec et al. (2005) and transcriptomics according to Welle et al. (2003).

Histology and image analyses revealed a pronounced reduction in fibre area, smaller myonuclear
domain, more centralised myonuclei and an increased proportion of apoptotic nuclei in old
gastrocnemius muscles. Histology further indicated profound alterations in the extracellular matrix
(ECM), as evidenced by increases in ECM area (fibrosis), in proteoglycan immunolabelling, and
in density of fibroblasts and macrophages, all indicative of chronic inflammation. Aging was also
associated with fibre type grouping (Cox, SDH), suggesting fibre regeneration and neuromuscular
remodelling. Ultrastructural analysis further showed that 3-4% fibres exhibit myofibril thinning,
thickened basal lamina, mitochondrial aggregation, and lysosomal lipofuscin accumulation. To
gain more information on the molecular mechanisms underlying sarcopenia, transcriptome analyses
using Affymetrix arrays were performed. There were approximately 31 000 genes on the array,
of which 16 000 were expressed at all ages, and 1100 were differentially expressed in old rats.
Some of the highlighted changes are increased expression of mRNA encoding amyloid proteins,
cathepsin proteases, complement proteins, and decreased mRNA for glycolysis and Krebs cycle.
Classic sarcopenia markers including oxidative stress, lamin A, and metallothionein also changed

expression. Because mRNA levels do not predict changes in protein levels and in post-translational
modifications, we also undertook high resolution differential proteomic analyses in young, middle-
age and old rat muscles. Two-dimensional gel electrophoresis and subsequent MALDI-TOF mass
spectrometry led to the identification of 40 differentially expressed proteins. A Western blot plateform
(BD Biosciences) analysis of 744 minor proteins further identified 18 sarcopenia markers. Most
differences characterised old animals, whereas young and mature adults exhibited similar patterns
of expression. Our proteomic studies underline important modifications in contractile (actin, myosin
LC, troponins-T) and in essential regulatory proteins (MyBP, CapZ-β, telethonin), which likely
account for dysfunctions in old muscle contractile properties. They show that this disorganisation
of myofibrillar structures is compensated for by an increase in cytoskeletal proteins (desmin,
β-tubulin, gelsolin, annexin-II, M-cadherin). Differential proteomics also point to an analogous
decline of glycolytic (triose-P isomerase, enolase, glycerol-3-P-DH), Krebs cycle (isocitrate-DH)
and respiratory chain (ATP synthase α, Cox) enzymes, indicating profound perturbations in the
energy metabolism of old muscles. The old gastrocnemius is challenged with the detoxification of
cytotoxic aldehydes (increased aldehyde-DH, glutathione transferase, glyoxalase) and overexpresses
molecular chaperones, which counteract protein misfolding (hsp20, hsp27, Er60). We further noticed
up-regulation of proteins implicated in transcriptional elongation (RNA capping protein), alternative
splicing (YT521-B) and RNA-editing (Apobec2). Finally, another important observation was the
overexpression of galectin-1, which regulates essential functions, such as neuronal path-finding or
myoblast spreading and fusion.
In conclusion, these studies support that chronic inflammation (‘InflamAging’), re-innervation,

perturbed energy metabolism, altered antioxidant and contractile systems and up-regulation of
cytoskeletal structures are typical of the aging muscle physiology. The majority of our biomarkers
were previously unrecognised as differentially expressed in old muscles, and they represent novel
starting points for elucidating the mechanisms of muscle aging.
References
Balagopal, P., O.E. Rooyackers, D.B. Adey, P.A Ades and K.S. Nair, 1997. Effects of aging on in vivo synthesis of
skeletal muscle myosin heavy-chain and sarcoplasmic protein in humans. Am. J. Physiol. 273, E790-E800.
Carmeli, E., R. Coleman and A.Z. Reznick, 2002. The biochemistry of ageing muscle. Exp. Gerontol. 37, 477-489.
Jubrias, S.A., L.R. Odderson, P.C. Esselman and K.E. Conley, 1997. Decline in isokinetic force with age: muscle
cross-sectional area and specific force. Pflugers Arch. 434, 246-253.
Kamel, H.K., 2003. Sarcopenia and aging. Nutr. Rev. 61, 157-167.
Lexell, J., C.C. Taylor and M. Sjostrom, 1988. What is the cause of the ageing atrophy? Total number, size and
proportion of different fiber types studied in whole vastus lateralis muscle from 15- to 83-year-old men. J. Neurol.
Sci. 84, 275-294.
Piec I., A. Listrat, J. Alliot, C. Chambon, R.G. Taylor and D.M. Bechet, 2005. Differential proteome analysis of aging
in rat skeletal muscle. FASEB J. 19, 1143-1145.
Renault, V., L.E. Thornell, P.O. Eriksson, G. Butler-Browne and V. Mouly, 2002. Regenerative potential of human
skeletal muscle during aging. Aging Cell 1, 132–139.
Welle S., A.I. Brooks, J.M. Delehanty, N. Needler, C.A. and Thornton, 2003. Gene expression profile of aging in human
muscle. Physiol. Genomics 14, 149-159.

Intestinal mucosa proteome analysis in goat kids fed milk or soy protein
based diets
S. Kuhla1, P.E. Rudolph2, D. Albrecht4, U. Schönhusen1, R. Zitnan5, W. Tomek3, K. Huber6, J. Voigt1
and C.C. Metges1
1Research Units Nutritional Physiology ‘Oskar Kellner’, Research Institute for the Biology of Farm
Animals (FBN), D-18196 Dummerstorf, Germany
2Genetics and Biometry, Research Institute for the Biology of Farm Animals (FBN), D-18196
Dummerstorf, Germany
3Reproductive Biology, Research Institute for the Biology of Farm Animals (FBN), D-18196
4Institute of Microbiology, Ernst-Moritz-Arndt-University Greifswald, D-17489 Greifswald,
Germany
5Institute of Animal Nutrition, Slovak Agricultural Research Centre Nitra, SK-94992 Nitra, Slovak
Republic
6Department of Physiology, School of Veterinary Medicine Hannover, D-30173 Hannover,
Germany
Introduction
Alternate-protein milk replacer formulas for suckling pre-ruminants containing 50% or less total
protein as soy protein (SP) have been shown to reduce growth and feed efficiency in young ruminants
when compared to whole milk or casein diets (Drackley et al., 2006). This might be associated
to changes of small intestinal mucosa with decreased villous height and crypt cell depth with
consequences on absorptive function. A known deficit of indispensable amino acids (AA) can be
responsible for these effects. We recently found an altered RNA metabolism in the small intestinal
mucosa of young goats fed a diet partly containing SP supplement compared to a whole milk/casein
based diet (Schoenhusen et al., 2007). We therefore were interested in exploring whether this diet also
changes protein expression patterns in jejunal protein, bearing in mind that specific proteins must be
involved in the processes leading to the above described structural and functional alterations.

Fourteen male 2 wk old white German dairy goat kids were fed comparable diets (isonitrogenous
and isocaloric) based on whole cows` milk in which 35% of the crude protein was either casein (milk
protein group; MP) or soy protein supplemented by indispensable AA (SPAA) for 34 d (n=7/group).
Energy and protein supply was 0.7 MJ/(kg BW0.75 × d) and 12 g/(kg BW0.75 × d), respectively.
Jejunal tissue was collected to determine intestinal morphology by microscopy and protein composition
by two-dimensional gel electrophoresis and mass spectrometry.
The morphology of intestinal villi and crypts was determined by the computer-operated Image C
picture analysis system (Intronic GmbH, Berlin, Germany) and the IMES analysis program using a
colour video camera (SONY 3 CCD) microscopy analysis.
Two-dimensional gel electrophoresis (2-DE) was performed as described (Görg et al., 2000). In
the first dimension, proteins were separated by isoelectric focussing and in the second dimension
according to molecular mass by SDS-PAGE. The segregated proteins were visualised by Coomassie
Brilliant Blue R 250 staining. The evaluation of the 2-DE gels was performed with 2-DE gel analysis
software Delta2D (DECODON, Greifswald, Germany). The protein spot abundance in each gel was
quantified in terms of % volume. Mean values of each spot were calculated. Those proteins which

differ in spot volume between the feeding groups at P<0.05 were considered to be differentially
expressed. Protein spots were excised using an automated spot cutter (Proteome WorksTM, Biorad)
and digested with trypsin. The molecular masses of tryptic digest were measured by matrix-assisted
laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) (4800 MALDI
TOF/TOF Analyzer; Applied Biosystems, Foster City, CA, USA). For the identification of proteins,
data base search with peptide mass fingerprinting of the analyte was performed against the databases
NCBInr and Swiss-Prot using the Mascot search engine version 2.1 (Matrix Science Ltd, London,
UK). Data evaluation was performed using the Student t-test.

The crypt depths in the proximal jejunum (prox) tended to be lower (P<0.1) whereas the villus
height to crypt depth ratios were higher (P<0.05) in SPAA compared to MP. In the medial jejunum
(med) the villus heights in the SPAA group tended to be decreased (P<0.1). This finding suggests
a reduced absorptive capacity. Electron microscopy indicated that goat kids fed the MP diet had
intestinal villi that were finger-shaped, long, round, and uniform. In contrast, kids fed SPAA had
villi that were less uniform, and showed a tendency to bend.
Twenty-five proteins were found to be differently expressed in both groups. In SPAA, 14 down-
regulated jejunal proteins were involved in processes related to cytoskeleton generation, protein,
lipid, and energy metabolism. Eleven proteins were up-regulated in SPAA and were involved in
cytoskeleton assembly, proteolysis and carbohydrate breakdown.
In terms of specific proteins possibly related to the jejunal morphology, we found a down-regulation
of 3 proteins related to the cytoskeleton and muscle contraction in goat kids fed the SPAA diet:
Tropomyosin 1 alpha chain (prox, med), Tropomyosin alpha 4 chain (med), PDZ and LIM domain
protein 1 (prox).
Proteasome subunit alpha type 2 and proteasome subunit alpha type 4 belonging to the major
cellular proteolytic system were up-regulated, whereas 40S ribosomal protein S12 involved in the
protein synthesis machinery and the proteins histidine triad nucleotide-binding protein 1, rho GDP-
dissociation inhibitor 2 and brain creatine kinase related to energy and signal transduction were
concurrently down-regulated. This was in agreement with a lower protein content found in the small
intestinal mucosa of goat kids fed SPAA, and may be related to a local difference in intestinal AA
availability with effects on protein turnover.
In conclusion, an SPAA diet as compared to a milk diet was related to changes in morphology and
cell metabolism in intestinal tissue with no apparent impact on animal growth.
References
Drackley, J.K., P.M. Blome, K.S. Bartlett and K.L. Bailey, 2006. Supplementation of 1% L-Glutamine to milk replacer
does not overcome the growth depression in calves caused by soy protein concentrate. J. Dairy Sci. 89, 1688-
1693.
Görg, A., C. Obermaier, G. Boguth, A. Harder, B. Scheibe, R. Wildgruber and W. Weiss, 2000. The current stand of
two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 21, 1037-1053.
Schoenhusen, U., S. Kuhla, R. Zitnan, K.D. Wutzke, K. Huber, S. Moors and J. Voigt, 2007. Effect of a soy protein
based diet on the ribonucleic acid metabolism in the small intestinal mucosa of goat kids. J. Dairy Sci., in press.

Influence of catechin on urinary metabolic profiles and antioxidant status in
hyperlipidic diets-fed rats by using a LC-Qtof-based metabonomic approach
A. Fardet, R. Llorach, J.-F. Martin, C. Besson, E. Pujos, C. Obled and A. Scalbert
INRA, Unité de Nutrition Humaine, Centre de Theix, 63122 Saint-Genès-Champanelle, France
Introduction
Unbalanced diets are known to produce oxidative stress which is a risk factor for developing
metabolic diseases such as atherosclerosis, diabetes and cancer (Sies et al., 2005). Polyphenols have
been shown to protect from metabolic disorders such as hyperlipidemia or aortic atherosclerosis
(Auger et al., 2002). However, the mechanisms of action of antioxidant flavonoids on metabolic
effects of such diets remains poorly understood. Due to the complexity of the metabolic response
to complex diets and of metabolic effects of polyphenols, we used a metabonomic approach to
study the influence of catechin (a common antioxidant polyphenol) on the metabolic disturbances
provoked by hyperlipidic diets in rats.

Six groups of eight male Wistar rats were fed during 6 wk normo- (5% of lipids, w/w) or hyperlipidic
diets (15 and 25%) with or without a supplementation of catechin (0.2%). Lipids were 70% lard, 15%
peanut oil and 15% colza oil. Oxidative stress markers were determined in urine (malondihaldehyde,
MDA), plasma (FRAP), and tissues (glutathione - GSH -, GSH-related enzymes and MDA). Plasma
triglycerides and cholesterol concentrations were also determined. Significant differences among the
six groups of rats were determined by one-way ANOVA followed by the PLSD Fisher test. Urine
metabolome (6 rats/group) was analysed 17 and 38 d after the diet consumption by mass spectrometry
(LC-QTof) using full scan mode (m/z 100-1000) and positive ionisation. Multivariate analyses
(ANOVA, PCA and PLS-DA) were applied to LC-QTof data in order to discriminate biological
variations between each group of rats. Finally the markers obtained were tentatively identified on the
basis of their exact mass, MS fragments and relative retention time according to both bibliography
and database information (KEGG and Human metabolome databases).

Weight gain during the 6 wk of diet was not significantly different among the six groups of rats.
Hyperlipidic diets induced oxidative stress at the liver (29 to 80% increased MDA and 14 to 21%
decreased GSH; P<0.05) and aorta (23-31% decreased GSH; P<0.05) levels, which was not reversed
by catechin. In the liver, hyperlipidic diets had no significant effect on GSH-related enzyme activities
(GSH-Px, GSH S-transferase and GSH-reductase). In plasma, triglycerides, cholesterol and FRAP
were not significantly modified by the lipid contents of the diets and catechin supplementation.
Multivariate analyses applied to data acquired through LC-Qtof analyses clearly discriminated
diets with or without catechin (PCA), and normo- and hyperlipidic diets (PLS-DA), at both d 17
and 38. Variables were then classified according to their dependence on lipid and catechin intake.
ANOVA showed that 32% of the variables were significantly affected by catechin intake. Among the
variables affected by catechin supplementation, the phase II metabolites (methyl and/or glucuronide
derivatives of catechin) and some microbial metabolites (e.g. hydroxyhippuric acid, 3-methoxy-4-
hydroxyphenyl-valerolactone glucuronide - described here for the first time, etc.) were identified.
Other variables related with endogenous changes were also detected and their identification is still in
progress. Concerning the lipid content, 18% of the variables were significantly affected. Among these
variables, 15 were increased with hyperlipidic diets (r>0.8; P<0.05) whatever the sampling time or

the catechin content. A variable was identified as being possibly 3-Hexenedioic acid (m/z 145.052),
whose excretion is increased in conditions of fatty acid metabolism disorder. This metabolite, and
those not yet identified, could be robust biomarkers of early metabolic disorders associated to
hyperlipidic diets. A large fraction of the variables affected by the lipid content in the diet (between
70 and 90%) were no longer affected after adding catechin to the control and high-fat diets. Among
these variables, 164 were fully reversed by catechin supplementation, i.e. there was no significant
difference when comparing the control diet without catechin vs. high-fat diets with catechin. Two
variables were identified as deoxycytidine (m/z 228.101) and its fragment cytosine (m/z 112.053)
according to the MS/MS fragmentation pattern (Bond et al., 2006). Another variable was likely to
be 8-Hydroxyguanine (m/z 168.050), a mutagenic base which is a marker for OH-mediated DNA
damage through oxidative stress. Both metabolites have been associated with cancers of various
tissues (Rozalski et al., 2002; Dudley et al., 2003), emphasising a possible relation between high-
fat consumption and early DNA oxidative damages, upon which the action of catechin remains to
be elucidated.
Conclusion
Despite the fact that rats exhibited the same weight gain and no major effects on common markers of
oxidative stress and lipemia, more metabolic changes upon high-fat diets could be seen at the urine
level. Evidence of the partial reversal of these metabolic changes by a phenolic antioxidant has been
shown by the metabonomic approach. Further identification of biomarkers will therefore contribute
to unravel the mechanisms associated with the protective effects of catechin against the deleterious
impact of hyperlipidic diets. This study also showed the great potential of the metabonomic approach
to study the metabolic fate of food phytochemicals.
References
Auger, C., B. Caporiccio, N. Landrault, P.L. Teissedre, C. Laurent, G. Cros, P. Besancon and J.M. Rouanet, 2002. Red
wine phenolic compounds reduce plasma lipids and apolipoprotein B and prevent early aortic atherosclerosis in
hypercholesterolemic golden Syrian hamsters (Mesocricetus auratus). J. Nutr. 132, 1207-1213.
Bond, A., E. Dudley, F. Lemière, R. Tuytten, S. El-Sharkawi, A. Gareth Brenton, E.L. Esmans and R.P. Newton, 2006.
Analysis of urinary nucleosides. V. Identification of urinary pyrimidine nucleosides by liquid chromatography/
electrospray mass spectrometry. Rapid Commun. Mass Spectrom. 20, 137-150.
Dudley, E., F. Lemiere, W. Van Dongen, J.I. Gridge, S. El-Sharkawi, D.E. Games, E.L. Esmans and R.P. Newton,
2003. Analysis of urinary nucleosides. III. Identification of 5’-deoxycytidine in urine of a patient with head and
neck cancer. Rapid Commun. Mass Spectrom. 17, 1132-1136.
Rozalski, R., D. Gackowski, K. Roszkowski, M. Foksinski and R. Olinski, 2002. The level of 8-Hydroxyguanine, a
possible repair product of oxidative DNA damage, is higher in urine of cancer patients than in control subjects.
Cancer Epidemiol. Biomarkers Prev. 11, 1072-1075.
Sies, H., W. Stahl and A. Sevanian, 2005. Nutritional, dietary and postprandial oxidative stress. J. Nutr. 135, 969-972.

Impact of the ingestion of polyphenols and fruits rich in polyphenols on
atherosclerosis studied on apoE deficient mice by a transcriptomic approach
D. Milenkovic, S. Auclair, C. Besson, A. Mazur and A. Scalbert
UNH, INRA, Centre de Clermont-Fd/Theix, 63122 St Genès Champanelle, France
Introduction
Atherosclerosis is a complex and progressive cardiovascular disease characterised by the accumulation
of lipids and fibrous elements in the large arterial (Lusis, 2000). Knowledge on atherosclerosis
development and its prevention by polyphenols derives mainly from studies in animal models. Mice
deficient in apolipoprotein E (ApoE), a glycoprotein present in all lipoproteins, was created in 1992.
Sequential events related to lesion formation observed in these mice are strikingly similar to those
in humans (Jawien et al., 2004). This model is the most largely used in genetic and physiological
studies of atherosclerosis (Lusis, 2000). This model has also been used to study the impact of
polyphenol consumption on atherosclerosis development. Feeding Apo E deficient mice with caffeic
acid phenethyl ester, a natural flavonoid, for 12 wk, significantly reduced aortic atherosclerosis
development (Hishikawa et al., 2005). Reduction in atherosclerosis lesion size has also been
described after feeding mice with grape powder polyphenols; green tea polyphenols; red wine and
its major polyphenols (Hayek et al., 1997). Most authors have explained these protective effects of
polyphenols by their antioxidant properties and their capacity to inhibit lipid peroxidation. However,
it appears today that the mechanisms of action could be much more varied and could depend on gene
expression variations. The goal of our study was to identify the impact of the ingestion of major
fruit and wine polyphenols on the development of atherosclerosis in apoE deficient mice. Lesion
size is assessed by histological analyses of thin sections of the aortic root. In order to understand the
key metabolic pathways involved in the prevention of atherosclerosis by polyphenols, global gene
expression variations in the aorta are measured using mouse pangenomic microarrays.

Apolipoprotein deficient male and female mice were acquired from Jackson laboratory and bred
in our animal husbandry facility. At the age of 10 wk, male mice were weighed and divided into 3
groups of 15. The control group received a standard diet, the other groups received the same diet
supplemented with 0.02% of catechin or 0.06% for proanthocyanidins (isolated from grape seeds).
After these periods of supplementation, the mice were euthanised by i.p. injection of sodium
pentobarbital solution (40 mg/kg of body weight). The blood was collected from the vena cava into
tubes containing heparin and centrifuged immediately at 14000 rpm for 2 min. Plasma was collected
and stored at -20 °C. Concentrations of total cholesterol and triglyceride were analysed using
enzymatic kits. Total plasma antioxidant capacity was determined using the ferric reducing ability
of plasma (FRAP) method. Quantification of atherosclerotic lesions was done by calculating the
lipid deposition size in the aortic sinus. Briefly, the heart with the aortic arch was dissected under a
microscope and frozen (in liquid nitrogen) in OCT (optimal cutting temperature) embedding medium
for serial cryosectioning. The heart (stored at -80 °C) was cut in a microtome (Reichert-Jung Frigocut
2800-San Marco) at -20 °C. Sections (10 µm thick) were taken at every 100 µm throughout the
aortic sinus (300 µm of the distal portion) and analysed. Obtained sections were evaluated for fatty
streak lesions after staining with Oil red O and counterstaining with hematoxylin. Image analysis
was determined using ImageJ software. Immediately after animals were euthanised, liver and aorta
were collected and put into liquid nitrogen. The organs were stored at -80 °C until use. Aorta were
cleaned from surrounding tissues (muscle and fat tissues) and total RNA were extracted using SV
total RNA extraction kit (Promega). Messenger RNA were amplified, fluorescently labelled with
cyanine 3 and 5 (Ambion) and hybridised on glass microarrays containing 25 000 oligonucleotides

corresponding to about 22 000 different genes (acquired from the National Genopole Network).
Reciprocal hybridisations (Dye Swap) were systematically carried out. After image acquisition
using Imagene software, data were normalised (Lowess) and differentially expressed genes were
identified using a test t followed by Bonferonni correction for false positives.
Results
No differences in weight were observed between groups all along the study. Differences in weight
were observed between animals at the beginning of the study and 1 mo later as well as after 2 mo. No
changes were observed between mo 2 and mo 3. Comparison of plasma and blood parameters between
control and catechin groups showed no differences for total triglycerides and cholesterol concentration.
A weak increase of total plasma antioxidant capacity was observed after 6 wk of supplementation
of catechin. Aortic crosses were collected from all mice (15 control and 15 supplemented) and 30
sections (10 µm each) were taken for each aortic root for histological analyses. A decrease in the
surface of fatty streak lesions were observed between mice supplemented with catechin during 6 wk
and control mice. Therefore, supplementation with catechin for 6 wk at nutritional dose (0.02%) does
reduce atherosclerosis development in ApoE deficient mice. The analyses of the sections obtained
from mice after 12 wk of supplementation with either catechin or proanthocyanidin are in progress.
The expression of genes in aortas of mice fed a control diet and mice fed diets supplemented by
catechin and proanthocyanidins are being compared. First analyses suggest that genes involved in
lipid metabolism, energy metabolism and immune system could be regulated by polyphenols, as well
as the extracellular matrix, and are down regulated. The expression of genes whose expression is
modified in the presence of catechin seems to be under control of MAPK pathway and PPAR. These
factors induce atherosclerosis development. The results obtained using microarray will be verified
using quantitative real-time PCR. Combining histological and transcriptomic results will allow us
to identify cellular pathways modulated by these polyphenols that are important in the prevention of
atherosclerosis. Together with this work, the study on the effect of whole fruits rich in polyphenols
on atherosclerotic lesion development is in progress.
References
Hayek, T., B. Fuhrman, J. Vaya, M. Rosenblat, P. Belinky, R. Coleman, A. Elis and M. Aviram, 1997. Reduced
progression of atherosclerosis in apolipoprotein E-deficient mice following consumption of red wine, or its
polyphenols quercetin or catechin, is associated with reduced susceptibility of LDL to oxidation and aggregation.
Arterioscler. Thromb. Vasc. Biol. 17, 2744-2752.
Hishikawa, K., T. Nakaki and T. Fujita, 2005. Oral flavonoid supplementation attenuates atherosclerosis development
in apolipoprotein E–deficient mice. Arterioscler. Thromb. Vasc. Biol. 25, 442-446.
Jawien, J., P. Nastalek and R. Korbut, 2004. Mouse models of experimental atherosclerosis. J. Physiol. Pharmacol.
55, 503-517.
Lusis, A.T., 2000. Atherosclerosis. Nature 407, 233-241.
This research was carried out as part of the EU-funded project FLAVO (Food CT-2004-513960).

AGENAE – GENANIMAL: the French research program in animal
genomics
C. Chevalet1, J.F. Hocquette2, P. Sellier3 and P. Monget4
1INRA, Laboratoire de Génétique Cellulaire, 31326 Castanet, France
2INRA, Unité de Recherches sur les Herbivores, 63122 Saint-Genès Chamanelle, France
3INRA, Station de Génétique Quantitative et Appliquée, 78352 Jouy en Josas, France
4INRA, Physiologie de la Reproduction et des Comportements, 37380 Nouzilly, France
Introduction
At the turn of the century, the advent of genomics led to a change of scale in the investigations
pertaining to human biology and health and genomics became a central and cohesive discipline of
biomedical research. During the yr 1999-2000, it was clear that genomics should foster research
activities dealing with farm animals, whatever the field of interest: reproductive capacity, growth and
development, health status, behaviour and welfare, milking ability, quality of animal products, etc.
This novel scientific context has stimulated efforts and coordinated initiatives for facing the grand
challenge of genomics research in the area of animal production. The French National Institute for
Agricultural Research (INRA) launched its own animal genomics program, focusing on high density
gene mapping and large scale sequencing of expressed genes in four main species (cattle, pig, chicken,
and trout). Moreover, discussions resulted in the spring of 2002 in the formation, for a five-yr period,
of a « group of scientific interest » – Groupement d’Intérêt Scientifique (G.I.S.) in French – consisting
of two categories of partners, i.e. State-supported research organisations, on the one hand, and private
associations representing the various components of the animal industry, on the other hand. The name
of the cooperative research programme piloted by this group is AGENAE, an acronym for Analyse
du GENome des Animaux d’Elevage (Analysis of the genome of farm animals).
Governance and funding

The aims of the AGENAE consortium are to share decisions on scientific objectives, to share
financial supports to targeted projects, and to share industrial property on the results of joint projects.
The funding of the projects (excluding permanent staff salaries, infrastructure and equipments
brought about by State research organisations) originates according to the following general rules.
Firstly, any funding is conditioned on a positive evaluation of a project application submitted to the
‘GENANIMAL’ call launched by the Ministry of Research in 2003-2004 and the National Agency for
Research since 2005. Secondly, depending on the scope of the project, projects are either supported
by public funds only for basic research, or supported by joint contributions from the State and from
animal industry associations for applied research projects. Important European funding provided to
fish research is also to be mentioned.
First results
From 2003 to 2006, the GENANIMAL calls allowed 30 applied projects and 19 basic research
projects to be supported in different fields, as shown in Table 1.
Among significant results obtained in the framework of the public-private collaboration set up with
AGENAE, the identification of the mutation responsible for syndactyly in cattle, the mechanism of
action of the ‘double muscled’ gene in the Texel sheep breed, and new QTL for male fertility in cattle
and disease resistance in trout are worth mentioning. New biological markers of sperm maturation,
meat quality, and early infection are also examples of the efficiency of the new integrative approaches
carried out in the program.

Table 1. Numbers of projects supported by the 2003 to 2006 GENANIMAL calls (Applied projects
+ Basic research projects).
Species Cattle, sheep Pigs Chicken and Rainbow trout

and goats avian species and other fishes
Reproduction: gamete fertility, 7+2 0+1 - 3+2

determinism of sex, embryo
development
Meat production, 3+1 1+0 2+1 1+0
growth, product quality
Immunity, animal health 1+2 0+3 1+1 2+1
Milk yield, milk composition 2+0 - - -
Nutrition - - - 3+0
Genetic abnormalities 1+0 - - -
Evolution, domestication 0+1 - 0+1 -
Biotechnology and methods 2+1 - 0+2 1+0
In association with joint support from the CNRG (the French National Consortium for Genomic
Research) and from research institutions, the GENANIMAL calls also allowed French groups to
contribute to the international consortia for the sequencing of complete genomes (physical mapping
in cattle and pigs, irradiation mapping in the chicken, expressed gene sequencing in trout, search for
gene polymorphisms in the pig) and to set up national infrastructures (GADIE: National Centre for
Animal Genomic Resources, SIGENAE: bioinformatics platform) which are able to provide research
groups with genomic materials (gene libraries, DNA chips) and computer resources (comparative
genomic tools, statistical tools for expression analysis, project management).
New trends
The scope of the French national program for animal genomics is now changing in two directions.
Firstly, the GENANIMAL call is open since the 2005 edition to any animal species of economic
value (and not only to the four species of the first yr). As an example, a new large research program
is being launched on the genome of oysters. Also, focus is put on animal health issues, with two
projects starting on emergent diseases (avian influenza and ‘bluetongue’ disease). Secondly, following
discussions with the German FUGATO program (an equivalent of the French AGENAE program),
joint scientific meetings are scheduled in 2007 in order to identify specific topics that could be
included in a joint call for projects.
A general description of the program is available at: http://www.inra.fr/agenae/

FUGATO - Functional genome analysis in animal organisms
K. Sanders1 and G. Ostermann2
1FUGATO secretariat, Adenauerallee 174, 53113, Bonn, Germany
2Project Management Organisation Jülich (PtJ), Division BIO, Forschungszentrum Jülich GmbH,
52425 Jülich, Germany
Introduction
In the year 2004, the German Federal Ministry of Education and Research (BMBF) implemented
the German genome analysis program FUGATO (Functional genome analysis in animal organisms),
which complements the National Genome Research Network. The funding initiative aims to clarify
the molecular basis of traits with economic importance and to develop processes and technologies that
enhance classical farm animal breeding. The main research focus is on animal health, animal welfare,
and product quality. FUGATO, as a public-private partnership, is supported by the business platform
FUGATO (IVF), which represents the interests of the industry and guarantees the implementation of
the research results into practice. The involved projects are financed both by public funds (BMBF)
and third-party funds. The first supporting phase started in the middle of 2005. On the long term,
FUGATO is planned for eight years.
The aims of FUGATO

Within the funding initiative FUGATO, the molecular-genetic background of important functions in
farm animal organisms shall be clarified with the aid of functional genome analysis and molecular
biology. FUGATO hopes to realise new scientific and economic aims and additionally, to focus
the critical body of expertise at the research institutions and industrial companies involved. The
combination of modern experimental methods with conventional breeding will enhance the expertise
in farm animal breeding and the knowledge transfer of research results into practical breeding
programs.
FUGATO strengthens the networking of national responsibilities in the field of the animal genome
analyses, including disciplines such as animal breeding, veterinary medicine and bioinformatics,
as well as networking with other national genome programs. The efforts focus on the durable co-
operation between industry and sciences. This supports an efficient technology and knowledge
transfer and enhances patent protection for the results of the different consortia projects within the
FUGATO program. Through a better value creation, the position of the business location in Germany
will be improved. Besides this, FUGATO aims to profile national animal breeding research on a
European level. Thus, the program will be strengthened for co-operations with research programs
in other European countries.
Research areas
The main research focus of FUGATO is the improvement of animal health, animal welfare, and
product quality through the combination of classical animal breeding methods and molecular
genetics. Beyond these, the development of new vaccines, pharmaceuticals or other new products
is expected.
Animal health and welfare
In the research area of animal health, the most important role plays the investigation of genetic factors
for infection defense, vitality, fertility, and metabolism. With the knowledge of the genetic background

and the functional interrelation of these characters, it is possible to implement arrangements, which
assure the health and the well-being of the livestock in the fields of animal breeding and husbandry.
In the area of animal welfare, the knowledge about the genetic background of hereditary diseases
can ensure the exclusion of carrier-animals out of the breeding scheme.
Sustainability and product quality
The influence of the genotype-environment-interaction is an important factor of the product quality.

Through the enlightenment of molecular mechanisms of metabolism it will be possible to optimise the
feeding management in view of an adapted nutrient absorption. Another aspect is the contribution to
sustainable production, which can be reached through an optimisation of economical and ecological
metabolism effects.
The FUGATO projects

Until now, six cooperative projects are established in the fields of host-pathogen interactions
(cattle, pigs, and chicken), biology of reproduction (cattle) as well as hereditary diseases (pigs).
The first consortium, IRAS, aims the development of genetic markers for immune defense and
resistance in the porcine respiratory tract. The main focus of the E. coli-chick project is to study
host-pathogen interactions in E. coli-resistance in chickens and its application in breeding programs.
The identification of genes causing hereditary diseases like inverted teats, atresia ani, and splaylegs
and their avoidance is the objective of the HeDiPig consortium. The fourth project, QuaLIPID,
deals with the functional analysis of genes involved in lipid metabolism in cattle and swine to
identify relevant DNA variation, especially for product quality. Fertilink investigates the molecular
mechanisms of fertility problems in cattle with a view to an early diagnosis of fertility problems.
Finally, the M.A.S.-Net project analyses the genetic mechanisms determining the variability of
protective ability and resistance against mastitis in cattle.
Outlook
The involved partners of the different cooperative projects establish a network of the leading academic
institutes and the industry, mainly the livestock breeding economy, in Germany. This network
will be strengthened by new projects and new partners which will be established in 2007 within
FUGATO-plus, the follow-up initiative of the German Federal Ministry of Education and Research.
FUGATO-plus builds on the successful funding program FUGATO and takes its research to a new
qualitative and quantitative level in the areas of the quality of animal food products, animal health,
and animal welfare. FUGATO-plus opens the program for the species sheep, goats, horses, and bees.
Furthermore, it will fund junior research groups to give new impetus to research in the area of animal
breeding and to improve career prospects for qualified young scientists within Germany.
In the field of international co-operations, the implementation of a bilateral announcement for joint
projects between the French AGENAE and the German FUGATO program is planned for the year
2007.

Nutrigenomics New Zealand – tailoring New Zealand foods to match
people’s genes; a case study
N.C. Roy, M.P.G. Barnett and W.C. McNabb
Food, Metabolism & Microbiology, Food & Health Group, AgResearch Grasslands, Tennent Drive,
Introduction
Nutrigenomics New Zealand (NuNZ; http://www.nutrigenomics.org.nz) is a strategic alliance that
has established a food-focussed Nutrigenomics Research Centre for NZ. The alliance is between The
University of Auckland and three Crown Research Institutes: Crop & Food Research, HortResearch,
and AgResearch Limited, and is largely funded by the NZ Foundation for Research, Science &
Technology. The initial focus that will enable NuNZ to establish nutrigenomics capabilities for NZ
is Crohn Disease (CD), one of the Inflammatory Bowel Diseases (the other being Ulcerative Colitis)
which causes intestinal inflammation. The cause of CD is still unclear, but it is likely to involve
an irregular immune response to normal gut bacteria. CD will be used as a ‘proof-of-concept’ to
establish technologies for NuNZ to ‘tailor NZ foods to match people’s genes’.
Nutrigenomics is the study of how dietary components interact with genes and gene products
to alter phenotype (Müller and Kersten, 2003; Kaput et al., 2005). Diet can alter gene-nutrient
interactions and gene expression in ways that increase the risk of developing chronic disease. Due
to the complex interplay of human genetic variation and environmental factors, identifying causative
genes and nutrients requires a multi‑disciplinary or ‘systems biology’ approach.It is possible using
new technologies such as microarrays, proteomics and metabolomics to analyse the effects of diet
on the activity of an individual’s genes and the implications of this interaction for the health and
wellbeing of that individual.

In on-going studies, the genetic make-up of NZ populations with CD is being determined by
scientists in NuNZ, with a view to identifying genetic variants predisposing these individuals to the
disease phenotype. A comprehensive nutritional questionnaire is also being filled out by patients
taking part in these genetic studies, in order to link the genetic component to diet, and to provide
potential food targets.
Extracts from various NZ foods, initially those where there is literature evidence of a beneficial effect
in CD, or those identified in the nutritional questionnaires, are being tested in vitro using cell-based
high-throughput screens that utilise the genetic variations relevant to CD. This will enable NuNZ
to identify food components that can ‘correct’ for the genetic variation and thereby, ameliorate the
inflammation common to CD.
Food components identified in this way are further tested using mouse models of human gut
inflammation. NuNZ currently uses two mouse models; IL10 (Roy et al., 2007) and mdr1a (Dommels
et al., 2007) gene-deficient mice, while a further model (NOD2Δ33, Maeda et al., 2004) is currently
being developed. Food components that reduce intestinal inflammation in these mouse models were
investigated in more detail to identify pathways that are involved in intestinal inflammation, and
are beneficially responsive to specific food components. Food components showing promise in
mouse models will also be considered for inclusion in human clinical trials, leading to two possible
outcomes: dietary advice linking a particular food component to a particular genotype for CD, or
the development of CD-specific foods.

To date, NuNZ has identified a number of genetic associations in samples from NZ patients with
CD (Browning et al., unpublished data). Extracts from blueberries, strawberries, kiwifruit, chillies
and black tea have shown promise using in vitro assays. Mouse models are well established (Roy
et al., 2007; Dommels et al., 2007) and polyunsaturated fatty acids (n-3 and n-6) and/or flavonoids
(green tea, curcumin and rutin) have already been tested using these animal models of human gut
inflammation. The development of technology to incorporate promising components into foods
ready for the market is also well underway.
While the development of novel nutrigenomics foods is some way off, it is worth considering some of
the implications of such developments. First is the issue of safety; it is not just which ingredients are
functional in the amelioration of symptoms, but what dose is appropriate to provide benefits without
side effects. It will also be necessary to determine efficacy in humans, and deal with legislation with
respect to labelling, health claims and regulations. For example, can the product be marketed as a
food, or does it cross into the somewhat more problematic world of ‘nutraceuticals’? It is likely that
the development of such foods will require a mix of small innovative companies and large global
companies; the former to engage in the potentially risky initial development, the latter to grow the
market once a product has been established. Finally, these foods must have consumer appeal. The
days when healthy food was unpalatable have long gone.
References
Dommels, Y.E.M., C. Butts, S.T. Zhu, M. Davy, S. Martell, D. Hedderly, M.P.G. Barnett, W.C. McNabb and N.C.
Roy, 2007. Characterization of intestinal inflammation and identification of related gene expression changes in
mdr1a-/- mice. Gene Nutr., in press.
Kaput, J., J.M. Ordovas, L.R. Ferguson et al., 2005. The case for strategic international alliances to harness nutritional
genomics for public and personal health. Br. J. Nutr. 94, 623-632.
Maeda, S., L.-C. Hsu, H. Liu, L.A. Bankston, M. Iimura, M.F. Kagnoff, L. Eckmann and M. Karin, 2005. Nod2 mutation
in Crohn’s Disease potentiates NF-kB activity and IL-1β processing. Science 307, 734-738.
Müller, M. and S. Kersten, 2003. Nutrigenomics: goals and strategies. Nat. Rev. Genet. 4, 315-322.
Roy, N.C., M.P.G. Barnett, B. Knoch, Y.E.M. Dommels, R.C. Anderson and W.C. McNabb, 2007. Animal Models of
Inflammatory Bowel Diseases: developing nutrigenomics foods for intestinal health using transcriptomic analysis.
Mutat. Res., in press.
Nutrigenomics New Zealand (http://www.nutrigenomics.org.nz) is a collaboration between

AgResearch Limited, Crop & Food Research, HortResearch and The University of Auckland and
is largely funded by the Foundation of Research, Science and Technology (FRST). M.P.G. Barnett
is funded by FRST Postdoctoral Fellowship AGRX0504. B. Knoch PhD Fellowship is funded by
Nutrigenomics New Zealand.

Part 4. Coordination between tissues for the metabolic
utilisation of nutrients
4. Splanchnic metabolism and interorgan exchanges of

nutrients
Interorgan exchange of amino acids: what is the driving force?
Y.C. Luiking and N.E.P. Deutz
Center for Translational Research on Aging & Longevity, Donald W. Reynolds Institute on Aging,
University of Arkansas for Medical Sciences, Little Rock, USA
Abstract
Interorgan exchange of amino acids is a highly active and regulated process that provides and delivers
amino acids to all organs and tissues for protein synthesis and for specific metabolic functions. The
gut is a major organ that controls the digestion and absorption rate of dietary protein and amino acids
and the subsequent availability of substrate to the liver, the muscle and other organs. In addition, the
gut utilizes amino acids for its own metabolism. The process of interorgan amino acid exchange after
food intake can be affected by the protein quality of a protein meal, which is related to the digestion
rate of the protein or a deficiency of specific amino acids. The addition of deficient amino acids to
the meal, but also addition of carbohydrates to a protein meal improves the ‘protein quality’ of a diet.
Moreover, during disease, amino acids and protein requirements increase and the muscle and gut
become organs that serve the liver and immune cells. This involves the delivery of amino acids for
gluconeogenesis as energy supply, but also for specific functions such as the production of protein (or
peptides) needed for the metabolic response to disease. Feeding under these conditions is important to
support protein metabolism in the organs, to stimulate the transamination process and production of
glutamine in the muscle, and to enhance the metabolic response to disease with improved recovery.
Keywords: gut, muscle, liver, amino acid, protein
Introduction
Interorgan exchange of amino acids is a highly active and regulated process that provides and delivers
amino acids to all organs and tissues for protein synthesis and for specific metabolic functions.
Moreover, it contributes to maintain plasma amino acid homeostasis through either net uptake or net
release by different tissues. Main determinants for the transport of amino acids between organs are
the physiological and nutritional state. After feeding and in the initial step of fasting, amino acid flux
is predominantly from the intestine to other tissues, while during prolonged fasting and starvation
and in many diseases the dominant flux is from muscle to the liver and kidney (for reviews: Tessari
and Garibotto, 2000; Brosnan, 2003). This paper will focus on interorgan exchange of amino acids
and the importance of the gut in normal physiology, during feeding of high and low quality protein,
and during disease with sepsis in particular.
Interorgan exchange of amino acids

Interorgan metabolism and flux of amino acids have been studied mainly in animal models, using
multicatheter techniques, which enable the sampling of blood entering and leaving the organ and
the study of organ blood flow (Ten Have et al., 1996). In humans, possibilities to study interorgan
metabolism are limited to peroperative measurement, when blood vessels are accessible for the
implantation of sampling catheters. In conscious humans, measurements are largely limited to the
study of splanchnic extraction (Biolo et al., 1992; Boirie et al., 1997b), which represents gut and
liver amino acid uptake and utilization, or the study of muscle metabolism across the leg or arm
(Biolo et al., 1995; Tessari et al., 1995). Application of stable isotope techniques has enabled a more
detailed study of metabolic conversions and amino acid utilization. In this review, we will mainly
focus on interorgan data obtained in pig studies.

Flow of amino acids from the gut to the rest of the body
Amino acids from dietary protein first pass the gut, which controls amino acid absorption and local
amino acid extraction for metabolism, the latter including oxidation, protein synthesis and specific
conversions. Accordingly, the gut therefore also controls the degree and rate of transport of amino
acids via the portal vein to the liver and subsequent other organs (Figure 1).
Besides the organ that has direct contact with ingested food, the gut is also a metabolically highly
active organ (Wu, 1998) and supply of amino acids does not rely solely on luminal supply. There is
substantial use from arterial source and as such the gut is ‘competing’ with other tissues for amino
acid utilization. The high level of metabolic activity in the gut is also demonstrated by the fact that
rates of protein synthesis in the intestinal mucosa are among the highest in the body (McNurlan
and Garlick, 1980), related to its organ size and metabolic activity. As a result, feeding rapidly
stimulates protein synthesis in the gut. Malnutrition or starvation, protein depletion or deficiencies
of specific nutrients all inhibit the growth and turnover of the intestinal mucosa (Ziegler et al., 2003).
Besides nutrition in general, luminal contact with nutrients specifically is important in regulation
of intestinal protein metabolism, regarding the profound intestinal atrophy and reduced intestinal
protein synthesis during intravenous nutrition (Dudley et al., 1998). Finally, individual amino acids
have been suggested for their beneficial effects on the intestine, of which glutamine and arginine
have been studied most intensively (for reviews: Duggan et al., 2002; Ziegler et al., 2003). More
recently it was observed that the gut also uses threonine, sulphur amino acids (cysteine, methionine)
and branched-chain amino acids (leucine, isoleucine, valine) to a high extent. Threonine is important
for the structural protein mucus layer (Schaart et al., 2005), while cysteine is the substrate for the
antioxidant glutathione (Shoveller et al., 2005). Branched-chain amino acids (BCAA) needs are
probably also higher than thought in the intestine as the enterally fed BCAA requirements in pigs
are much higher than the parenterally fed requirements (Elango et al., 2002). The involvement of
these amino acids in metabolic pathways in the gut is also reflected by their large intestinal first pass
extraction during enteral feeding as calculated by isotopically-labelled amino acid extraction as well
as a percentage of portal appearance from defined meals (e.g. glutamate (96%), glutamine (64%),
Figure 1. Interorgan flow of amino acids between the gut, liver and muscle during continuous enteral
feeding (24 h) in young (growing) pigs. Numbers indicate net balances (i.e. net uptake or release from
the organ) and enteral intake as µmol/kg.min for glutamine (GLN), glutamate (GLU), alanine (ALA),
and branched-chain amino acids (BCAA). Based on data published by Bruins et al. (2000).

threonine (57%), arginine (65%), cysteine (44%), BCAA (50-60%) (Deutz et al., 1998; Reeds and
Burrin, 2000; Luiking et al., 2003; Burrin and Davis, 2004).
Flow of amino acids across the liver

The liver is responsible for deamination of most amino acids and is the major organ for amino acid
disposal. However, the capacity of the liver to metabolize branched chain amino acids (BCAA) is
limited, and this occurs predominantly in the muscle. The liver oxidizes amino acids as one of its
main energy sources, as well as large amounts of plasma glutamine and alanine for gluconeogenesis.
During starvation, gluconeogenesis in the liver served other obligatory glucose-using organs like the
brain. Moreover, amino acids are used for protein synthesis in the liver (Brosnan, 2000). However,
net flux (being net retention or loss) of an amino acid across a tissue is determined by the difference
between uptake for protein synthesis and oxidation and release of amino acids by protein breakdown
or export of proteins. In the case of the liver, the rate of protein synthesis and breakdown can be
stimulated by inflammation that occurs in disease states like COPD, cancer, sepsis. Especially,
the synthesis rate of plasma proteins like albumin and the acute phase proteins are important in
determining liver protein synthesis. Following extraction of amino acids by the gut and liver, the
so-called first pass splanchnic extraction; amino acids that enter the systemic circulation become
available for other organs.
Flow of amino acids across skeletal muscle

The muscle is a major organ for net uptake of amino acids during feeding, and amino acid availability
is a potent regulator of muscle protein synthesis. It was observed that non-essential amino acid
ingestion is not needed to stimulate muscle protein synthesis, and that plasma essential amino
acids can be considered the main regulators (Volpi et al., 2003). The intake of amino acids with
carbohydrates shows a synergistic effect. In general, the magnitude and duration of the response on
muscle protein synthesis depends on the change in plasma amino acid concentration, and the degree
of the insulin response (Wolfe, 2002).
While during feeding muscle metabolism is in general characterized by net amino acid uptake, this
changes to net amino acid release and flow of amino acids from muscle to the spanchnic area and
liver during fasting in healthy subjects. These aspects make the role of the muscle important for
health and disease states (Wolfe, 2006). At all times, however, there is a net output of alanine and
glutamine from muscle, representing the disposal of the amino groups from the BCAA (Brosnan,
2003). Glutamine is utilized as a major energy substrate by intestinal mucosal and immune cells. In
addition, glutamine is important for maintenance and repair of intestinal mucosa, it can be metabolized
to pyruvate, which is then transaminated and exported to the liver as alanine, and glutamine is a
substrate for production of the antioxidant glutathione and the amino acid citrulline (Tapiero et al.,
2002). Citrulline, on its turn, is important for endogenous renal production of arginine (Featherston
et al., 1973; Tizianello et al., 1980; Windmueller and Spaeth, 1981), which has an important role
through its metabolites ornithine and nitric oxide on cellular growth and differentiation, local
perfusion and intestinal motility (Wu and Morris, 1998).
While the splanchnic area is considered a first pool of protein during short-term starvation, ongoing
inadequate amino acid supply results in breakdown of other protein reserves, with the muscle as the
largest pool. Long-term starvation or increased protein and amino acid needs, the latter of which is
common in disease, therefore result in enhanced breakdown of muscle proteins.

Flow of amino acids across the kidney
Other organs involved are the kidney and brain. The kidney is a prime site for glutamine deamination,
producing ammonium to maintain acid-base balance, and glutamine also serves as a substrate
for gluconeogenesis in the kidney (van de Poll et al., 2004). The kidneys play a major role in
the interorgan metabolism of citrulline, arginine, glycine, serine and glutamine (Brosnan, 1987).
Moreover, the high protein turnover rate in the kidneys contributes to regulation of whole body
protein turnover (Tessari et al., 1996).
Impact of the diet on gut amino acid metabolism and interorgan

exchanges
The amount of protein in the diet is important in determining the magnitude of change in protein
metabolism during feeding. Regarding the net protein synthesis following a meal and the net protein
loss in the postabsorptive state, it has been suggested that the body accumulates protein in a so-called
labile protein pool during feeding (Quevedo et al., 1994; Waterlow, 1995; Soeters et al., 2001). This
pool may be present in the splanchnic area and probably serves to maintain anabolism in other organs
(Soeters et al., 2001). Besides the amount of protein, also the protein source in the meal determines
the anabolic capacity of a meal, which is considered an indicator of protein quality.
With an increasing protein content in the diet and increased plasma amino acid levels, whole body
endogenous protein breakdown is more markedly inhibited and protein synthesis stimulated, although
the latter occurs to a lesser extent and is dependent on the amount of protein ingested (Motil et
al., 1981; Pacy et al., 1994; Gibson et al., 1996; Giordano et al., 1996). Moreover, the nutritional
value of dietary protein is related to both the bio-availability of ingested nitrogen and amino acids
and the efficiency of their metabolic utilization to meet nitrogen and amino acid requirements for
growth and renewal of body proteins (Pellet, 1990; Young and Marchini, 1990; Fuller and Garlick,
1994; Hiramatsu et al., 1994; Millward, 1994). Protein digestibility and naturally occurring growth
depressing or anti-nutritional factors in proteins affect both the bio-availability of nitrogen and amino
acids (Pellett et al., 1990; Sarwar, 1997) and postprandial protein kinetics. Metabolic utilization also
depends on the composition of the meal with respect to the presence or absence of essential amino
acids. The lack of an amino acid in a protein meal therefore makes the protein of low quality. Ingestion
of an isoleucine-poor blood protein in pigs for example, resulted in an elevated urea production, while
concomitant intravenous isoleucine infusion lowered the increase of urea and probably promotes
amino acid retention in the portal drained viscera (Deutz et al., 1991). In addition, addition of
carbohydrates to a protein meal results in increased intestinal amino acid retention and lower urea
production (Deutz et al., 1995) and modifies metabolic utilization of dietary protein in the phase of
nitrogen gain (Gaudichon et al., 1999). The addition of limiting amino acids or carbohydrates to the
meal therefore improves the quality of a protein meal.
A difference in the speed of protein digestion and absorption from the gut following protein intake,
which is related to the slow vs. fast concept of protein, also affects the quality of a protein. When
comparing whey and casein protein, the postprandial plasma amino acid profile showed a rapid
high increase with whey protein and a prolonged plateau of moderate hyperaminoacidemia with
casein protein (Boirie et al., 1997a). In addition, a more prolonged positive net protein balance was
observed with casein protein (Boirie et al., 1997a) or with repeated meals of whey protein to mimic
a slow digestion rate (Dangin et al., 2001). In contrast, uptake and metabolism across organs was
found to be equal for elementary, partially hydrolyzed or intact whey protein in a study in healthy
young pigs (Deutz et al., 1996), which is probably due to the fact that whey protein already is a fast
protein with little impact of hydrolysis on digestion rate, portal net release and liver metabolism.
Protein digestion rate therefore seems an independent factor that determines postprandial protein
deposition (Dangin et al., 2001).

Another protein with a fast digestion rate is soy. In a pig study comparing soy and casein protein,
liver urea production and release of essential amino acids by the portal drained viscera was higher
with soy compared with casein protein (Deutz et al., 1998). This was confirmed in a study in
healthy subjects, demonstrating a lower net protein synthesis and higher ureagenesis after a soy
containing meal (Luiking et al., 2005). Both studies were performed under continuous enteral feeding.
Therefore, the biological value of soy protein may be considered inferior to casein with respect to
protein metabolism, which may be due to the fact that soy is deficient in the essential amino acids
methionine and lysine and contains less branched-chain amino acids. Another explanation is that the
lower postprandial nitrogen retention with soy is due to difference in both digestion rate and amino
acid composition of both proteins (Bos et al., 2003). The mechanistic hypothesis is that a dietary
protein with an unbalanced amino acid composition results, after digestion, in an unbalanced amino
acid mixture for gut protein synthesis with subsequent elevated free amino acid levels that reach
the liver via the portal vein where urea synthesis is stimulated (Soeters et al., 2001). Alternatively,
it may be hypothesized that casein digestion releases peptides having a local effect on the gut, due
to a trophic effect or increased mucine production (Claustre et al., 2002).
However, recent evidence indicates that the benefits of the appropriate dietary protein source and
pattern probably change with age, and that stimulation of muscle protein in the elderly is higher with
fast proteins like whey, with protein hydrolysates or with protein feeding pulse pattern (Walrand
and Boirie, 2005). These observations most likely are linked to reduced response of muscle in the
elderly to amino acids delivered.
Impact of physico-pathological state on interorgan exchange of amino

acids
Disease is often characterized by a change in protein metabolism. During inflammation in general,
but sepsis more severely, protein and amino acid metabolism are characterized by catabolism and the
amino acid demand therefore increases (Biolo et al., 1997). Sepsis, a severe systemic inflammatory
response to an infection, is characterized by severe whole body protein loss (catabolism) and reduced
plasma levels of glutamate (48%), glutamine (33%), threonine (38%), valine (28%), ornithine
(28%), lysine (30%), arginine (47%) and citrulline (56%) in humans (Luiking et al., unpublished
results). The large reduction in plasma citrulline probably reflects reduced mucosal mass with
intestinal failure (Crenn et al., 2000) that may subsequently affect dietary protein digestion and
absorption (Sodeyama et al., 1993). However, enteral nutrition may be a preventive therapeutic
means to maintain gut integrity and can provide substrate for protein synthesis and transamination
processes in the various organs. Moreover, while the carbon cycle of alanine and lactate/glucose to
provide energy is probably more important during fasting, the nitrogen cycle of BCAA/glutamate
and glutamine/alanine between muscle and gut on the one hand and liver on the other hand becomes
more important during feeding. However, even during feeding, the nitrogen output from the gut was
64-73% higher after endotoxemia in pigs, probably as a result of impaired retention by the gut and
increased transamination (Bruins et al., 2000)
Interorgan amino acid exchange in sepsis during fasting

Skeletal muscle protein wasting is a prominent feature of the metabolic response to sepsis, and
ultimately results in muscle dysfunction and prolonged recovery. Inflammatory mediators and
hormones are considered important factors that modulate protein catabolism, anabolism, amino acid
supply and uptake. Increased protein degradation results in substrate supply for energy consuming
metabolic processes in the liver, other visceral organs, immune cells and the muscle itself. In sepsis,
the skeletal muscle predominantly accounts for the provision of substrates including arginine,
glutamine, glutamate, leucine and alanine, fully or in part resulting from accelerated protein
breakdown. The driving force is probably both energy demand and protein synthesis. The enhanced

alanine and glutamine efflux from muscle is largely taken up by the liver for gluconeogenesis.
The liver can be considered as the major consumer of amino acids during sepsis. These include
non-essential gluconeogenic amino acids (including glutamine, glycine and alanine) and essential
gluconeogenic amino acids (including BCAA, tyrosine and phenylalanine). Also, the liver utilizes
amino acids for export protein synthesis, especially acute phase proteins during sepsis, which are
involved in the host-defense response. Sepsis in humans leads to enhanced splanchnic BCAA use
and enhanced leucine oxidation (Fong et al., 1994). The enhanced glutamate release from the liver
in sepsis constitutes an important nitrogen-sparing mechanism, since glutamate serves as a nitrogen
intermediate for the synthesis of alanine and glutamine in the muscle (Bruins et al., 2003). Enhanced
release of arginine from muscle, may serve to increase substrate for nitric oxide synthesis and protein
synthesis in the liver (Bruins et al., 2002a) (Figure 2).
Interorgan amino acid exchange in sepsis during feeding

Under fed conditions post-endotoxemia, liver metabolism is characterized by a higher protein
synthesis (measured as disposal of valine) and a lower protein breakdown (measured as a production
of valine), resulting in net protein synthesis (net uptake of valine across the liver). A shift from
efflux into influx of glutamine, valine and isoleucine postendotoxemia was observed and a higher
net uptake of arginine was observed (Bruins et al., 2000) (Figure 3).
Sepsis accelerates protein degradation from muscle, but no muscle catabolism was manifest during
nutritional intervention after endotoxemia (Bruins et al., 2000). Continuous enteral feeding did not
affect muscle protein metabolism at 1 day after endotoxemia, but in a more prolonged phase post-
endotoxemia (day 4) the increase in protein synthesis exceeded the increase in protein breakdown
with net protein anabolism. Muscle protein metabolism was evaluated using stable isotope techniques.
This probably represents replenishment of wasted muscle protein. A higher net influx of arginine was
observed during feeding after endotoxemia, which may reflect replenishment of the depleted muscle
arginine pool and may add to protein synthesis (Bruins et al., 2000). At 1 day after endotoxemia, the
amount of valine disposal used in transamination was elevated. Together with an increased glutamine
efflux from muscle and net glutamate consumption this suggests an increased transamination activity
in muscle during the acute and prolonged post-endotoxemia phase. In the anabolic state, muscle
may use both glutamate and BCAA from increased inward transport and from increased protein
breakdown preferably for transamination to glutamine in muscle, instead of alanine, related to the
fact that glucose is no longer a limiting fuel for the liver during feeding (Bruins et al., 2000).
Figure 2. Interorgan flow of amino acids during fasting in health (left) and disease (24 h after
infusion of LPS, right) in young (growing) pigs. Numbers indicate net balances (i.e. net uptake or
release from the organ) as µmol/kg.min for glutamine (GLN), glutamate (GLU), alanine (ALA), and
branched-chain amino acids (BCAA). Adapted from Bruins et al. (2003).

Figure 3. Interorgan flow of amino acids during feeding in disease 24 h (left) and 96 h after infusion
of LPS (right) in young (growing) pigs. Numbers indicate net balances (i.e. net uptake or release from
the organ) and enteral intake as µmol/kg.min for glutamine (GLN), glutamate (GLU), alanine (ALA),
and branched-chain amino acids (BCAA). Based on data published by Bruins et al. (2000).
Administration of arginine enhanced protein turnover (synthesis and degradation) of the muscle,
but without an effect on net protein synthesis (Bruins et al., 2002b). In the liver, protein degradation
and synthesis were reduced, without affecting the protein net balance (Bruins et al., 2002b), while
liver NO production was increased (Bruins et al., 2002c).
Conclusion
The crucial role of the gut not only applies to the postprandial state, related to meal digestion and
absorption of nutrients, but also to the postabsorptive state as a pool of amino acids for other organs.
This requires a very active metabolism and interaction with other organs, of which the liver and muscle
can be considered the major organs involved. Differences between protein diets and postprandial
protein kinetics are present under healthy conditions. After sepsis (disease), organ protein metabolism
is modified in such a way that both the gut and the muscle serve the liver and possibly the immune
system by releasing amino acid precursors used for recovery. Substrate supply in general becomes
important for rapid recovery, while an adequate amount and quality of dietary protein probably can
add even further in the recovery process. In addition, supplementation of deficient amino acids may
also support metabolism.
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Wolfe, R.R., 2002. Regulation of muscle protein by amino acids. J. Nutr. 132, 3219S-3224S.
Wolfe, R.R., 2006. The underappreciated role of muscle in health and disease. Am. J. Clin. Nutr. 84, 475-82.
Wu, G., 1998. Intestinal mucosal amino acid catabolism. J. Nutr. 128, 1249-1252.
Wu, G. and S.M. Morris, Jr., 1998. Arginine metabolism: nitric oxide and beyond. Biochem. J. 336 (Pt 1), 1-17.
Young, V.R. and J.S. Marchini, 1990. Mechanisms and nutritional significance of metabolic responses to altered intakes
of protein and amino acids, with reference to nutritional adaptation in humans. Am. J. Clin. Nutr. 51, 270-289.
Ziegler, T.R., M.E. Evans, C. Fernandez-Estivariz and D.P. Jones, 2003. Trophic and cytoprotective nutrition for
intestinal adaptation, mucosal repair, and barrier function. Annu. Rev. Nutr. 23, 229-261.

Intake of fermentable fiber stimulates mucin and mucosal protein
synthesis in the pig colon
A.J. Libao-Mercado1,2, C.L. Zhu1 and C.F.M. de Lange1
1Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario, Canada
2Cargill Animal Nutrition Phils., Pulilan, Bulacan, Philippines
Introduction
In a previous study, a reduced efficiency of using ileal digestible threonine intake for protein deposition
(PD) was observed in growing pigs fed fermentable fiber (Zhu et al., 2005). This reduced efficiency
can not be attributed to fiber-induced increase in endogenous protein loss in the upper gut (Zhu et
al., 2005). It was hypothesized that the synthesis of mucosal proteins, especially the threonine-rich
mucins, in the hindgut of pigs is increased with dietary inclusion of fermentable fiber. Synthesis
and secretion of endogenous protein in the colon contributes to dietary threonine requirements and
may increase as a consequence of colonic mucins facilitating the movement of indigestible bulk
through the gut, serving as substrate for microbial fermentation, or as part of host defense against
microbes. An experiment was conducted to determine the impact of feeding additional fermentable
fiber (pectin) on mucosal and mucin protein synthesis in the gut of growing pigs.

Twelve Yorkshire barrows (21.0 kg) were surgically fitted with catheters in the external jugular
veins, and were given either a cornstarch and soybean meal-based diet (Control) or the control diet
with 12% additional pectin (Pectin). Diets were fed for 10 d prior to administration of a flooding
dose of valine (1.5 mmol/kg BW) to measure fractional synthesis rates (FSR; %/d) of mucin and
mucosal proteins in the jejunum and colon. Pure mucosal samples were obtained by gently scraping
the surface of previously washed jejunum and colon tissues using a microscope slide. Mucins were
purified from mucosal samples according to Libao-Mercado and de Lange (2007). Total mucosal
mass and protein content were estimated from mucosal scrapings and used to calculate the absolute
synthesis rates (ASR; g/d) of mucosal proteins.

Inclusion of fermentable fiber (12% pectin) in the diet tended to increase the FSR of mucins (P=0.069)
and mucosal proteins in the colon (P≤0.05; Table 1), but not in the jejunum (P>0.10). Mass of mucosal
protein in the jejunum and colon were not influenced by dietary treatment (P>0.10); these estimates
were much lower than previous observations, when a different and more appropriate methodology
was used to measure mucosal protein mass (Nyachoti et al., 2000). The ASR of mucosal proteins
in the jejunum and colon were numerically increased by 26 and 32%, respectively. The difference
between dietary treatments, however, was only statistically significant for the colon (P<0.05).
Amino acids that are secreted into the gut of pigs as part of mucins or other mucosal proteins, and
not reabsorbed contribute to dietary requirements for amino acids. Based on an assumed threonine
content of 15% in mucosal proteins and 3.7% in PD, the pectin-induced increase in mucosal protein
synthesis (0.35 g/d) will reduce PD by about 1.4 g/d, which is lower than the predicted reduction in
PD with pectin supplementation (~6.4 g/d; Zhu et al., 2005). If, however, the amount of mucosal
protein is predicted from intestinal mass according to Nyachoti et al. (2000), then the pectin-induced
increase in mucosal protein synthesis is estimated at 1.5 g/d, which corresponds to a reduction in
PD of approximately 6.0 g/d. Thus, in order to accurately determine dietary amino acids needed to
support the fermentable fiber-induced increase in intestinal mucosal protein (or mucin) synthesis,

an accurate measurement of mucosal or mucin mass is needed. The possibility of amino acids being
lost through other mechanisms should not be discounted either.
Table 1. Fractional (FSR) and absolute synthesis rates (ASR) of mucin and mucosal proteins in the
jejunum and colon of growing pigs fed diets without (Control) or with additional fermentable fiber
(Pectin)1.
Control Pectin
mean SE mean SE P value
Mucin protein
Jejunum (FSR, %/d) 135.3 8.8 143.1 29.0 0.807
Colon (FSR, %/d) 108.5 4.8 127.7 8.1 0.069
Mucosal protein
Jejunum (FSR, %/d) 79.6 3.7 85.2 15.9 0.745
(ASR, g/d) 1.13 0.15 1.43 0.17 0.239
Colon (FSR, %/d) 65.8 4.7 83.5 4.6 0.025
(ASR, g/d) 0.16 0.02 0.21 0.01 0.051
1Enrichment of tissue free valine was used to estimate enrichment at the site of protein synthesis.
Conclusions and implications

Feeding additional fermentable fiber (12% pectin) to pigs increases the fractional synthesis rate
of mucin and mucosal proteins in the colon, but not in the jejunum. The absolute synthesis rate of
mucosal proteins in the colon was also increased with pectin feeding. The observed increase in mucin
and mucosal protein synthesis contributes to the reduced utilization of ileal digestible threonine
intake for PD in growing pigs when feeding fermentable fiber.
References
Libao-Mercado, A.J. and C.F.M. de Lange, 2007. Refined methodology to purify mucins from pig colonic mucosa.
Livest. Sci., in press.
Nyachoti, C.M., C.F.M. de Lange, B.W. McBride, S. Leeson and H. Schulze, 2000. Dietary influence on organ size
and in vitro oxygen consumption by visceral organs of growing pigs. Livest. Prod. Sci. 65, 229-237.
Zhu, C.L., M. Rademacher and C.F.M. de Lange, 2005. Increasing dietary pectin level reduces utilization of digestible
threonine intake, but not lysine intake, for body protein deposition in growing pigs. J. Anim. Sci. 83, 1044-
1053.

Impact of a low dietary threonine supply on protein synthesis, amino
acid deposition and composition of the intestine and the carcass of piglets
A. Hamard1, B. Sève1, D. Melchior2 and N. Le Floc’h1
1INRA, UMR1079 Système d’Elevage, Nutrition Animale et Humaine, 35590 Saint-Gilles, France
2Ajinomoto Eurolysine S.A.S., 153 rue de Courcelles 75817 Paris, France
Introduction
In pigs, threonine (Thr), an essential amino acid (AA), is the first limiting AA for maintenance.
It is now well established that high Thr requirements for maintenance are mainly associated to
intestinal metabolism. Indeed, Le Floc’h and Sève (2005) showed that about 50% of dietary Thr
was extracted in first pass by the portal-drained viscera of continuously fed piglets. The high
dietary Thr extraction by the digestive tract suggests that it contributes to maintain gut integrity and
functionality. We previously showed that a 30% reduction of Thr supply induced villous hypotrophy
associated with a reduction in aminopeptidase N activity in the ileum of early-weaned piglets.
We hypothesised that these structural and functional modifications may be explained by protein
metabolism disturbances in the intestine. In this paper, we compared the rate of protein synthesis,
AA deposition and composition in the intestine of early-weaned piglets fed either a well-balanced
or a low Thr diet for 2 wk. Protein synthesis and AA composition were also measured in the muscle
and the carcass respectively. Indeed the response of these tissues to variation of dietary Thr level
could be different from that of the intestine.
Methods
Two studies using the same experimental design were conducted. Eight and ten pairs of littermate
Piétrain×(Landrace×Large White) piglets weaned at 7 d of age were used in the first and the second
study, respectively. Within pairs, piglets were pair-fed either a well-balanced control diet (C group:
9.3 g Thr/kg) or a low Thr diet (LT group: 6.5 g Thr/kg) for two wk. The experimental diets were
composed of skimmed-milk powder and soluble fish protein concentrate (25% of protein, N×6.25).
A free AA mixture was added to meet AA requirements, except for Thr in the LT diet. Different
measurements were performed on tissues collected at slaughter:
• Study 1. The fractional synthesis rate (Ks; % per d) was measured in the semitendinosus muscle
and the mucosa of the proximal jejunum and the ileum after piglets were injected with a flooding
dose of 15N-valine according to the procedure described by Sève et al. (1993).
• Study 2. Total AA composition was determined in the whole small intestine (from the Treitz
ligament to the ileoceacal junction) and in the carcass (eviscerated, no scalded, with head and
feet). AA deposition was calculated according to the comparative slaughter technique.

Thr first pass extraction by the intestine has been mainly associated to incorporation into mucosal
protein since Thr is not degraded by the intestine (Le Floc’h and Sève, 2005). In our study, growth
performances did not differ between the 2 groups. Dietary Thr supply did not modify the fractional
protein synthesis rate measured in the mucosa of the proximal jejunum and of the ileum (average
values of the C and LT groups: 70.3 ± 2.0%/d and 70.9 ± 2.5%/ d, respectively). Moreover, AA
deposition and AA composition (average value of the C and LT groups: 3.84 ± 0.01 g Thr/16 g N)
in the whole small intestine did not differ between piglets of the two groups. These results suggest
that an inadequate dietary Thr supply for two wk did not alter protein metabolism in the intestine.
This supports previous results showing that intestinal protein metabolism was preserved under non-

optimal nutritional conditions such as AA imbalance (Ponter et al., 1994) or undernutrition associated
to weaning (Sève et al., 1986). This also suggests that villous hypotrophy we previously observed
in the ileum of LT piglets does not result from a modification of protein metabolism. Nevertheless,
we can not exclude that villous hypotrophy could result from protein metabolism modifications
occurring earlier as well as from alteration of cell renewal (cell proliferation and migration).
The small intestine appears to cover its Thr requirements despite the decrease in dietary Thr supply.
This suggests that Thr could be less available for peripheral tissues. Muscle protein synthesis
(average value of the C and LT groups: 20.7 ± 0.5%/d) and AA deposition in the carcass were not
significantly affected by dietary Thr intake. However low dietary Thr supply induced modification
in AA composition of the carcass compared to the C group. Thr (3.52 ± 0.14 for the LT group vs.
3.66 ± 0.09 g/16 g N for the C group; P<0.01), leucine and tyrosine (P<0.05) contents of protein
were lower in the LT than in C piglets suggesting deposition of protein less rich in Thr. This may
be attributed to an adaptation of the piglets to an inadequate Thr supply in order to preserve body
growth of the piglet.
Conclusion
Our results show that mucosal protein metabolism is not impacted by a 30% reduction of Thr supply.
This could be due to the priority of the intestine over the carcass for Thr utilisation in young piglets.
However compensatory mechanisms associated with this priority failed to maintain functional
properties of the ileal mucosa. These modifications are probably associated with protein qualitative
modifications without change in overall amino acid profile at the mucosal level. Body growth is
preserved probably through a modification in the profile of the protein deposited in the carcass.
The impact of a low Thr supply on growth performance and intestinal physiology on longer term
deserves further work.
References
Le Floc’h, N. and B. Sève, 2005. Catabolism through the threonine dehydrogenase pathway does not account for the
first-pass extraction rate of dietary threonine by the portal drained viscera in pigs. Br. J. Nutr. 93, 447-456.
Ponter, A. A., N.O. Cortamira, B. Seve, D.N. Salter and L.M. Morgan, 1994. The effects of energy source and tryptophan
on the rate of protein synthesis and on hormones of the entero-insular axis in the piglet. Br. J. Nutr. 71, 661-674.
Sève, B., P.J. Reeds, M.F. Fuller, A. Cadenhead and S.M. Hay, 1986. Protein synthesis and retention in some tissues
of the young pig as influenced by dietary protein intake after early-weaning. Possible connection to the energy
metabolism. Reprod. Nutr. Dev. 26, 849-861.
Sève, B., O. Ballèvre, P. Ganier, J. Noblet, J. Prugnaud and C. Obled, 1993. Recombinant porcine somatotropin and
dietary protein enhance protein enhance protein synthesis in growing pigs. J. Nutr. 123, 529-540.

Intestinal valine irreversible loss rate during an established
Trichostrongylus colubriformis infection in lambs
E.N. Bermingham, W.C. McNabb, B.R. Sinclair, M. Tavendale, B.P. Treloar and N.C. Roy
Food, Metabolism & Microbiology Section, Food & Health Group, AgResearch Grasslands,
Palmerston North, 4474, New Zealand
Introduction
Sub clinical Trichostrongylus colubriformis infections increase irreversible loss rate (ILR) and
oxidation of leucine in the small intestine and endogenous protein secretion into the gastro‑intestinal
tract (Yu et al., 1999; Yu et al., 2000). Whilst the gastro-intestinal tract uses amino acids (AA) from
both arterial and luminal sources, more than 80% of essential AA used by the gastro‑intestinal tract
are extracted from circulating blood (MacRae et al., 1997). During an intestinal parasitic infection,
AA from luminal sources are likely to be used to meet the increased AA requirement at the site
of infection. Our hypothesis was that an established Trichostrongylus colubriformis infection will
increase the ILR of valine across the mesenteric drained viscera (MDV) and portal drained viscera
(PDV; MDV plus rumen, large intestine and mesenteric lymph nodes) through increased reliance
on luminal AA and arterial supply. The effects of such parasitic infection on the ILR of AA across
the MDV and PDV of sheep were studied using valine as AA tracers.

Twelve multi-catheterised (mesenteric artery, cranial mesenteric vein and portal vein) lambs were
fed fresh Sulla (Hedysarum coronarium; 800 g DM/d) throughout the experimental period. Two wk
after surgery (d0), the lambs were dosed with 6000 Trichostrongylus colubriformis each d for 6 d
(Bermingham et al., 2006). On d 48, the lambs received a continuous 8 h infusion of [3, 4-3H]-valine
(5.8 MBq/h plus 6000 iu heparin/h) into the jugular vein and [1‑13C]‑valine (99 atom percent; 101
mg/h) and 35S-cysteine (2.4 MBq/h) into the abomasum. Plasma flows across the MDV and PDV
were measured using a continuous 8-h infusion of para‑aminohippuric acid (PAH; 723 mg/h; 0.14
mmol/L, Na form) into the mesenteric vein. Blood was continuously withdrawn every 2 h over the
last 4 h from the mesenteric artery, the mesenteric vein and portal vein. Plasma PAH and valine
concentration and valine isotopic activity were determined by spectrophotometry, HPLC with an
in line scintillation detector (3H) or GCMS (13C), respectively (Bermingham et al., 2006). Valine
kinetics were calculated as outlined by Neutze et al. (1997) and Yu et al. (2000). Statistical analysis
used a general linear model (SAS version 8, 1999).

This study showed that an established parasitic infection in lambs fed Sulla triggered shifts in AA
metabolism in the MDV, such as a decrease (P<0.05) in valine luminal ILR (Table 1), despite similar
feed intakes (Control: 769 vs. Parasite: 689 (SD 47) g DM/d, P>0.05). Changes in MDV plasma
flows were not responsible for this effect since no difference was seen between treatments (Control:
533 vs. Parasite: 663 (SD 286) mL/min, P>0.05). The presence of an established parasitic infection
had no effect on the MDV valine arterial ILR (Table 1), suggesting no repartitioning of AA from the
hindquarters in these sheep (Bermingham, 2004). As a result, total valine ILR (arterial plus luminal)
across the MDV was similar between control and infected lambs (P>0.05). In the PDV, plasma flow
(Control: 1518 vs. Parasite: 1398 (SD 369) mL/min) as well as arterial, luminal and total valine ILR
were also similar (P>0.05) between control and infected lambs (Table 1). This study shows that,
while the luminal ILR was decreased by parasitic infection, the overall ILR was not.

Table 1. Valine irreversible loss rate (ILR; mmol/d) across the mesenteric drained viscera (MDV)
and portal drained viscera (PDV) in lambs with (Parasite; n=6) or without (Control; n=6)
Trichostrongylus colubriformis infection using a continuous 8 h infusion of [3, 4-3H]-valine into the
jugular vein (arterial ILR) and of [1-13C]-valine (luminal ILR). Least squares means and associated
pooled standard deviation (SD) are shown.
MDV PDV
Control Parasite Pooled P Control Parasite Pooled P

SD SD
Apparent absorption1 35.0 26.2 4.4 0.01 - - - -

Net release -38.3 -26.3 15.8 0.37 -39.5 -16.5 16 0.05
Arterial ILR 8.9 32.3 15.6 0.25 64.6 43.7 31.9 0.34
Ileal endogenous loss 4.9 5.7 3.0 0.69 - - - -
Luminal ILR 29.1 15.7 1.3 0.01 32.3 24.5 6.7 0.25
Total ILR 38.0 48.0 16.9 0.69 102.2 68.3 38.6 0.45
1 Apparent absorption from the small intestine (valine flux at abomasum – valine flux at ileum).
References
Bermingham, E.N., 2004. The metabolic cost of an intestinal parasite infection on amino acid kinetics in sheep fed
fresh forages. PhD Thesis, Massey University, New Zealand.
Bermingham, E.N., W.C. McNabb, I.A. Sutherland, G.C. Waghorn, B.R. Sinclair, B.P. Treloar and N.C. Roy, 2006.
Whole-body valine and cysteine kinetics in lambs fed Sulla and infected with or without Trichostrongylus
colubriformis. Br. J. Nutr. 96, 28-38.
MacRae, J.C., L.A. Bruce, D.S. Brown and A.G. Calder, 1997. Amino acid use by the gastrointestinal tract of sheep
given lucerne forage. Am. J. Physiol. 36, G1200-G1207.
Neutze, S.A., J.M. Gooden and V.H. Oddy, 1997. Measurement of protein turnover in the small intestine of lambs. 1.
Development of an experimental model. J. Agr. Sci. Camb. 128, 217-231.
Yu, F., L.A. Bruce, R.L. Coopand and J.C. MacRae, 1999. Losses of non-resorbed endogenous leucine from the
intestine of lambs exposed to the intestinal parasite Trichostrongylus colubriformis. VIIIth Int. Symp. Prot. Metab.
Nutr. 48.
Yu, F., L.A. Bruce, A.G. Calder, E. Milne, R.L. Coop, F. Jackson, G.W. Horgan and J.C. MacRae, 2000. Subclinical
infection with the nematode Trichostrongylus colubriformis increases gastrointestinal tract leucine metabolism
and reduces availability of leucine for other tissues. J. Anim. Sci. 78, 380-390.

Contribution of the digestive tract and the liver to the whole body
metabolism of phenylalanine and leucine in growing lambs
Unité de Recherche sur les Herbivores, Equipe Nutriments et Métabolismes, INRA Clermont Ferrand
Theix, 63122 Saint Genès Champanelle, France
Introduction
The aim of the present study was to measure the contribution of the liver and the portal drained viscera
(PDV) to the metabolic fluxes of amino acids (AA) in ruminants when animals were submitted to a
diet deficient in nitrogen (N). A better understanding of the regulation of AA and N utilisation at the
splanchnic level can give important tools to improve the efficiency of production of muscle or milk
proteins. However, previous work (Lobley et al., 1996) has shown that even if most studies are using
one single AA as a tracer (and make extrapolations to whole body kinetics), the differences in the
utilisation of the AA by the various tissues and organs and metabolic routes can lead to misleading
estimates. Consequently, the present study used two AA as tracers: leucine (Leu) and phenylalanine
(Phe) to assess the metabolic fluxes of the whole body and through the PDV, liver and the splanchnic
tissues. These two tracers were chosen because of their different metabolic fates (and intensity of
utilisation) in the splanchnic and the peripheral tissues (Reynolds, 2006).

Six growing lambs (41.5 ± 2.6 kg) were surgically fitted with catheters placed in the splanchnic
area (aorta, portal vein, hepatic vein) and the vena cava, an ultrasonic flow probe (portal vein), for
blood sampling, blood flow measurement and tracer infusion. The animals were fed two diets made
of hay (30%) and of an experimental concentrate mix (70%) which differed in composition. The
control diet (C) was designed to offer a balanced level of nitrogen and energy contents allowing
a 200g/day growth rate. The N deficient diet (N) was designed to reduce the dietary level of N by
23% (P<0.001). Plasma samples were collected from 3 to 9 h after the beginning of the 13C Leu and
2H Phe infusion for isotopic enrichment (IE) and AA concentration measurement. The Phe and Leu
5
fluxes measured at the whole body, portal drained viscera (PDV) and liver levels were calculated
in order to estimate the relative utilisation of leucine and phenylalanine by the various tissues and
organs and the contribution of this utilisation to whole body AA fluxes. The calculations of the AA
fluxes at the different sites are detailed elsewhere (Lobley et al., 1996). For sake of homogeneity in
the results, the precursor pool used for flux calculation was the artery. Consequently, the liver ILR
may be underestimated. The results were analysed by analysis of variance.

In the C diet, the TSP represented 40% and 84% of whole body Leu and Phe fluxes respectively.
This was consistent with results from Lobley et al. (1996). This confirmed the intense utilisation of
Phe in the splanchnic area. More precisely, Leu is utilised by the PDV (32% of the WB flux) and
not by the liver (8% of the WB flux) whereas the utilisation of Phe is intense in the liver (66% of
the WB flux).
The N diet induced a similar reduction of the WB flux of Leu (-12%, P=0.09) and Phe (-10%,
P=0.08). However, the regulation of this reduction of flux at the whole body level was linked to a
reduction of the PDV flux (-38%, P=0.04) for Leu whereas it is due to a reduction of the liver flux
(-47%, P=0.04).

Table 1. Irreversible loss rate (ILR, mmol/h) at the whole body (WB), portal drained viscera (PDV),
liver and total splanchnic tissue (TSP) levels for leucine and phenylalanine in lambs fed a control
(C) or nitrogen deficient (N) diet.
Diet SEM Probability
C N C vs. N
Leucine ILR WB 7.70 6.74 0.28 0.09

PDV -2.48 -1.54 0.18 0.04
Liver -0.61 -0.81 0.24 0.60
TSP -3.09 -2.35 0.30 0.18
Phenylalanine ILR WB 3.21 2.90 0.08 0.08
PDV -0.57 -0.57 0.11 0.99
Liver -2.13 -1.12 0.20 0.04
TSP -2.71 -1.70 0.11 0.01
Since systemic Leu is known to be oxidised across the PDV (Lobley et al., 2003), the N diet must
have induced a reduction in the oxidation of Leu (and maybe other AA oxidised at the PDV level
such as methionine, (Lobley et al., 2003)). In the liver, the utilisation of Phe is strongly reduced
(through a reduction of oxidation or protein metabolism, since Phe participates in both processes)
whereas Leu is poorly extracted by the liver, on a net basis.
In conclusion, these results clearly show the intense utilisation of AA in general and Phe particularly
by the splanchnic tissues (Lobley et al., 2003). They also clearly prove all the limitations associated
with the choice of the tracer in a study of whole body amino acid fluxes (Lobley et al., 1996).
Indeed, a reduction of the N supply in the diet can lead to a similar decrease in the whole body flux
measured by two different tracers such as Leu and Phe. However, the mechanisms of regulation of
this decrease can be different as well as the tissues involved in the decreased utilisation of the amino
acids observed in the N diet. Leu is spared through a probable decreased PDV oxidation whereas
Phe is spared by a decreased utilisation of this AA by the liver (the metabolic pathway involved in
this case remains to be determined).
References
Lobley, G.E., A. Connell, D.K. Revell, B.J. Bequette, D.S. Brown and A.G. Calder, 1996. Splanchnic-bed transfers
of amino acids in sheep blood and plasma, as monitored through use of a multiple U-13C-labelled amino acid
mixture. Br. J. Nutr. 75, 217-235.
Lobley, G.E., X. Shen, G. Le, D.M. Bremner, E. Milne, A.G. Calder, S.E. Anderson and N. Dennison, 2003. Oxidation
of essential amino acids by the ovine gastrointestinal tract. Br. J. Nutr. 89, 617-629.
Reynolds, C.K., 2006. Splanchnic amino acid metabolism in ruminant. In: Sejrsen K., T. Hvelplund and M.O. Nielsen
(editors), Ruminant Physiology: digestion, metabolism and impact of nutrition on gene expression, immunology
and stress. Wageningen Academic Publishers, Wageningen, the Netherlands.

Net flux of amino acids across splanchnic tissues of ewes during
abomasal protein and glucose infusion
H. Freetly1, C. Ferrell1 and S. Archibeque2
1USDA, ARS, US Meat Animal Research Center, P.O. Box 166, 68933, Clay Center, USA
2Colorado State University, Mailstop 1171, 80523, Fort Collins, USA
Introduction
Studies in sheep (MacRae et al., 1997) and in pigs (Stoll et al., 1998) indicated that approximately
one-third of the amino acids absorbed by the enterocytes are metabolized within the enterocyte and are
never released into the blood. Amongst those amino acids that are metabolized, 60% are apparently
catabolized (Stoll et al., 1998) suggesting that 20% of the absorbed amino acids are catabolized for
energy within the enterocyte. A potential mechanism to reduce amino acid catabolism by enterocytes
is to provide an alternative energy source. We hypothesized that amino acid catabolism within
enterocytes can be reduced by elevating glucose availability to enterocytes, resulting in increased
net appearance of amino acids in portal vein blood.

Eighteen Dorset ewes (71.7 ± 0.7 kg) were individually penned and fed a pelleted diet (95% brome
grass hay and 5% soybean meal, as DM) at 52.2 g/BW kg0.75. The diet was 10% CP and 1.96 Mcal
ME/kg, as DM. Ewes were fed a single meal daily at 15.00 h. Catheters were placed in the hepatic
portal vein, a hepatic vein, a mesenteric vein, and the abdominal aorta (Freetly and Ferrell, 1998).
Glucose and protein were infused into the abomasum via a cannula. Ewes either received glucose
(3.84 g/h) or glucose-free (Control) infusions along with one of five protein infusions. Each ewe
remained on assigned glucose treatments and received all five protein infusions in a replicated Latin
square design within treatment. The protein infusions consisted of isolated soy protein (Ardex® F
Dispersible, Archer Daniels Midland Company, Decatur, IL 62525) and cysteine 8.26% by weight).
The infusion levels were 0, 2.616, 5.232, 7.848, and 10.464 g/h Ardex + cysteine. The calculated
amino acid equivalents were 0, 18.1, 36.3, 54.4, and 72.6 mmol/h. Abomasal infusions were started
17 h after the meal. Para-amino hippuric acid (ρAH, 0.15 M) was infused into the mesenteric vein
(0.8 mL/min). Four hours after abomasal infusions were initiated, blood samples were drawn at
30-minute intervals for a total of five sets of samples. Whole blood was analyzed for ρAH and
glucose (Freetly and Ferrell, 1998). Glutamate and glutamine concentrations were determined on
a membrane-immobilized system. Blood amino acids were analyzed according to the procedure of
Calder et al. (1999). Net fluxes were calculated as described by Freetly and Ferrell (1998). Data
were analyzed by use of a split-plot model. All fluxes were tested with a full model that included
treatment, animal nested within treatment, protein level, (protein level)2, treatment × (protein level),
and treatment × (protein level)2. Protein level was treated as a continuous effect. A step-down
regression approach was used to determine the model that best described the regression coefficients
for net fluxes on protein level.

Net portal-drained viscera (PDV) lysine release (P=0.05; 2.33 ± 0.48 vs. 1.04 ± 0.49 mmol/h) and
net histidine release (P=0.04; 1.29 ± 0.43 vs. 0.24 ± 0.43 mmol/h) increased in glucose-infused ewes
compared to controls. Net PDV release of other amino acids did not differ with glucose infusion
(P>0.13). Net PDV release of alanine (2.11 ± 0.23 mmol/h; P=0.10) glycine (2.03 ± 0.17 mmol/h;
P=0.69), histidine (P=0.84), lysine (P=0.21), threonine (0.84 ± 0.09 mmol/h; P=0.11) and valine
(1.07 ± 0.09 mmol/h; P=0.13) did not differ with abomasal amino acid infusion; however, net release
of other amino acids increased linearly (Table 1).

Table 1. Net portal-drained viscera release (mmol/h) = f(x) = b1x + b0 where x = mixed amino acid
abomasally infused (mmol/h).
Amino acid b1 ± SE b0 ± SE PAA1 PGlucose2 R2
Leucine 0.0148 0.0041 0.84 0.19 0.001 0.38 0.25

Isoleucine 0.0094 0.0026 0.60 0.12 0.001 0.40 0.25
Methionine 0.0018 0.0007 0.24 0.33 0.02 0.13 0.25
Phenylalanine 0.0109 0.0027 0.70 0.12 0.0001 0.22 0.33
Tyrosine 0.0052 0.0026 0.61 0.12 0.05 0.26 0.23
EAA3 0.0736 0.0326 5.39 1.42 0.04 0.14 0.06
Glutamate 0.0160 0.0034 -0.86 0.15 <0.0001 0.42 0.48
Glutamine 0.0870 0.0119 -8.58 0.53 <0.0001 0.96 0.57
Proline 0.0126 0.0030 0.38 0.13 <0.0001 0.43 0.30
Serine 0.0152 0.0038 0.99 0.17 0.0002 0.21 0.32
Aspartate 0.0115 0.0036 0.21 0.16 0.002 0.45 0.32
1Probability that the net flux increased with amino acid infusion; 2Probability that net fluxes differed with glucose
infusion; 3His+Ile+Leu+Lys+Met+Phe+Thr+Val.
Net PDV release of glucose was greater (P<0.001) in ewes that received the glucose infusion (1.6
± 1.5 mmol/h) compared to controls -12.9 ± 2.8 (mmol/h). Net glucose release increased in both
glucose treatments with increased amino acid infusion
[P = 0.03, f(x) = -0.0026x2 + 0.161x].
We found that for most amino acids, increased lumenal glucose did not improve net flux across the
PDV. These findings suggest that glucose does not spare amino acids; however, amino acids may
improve net PDV glucose release.
References
Calder, A.G., K.E. Garden, S.E. Anderson and G.E. Lobely, 1999. Quantitation of blood and plasma amino acids using
isotope dilution electron impact gas chromatography/mass spectrometry with U-13C amino acids as internal
standards. Rapid Commun. Mass Spectrom. 13, 2080-2083.
Freetly, H.C. and C.L. Ferrell, 1998. Net flux of glucose, lactate, volatile fatty acids, and nitrogen metabolites across
the portal-drained viscera and liver of pregnant ewes. J. Anim. Sci. 76, 3133-3145.
MacRae, J.C., L.A. Bruce, D.S. Brown and A.G. Calder, 1997. Amino acid use by the gastrointestinal tract of sheep
given lucerne forage. Am. J. Physiol. 273, G1200-G1207.
Stoll, B., J. Henry, P.J. Reeds, H. Yu, F. Jahoor and D.G. Burrin, 1998. Catabolism dominates the first-pass intestinal
metabolism of dietary essential amino acids in milk protein-fed piglets. J. Nutr. 128, 606-614.

Effect of abomasal glucose infusion on splanchnic glucose metabolism in
freshening dairy cows
Faculty of Agricultural Sciences, University of Aarhus, DK-8830 Tjele, Denmark
Introduction
Nutritional management of fresh cows is of critical importance to the success of a dairy operation.
Fresh cows often suffer from hypoglycaemia and hyperlipidemia that might be part of the reason
for the health problems associated with the transition from pregnancy into lactation. Feeding diets
containing bypass starch is in theory an attractive strategy to overcome some of the nutritional
shortcomings. However, the efficacy of increasing glucose supply to the peripheral tissues through
increased small intestinal glucose absorption in freshening dairy cows is not known. The aim of
the present experiment was to investigate the effect of continuous abomasal glucose infusion on
splanchnic glucose metabolism in freshening dairy cows.

Six primiparous Danish Holstein cows were blocked according to their expected calving date and
randomly assigned to either no infusion (Control) or continuous abomasal infusion of 1500 g glucose/
d from the day of calving (Infusion). Ruminal cannulas and permanent indwelling catheters in the
mesenteric vein, mesenteric artery (n=3), intercostal artery (n=3), hepatic portal vein, and hepatic vein
(n=5; one cow on Control treatment did not have a functional hepatic vein catheter) were implanted
at least 6 wk before their expected second calving. All cows were offered the same dry-ration pre
partum and the same lactation-ration post partum. Cows were fed restricted pre partum, but fed ad
libitum post partum. Feed was offered as a TMR and fed in equally sized portions at 08.00, 16.00
and 24.00 h. The lactation-ration contained (percent of DM): maize silage, 41; clover grass silage,
20; rolled barley, 15; rape seed cake, 9.5; sugar beet molasses, 7.5; sugar beet pulp, 2.5; soy meal,
1.75; sodium bicarbonate, 0.75; fat supplement (Leci-E), 0.75; mineral and vitamin premixes, 1.75.
An infusion device was placed in the abomasum via the rumen cannulae at the day of calving and
the glucose infusion was stepped up over three days. The infusion device was only placed with
Infusion, since preliminary experiments showed no effect of the infusion device on cow performance.
Feed intake and milk yield were recorded daily. Eight hourly arterial, portal vein, and hepatic vein
samples were collected simultaneously starting 30 min before feeding at 08.00 h on day 4, 14, and
28 post partum. Blood plasma flows were measured by down stream dilution of para-aminohippuric
acid infused into the mesenteric vein. Samplings within day (i.e. time) were considered as repeated
measures and analyzed using the autoregressive order 1 structure in the Mixed Procedure of SAS.
The model included the effects of block, treatment, DIM (days in milk), time and the interactions:
treatment × DIM, treatment × time, DIM × time, and treatment × DIM × time. The cow × DIM
interaction was designated as a random effect.

The actual abomasal infusion rate of glucose with the Infusion treatment was 349 ± 6 mmol/h. Dry
matter intake post partum tended (P=0.09) to decrease with Infusion (Table 1). A tendency (P=0.06)
for treatment by DIM interaction was observed for milk yield reflecting lower milk yield with Infusion
from about d 4 post partum. The arterial plasma concentration of glucose increased (P=0.02) with
Infusion. The net portal flux of glucose increased (P<0.01) with Infusion. The increased net portal
flux of glucose accounted for 72% of the glucose infused into the abomasum. The portal-drained
visceral uptake of arterial glucose might have increased with Infusion and glucose absorption from

small intestinal digestion of dietary starch might have decreased with decreasing DMI. Therefore
the estimated portal recovery of infused glucose is most likely underestimating the true increase in
glucose absorption with infusion. These data indicate that glucose originating from small intestinal
starch digestion will be efficiently absorbed to the portal blood of freshening cows. Starch sources
that bypass the rumen and have high intestinal digestibility might therefore be used to reduce
hypoglycemia of freshening cows.
Table 1. Dry matter intake, milk yield, arterial concentration, net portal flux, net hepatic flux, and
net splanchnic flux of glucose in dairy cows sampled 4, 14, and 28 d post partum.
Item Treatment1 SEM2 P
Control Infusion Treat DIM3 Time4
Dry matter intake, kg/d 17 11 1 0.09 <0.01 -

Milk yield, kg/d5 27 20 2 0.16 <0.01 -
Arterial plasma glucose, mM 3.38 3.92 0.15 0.02 0.89 0.41
Plasma glucose fluxes, mmol/h
Net portal flux -52 201 25 <0.01 0.23 0.52
Net hepatic flux 865 536 88 0.03 0.14 0.84
Net splanchnic flux 770 741 76 0.77 0.19 0.74
1Treatments were no infusion (Control) or continuous infusion of 349 ± 6 mmol glucose/h into the abomasum from
d 1 to 28 post partum; 2Standard error of the mean (n=3; except for net hepatic flux and net splanchnic flux with
the Control treatment where n=2); 3Days in milk; 4Sampling time within sampling day; 5Treatment by DIM effect
(P=0.06).
The net hepatic flux of glucose decreased (P=0.03) with Infusion. The reduction in net hepatic
glucose flux was equivalent to 94% of the infused amount of glucose. However, the net hepatic flux
of glucose per kg DMI was not affected (P=0.50) by treatment. The net splanchnic flux of glucose
was not affected (P=0.77) by treatment.
In conclusion, glucose available in the lumen of the small intestine is absorbed by the portal blood
and only a relatively small fraction (maximum 28%) is metabolised by the enterocytes. Glucose
infusion tended to decrease both feed intake and milk yield. The liver output of glucose decreased
with glucose infusion and the result of increased absorption and decreased hepatic output was that
the net splanchnic flux remained unaffected. Reduced milk yield with the same net splanchnic flux
of glucose will result in increased supply of glucose to the peripheral tissues of the cow which were
in agreement with the observed increase in arterial glucose concentration. The profound metabolic
effects of glucose supply in the present study indicate that manipulation of the glucogenic properties
of the feed could be used to reduce metabolic stress in freshening cows.

Portal recovery of glucose infused into the abomasum of lactating dairy cows
Introduction
Feeding diets containing bypass starch to fresh cows is in theory an attractive strategy to overcome
hypoglycaemia in early lactation. However, glucose sequestration by enterocytes might limit the
efficacy of starch digested in the small intestine. Therefore, when starch is bypassed to the small
intestine and hydrolysed into glucose, the absorbed glucose might not be released into the blood stream
because of metabolism in the enterocytes. Very limited data are available on glucose metabolism by
enterocytes of dairy cattle. The aim of the present study was to investigate glucose sequestration by
enterocytes in dairy cattle by measuring portal recovery of glucose infused into the abomasum.

Six multiparous lactating (27 ± 4 kg milk/d) Danish Holstein cows implanted with ruminal cannulas
and permanent indwelling catheters in the mesenteric vein, mesenteric artery (n=3), intercostal
artery (n=2), carotid artery (n=1), and hepatic portal vein were used. Cows were fed a commercial
compound feed (16 ± 3 kg dry matter/d; Danko Grøn, DLG, Copenhagen, Denmark) and grass hay
(3 ± 2 kg dry matter/d). The experimental period was 14 d and samplings were conducted on d 14.
Cows were not fed 15-19 h prior to samplings to avoid small intestinal glucose absorption from
dietary starch. An infusion line was placed in the abomasum via the rumen cannula and 40 g of
glucose was infused in 500 mL of saline. The infusion line was flushed with another litre of saline.
Arterial and portal blood samples were collected simultaneously according to the following scheme:
-60, -50, -30, 10, 20, 40, 60, 90, 120 and 150 min relative to infusion. The sampling scheme was
extended with -40, 5, 15, 25, 30, 35, 45, 50 and 75 min relative to infusion for 3 cows. Portal blood
plasma flow was measured by down stream dilution of para-aminohippuric acid infused into the
mesenteric vein. The portal recovery of infused glucose was calculated for each cow as the area
under the net portal flux curve corrected for the baseline. The baseline was defined by the net portal
flux of glucose -60 to -30 as well as +90 to +150 min relative to abomasal infusion. Samplings
within d (i.e. time) were considered as repeated measures and analysed using the autoregressive
order 1 structure in the Mixed Procedure of SAS. The model included the effect of time. The cow
was considered as a random effect.

The abomasal infusion of 40 g of glucose increased the arterial (P<0.01) and portal (P<0.01) glucose
concentration (Figure 1a). The portal-arterial concentration difference for glucose increased (P<0.01)
after abomasal glucose infusion. The net portal flux of glucose increased (P<0.01) after the infusion
(Figure 1b). For all cows, the net portal flux of glucose returned to the baseline within 90 min after
infusion. Portal recovery of abomasally infused glucose was estimated to 90 ± 10%, indicating a
small first pass metabolism of glucose by the enterocytes.
Some of the infused glucose might have been fermented by microbes in the abomasum and
small intestine, and part of the infused glucose could have passed the small intestine unabsorbed.
Consequently the true portal recovery of absorbed glucose could have been higher than estimated
from our data. In steers, the portal recovery of abomasally infused glucose has been estimated in the
range from 73 to 94% (Kreikemeier et al., 1991; Kreikemeier and Harmon, 1995). These estimates
are in good agreement with the results of the present study. The cows in the present study were fed a

Figure 1. a) Arterial (■) and portal (□) plasma concentrations of glucose in dairy cows before and
after abomasal infusion of 40 g of glucose (infusion at time=0). b) Net portal flux (r) of glucose
in dairy cows before and after abomasal infusion of 40 g of glucose. Baseline (--) fitted by linear
regression using values obtained before infusion and values from 90 to 150 min after infusion. Each
data point is the mean of 6 observations ± SE.
diet containing approximately 10% starch in dry matter and this diet probably resulted in only small
amounts of starch bypassing the rumen. Our data therefore indicate that even ruminants not adapted to
large small intestinal digestion of dietary starch have a relatively large capacity for glucose absorption
and a sudden increased presence of glucose in the small intestine is efficiently transferred into the
portal blood. Our data point to the conclusion that starch sources that contribute to small intestinal
starch digestion will provide dairy cows with glucose available for intermediary metabolism.
Conclusion
Glucose absorption into the portal blood could account for 90 ± 10% of the glucose infused into
the abomasum of lactating dairy cows. Enterocytes metabolise small amounts/none of the glucose
absorbed from the small intestine and the capacity for glucose absorption in the small intestine is
considerable even in cows fed low amounts of dietary starch.
References
Kreikemeier, K.K, D.L. Harmon, R.T. Brandt Jr, T.B. Avery and D.E. Johnson, 1991. Small intestinal starch digestion
in steers: Effect of various levels of abomasal glucose, corn starch and corn dextrin infusion on small intestinal
disappearance and net glucose absorption. J. Anim. Sci. 69, 328-338.
Kreikemeier, K.K. and D.L. Harmon, 1995. Abomasal glucose, maize starch and maize dextrin infusions in cattle:
Small-intestinal disappearance, net portal flux and ileal oligosaccharide flow. Br. J. Nutr. 73, 763-772.

Amino acid and energy metabolism by the portal-drained viscera of beef
steers: quantitative relationships with metabolizable energy intake
K.R. McLeod, S.W. El-Kadi, D.L. Harmon and E.S. Vanzant
University of Kentucky, Lexington, KY 40546, USA
Introduction
In addition to changes in nutrient supply to the small intestine, previous studies have shown that feed
intake also influences gastrointestinal tract (GIT) mass (Burrin et al., 1990; McLeod and Baldwin,
2000). Such changes affect our ability to predict nutrient availability to post-absorptive tissues.
To date, most existing data regarding ME intake and its effect on energy metabolism and nutrient
absorption by the GIT of cattle stems from studies in which two ME levels of the same diet were fed
(e.g. Reynolds et al., 1992). In studies where more than two levels of ME intake were used, feeding
has been limited to mixed high concentrate diets (e.g. Lapierre et al., 2000). However, differences
in tissue mass were reported when sheep were on high forage as compared to high concentrate diets,
even when ME intake was constant (McLeod and Baldwin, 2000). The goal of the current study was
to determine how increments of forage intake, and thus, increments of ME intake, affect nutrient and
energy utilization by the GIT of beef cattle and to establish response relationships between intake
and nutrient net availability to post-absorptive tissues.

Eight Angus (328 ± 40 kg BW) steers were surgically fitted with portal, and mesenteric arterial
and venous catheters, and were fed alfalfa cubes in a replicated 4×4 Latin square design with
4 levels of energy intake between 1 and 2× maintenance energy requirements. On d 28 of each
experimental period, p-aminohippuric acid was infused to measure plasma flow, and blood samples
were simultaneously collected from arterial and venous catheters. Plasma flow rates across the GIT
were calculated using the Fick principle, and plasma AA concentrations were determined by isotope
dilution gas chromatography-mass spectrometry as previously described by Calder et al. (1999). Net
fluxes of nutrients across the GIT were calculated as the product of arterio-venous concentration
difference and plasma flow. The relationships between ME intake and AA and oxygen net fluxes
were tested using a backward stepwise regression, where a third order model was tested, and if not
significant, the analysis was repeated with a lower order model until significance was reached.

Oxygen utilization linearly increased (815 to 1,250 mmol/h) in response to increased ME intake
(P<0.05), and therefore equated to an increase (2.321 to 3.834 Mcal/d) in energy utilization by
the GIT (P<0.05). For all AA, except for tryptophan, glutamate, and glutamine, the relationship
between AA net flux across the GIT and ME intake was significant (Table 1), where portal net flux
increased (P<0.05) in response to ME intake. Given that the glutamate and glutamine net fluxes
were constant despite the increased small intestinal supply, implies that GIT utilization was greater
as ME intake increased.
Previous reports have suggested that oxygen utilization across the GIT is a function of tissue mass
(McLeod and Baldwin, 2000). The current study provides response relationships between ME
intake and GIT energy expenditure and AA net release in steers fed a forage diet. Net portal flux of
the essential AA was also compared to predicted AA supply to the small intestine using the Cornel
Net Carbohydrate and Protein System. The slopes of equations describing the relationship between
predicted supply and AA net flux across the GIT were between 0.44 and 0.88, indicating that GIT

utilization occurred at a constant proportion of an increasing supply. It is likely that GIT mass
increased in response to increased ME intake, and thus increased AA and energy utilization. While
the increased AA utilization may have resulted in part from larger requirements for structural and
secretory proteins, AA may have also played a role as energy substrates for GIT cells.
Table 1. Amino acid portal net flux (mmol/h) of steers fed increments of ME intake.
ME intake, Mcal/(kg BW0.75×d) Linear regression
0.117 0.156 0.195 0.234 SEM P-value P-value R2
Valine 0.67a 2.11a 3.91a 4.22a 1.087 0.033 <0.001 0.59

Leucine 1.47b 3.26 ab 5.19ab 6.05a 1.249 0.024 <0.001 0.56
Isoleucine 1.00b 2.16 ab 3.51ab 4.37a 0.920 0.024 <0.001 0.61
Methionine 0.50b 1.03ab 1.73a 2.23a 0.416 0.003 <0.001 0.57
Threonine 0.87b 1.80ab 3.47ab 4.26a 0.940 0.032 <0.001 0.58
Phenylalanine 1.39b 2.56ab 4.00a 4.77a 0.873 0.003 <0.001 0.67
Lysine 2.02b 3.17 ab 4.84ab 6.36a 1.108 0.012 <0.001 0.66
Histidine 0.48c 0.83bc 1.41ab 1.65a 0.300 0.001 <0.001 0.82
Tryptophan 0.27 -0.11 0.87 0.52 0.405 NS1 NS 0.43
Alanine 2.99b 6.44ab 10.70a 12.71a 2.460 0.010 <0.001 0.64
Glycine 3.91 5.10 7.26 7.18 1.511 NS 0.004 0.69
Proline 0.76b 1.41ab 2.45a 2.79a 0.555 0.008 <0.001 0.65
Serine 2.52b 3.82ab 5.64ab 7.24a 1.244 0.016 <0.001 0.34
Aspartate 0.33b 0.46ab 0.58ab 0.75a 0.113 0.041 0.001 0.50
Glutamate 0.55 1.88 -0.61 0.50 0.875 NS NS 0.02
Glutamine -3.63 -4.91 -4.18 -5.07 0.970 NS NS 0.52
a-b Means within a row with different superscripts are significantly different from one another (P≤0.05).
1 NS = not significant (P>0.05).
References
Burrin, D.G., C.L. Ferrell, R.A. Britton and M. Bauer, 1990. Level of nutrition and visceral organ size and metabolic
activity in sheep. Br. J. Nutr. 64, 439-448.
Calder, A.G., K.E. Garden, S.E. Anderson and G.E. Lobley, 1999. Quantitation of blood and plasma amino acids using
isotope dilution electron impact gas chromatography/ mass spectrometry with U-13C amino acids as internal
standards. Rapid Commun. Mass Spectrom. 13, 2080-2083.
Lapierre, H., J.F. Bernier, P. Dubreuil, C.K. Reynolds, C. Farmer, D.R. Ouellet and G.E. Lobley, 2000. The effect of
feed intake level on splanchnic metabolism in growing beef steers. J. Anim. Sci. 78, 1084-1099.
McLeod, K.R. and R.L. Baldwin, 2000. Effects of diet forage: concentrate ratio and metabolizable energy intake on
visceral organ growth and in vitro oxidative capacity of gut tissues in sheep. J. Anim. Sci. 78, 760-770.
Reynolds, C.K., H. Lapierre, H.F. Tyrrell, T.H. Elsasser, R.C. Staples, P. Gaudreau and P. Brazeau, 1992. Effects
of growth hormone-releasing factor and feed intake on energy metabolism in growing beef steers: net nutrient
metabolism by portal-drained viscera and liver. J. Anim. Sci. 70, 752-763.

Effect of polyethylene glycol on the net flux of amino acids in the
mesenteric- and portal-drained viscera in lactating ewes fed Sulla, a
condensed tannin forage
D.L. Deighton, W.C. McNabb, B.R. Sinclair, B.P. Treloar and N.C. Roy
Food, Metabolism & Microbiology Section, Food & Health Group, AgResearch Grasslands Research
Centre, Palmerston North 4442, New Zealand
Introduction
Moderate dietary levels of condensed tannins (CT) can reduce protein degradation in the rumen
(McNabb et al., 1996) and increase the apparent intestinal absorption of amino acids (AA), especially
the essential AA (EAA; Bermingham et al., 2001). Polyethylene glycol (PEG) binds to and inactivates
CT. The effects of CT can be elucidated by comparing the net flux of AA across a tissue bed in ewes
fed fresh Sulla and dosed with PEG (CT inactive) with ewes that had not received PEG (CT active).
The hypothesis of this study was that the CT in Sulla (Hedysarum coronarium) would increase the
net portal absorption of EAA in lactating ewes. The aim of this study was to quantify the effect of
PEG on the net flux of AA across the mesenteric-drained viscera (MDV) and portal-drained viscera
(PDV) in lactating ewes fed fresh Sulla.

Twelve ewes had catheters inserted in the mesenteric artery and portal and cranial mesenteric veins.
The infusion catheter tip was 10 cm distal to the sampling catheter tip in the cranial mesenteric vein,
and the sampling catheter was located upstream of the junction with the caudal mesenteric vein.
Four wk post partum, ewes were fed fresh Sulla (1500 g DM/d; 80 g CT/d) and orally drenched
each d with either water (control n=6; CT active) or PEG (n=6; 160 g/d; CT inactive). At 6 wk post
partum (48.9 kg body weight), sodium para‑aminohippurate (pAH) was infused for 7 h into the
mesenteric vein to measure plasma flow. Plasma AA concentrations were measured by HPLC and
pAH by spectrophotometry. Net AA flux (arterio-venous concentration difference of AA (μmol/mL) ×
plasma flow (mL/min)) were analysed according to a randomised design (SAS 9.1 GLM). Treatment
differences were declared significant at a probability less than 0.05 and a trend at a probability less
than 0.10.

The dry matter intake of Sulla was not affected by the PEG treatment and averaged 1357 (SD 68)
g/d. Six wk post partum (i.e. after 3 wk of ± PEG treatment), MDV (727 (SD 308) mL/min) and
PDV (2171 (SD 249) mL/min) plasma flows were not affected by PEG. A net release of EAA and
non essential AA (NEAA) by the MDV was seen in both treatments except for glutamine which
was taken up (Table 1). Only the net release of tyrosine tended (P<0.10) to decrease with the
PEG treatment (Table 1). Glutamate and aspartate were released at a lower rate (P<0.05) in the
mesenteric drainage in the PEG ewes (Table 1). The net appearance of EAA, NEAA and total AA
in the mesenteric drainage was similar between the PEG and control ewes (Table 1). Appearance of
arginine, aspartate and methionine in the portal drainage was also lower (P<0.05) in the PEG ewes
(Table 1). No effect of PEG was observed on the net appearance of other individual AA across the
PDV (Table 1). The net appearance of EAA, NEAA and total AA in the portal drainage was similar
between treatments (Table 1). The MDV and PDV values for individual AA were within the same
range as those reported by Rémond et al. (2003). These results show that the intra-luminal addition
of PEG in lactating ewes fed the CT containing Sulla decreased the mesenteric and portal appearance
of certain AA. This is consistent with reports that CT increased intestinal AA absorption and this

may contribute to the increase in lactational performance that has been observed in ewes and dairy
cows fed fresh CT-containing forages (Woodward et al., 1999).
Table 1. Effect of polyethylene glycol on the net flux of amino acids (AA; ± SED, μmol/min) across
the mesenteric- and portal-drained viscera in lactating ewes fed Sulla1.
Mesenteric-drained viscera Portal-drained viscera
Control PEG Control PEG
Total EAA total -532.2 (±90.0) -412.5 (± 82.1) -338.9 (± 32.7) -279.4 (± 32.7)
Arginine -43.8 (± 7.7) -30.8 (± 7.0) -26.5 (± 2.3) -15.6 (± 2.1)**
Histidine -25.9 (± 4.2) -19.7 (± 3.8) -18.3 (± 1.8) -13.8 (± 1.7)†
Isoleucine -70.9 (± 12.2) -56.3 (± 11.2) -45.6 (± 4.7) -35.7 (± 4.3)
Leucine -75.1 (± 15.1) -62.9 (± 13.8) -48.2 (± 6.6) -35.3 (± 6.0)
Lysine -63.5 (± 11.0) -45.8 (± 10.1) -41.0 (± 5.2) -26.7 (± 4.8)†
Methionine -25.0 (± 4.4) -18.6 (± 4.1) -16.9 (± 1.7) -10.9 (± 1.5)*
Phenylalanine -35.5 (± 6.7) -28.9 (± 6.1) -27.5 (± 3.1) -20.4 (± 2.8)
Threonine -56.6 (± 10.5) -40.7 (± 9.6) -28.6 (± 3.1) -26.9 (± 3.1)
Tyrosine2 -42.0 (± 6.4) -25.2 (± 5.8)† -27.8 (± 5.7) -21.2 (± 5.2)†
Valine -93.8 (± 18.6) -83.6 (± 16.9) -58.6 (± 6.7) -47.6 (± 6.1)
Total NEAA total -529.0 (± 90.5) -414.4 (± 82.6) -262.6 (± 34.8) -203.8 (± 31.8)
Alanine -132.8 (± 24.1) -118.8 (± 22.0) -88.4 (± 11.2) -73.7 (± 10.2)
Asparagine -64.4 (± 13.3) -54.6 (± 12.1) -27.6 (± 7.0) -24.2 (± 6.4)
Aspartate -10.6 (± 2.4) -2.7 (± 2.2)* -7.6 (± 1.7) -1.5 (± 1.5)*
Glutamate -25.9 (± 1.9) -13.4 (± 1.7)** -13.8 (± 5.4) -3.6 (± 4.9)
Glutamine 1.1 (± 6.8) 8.8 (± 6.2) 79.0 (± 16.0) 58.3 (± 14.6)
Glycine -83.8 (± 14.7) -64.1 (± 13.4) -70.8 (± 9.8) -57.3 (± 8.9)
Ornithine synthesis -11.2 (± 2.0) -7.0 (± 1.8) -12.8 (± 1.8) -7.8 (± 1.6)†
Proline -67.5 (± 11.8) -54.0 (± 10.8) -38.2 (± 4.2) -28.6 (± 3.8)
Serine -63.5 (± 11.3) -51.4 (± 10.3) -43.1 (± 5.7) -35.3 (± 5.2)
Total AA -1061.2 (± 180.1) -826.94 (± 164.4) -601.5 (± 48.9) -517.6 (± 48.9)
1 Positive
values indicate net uptake and negative values, net appearance; 2 Conditionally essential; † 0.10<P>0.05,
*P<0.05, **P<0.01.
References
Bermingham, E.N., K.J. Hutchinson, D.K. Revell, I.M. Brookes and W.C. McNabb, 2001. The effects of condensed
tannins in sainfoin (Onobrychis viciifolia) and sulla (Hedysarum coronarium) on the digestion and absorption of
amino acids in sheep. Proc. NZ Soc. Anim. Prod. 61, 116-119.
McNabb, W.C., G.C. Waghorn, J.S. Peters and T.N. Barry, 1996. The effect of condensed tannins in Lotus pedunculatus
on the solubilization and degradation of ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39; Rubisco) protein in
the rumen and the sites of Rubisco digestion. Br. J. Nutr. 76, 535-549.
Rémond, D., L. Bernard, B. Chauveau, P. Noziere and C. Poncet, 2003. Digestion and nutrient net fluxes across the
rumen, and the mesenteric- and portal-drained viscera in sheep fed with fresh forage twice daily: net balance and
dynamic aspects. Br. J. Nutr. 89, 649-666.
Woodward, S.L., M.J. Auldist, P.J. Laboyrie and E.B.L. Jansen, 1999. Effect of Lotus corniculatus and condensed
tannins on milk yield and milk composition of dairy cows. Proc. NZ Soc. Anim. Prod. 59, 152-155.

Splanchnic net release and body retention of nitrogen in growing lambs
fed diets unbalanced for energy and protein
G. Kraft, I. Ortigues-Marty and I. Savary-Auzeloux
INRA, Centre de Theix, UR1213 Herbivores, Nutrients and Metabolism Group, 63122 Saint-Genès-
Champanelle, France
Introduction
Nutritional allowances to Ruminants are defined for both protein and energy in the diet, but animal
metabolic responses to unbalanced diet, either in protein or in energy, are difficult to predict. They
have generally been studied using an excess supply in one or another nutrient (Savary-Auzeloux
et al., 2003). The present work focusses on the influence of a nutritional deficit in either energy or
protein. The objective was to quantify the net fluxes of the major nitrogenous nutrients across the
splanchnic tissues (TSP) and compare them with nitrogen retention data to identify the tissues/organs
responsible for metabolic adaptation.

Six growing lambs (41.5 ± 2.6 kg) were surgically fitted with catheters placed in the splanchnic area
(aorta, portal and hepatic veins) and an ultrasonic probe on the portal vein for blood flow measurement.
The animals were fed three diets made of 30% hay and 70% of one of three experimental concentrates,
according to a duplicated 3 × 3 Latin Square design. The control diet (C) was designed to offer
a balanced supply of protein and metabolisable energy (ME) for growing lambs (total nitrogen
ingested=1.43gN/d/BW0.75 and ME=204 kcal/d/BW0.75). The protein (N-) and the energy deficient
(E-) diets presented a 23% deficit of protein and a 20% deficit in ME supply compared to the C
diet, respectively. Nitrogen (N) retention was determined by urine and feces collection over 6 d and
Kjeldahl N analyses. Blood samples were then collected simultaneously from the three vessels for
analysis of blood ammonia, plasma urea and 18 individual amino acids (AA) in order to calculate
the net fluxes of N nutrients across the TSP (Savary-Auzeloux et al., 2003).

Apparent dietary N digestibility was very slighty altered by the diet (70, 66 and 72% for C, N- and
E- respectively). The proportion of dietary N which was retained by the body (34.8% with the C
diet) tended to be improved with N- (39.1%, P<0.1) but was reduced with E- (24.2%, P<0.01). These
effects were due to significant changes in urinary N losses (35, 27 and 48% of N intake lost in the
urine, for C, N- and E- respectively; P<0.001) emphasising the important role of the liver.
In the portal-drained viscera (PDV), the N- diet reduced the proportion of N-ingested recovered
as AA in the portal vein (39.6% vs. 53.4% for N- and C, P=0.03) despite a reduced amount of
ammonia absorbed in the PDV (-40%, P<0.001) and a significantly greater proportion of urea-N
recycled to PDV (68 vs. 53% of net hepatic urea flux for the N- and C diets, P=0.005). However,
the E- diet did not modify the proportion of ingested-N appearing as AA in the portal vein (52.1
vs. 53.4% for E- and C, P=0.82) nor that appearing as ammonia (37.3 vs. 41.5%, P=0.16), nor the
proportion of net hepatic urea release recycled to the gut (46 vs. 53% for E- and C, P=0.14). In the
liver, net total AA removal represented 73 and 74% of net portal appearance of AA for the C and
E- diets, and was reduced in the N- diet (-39%, P=0.002). Consequently, net AA TSP emission did
not differ between the three diets (P=0.51). Interestingly, the potential ammonia contribution to net
urea hepatic release was 89 and 87% with C and N- respectively, and 71% with E- (P=0.03) due to
an increased AA catabolism.

Table 1. Whole body nitrogen balance and net splanchnic fluxes of nitrogenous compounds in growing
lambs fed control, protein (N-) and energy (E-) deficient diets.
Experimental diets SEM P Contrast
C N- E- C vs. N C vs.
E
Nitrogen, g/d Intake 23.3 18.7 20.4 0.80 0.01 0.004 0.04
Fecal 7.1 6.3 5.7 0.25 0.02 0.06 0.01
Urinary 8.1 5.1 9.7 0.28 0.001 0.001 0.01
Retained 8.1 7.3 5.0 0.51 0.01 0.28 0.003
Net fluxes g N/d
Portal Ammonia 9.65 5.66 7.61 0.51 0.002 0.001 0.02
Urea -5.75 -4.72 -5.37 0.47 0.33 0.16 0.58
Amino acids 12.44 7.43 10.74 0.91 0.01 0.005 0.23
Hepatic Ammonia -9.66 -5.82 -8.13 0.40 0.0004 0.0001 0.03
Urea 10.92 6.90 11.57 0.46 0.0002 0.0003 0.35
Amino acids -8.99 -5.45 -8.02 0.55 0.005 0.002 0.24
Splanchnic Ammonia -0.01 -0.16 -0.52 0.21 0.28 0.63 0.13
Urea 5.17 2.18 6.20 0.38 0.001 0.001 0.09
Amino acids 3.44 1.98 2.72 0.85 0.51 0.26 0.56
It is noteworthy that AA TSP release was lower than N retention in all diets probably due to N
compounds not being accounted for (peptides, proteins) (Reynolds et al., 1992; Savary-Auzeloux
et al., 2007). Still, the relative comparison among treatments indicated that with the N- diet the
decrement in net AA TSP release (1.46 g N/d, compared with the C diet) was larger than the decrement
in N retention (0.8 g N/d), suggesting an improved efficiency of N utilisation by peripheral tissues. The
opposite was noted with the E- diet (the reduction in net AA TSP release was 0.72 g N/d whereas the
reduction in N retention was 3.1gN/d), suggesting a reduced efficiency of N utilisation by peripheral
tissues. These data indicate that the safety margin included in the nutritional N recommendations to
growing lambs may be too high. Consequently, in order to make these recommendations in ruminants
clearer, the modulation of the TSP metabolism (as shown here) as well as the adaptations of N
utilisation by peripheral tissues (probable in this study) will require further investigations.
References
Reynolds, C.K., H. Lapierre, H.F. Tyrrell, T.H. Elsasser, R.C. Staples, P. Gaudreau and P. Brazeau, 1992. Effects
of growth hormone-releasing factor and feed intake on energy metabolism in growing beef steers: net nutrient
metabolism by portal-drained viscera and liver. J. Anim. Sci. 70, 752-763.
Savary-Auzeloux, I.C., L. Majdoub, N. LeFloc’h and I. Ortigues-Marty, 2003. Effects of intraruminal propionate
supplementation on nitrogen utilisation by the portal-drained viscera, the liver and the hindlimb in lambs fed
frozen rye grass. Br. J. Nutr. 90, 939-952.
Savary-Auzeloux, I. and I. Ortigues-Marty, 2007. Can liver synthesis be affected by an imbalanced dietary supply
of energy or nitrogen in growing lambs? Int. Symp. on Energy and Protein Metab. and Nutr., Vichy, France, in
press.

Partitioning of nitrogen net fluxes across the portal-drained viscera in
sheep: effect of dietary protein rumen degradability
D. Rémond1 and C. Poncet2
1Unité de Nutrition Humaine-UMR1019, INRA de Clermont-Ferrand/Theix, 63 122 St Genès-
Champanelle, France
2Unité de Recherches sur les Herbivores, INRA de Clermont-Ferrand/Theix, 63 122 St Genès-
Champanelle, France
Introduction
Increasing dietary protein supply usually results in a greater portal absorption of amino acids, as long
as the energy supply is adequate to support rumen microbial protein synthesis (Raggio et al., 2004).
The specific effect of dietary protein rumen degradability is less documented. Initial studies failed
in demonstrating a significant effect of an increase in rumen undegradable protein (RUP) on portal
α-amino N net release in steers (Huntington, 1987). More recently, an increase in RUP content of
the diet increased the portal absorption of amino acids in lactating cows (Blouin et al., 2002), but
decreased it in steers (Han et al., 2001). The present study was aimed at determining the effect of a
shift in dietary protein digestion from the rumen to the small intestine on N transactions across the
different segments of the gut.

Four Texel wethers (58 ± 3 kg) were surgically fitted with catheters in the right ruminal vein, the
cranial mesenteric vein (upstream of the ileocaecocolic vein), the portal vein and a mesenteric artery.
Blood flow probes were implanted around the portal and mesenteric vein, and the right ruminal artery
(Rémond et al., 2003). Sheep were fed 1150 g of DM/d in two equal meals at 09.00 and 21.00 h.
The diets contained 66% chopped orchard grass hay and 34% of pelleted pea seeds. Pea seeds were
used raw (RP) and extruded (EP). The extrusion procedure was shown to decrease in vivo ruminal
digestibility of pea proteins from 80 to 47% (Poncet and Rémond, 2002). RUP accounted for 23 and
40% of dietary crude protein for RP and EP, respectively. Diets were tested according to a cross-over
design. The animals were allowed to adjust to the diet during 15 d before sampling. Blood samples
were simultaneously withdrawn through the four catheters, at 06.00, 08.00, 09.00, 10.00, 12.00,
14.00, and 16.00 h. They were collected into cold syringes, and rapidly placed on ice. Whole blood
was used for immediate analyses of packed cell volume, haemoglobin, urea and ammonia. Blood
and plasma samples were deproteinised with sulfosalicylic acid and extracts were stored frozen
(- 80 °C) for later analysis of blood free (FAA) and plasma peptide (PAA) amino acids. Laboratory
analysis and flux calculations were described previously (Rémond et al., 2003).

Rumen and PDV ammonia absorption (Table 1), as also uraemia, decreased with the EP diet.
Although urea transfer across the rumen wall was not significantly affected, the extraction rate of
arterial urea by the rumen wall was nearly doubled with extruded pea. The net balance of urea and
ammonia fluxes across the rumen wall showed the lowest value of N losses from the rumen with
the EP diet (0.9 vs. 4.3 g of N/d). Daily means of arterial FAA (2346 ± 70 mM) and PAA (272 ± 18
mM) were not affected by dietary RUP. In agreement with the increase in duodenal flow of amino
acids (+30%) observed with pea extrusion by Poncet and Rémond (2002), FAA net flux across the
MDV increased with the EP diet (+20%). Conversely to Han et al. (2001), the increase in RUP
content of the diet with extruded pea did not affect PAA net flux across the PDV. It accounted for
20 and 17% of non-protein amino acid net release into the portal vein for RP and EP, respectively.

The sum of FAA and PAA net fluxes across the PDV accounted for 40% and 47% of N intake for the
RP and EP diet respectively. All indispensable amino acid net fluxes across the PDV were greater
with the EP diet except that of lysine, for which the ratio (free + peptide) lysine net flux across the
PDV/predicted duodenal flux (estimated from Poncet and Rémond, 2002) fell from 0.88 to 0.64.
In conclusion, although portal N balance (urea + ammonia + FAA + PAA fluxes) was not affected,
the decrease in rumen protein degradability decreased N loss from the rumen, and increased amino
acid intestinal absorption and portal delivery.
Table 1. Portal-drained viscera (PDV), mesenteric-drained viscera (MDV), and rumen net flux of
nitrogen (g of N/d) in sheep (n=4) fed raw or extruded pea.
Item Raw pea Extruded pea P
Mean SE Mean SE
Ammonia PDV 21.2 2.7 16.3 2.2 0.003

MDV 4.9 0.8 3.5 0.5 0.027
Rumen 8.0 0.7 5.2 0.9 0.009
Free amino acids PDV 10.0 0.7 12.1 1.1 0.019
MDV 17.8 1.3 20.1 1.2 0.036
Rumen -1.7 0.2 -3.0 1.0 0.307
Peptide amino acids PDV 2.6 0.7 2.6 0.5 0.992
MDV 1.2 0.7 1.2 0.4 0.891
Rumen1 ND ND
Urea PDV -7.1 0.5 -6.0 0.6 0.037
MDV -2.2 0.4 -1.0 0.4 0.001
Rumen -3.7 0.5 -4.3 0.2 0.419
1ND=not different from zero.
References
Blouin, J.P., J.F. Bernier, C.K. Reynolds, G.E. Lobley, P. Dubreui and H. Lapierre, 2002. Effect of supply of metabolizable
protein on splanchnic fluxes of nutrients and hormones in lactating dairy cows. J. Dairy Sci. 85, 2618-2630.
Han, X.T., B. Xue, L.H. Hu and J.Z. Du, 2001. Effect of dietary protein degradability on net fluxes of free and peptide
amino acids across the portal-drained viscera of steers. J. Agric. Sci. Cambridge 137, 471-481.
Huntington, G.B., 1987. Net absorption from portal-drained viscera of nitrogenous compounds by beef heifers fed on
diets differing in protein solubility or degradability in the rumen. Br. J. Nutr. 57, 109-114.
Poncet, C. and D. Rémond, 2002. Rumen digestion and intestinal nutrient flows in sheep consuming pea seeds: the
effect of extrusion or chestnut tannin addition. Anim. Res. 51, 201-216.
Raggio, G., D. Pacheco, R. Berthiaume, G.E. Lobley, D. Pellerin, G. Allard, P. Dubreuil and H. Lapierre, 2004. Effect
of level of metabolizable protein on splanchnic flux of amino acids in lactating dairy cows. J. Dairy Sci. 87,
3461-3472.
Rémond, D., L. Bernard, B. Chauvau, P. Nozière and C. Poncet, 2003. Digestion and nutrient net fluxes across the
rumen, and mesenteric- and portal-drained viscera in sheep fed with fresh forage twice daily: net balance and
dynamic aspects. Br. J. Nutr. 89, 649-666.

Can liver protein synthesis be affected by an imbalanced dietary supply
of energy or nitrogen in growing lambs?
Unité de Recherche sur les Herbivores, Equipe Nutriments et Métabolismes, INRA Clermont Ferrand
Theix, 63122 Saint Genès Champanelle, France
Introduction
The aim of the present study was to measure the regulation of liver exported protein synthesis in
growing ruminants. A better understanding of the regulation of some of the major pathways of
utilisation of amino acids (AA) at the splanchnic level can give important tools to improve the
efficiency of AA supply to and utilisation by muscles (Connell et al., 1997).

Six growing lambs (41.5 ± 2.6 kg) were surgically fitted with catheters placed in the splanchnic area
(aorta, portal vein, hepatic vein) and the vena cava and an ultrasonic flow probe (portal vein), for
blood sampling, blood flow measurement and 1-13C Leu infusion respectively. Three diets made
of hay (30%) and of an experimental concentrate mix (70%) were offered for 2 wk each according
to a duplicated 3×3 Latin Square design. Diets differed by the composition of the concentrate
mix. The control diet (C) was designed to offer a balanced level of dietary nitrogen (24.6 g/d) and
metabolisable energy (ME, 14.1 MJ/d) contents for lambs to allow a growth rate of 200 g/d. The
nitrogen deficient diet (N-) and the energy deficient diet (E-) presented a 23% deficit of nitrogen
supply and a 20% deficit in ME supply in comparison to the C diet, respectively. Plasma samples
were collected from 3 to 9 h after the beginning of the 13C Leu infusion to determine plasma AA
and protein concentrations and isotopic enrichment (IE) of 13C Leu. The plasma proteins and TAA
concentrations, 13C Leu IE in plasma free Leu and plasma proteins as well as the calculation of the
fractional synthesis rate (FSR) and absolute synthesis rate (ASR) of plasma proteins were determined
as previously described (Connell et al., 1997; Raggio et al., 2007). Data were analysed by ANOVA
according to a duplicated Latin Square design.

The net hepatic flux of EAA in N- and, to a lesser extent E- was significantly decreased by comparison
with the C diet (-30% and -25%, P<0.03 respectively). These decreased net hepatic uptake of EAA
were partially associated with changes in the net portal appearance of EAA (Table 1). This was
accompanied by modifications in the partition of the EAA among the various metabolic pathways
within the liver, among which protein synthesis.
A simple index of the activity of protein synthesis within the liver is the concentration of plasma
proteins. Their arterial concentrations, whether total or specific (albumin or fibrinogen) were not
significantly modified by a 23-20% decrease in the N or ME supply (Table 1). Similar mild alterations
of the diet composition (+30% N supply) also lead to small alterations of the plasma protein
concentrations (Raggio et al., 2007). Longer-term denutrition or fasting are necessary to significantly
decrease albuminemia in sheep (Connell et al., 1997), rodents or human beings (Ruot, 2001). Because
the arterial concentration cannot show the complex interactions between the tissues and organs for
the utilisation and synthesis of exported hepatic proteins, their synthesis rates (expressed as FSR or
ASR) at the whole body level were also measured (Table 1).

Table 1. Net portal appearance (NPA) and net hepatic flux (NHF) of essential AA (EAA) (mmol/h), as
well as arterial concentrations (A conc, g/L) of total proteins, albumin and fibrinogen, and synthesis
rate (ASR (g/d), FSR (%/d)) of total proteins and in growing lambs fed C, N- and E- diets.
Diet SEM P Contrast
C N- E- C vs. N- C vs. E-
EAA NPA 15.92 11.11 13.37 1.19 0.06 0.02 0.17

NHF -6.97 -4.91 -5.23 0.46 0.03 0.01 0.03
Total protein A conc. 65.8 65.5 65.8 0.5 NS NS NS
ASR 9.77 9.12 7.28 0.26 <0.01 0.12 <0.01
FSR 7.93 7.95 6.81 0.22 0.02 0.95 0.01
Albumin A conc. 31.4 31.2 32.4 0.34 <0.1 NS <0.1
Fibrinogen A conc. 3.59 4.21 3.29 0.26 <0.1 NS NS
With the N- diet, the FSR and ASR of total proteins were not significantly modified, as already noted
by Raggio et al. (2007) where the nitrogen level in the diet did not have any effect on the synthesis
of total plasma proteins in lactating cows. Thus, the decrease in net EAA hepatic uptake observed
with the N- diet cannot be explained by changes in protein synthesis. The contribution of ASR to
this net EAA uptake would rather be increased in this case. Modifications of other pathways (protein
breakdown, ureagenesis, neoglucogenesis) (Ortigues Marty et al., 2003) can be hypothesised.
On the contrary, with the E- diet, the ASR and FSR of total proteins were significantly reduced.
Similar effects have already been shown in extreme nutritional states such as fasting in ruminants
by Connell et al. (1997). This decreased protein synthesis can explain, partially, the decreased net
hepatic flux induced by the energy deficit.
Consequently, a reduction in dietary ME supply was responsible for the reduction of FSR of the
total plasma proteins whereas a reduction of the N supply had no effect in growing lambs. These
preliminary results clearly show that studying the metabolism of total and specific plasma proteins is
essential in the understanding of the regulation of hepatic AA metabolism in ruminants, particularly
in the situation of nutritional imbalances.
References
Connell A., A.G. Calder, S.E. Anderson and G.E. Lobley, 1997. Hepatic protein synthesis in sheep: effect of intake as
monitored by use of stable-isotope-labelled glycine, leucine and phenylalanine. Br. J. Nutr. 77, 255-271.
Ortigues-Marty I., C. Obled, D. Dardevet and I. Savary-Auzeloux, 2003. Role of the liver in the regulation of energy and
protein status. In: Souffrant, W.B. (editor), Progress in Research on Energy and Protein Metabolism. Wageningen
Academic Publishers, Wageningen, The Netherlands, EAAP Publ. 109, 83-98.
Raggio, G., G.E. Lobley, R. Berthiaume, D. Pellerin, G. Allard, P. Dubreuil and H. Lapierre, 2007. Effect of protein
supply on hepatic synthesis of plasma and constitutive proteins in lactating dairy cows. J. Dairy Sci. 90, 352-
359.
Ruot, B., 2001. Synthèse des protéines de la réaction inflammatoire en réponse à l’infection. Thèse de l’Université
Blaise Pascal, Clermont-Ferrand, France.

A stimulation of protein metabolism in the whole intestine is the main
cause of the decreased nutritional value of a pectin supplemented diet
T. Pirman1, M.C. Ribeyre2, D. Rémond2, L. Mosoni2, C. Buffière2, A. Lavrenčič1, A. Pogačnik3, M.
Vrecl3, J. Salobir1 and P. Patureau Mirand2
1University of Ljubljana, Biotechnical Faculty, Zootechnical Department, Chair of Nutrition, Groblje
3, 1230 Domžale, Slovenia
2INRA Clermont-Ferrand – Theix, Human Nutrition Unit, 63122 Saint Genès Champanelle,
France
3University of Ljubljana, Veterinary Faculty, Institute for Anatomy, Histology and Embryology,
1000 Ljubljana, Slovenia
Introduction
Soluble fibres, like pectin escape digestion in the upper part of the gastrointestinal tract and are
fermented in short chain fatty acids (SCFA) by intestinal microflora in the large intestine. Pectin
has been reported to increase brush border membrane enzyme activities, to stimulate enterocyte
proliferation (Chun et al., 1989) and to alter nitrogen (N) excretion pattern (Pastuszewska et al., 2000).
Our hypothesis was that pectin would also stimulate protein metabolism (synthesis and degradation)
in intestinal tissues, and thus lower nutritional utilisation of dietary proteins.

Two 14-d experiments were performed in 48 growing rats (120 ± 5 and 157 ± 9 g of body weight
in experiments 1 and 2 respectively). In both experiments, two isocaloric and isoproteic diets (119
g and 117 g of crude protein in dry matter in Control and Pectin group, respectively) designed to
meet all their nutritional requirements were prepared, the Control diet and the Pectin diet in which a
fraction of wheat starch was replaced with pectin (8%) from citrus peel. Food intake and growth rate
were registered. The animals were killed at the end of both experiments and intestines were taken for
tissue and luminal content masses. The first experiment included a balance trial performed between
d 6 and d 11. In the second experiment, villi characteristics, in vivo intestinal tissue protein synthesis
rates in the fed state by the flooding dose method, and short chain fatty acid (SCFA) amounts in
digestive contents were determined.

In both experiments, the growth rate and dry matter intake were significantly lower in the pectin
group than in the control (Table 1). N excretion in faeces was increased by pectin feeding, which
resulted in lower apparent protein digestibility (83.0 ± 2.3 and 91.3 ± 1.1%, respectively) but urine
N excretion was significantly lowered. Nitrogen balance was lower, which was in close agreement
with the lower growth rate of the rats fed the Pectin diet, suggesting the decreased nutritional value
of this diet (Figure 1A).
Pectin fermentation in the caecum resulted in higher SCFA levels (Table 2). Its recognised trophic
effect for distal intestinal tissues caused an increase in villi size not only in those tissues, but also
in the duodenum (Figure 1B). In the whole intestine, protein absolute synthesis rates were higher
in the Pectin group than in the Control group (Table 2).

Table 1. Growth rate, food intake and whole intestine weights in both experiments.
Pectin (12) Control (12) Pectin (12) Control (12)
Food intake, g DM/d 14.1 ± 0.3** 15.8 ± 1.2 15.65 ± 0.43** 16.62 ± 0.68
Growth rate, g/d 4.57 ± 0.35** 5.24 ± 0.58 4.52 ± 0.40** 5.19 ± 0.47
Whole intestine, g/100 g BW 4.97 ± 0.88** 3.97 ± 0.33 6.64 ± 0.58*** 3.82 ± 0.15
Mean ± SD (n). DM: dry matter. Within each experiment, significantly different from control: ** P<0.01,
*** P<0.001.
** 1200 Villi height µm

12 ****
mg N / g DM intake
800
*
8 Pectin
400
**** Control
4 0
0 -400 Crypt depth µm **** **

Faeces Urine Balance Duodenum Jejunum
Figure
Figure 1. (A)1.Nitrogen
Nitrogenexcretion
excretion and
and balance;
balance. (B)
Figure 2. intestine
Small Small intestine villi characteristics.
villi characteristics.
Table 2. Caecal SCFA and intestinal protein synthesis.
Pectin (10) Control (10)
Caecal SCFA, µmoles/rat 295 ± 95**** 130 ± 25

Small intestinal protein synthesis, g/d 1.35 ± 0.15**** 0.63 ± 0.16
Large intestine protein synthesis, g/d 0.123 ± 0.030**** 0.057 ± 0.008
Mean ± SD (n). Significantly different from control: **** P<0.0001.
Whereas nitrogen loss per g of dry matter intake was not different in urine (5.11 ± 0.45 and 4.77 ±
0.52 mg for Pectin and Control rats, respectively), it was considerably higher in faeces of Pectin than
in Control rats (3.18 ± 0.42 and 1.65 ± 0.21 mg, P<0.0001). Consequently, the determinant factor of
food efficiency was faecal nitrogen loss, which was proportional to the amount of intestinal protein
synthesised (34.3 mg/g intestinal protein synthesis in the Pectin and Control rats).
Thus the apparent lower nutritional value of a pectin diet during the first 14-d appears to be the
consequence of the increase of intestinal protein turnover induced by pectin feeding resulting in an
increase in faecal endogenous N losses.
References
Chun, W., T. Bamba and S. Hosoda, 1989. Effect of pectin, a soluble dietary fiber, on functional and morphological
parameters on the small intestine in rats. Digestion 42, 22-29.
Pastuszewska, B., J. Kowalczyk and A. Ochtabinska, 2000. Dietary carbohydrates affect caecal fermentation and modify
nitrogen excretion patterns in rats. I. Studies with protein-free diets. Arch. Tierernahr. 53, 207-225.
This study is a part of the bilateral project ‘Proteus FR – 2002/7’ between Slovenia and France.

Ruminal fermentation, portal absorption and hepatic metabolism of
glycerol infused into the rumen of lactating dairy cows
N.B. Kristensen and B.M.L. Raun
Introduction
Increased availability and decreased costs of glycerol have increased the interest for using glycerol
as a feed ingredient for dairy cows. Glycerol has previously been considered as a glucogenic feed
additive, however, glucogenic effects and the relative importance of ruminal versus intermediary
pathways in metabolism of dietary glycerol have not been fully elucidated in lactating dairy cows.
The aim of the present experiment was to investigate the ruminal metabolism, portal absorption
and hepatic metabolism of glycerol following an intraruminal dose of glycerol in cows not adapted
to dietary glycerol.

Three multiparous lactating Danish Holstein cows implanted with ruminal cannulas and permanent
indwelling catheters in the hepatic portal vein, hepatic vein, mesenteric vein, and mesenteric artery
were used in the study. Cows were fed a ration containing (percent of dry matter): corn silage,
54.4; artificially dried hay, 18.1; rape seed cake, 12.4; sugar beet pulp, 12.1; urea, 1.4; minerals and
vitamins, 1.6. Samplings with no infusion (Control) and samplings with glycerol infusion (Glycerol)
were separated by at least 14 d. Continuous infusion of para-aminohippuric acid (31 ± 2 mmol/h)
into the mesenteric vein was initiated 1 h before first blood sampling. Ten sets of samples were
obtained simultaneously from the rumen, artery, portal vein, and hepatic vein -30, 30, 90, 150, 210,
300, 390, 480, 570, 660 min relative to feeding. With the Glycerol treatment, infusion of glycerol
(925 g 85% glycerol in 10 L warm tap water; 8.54 mol glycerol) was done at the time of feeding in
the morning (07.00 h). Dry matter intake and milk yield did not differ (P>0.10) between Control and
Glycerol and were on average 15 ± 1 kg dry matter/d and 22 ± 2 kg milk/d, respectively. Samplings
within d (i.e. time) were considered as repeated measures and analysed using the autoregressive
order 1 structure in the Mixed Procedure of SAS. The model included the effects of treatment (Trt),
cow, sampling time (Time), and the interaction between treatment and sampling time. The cow ×
treatment interaction was designated as a random effect.

All ruminal variables were affected by sampling time (P<0.01; Table 1). Interactions between
treatment and sampling time were observed for ruminal pH as well as ruminal concentrations of
glycerol, acetate, and butyrate (P≤0.05). The zenith for glycerol concentration (97 ± 10 mM) was
observed 30 min after dosing. The observed interactions reflected decreased molar proportion of
ruminal acetate and increased molar proportion of ruminal butyrate with Glycerol. The total ruminal
concentration of volatile fatty acids (VFA) and the molar proportion of propionate were not affected
by treatment (P>0.10).
The arterial concentration of glycerol increased (treatment by time, P=0.02) with Glycerol, the
zenith was observed 30 min after dosing. The arterial concentration profile reflected the ruminal
concentration profile, however, the arterial response was relatively small since the highest observed
arterial concentration was 0.25 ± 0.10 mM. A treatment by time effect was observed for arterial
glucose (P<0.01) reflecting a reduced glucose concentration with Glycerol from approximately 2.5
h after dosing. The arterial concentration of insulin was not affected by treatment (P=0.91)

Table 1. Mean concentrations of metabolites in ruminal fluid and arterial plasma as well as net
portal, net hepatic, and net splanchnic flux of glycerol in lactating dairy cows.
Item Treatment1 SEM2 P
Control Glycerol Trt Time Trt×Time
Ruminal variables
pH 6.56 6.50 0.04 0.25 <0.01 0.02
Glycerol, mM <0.1 17.4 2 <0.01 <0.01 <0.01
Total VFA, mM 99 91 3 0.18 <0.01 0.32
Acetate, mol/100 mol 0.65 0.61 0.01 0.18 <0.01 0.05
Propionate, mol/100 mol 0.22 0.23 <0.01 0.24 <0.01 0.76
Butyrate, mol/100 mol 0.10 0.12 0.01 0.10 <0.01 <0.01
Arterial variables
Glycerol, mM 0.004 0.038 0.01 0.18 0.01 0.02
Glucose, mM 4.05 3.85 0.07 0.33 <0.01 <0.01
Insulin, pM 88 87 30 0.91 0.14 0.33
Net portal flux
Glycerol, mmol/h 1 70 17 0.04 <0.01 <0.01
Net hepatic flux
Glycerol, mmol/h -5 -60 10 0.03 <0.01 <0.01
Net splanchnic flux
Glycerol, mmol/h -4 10 8 0.30 0.19 0.25
1Treaments were no infusion (Control) or infusion of 925 g 85% glycerol (Glycerol) at time of feeding in the
morning.
2Standard error of the mean (n=3).
The net portal flux of glycerol increased (treatment by time interaction; P<0.01) with Glycerol and
accounted for 10 ± 3% of the glycerol dose. The net hepatic uptake of glycerol increased (treatment
by time interaction; P<0.01) with Glycerol and the increased hepatic uptake accounted for 8 ± 2%
of the dose. The net splanchnic flux of glycerol was not affected by treatment (P>0.10), however,
data indicated a numerical shift from a net splanchnic uptake of glycerol with Control to a net output
of glycerol with Glycerol.
Conclusion
Intraruminal metabolism dominated compared with intermediary metabolism of glycerol in dairy
cows dosed with glycerol in the rumen. Glycerol dosing decreased the plasma concentration of
glucose and did not affect the plasma concentration of insulin.

Identification of a vacuolar H+-ATPase as a new energy consuming
mechanism in rumen epithelium of sheep and cattle
M. Schweigel1, K.S. Heipertz2, M. Kolisek2, W. Jähme1, E. Albrecht1 and R. Zitnan3
1Research Institute for the Biology of Farm Animals (FBN), Wilhelm-Stahl-Allee 2, 18196,
2Free University Berlin, Oertzenweg 19b, 14163, Berlin, Germany
3Research Institute of Animal Production, Rastislavova 48, 04001, Kosice, Slovakia
Introduction
Ruminal transport processes are long known to be energised by a Na+/K+-ATPase, which contributes
substantially to the tissue energy expenditure. Recently we identified a second active transport
mechanism namely a vacuolar-type H+-adenosine triphosphatase (vH+-ATPase) to be existent in
ruminal epithelial cells, REC (Etschmann et al., 2006). The expression, localisation and activity of
vH+-ATPase in rumen epithelium and in isolated REC were explored in the present study to clarify
its possible role in the functional adaptation (growth and transport capacity) of the epithelium to
various feeding levels.

Ovine and bovine REC were isolated by fractional trypsination as described by Galfi et al. (1980).
Protein and RNA samples of freshly isolated or cultured REC were prepared using commercial kits
(MEM-Per, Pierce; NucleoSpin RNA II, Macherey-Nagel) and analysed for vH+-ATPase abundance
by the use of Western blotting and RT-PCR as described by Etschmann et al. (2006). The primer
sets of subunits E and B of vH+-ATPase were 5’-gccaatgagaaagcaga-3’ / 5’-ggtccagccgactttcg-3’
and 5’-gaggagatgattcagactgg-3’ / 5’-ttcatggcttgtacatcctt-3’, respectively. For immunocytochemistry,
complete rumen papillae were isolated with scissors, snap-frozen in liquid nitrogen and stored at -80
°C until cryosectioning. In both Western blot and immunofluorescence studies, a monoclonal mouse
antibody (13D11-B2, Molecular Probes) directed against the yeast vH+-ATPase 60-kDa subunit was
used to detect the vH+-ATPase protein. In parallel experiments, the Na+/K+-ATPase expression was
investigated using a monoclonal mouse anti-sheep Na+/K+-ATPase antibody (MA3-928; ABR Affinity
BioReagents, Golden). The functional activity of the vH+-ATPase was determined by measuring the
intracellular pH (pHi) of isolated REC in the absence or presence of the specific vH+-ATPase inhibitor
foliomycin. This was done by the use of the H+ sensitive fluorescence probe BCECF. Ouabain was
used to differentiate vH+-ATPase and Na+/K+-ATPase related effects.

RT-PCR and immunoblot of REC vH+-ATPase
The mRNA transcripts of the E and B subunits of vH+-ATPase were detectable in RNA from freshly
isolated and primary cultured REC. By Western blot analyses, the expected 60-kDa immunoreactive
band was detected in protein lysates of ovine and bovine REC proving the presence of the subunit
B of the vH+-ATPase. In addition, the anti-Na+/K+-ATPase antibody labels a 110- to 120-kDa
protein representing the α1 subunit of the protein. Parallel determinations of the relative abundance
of vH+- and Na+/K+-ATPases in bovine REC demonstrate an about 12-fold stronger expression of
the latter protein.

Localisation of REC vH+-ATPase by immunocytochemistry
Immunostaining clearly confirmed the presence of a vH+- and Na+/K+-ATPase in the rumen
epithelium. In rumen papillae, obtained from high yielding dairy cows, both proteins were localised
in the plasma membrane. In contrast, dominant cytoplasmatic staining of vH+-ATPase has been
observed in rumen tissue from animals at lower performance and feeding levels.
Effect of vH+- and Na+/K+-ATPase inhibitors on the pHi of REC
Application of the vH+- and Na+/K+-ATPase inhibitors foliomycin (2 µM/L) and/or ouabain (500
µM/L) induced a significant pHi decrease in freshly isolated bovine rumen epithelial cells. Figure
1 gives a summary of the results. At the end of the 10-min measuring period, the pHi difference
between control and ouabain- or foliomycin-treated REC amounted to -0.15 ± 0.07 and -0.11 ± 0.05
pH units, respectively. A combined application of both inhibitors showed that the inhibitory effect
was additive (-0.26 ± 0.03 pH units).
0.0
*
*
change of pHi (pH units)
-0.1
-0.2
*
-0.3 ouabain +
ouabain foliomycin foliomycin
Figure 1. Effects of the vH+-ATPase inhibitor foliomycin (2 µM/L) and the Na+/K+-ATPase inhibitor
ouabain (500 µM/L) on the pHi of freshly isolated bovine REC. The mean pHi change from pHi
measured in control medium (HEPES-buffered NaCl-solution with 20 mM butyrate) without inhibitors
was calculated. Bars represent means ± SE of 4 single experiments. *P<0.05 vs. control.
Figure 1. Effects of the vH+-ATPase inhibitor foliomycin (2 µM/L) and the Na+/K
Conclusion
inhibitor ouabain (500 µM/L) on the pHi of freshly isolated bovine REC. The mea
change
The results fromthepH
demonstrate i measured
presence in control
of a functionally medium
active (HEPES-buffered
vH+-ATPase NaCl-solution w
in REC cell membrane
butyrate)
and suggest without
an important role ofinhibitors was
the enzyme for calculated.
feed-induced Bars represent
adaptation means
of forestomach ± SE of 4 singl
function.
experiments. *P<0.05 vs control.
References
Etschmann, B., K.S. Heipertz, A. von der Schulenburg and M. Schweigel, 2006. A vH+-ATPase is present in cultured
sheep ruminal epithelial cells. Am. J. Physiol. Gastrointest. Liver Physiol. 291, G1171-G1179.
Galfi, P., S. Neogrady and F. Kutas, 1980. Culture of ruminal epithelial cells from bovine ruminal mucosa. Vet. Res.
Commun. 4, 295-300.
This study was supported by the Deutsche Forschungsgemeinschaft (M. Schweigel, Schw642/5).

4A. Regulations by nutrients, hormones and nervous system

Reduced insulin responses by asynchronous protein and lactose intake in
veal calves despite high plasma glucose levels
T. Vicari1, J.J.G.C. van den Borne2, W.J.J. Gerrits2, Y. Zbinden1 and J.W. Blum1
1Veterinary Physiology, Vetsuisse Faculty, University of Bern, CH-3012 Bern, Switzerland
2Animal Nutrition Group, Wageningen University, NL-6700 AH Wageningen, The Netherlands
Introduction
Heavy veal calves often exhibit postprandial hyperglycemia, glucosuria and insulin (I) resistance if
the total milk or milk replacer is fed in a small number of daily meals (Blum and Hammon, 1999).
I resistance can be associated with decreased protein (P) and fat deposition. Nutrient asynchrony
by concentrating lactose (L) in one and P in the other daily meal did not decrease performance,
but reduced heat production and consequently increased fat retention without affecting P retention
in veal calves (Van den Borne et al., 2006). The current study was aimed at investigating the
underlying endocrine mechanisms that mediate the effects of asynchronic L and P supply on nutrient
partitioning and at assessing the effects of nutrient asynchrony on glucose (G) homeostasis in heavy
veal calves.

Holstein-Friesian calves (n=36) were fed a milk replacer at 06.00 and 18.00 h by bucket. Daily
nutrient intakes and amounts of fat for each meal were identical, but P and L intakes were gradually
separated over two daily meals. Calves were assigned to one of six degrees of nutrient synchrony
(SYN 1-6; 6 calves/treatment). They were fed a P-rich (P-)meal and an L-rich (L-)meal at 06.00
and 18.00 h, respectively (sequence A), or vice versa (sequence B). Protein/lactose ratios at SYN
1, 2, 3, 4, 5 and 6 in sequence A were 50/50, 57/44, 64/38, 71/32, 78/26 and 85/20, respectively,
and in sequence B were 50/50, 43/56, 36/62, 29/68, 22/74 and 15/80, respectively P and L were
iso-energetically exchanged between the two daily meals from SYN 1 to 6. Calves were adapted to
the treatment for 28 d, followed by a balance period of 8 d. Blood samples were collected every h
during 24 h through a jugular vein catheter on d 5 of the balance period. Urine was collected during
the complete balance period. Metabolites were measured by enzymatic assays, hormones by specific
radioimmunoassays. The 24-h means and the integrated 5-h postprandial changes of metabolites and
hormones were evaluated by calculation of areas under concentration curves (AUC).

Nutrient intake was similar across treatments. The digestibility of dry matter, P, L and energy of
calves at SYN 5 and SYN 6 were, however, lower than those of calves at SYN 1-4 (Van den Borne
et al., 2006). Because the goal was to compare degrees of nutrient asynchrony at identical intakes
of digestible nutrients, regression analyses were performed separately for SYN 1-6 and SYN 1-4.
Mean body weights at the end of the experiments (152 kg) and average daily gains (1.20 kg) were
not affected by nutrient asynchrony.
Plasma G concentrations transiently increased after feeding, AUC were similar after P- and L-meals
for SYN 1, but mean 24-h G concentrations and AUC increased along with nutrient asynchrony
after the L-meal, whereas AUC decreased with increasing nutrient asynchrony after the P-meal, as
expected. The 24-h urinary G excretion increased for SYN 1-6, but was unaffected for SYN 1-4.
Mean 24-h I concentrations decreased with increasing nutrient asynchrony. Postprandial I responses
(AUC) decreased with increasing nutrient asynchrony after both P- and L-meals (Figure 1). Plasma
I responses decreased with increasing nutrient asynchrony, demonstrating that a synchronous

provision of P and L in a meal was needed to induce high I responses. Possible explanations for the
low I response with increasing nutrient asynchrony include a reduced pancreatic I secretion and
an increased I clearance rate. The 24-h I/G ratios decreased with increasing nutrient asynchrony,
which may be associated with enhanced I sensitivity and (or) responsiveness (Mari et al., 2005).
This possibly contributed to enhanced fat retention with increasing nutrient asynchrony in SYN 1-4.
Mean 24-h non-esterified fatty acid concentrations and AUC0-5h were similar for P- and L-meals.
Urea concentrations decreased after the L-meal, whereas they increased after the P-meal. Although
within-d variation in plasma urea concentrations increased along with separation of L and P intake,
24-h urea concentrations were unaffected. This corresponded with the unaffected P retention (Van
den Borne et al., 2006). Interestingly, nutrient asynchrony did not affect plasma concentrations of
insulin-like growth factor-1, glucagon, growth hormone, leptin, 3.5.3´-triiodothyronine and thyroxine.
In conclusion, separation of P and L intake over meals affected postprandial responses of urea, G and
I, and inhibited I responses despite high plasma G concentrations after high L and low P meals.
A B
Figure 1. Plasma glucose (-----) and insulin (——) concentrations after a high protein (□) and a
high lactose (●) meal in synchronously (Left: 50% of daily protein and 50% of daily lactose in each
meal) and asynchronously fed veal calves (Right: 85% of daily protein and 20% of daily lactose in
the high protein meal and the remainder in the high lactose meal).
References
Blum, J. W. and H.M. Hammon, 1999. Endocrine and metabolic aspects in milk-fed calves. Domest. Anim. Endocrinol.
17, 219-230.
Mari, A., B. Ahren and G. Pacini, 2005. Assessment of insulin secretion in relation to insulin resistance. Curr. Opin.
Clin. Nutr. Metab. Care 8, 529-33.
Van den Borne, J.J.G.C., M.W.A. Verstegen, S.J.J. Alferink, F.H.M. van Ass and W.J.J. Gerrits, 2006. Synchronizing
the availability of amino acids and glucose decreases fat retention in heavy preruminant calves. J. Nutr. 36,
2181-2187.

Modulation of adipose tissue metabolism in periparturient dairy cattle
through pre partum administration of thiazolidinediones
T.R. Overton and K.L. Smith
Department of Animal Science, Cornell University, Ithaca, NY 14853 USA
Introduction
Thiazolidinediones (TZD) are potent ligands for Peroxisome Proliferator-Activated Receptor gamma
(PPAR-γ) in mammalian species, and have been shown to modulate responses of fatty acid and
glucose metabolism to insulin in several species (Houseknecht et al., 2002). Research conducted
in our laboratory several yr ago suggested that adipose tissue in periparturient dairy cows may be
more refractory to insulin during the pre partum period than during the post partum period (Smith,
2004). We hypothesized that this reduction in response of nonesterified fatty acid (NEFA) release
to insulin in adipose tissue in pre partum cows was in part responsible for the large increase in
circulating NEFA as cows approach parturition, which might contribute to the dramatic decrease in
dry matter intake (DMI) that also occurs as cows approach parturition (Ingvartsen and Andersen,
2000). Given that PPAR-γ is expressed in bovine adipose tissue and not skeletal muscle (Sundvold
et al., 1997), we hypothesized that activation of PPAR-γ by TZD administration to dairy cows during
the pre partum period would decrease circulating NEFA and potentially increase DMI during the
periparturient period without compromising the important homeorhetic adaptations that occur in
insulin-dependent glucose utilization in skeletal muscle (Bell, 1995).

All procedures involving animals were approved by the Cornell University Institutional Animal
Care and Use Committee prior to the onset of the experiment in September 2005. Holstein dairy
cows (n=9) entering second or greater lactation were used in this experiment. Beginning at 25 d
before expected parturition, cows were administered either saline (control; n=5) or TZD (n=4; 2
mg/kg of body weight; Sigma Chemical Co., St. Louis, MO) via a jugular catheter. Treatments
were administered once daily until parturition and ceased upon parturition. Cows were fed a pre
partum total mixed ration limited to 130% of their predicted energy requirement and a separate total
mixed ration for ad libitum intake during the post partum period (calving through d 8 post partum).
Blood was sampled daily throughout the experimental period and cows were subjected to insulin
challenges (0.8 and 2.4 μg/kg of body weight) followed by serial blood sampling on d 10 and 9 before
expected parturition and on d 6 and 7 post partum. Data were subjected to analysis of variance using
the MIXED procedure of SAS (2001) with repeated measures where appropriate. Data collected
before assignment to treatment for plasma variables and DMI were used as covariates. Statistical
significance was declared at P<0.05 and trends were discussed at 0.05<P<0.10.
Results
Cows administered TZD during the pre partum period tended (P<0.06) to have decreased peripartal
NEFA concentrations (Figure 1) and tended (P<0.08) to have increased DMI during the peripartal
period (Figure 2). Insulin-dependent glucose utilization was not affected by treatment (data not
shown) during either the pre partum or post partum periods, suggesting that TZD effects were specific
to adipose tissue. Overall, the results suggest that pre partum TZD administration may change the
dynamics of NEFA and DMI in periparturient dairy cows.

Figure 1. Least squares means and standard errors for peripartal concentrations of plasma NEFA
for cows administered TZD or saline during the pre partum period.
Figure 2. Least squares means and standard errors for peripartal DMI of cows administered TZD
or saline during the pre partum period.
References
Bell, A.W., 1995. Regulation of organic nutrient metabolism during transition from late pregnancy to early lactation.
J. Anim. Sci. 73, 2804-2819.
Houseknecht, K.L., B.M. Cole and P.J. Steele, 2002. Peroxisome proliferator-activated receptor gamma (PPARgamma)
and its ligands: a review. Domest. Anim. Endocrinol. 22, 1-23.
Ingvartsen, K.L. and J.B. Andersen, 2000. Integration of metabolism and intake regulation: a review focusing on
periparturient animals. J. Dairy Sci. 83, 1573-1597.
SAS, 2001. User’s Guide: Statistics, Version 8 edition. SAS Inst. Inc., Cary, NC.
Smith, K.L., 2004. Effects of pre partum carbohydrate source and chromium supplementation in dairy cows during
the periparturient period. MS Thesis, Cornell Univ., Ithaca, NY.
Sundvold, H., A. Brzozowska and S. Lien, 1997. Characterisation of bovine peroxisome proliferator-activated receptors
gamma 1 and gamma 2: genetic mapping and differential expression of the two isoforms. Biochem. Biophys.
Res. Commun. 239, 857-861.

Glucagon-like peptide 2 inhibits intestinal lysosomal proteolysis and
improves small intestinal recovery in refed starved rats
A. Codran1, S.E. Samuels2, S. Ventadour1, A. Claustre1, M.-P. Roux1, D. Taillandier1, D. Béchet1,
D. Attaix1 and L. Combaret1
1Human Nutrition Research Center, Clermont-Ferrand, France, and Human Nutrition Unit, French
National Institute for Agricultural Research, Ceyrat, France
2Food, Nutrition and Health, University of British Columbia, Vancouver, BC, Canada
Introduction
The small intestine protein mass is highly dynamic and extensively sensitive to stress. This organ
is rapidly wasted in response to a number of catabolic stimuli but also has the ability to rapidly
regenerate. Most studies have shown that protein synthesis in the small intestine of starved rats
either slightly decreased or did not change (Burrin et al., 1991) implying that increased degradation
plays a major role in small intestinal atrophy. However, the relative role of the different proteolytic
pathways (i.e. lysosomal and ubiquitin-proteasome-dependent proteolytic systems) in the small
intestine is poorly defined.
Moreover, the small intestine is responsible for the digestion and absorption of nutrients, serves
as a physical barrier to pathogens and bacteria, and is involved in mucous secretion and immune
function (Mc Burney et al., 1994). Compromises to the integrity and function of the small intestine
may interfere with these processes and may also have an impact on nutritional, immune and health
status. Our aim was to study intestinal proteolysis and the role of the trophic factor glucagon-like
peptide-2 (GLP-2) on small intestinal recovery following fasting.

Young male Wistar rats (50-60 g) were randomly divided into control and GLP-2 treated groups.
Animals received a subcutaneous injection of saline or GLP-2 twice a d (40 µg/kg BW/d) from d
0 until the end of protocol. At d 4, animals were either fed or starved for the next 2 d. For recovery
studies, rats were refed for 6 or 24 h after the 2 d of starvation. At the end of the protocol, animals
were anesthetized and sacrificed. Small intestine and jejunum were weighed. Fragments of the
jejunum were used to determine peptidase activities (proteasome and cathepsins), and for histology
and morphometric measurements (villus height and crypt depth). A two-way ANOVA and an ‘a
posteriori PLSD Fisher’ test were performed to assess statistical differences (P<0.05).
Results
Starvation induced a strong body weight loss in both untreated and GLP-2 treated groups (-25% and
-22%, respectively). GLP-2 treatment limited small intestinal atrophy and the reduction in villus
height and crypt depth in the jejunum from starved rats. In addition, GLP-2 blocked the increased
fasting-induced cathepsin B activity in the jejunum without any effect on increased proteasome
activities (Table 1).
Table 2 showed that GLP-2 treatment improved the recovery of small intestinal mass in refed starved
animals. The small intestine completely recovered after 24 h of refeeding in GLP-2 treated rats, but
was still atrophied by 27% (P<0.0001) in saline injected rats.

Table 1. Effect of GLP-2 on cathepsin B activity in the jejunum.
Cathepsin B activity Chymotrypsin-like activity
Saline GLP-2 treated Saline GLP-2 treated
Fed 100 ± 8ab 89 ± 12a 100 ± 7a 124 ± 7a

Starved 125 ± 10b 90 ± 10a 198 ± 20b 189 ± 11b
Data are % of saline-injected fed rats and are expressed as means ± SEM. Numbers within a column with different
letters are significantly different (P<0.05).
Table 2. Effect of GLP-2 on small intestinal recovery.
Small intestine mass, % of fed animals
Saline GLP-2 treated
Fed 100 ± 4a 101 ± 2a

Starved 63 ± 3b 78 ± 3b,*
Refed 6 h 68 ± 1bc 85 ± 3bc,*
Refed 24 h 73 ± 4c 93 ± 4ac,*
Data are % of saline-injected fed rats and are expressed as means ± SEM. Numbers within a column with different
letters are significantly different (P<0.05). * P<0.05 vs. saline-injected rats.
Conclusion
We have shown that intestinal atrophy induced by fasting correlated with increased 20S proteasome and
cathepsin activities in the jejunum. The results suggest that both lysosomal and ubiquitin-proteasome-
dependent proteolysis are involved in the regulation of small intestinal protein metabolism. This
study demonstrated that GLP-2 treatment can efficiently reduce the deleterious effects of fasting in
the small intestine, and more precisely in the jejunum. This is of obvious interest since this tissue
atrophies during catabolic conditions and contributes to the cachexia. This work should contribute
to develop nutritional and/or hormonal strategies for limiting intestinal atrophy and/or accelerating
the recovery of this organ in clinical settings.
References
Burrin, D.G., T.A. Davis, M.L. Fiorotto and P.J. Reeds, 1991. Stage of development and fasting affect protein synthetic
activity in the gastrointestinal tissues of suckling rats. J. Nutr. 121, 1099-1108.
McBurney, M.I., 1994. The gut: central organ in nutrient requirements and metabolism. Can. J. Physiol. Pharmacol.
72, 260-265.

Postprandial blood hormone and metabolite responses influenced by
feeding frequency and feeding level in heavy veal calves
T. Vicari1, J.J.G.C. van den Borne2, W.J.J. Gerrits2, Y. Zbinden1 and J.W. Blum1
1Veterinary Physiology, Vetsuisse Faculty, University of Bern, CH-3012 Bern, Switzerland
2Animal Nutrition Group, Wageningen University, NL-6700 AH Wageningen, The Netherlands
Introduction
It is known that heavy veal calves frequently have postprandial problems with glucose (G) homeostasis
(hyperglycemia, glucosuria, hyperinsulinemia) and insulin (I) resistance (Hostettler-Allen et al., 1994;
Hugi et al. 1997, 1998; Blum and Hammon, 1999). Feeding frequency (FF) and feeding level (FL)
influence calf performance and health. It is common practice to feed veal calves twice daily at a high
FL. Kaufhold et al. (2000) showed that increasing the FF reduced postprandial G and I responses
in heavy milk-fed calves. They did, however, use skimmed milk powder as the sole protein source.
Producers of milk replacers are increasingly using non-clotting protein sources, for example, whey
and soluble wheat and soy proteins, leading to more transient absorption rates of amino acids, fatty
acids. G. Van den Borne et al. (2006) investigated the effect of increasing FF and FL on protein
and fat retention, using whey proteins as the only protein source. They revealed that increasing FF
increased both nitrogen (N) and fat retention. The present paper deals with postprandial endocrine and
metabolite responses of the calves in the study of Van den Borne et al. (2006). It was hypothesized
that an increase in FF decreases problems with G homeostasis more at a high than at a low FL in
heavy veal calves. In addition, it was hypothesized that the greater protein and fat deposition with
increasing FF in heavy veal calves are associated and mediated by endocrine changes.

Effects of FF and FL on pre- and postprandial hormone and metabolite concentrations were studied
in 15 heavy veal calves (body weight >100 kg) fed 1× (FF1; at 12.00 h), 2× (FF2; at 12.00 and
24.00 h) or 4× daily (FF4; at 06.00, 12.00, 18.00 and 24.00 h) as described by Van den Borne et
al. (2006). The adaptation period lasted for 28 d, followed by the experimental (balance) period of
14 d. Blood samples were collected during the last 2 d of the experimental period. Each calf was
studied in 2 periods (P1, P2). In P1, all calves were fed at a low FL (FLlow; 1.5 × metabolisable
energy requirements for maintenance, MEm). In P2, FF2 and FF4 calves were fed at a high FL
(FLhigh; 2.5 × MEm), although FF1 calves remained at FLlow. Blood was sampled every 30 min
from 12.00 to 18.00 h from a jugular vein. Postprandial integrated plasma hormone and metabolite
concentrations (AUC12-18h) were calculated and for growth hormone (GH) the pulsatile secretory
pattern was evaluated. For 3.5.3’-triiodothyronine (T3) and thyroxine (T4), plasma samples from
10.30-18.00 h were pooled. Metabolites were measured enzymatically and hormones by specific
radioimmunoassays.
Results
Concentrations of plasma G, non-esterified fatty acids (NEFA), urea, I, glucagon, and leptin changed
significantly after feed intake, whereas concentrations of insulin-like growth factor-1 (IGF-1)
remained stable and those of GH did not change in a consistent manner. AUC12-18h of G increased
with increasing FL, but decreased with increasing FF; AUC12-18h of urea increased with increasing
FL, but was unaffected by FF; AUC12-18h of NEFA was unaffected by both FL and FF. AUC12-18h of
I decreased with increasing FF and increased with increasing FL. AUC12-18h of glucagon increased
with increasing FL and decreasing FF. AUC12-18h of GH decreased, whereas AUC12-18h of IGF-1
and leptin increased with increasing FL, but neither GH, IGF-1 and leptin AUC12-18h were affected

by FF. There were no significant effects of FF or FL on basal concentrations, peak amplitudes and
peak frequencies of GH. Mean plasma concentrations of T3 and T4 were enhanced by increasing FF
and FL (P<0.01) and were higher in FF4 at FLhigh than at FLlow. There were no FF × FL interactions,
except for plasma glucose (smaller AUC12-18h at FLhigh at FF4 than at FF2). Plasma I/G ratios were
greater at FLhigh than at FLlow and decreased with increasing FF.
Discussion
The present study was performed under conditions in which enhanced FF did not affect dry matter
intake, gross energy intake and N intake, but positively affected energy retention and N retention
(Van den Borne et al., 2006). An increase in FL increased average daily gain, N and energy retention.
The study showed that metabolic and endocrine factors were differently affected by FF and FL.
Except for plasma G, there were no FF × FL interactions, suggesting separate effects of FF and FL.
An increase in FF decreased postprandial plasma concentrations of G and I, and resulted in lower
G/I ratios, which indicates enhanced I sensitivity and (or) responsiveness and therefore may explain
the increase in growth performance. Overloading of G metabolism (hyperglycemia) and excessive
postprandial I responses and I resistance can be prevented by decreasing FL and (or) increasing FF
in heavy veal calves.
References
Blum, J.W. and H.M. Hammon, 1999. Endocrine and metabolic aspects in milk-fed calves. Domest. Anim. Endocrinol.
17, 219-230.
Hostettler-Allen, R.L., L. Tappy and J.W. Blum, 1994. Insulin resistance, hyperglycemia, and glucosuria in intensively
milk-fed calves. J. Anim. Sci. 72, 160-173.
Hugi, D.P., R.M. Bruckmaier and J.W. Blum, 1997. Insulin resistance, hyperglycemia, glucosuria, and galactosuria in
intensively milk-fed calves: dependency on age and effects of high lactose intake. J. Anim. Sci. 75, 469-482.
Hugi, D., L. Tappy, H. Sauerwein, R.M. Bruckmaier and J.W. Blum, 1998. Insulin-dependent glucose utilization and
kinetics in intensively milk-fed veal calves is modulated by supplemental lactose in an age-dependent manner.
J. Nutr. 128, 1023-1030.
Kaufhold, J.N., H.M. Hammon, R.M. Bruckmaier, B.H. Breier and J.W. Blum, 2000. Postprandial metabolism and
endocrine status in veal calves fed at different feeding frequencies. J. Dairy Sci. 83, 2480-2490.
Van den Borne, J.J.G.C., M.W.A. Verstegen, S.J.J. Alferink, R.M.M. Giebels and W.J.J. Gerrits, 2006. Effects of feeding
frequency and feeding level on nutrient utilization in heavy preruminant calves. J. Dairy Sci. 83, 3578-3586.

The role of feeding regimens in regulating metabolism of broiler
breeders: hepatic lipid metabolism, plasma hormones and metabolites
M. de Beer1, R.W. Rosebrough2, B.A. Russell2, S.M. Poch2, M.P. Richards2, J.P. McMurtry2, D.M.
Brocht2 and C.N. Coon1
1University of Arkansas, Center of Excellence for Poultry Science, Fayetteville, Arkansas 72701,
USA
2Growth Biology Laboratory, USDA-ARS, 10200 Baltimore Ave., Beltsville, Maryland 20705
Introduction
Broiler breeders are feed restricted to reduce the occurrence of reproductive problems associated
with obesity. To improve uniformity, skip-a-day (SK) feed restriction programs are commonly used
in preference to everyday (ED) programs. While differences between restricted and ad libitum fed
birds have received much attention, less biological information is available comparing ED and SK
programs. Feed restriction can alter metabolic characteristics of the hen, such as its capacity for
lipogenesis (Richards et al., 2003). Programs like SK essentially represent a constant cycle of fasting
and refeeding, which may stimulate expression of lipogenic genes. Several authors have shown
that practical levels of feed restriction can lead to chronic stress in broiler breeders (Hocking et al.,
1996; De Jong et al., 2002). Understanding the potential effects of ED or SKIP feeding on lipogenic
gene expression, metabolic hormones and plasma metabolites may provide insight into the long
term effects of such feeding programs. The aim of the study reported here was to determine what
effect different feed restriction programs have in regulating hepatic lipid metabolism and altering
endocrine status of broiler breeder pullets.

A flock of 350 Cobb 500 breeder pullets was divided in two at 4 wk of age and fed either everyday
(ED) or skip-a-day (SKIP) from 4 to 16 wk of age. Total feed intake did not differ between the two
groups. At 112 d, 52 randomly selected pullets from the ED fed pullets, and 76 from the SKIP fed
pullets were individually caged and fed a meal of 74 g (ED) or 148 g (SKIP) of the same standard
pullet grower ration. Four pullets from each group were sacrificed at several intervals after feeding
and livers were collected, weighed and snap frozen for determination of lipogenic gene expression.
Blood samples were also collected for hormone and metabolite analysis. ANOVA and Tukey
Studentized range test were performed using JMP IN 5.1 software with significance based on testing
at P≤0.05.
Hepatic gene expression
Total RNA was isolated from livers using Trizol® reagent and quantitatively measured by noting
the OD 260/280 ratio and qualitatively by gel electrophoresis. The expression of certain regulatory
genes in metabolism (acetyl CoA carboxylase, ACC; fatty acid synthase, FAS; malic enzyme, ME;
isocitrate dehydrogenase, ICDH and aspartate aminotransferase, AAT) was determined by real-time
RT-PCR. Remaining liver portions were analyzed for enzyme activity of ME, ICDH and AAT as
well as glycogen and lipid content.
Hormone and metabolite assays
Plasma was analyzed for insulin, glucagon, insulin-like growth factor-I and II, triiodothyronine (T3)
and thyroxine (T4), corticosterone, leptin, glucose, non-esterified fatty acids (NEFA), triglycerides
and uric acid. The rate of crop emptying was also noted.

Liver weight was higher in SKIP than ED birds. Feeding caused dramatic increases in liver weight
of SKIP birds, as well as liver glycogen and lipid. Expression of ACC, FAS and ME genes were
increased in SKIP birds 12 and 24 h after feeding, with the increases in ME expression from 0 to 24
h after feeding being of the greatest magnitude. In contrast, SKIP decreased ICDH and AAT gene
expression, paralleling the findings noted in fasting-refeeding experiments conducted with much
younger birds. SKIP feeding resulted in far greater changes in gene expression compared to ED,
which was indicative of the inconsistent supply of nutrients in such regimens. Enzyme activity of
ME, ICDH and AAT was reflective of noted changes in gene expression. In ED birds the crop was
empty by 12 h and SKIP birds by 24 h after feeding. The alterations in metabolic hormone and
metabolite levels after feeding and during the subsequent fasting period were similar to those reported
by several authors in true fasting and refeeding experiments. The physiological responses to fasting,
such as increased glucagon and corticosterone, and reduced plasma triglyceride, occurred at times
coincidental with crop emptying in both ED and SKIP birds. Overall mean IGF-I levels were higher
in ED birds providing a further possible explanation for improved feed utilization in such birds. T3
was higher (P=0.09) in SKIP birds, which may explain the observed elevations in hepatic lipogenic
gene expression. Overall mean plasma corticosterone was two-fold higher in SKIP birds which may
be related to the increased length of fasting periods, hunger and stress. Plasma leptin was consistently
higher in ED fed birds which was indicative of their more consistent food supply and more stable
energy status. In summary, the experiment reported here shows that different feeding regimens can
alter gene expression, hormone and metabolite profiles, in spite of total feed intakes being equal.
References
De Jong, I.C., A. Sander Van Voorst, D.A. Ehlhardt and H.J. Blokhuis, 2002. Effects of restricted feeding on physiological
stress parameters in growing broiler breeders. Br. Poult. Sci. 43:157-168.
Hocking, P.M. M.H. Maxwell and M.A. Mitchell, 1996. Relationships between the degree of food restriction and
welfare indices in broiler breeder females. Br. Poult. Sci. 37, 263-278.
Richards, M.P., S.M. Poch, C.N. Coon, R.W. Rosebrough, C.M. Ashwell and J.P. McMurtry, 2003. Feed restriction
significantly alters lipogenic gene expression in broiler breeder chickens. J. Nutr. 133, 707-715.

The relation between circulating ghrelin and vitamin A during the
fattening period in Japanese Black steers
M. Hayashi, K. Kido and K. Hodate
National Institute of Livestock and Grassland Science, Tsukuba, Ibaraki, 305-0901 Japan
Introduction
In Japan, the market value of beef carcass depends greatly on marbling scores; meat with a high
degree of marbling score is expensive. Low serum vitamin A concentrations produce a lot of marbling
(Oka et al., 1998), but sometimes this causes a reduction of feed intake.
Anorexia is well known as a symptom of vitamin A deficiency in many species, but the mechanism
remains to be established. Wertz-Lutz et al. (2006) reported that ghrelin, a peptide hormone synthesised
by the abomasal and ruminal tissues of cattle, probably works as an appetite regulator in cattle.
The objective of this study was to establish the relationship between plasma ghrelin concentrations
and vitamin A status in Japanese Black steers.

Nine half-sib Japanese Black steers at 14 mo of age (324 kg initial body weight) were randomly
assigned into three treatments. Three steers were supplemented with 42.4 IU/kg vitamin A (S group),
three of them were restricted vitamin A (R group), and the last 3 steers were restricted until 24 mo
of age and then supplemented with 42.4 IU/kg vitamin A (RS group). Feed intake was measured
daily and body weight was recorded biweekly. Feed for 14-20 mo old steers consisted of concentrate
(75% of weight) and timothy hay (25%) as roughage (the growing phase). Concentrate (91%) and
rice straw (9%) were used for the 21-28 mo old steers (the finishing phase). Steers had a once-daily
feed offering at 09.00 h.
Blood samples in each steer were obtained every 4 wk during the experimental period before
feeding. Blood samples were added aprotinin 500 U/mL blood and then centrifuged. An amount of
0.1 M HCl equal to 10% of the blood plasma volume was added for ghrelin measurement. Ghrelin
concentrations were measured by ghrelin (Active) RIA kit (Linco). Concentrations of retinol in
plasma were determined using HPLC.
Ghrelin concentrations, vitamin A concentrations and dry matter intake (DMI) were analysed as
repeated measures in time by using the SAS MIXED procedure (Littell et al., 1998). The model
included sampling time, treatment (vitamin A supplemented vs. restricted), steer, and the interaction
of sampling time and treatment as independent variables.

Plasma vitamin A of the R groups was significantly smaller than that of the S group (P<0.05) when
steers were fed rice straw as roughage in the finishing phase. In the RS group, plasma vitamin A
concentration of the supplemented period was significantly greater than that of the restricted period
(Figure 1A).
Plasma ghrelin of the supplemented period was significantly greater than that of the restricted period
in the RS group (Figure 1B).

Figure 1. (A) Plasma vitamin A concentration. (B) Plasma ghrelin concentration.
In statistical analysis, there was a significant effect of vitamin A supplement or restricted (P<0.001),
sampling time (P<0.001), and their interaction (P<0.05) on Vitamin A concentration. There was
a significant effect of vitamin A supplement (P<0.01), and sampling time (P<0.001) on ghrelin
concentration. Vitamin A had no significant effect on DMI.
This result suggests that the supplementation of vitamin A in vitamin A restricted steers increases
plasma ghrelin levels. It is possible that no difference of ghrelin concentration in the growing phase
is because of no significant difference in plasma vitamin A concentration.
References
Oka, A., Y. Maruo, T. Miki, T. Yamasaki and T. Saito, 1998. Influence of vitamin A on the quality of beef from the
Tajima strain of Japanese black cattle. Meat Sci. 48, 159-167.
Littell R.C., P.R. Henry and C.B. Ammerman, 1998. Statistical Analysis of Repeated Measures Data Using SAS
Procedures. J. Anim. Sci. 76, 1216-1231
Wertz-Lutz, A.E., T.J. Knight, R.H. Pritchard, J.A. Daniel, J.A. Clapper, A.J. Smart, A. Trenkle and D.C. Beitz, 2006.
Circulating ghrelin concentrations fluctuate relative to nutritional status and influence feeding behavior in cattle.
J. Anim. Sci. 84, 3285-3300.

Effects of rumen protected choline on selected metabolites and liver
constituents in dairy goats within the first month of lactation
L. Pinotti1, A. Campagnoli1, F. D’Ambrosio1, C. Pecorini1, R. Rebucci1, D. Magistrelli2, V. Dell’Orto1
and A. Baldi1
1Department of Veterinary Sciences and Technology for Food Safety,Veterinary Faculty, University
of Milan, Via Celoria 10, 20133, Milano, Italy
2Department of Animal Science, Agricultural Faculty, University of Milan, Via Celoria 2, 20133,
Milano, Italy
Introduction
Choline has been classified as a vitamin-like compound. Two types of choline functions are known:
as choline per se, for which the choline moiety is required, and functions as a methyl donor. Choline
per se plays a major role in lipid metabolism, particularly in lipid transport, as a lipotropic agent.
Choline is also an important source of labile methyl groups for the biosynthesis of other methylated
compounds. Based on this second function choline and methionine are interchangeable, as sources
of methyl groups. According to these functions choline occupies a key position between energy and
protein metabolism. In adult ruminants, choline is extensively degraded in the rumen, and it has been
suggested that choline can be a limiting nutrient for milk production. Based on these assumptions we
investigated the effects of rumen protected choline administration on milk production and metabolic
profile during the periparturient period of dairy goats. Milk production and metabolic profile data
have been partially reported previously (Pinotti et al., 2006), while the present experiment was
undertaken to determine the effects of rumen protected choline on metabolic profile and selected
liver constituents in dairy goats within the first month of lactation.

Eight pregnant multiparous Saanen goats were assigned to one of the two following experimental
groups: CTR, control group, no choline supplementation; RPC, supplemented with 4 g/d choline
chloride in rumen-protected form (Sta-Chol 50%, Ascor Chimici, Forlì, Italy). The quantity of
choline given was based on experiments in dairy cows (Pinotti et al., 2003) and metabolic BW at the
beginning of the experiment. Treatment was administered individually before the morning feeding
to ensure complete consumption, starting from 30 d prior to the expected kidding and continued
for 30 d after parturition. During the experiment the goats were fed with a basal diet formulated
to provide ME 2.00, 2.40 Mcal/kg DM, CP 11.50%, 14.30% kg DM, for pre-kidding and lactation
phase, respectively. Lactation diet contained 1.54% methionine of metabolisable protein. Through
the first 30 d of lactation, milk yield and composition were measured weekly. Blood samples were
taken on d 0, 7, 14, 21 and 28 post partum. The plasma obtained was analysed for glucose, β-
hydroxybutyrate, non-esterified fatty acids (NEFA), and cholesterol. On d 28 in milk, liver samples
were obtained from the same animals. Hepatic tissue was analysed for total lipid (Munro and Fleck,
1969), phospholipids (Munro and Fleck, 1969) and DNA contents (Munro and Fleck, 1969). Blood
measurements were analysed using the PROC MIXED procedure of SAS. Liver lipid, DNA and
their ratio were analysed by the general linear model procedure of SAS. Differences between means
were determined by contrasts.

Milk yield, milk composition, and plasma metabolites recorded from wk 1 to 4 after kidding according
to treatment group are reported in Table 1. At comparable milk yield in CRT and RPC group during
the first mo of lactation, choline supplemented goats showed a lower (P<0.05) level of plasma β-

hydroxybutyrate goat. Liver total lipid, DNA, total lipids/DNA and phospholipids in control and
RPC goats, were as follows: total lipid 46.03 vs. 45.35 mg/g of wet tissue (P=0.89), DNA 2.89 vs.
4.10 mg/g of wet tissue (P<0.05), total lipid/DNA 15.87 vs. 11.14 (P<0.05), phospholipids 2.61 vs.
2.03 mg/g of wet tissue (P=0.30).
Table 1. Milk yield, milk composition, and plasma metabolites from wk 1 to 4 of lactation according
to treatment group.
CTR RPC SEM
Milk
Yield, kg/d 3855 3805 360
Fat, % 3.38 3.45 0.40
Protein, % 3.30 3.36 0.32
Plasma
β-hydroxybutyrate, mmol/L 0.71a 0.39b 0.18
NEFA, mmol/L 0.53 0.47 0.36
Glucose, mmol/L 3.50 3.48 0.80
Cholesterol, mmol/L 2.45 2.83 0.81
a, b Means within row with different superscript differ at P<0.05.
Therefore, although liver lipid content on 28 d in milk was unaffected by the treatment, data on DNA
seems to indicate a higher cellularity in RPC goats. Furthermore, a reduction by 35% in total lipid/
DNA ratio in choline supplemented goats suggest a lower cellular lipid accumulation, as reported
in dairy cows receiving choline (Grummer, 2006). Overall these results suggest that greater choline
availability seems to be essential for optimising metabolic health in dairy ruminants, particularly
in animals fed basal diets that limit post-ruminal methionine supply. Reduction of methyl group
demands from the methionine pool may offer production benefits to dairy ruminants that might be
achieved by nutritional manipulation other than alteration of the methionine supply. Moreover, the
role of choline as a methyl donor, does not exclude its function per se, mainly in lipid metabolism
as suggested by liver data. However, the present results are based on a limited number of goats, thus
choline needs and functions in lactating dairy ruminants deserve further investigation.
References
Grummer, R.R., 2006. Etiology, pathophysiology of fatty liver in dairy cows. In: Joshi, N.P. and T.H. Herdt (editors),
Production diseases in farm animals. Wageningen Academic Publishers, Wageningen, The Netherlands, 141-
153.
Munro, H.N. and A. Fleck, 1969. Analysis for costituents. In: Munro, H.N. (editor), Mammalian Protein Metabolism,
Volume III. Academic Press, London, UK, 484-485.
Pinotti, L., A. Baldi, I. Politis, R. Rebucci, L. Sangalli and V. Dell’Orto, 2003. Rumen protected pholine administration
to transition cows: effects on milk production and vitamin E status. J. Vet. Med. A 50, 18-21.
Pinotti L., A. Campagnoli, F. D’Ambrosio, F. Susca, G. Savoini and A. Baldi, 2006. Rumen-protected choline and
vitamin E supplementation in transition dairy goats. Rivista de Ciêcias Veterinárias 4 (1), 32.

Rare earth elements (REE) in piglets feeding – performance and thyroid
hormone status
D. Förster1, A. Berk1, H.-O. Hoppen2 and G. Flachowsky1
1Institute of Animal Nutrition, Federal Agricultural Research Centre (FAL), Bundesallee 50, D-
2Institute for Food Toxicology and Chem. Analytics, University of Veterinary Medicine Bischofsholer
Damm 15, D-30173 Hannover, Germany
Introduction
Rare earth elements (REE) comprises the elements scandium, yttrium, lanthanum and the 14 chemical
elements following lanthanum (order numbers 58 – 71) called lanthanides. REE have been used in
China in plant production and farm animal feeding to promote growth. Under Western production
conditions, it was reported that REE in the diets can improve animal performance significantly
as recently summarised by Redling (2006). Most of the experiments with piglets and pigs were
carried out with dosages between 100 and 300 mg REE mixtures per kg feed (Redling, 2006).
Higher performances were mostly accompanied by decreased thyroid hormone levels (Knebel,
2004; Schuller et al., 2002). The objective of the present study was to carry out a dose-response
experiment to measure the influence of wide-range REE-dosages on piglet performance as well as
their influences on thyroid hormone concentration.

A total of 80 piglets (40 females/40 castrated males), weaned at the age of 21 d with an average body
weight of about 7.2 kg, were divided into five groups (4 piglets in 4 boxes, 16 piglets per group).
The feed of group 1 was a cereal-soybean meal based diet that served as the control. This diet was
supplemented with 100, 200, 400 or 800 mg/kg feed (groups 2 to 5) of citrate bound REE consisting
of 30% La, 55% Ce, 5% Pr and 10% Nd. The trial duration was 35 d. Feed, in mash form, and water
were offered ad libitum. Blood samples were taken from the Vena cava cranialis at 2 times (Table 2)
and the serum was analysed for trijodothyronine (T3) and thyroxine (T4) by radioimmunoassay. Mean
comparisons were conducted by the student t-test with the SAS (2002) univariate procedure.

The daily weight gain of piglets of group 2 (100 mg REE/kg) was significantly higher (P<0.05) than
those of group 3 (200 mg REE/kg), but did not show significant differences to all other treatments
(Table 1). The animals of group 3 (200 mg REE/kg) need significant more feed per kg weight gain
than the piglets of all other groups (P<0.05, Table 1).
Efficient effects of low dosages of REE (up to 300 mg/kg) on weight gain were described by (Rambeck
et al., 1999; He et al., 2001; Borger, 2003; Knebel, 2004). In agreement with a recent study by Kratz
et al. (2006) the present study did not show such significant effects.
Both thyroid hormones showed a lower concentration in blood serum after five wk in comparison
to one wk. Apart from the T3-concentration after one wk, thyroid hormones did not show significant
differences (P>0.05) between treatments (Table 2). At present, the effects of REE-supplementation
on thyroid hormone concentration are not quite clear. On the contrary to the present study, thyroid
hormone activities decreased with higher animal performances in some cases (He et al., 2001;
Schuller et al., 2002; Eisele 2003).

In conclusion, more studies are necessary to understand the mode of action of supplemented REE
and to learn conditions for a sure reproduction of results.
Table 1. Animal performance in dependence on REE-supplementation.
Groups 1 2 3 4 5
REE, mg/kg - 100 200 400 800
Feed intake, g/d 467 ± 39 494 ± 61 461 ± 52 447 ± 33 433 ± 44

Daily weight gain, g 283ab ± 47 301ab ± 65 243b ± 101 258ab ± 34 265ab ± 71
Feed/gain ratio, kg/kg 1.67b ± 0.09 1.64b ± 0.15 1.93a ± 0.13 1.74b ± 0.10 1.64b ± 0.04
Table 2. Influence of REE-supplements on the thyroid hormone activities in serum, nmol/L.
Groups 1 2 3 4 5
REE, mg/kg - 100 200 400 800
1st wk T4 57.6 ± 5.9 56.0 ± 9.4 60.9 ± 5.5 57.0 ± 7.4 52.2 ± 13.3
T3 1.27bc ± 0.53 0.85c ± 0.16 1.49b ± 0.21 1.00bc ± 0.24 2.09a ± 0.35
5th wk T4 35.7 ± 3.4 42.2 ± 3.8 42.8 ± 6.2 42.2 ± 2.8 41.2 ± 7.3
T3 0.73 ± 0.14 1.20 ± 0.21 1.16 ± 0.64 1.20 ± 0.25 1.31 ± 0.53
References
Borger, C., 2003. Alternative Methoden in der Schweinemast. Untersuchungen zum leistungssteigernden Potential
Seltener Erden und zur Jodanreicherung im Gewebe durch die Verfütterung von Meeresalgen. Diss. Ludwig-
Maximilians-Universität München, Germany.
Eisele, N., 2003. Untersuchungen zum Einsatz Seltener Erden als Leistungsförderer beim Schwein. Diss. Ludwig-
Maximilians-Universität München, Germany.
He, M.L., D. Ranz and W.A. Rambeck, 2001. Study on performance enhancing effect of rare earth elements in growing
and fattening pigs. J. Anim. Physiol. Anim. Nutr. 85, 263-270.
Knebel, C., 2004. Untersuchungen zum Einfluss Seltener Erd-Citrate auf Leistungsparameter beim Schwein und die
ruminale Fermentation im künstlichen Pansen (RUSITEC). Diss. Ludwig-Maximilians-Universität München,
Germany.
Kraatz, M., D. Taras, K. Männer and O. Simon, 2006. Weaning pig performance and faecal microbiota with and without
in-feed addition of rare earth elements. J. Anim. Physiol. Anim. Nutr. 90, 361-368.
Rambeck, W.A., M.L. He, J. Chang, R. Arnold, R. Henkelmann and A. Süss, 1999. Possible role of rare earth elements
as growth promoters. Symposium ’Vitamine und Zusatzstoffe in der Ernährung von Mensch und Tier’, 22-23
September 1999, Jena, Germany, 311-317.
Redling K., 2006. Rare earth elements in agriculture with emphasis on animal husbandry. Diss. Ludwig-Maximilians-
Universität München, Germany.
Schuller S., C. Borger, M.L. He, R. Henkelmann, A. Jadamus, O. Simon and W. Rambeck, 2002. Untersuchungen zur
Wirkung von Seltenen Erden als mögliche Alternative zu Leistungsförderern bei Schweinen und Geflügel. Berliner
Münchner Tierärztliche Wochenschrift. 115, 16-23.

Role of the 5’AMP-activated protein kinase in reproduction: a possible
involvement in the central regulation
L. Tosca, G. Ferreira, E. Jeanpierre, S. Coyral-Castel, A. Caraty, D. Lomet, C. Chabrolle and J.
Dupont
INRA, Unité de Physiologie de la Reproduction et des Comportements, Centre de Tours, 37380
Nouzilly, France
Introduction
The 5’AMP-activated protein kinase (AMPK) is an enzyme regulating cellular energy homeostasis
(Carling et al., 1987). It allows cells to adapt to nutrient deprivation in vitro. We have recently
characterised AMPK in mammalian and avian ovary and showed that its activation by some
pharmacological agents such as 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR)
or Metformin modulated granulosa cell steroidogenesis (Tosca et al., 2005, 2006 and 2007). Thus,
AMPK could act directly at the ovarian level in order to regulate the functions of reproduction and
the interactions reproduction/nutrition. Since AMPK is present and regulates food intake in the
hypothalamus (Kim et al., 2004), we hypothesised that this kinase could also act upstream of the
gonadal axis at the central level (brain and pituitary). We checked this hypothesis by using in vivo
and in vitro approaches.

In vivo, 10 wk old female rats were submitted to brain surgery and then received an intracerebroventricular
injection of AICAR (1, 10 or 50 µg) or saline. Food intake, body weight and ovarian cyclicity were
evaluated every day during three weeks before and three weeks after the injection. Then, animals
received a last injection of AICAR 50 µg and were killed 30 min after. Different tissues (ovaries,
liver and several brain regions) were dissected in order to assess the phosphorylation levels of AMPK
on Thr172 and of Acetyl CoA Carboxylase (ACC) on Ser 79 (a well-known target of AMPK) by
western-blotting. In vitro, we determined by radioimmunoassay the effects of Metformin (1-10 mM)
on GnRH secretion for 4 h or 16 h by the hypothalamic GT1-7 cells and LH and FSH releases for
48 h by rat primary pituitary cells, and by immunoblot the levels of AMPK phosphorylation (0-2
h) in these cells.

In good agreement with other studies, AICAR (50 µg) increased food intake of rats just after the
injection (data not shown). Furthermore, the drug also decreased the interval between 2 oestrus
after the injection (Figure 1). Protein analysis by immunoblotting showed an increase in the
phosphorylation levels of AMPK and those of ACC in the hypothalamus 30 min after the injection.
AMPK activation was not detected in the other analysed tissues (frontal cortex, olfactive bulbs,
cerebellum, pituitary and ovary, data not shown). In vitro, AMPK phosphorylation was increased
after Metformin treatment (5-10 mM, 2 h) in GT1-7 and rat pituitary cells (data not shown). In GT1-7
cells, basal GnRH secretion was significantly reduced by about 20% after 16h of stimulation with
Metformin (5-10 mM) (Figure 2A). In rat pituitary cells, GnRH stimulation (10-8 M, 48 h) increased
LH and FSH secretions by about 2.5-fold and Metformin (5-10 mM, 48 h) decreased by 1.5-fold
and 2-fold GnRH (10-8 M)-stimulated LH and FSH secretions (Figure 2B).

6,5
a
6
Interval between two

a a Before injection
5,5
oestrus (days)
a After injection
5
4,5 b
4
3,5
3
Control Saline AICAR
50 µg
Figure 1. In vivo effect of an intracerebroventricular injection of AICAR (50 µg) or saline on the
interval between two oestrus in rat. Different letters indicate significant differences. (ANOVA test,
P<0.05).
A. B.
FSH (% / unstimulated stade)

LH (% / unstimulated stade)
120 b bc
(% / unstimulated state)
a a b bc
100 300 cd 300
b c
GnRH secretoin
b
80 Figure200
1 d
200
a
60 a a
40 100 100
20
0 0 0
Metformin (mM) 0 1 5 10 0 0 1 5 10 Metformin (mM) 0 0 1 5 10

_ _
+ + + + GnRH (10-8 M) + + + +
Figure 2. In vitro effects of different doses of Metformin (1-5 mM) on basal GnRH secretion by
GT1-7 cells (A) and on GnRH-stimulated LH and FSH releases by rat pituitary cells (B). Different
letters indicate significant differences. (ANOVA test, P<0.05).
Figure 2
Thus, we showed in vivo that a pharmacological activation of AMPK in the hypothalamus can
regulate ovarian cyclicity. In vitro, Metformin increased AMPK phosphorylation and decreased
basal GnRH (in GT1-7 cells) and GnRH-stimulated LH and FSH (rat pituitary cells) secretions.
Thus, AMPK could act directly at the ovarian level but also more centrally at the hypothalamus-
pituitary axis level. These data are being confirmed using an adenovirus-mediated expression of
dominant-negative AMPK.
References
Carling, D., V.A. Zammit and D.G. Hardie, 1987. A common bicyclic protein kinase cascade inactivates the regulatory
enzymes of fatty acid and cholesterol biosynthesis. FEBS Lett. 223, 217-222.
Kim, E.K., I. Miller, S. Aja, L.E. Landree, M. Pinn, J. McFadden, F.P. Kuhajda, T.H. Moran and G.V. Ronnett, 2004.
C75, a fatty acid synthase inhibitor, reduces food intake via hypothalamic AMP-activated protein kinase. J. Biol.
Chem. 279, 19970-19976.
Tosca, L., P. Froment, P. Solnais, P. Ferre, F. Foufelle and J. Dupont, 2005. Adenosine 5’-monophosphate-activated
protein kinase regulates progesterone secretion in rat granulosa cells. Endocrinology 146, 4500-4513.
Tosca, L., S. Crochet, P. Ferre, F. Foufelle, S. Tesseraud and J. Dupont, 2006. AMP-activated protein kinase activation
modulates progesterone secretion in granulosa cells from hen preovulatory follicles. J. Endocrinol. 190, 85-97.
Tosca, L., C. Chabrolle, S. Uzbekova and J. Dupont, 2007. Effects of metformin on bovine granulosa cells steroidogenesis:
possible involvement of adenosine 5’ monophosphate-activated protein kinase (AMPK). Biol. Reprod. 76, 368-
378.

Chicken liver glucokinase is activated by a glucokinase activator
N. Rideau1, A. Picard1 and J. Grimsby2
2Roche, Department of Metabolic Diseases, 340 Kingsland Street Nutley, NJ 07110-1199, USA
Introduction
In order to further understand the peculiar glucose homeostasis in chickens (high basal plasma glucose
level (2g/L) with plasma insulin level similar to mammals; relative insensitivity of the pancreatic
B-cell to glucose) we focussed our attention on glucokinase (GK), a major regulator of insulin
release and liver glucose utilisation in mammals. We recently provided evidence for the presence
of the chicken GK gene, pancreas and liver protein as well as hepatic GK-like activity (Berradi et
al., 2005). In the present study, we used a small molecule activator of mammalian GK to further
characterise and confirm the identity of hepatic chicken GK-like activity.

Animals
Male broiler chickens (Ross) randomly allocated to individual cages were fed a regular, ad libitum
balanced diet based on corn, wheat and soybean meal (11.8 MJ/kg containing ~ 37% carbohydrates
and 22% proteins). On d 38 chickens were sacrificed in the fasted (24 h) and the fed states. They
were killed by cervical dislocation. Livers were quickly removed, frozen, powdered into liquid
nitrogen and stored at –80 °C.
GK enzymatic assay
All measurements were done on 900 g supernatant from frozen chicken liver within 2 h after
extraction as described previously (Berradi et al., 2005). Enzyme activities were measured at 37 °C
by coupling ribulose-5-phosphate formation from glucose-6-phosphate to the reduction of NADP
using purified glucose-6-phosphate dehydrogenase (Sigma) as the coupling enzyme using the
Microplate reader ARGUSTM 300, Packard) and KC4 software (Biotek® instruments, Inc.). GK
activity of the crude homogenate was estimated by the standard method of subtracting the rate of
NADPH formation (at 340 nm) in the presence of 0.5 mM glucose (scoring low-Km HK activities)
from that at 100 mM glucose (scoring total HK activities including GK). Dose response curves for
chicken liver GK-like activity were established on liver homogenates from fasted (n=6) and fed
(n=5) chickens using 5 glucose concentrations (100 mM to 0.5 mM glucose final) in the absence
or the presence of GKA (RO0281675, Roche) at 4 concentrations from 1 µM to 20 µM. Apparent
substrate concentration at 0.5 Vmax (SO.5a) for glucose and apparent Vmax (Vmaxa) were calculated
using the Hanes-Woolf plot.
Results
Glucose dose response curves for chicken liver GK-like activity and the resulting S0.5a and Vmaxa
are presented respectively on Figure 1 and Table 1. GK’s Vmaxa was lower in the fasted state as
compared to the fed state in the presence and in the absence of the GKA. RO0281675 clearly activates
chicken GK activity in both the fasted and the fed states with a dose dependent effect more obvious
in the fed state as compared to the fasted state (Figure 1). RO0281675 decreased chicken GK’s S0.5a
for glucose dose dependently and increased GK’s Vmaxa maximally at 1 µM (Table 1).

None None
A (fasted state) GKA 1 µM
GKA 10 µM
B (fed state) GKA 1 µM
GKA 10 µM
GKA 20 µM GKA 20 µM
3,0 3,0
(mU/mg protéin)
(mU/mg protéin)
2,0 2,0
Glucokinase
Glucokinase
1,0 1,0
0,0 0,0
0 25 50 75 100 0 25 50 75 100
[glucose] (mM) [glucose] (mM)
Figure 1. Glucose dose-response curve for chicken liver GK-like activity in the absence (none) or
the presence of RO0281675 (GKA, 1 to 20 µM) (mean ± SEM; (A) fasted state: n= 6, (B) fed state:
n=5).
Table 1. Chicken GK S0.5a (mM glucose) and Vmaxa (mU/mg protein) as a function of RO0281675
concentration (GKA, 1 to 20 µM). S0.5a and Vmaxa are calculated according to the Hanes-Woolf
plot, (mean ± SEM, fasted n=6 and fed states n=5).
Fasted state Fed state
None GKA GKA GKA None GKA GKA GKA

1 µM 10 µM 20µM 1 µM 10 µM 20µM
S0.5a, mM 51.1 ± 7.2 18.4 ± 5.2 11.4 ± 2.8 6.2 ± 2.2 16.3 ± 4.3 19.5 ± 3.9 8.4 ± 2.3 2.5 ± 1.0
Vmax a, mU/mg 0.59 ± 0.09 1.07 ± 0.21 0.91 ± 0.19 0.71 ± 0.17 1.46 ± 0.21 3.02 ± 0.56 2.64 ± 0.44 2.19 ± 0.64
Conclusion
The glucokinase activator RO0281675 decreases chicken liver GK’s S0.5a for glucose dose-
dependently and increases its Vmaxa. It was previously reported that RO0281675 increased the
enzymatic activity of recombinant human GK in a dose dependent manner. At a concentration
of 3 µM RO0281675 increased the Vmax of GK by a factor of about 1.5 and decreased S0.5 for
glucose from 8.6 mM to 2.0 mM (Grimsby et al., 2003). Our results provide unequivocal evidence
for the presence of hepatic GK in chickens. GKA may be a useful tool to further examine the role
of glucokinase on plasma glucose and insulin levels.
References
Berradi, H., M. Taouis, S. Cassy and N. Rideau, 2005. Glucokinase in chicken (Gallus gallus). Partial cDNA cloning,
immunodetection and activity determination. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 141, 129-39.
Grimsby, J., Sarabu R., W.L. Corbett, N.E. Haynes, F.T. Bizzarro, J.W. Coffey, K.R. Guertin, D.W. Hilliard, R.F.
Kester, P.E. Mahaney, L. Marcus, L. Qi, C.L. Spence, J. Tengi, M.A. Magnuson, C.A. Chu, M.T. Dvorozniak,
F.M. Matschinsky and J.F. Grippo, 2003. Allosteric activators of glucokinase: potential role in diabetes therapy.
Science 301 (5631), 370-3.

Effects of in ovo amino acids or glucose administration on hexokinase
activity in hatching muscle of broiler embryos
Y. Ohta, Y. Furuya, I. Yoshimura, H. Furuta and M. Sugawara
Nippon Veterinary and Life Science University, 1-7-1, Kyonan-cho, Musashino-shi, Tokyo, Japan,
180-8602
Introduction
In ovo amino acid (AA) injection improves the AA and nitrogen utilisation in the broiler breeder
embryo (Ohta et al., 2001). However, the mechanisms of that improvement have never been
studied. One of the reasons might be that the injected AA affected glucose metabolism, because
the glucose derived from AA is important as the energy source to hatch and to maintain the high
blood glucose level in the Avian embryo (John et al., 1988). Therefore, in the present study, in
order to clarify the effects of in ovo AA injection on glucose metabolism, the effects of in ovo AA
or glucose administration on hexokinase (HK) activity were studied in the hatching muscles and
liver of broiler embryos.

Two experiments were conducted utilising 24 and 32 fertilised commercial broiler eggs of a Cobb
strain in Experiments 1 and 2, respectively. Eggs were incubated at 37.8 °C and 60% relative humidity.
In Experiment 1, on d 17, 18, and 19 of incubation, 6 eggs were selected randomly, crashed, and
the hatching muscles of the neck (M. complexus) and leg (all of thigh muscles), and the liver were
collected immediately. In Experiment 2, eggs were divided into four groups. The control group,
water, AA, or glucose solution injection groups with 5 eggs were prepared. A 0.5 mL of water, AA,
with an identical AA pattern as the whole egg, or glucose solution at 106 mg/mL was injected into
eggs of the water, AA and glucose injection groups on d 17 of incubation, respectively, as described
previously (Ohta et al., 1999). On d 19 of incubation, tissues were collected in the same way as
Experiment 1. Total HK and glucokinase (GK) activities were determined. Treatments were analysed
by ANOVA using the General Linear Models procedure.

The activity of HK and GK were low in all tissues, and there were no significant differences in enzyme
activities among all incubation periods in Experiment 1 (Table 1). In addition, there were no significant
differences in HK and GK activities among all treatment groups in each tissue in Experiment
2 (Table 2). Uni et al. (2005) suggested that in ovo administration of carbohydrate improved
embryonic development, while Sato et al. (2006) showed that, between 12 to 18 d of embryonic
development, the respiratory coefficient of chicken embryos is approximately 0.7, suggesting that
chicken embryos mainly use lipid as energy substrate. Those facts indicate that although glucose
is important for development of the chicken embryo, it is not the main energy source for hatching.
The obtained results showed that AA injection in broiler eggs did not affect glucose metabolism in
the liver and muscles of chicken embryos, and AA injection might improve other functions such as
protein turnover, immune development, or something else in broiler embryos.

Table 1. Hexokinase and glucokinase activities in the liver and hatching muscles of broiler breeder
embryos on d 17, 18, and 19 of incubation (μmol/min/g tissue)1.
d of incubation Hexokinase Glucokinase
Liver 17 0.70 ± 0.16 0.01 ± 0.07

18 0.55 ± 0.10 0.01 ± 0.21
19 0.66 ± 0.09 0.04 ± 0.07
Neck muscle 17 0.18 ± 0.07 0.04 ± 0.10
18 0.24 ± 0.11 0.02 ± 0.04
19 0.26 ± 0.10 0.06 ± 0.04
Thigh muscle 17 0.24 ± 0.06 0.03 ± 0.15
18 0.30 ± 0.10 0.04 ± 0.14
19 0.26 ± 0.14 0.04 ± 0.08
1Values are means ± SD for 6 embryos.
Table 2. Effects of in ovo injection of amino acids or glucose on hexokinase (HK) and glucokinase
activity (GK) (μmol/min/g)1.
Injection
- Water Amino acids Glucose
Liver HK 0.81 ± 0.11 0.76 ± 0.11 0.76 ± 0.11 0.76 ± 0.11

GK 0.04 ± 0.04 0.04 ± 0.04 0.04 ± 0.03 0.04 ± 0.00
Neck muscle HK 0.13 ± 0.04 0.12 ± 0.02 0.11 ± 0.02 0.10 ± 0.02
GK 0.02 ± 0.01 0.02 ± 0.01 0.02 ± 0.00 0.02 ± 0.01
Thigh muscle HK 0.13 ± 0.05 0.14 ± 0.05 0.15 ± 0.04 0.13 ± 0.02
GK 0.02 ± 0.02 0.02 ± 0.02 0.02 ± 0.02 0.02 ± 0.02
1Valuse are means ± SD for 5 embryos.
References
John, T.M., J. C. George and E. T. Moran Jr., 1988. Metabolic changes in pectoral muscle and liver of turkey embryos
in relation to hatching: influence of glucose and antibiotic-treatment of eggs. Poultry Sci. 67, 463-469.
Ohta, Y., N. Tsushima, K. Koide, M. T. Kidd and T. Ishibashi, 1999. Effect of amino acid injection in broiler breeder
eggs on embryonic growth and hatchability of chicks. Poultry Sci. 78, 1493-1498.
Ohta, Y., M. T. Kidd and T. Ishibashi, 2001. Embryo growth and amino acid concentration profiles of broiler breeder
eggs, embryos, and chicks after in ovo administration of amino acids. Poultry Sci. 78, 1430-1436.
Sato, M., T. Tachibana and M. Furuse, 2006. Heat production and lipid metabolism in broiler and layer chickens during
embryonic development, Comp. Biochem. Phys. Part A, 143, 382-388.
Uni, Z., P. R. Ferket, E. Tako and O. Kedar, 2005. In ovo feeding improves energy status of late-term chicken embryos.
Poultry Sci. 84, 764-770.

Effect of conjugated linoleic acid on gluconeogenesis, glycogen turnover
and IGF-1 synthesis in primary culture of porcine hepatocytes
I. Fernández-Fígares, J.A. Conde-Aguilera, M. Lachica, R. Nieto and J.F. Aguilera
Department of Animal Nutrition, Estación Experimental del Zaidín, CSIC, Camino del Jueves s/n.
Introduction
Pigs fed CLA deposit less fat and have improved body composition (Dugan et al., 2004). These
activities appear to be associated with lipid and carbohydrate metabolism although the mechanisms
are poorly understood. Furthermore, the effects of CLA on hepatic metabolism in pigs have not
been elucidated. The aim of the present work was to study the effect of CLA on IGF-1 synthesis,
gluconeogenesis (GNG) and glycogen turnover in porcine hepatocytes.

Hepatocytes were isolated from pigs of approximately 15 kg BW and cultured as described previously
(Fernández-Fígares et al., 2004). Briefly, hepatocytes were seeded into T-25 flasks in William E
medium with 10% fetal bovine serum (FBS). Following a 3 h attachment period, William E containing
5% FBS was added to each flask. On the following d the medium was replaced with serum-free
William E amended with dexamethasone, dimethyl sulphoxide, β-mercaptoethanol, glutamine,
antibiotics and Na2SeO3 (basal medium). Linoleic acid (LA) and CLA (mixture of cis9,trans11
and trans10,cis12 CLA) were bound to fatty acid free albumin and used either at 0.1 or 0.05 mM.
A total of 4 pigs were used. All data were expressed per milligram cell protein determined by the
Lowry procedure.
Synthesis of IGF-1: Cells were cultured for 24 h in basal medium with high insulin (500 µg/mL)
and LA or CLA. IGF-1 was analysed in media using a two-site IRMA. IGF-1 was separated from
its binding proteins.
Synthesis of glycogen: Cells were cultured on d 1 for 3 h in basal medium with low insulin (10
µg/mL) and high glucagon (100 µg/mL) to deplete glycogen stores. Next, cells were cultured in
William E with high insulin, without glucagon, and LA or CLA for 21 h. Samples were incubated in
the absence or presence of amyloglucosidase to liberate glucosyl units from glycogen and glucose
determined with the glucose oxidase/peroxidase method.
Degradation of glycogen: Cells were cultured on d 1 for 24 h in William E with high insulin to
replenish glycogen stores and media changed to basal medium with low insulin, high glucagon (to
stimulate degradation) and LA or CLA for 3 h and glycogen was determined in cell homogenates.
GNG: Cells were cultured on d 1 for 24 h in basal medium amended with low insulin and high
glucagon to deplete glycogen stores. Next, media was changed to glucose free Dulbecco modified
Eagle medium with low insulin, high glucagon, lactate, pyruvate, alanine and LA or CLA for 40
min and glucose was determined in the media.
Factorial ANOVA was performed and differences (P<0.05) were determined by the Duncan t- test.

Compared to LA, CLA decreased GNG (14%) and glycogen synthesis (11%) without affecting
glycogen degradation. The magnitude of the effect is greater for GNG than for glycogen synthesis.
Similarly to our results, GNG in rat hepatocytes was decreased (Cantwell et al., 1999) by CLA
while GNG in bovine hepatocytes was unaltered (Mashek and Grummer, 2004). IGF-1 production
was diminished (22%) in pig hepatocytes cultured with CLA compared to LA. Ramsay et al. (2001)
found no altered IGF-1 serum level in growing pigs fed CLA supplemented diets. Increasing free
fatty acid (FFA) level from 0.05 to 0.1 mM increased glucagon stimulated GNG (16%) and glycogen
degradation (2%) while decreased insulin stimulated glycogen synthesis (10%). Taken together these
findings are compatible with an insulin resistance effect of FFA in porcine hepatocytes. The link
between fatty acid oxidation and GNG is conflicting in the suckling pigs (Duee et al., 1985). IGF-1
production was decreased by 61% when FFA level increased from 0.05 to 0.1 mM. In conclusion,
CLA decreased GNG, glycogen synthesis and IGF-1 production in pig hepatocytes.
Table 1. Effect of CLA and level of free fatty acid on IGF-1 synthesis, glycogen synthesis (Glyc synth)
and degradation (Glyc deg) and gluconeogenesis (GNG) in culture of porcine hepatocytes1.
Free fatty acid (FFA) Level of FFA SEM Level of significance
Item LA CLA 0.05mM 0.1mM FFA Level FFA×Level
IGF-1 544 423 597 371 38 0.030 0.000 0.49

Glyc synth 75.7 67.41 75.4 67.7 1.47 0.000 0.001 0.44
Glyc deg 145.5 143.4 142.3 146.6 0.88 0.108 0.002 0.33
GNG 53.8 46.5 46.54 53.8 0.89 0.000 0.000 0.012
1IGF-1 is expressed as pg/mg protein per 24 h; Glyc synth as ug/mg protein per 21 h; Glyc deg as µg/mg protein per
3 h and GNG as µg/mg protein per 40 min. Results are means of four replicated experiments.
References
Cantwell, H., R. Devery, M. Oshea and C. Stanton, 1999. The effect of conjugated linoleic acid on the antioxidant
enzyme defense system in rat hepatocytes. Lipids 34, 833-839.
Dugan, M. E., J. L. Aalhus and J. K. Kramer, 2004. Conjugated linoleic acid pork research. Am. J. Clin. Nutr. 79,
1212S–1216S.
Duee, P.H., J.P. Pegorier, J. Peret and J. Girard, 1985. Separate effects of fatty acid oxidation and glucagon on
gluconeogenesis in isolated hepatocytes from newborn pigs. Biol. Neonate 47, 77-83.
Fernandez-Figares, I., A.E. Shannon, D. Wray-Cahen and T.J. Caperna, 2004. The role of insulin, glucagon,
dexamethasone and leptin in the regulation of ketogenesis and glycogen storage in primary cultures of porcine
hepatocytes prepared from growing pigs. Domest. Anim. Endocrinol. 27, 125-140.
Mashek, D.G. and R.R. Grummer, 2004. Effects of conjugated linoleic acid isomers on lipid metabolism and
gluconeogenesis in monolayer cultures of bovine hepatocytes. J. Dairy Sci. 87, 67-72.
Ramsay, T.G., C.M. Evock-Clover, N.C. Steele and M.J. Azain, 2001. Dietary conjugated linoleic acid alters fatty acid
composition of pig skeletal muscle and fat. J. Anim. Sci. 79, 2152-2161.

Serum profile of metabolites and hormones of growing Iberian gilts fed
diets supplemented with betaine, conjugated linoleic acid or both
I. Fernández-Fígares, M. Lachica, R. Nieto and J.F. Aguilera
Department of Animal Nutrition, Estación Experimental del Zaidín, CSIC, Camino del Jueves s/n.
Introduction
Betaine (BET) and conjugated linoleic acid (CLA) have the potential to alter growth and body
composition in swine. Preliminary results in Iberian pigs have shown that BET and CLA have a
synergistic effect on growth (Fernández-Fígares et al., 2004) and carcass composition. Nevertheless,
little research has been conducted to investigate the pathways through which they mediate their effect
on growth and carcass traits. The aim of the present work was to evaluate changes in metabolic
hormones and the biochemical profile of growing Iberian pigs fed BET, CLA or BET + CLA
supplemented diets.

Twenty Iberian gilts (20 kg BW) were individually penned and fed barley-soybean meal based diets
(12% CP, 0.81% lysine and 14.7 MJ/ ME kg DM) containing either no added BET or CLA (control),
0.5% BET, 1% CLA, or 0.5% BET + 1% CLA, at 95% ad libitum energy intake, given in two meals
at 9.00 and 14.00h. At 30 kg BW, the pigs were fitted with jugular catheters. After recovery, on d
11, pigs were bled frequently (from 09.00 h to 05.00 h) and serum was frozen at -80 °C in aliquots.
The experimental protocol was approved by the Bioethical Committee of the CSIC. Insulin, IGF-1,
leptin and GH were measured by duplicate or triplicate using commercially available radio-immuno
assay or immuno-radiometric assay kits following directions of the manufacturer as previously
described (Fernández-Fígares et al., 2007). The intra- and interassay coefficients of variation were
13.1 and 15.5% for GH, 2.3 and 3.8% for IGF-1, 7.5 and 9.8% for insulin, 3.7 and 7.5% for leptin,
respectively. Serum glucose, creatinine, urea, total-cholesterol (chol), HDL-chol, LDL-chol and
triglycerides were determined colorimetrically using an automated Advia 1650 apparatus (Dublin,
Ireland). The area under the response curves were determined using trapezoidal geometry (0-20 h).
The experimental data were subjected to GLM ANOVA using as the covariate the basal concentration
of each parameter and differences (P<0.05) were determined by the Duncan t-test.

Compared to control pigs, gilts fed with BET+CLA supplemented diets had increased serum insulin
(79%), decreased urea (28.2 vs. 33.3 mg/dL/h; 15%) and decreased HDL-/total-chol (0.46 vs.
0.63; 27%) and HDL-/LDL-chol (0.66 vs. 1.07; 38%) concentrations, while pigs fed BET or CLA
supplemented diets had intermediate values. Furthermore, there was a trend towards increased serum
LDL-chol (101 vs. 78 mg/dL/h; 29%) in BET+CLA compared to control pigs. Compared to control
pigs, BET pigs had lower serum urea (30 vs. 33 mg/dL/h; 9%) and pigs fed CLA had increased
triglycerides (93 vs. 45 mg/dL/h; 107%). The rest of the hormones and biochemical parameters
studied were not affected by the treatments (P>0.1).

Table 1. Effect of betaine (BET) conjugated linoleic acid (CLA) or both on the area under the curves
for serum hormones (ng/mL/h except insulin µU/mL/h) in growing Iberian pigs.
Dietary treatments SEM
Item Control BET CLA BET+CLA
Insulin 36.3a 37.1a 48.6a 64.9b 3.3

IGF-1 527 518 507 550 24
GH 3.0 2.5 2.5 2.4 0.2
Leptin 20 18 24 22 2.2
There are indications that CLA may alter serological traits of lipid metabolism in pigs (Muller et
al., 2000) although not in all cases (Stangl et al., 1999). GH, insulin and IGF-1 did not change in
CLA fed pigs (Ramsay et al., 2001; Mitchell et al., 2005) while leptin increased in lactating sows
(Bontempo et al., 2004). In general, pigs fed BET had subtle changes in serum chol and triglycerides
(Matthews et al., 1998), urea and ammonia (Fernández-Fígares et al., 2002), the exception being a
recent work of Huang et al. (2006) with Chinese finishing pigs. No reports have been found on the
effect of BET+CLA on hormones and biochemical parameters. From these results it was concluded
that the association BET+CLA elicited changes in hormone and biochemical profiles in growing
pigs that may affect their protein and energy status. It could be recommended for young pigs when
lean carcasses are a goal.
References
Bontempo, V., D. Sciannimanico, G. Pastorelli, R. Rossi, F. Rosi and C. Corino, 2004. Dietary conjugated linoleic acid
positively affects immunological variables in lactating sows and piglets. J. Nutr. 134, 817-824.
Fernández-Fígares, I., D. Wray-Cahen, N.C. Steele, R.G. Campbell, D.D. Hall, E. Virtanen and T.J. Caperna, 2002.
Effect of dietary betaine on energy utilization and partitioning in the young growing feed restricted pig. J. Anim.
Sci. 80, 421-428.
Fernández-Fígares, I., M. Lachica, R. Nieto, E. González Sánchez and J.F. Aguilera, 2004. Use of betaine and conjugated
linoleic acid as growth promotants in growing iberian pigs. J. Anim. Sci. 82, 176.
Fernández-Fígares, I., M. Lachica, R. Nieto, M. Rivera-Ferre and J.F. Aguilera, 2007. Serum profile of metabolites
and hormones in obese (Iberian) and lean (Landrace) growing gilts fed balanced or lysine deficient diets. Livest.
Sci. (available on line November 2006).
Matthews, J.O., L.L. Southern, J.E. Pontif, A.D. Higbie and T.D. Bidner, 1998. Interactive effects of betaine, crude
protein, and net energy in finishing pigs. J. Anim. Sci. 76, 2444-2455.
Mitchell, A.D., V.G. Pursel, R.H. Elsasser, J.P. McMurtry and G. Bee, 2005. Effects of dietary conjugated linoleic acid
on growth and body composition of control and IGF-I transgenic pigs. Anim. Res. 54, 395-411.
Muller, H.L., M. Kirchgessner, F.X. Roth and G.I. Stangl, 2000. Effect of conjugated linoleic acid on energy metabolism
in growing-finishing pigs. J. Anim. Physiol. Anim. Nutr. 83, 85-94.
Ramsay, T.G., C.M. Evock-Clover, N.C. Steele and M.J. Azain, 2001. Dietary conjugated linoleic acid alters fatty acid
composition of pig skeletal muscle and fat. J. Anim. Sci. 79, 2152-2161.
Stangl, G.I., H. Muller and M. Kirchgessner, 1999. Cojugated linoleic acid effects on circulating hormones, metabolites and
lipoproteins, and its proportion in fasting serum and erythrocyte membranes of swine. Eur. J. Nutr. 38, 271-277.

4B. Health and metabolism

Association of plant extracts rich in polyphenols and vitamin E efficiently
prevents lipoperoxidation in plasma of sheep fed a n-3 PUFA rich diet
C. Gladine1, E. Rock2, C. Morand2, D. Gruffat1, D. Bauchart1 and D. Durand1
1INRA, Research Unit on Herbivores, Nutrients and Metabolisms Group, 63122 Saint-Genès-
Champanelle, France
2INRA, Research Unit on Human Nutrition, Metabolic Stress and Micronutrients Group, 63122
Saint-Genès-Champanelle, France
Introduction
In ruminants, incorporation of n-3 polyunsaturated fatty acids (n-3 PUFA) in the diet improves
the nutritional quality of meat (Bauchart et al., 2005). However, such a dietary practice favours
lipoperoxidation (Scislowski et al., 2005) which can be detrimental for the health and growth
performances of animals. In this context, addition of antioxidants in the animal’s diet is recommended
but the exclusive use of vitamin E becomes criticised since it has a limited efficiency when n-3
PUFA intake increases (Allard et al., 1997). To optimise the prevention of lipoperoxidation in n-3
PUFA fed animals, we hypothesised that the use of different types of antioxidants may complete the
protection already provided by vitamin E. In previous experiments, we investigated the bioefficiency
of four plant extracts rich in polyphenols (PERP) and we showed that they all efficiently prevented
lipoperoxydation in rat and sheep supplemented with n-3 PUFA (Gladine et al., 2007a and 2007b)
and exhibited complementary mechanisms of actions. In the present work, we aimed at investigating
(i) the bioefficiency of a mixture of the 4 PERP in comparison with vitamin E and (ii) the synergy
of action of polyphenols associated to vitamin E.

The experiment was carried out with Texel sheep (n=24) given a n-3 PUFA enriched diet for 7 wk
(12% of extruded linseed) without (Control) or with one of the three antioxidant supplements which
consisted in a mixture of the four PERP (PERP group, 10 g/kg DM; n=6), or an α-tocopheryl acetate
supplement (VitE group, 200 mg/kg DM; n=6) or both antioxidant supplements (PERP+VitE group;
n=6). Plasma was collected before and after a 7-wk supplementation period to measure the level
of one final product of lipoperoxidation (i.e., malondialdehyde or MDA) by HPLC coupled with
a fluorimetric detection. The susceptibility of plasma to lipoperoxidation was also evaluated by
monitoring the kinetics (lag phase and oxidation rate) of conjugated diene (CD) generation with a
double-beam spectrophotometer. The effect of antioxidant supplements was determined by analysing
contrasts between the four groups.

MDA production (Table 1) was not significantly reduced by the PERP mixture or the vitamin E
supplement used alone when compared to the control group. However, association of PERP and
vitamin E significantly reduced the concentration of MDA in plasma demonstrating a synergy
between these two types of antioxidants (P=0.04). This may be due to their complementary sites
and mechanisms of action. Indeed, polyphenols are known to act within aqueous environment as
scavengers of lipoperoxidation initiators whereas vitamin E is integrated within lipid membranes
where it reduces lipoperoxides produced from PUFA.

Table 1. Effect of supplementation with PERP (PERP, 10 g/kg of DM), vitamin E (VitE, 200 mg/kg of
DM) or a mixture of PERP and vitamin E (PERP+VitE) on plasma concentration1 of malondialdehyde
(MDA) and parameter indicators of lipoperoxydation (conjugated dienes, CD: lag phase and
oxidation rate) in the plasma of sheep (n=24) fed a n-3 PUFA rich diet.
Diet Contrast2 (P value)
Control PERP VitE PERP +VitE PERP VitE Synergy
MDA, 0.27 ± 0.17 0.18 ± 0.07 0.08 ± 0.11 -0.13 ± 0.13 0.30 0.17 0.04
µg/mg PUFA
CD lag phase, -77 ± 42 74 ± 50 25 ± 87 655 ± 573 0.02 0.16 0.09
min/mg PUFA
CD oxidation rate, 1.78 ± 1.89 4.81 ± 3.28 0.57 ± 2.06 -5.80 ± 5.72 0.22 0.34 0.06
A234/min/mg PUFA
1Values represent means (n=6, ± SE) of differences observed before and after the 7-wk supplementation period.
None of the means presented here was significantly different from zero; 2Contrasts were determined by comparing
the Control with PERP (contrast ‘PERP’) or VitE (contrast ‘VitE’) group or by comparing PERP and VitE groups
with PERP+VitE (contrast ‘Synergy’) using the Student t test.
This hypothesis is consistent with values of kinetic parameters of lipoperoxydation (production

of conjugated dienes) (Table1) showing a higher lag phase in the PERP group (P=0.02) compared
to the VitE group. This suggests that PERP’polyphenols were involved at the initiation of the
lipoperoxidation process explaining their synergy with vitamin E which usually plays a ‘chain-
breaking antioxidant’ role.
In conclusion, we demonstrated that, when administrated separately, neither PERP nor vitamin E can
efficiently prevent lipoperoxidation in the plasma of sheep given an n-3 PUFA rich diet. However,
when given together, these two types of antioxidants acted synergically leading to a significant
reduction of lipoperoxidation processes, presumably because of complementary sites/mechanisms
of action.
References
Allard, J., R. Kurian, E. Aghdassi, R. Muggli and D. Royall, 1997. Lipid peroxidation during n-3 fatty acid and vitamin
E supplementation in humans. Lipids 32, 535-541.
Bauchart, D., C. Gladine, D. Gruffat, L. Leloutre and D. Durand, 2005. Effects of diets supplemented with oil seeds
and vitamin E on specific fatty acids of rectus abdominis muscle in Charolais fattening bulls. In: Hocquette, J.F.
and S. Gigli (editors), Indicators of milk and beef quality. Wageningen Academic Publishers, The Netherlands,
431-436.
Gladine, C., C. Morand, E. Rock, D. Gruffat, D. Bauchart and D. Durand, 2007a. The antioxidative effect of plant
extracts rich in polyphenols is tissue specific in rats fed n-3 PUFA rich diets. Anim. Feed Sci. Technol., in press.
Gladine, C., E. Rock, C. Morand, D. Bauchart and D. Durand, 2007b. Bioavailability and antioxidant capacity of plant
extracts rich in polyphenols in ruminant fed n-3 PUFA rich diets. Br. J. Nutr., in press.
Scislowski, V., D. Bauchart, D. Gruffat, P.M.M. Laplaud and D. Durand, 2005. Effect of dietary n-6 or n-3 poyunsaturated
fatty acids protected or not against ruminal hydrogenation on plasma lipids and their susceptibility to peroxidation
in fattening steers. J. Anim. Sci. 83, 2162-2174.

Cardiac oxidative stress in high fat/high sucrose fed rats is mainly due to
NADPH oxidase overexpression. Protective effects of polyphenols
T. Sutra1, C. Feillet-Coudray2, G. Fouret2, G. Cabello2, J.-P. Cristol1,3 and C. Coudray2
1EA 2993 Human Nutrition and Atherogenesis Laboratory, University Institute of Clinical Research,
34093, Montpellier, France
2UMR 866 Growth and cellular differentiation, INRA, 34060, Montpellier, France
3Biochemistry Laboratory, Lapeyronie University Hospital, 34295, Montpellier, France
Introduction
Metabolic syndrome cluster is a recognised risk factor for atherosclerotic cardiovascular complications.
Oxidative stress resulting from NADPH oxidase activity and/or mitochondrial dysfunction is
implicated in the development of atherosclerosis in metabolic syndrome patients. The increase of
reactive oxygen species (ROS) could conspire with adipocytokines dysregulation to promote cardiac
disease (Fujita et al., 2006). Recently, it has been shown that antioxidants, such as tea and fruit
polyphenols, decrease fat accumulation in obese mice (Klaus et al., 2005 and Lin and Lin-Shiau,
2006). The aim of the present study was to characterise the main source of ROS production in the
heart and its prevention by polyphenols.
Methodology
Twenty-four Wistar rats were randomly divided into 3 groups. Rats were fed for 12 wk with control
diet or high fat/high sucrose (HF/HS) diet supplemented or not with polyphenols (2 g polyphenols/
kg diet). At the end of the experimental period, rats were sacrificed and oxidative stress indices
(AGEs, AOPP) and antioxidant defences (Thiols, SOD, and GPX) were performed on plasma. The
superoxide production rate was evaluated on the left ventricle by chemiluminescence. On the left
ventricle homogenate, the expression of NADH/NADPH oxidase was determined by Western blot
and mitochondrial enzyme activities (Cytochrome c oxidase and citrate synthase) were monitored.
Data are shown as the means±SD and statistical analyses were provided using StatView software by
one-way ANOVA followed by the Fisher’s Protected Least Significant Difference test. Differences
were considered significant at P<0.05.
Results
The plasma total cholesterol from the HF/HS rats was higher than the control group (P<0.05). Such
an increase was prevented by supplementation of wine polyphenols (P<0.05).
As shown in Table 1, plasma thiols decreased (P<0.05) and plasma AGE and AOPP increased
(P<0.05), whereas MDA level remained unchanged in the HF/HS group compared with the control
group (data not shown). Polyphenol intake had a slight effect only on AGE parameter (-15%). In
the left ventricle AOPP, Thiols and MDA were not modified by the HF/HS diet compared with the
control group.
Plasma GPx, erythrocyte SOD, Catalase and GPx activities were similar in the three groups (data
not shown). Only plasma SOD significantly decreased by HF/HS diet without any improvement by
polyphenols. We observed also similar results for the ventricular catalase, GPx, SOD and SOD-Mn
activities, only the SOD-Mn/SOD ratio decreased by polyphenols compared with control fed rats
(Table 1).

Table 1. Plasma and cardiac oxidative stress parameters.
Plasma SOD Plasma Thiols Plasma AOPP Plasma AGEs cardiac

U/mL µM µM OD at 340 nm MnSOD/SOD
Standard 19.2±1.6a 0.21±0.02a 58±39a 6.04±0.84a 0.80±0.04a

HF/HS 14.9±1.1b 0.17±0.02b 165±57b 7.84±1.04b 0.75±0.1a
HF/HS+PP 14.9±2.6b 0.17±0.03b 150±63b 6.68±1.01ab 0.68±0.12b
In the left ventricle, the NADPH oxidase expression and superoxide production significantly increased
in the HF/HS group compared with the control group. In both cases, polyphenol intake restored the
superoxide production and the NADPH oxidase expression at least to their basal state (Table 2).
Table 2. Ventricular NADPH oxidase expression and superoxide production.
NADPH oxidase expression (AU) cardiac superoxide count/mg protein
Standard 34.2±27.9a 13.16±1.59a

HF/HS 63.1±15.1b 17.79±1.83b
HFHS+PP 33.1±3.7a 9.60±1.44c
Discussion
HF/HS-induced oxidative stress is known to promote endothelial dysfunction limiting NO
bioavailability, myocardial lipid peroxidation (Vincent et al., 2001), and recently aorta NADPH
oxidase overexpression is implied in diet-induced oxidative stress (Roberts et al., 2006). According to
this, increased cardiac superoxide (O2°-)production and p22phox expression together with a decrease
of antioxidant mechanisms in HF/HS fed rats indicates that O2°- production is strongly associated
with HF/HS diet-induced cardiac complications. The slight effect of O2°- production observed after
12 wk of the HF/HS diet did not induce any cardiac oxidative stress without any increase of the
index of cardiac weight, as observed in other animal models. In conclusion, diet-induced NADPH
oxidase expression could be prevented by polyphenols supplementation, as previously reported in
other nutritional rodent models.
References
Fujita, K., H. Nishizawa, T. Funahashi, I. Shimomura and M. Shimabukuro, 2006. Systemic oxidative stress is associated
with visceral fat accumulation and the metabolic syndrome. Circ. J. 70, 1437-1442.
Klaus, S., S. Pultz, C. Thone-Reineke and S. Wolfram, 2005. Epigallocatechin gallate attenuates diet-induced obesity
in mice by decreasing energy absorption and increasing fat oxidation. Int. J. Obes. (Lond) 29, 615-623.
Lin, J.K. and S.Y. Lin-Shiau, 2006. Mechanisms of hypolipidemic and anti-obesity effects of tea and tea polyphenols.
Mol. Nutr. Food Res. 50, 211-217.
Roberts C.K., R.J. Barnard, R.K. Sindhu, M. Jurczak, A. Ehdaie and N.D. Vaziri, 2006. Oxidative stress and dysregulation
of NAD(P)H oxidase and antioxidant enzymes in diet-induced metabolic syndrome. Metabolism 55, 928-934.
Vincent, H.K., S.K. Powers, A.J Dirks and P.J. Scarpace, 2001. Mechanism for obesity-induced increase in myocardial
lipid peroxidation. Int. J. Obes. Relat. Metab. Disord. 25, 378-388.

The effect of sodium ascorbate, trolox and 3-hydroxy-3-methylbutyrate
on apoptosis induced by oxidative stress in C2C12 cells without
dystrophin
S.J. Berwid and P. Ostaszewski
Department of Physiological Sciences, Warsaw Agricultural University, Warsaw, Poland
Introduction
Duchenne Muscular Dystrophy (DMD) is one of the most common inherited muscle disorders
occurring in people. This X-linked myopathy is caused by lack of dystrophin (a major protein
composing the cytoskeleton of muscle cells) as a consequence of null mutations in the gene encoding
dystrophin. The animal model of DMD is the mdx mouse. Although it lacks functional dystrophin,
the regeneration mechanism is so successful that the model does not exhibit features of the disorder
that are similar to those in humans. That is why we decided to obtain a dystrophic muscle cell line
through a gene silencing technique by dystrophin knock down in the popular murine muscle cell
line C2C12.
We took two well known anti-oxidants (sodium ascorbate -VC and trolox- VE) and 3-hydroxy-3-
methylbutyrate (HMB) and compared their impact on cell viability after inducing cell death with
oxidative stress donors (sodium nitroprusside- SNP and hydrogen peroxide - H2O2). Smith et al.
(2004) noticed that the calcium salt of HMB (50μM) limits muscle cell loss in murine myotubes
through attenuation of the ubiquitin-proteasome proteolytic pathway. In in vivo studies in rats, Betters
et al. (2004) observed that trolox had a significant impact on the reduction of muscle proteolysis due
to oxidative stress and trolox treatment attenuated the induced rise in protease activity.
The aim of this study was to evaluate the molecular mechanism responsible for death of dystrophic
muscle cells and to determine an effect of chosen antioxidants and 3-hydroxy-3-methylbutyrate on
the viability of murine muscle cells with and without dystrophin.

All experiments were performed on the control C2C12 cell line and on the C2C12 cell line with
knocked down dystrophin. The dystrophic muscle cell line was obtained by a gene silencing technique
using commercial siRNA expression vector for mammalian transfection (GenScript, USA). This
myogenic cell line was cultured in standard culture conditions. The differentiation into myotubes
was conducted for 5 d prior to the experiment.
Antioxidants and HMB (used for the first time as a pure acid in vitro) were added to the medium
for 24 h pre-incubation before changing the medium to the experimental one (with oxidative stress
inducers). The concentration of reagents used were as follows: 0.5mM sodium ascorbate, 150 mM
trolox, 25 µM HMB, 0.75 mM H2O2, 0.5 mM SNP. The pre-incubation time was 24 h and the
experimental time was 0, 4, 8 and 16 h. After that time cells were prepared for estimation of the type
of death with one of the following methods: cells death determination by annexin and propidium
iodide test (Biosource, USA) rated by flow cytometry and the MTT viability test. We also introduced
specific antibodies for the chosen proteins (PARP, dystrophin, active-caspase 3) used in fluorescence
and confocal microscopy evaluation.
All the data sets were shown as the percent of control. They were presented as means from 3 separate
measurements (made always in 2 repetitions) with standard error of the mean (SEM). The one-way
ANOVA test was performed using the Tukey test (GraphPad Prism 3.00, USA) and statistically

significant differences between the control and investigated values were marked as significant
(P<0.05).

Our results show that cells reacted differently to oxidative stress induced by SNP (0.5 mM) and H2O2
(0.75 mM). Preincubation (24 h) with sodium ascorbate (0.5 mM) did not protect differentiated muscle
cells against apoptosis but significantly affected (P<0.05) proliferating culture viability (nearly 150%
of control), probably through gain in antioxidative potential as observed by other authors in vivo
(Ashton et al., 2003). It was demonstrated that trolox (150 mM) prevented better against apoptosis
(nearly 10% less mortality vs. control and other factors) and lowered the number of cells overcoming
with apoptosis and necrosis (about 25% less dead cells). These results were similar to other authors
who treated the trolox pre-incubated C2C12 cell line with different oxidative inducers (Usuki et al.,
2000). Therefore it had a significant influence on increase of dystrophic differentiated cells viability
under oxidative stress (HMB and trolox preincubated cells). HMB was employed for the first time in
the in vitro study and had a significant impact (P<0.05) on the viability of dystrophic differentiated
muscle cells (more than 10%). It was observed that HMB protected against apoptosis, presumably by
down-regulating the ubiquitin-proteasome pathway as previously suggested by Smith et al. (2004).
The mdx muscles undergo apoptosis and necrosis (Sandri et al., 1995), so it can be concluded that
these muscles express mixed types of cell death. In other similar catabolic disorders like cachexia
it was observed that ubiquitin-proteasome-dependent proteolysis is the predominant mechanism of
muscle protein loss in these conditions (Hasselgren et al., 2002) and also lead to various types of
cell death. The possibility of blocking ubiquitin-proteasome-dependent proteolysis is a new idea for
complementary treatment in patients with muscle wasting. In summary our data indicate that 24 h
pre-incubation with HMB protects against apoptosis both in the normal murine C2C12 muscle cell
line as well as with knocked-down dystrophin.
References
Ashton T., I.S. Young, G.W. Davison, C.C. Rowlands, J. McEneny, C. Van Blerk, E. Jones, J.R. Peters and S.K.
Jackson, 2003. Exercise-induced endotoxemia: the effect of ascorbic acid supplementation. Free Radic. Biol.
Med. 35, 284-291.
Betters J.L., D.S. Criswell, R.A. Shanely, D. Van Gammeren, D. Falk, K.C. Deruisseau, M. Deering, T. Yimlamai and
S.K. Powers, 2004. Trolox attenuates mechanical ventilation-induced diaphragmatic dysfunction and proteolysis.
Am. J. Respir. Crit. Care Med. 170, 1179-1184. Epub. 2004 Sep 16.
Hasselgren P.O., C. Wray and J. Mammen, 2002. Molecular regulation of muscle cachexia: it may be more than the
proteasome. Biochem. Biophys. Res. Commun. 290, 1-10.
Sandri M., U. Carraro, M. Podhorska-Okolov, C. Rizzi, P. Arslan, D. Monti and C. Franceschi, 1995. Apoptosis, DNA
damage and ubiquitin expression in normal and mdx muscle fibers after exercise. FEBS Lett. 373, 291-295.
Smith H.J., S.M. Wyke and M.J. Tisdale, 2004. Mechanism of the attenuation of proteolysis-inducing factor stimulated
protein degradation in muscle by beta-hydroxy-beta-methylbutyrate. Cancer Res. 64, 8731-8735.
Usuki F., N. Takahashi, N. Sasagawa and S. Ishiura, 2000. Differential signaling pathways following oxidative stress
in mutant myotonin protein kinase cDNA-transfected C2C12 cell lines. Biochem. Biophys. Res. Commun. 267,
739-743.

Effects of inflammation in peripartum dairy cows on milk yield, energy
balance and efficiency
E. Trevisi, A. Gubbiotti and G. Bertoni
Institute of Zootechnics, Faculty of Agriculture, U.C.S.C., 29100, Piacenza, Italy
Introduction
Post partum dairy cows are in a negative energy balance due to the sharp raise of milk yield and
to the slower increase of feed intake. Endocrine factors (i.e. insulin, growth hormone, leptin) are
involved in the regulation of body energy homeostasis and homeoresis (appetite, energy expenditure,
nutrient partitioning between tissues and mammary gland), but none seems to fully explain the
difference of feed intake and energy utilisation observed at the beginning of lactation. It has also
been demonstrated that transition dairy cows frequently show clear inflammatory phenomena
around calving, often without clinical symptoms (Bionaz et al., 2007). Inflammation is orchestrated
by specific cytokines (i.e. TNFα, IL-1), released by the immune system, that affect several organs
worsening the energy balance: promoting fever, anorexia, muscle catabolism and nutrient drainage
for the immune system function (Elsasser et al., 2000). The aim of our work was to estimate the
energy balance and efficiency in dairy cows with or without subclinical inflammation after calving
to better know their relationship.

Eleven Italian Friesian dairy cows were fed daily with 2 forage meals (corn-silage and good quality
hay) and with 2 (dry period) or 8 (lactation) concentrate meals; water was given ad libitum. Rations
were formulated to cover requirements according to INRA (energy) and NRC (protein). Cows were
monitored daily during the mo before and after calving for health status, rumination rate, feed and
water consumption and rectal temperature. In addition, body weight was measured and body condition
score (BCS) was estimated once a wk as well as the d after calving, while milk yield was measured
at each milking (5:00 a.m. and p.m.) and representative samples of both daily milking were collected
twice a wk for protein, fat and lactose determination. These data were used to evaluate the weekly
energy balance according to Walter and Mao (1989). Finally, blood samples were drawn from the
jugular vein before the morning feeding, twice a wk and daily around calving. Some biochemical
parameters have been measured with particular emphasis to the acute phase proteins (positive and
negative) to evaluate the inflammatory conditions. Namely, the cows were retrospectively divided
into 2 groups according to 3 liver functions (showed by albumin, lipoprotein and bilirubin; Bertoni et
al., 2006) which are reduced by inflammation; the groups were HI (5 cows with high inflammation)
and LI (6 cows with low inflammation). Data were statistically analysed as repeated measures of
variance model (procedure MIXED of SAS Institute, Cary, NC, release 8.0), including group (HI,
LI), DIM (ds in milk) and group by DIM interaction as fixed effects, and cow within group as the
random variable.

According to the criteria used to partition the cows, the LI group showed - in comparison to HI - the
lowest level of bilirubin in the 1st wk after calving (6.38 ± 2.82 vs. 10.73 ± 5.06 µmol/L; P<0.01),
the fastest raise of cholesterol during the 1st month of lactation (5.10 ± 0.43 vs. 4.36 ± 0.74 mmol/L
at 28th DIM; P<0.05) and the highest level of albumin (38.93 ± 1.3 vs. 36.38 ± 2.3 mmol/L at 28th
DIM, P<0.05). This could confirm a lower liver response to inflammatory conditions in the LI group,
because positive acute phase proteins (haptoglobin and ceruloplasmin) did not differ, although LI
showed a tendency to a faster reduction after the 7th DIM. The main blood indices of energy balance

Table 1. Average values of some parameters included in the study for cows with low (LI) or high
(HI) inflammation during the first 28 DIM.
Parameter BCS points Dry matter Milk yield Dairy UNEM

intake (DMI) (MY) efficiency %
0 DIM 28 DIM kg/d kg/d kg MY/kg DMI
LI 2.54 2.10 19.63 46.33 b 2.37 b 119.80 b

HI 2.69 2.18 19.71 41.95 a 2.14 a 104.80 a
SE 0.146 0.543 1.270 0.071 3.482
Note: UNEM (Utilisation of Net Energy for Milk) = conversion of net feed energy to milk, considering maintenance
and body reserve contribution; SE = standard error; a,b = significant difference between groups (P<0.05).
have shown few differences between the 2 groups: glucose remained lower in HI till 14th DIM,
while free fatty acids and β-OH-butyrate had a tendency to a quicker reduction in the HI group
from the 21st DIM. As expected, body weight and BCS (Table 1) were gradually reduced during
the first 28 DIM (P<0.01), but similarly in the 2 groups (about 2.34 kg/d of body weight losses and
-0.45 points of BCS). Nevertheless, the LI group showed a less pronounced body loss in the 1st wk
after calving and a higher one in the 4th wk (NS). DMI as well did not differ between the 2 groups,
while milk yield (Table 1) was higher in LI (P<0.05), but fat content was similar. Consequently, the
energy efficiency was about 10% lower in HI for the whole 1st month of lactation when calculated
as dairy efficiency (milk yield/dry matter intake) or evaluated as net efficiency (Table 1). It therefore
seems that in case of response to inflammation, a sensible depletion of the lean body mass and the
increase of energy expenditure occur as suggested by Elsasser et al. (2000). Although we only
considered subclinical inflammatory conditions after calving, we still observed a marked change
on energy balance, with a significant decrease of diet energy utilisation. This confirms that the
inflammatory phenomena at calving time were responsible for metabolic inefficiencies that cause
the marked increases of maintenance costs, likely to support the immune system activity. Therefore,
our results suggest the utility of tools that are able to reduce inflammatory responses around calving
as a necessary condition to improve the performance of dairy cows.
References
Bertoni, G., E. Trevisi, A.R. Ferrari and A. Gubbiotti, 2006. The dairy cow performances can be affected by inflammations
occurring around calving. Proc. of 57th EAAP Meeting, Antalya, Turkey, 325 pp.
Bionaz, M., E. Trevisi, L. Calamari, F. Librandi, A. Ferrari and G. Bertoni, 2007. Plasma paraoxonase, inflammatory
conditions, liver functionality and health poblems in transition dairy cows. J. Dairy Sci. 90, 1740-1750.
Elsasser, T.H., K.C. Klasing, N. Filipov and F. Thompson, 2000. The metabolic consequen-ces of stress: target for
stress and priorities of nutrient use. In: Moberg, G.P. and J.A., Mench (editors), The biology of animal stress.
CABI Publishing, New York, USA, 77-110.
Walter, J.P. and I.L. Mao, 1989. Modelling net energy efficiencies as quantitative characteristics in lactating cows. J.
Dairy Sci. 72, 2362-2374.

Dietary L-carnitine enhances the acute phase response in chickens
J. Buyse1, Q. Swennen1, T.A. Niewold1, K.C. Klasing2, G.P.J. Janssens3, M. Baumgartner4 and
B.M. Goddeeris1
1Laboratory of Livestock Physiology, Immunology and Genetics, Katholieke Universiteit Leuven,
Kasteelpark Arenberg 30, 3001 Leuven, Belgium
2Department of Animal Science, University of California, Davis, California, USA
3Laboratory of Animal Nutrition, Ghent University, Heidestraat 19, 9820 Merelbeke, Belgium
4Lonza Ltd., Muenchensteinerstrasse 38, CH-4002 Basel, Switzerland
Introduction
L-carnitine acts as a carrier for translocation of long-chain fatty acids from the cytosol into mitochondria
for β-oxidation. Dietary L-carnitine supplementation in animals was described to positively influence
growth and feed efficiency, although conflicting data exist in the chicken (Buyse et al., 2001). There
is also growing evidence that L-carnitine also plays a role in other physiological processes in humans
and animals. It enhances the humoral immune response in the chicken (Mast et al., 2000; Deng et al.,
2006) and other species. Very little is known on its effect on innate immunity, except in mammals
(Bellinghieri et al., 2005). We therefore investigated the response of two major positive acute phase
proteins hemopexin (HX) and alpha-1 acid glycoprotein (AGP) upon a lipopolysaccharide (LPS)
challenge in broiler chickens, in order to see if the responses of these mediators of the innate immune
system were altered when supplemental L-carnitine was present in the diet.

One hundred and eighty 1-d-old male broiler chicks (Ross) from a commercial hatchery were divided
over twelve pens in an environmentally controlled poultry house. All chickens received a commercial
broiler starter diet (d 0-14), after which the animals were divided into three pens each, receiving a
commercial broiler finisher diet containing 0, 15 or 100 mg of added L-carnitine/kg (Lonza Ltd.),
respectively. The basal L-carnitine content of the starter and finisher diet was 17.8 and 22.9 mg/kg,
respectively, calculated based on the L-carnitine content of the ingredients used (Baumgartner and
Blum, 1997). Body weight gain, feed intake, and plasma was collected regularly. After two wk on
the finisher diets, eight broiler chickens of each diet were injected subcutaneously with 300 µg E.
coli lipopolysaccharide (LPS), or vehicle alone (d 0). From the same birds, plasma HX and AGP d
0, 1, 3, and 7 post LPS, were quantified by rocket gel electrophoresis (Adler et al., 2001). Statistical
significance between groups or treatments was determined using Proc GLM for repeated measures,
SAS for Windows version 8e.

Body weight and feed intake were not affected by the dietary L-carnitine treatment, thus confirming
the majority of earlier results in chickens (as reviewed by Buyse et al., 2001).
LPS injection caused a significant (P<0.01) decrease in body weight gain compared to vehicle
controls. L-carnitine supplementation did not affect this (results not shown). The adverse effect
of LPS on growth rate was in accordance with previous findings (Humphrey and Klasing, 2004).
Extra L-carnitine in the diet of broiler chickens clearly enhances the (transient) acute phase protein
response as was evident on d one after LPS-injection (Figure 1), and without additional growth
depression. This observation was inconsistent with an L-carnitine induced generalised increase in the
proinflammatory response for the absence of associated weight loss. It rather suggests that L-carnitine
augments hepatic production of acute phase proteins. L-carnitine has recently been recognised to

be a glucocorticoid mimicker (Manoli et al., 2004). Glucocorticoids enhance the synthesis of most
acute phase proteins. It is concluded that extra L-carnitine in the diet of broiler chickens modulates
the innate immune response by enhancing the acute phase protein response. The exact mode of action
needs to be further elucidated, but the results obtained here are most consistent with the described
glucocorticoid-like effect of L-carnitine (Manoli et al., 2004).
0.9 *
0.8
0.7
*
0.6
0.5
*
mg/ml
0.4
0.3
0.2
0.1
0.0
0 LPS 15 CA LPS 100 CA LPS
Figure 1. Dietary supplementation of L-carnitine (CA: 15 and 100 mg/kg diet) causes enhancement
of the LPS induced acute phase proteins hemopexin (filled bars) and alpha-1 acid glycoprotein (open
bars) after 24 h. An asterisk indicates a significant difference with the untreated control (0).
References
Adler, K.L., P.H. Peng, R.K. Peng and K.C. Klasing, 2001. The kinetics of hemopexin and α-acid glycoprotein levels
induced by injection of inflammatory agents in chickens. Avian Dis. 45, 289-296.
Baumgartner, M. and R. Blum, 1997. Typical L-carnitine contents in feedstuffs. In: Baumgartner, M. (editor), L-carnitine
in Animal Nutrition. Lonza Ltd, Basel.
Bellinghieri, G., D. Santoro and M.V. Calvani, 2005. Role of carnitine in modulating acute-phase protein synthesis in
hemodialysis patients. J. Ren. Nutr. 15, 13-17.
Buyse, J., G.P.J. Janssens and E. Decuypere, 2001. The effects of L-carnitine supplementation on the performance,
organ weights and circulating hormone and metabolite concentrations of broiler chickens reared under a normal
or low temperature schedule. Br. Poult. Sci. 42, 230-241.
Deng, K., C.W. Wong and J.V. Nolan, 2006. Long-term effects of early-life dietary L-carnitine on lymphoid organs
and immune responses in Leghorn-type chickens. J. Anim. Phys. Anim. Nutr. 90, 81-86.
Humphrey, B.D. and K.C. Klasing, 2004. Modulation of nutrient metabolism and homeostasis by the immune system.
Worlds Poult. Sci. J. 60, 90-100.
Manoli, I., M.U. De Martino, T. Kino and S. Alesci, 2004. Modulatory effects of L-carnitine on glucocorticoid receptor
activity. Ann. NY Acad. Sci. 1033, 147-157.

Dietary β-hydroxy-β-methylbutyrate supplementation influences
performance differently after immunisation in broiler chickens
J. Buyse1, Q. Swennen1, B.M. Goddeeris1, F. Vandemaele1, K.C. Klasing2, M. Baumgartner3 and
T.A. Niewold1
1Laboratory of Livestock Physiology, Immunology and Genetics, Katholieke Universiteit Leuven,
Kasteelpark Arenberg 30, 3001 Leuven, Belgium
2Department of Animal Science, University of California, Davis, California, USA
3Lonza Ltd, Muenchensteinerstrasse 38, CH-4002 Basel, Switzerland
Introduction
Dietary β-hydroxy-β-methylbutyrate (HMB) promotes growth in various (farm) animal species
as well as in humans. Multiple positive effects of HMB have been reported such as an increase in
muscle mass with less body fat deposition, increased milk fat production, reduced mortality and
morbidity, and reduced plasma LDL-cholesterol levels (e.g. Nissen and Abumrad, 1997; Nissen and
Sharp, 2003). Furthermore, dietary HMB supplementation has enhancing properties on immune
function in poultry (Peterson et al., 1999). The mechanism by which HMB acts are less clear, yet
several studies indicated that HMB attenuates immune responses mediated by the NF-κB pathway
(Tisdale, 2005; McCarty and Block, 2006). This contrasts with the described enhanced immune
responses, which are associated with metabolic costs. The apparent inconsistency was investigated
in an immunization model in broilers using the following parameters: zootechnical performance,
and the immune response after injection with human serum albumin (HSA). The chickens received
commercial feeds either supplemented or not with 300 mg HMB/kg feed.

One hundred and eighty 1-d-old male broiler chicks (Ross) from a commercial hatchery were divided
over twelve pens in an environmentally-controlled poultry house. Half of the chicks (six pens)
received commercial broiler diets (starter d1-14, finisher d14-39), whereas the other six pens received
the same diets supplemented with 300 mg HMB/kg feed (Lonza Ltd). Body weights and feed intake
per pen were recorded at 14, 29 and 39 d of age. Before weighing, a plasma sample was collected
from ten chickens per diet for analysis. At 3 wk of age, twelve animals of the HMB supplemented
diet and twelve chickens reared on the control diet were weighed, and plasma collected, immunized
with 100 µg HSA in Freund complete adjuvant, or vehicle alone (d 0). During the next 18 d, plasma
was taken and individual body weights were recorded regularly. A second immunization with HSA
in incomplete Freund adjuvant followed at d 14. Plasma hemopexin (HX) and α-1 acid glycoprotein
(AGP) were quantified by rocket gel electrophoresis (Adler et al., 2001). Anti-albumin (HSA)
antibodies were quantified by ELISA. Statistical significant differences between groups/treatments
were determined using Proc GLM for repeated measures; SAS for Windows, version 8e.

Body weight was significantly higher in HMB supplemented chickens over the first two wk pre-
immunization. Furthermore, HMB significantly (P<0.005) attenuated the post-immunization (d 1)
acute phase protein (HX and AGP) response (results not shown). Both phenomena are consistent
with inhibiting NF-κB in both macrophages and in hepatocytes (Grimble, 1998). After one wk
post-immunization, no significant differences in growth were seen in the unsupplemented controls.
However, in the supplemented animals, HMB caused a significant difference between the groups,
depressed body weight gain (Figure 1), and enhanced the anti-HSA IgG response (results not shown).
This suggests NF-κB stimulation, as opposed to the results obtained concerning the early responses.

This indicates a complex mechanism for HMB (Siebenlist et al., 2005). However, HMB is clearly
beneficial for growth under normal circumstances, but appears to play a growth depressing role
under immunological challenge.
*
*
1850 C
C-HSA
1600 HMB *
Growth (g)
HMB-HSA
1350
1100
850
*
600
8 14 16 18
Days post immunization
Figure 1. Immunization with HSA causes growth retardation in the HMB supplemented group,
but not in the non-supplemented controls (C). Significant differences (P<0.05) within one diet are
indicated with an asterisk.
References
Adler, K.L., P.H. Peng, R.K. Peng and K.C. Klasing, 2001. The kinetics of hemopexin and α-acid glycoprotein levels
induced by injection of inflammatory agents in chickens. Avian Dis. 45, 289-296.
Grimble, R.F., 1998. Nutritional modulation of cytokine biology. Nutrition 14, 634-640.
McCarty, M.F. and K.I. Block, 2006. Toward a core nutraceutical program for cancer development. Integr. Cancer
Ther. 5, 150-171.
Nissen, S.L. and N.N. Abumrad, 1997. Nutritional role of the leucine metabolite β-hydroxy-β-methylbutyrate. Nutr.
Biochem. 8, 300-311.
Nissen, S.L. and S.L. Sharp, 2003. Effect of dietary supplements on lean mass and strength gain with resistance exercise:
a meta-analysis. J. Appl. Physiol. 94, 651-659.
Peterson, A.L., M.A. Qureshi, P.R. Ferket and J.C. Fuller,1999. Enhancement of cellular and humoral immunity
in young broilers by the dietary supplementation of beta-hydroxy-beta-methylbutyrate. Immunopharmacol.
Immunotoxicol. 21, 307-330.
Siebenlist, U., K. Brown and E. Claudio, 2005. Control of lymphocyte development by nuclear factor-KB. Nat. Rev.
Immunol. 5, 435-445.
Tisdale, M.J., 2005. The ubiquitin-proteasome pathway as a therapeutic target for muscle wasting. J. Support. Oncol.
3, 209-217.

The comparative study of effects of Immunowall® (prebiotics) and
Avilamycin on amounts of humoral immunity of broiler chickens
A. Zakeri1, M. Fadaei2, S. Charkhkar3 and S. Zakeri4
1Department of veterinary science, faculty of agriculture, Islamic Azad University branch Tabriz,
Tabriz, Iran
2Department of mathematics, Tehran, Iran
3Department of Clinical Science, Faculty of Specialized Science, Islamic Azad University, Science
& Research Campus, Tehran, Iran
4Faculty of Veterinary Medicine, Islamic Azad University branch Tabriz, Tabriz, Iran
Introduction
Nowadays, one of the important goals of the poultry industry and especially with broiler chickens is
increasing the amount of humoral immunity. Chickens with good immunity are resistant to diseases
and respond to vaccination programs perfectly, with at last increased weight gain and improved
FCR. Avilamycin is an antibiotic growth promoter (AGP) that increases weight gain and humoral
immunity but Immunowall is a mannan oligosaccharide (MOS) from prebiotics growth promoter
substance that stimulates weight gain and humoral immunity (Vegad, 2004).

In this study 360 Cobb 500 broiler chickens were divided into three similar groups with 120 chickens
in four replicates of 30 chickens in each group. Immunowall® (1 kg MOS/ton) and avilamycin (100
g/ton) were added to the basal diets in experimental groups Ι and ΙΙ, respectively. The control group
of chickens was fed with the basal diet only. The composition of the basal diet is shown on Table
1. On d 9, 17 and 25 of growth (1 d before and 7 and 14 d after first Newcastle B1 vaccination),
40 chickens were chosen randomly from each group each time and serum antibody titers of these
chickens were measured against Newcastle vaccine by the haemagglutination inhibition (HI) test.
The results were analyzed by Anova, Duncan and LSD with SPSS (version 12).
Table 1. The composition of the basal diet.
Growth periods Composition

(d)
Men, CP, CF, Fat, Met, Lys, Met+Cys, avail. P, Ca,
kcal/kg % % % % % % % %
Starter (0-15) 2900 21.3 3.6 4.5 0.5 1.4 0.91 0.6 1.1
Grower (16-28) 2985 20.2 4.9 5.4 0.42 1.2 0.82 0.6 0.9
Finisher (29-42) 3095 19.2 5.4 6.2 0.59 0.9 0.99 0.52 0.75

Serum antibody titers at the first HI test at d 9 indicated a non statistical difference (P>0.05) between
the three groups because chickens of each three groups were from a same parent stock. Serum
antibody titers by the second (d 17) and third (d 25) HI test indicated a significant effect of the
treatments (P<0.01) (Table 2). Serum antibody titers were higher with the Immunowall treatment
as compared to the avilamycin treatment, both being higher than with the control.

Table 2. Effect of dietary supplementation on specific serum antibody titers after Newcastle B1
vaccination as determined by haemagglutination inhibition (HI) (means and SEM).
Age, d/day post- Treatment P

vaccination
Control Immunowall® Avilamycin
9 (d - 1) 2.08 (0.28) 2.03 (0.23) 2.03 (0.23) 0.535

17 (d + 7) 5.01 (0.32) c 5.27 (0.27) a 5.13 (0.20) b <0.001
25 (d + 14) 5.04 (0.44) c 5.87 (0.47) a 5.61 (0.41) b <0.001
a,b,c Means with unlike superscript letters differ (P<0.05).
Discussion
Significant differences in serum antibody titers between the three groups 7 and 14 d after Newcastle
B1 vaccination indicated that using avilamycin or prebiotics improved humoral immunity. However,
Immunowall revealed to be more immunostimulant than avilamycin. MOS prevents attachment
and colonization of some enteric bacteria, but dose not kill them and it has specific effect on Gram-
negative bacteria with mannose-specific Type-1 fimbriae (Macfarlance and Cummings, 1999). MOS
stimulates gut associated and immunity system by acting as a non-pathogenic microbial antigen and
also it improves non-specific mucosal protection by increasing relative goblet cell numbers and mucus
secretion and increasing colonization of beneficial bacteria (Roberfoid, 2000a and b). Portions of the
cell wall structure of the yeast organism, Saccharomyces contained in MOS has been shown to elicit
powerful antigenic properties. From June 1999, the use of many growth promotor antibiotics were
forbidden while MOS (Immunowall) is a natural substance that dose not have any drug residual
in meat of poultry and it is a suitable alternative for growth promoter antibiotics.
References
Macfarlance, G.T. and J.H. Cummings, 1999. Probiotics and Prebiotics: Can regulating the activities of intestinal
bacteria benefit health. Est. J. Med. 171, 187–191.
Roberfroid, M.B., 2000a. Health benefits of non-digestible oligosaccharides. Adv. Exp. Med. Bull. 427, 211–219.
Roberfroid, M.B., 2000b. Prebiotics, Probiotic: are they functional foods. J. Am. Clin. Nutr. 71 (Suppl. 6), 1682S-
1687S.
Vegad, J.L., 2004. Prebiotics, Probiotics, Acidifiers and Antibiotic growth Promotors. In: Vegad, J.L. (editor), Poultry
diseases a guide for farmers and poultry professional. First edition, 339-346. International Book Distribution Co,
India.

Non-specific resistance state in rats fed a protein free diet
E. Sawosz1, A. Chwalibog2, I. Kosieradzka1, M. Grodzik1, T. Niemiec1 and J. Skomiał1
1Department of Animal Nutrition and Feed Science, Warsaw Agricultural University, Ciszewskiego
8, 02-786 Warsaw, Poland
2Department of Basic Animal and Veterinary Sciences, University of Copenhagen, Groennegaardsvej
3, 1870 Frederiksberg C, Denmark
Introduction
Protein low or free diets used in some slimming procedures affect the immune system. Protein
malnutrition, can affect the metabolism via modification of the hormonal profiles. Dysfunction of
the enzymatic system of cellular integrity may induce apoptosis or necrosis symptoms. The liver
microsomal detoxification enzyme activities and inducibility were shown to be reduced by low
protein diets (Kato et al., 1980) and higher levels of apoptosis were observed in rats fed low protein
diets (Shin, 2003).
The objective of the present study was to determine the effect of short-term application of a protein
free diet on selected parameters of non-specific resistance in rats. These parameters characterise
the effectiveness of killing properties of neutrophils and monocytes, using oxygen dependent and
independent mechanisms.

Twenty-four Wistar male rats (200-250 g) were divided into two groups, fed ad libitum two semi-
synthetic, isoenergetic diets (14.9 MJ ME/kg diet); the control (192 g crude protein/kg diet) and
protein-free diet. The rats were kept in individual cages for 14 d at 22 °C, 60-70% relative humidity
and 12 h lighting. At the end of the experiment the rats were fasted for 12 h and then sedated by
intramuscular ketamine. Blood was sampled from the heart into heparinised tubes, cooled to 4 °C
and analysed. The animals were euthanised by ketamine overdose. Phagocytic activity of neutrophils
and monocytes of whole blood and oxidative burst activity of neutrophils were determined using
Phagotest and Bursttest (Orpegen Pharma, D-69115 Heidelberg, Germany) by flow cytometry.
The results were analysed by one-way analysis of variance using the Statgraphics Plus 4.1. The
significance of differences was evaluated with the Duncan test.

In the present experiment, the number of phagocytising monocytes and neutrophils (percent of
the total number) as well as intensity of phagocytosis (mean fluorescence intensity) via oxygen-
independent mechanisms of killing were not affected (Table 1). However, there was a significant
increase in the number of neutrophils with oxidative burst and enlargement of oxidative burst intensity
after E. coli stimulation (Table 2). Neutrophils play a key role in response to bacterial infection.
Production of O2− and H2O2 into both the intracellular and extracellular milieu is an important
activity of bacterial killing by neutrophils (Hampton et al., 1998). However, reactive oxygen species
released by neutrophils in extended amounts can lead to cell, tissue and organ damage (Clark, 1999).
Furthermore, it was demonstrated that increased intracellular O2− induces proinflammatory cytokine
production in neutrophils through activating p38 MAP kinase and enhancing nuclear translocation
of NF-κB (Mitra and Abraham, 2006).

Table 1. Phagocytic activity of neutrophils and monocytes in peripheral blood of control and protein-
free groups of rats (n = 2 × 12).
Cells Parameters Groups ANOVA
Control Protein free SEM P-value
Neutrophils Phagocytising cells, % 69.8 64.9 4.53 NS

Mean fluorescence intensity, FU1 495.8 595.8 39.73 NS
Monocytes Phagocytising cells, % 16.9 17.3 1.87 NS
Mean fluorescence intensity, FU1 196.3 240.9 31.99 NS
1FU- fluorescence units (4 decades, 1025 channels, log).
Table 2. Oxidative burst activity of neutrophils in peripheral blood of control and protein-free groups
of rats (n = 2 × 12).
Activators Parameters Groups ANOVA
Control Protein free SEM P-value
E. coli Phagocytising cells, % 21.5 39.5 5.61 0.037

Mean fluorescence intensity, FU1 45.6 121.7 9.40 0.000
PMA2 Phagocytising cells, % 35.6 40.2 4.84 NS
Mean fluorescence intensity, FU1 93.2 139.1 8.51 0.001
1FU- fluorescence units (4 decades, 1025 channels, log); 2PMA - phorbol 12-myristate 13-acetate
It can be concluded that short term protein deprivation does not affect monocyte and neutrophil
phagocytosis via an oxygen independent system, but may increase oxidative burst in activated
neutrophils.
References
Clark, R.A., 1999. Activation of the neutrophil respiratory burst oxidase. J. Infect. Dis. 179, S309-S317.
Hampton, M.B., A.J. Kettlt and C.C. Winterbourn, 1998. Inside the neutrophil phagosome: oxidants, myeloperoxidase
and bacterial killing. Blood 92, 3007-3017.
Kato, N., T. Tani and A. Yoshida, 1980. Effect of dietary level of protein on liver microsomal drug-metabolizing
enzymes, urinary ascorbic acid and lipid metabolism in rats fed PCB. J. Nutr. 110, 1686-1694.
Mitra, S. and E. Abraham, 2006. Participation of superoxide in neutrophil activation and cytokine production. BBA
– Molecular basis of Diseases 1762, 732-741.
Shin, S.J., 2003. High levels of apoptosis induced by total body irradiation in mice fed a low protein–low vitamin E
diet. Food Chem. Toxicol. 41, 665-670.

Feeding a lower-protein, amino-acid supplemented diet has no effect on
growth performance but reduces post-weaning diarrhoea in pigs
J.M. Heo1, J.C. Kim2, B.P. Mullan2, D.J. Hampson1, R.H. Wilson3, J. Callesen4, C.F. Hansen4 and
J.R. Pluske1
1School of Veterinary and Biomedical Science, Murdoch University, Murdoch, WA 6150,
Australia
2Animal Research and Development, Department of Agriculture and Food of Western Australia,
Locked Bag No. 4, Bentley Delivery Centre, WA 6983, Australia
3Wandalup Farms, PO Box 642, Mandurah, WA 6210, Australia
4Danish Pig Production, Axeltorv 3, 1609 Copenhagen V, Denmark
Introduction
Diets for piglets are usually formulated to contain 20% to 23% crude protein (CP) (NRC, 1998).
However, average small intestinal digestibility of CP in healthy piglets is between 60-80%, and
this figure can be even lower in unhealthy piglets (Högberg and Lindberg, 2004). Increased protein
entering the large intestine has been associated with a higher incidence of post-weaning diarrhoea
(PWD) (Aumaitre et al., 1995), and one strategy to reduce the incidence of PWD is feeding a diet
with a lower protein content. However, a potential problem when feeding a lower protein diet to
weaner pigs is a reduction in growth (Nyachoti et al., 2006), either because the diets are fed for
too long after weaning and (or) the low CP diets are not balanced for essential amino acids (AA).
The purpose of this study was to investigate the effects of feeding a diet low in protein (18%), but
supplemented with essential AA, for different periods of time after weaning on growth performance
and PWD.

Sixty mixed-sex pigs (Large White × Landrace) aged 21 d and weighing 6.1 ± 0.13 (SEM) kg were
used. Pigs were individually allocated in a completely randomised design to one of five treatments,
with 12 pigs per treatment. Pigs were housed in an environmentally controlled room in wire-mesh
crates with slatted flooring. The five treatments were the following: (i) high protein diet (24% CP)
for 14 d (HP); (ii) low protein AA supplemented diet (18% CP) for 5 d (LP5); (iii) LP for 7 d (LP7);
(iv) LP for 10 d (LP10) and (v) LP for 14 d (LP14). A Phase-II diet (20% CP) was fed to pigs at
the conclusion of each treatment. None of the diets contained antimicrobial compounds. The low
protein diet contained lysine, methionine, tryptophan, threonine, isoleucine and valine up to the
levels recommended by NRC (1998). The performance of pigs was monitored after weaning and
diarrhoea was recorded in the first 14 d. Repeated-measures ANOVA using GLM procedures was
used to analyse the results.

Lowering the dietary CP level while formulating diets for six essential AA had no negative effects
on growth performance (P>0.05) after weaning, or up to d 106 of age (mean weight 81.7 ± 1.03
(SEM) kg for all treatment groups; P>0.05). Nyachoti et al. (2006) reported that growth performance
decreased after weaning as dietary CP was lowered (19% to 17%), however it has been suggested
that supplementation of isoleucine and valine to the diets, as was done in the present study, is needed
to maintain optimal performance (Figueroa et al., 2002). Pigs fed LP5, LP7, LP10 and LP14 had
less diarrhoea at d 8 and 9 (P=0.04) after weaning. Furthermore, feeding a low protein diet was
associated with less diarrhoea between d 8 and 12 (HP vs. LP; P<0.01 and P=0.04, respectively).
No significant differences (P>0.05) were found between male and female pigs. Our results suggest

that feeding a low-protein AA supplemented diet for a short period of time after weaning can be
used as a strategy to minimise diarrhoea without depression of growth performance.
Table 1. Effect of a low-protein diet on pig performance and incidence of diarrhoea at various time
points after weaning.
Item Diets SEM P value (Contrasts)
HP LP5 LP7 LP10 LP14 T1 HP HP HP HP HP

vs. vs. vs. vs. vs.
LP5 LP7 LP10 LP14 LP
Average daily gain, g/d

d 1-7 59 60 63 36 53 8.5 0.86 0.97 0.86 0.40 0.83 0.79
d 8-14 237 257 214 190 234 9.7 0.23 0.51 0.45 0.12 0.92 0.57
d 1-28 293 298 305 283 282 7.9 0.88 0.85 0.62 0.69 0.68 0.97
Average daily feed intake, g/d
d 1-7 131 115 109 92 103 9.7 0.79 0.61 0.48 0.22 0.39 0.28
d 8-14 326 327 304 258 315 10.5 0.20 0.98 0.50 0.04 0.74 0.33
d 1-28 450 451 470 425 440 11.1 0.80 0.96 0.57 0.50 0.80 0.92
Feed conversion, g/g
d 1-7 1.68 1.78 1.71 1.71 1.67 0.023 0.58 0.18 0.74 0.78 0.82 0.56
d 8-14 1.42 1.31 1.47 1.37 1.42 0.033 0.60 0.26 0.68 0.65 0.94 0.69
d 1-28 1.55 1.53 1.54 1.47 1.56 0.016 0.42 0.58 0.78 0.11 0.87 0.49
Incidence of diarrhoea
d 1-3 0.0 0.0 0.0 0.0 0.0 0.00
d 4-7 0.3 0.0 0.0 0.1 0.3 0.04 0.12 0.06 0.06 0.12 1.00 0.11
d 8/9 0.4 0.0 0.2 0.1 0.0 0.05 0.04 0.01 0.10 0.03 0.01 <0.01
d 10-12 0.2 0.0 0.0 0.0 0.0 0.03 0.42 0.12 0.12 0.12 0.12 0.04
1T = treatment effect.
References
Aumaitre, A., J. Peiniau and F. Madec, 1995. Digestive adaptation after weaning and nutritional consequences in the
piglet. Pig News Info 16, 73N - 79N.
Figueroa, J.L., A.J. Lewis, P.S. Miller, R.L. Fischer, R.S. Gomez and R.M. Diedrichsen, 2002. Nitrogen metabolism and
growth performance of gilts fed standard corn-soybean meal diets or low-crude protein, amino acid-supplemented
diets. J. Anim. Sci. 80, 2911-2919.
Högberg, A. and J.E. Lindberg, 2004. Influence of cereal non-starch polysaccharides and enzyme supplementation on
digestion site and gut environment in weaned piglets. Anim. Feed Sci. Technol. 116, 113-128.
NRC, 1998. Nutrient requirements of swine. National Academy Press, Washington, DC.
Nyachoti, C.M., F.O. Omogbenigun, M. Rademacher and G. Blank, 2006. Performance responses and indicators of
gastrointestinal health in early-weaned pigs fed low-protein amino acid-supplemented diets. J. Anim. Sci. 84,
125-134.

Protein balance of lambs infected with Haemonchus contortus and fed
tanniniferous sainfoin (Onobrychis viciifolia)
A. Scharenberg1, Y. Arrigo1, F. Heckendorn2, H. Hertzberg2, A. Gutzwiller1, H.D. Hess1, M. Kreuzer3
and F. Dohme1
1Agroscope Liebefeld-Posieux Research Station ALP, Tioleyre 4, 1725 Posieux, Switzerland
2Research Institute for Organic Farming (FiBL), Ackerstrasse, 5070 Frick, Switzerland
3ETH Zurich, Institute of Animal Science, 8092 Zurich, Switzerland
Introduction
By decreasing ruminal proteolysis and increasing the level of free essential amino acids in the
blood plasma, condensed tannins (CT) can have a positive influence on protein supply in ruminants
(Waghorn et al., 1994). This effect could improve the resilience of sheep infected with gastrointestinal
nematodes by compensating for the associated protein loss (Coop and Holmes, 1996). In addition,
CT as plant secondary compounds, and CT-containing plants, could have direct anthelmintic
properties (Heckendorn et al., 2006). The lack of knowledge concerning the mode of action of CT
in ruminants infected with gastrointestinal nematodes represents one constraint for this alternative
strategy controlling parasitism in ruminant livestock to be applied. The aim of the present experiment
was, therefore, to evaluate the effect of a CT-plant (sainfoin) with known anthelmintic properties
on the nitrogen balance of lambs artificially infected with Haemonchus contortus. In order to be
able to separate between CT and protein effects, sainfoin (197 g crude protein/kg dry matter) was
also tested when treated with the CT-binding agent polyethylene glycol (PEG), and was compared
with a low-CT lower protein (132 g/kg) grass-clover diet. Uninfected lambs fed the grass-clover
mixture served as control.

Twenty-four female lambs of the Swiss White Hill breed (30.5 ± 2.2 kg) were assigned to four
treatment groups differing in infection and forage type (n=6). At the start of the experiment, 3 groups
of lambs were infected with larvae of the blood-sucking abomasal nematode H. contortus. During the
first 4 wk, when the infection was allowed to fully establish, all animals received a mixture of grass
and white clover. Subsequently, infected lambs were fed dehydrated Onobrychis viciifolia (sainfoin;
CT-content: 36 g/kg dry matter) without and with PEG (PEG: 100 g/d), or continued to receive the
grass-clover mixture. Uninfected lambs also continued to receive the grass-clover mixture. The
daily ration consisted of 66 g organic matter from forage per kg metabolic body weight and of 20 g
of a mineral mix per d. During wk 7 post infection (p.i.) and after an adaptation period of 2 wk to
the experimental diets, feed refusals and faecal and urinary excretions were individually recorded
and samples were taken and pooled over the week for analysis of nitrogen. Blood was sampled
twice and plasma was analysed for the concentration of free amino acids. The development of the
H. contortus infection was recorded through weekly determinations of the egg count in the faecal
dry matter (FEC). The packed cell volume in blood (PCV) was measured to monitor the severity
of infection. The effects of CT, protein level, and infection were evaluated with a contrast model of
SAS (GLM). In detail, contrasts compared differences (i) between sainfoin with and without PEG,
(ii) between PEG-treated sainfoin and grass-clover mixture in infected lambs, and (iii) between
infected and uninfected lambs both receiving the grass-clover mixture.

Before the feeding of the experimental diets started (wk 4 p.i.), the FEC of the infected lambs averaged
at 15.9 ± 5.94 ×103 eggs/g while the uninfected lambs remained free of H. contortus. The PCV of

the infected groups averaged 0.27 ± 0.022 L/L and was lower (P<0.001) compared to that of the
uninfected group (0.32 ± 0.015 L/L). The FEC and PCV showed no significant differences among
the 3 treatments after feeding the experimental diets and accounted for 10.5 ± 8.21 ×103 eggs/g
and 0.31 ± 0.032 L/L on average at the end of wk 7 p.i.. Dry matter intake did not differ among
treatments, resulting in a higher N-intake for the lambs fed PEG-treated sainfoin compared to the
grass-clover fed lambs due to the differences in feed N-content. The feeding of the sainfoin with
PEG in comparison to grass-clover increased (P<0.001) urinary and total N-excretion, but also N-
retention. Treating sainfoin with PEG lowered faecal N-excretion (P<0.05) but had no influence on
urinary N-excretion. However, the proportion of urinary N (mg/g N excreted) was higher (P<0.05)
with PEG-treated sainfoin than with untreated sainfoin. Between infected and not infected lambs
fed with grass-clover, no difference (P>0.05) in N-balance was found. Plasma levels of arginine
were lower (P<0.05) and those of threonine and tryptophan were higher (P<0.05) in uninfected
compared to infected lambs fed grass clover. The CT-effects (+/-PEG) in sainfoin-fed lambs were
very low compared to those observed in other experiments (e.g., Waghorn, et al., 1994), which
may suggest that the CT in the tested sainfoin were of low activity. This could also have reduced
the anthelmintic potential recently demonstrated for sainfoin (Heckendorn et al., 2006). The lack
of differences in the N-balance between infected and uninfected lambs contrasts clearly with the
effects of gastrointestinal nematode infections on protein metabolism described by Louvandini et al.
(2006). A possible explanation for these contrasting results could be that in the study of Louvandini
et al. (2006) two parasites (Trichostrongylus colubriformis and H. contortus) were investigated.
Furthermore, the infection might not affect absorption but utilisation of the absorbed N-compounds
(Colditz, 2003).
In conclusion, the unexpected lack of an influence of feeding sainfoin to infected lambs suggests
that the possibilities of enforcing the resilience of hosts infected with H. contortus by improving the
supply with protein, either directly or via feeding CT, are not always efficient. However, it might be
that the exertion of the effect needs a longer period of time of feeding a CT-rich plant.
References
Colditz, I.G., 2003. Metabolic effects of host defence responses during gastrointestinal parasitism in sheep. Aust. J.
Exp. Agr. 43, 1437-1443.
Coop, R.L. and P.H. Holmes, 1996. Nutrition and parasite interaction. Int. J. Parasitol. 26, 951-962.
Heckendorn, F., D.A. Häring, V. Maurer, J. Zinsstag, W. Langhans and H. Hertzberg, 2006. Effect of sainfoin (Onobrychis
viciifolia) silage and hay on established populations of Haemonchus contortus and Cooperia curticei in lambs.
Vet. Parasitol. 142, 293-300.
Louvandini H., C.F.M. Veloso G.R. Paludo, A. Dell’Porto, S.M. Gennari and C.M. McManus, 2006. Influence of
protein supplementation on the resistance and resilience on young hair sheep naturally infected with gastrointestinal
nematodes during rainy and dry seasons. Vet. Parasitol. 137, 103-111.
Waghorn, G., I.D. Shelton, W.C. McNabb and S.N. McCutcheon, 1994. Effects of condensed tannins in Lotus
pedunculatus on its nutritive value for sheep. 2. Nitrogenous aspects. J. Agr. Sci. 123, 109-119.

Venous blood gas in Holstein steers fed diets with different concentrate
to alfalfa hay ratios
A.R. Vakili, M. Danesh Mesgaran, A.R. Heravi Mousavi and S. Danesh Mesgaran
Dept. of Animal Science, Excellence Center for Animal Science, Faculty of Agriculture, Ferdowsi
University of Mashhad, P O Box 91775-1163, Mashhad, Iran
Introduction
In ruminants, feeding diets high in grain and other highly fermentable carbohydrates increases the
risk of ruminal and blood acidosis. Although, ruminal pH varies considerably within a d, cows
possess a highly developed system to maintain ruminal pH within a physiological range. However,
if the acid production from fermentation is more than the system which can be buffered, ruminal
pH compensation fails and it may drop drastically (Krause and Oetzel, 2005). An arterial and/or
venous blood gas (ABG and VBG, respectively) is a clinical tool for determining pulmonary and
metabolic status in animals. However, most blood tests are done on a sample of blood taken from
a vein due to the following: (i) Collecting blood from an artery is more painful than collecting it
from a vein because the arteries are deeper and have more nerves, (ii) Artery may be inaccessible
due to periarterial tissues (overlying muscle, connective tissue, or fat). The importance of venous
blood gas measurements in the ruminant is useful to predict some health problems such as ruminal
parakeratosis, erosion ad ulceration of the ruminal epithelium (Garry, 2002), laminitis, sole abscesses
and ulcer (Nocek, 1997). Therefore, the determination of ABG or VBG in ruminal acidosis can
prevent some health problems (Garry, 2002). ABG and VBG provide a direct measurement of
partial blood pressures of carbon dioxide (PCO2) and oxygen (PO2), hydrogen ion activity (pH),
total hemoglobin (Hb total), oxyhemoglobin saturation (HbO2) and bicarbonate ion concentration
(HCO3-). The objective of the present experiment was to investigate the effect of diets providing
different concentrate to alfalfa hay ratios on partial blood pressures of carbon dioxide (PaCO2) and
oxygen (PO2), hydrogen ion activity (pH), oxyhemoglobin saturation (HbO2) and bicarbonate ion
concentration (HCO3-) in Holstein steers.

Holstein steers (initial body weight= 261 ± 15 kg, n=30) were adapted to experimental diets for
one wk. Then, for 120 d, steers were fed 10 kg of DM of diets differing in concentrate (155 g
CP/kg of DM; 30% maize, 34% barley, 8% soybean meal, 5% sugar beet pulp, 10% wheat bran,
12% cottonseed meal, 0.3% CaCO3, 0.5% mineral and vitamin premix, 0.2% salt) to alfalfa hay
(155 g CP/kg of DM) ratios as 60:40 (C60:L40) and 80:20 (C80:L20) in a completely randomized
design. The animals were housed in individual pens, and fed the experimental diets as total mixed
rations twice daily at 08.00 and 20.00 h. They had free access to drinking water. At d 60 and 120
of the experiment, blood samples were taken from the jugular vein 4 h after the morning feeding.
Samples were analyzed for VBG (blood pH, PO2, PCO2, HbO2 and HCO3-) by an automatic blood
gas system (AVL 995, Switzerland). Data of sampling d were analyzed as repeated measures using
the PROMIX of SAS (y = Mean + Treatment + Animal (Treatement) + Time + Treatment × Time
+ residual) and the means were compared by the Duncan (P<0.05) test.

Data of pH and venous blood gases in Holstein steers fed diets differing in concentrate: alfalfa hay
ratios are presented in Table 1. Blood pH, PO2, PCO2, HbO2 and HCO3- were all similar among the
diets at each sampling d (P>0.05). However, PO2 was numerically higher in animals fed C80:L20
compared with those fed C60:L40 on d 120 of the experiment (35.33 vs. 38.37 mmHg, P=0.05). The

results of the present study demonstrated that the increasing of concentrate in the diets of Holstein
steers did not significantly affect blood pH. In mixed metabolic acidosis the rate of increasing in
plasma bicarbonate appears to be a function of the rate of increasing in plasma PCO2. In addition, for
compensating respiratory acidosis, increasing in plasma [HCO3−] paralleled the increase in plasma
[PCO2]. The results of the present study indicate that blood HCO3− (mEq/L) and PCO2 (mmHg)
did not significantly change when steers were fed high concentrate diets. However, HCO3- was
significantly affected by time (P<0.05). Therefore, it was concluded that the increasing of concentrate
from 60 to 80 percent could not cause a mixed metabolic acidosis in steers in our conditions.
Table 1. Venous blood gases in Holstein steers fed diets differing in concentrate: alfalfa hay
ratios.
Item Concentrate:alfalfa hay ratio1 Treatment effect Time effect
C60:L40 C80:L20 60 d 120 d
60 d 120 d 60 d 120 d SEM2 P3 SEM P
Blood pH 7.33 7.38 7.35 7.36 0.01 0.97 0.01 0.05

PO2, mHg 37.31 35.33 35.41 38.37 1.17 0.76 1.02 0.67
PCO2, mmHg 56.95 55.85 57.28 65.80 2.63 0.19 2.35 0.22
HCO3-, mEq/L 29.61 31.50 30.80 35.24 1.23 0.19 1.1 0.03
O2 saturation, % 62.34 60.23 60.67 64.16 1.84 0.74 1.46 0.05
1C :L = 60% concentrate + 40% alfalfa hay, C :L = 80% concentrate + 20% alfalfa hay
60 40 80 20
2SEM= Standard Error of Mean; 3 P=Probability
References
Garry, F.B., 2002. Indigestion in ruminants. In: Smith, B.P. (editor), Large Animal Internal Medicine, Mosby-Year
Book. Mosby, St. Louis, Missouri, 722–747.
Krause, M.K. and G.R. Oetzel, 2006. Understanding and preventing sub acute ruminal acidosis in dairy herds: A review.
Anim. Feed Sci. Technol. 126, 215–236.
Nocek, J.E., 1997. Bovine acidosis: implications on laminitis. J. Dairy Sci. 80, 1005–1028.
This research was supported by a grant from Ferdowsi University of Mashhad and Excellence
Center for Animal Science.

Long-term physical activity does not influence the glycemic index in women
S. Mettler, P. Vaucher, P.M. Weingartner, C. Wenk and P.C. Colombani
Department of Agricultural and Food Sciences, ETH Zurich, Universitaetstr. 2, LFW B 57, 8092
Zurich, Switzerland
Introduction
The glycemic index (GI) of a food is a percentage representing the incremental area under the blood
glucose response curve (IAUC) relative to the blood glucose response curve of a reference food
(glucose or white bread) (FAO/WHO, 1998)). One of the characteristics of the GI is that it describes
a specific food and is believed not to be related to subject-specific factors such as age, gender, body
mass index (BMI), ethnicity or absolute glycemic response (Wolever, 2003). An important subject-
specific factor, physical activity, however, had not been investigated since the introduction of the
GI. The aim of this study was to investigate if the GI is independent of the physical activity state
of the subjects.
Methods
Seventeen sedentary (SE, age 24.2 ± 4.6 y, 0 ± 0 training session per wk) and 19 endurance trained
(ET, age 23.7 ± 4.7 yr, 6.8 ± 2.4 training sessions per wk) women participated in this study. The
study was carried out in a random cross-over order and consisted of a duplicate assessment of the
reference food glucose and of a commercially available breakfast cereal. Each test meal provided
50 g carbohydrates. After measuring the fasting (time = 0 min) values (capillary and venous glucose
and insulin) the subjects consumed the test meals. Further values were taken after 15, 30, 45, 60,
90 and 120 min and the incremental area under the curve (IAUC) was calculated according to the
standard procedure (Brouns et al., 2006).
Results
The GI calculated from both the capillary (mean ± standard error: 61.4 ± 4.3 and 69.5 ± 4.7 for SE
and ET respectively, P=0.21) and venous blood glucose (60.8 ± 8.1 and 64.4 ± 6.2, P=0.72) did not
differ significantly between SE and ET, nor was there a difference with the insulin index (P=0.75).
The capillary and venous glucose and the insulin kinetics showed a significant group × time effect
(P<0.001), indicating that the kinetics of all metabolites differed significantly between the groups
(Figure 1). The fasting values did not differ between the groups for any analyte. A significant group
× time effect was found for all analytes (P<0.001).
Discussion
The GI did not differ between the groups, indicating that the GI was independent of the physical
activity state of the subjects. This was in contrast to previous results obtained with male subjects
(Mettler et al., 2006; Englert et al., 2006), but in agreement with the original assumption that the
GI is independent of subject specific factors (Wolever, 2003; Brouns et al., 2006). The lack of a GI
difference according to the training state with the female subjects was clear and cannot be related to
a poor statistical power. With 17 and 19 subjects in the two groups of the present study it was better
powered than the studies with male subjects (10 to 12 subjects per group). Recent results indicate that
acute exercise the day before a test does not influence GI determination (Englert et al., 2006). The
results of the present study indicate that the long-term endurance training behaviour of the subjects
does not influence the GI too, at least with women. However, when regarding the absolute values
and the kinetics of glucose and insulin, the ET subjects show significantly blunted values compared

to the SE subjects. This might not be without clinical relevance with respect to developing glucose
intolerance or diabetes in advanced age of the presently young subjects.
Figure 1. Relative values (standardised against fasting value) of capillary and venous blood glucose
and insulin.
References
Brouns, F., I. Bjorck, K.N. Frayn, et al., 2006. Glycaemic index methodology. Nutr. Res. Rev. 18, 145-171.
Englert, V., K. Wells, W. Long, M.S. Hickey and C.L. Melby, 2006. Effect of acute prior exercise on glycemic and
insulinemic indices. J. Am. Coll. Nutr. 25, 195-202.
FAO/WHO, 1998. Carbohydrates in human nutrition: Report of a Joint FAO/WHO Expert Consultation. Rome,
FAO/WHO.
Mettler, S., C. Wenk and P.C. Colombani, 2006. Influence of training status on glycemic index. Int. J. Vitam. Nutr.
Res. 76, 39-44.
Mettler, S., F. Lamprecht-Rusca, N. Stoffel-Kurt, C. Wenk and P.C. Colombani, 2007. The influence of the subjects’
training state on the glycemic index. Eur. J. Clin. Nutr. 61, 19-24.
Wolever, T.M., 2003. Carbohydrate and the regulation of blood glucose and metabolism. Nutr. Rev. 61, S40-S48.

4C. Nitrogen digestion and recycling

Source of nitrogen for pig gut microbes: effect of feeding fermentable fiber
A.J. Libao-Mercado1,2, C.L. Zhu1, J.P. Cant1, H.N. Lapierre3, J.N. Thibault4, B. Sève4, M.F. Fuller5
and C.F.M. de Lange1
1Department of Animal and Poultry Science, University of Guelph, Guelph, ON, Canada
2Cargill Animal Nutrition Phils. Inc, Pulilan, Bulacan, Philippines
3Agriculture and Agri-Food Canada, Sherbrooke, Quebec, J1M 1Z3 Canada
4INRA, UMR SENAH, Centre de Recherches de Rennes, 35590 Saint Gilles, France
5State University of New York, Stony Brook, NY, USA
Introduction
The presence of microbes in the digestive tract has implications for amino acid (AA) utilization in
pigs. Pigs are capable of absorbing AA synthesized by microbes in the upper gut (Torrallardona et
al., 2003). Whether microbes make a net contribution to the AA supply of the host depends on what
sources of nitrogen are used to synthesize microbial protein, and the metabolic costs associated with
carriage of gut microbes. These costs are related to degradation of AA via microbial fermentation
in the upper gut and AA losses associated with synthesis and secretion of endogenous gut proteins.
Quantification of these AA losses becomes more important when microbial activity is stimulated,
such as when feeding highly fermentable fibers to pigs. Our objectives were to estimate the relative
contributions of various sources of nitrogen (urea, ammonia (NH3), endogenous protein (EP) and
dietary protein (DP)) to microbial protein production, to estimate the relative contribution of urea
and non-urea sources (DP plus EP) to ileal digesta NH3, and to determine if feeding additional
fermentable fiber has an effect on these aspects of nitrogen metabolism in the pig.

Eight Yorkshire barrows, in 2 equal batches, were fitted with T cannulas at the distal ileum and
catheters in the jugular veins, and were assigned to either a cornstarch and soybean meal-based
diet (Control; 19% CP) or the control diet with 12% pectin (Pectin). Pigs were adapted to diets for
11 d prior to a 4-d continuous intravenous infusion of a saline solution that provided 0.60 and 0.39
mmoles/kg BW per d of L-1-13C valine and 15N2 urea, respectively. Microbes were isolated from ileal
digesta according to Li et al. (2003). Relative contributions of urea, NH3, and EP to microbial valine
at the ileum were estimated by relating enrichment of microbial valine (13C or 15N) to enrichment of
plasma urea, digesta NH3 or plasma valine, respectively. The relative contribution of DP to microbial
valine was estimated as the difference between unity and the sum of the relative contributions of
NH3 and EP. The proportion of digesta NH3 produced by microbial fermentation of DP and EP was
also estimated as the difference between unity and the relative contribution of urea to digesta NH3,
estimated by relating 15N enrichment of digesta NH3 to plasma 15N2 urea enrichment. Values were
analyzed using the GLM procedure of SAS with diet and batch as sources of variation.

Valine synthesized by enteric microbes, based on utilization of 15NH3, contributed 7.2% or less to
microbial valine (Table 1). For both dietary treatments, more than 92% of microbial valine was derived
directly from preformed valine from EP and DP. Across dietary treatments, DP contributed more
to microbial valine than EP (64.5 vs. 30.8%; P<0.05). The addition of pectin to the diet increased
the contribution of digesta NH3 and reduced the contribution of DP to microbial valine (P<0.05).
A large proportion of NH3 in ileal digesta was produced from fermentation of DP or EP (71.5%
for Control and 80.9% for Pectin; SE=4.3; P=0.19); the remainder was generated from microbial
hydrolysis of urea.

Based on the approach used in this study, the synthesis of valine by microbes is likely overestimated,
due to the potential contribution of transamination to 15N in pre-formed valine. Synthesis of valine by
microbes is underestimated only if there is substantial channeling of NH3 from DP or EP to microbial
valine synthesis. Regardless of these considerations, the current data provide evidence that DP and
EP contribute substantially to microbial AA in the upper gut of pigs. This was consistent with in
vitro studies with rumen microbes that have shown large reductions in the use of NH3 for microbial
AA synthesis, when peptides or AA are available (Atasoglu et al., 1999). The current study also
demonstrates that AA are lost via microbial degradation of AA in the upper gut. The latter is not
considered when using ileal digestible AA intake as an estimate of available AA intake in pigs.
Table 1. Relative contributions (%) of various sources of nitrogen to microbial valine at the distal
ileum of pigs fed diets without (Control) or with additional fermentable fiber (Pectin).
Control (n=3) Pectin (n=4) SD P value
Ammonia 3.2 7.2 1.4 <0.05

Urea 0.8 1.4 0.3 <0.10
Dietary + endogenous 2.4 5.9 1.3 <0.05
Pre-formed valine 96.8 92.8 1.4 <0.05
Endogenous 27.8 32.7 3.1 0.10
Dietary 69.0 60.0 3.9 <0.05

Microbial protein in the upper gut of pigs is synthesized mainly from AA that are derived directly
from DP and EP. A large proportion of NH3 in ileal digesta is produced via microbial fermentation of
DP and EP. In the present study, dietary inclusion of fermentable fiber (pectin) led to minor changes
in the relative contributions of the different sources of nitrogen to microbial protein synthesis, or to
NH3 in ileal digesta. It is, however, important to consider that the absolute rate of microbial protein
synthesis and fermentation may have increased, and thus AA losses, when feeding pectin. These AA
losses should be quantified and considered when estimating the available AA supply for pigs.
References
Atasoglu, C., C. Valdés, C.J. Newbold and R.J. Wallace, 1999. Influence of peptides and amino acids on fermentation
rate and de novo synthesis of amino acids by mixed micro-organisms from the sheep rumen. Br. J. Nutr. 81,
307-314.
Li, M., J. Gong, M. Cottrill, H. Yu., C.F.M. de Lange, J. Burton and E. Topp, 2003. Evaluation of QIAamp DNA Stool
Mini Kit for ecological studies of gut microbiota. J. Microbiol. Methods 54, 13-20.
Torrallardona, D., C.I. Harris and M.F. Fuller, 2003. Lysine synthesized by the gastrointestinal microflora of pigs is
absorbed mostly in the small intestine. Am. J. Physiol. Endocrinol. Metab. 284, E1177-E1180.

Effects of metabolizable protein supply on nitrogen metabolism and
recycling in lactating dairy cows
D. Valkeners1, H. Lapierre1, J. Marini2 and D.R. Ouellet1
1Agriculture and Agri-Food Canada, STN Lennoxville, J1M 1Z3, Sherbrooke, QC, Canada
2Baylor College of Medecine, Mail Stop BCM320, Houston, TX 77030-2600, USA
Introduction
Farm operations need to be carried out with minimum environmental impacts. In dairy cows, an
achievable target is to increase the transfer of N intake to milk protein thereby diminishing excretion
of N. The objective of the present study was to determine the effect of metabolizable protein (MP)
supply on N metabolism, including N balance, duodenal N flows and urea kinetics.

Four cows fitted with closed-T duodenal cannula were used in an incomplete replicated 3×3 Latin
square design (28-d experimental periods). Cows were fed a total mixed ration (49.4% grass silage
and 50.6% concentrate on DM basis) in 12 equal meals per d. Three concentrates were formulated
to provide NEL according to requirements (126 MJ/d) and to supply incremental amounts of MP:
1430 (Low), 1920 (Medium) and 2160 g MP/d (High), which correspond to 72, 98 and 111% of
the MP requirements for a cow producing 30 kg/d of milk (NRC, 2001). After a 13 d-adaptation
period, total collection of urine and faeces were performed from d 14 to 20. On d 13, cows were
catheterized in one jugular vein and, on d 14, were infused with 15N2-urea (0.5 mmol/h) for 72 h
to determine urea kinetics. Samples were collected on d 14 to measure the natural abundance, and
on d 16 to measure the isotopic enrichment of 15N15N and 15N14N urea in urine and total 15N in
faeces. Urea kinetics were calculated as described by Lobley et al. (2000). On d 27 and 28, samples
of duodenal digesta (4 per d) were collected through a closed-T cannula to determine total digesta-
N and microbial-N flows, using chromium sesquioxide as an indigestible marker and purines as a
microbial marker. Data were analysed using the MIXED procedure of SAS (SAS Inst. Inc., Cary,
NC), with period and MP level as fixed factors, and cow as random factor. Linear and quadratic
orthogonal polynomial contrasts (estimated to account for the unequal interval between treatments)
were used to compare treatments.
Results
Increasing MP supply increased linearly urinary and faecal N excretion, whereas milk N increased
only from Low to Medium. Indeed, 0.61 and 1.25 of the incremental supply of N was excreted
between Low vs. Medium and Medium vs. High, respectively. Proportion of MP transferred into
milk protein decreased linearly from 0.61 to 0.43, with increasing MP supply. Increasing MP
increased linearly total N and microbial N duodenal flows. Urea entry rate (UER) increased linearly
with the MP supply and is a major cross-road of N metabolism as it represented a high proportion
of digested N, increasing from 0.46 to 0.73. Only 0.086 of UER was excreted in the urine of cows
receiving the lower level of MP and this proportion increased with increasing MP supply (0.249
and 0.341 for Medium and High, respectively). Increasing MP supply increased the amount of urea
recycled in the gastrointestinal tract (GER). Contribution of GER to the gut-N pool was important
since it represented between 0.42 and 0.48 of the digested N, but this proportion was not affected
by treatments. The proportion of GER that returned to the ornithine cycle increased, however, with
increasing MP supply, but since the proportion excreted in faeces decreased, the fraction of N recycled
used for anabolism remained fairly constant across diets and averaged 0.51.

Table 1. Effect of metabolizable protein (MP) supply on urinary and faecal N excretion, milk N,
duodenal flows of total and microbial N, and urea kinetics in lactating dairy cows.
Item MP levels1 SEM Contrasts
Low Medium High Linear Quadratic
N intake, g/d 350.3 459.8 505.1 8.98 <0.001 0.051

Urinary N, g/d 63.9 119.8 161.1 4.59 <0.001 0.121
Faecal N, g/d 139.0 149.6 164.8 5.38 0.002 0.087
Milk N, g/d 135.1 146.3 146.4 2.85 0.014 0.230
N balance, g/d 12.3 44.1 32.8 6.57 0.020 0.037
Duodenal flow:
- Total N, g/d 383.5 509.5 574.2 9.48 <0.001 0.933
- Microbial N, g/d 304.9 360.5 376.9 8.07 0.003 0.495
UER2, g/d 97.4 200.2 249.8 10.67 <0.001 0.909
UUN3, g/d 7.4 50.1 85.4 5.78 0.001 0.255
u4 0.086 0.249 0.341 0.0157 <0.001 0.740
GER5, g/d 90.0 150.1 164.3 6.80 <0.001 0.151
ROC6, g/d 27.8 61.7 68.0 5.31 0.002 0.226
UFE7, g/d 13.2 15.5 14.7 1.41 0.152 0.240
ANABOL8, g/d 48.9 72.9 81.6 2.89 0.001 0.565
r9 0.311 0.408 0.413 0.0203 0.011 0.247
f10 0.146 0.103 0.088 0.0069 <0.001 0.061
a11 0.543 0.489 0.499 0.0232 0.096 0.291
11430, 1920 and 2160 g MP/d, respectively for Low, Medium and High; 2urea entry rate; 3urinary urea-N excretion;
4proportion of the urea produced excreted in the urine; 5urea gastrointestinal entry rate; 6N from urea returning to
the ornithine cycle; 7N from urea excreted in the faeces; 8N from urea used for anabolic purposes; 9proportion of
GER returned to the ornithine cycle; 10proportion of GER excreted in faeces; 11proportion of GER used for anabolic
purposes.
Conclusion
The increased efficiency of transfer of MP into milk protein at lower MP supply was achieved through
a decreased UER and a higher fraction of GER/UER, leading to minimal excretion of urea-N. Despite
this decreased N excretion, feeding Low MP diet yet decreased lactation performance. To maintain
production when low MP diets are fed, further research is needed to define strategies to increase the
rumen capture of GER with decreasing ROC and/or UFE.
References
Lobley, G.E., D.M. Bremner and G. Zuur, 2000. Effects of diet quality on urea fates in sheep as assessed by refined,
non-invasive [15N15N]urea kinetics. Brit. J. Nutr. 84, 459-468.
National Research Council, 2001. Nutrient Requirements of Dairy Cattle. 7th edition. Natl. Acad. Press, Washington,
DC.

Effect of ruminal degradable nitrogen deficit on nitrogen metabolism in
growing double-muscled Belgian Blue bulls fed beet pulp silage based diet
D. Valkeners, Y. Beckers, A. Lindebrings and A. Théwis
Gembloux Agricultural University, Passage des Déportés 2, 5030 Gembloux, Belgium
Introduction
In ruminants, urea recycling provides a mechanism by which N may be salvaged into bacterial
matter that may be digested by the animal to supply amino acids for production purposes instead
of excreting it in the urine. Therefore, the urea-N salvage mechanism could be a very important
key to increase N efficiency in ruminants (Stewart and Smith, 2005). The objective of the present
study was to determine the effect of ruminal degradable N (RDN) level on nutrient digestion and N
metabolism in growing bulls fed beet pulp silage based diet in order to minimise N excretion in the
environment and maximise N retained by the animals.

Six double-muscled Belgian Blue bulls initially weighing 359 ± 19 kg and fitted with a ruminal
cannula and a T-type cannula at the proximal duodenum were used in the study. Bulls received a
diet made up of 55% beet pulp silage and 45% concentrate on DM basis at an intake level of 85 g
DM/kg0.75 in two equal meals (08:30 and 20:30). Three concentrates were formulated to provide
similar amounts per kg DM of intestinal digestible protein (84 g DVE), NE for fattening (7.2 MJ),
and fermentable OM (577 g FOM) according to the Dutch system (CVB, 2000), but different levels
of RDN by a modification of the urea contribution between treatments. In this way, 2 diets, one
characterised by an RDN deficit (LRDN) and the other by an RDN excess (HRDN), were compared
to a diet providing the optimal RDN supply according to the Dutch system (Control). The RDN:FOM
ratios of the 3 diets reached 19.8, 24.0 and 28.1 g/kg, for LRDN, Control and HRDN, respectively
and CP contents 133, 142 and 151 g/kg DM. The 3 experimental diets satisfied more than 95% of
the requirements for double-muscled Belgian Blue bulls with an average daily gain of 1.5 kg/d (De
Campeneere et al., 2001). The bulls were allocated to three treatment periods in a replicated 3×3
Latin square design. After a 21-d adaptation period, N balance and digesta flows (Cr2O3 marker) at
the duodenum and the faeces were determined at each period according to Valkeners et al. (2006).
Rumen ammonia and plasma urea concentrations were also monitored. Data were analysed using
the GLM procedure of SAS (SAS Inst. Inc., Cary, NC). Means were separated into significant main
effects by the SNK option of SAS.
Results
Organic matter intake (6.78 kg/d on average) was not affected by dietary RDN level. Ruminal
ammonia concentration and plasma urea-N level were highly influenced by the diet (Table 1) and
were the lowest when LRDN was fed compared to Control and HRDN. Organic matter and NDF
digestibility were not affected by RDN level and reached on average 78.0% and 74.0%, respectively.
Since dietary CP contents were different, N intake was highly influenced by treatments. Nitrogen
faecal excretion was not found to differ and reached on average 50.7 g/d. Contrariwise, urinary
N output increased significantly with dietary RDN. Compared to the Control, urinary N outputs
measured with LRDN and HRDN were 72.2% lower and 123.0% higher. Nitrogen retention,
expressed in g/d, was not affected by treatments and reached 56.6 g/d on average. Although N intake
increased with dietary RDN level, duodenal N flows were similar (Table 1). Such results involve
that N balance across the forestomach (Duodenal N flow minus N intake) amounted to 24.4, 36.0
and 6.5 g/d, respectively for the Control, LRDN and HRDN. Taking into account the endogenous

N at the duodenum (2.3 g of N/kg DM intake, Ouellet et al., 2002), the net N balances across the
forestomach were 7.1, 19.0 and –10.6 g/d.
Table 1. Effect of dietary ruminal degradable N (RDN) level on ruminal ammonia concentration,
plasma urea-N, N digestibility, and balance in double-muscled Belgian Blue bulls.
Item Level of RDN
LRDN Control HRDN SEM P
Ruminal N-NH3, mg/dL 3.1a 7.1b 12.4c 4.65 <0.001

Plasma urea-N, mg/dL 3.6a 6.4b 8.5c 0.56 <0.001
N digestibility, % 68.4 68.6 72.2 1.00 0.151
N intake, g/d 154.5a 168.1b 181.0c 1.65 <0.001
N faecal, g/d 48.9 52.7 50.1 1.63 0.385
N urinary, g/d 44.4a 61.7b 75.9c 2.06 <0.001
N retained
g/d 61.2 53.7 54.9 2.50 0.243
% N ingested 39.8a 32.3b 30.2b 1.32 0.018
% N digested 58.1a 47.1b 41.9b 1.75 0.008
Duodenal N flow, g/d 190.5 192.5 187.5 9.95 0.959
a,b,c within row, means with different superscripts differ significantly (P<0.05).
Our results indicate that feeding beet pulp silage based diets supplying similar contents of intestinal
digestible protein and NE for fattening, but with ruminal degradable N deficit significantly reduced
the N excretion of growing double-muscled Belgian Blue bulls mainly in urine but were without an
effect on the absolute amounts of N retained by the animals.
References
CVB, 2000. Veevoedertabel. Centraal Veevoederbureau, Lelystad, The Netherlands.
De Campeneere S., L.O. Fiems and C.V. Boucqué, 2001. Energy and protein requirements of Belgian Blue double-
muscled bulls. Anim. Feed Sci. Technol. 90, 153-167.
Ouellet D.R., M. Demers, G. Zuur, G.E. Lobley, J.R. Seoane, J.V. Nolan and H. Lapierre, 2002. Effect of dietary fiber
Stewart, G.S. and C.P. Smith, 2005. Urea nitrogen salvage mechanisms and their relevance to ruminants, non-ruminants
and man. Nutr. Res. Rev. 18, 49-62.
Valkeners D., A. Théwis, S. Amant and Y. Beckers, 2006. Effect of various levels of imbalance between energy and
nitrogen release in the rumen on microbial protein synthesis and nitrogen metabolism in growing double-muscled
Belgian Blue bulls fed a corn silage-based diet. J. Anim. Sci. 84, 877-885.
Research was funded by Direction générale de l’Agriculture, Ministère de la Région wallonne,

Belgium. The feedstuffs provision by SCAM is gratefully acknowledged.

Methodological considerations for the determination of standardised
ileal digestibilities of amino acids in newly weaned pigs
M. Eklund1, H.P. Piepho2, M. Rademacher3 and R. Mosenthin1
1Institute of Animal Nutrition, University of Hohenheim, 70599 Stuttgart; 3Degussa GmbH, 63457
Hanau-Wolfgang, Germany
2Bioinformatics Unit, University of Hohenheim, 70599 Stuttgart; 3Degussa GmbH, 63457 Hanau-
Wolfgang, Germany
Introduction
Standardised ileal digestibilities (SID) of crude protein (CP) and amino acids (AA) have been
introduced into diet formulation for pigs but barely any reports exist on the measurement of SID in
newly weaned pigs. Either regression analysis techniques or correction of apparent ileal digestibilities
(AID) for basal ileal endogenous CP and AA losses (IAALB) may be used to obtain SID values.
Moreover, different methods, including, regression analyses or feeding protein sources with assumed
CP and AA digestibilities of 100%, such as casein, can be used to obtain estimates of IAALB. In
newly weaned pigs, however, a limited capacity for CP digestion and AA absorption may affect the
determination of SID and IAALB. Therefore, the aims of the present study were to estimate IAALB
and SID in newly weaned pigs, which were fed graded levels of CP originating from casein.

Fourteen 3-wk old barrows (12+2 for replacement; German Landrace×Piétrain) were surgically fitted
with simple T-cannulas at the distal ileum. The piglets were randomly assigned to 6 semi-synthetic
cornstarch-based diets (including 10% dextrose, 7% cellulose, 2% soybean oil, 4% mineral and
vitamin premix, 0.1% titanium dioxide) with graded inclusion levels of casein, crystalline threonine
and cystine, which were added to the diets at the expense of cornstarch (9.0, 15.5, 22.0, 28.5, 35.0,
41.5% CP, as-fed basis). Four weekly repeated measurements of AID of CP and AA were conducted
(n=2 piglets, resulting in 8 observations per CP level). Starting with 28 d of age, the diets were fed
at a level of 3% of individual body weight, which was determined weekly to keep the feed intake in
relation to body weight at a constant level for all animals throughout all 4 experimental periods. To
estimate SID (%) in casein, the following methods were used: (i) Ileal recoveries of CP and AA (%)
originating from casein were estimated by linear regression analysis between total ileal recoveries
and dietary intakes of CP and AA (g/kg DMI); (ii) The SID of CP and AA (%) originating from
casein were estimated by linear regression analysis between apparent ileal digestible and total dietary
contents of CP and AA (g/kg DMI); (iii) The AID of CP and AA (%) were corrected for IAALB (g/kg
DMI). To estimate IAALB (g/kg DMI) the following methods were used: (i) Extrapolation from
linear regression analysis between apparent ileal digestible and total dietary contents of CP and AA
(g/kg DMI); (ii) Ileal recoveries from piglets fed casein were considered to represent IAALB (g/kg
DMI) and were expressed as a function of the dietary CP level (% DM). The data were subjected to
mixed model analysis using the MIXED procedure of SAS. The errors of repeated measurements
on the same subject (animal within CP level) were assumed to be serially correlated and different
correlation structures were fitted.

Using regression analysis between total ileal recoveries and dietary intakes of CP and AA (g/kg
DMI) for the ranges of graded dietary CP levels from 9.0 to 22.0, 22.0 to 35.0 and 28.5 to 41.5%
CP showed that ileal CP and AA recoveries originating from casein (%) are not different from zero
(slope, P>0.05). Thus, there is no evidence for a limited capacity for CP digestion and AA absorption

in newly weaned pigs and ileal recoveries from casein-fed piglets can be considered to represent
IAALB. For the total range of dietary CP levels (9.0 to 41.5%) there were linear increases (P<0.05)
in IAALB with increasing dietary CP levels. These increases in IAALB affected the linearity of the
regression between apparent ileal digestible and total dietary contents of CP and AA (g/kg DMI).
The slopes of the regression between apparent ileal digestible and total dietary contents of CP and
AA (g/kg DMI), which represent SID, decrease when higher graded dietary CP levels were included
in the regression analysis. The SID of CP, for example, decreases from 98.0 to 95.4%. The highest
estimates of SID and IAALB by regression analysis between apparent ileal digestible and total dietary
CP and AA contents (g/kg DMI) were obtained in the range from 9.0 to 22.0% CP. In this range
SID ranged between 96.2 for Trp and 103.6% for Gly, and IAALB ranged between 9.3 for CP and
0.1 g/kg DMI for Met and Trp. These values were in close agreement to values in grower-finisher
pigs. The correction of AID for IAALB resulted in SID values close to 100%, as well (Table 1).
Using IAALB obtained by extrapolation from apparent ileal digestible and total dietary content of
CP and AA (g/kg DMI; 9.0 to 22.0% CP) resulted for Ala, Pro and Ser (Table 1) in linear decreases
(P<0.05) in SID with increasing dietary CP levels. This decrease may be compensated for when the
linear increase in IAALB with increasing dietary CP levels is considered in the correction of AID for
IAALB (Table 1). However, for Thr, SID was significantly lower at 9% dietary CP content (Table
1). It can be concluded that both, regression analysis and correction of AID for IAALB are suitable
to obtain SID values in newly weaned pigs. For regression analysis, the dietary range from 9.0 to
22.0% CP provides the most reliable estimates of SID, whereas the correction of AID for IAALB
is more reliable at higher dietary CP levels with consideration of the linear increase in IAALB with
increasing dietary CP levels.
Table 1. Effect of dietary crude protein level and method for estimating basal ileal endogenous amino
acid losses on standardized ileal crude protein and amino acid digestibilities in casein.
Dietary CP level (% as-fed)
9.0 15.5 22.0 28.5 35.0 41.5
CP A 97.5 98.3 97.7 96.1 96.9 96.6

B 98.6 100.6 100.4 99.1 100.1 99.8
Thr A 97.1ab 98.3a 97.9ab 95.3b 96.7ab 95.8ab
B 98.6a 101.0b 100.9b 98.8ab 100.2ab 100.4ab
Ser A1 99.9 99.4 99.5 98.9 98.8 98.6
B 99.6 100.0 100.4 99.9 100.0 100.0
A: IAALB based on regression analysis (range: 9.0-22.0% CP); B: Linear increase in IAALB with increasing dietary
CP level; a,b within a row LSmean values with a common superscript are not significantly different at α=0.05; 1 linear
decrease (P<0.05).

Effects of feeding duration and ruminal nitrogen and energy release
rates on nitrogen balance and microbial synthesis in sheep
T. Ichinohe and T. Fujihara
Faculty of Life and Environmental Science, Shimane University, Matsue-shi, 690-8504, Japan
Introduction
An arrangement of synchronisation between nitrogen (N) and energy supply in the rumen is
theoretically regarded to improve microbial synthesis and its efficiency. However, current studies
assessing this hypothesis have produced conflicting results. Moreover, there is little information
whether lacking the rumen synchronicity could lead to some adaptive response by the host animal
or rumen microbes with respect to their nutrient metabolism. The objectives of this study were to
determine the effect of the degree of ruminal synchronisation between N and organic matter (OM)
release on N utilisation and microbial supply in sheep, and to investigate the changes in N utilisation
and rumen microbial synthesis during a long term feeding duration.

The ruminal degradation characteristics of OM and N for timothy hay, alfalfa hay, wheat middlings and
rolled barley were determined using three rumen-cannulated wethers by the nylon bag technique.
Six intact rams, aged 1-yr-old and mean body weight of 45.3 kg, were used for the feeding study.
The rams were divided into 2 groups and were allocated to 1 of 2 dietary treatments. The 2 diets
were formulated to provide maintenance requirements of metabolisable energy (ME) and rumen
degradable N (RDN). Moreover, diet formulations and feed distribution were arranged to yield
different synchronisation index between N and OM degradation (Sinclair et al., 1993), applying
the figures of chemical composition and rumen degradation parameters of the feeds. The diets were
formulated to yield a low degree of synchronisation index of 0.5 (asynchronous diet: ASYNC) and
a high degree of synchronisation index of 0.7 (synchronous diet: SYNC). The animals were fed two
equal sized meals at 08.00 and 18.00 h throughout the experiment. The mean ratio of degraded N
(g) to OM (kg) over the feeding regimen was arranged to be greater for ASYNC (ca 42 g/kg) than
that for SYNC (ca 30 g/kg).
The feeding study lasted for 3 mo, during which 5-d of balance trial was conducted on d 30, 60 and
90. Total urine was collected in order to estimate microbial N (MBN) supply to the small intestine
and urinary N excretion. On the final day of total collection, rams were blood-sampled at 4 h post
morning feeding. Blood plasma concentration of urea N (BUN) and glucose was determined using
a commercial kit. Data were analysed as repeated measures for a randomised complete block design
using the MIXED procedure. The general linear model included the effects of diets, day of sampling
and diet × day interaction.

The N intake (g N/kg BW0.75) was almost similar between ASYNC and SYNC throughout the
measurement. There was a significant sampling day effect on N retention (P<0.05) together with
a tendency of diet × day interaction (P≤0.1) without dietary effect. The sampling day effect on N
retention indicated an improvement in the efficiency of N utilisation with advancing feeding duration
with both of the diets. The BUN concentration decreased linearly with lengthening feeding period
(P≤0.01), while plasma glucose concentration tended to increase (P<0.1). The lengthening feeding
duration also changed the utilisation status of ME and metabolisable protein (MP) as evidenced

by the changes in BUN and plasma glucose concentration together with concomitant decrease in
urinary N excretion.
Unexpectedly, MBN supply was significantly greater for ASYNC than that for SYNC (0.64 vs.
0.54 g/kg BW0.75, P≤0.01); and MBN supply was also greater on d-60 and -90 than that on d-30
(0.55 vs. 0.63 g/kg BW0.75, P≤0.01). In contrast to the N retention, diet × day interaction was not
observed for MBN supply. Neither diets nor periods had a significant effect on the efficiency of
MBN synthesis (g N/kg ruminally degraded OM). The differences in dietary ingredients between
ASYNC and SYNC might have caused an associative effect on rumen degradation of them, which,
in turn affected the degradation synchronicity over feeding time. It seems that when RDN and
fermentable energy are provided in a day to meet animal requirement with an overall balance, there
will be a certain degree of degradation synchronisation index above which no further advantage can
be obtained. The upgraded rumen synchronicity for both diets appeared to have nullified any effect
by rectifying dietary synchronicity index as is shown in this study.
Table 1. Urinary nitrogen (N), N retention, concentration of blood metabolites, microbial N (MBN)
supply and microbial efficiency (MBE) in sheep fed diet with a differing synchronisation index.
Diet Sampling day P -value
ASYNC SYNC d-30 d-60 d-90 Diet Day Diet×Day
Urinary N 0.42 0.40 0.50 0.36 0.37 0.34 0.01 0.18

N retention 0.16 0.17 0.11 0.20 0.19 0.79 0.04 0.10
BUN 13.34 14.34 16.84 12.82 11.85 0.28 0.01 0.65
Glucose 75.65 74.48 67.97 71.76 85.47 0.81 0.09 0.40
MBN 0.64 0.54 0.55 0.62 0.64 0.01 0.01 0.14
MBE1 35.05 31.40 31.56 35.52 32.58 0.27 0.13 0.13
1 Efficiency of microbial synthesis (g N/kg ruminally degraded OM).
References
Sinclair, L.A., P.C. Garnsworthy, J.R. Newbold and P.J. Buttery, 1993. Effect of synchronizing the rate of dietary
energy and nitrogen release on rumen fermentation and microbial protein synthesis in sheep. J. Agric. Sci. Camb.
120, 251-263.

Timing of herbage and fasting allocation alters nutrient supply in
grazing cattle
P. Gregorini, S.A. Gunter and P.A. Beck
University of Arkansas Division of Agriculture, Southwest Research & Extension Center, 362 Highway
174 North, Hope, Arkansas 71801, USA
Introduction
The outcome of a grazing strategy results from an interaction among herbage, ingestion, digestion,
and nutrient absorption (Gregorini et al., 2006). Herbage is characterized by an increase in nutritive
value during the day (Mayland et al., 2005; Griggs et al., 2005), which is attributed to moisture
loss, non‑structural carbohydrate accumulation, NDF and crude protein dilution, and an increase in
IVOMD (Gregorini et al., 2006). This diurnal variation in herbage quality coincides with the daily
grazing pattern of cattle, which maximizes herbage intake at dusk (Gibb et al., 1998). Afternoon
herbage allocation has demonstrated increased intake at dusk (Gregorini et al., 2006), but it may not
be maximized. Greenwood and Demment (1988) found that fasting steers increases the intake rate
by up to 62%. Hence, delaying herbage allocation time and fasting steers might maximize nutrient
intake.
Intake pattern dictates the dynamics of fermentation and particulate passage rate through the rumen;
hence, a change in grazing pattern might alter patterns of ruminal metabolism. This latter idea leads
us to ask if the timing of forage allocation is modified to capitalize on evening grazing and can intake
and ruminal metabolism be augmented by daytime fasting?

This experiment was conducted at the Southwest Research & Extension Center in Hope, Arkansas
(33°42'N, 93°31'W) from March to June 2006. Four ruminally and duodenally cannulated Angus
heifers (279 kg = body weight [BW]) individually strip‑grazed a wheat (Tritucum aestivum) pasture
divided into 16 paddocks with treatments applied in a 4×4 Latin‑square design. Treatments were daily
allocations of herbage (6% of BW on a dry matter [DM] basis) in the afternoon (15.00 h, AHA),
morning (08.00 h, MHA), AHA or MHA after 20 h of fasting (AHAF and MHAF, respectively). Heifers
were adapted to treatment for the first 8 d in each period then data was collection the last 4 d.
On d 9, ruminal DM pools were measured four times daily (08.00, 12.00, 15.00, and 19.00 h) by
removing and weighing the ruminal contents and sampled to determine DM concentration; ruminal
liquor was collected at these same times plus 23.00 h for pH and ammonia analysis. From these
data, grazing dynamics and total DM intake was estimated from diurnal changes in the ruminal DM
pools (Taweel et al., 2005). Twelve duodenal samples were collected over a 48-h period on d 10 and
11 so that diurnal variation in flow was estimated every 2 h over a 24‑h period. Duodenal samples
were analyzed for DM, organic matter (OM), and ribonucleic acid to determined ruminal microbial
protein yield. Eating, ruminating and idling behaviors were recorded every 2 min and bite rate every
h on d 12 of each period. Eating rate, bite rate and herbage intake rates were summarized into three
periods: morning (08.00 to 11.59 h), afternoon (12.00 to 14.59 h) and evening (15.00 to 18.59 h) to
determine their diurnal patterns.

The eating pattern was modified by treatment, resulting in longer (P<0.05) and more intensive
(P<0.05; higher herbage intake rate, bite mass and bite rate) evening bouts for AHA and AHAF

than MHA and MHAF (data not shown); however, daily herbage DM intake did not differ (P>0.05;
average = 15.7 g/kg of BW) among treatments. The ruminal pH and ammonia concentration patterns
followed the pattern of herbage intake and ingestive behavior; ruminal pH was lower and ammonia
was higher (P>0.05) for AHA and AHAF during the evening (data not shown). Interestingly, the
flow of OM, N, and microbial N to the duodenum was greater (P<0.05) in AHA than MHA, MHAF,
and AHAF. Total tract digestibility of OM did not differ (P>0.05) for MHA, AHA and AHAF, but
digestibility was lower (P<0.05) for MHAF. True ruminal digestibility of OM did not differ (P>0.05)
among NHA, MHAF and AHAF, but digestibility was higher (P<0.05) for AHA and resulted in
increased (P<0.05) microbial N yield from the rumen.
Table 1. Nutrient flow at the duodenum and OM digestibility in heifers strip grazing wheat pasture
(least-squares means with uncommon superscripts differ; P<0.05).
Treatments P-values†
Items MHA MHAF AFA AHAF SE T P T×P
Duodenum, kg/d
Total OM flow 3.78a 3.33a 4.46b 3.36a 0.043 0.03 0.03 0.08
Total N 0.14a 0.14a 0.18b 0.13a 0.002 0.07 0.04 0.09
Microbial N 0.17 a 0.16a 0.21b 0.17a 0.033 0.05 0.19 0.12
Non‑microbial OM 2.09 ab 1.89a 2.55c 2.04a 0.044 0.06 0.04 0.11
Microbial OM 1.64b 1.30a 2.04c 1.61b 0.020 0.06 0.03 0.10
True ruminal OM 47.8a 52.3a 67.3b 44.0a 0.811 0.05 0.03 0.10
digestibility, %
Total tract OM 48.7b 37.7a 46.4b 48.3b 0.866 <0.01 <0.01 0.08
digestibility, %
†T = the effect of treatment; P = the effect of period; T×P = the interaction between T and P.
These results demonstrate that grazing management can alter ingestive‑digestive patterns and nutrient
supply to grazing cattle can be modified through simple grazing management, at the same level of
resource allocation and herbage intake.
References
Gibb, M.J., C.A. Huckle and R. Nuthall, 1998. Effect of time of day on grazing behavior by lactating dairy cows.
Grass Forage Sci. 53, 41-46.
Greenwood G.B. and M.W. Demment, 1988. The effect of fasting on short-term cattle grazing behavior. Grass Forage
Sci. 43, 377-386.
Gregorini, P., M. Eirin, R. Refi, M. Ursino, O.A. Ansin and S.A. Gunter, 2006. Timing of herbage allocation in strip
grazing: Effects on grazing pattern and performance of beef heifers. J. Anim. Sci. 84, 1943-1950.
Griggs T.C., J.W. MacAdam, H.F. Mayland and J.C. Burns, 2005. Nonstructural carbohydrate and digestibility
patterns in orchardgrass swards during daily defoliation sequences initiated in evening and morning. Crop Sci.
45, 1295-1304.
Mayland H., D. Mertens, T. Taylor, J. Burns, D. Fisher, P. Gregorini, T. Ciavarella, K. Smith, G. Shewmaker and T.
Griggs, 2005. Diurnal changes in forage quality and their effects on animal preference, intake, and performance.
California Alfalfa Symposium, 12-20.
Taweel, H.Z., B.M. Tas, J. Dijkstra and S. Tamminga, 2005. Intake regulation and grazing behavior of dairy cows
under continuous stocking. J. Dairy Sci. 87, 3417-3427.

Effect of water-soluble carbohydrate on rumen nitrogen kinetics of steers
given perennial ryegrass silage measured by 15N-tracer methodology
E.J. Kim1, N.D. Scollan1 and J.V. Nolan2
1Institute of Grassland and Environmental Research, Aberystwyth, SY23 3EB, United Kingdom
2University of New England, Armidale, NSW, 2351, Australia
Introduction
The efficiency of use of dietary nitrogen (N) in cattle is influenced by the rate of degradation of
dietary protein to peptides, amino acids and ammonia, and of assimilation of these products by
rumen micro-organisms for protein synthesis. The latter depends heavily on fermentable energy
availability (FME). Ammonia that is not assimilated is mainly absorbed across the rumen wall and
much of it is excreted as urea in urine, which represents inefficient use of dietary N. The stable
isotope, 15N, provides a useful means of making quantitative estimates of microbial synthesis and
N utilisation in the rumen. Our objective was to use 15N-tracer methodology to examine the effect
of water-soluble carbohydrate (WSC) on N kinetics in the rumen of steers given silages made from
standard and high-sugar perennial ryegrass.

Four Holstein-Friesian steers (mean liveweight 465 kg) equipped with rumen and duodenal cannulae
were used in a 4×4 Latin Square experiment. First-cut perennial ryegrass silages made from two
cultivars (Fenemma (FEN) and Aberdove (DOVE) for control and high sugar grass, respectively) were
offered alone or supplemented with sucrose (40 g/kg dry matter intake; FEN+S and DOVE+S). Diets
were offered ad libitum in equal portions each h. After two weeks adaptation, a solution containing
(15NH4)2SO4 (99.3 mol %, CK Gas products Ltd., UK) was administered into the rumen in two 500
mL portions (i.e. 20 mmol 15N), after which filtered rumen fluid (RF) was collected at intervals for
about 24 h. RF samples were centrifuged at 20,000×g (25 min, 4 °C) to give pure fluid-phase bacteria
that were washed free of ammonia by re-suspension in water and re-centrifugation. Supernatant (8
mL) was mixed with 0.8 mL 65% (w/v) trichloroacetic acid to precipitate soluble protein. Ammonia-
N was isolated from supernatant by diffusion at 20 °C. 15N enrichments of RF ammonia, bacteria
and soluble protein were determined by combustion (Dumas) and mass spectrometry; double-
exponential equations were fitted to the enrichment vs. time data using SigmaPlot (version 9, Systat
Inc.). Ammonia flux rates, compartment sizes and fractions of N in secondary compartments arising
from ammonia were determined (Nolan and Leng, 1974). Statistical analysis was done using GenStat
(Release 9.1) with REML for missing animals.

The silages used contained 241 and 224 g dry matter (DM)/kg, 18.5 and 18.4 g total-N/kg DM,
49.6 and 68.2 g WSC/kg DM, 639 and 597 g NDF/kg DM, 113 and 100 g ammonia-N/kg total-N
for FEN and DOVE, respectively. N intakes were higher for DOVE than for FEN, reflecting higher
DM intake for DOVE (Table 1). The RF ammonia-N concentrations were higher (P<0.001) on
FEN than DOVE; FEN+S had lower concentrations than FEN but DOVE+S did not differ from
DOVE. Other measures of N kinetics did not differ across diets (P>0.05) and are given as means;
estimates of RF ammonia compartment size, total flux and net flux of ammonia were 5.42 g N,
158 g N/d and 102 g N/d, respectively. The net flux was 0.90 of N intake indicating that the crude
protein from both cultivars was extensively degraded. The total ammonia flux was 1.6×N intake
and 1.4×net flux, further indicating that an appreciable fraction (0.35) of the ammonia-N leaving
the RF ammonia compartment was then returned to it. As the likelihood of 15N recycling via body

tissues is low, most 15N cycling must have occurred within the rumen, e.g. by bacterial assimilation
of ammonia followed by predation and ammonia release from protozoa or natural cell death and
fermentation of microbial debris. Across the treatments, 0.85 of the N in fluid phase bacteria (Table
1) was derived from RF ammonia, with only 0.15 apparently being derived from peptides or amino
acids or both. This was probably because little non-ammonia N was available to bacteria on these
silage diets. Labelled soluble protein was synthesised, presumably by rumen micro-organisms, and
secreted into RF probably partly as microbial enzymes; however, only 0.58 of this soluble N was
derived from ammonia, so most (0.62) was from dietary or endogenous sources. Both build-up
and decay rate constants of the fitted bacterial-N enrichment curve were faster (P<0.01) for DOVE
than for FEN, suggesting that bacterial synthesis and outflow from the rumen were higher in steers
ingesting DOVE silage.
Table 1. N intake and ammonia-N kinetics in the rumen of steers given the experimental diets.
Diets SED Significance†
FEN FEN+S DOVE DOVE+S V S I
N intake, g/d 111.0 110.8 110.1 117.6 3.27 * NS *

Ammonia, mg N/L 83.0 70.1 56.5 63.3 0.96 *** *** ***
Ammonia pool size, g N 7.12 5.03 4.24 5.28 1.408 NS NS NS
Total flux, g N/d 162.3 147.7 156.3 163.6 38.79 NS NS NS
Net flux, g N/d 100.8 97.2 98.5 110.2 16.14 NS NS NS
Recycling, g N/d 62.0 50.0 59.7 55.1 20.05 NS NS NS
Areas: Bacteria/NH3§ 0.870 0.804 0.856 0.887 0.1031 NS NS NS
Areas: Protein/NH3§ 0.618 0.557 0.596 0.577 0.0593 NS NS NS
Bacterial k1 ×103/minψ 0.126 0.138 0.157 0.169 0.0178 ** NS NS
Bacterial k2 ×103/minψ 0.679 0.743 0.837 0.795 0.0248 *** NS ***
† V,S and I represent variety, sugar supplement and interactions; *, ** and *** for P<0.05, P<0.01 and P<0.001,
respectively; NS = not significant; § Ratios of areas under fitted curves, fraction of N in bacteria or soluble protein in
RF arising from ammonia; ψBacterial k1 represent build-up rate constants and k2 terminal rate constants of the fitted
bacterial enrichment vs. time curves.
Conclusions
Tracer studies using 15N indicated that ammonia fluxes and intraruminal ammonia-N cycling were
high relative to N intake in steers on all diets. A more rapid incorporation of ammonia into bacterial
crude protein in steers ingesting DOVE vs. FEN silage (higher k1) helps explain their lower RF
ammonia concentrations; higher fractional removal of bacterial N each d (higher k2), also suggests
that they generated a higher net outflow of bacterial protein from the rumen. Dietary sucrose generally
had little effect on rumen kinetics but rumen ammonia concentrations in steers given FEN+S were
lower than in steers on the FEN control diet.
References
Nolan, J.V. and R.A. Leng, 1974. Isotope techniques for studying the dynamics of nitrogen metabolism in ruminants.
Proc. Nutr. Soc. 33, 1-8.

Effect of toasting organic field beans on starch and NDF digestibility and
rumen microbial protein synthesis in dairy cows
P. Lund, M.R. Weisbjerg, T. Hvelplund, M. Larsen and T. Kristensen
University of Aarhus, Faculty of Agricultural Sciences, Research Centre Foulum, P.O. Box 50,
DK-8830 Tjele, Denmark
Introduction
Organic milk production constitutes about 10% of the Danish milk production. Organic feed rations
for dairy cows are mainly based on home-grown grain and grass-clover silage, and consequently
often rich in starch and rumen degradable protein. A major challenge has therefore been to secure an
optimal rumen environment for fibre digestion and cow health, and a sufficient level of metabolisable
protein. Rumen microbial protein synthesis is both quantitatively and qualitatively important for the
amino acid supply of the ruminant animal. Feed processing may increase the level of metabolisable
protein by reducing rumen protein degradation in situations with surplus of rumen degradable protein
compared to rumen microbial energy supply. Optimised partition of starch digestion between the
rumen and the small intestine is essential for energy utilisation on the animal level. Field beans is a
potential protein source, which can be organically grown under Danish conditions. The purpose of
the present experiment was therefore to examine if processing of field beans could improve energy
supply, by increased rumen outflow of undegraded starch, rumen NDF digestibility and intestinal
starch digestion.

A 3×3 Latin square experiment, which complied with the guidelines of the Danish Ministry of Justice,
was conducted with three rumen and duodenal fistulated lactating Holstein Friesian dairy cows.
Each period consisted of 14 d. In a total mixed ration either untreated, 120 °C toasted, or 150 °C
toasted organic field beans (29% of DM) were fed together with grass-clover silage (67% of DM),
soya bean meal (3% of DM) and minerals (1% of DM). Field beans were rolled after cooling. Feed
offer and refusals were recorded daily. The cows were on average 229 d in milk and weighed 650
kg. The cows were milked twice daily (06.00 h; 17.00 h), and had an average yield of 25 kg milk.
Ten grams of chromic oxide was administrated via the rumen cannula as flow marker before each
of the two daily feedings (07.30 h; 17.30 h), and during the collection procedure on d 10 to 14, 12
samples of duodenal chyme and faeces were taken from each cow and pooled. The protocol was
arranged to give a representative sample of the diurnal flow. Rumen liquid bacteria were isolated
by sequential centrifugation and microbial protein synthesis was determined based on RNA:N and
DAPA:N ratios in isolated bacteria and duodenal chyme. Statistical analysis was done using the
GLM procedure in SAS 8.e using cow, period and treatment as class variables.

In contradiction to heat treatment at 120º°C, heat treatment at 150 °C decreased rumen starch
digestibility. However, also post-duodenal and total tract starch digestibility were decreased (Table 1),
indicating that the increase in rumen by-pass starch was counterbalanced by a reduction in intestinal
digestibility and an increased faecal loss. NDF digestibility and rumen microbial N-synthesis were
not significantly affected by heat treatment. The diurnal profile in rumen pH is illustrated in Figure 1.
Heat treatment at 120 °C did not affect rumen pH in agreement with the absence of an effect on rumen
starch digestibility, whereas heat treatment at 150 °C numerically increased rumen pH in agreement
with the reduction in rumen starch digestibility. Rumen NDF digestibility was not improved despite
that heat treatment at 150 °C decreased rumen starch digestibility. This was probably due to the

relatively high rumen pH level already obtained for untreated field beans, indicating that the fibrolytic
bacteria were not constrained. Milk yield was not significantly affected (P=0.55). The reduction in
rumen starch digestibility was confirmed in an accompanying in situ study, where toasting of field
beans at 140 °C decreased rumen starch degradability from 86 to 70%, primarily due to a decrease
in fractional rate of degradation from 26 to 12%/h.
Table 1. Intake of dry matter and starch (kg/d), digestibility (%) of starch and NDF in different parts
of the GI-tract, and rumen microbial N-synthesis (g/d).
Treatment
Untreated 120 °C 150 °C SEM P-value
Dry matter intake 20.5 19.3 20.3 0.62 0.47

Starch intake 2.81 2.52 2.82 0.084 0.20
Rumen starch digestibility 75.5 77.5 62.7 2.4 0.08
Post-duodenal starch digestibility 85.9 83.1 71.3 3.6 0.17
Total tract starch digestibility 96.7 96.2 89.6 0.77 0.04
Rumen NDF digestibility 60.9 60.9 61.5 2.9 0.99
Total tract NDF digestibility 61.5 65.1 62.6 1.2 0.23
Microbial N-synthesis 288 256 289 11 0.25
6,5 Untreated 120ºC 150ºC

6,4
6,3
6,2
6,1
6
5,9
5,8
0 4 8 12 16 20 24
Time
Figure 1. Diurnal profile of rumen pH, vertical arrows indicate feeding times.
Conclusion
To increase the amount of starch digested in the small intestine, the optimal temperature for toasting
field beans seems to be in the interval 120-150 °C. At 120 °C rumen starch digestibility and post-
duodenal digestibility were unaffected by heat treatment, but at 150 °C heat treatment decreased
rumen starch digestibility, although also post-duodenal digestibility was decreased. An important
future research area is therefore to find ways to decrease rumen starch digestibility without hampering
small intestinal digestibility of by-pass starch.

Pattern and rate of urea infusion into the rumen alter N balance and
plasma NH4+ in wethers
M.I. Recavarren and G.D. Milano
Facultad de Ciencias Veterinarias, UNCPBA, Campus Universitario, 7000, Tandil, Argentina
Introduction
In ruminants, diets with high content of rapidly available N in the rumen are linked to reduced N
retention (Ragland-Gray et al., 1997). It is not certain, however, whether this is the result of the
relative excess of rumen available N, the asynchrony of N and energy release in the rumen or both.
Also, the mechanisms involved, including low microbial protein production (Sinclair et al., 1993),
increased liver energy expenditure and amino acid disposal by NH4+-stimulated ureagenesis (Milano
et al., 2000) and hormonal responses to hyperammonemia (Choung and Chamberlain, 1995), are still
subject of debate. The objective of this experiment was to quantify the effect of a change in pattern
and rate of non-protein N (NPN) release in the rumen on N balance, urinary excretion of purine
derivatives (PD) and plasma concentration of NH4+, urea, glucose, insulin and IGF-1.

Animals, diet, design and infusions
Four Corriedale wethers (37 ± 2.6 kg body weight), fitted with permanent catheters in the rumen,
were housed in individual pens under continuous lighting and fed 850 g dry matter (DM)/d of lucerne
hay (600 kJ ME/kg0.75/d; 23 g N/d; 7 g soluble N/d), in equal meals at 02.00, 06.00, 10.00, 14.00,
17.30 and 22.00 h during, and for 80 d previous to, a 17-d experiment. Water (2.5 L/d) was offered
twice a d. On 2 consecutive experimental periods, urea was infused into the rumen with peristaltic
pumps, first continuously (CT; 10 d; 9.5 g urea/d; 0.0032 g N/min) and then discontinuously (DT;
7 d; 8.1 g urea/d in 6 doses/d at 0.16 g N/min, for 4 min, with each meal). Urea-N infusion rate on
DT is close to the maximum soluble N intake in sheep grazing local winter grasses (7 g DM/min;
19.2 mg soluble N/g DM).
Samples
On d 10 of CT and 2, 4 and 6 of DT, jugular blood samples were collected into heparinised syringes
1.5 h after the 14.00-h meal, mixed, centrifuged at 4 °C, and the plasma was analysed for NH4+,
urea, glucose, insulin and IGF-1. Hay samples, faeces and urine were collected daily from d 7 to 10
of CT and 2 to 7 of DT, weighed and aliquots were analysed for DM (hay and faeces), total-N (hay,
urine and faeces; Kjeldahl) and PD (urine; Chen et al., 1990). N balance and urinary excretion of
PD were calculated from urine and faeces samples pooled across 4 d in CT (CT7 to CT10) and 2-d
periods (DT2 to DT3, DT4 to DT5 and DT6 to DT7) in DT.
Statistics
The results were analysed as repeated measurements. Intercepts (β0), linear (β1) and quadratic (β2)
coefficients for temporal changes in all variates during DT were estimated by regression analysis,
with period CT considered as d 0 of DT (PROC MIXED; SAS 9.1.3, 2006).

Plasma urea (Mean ± SE; 13.0 ± 2.0 mM), glucose (3.33 ± 0.30 mM), insulin (0.349 ± 0.09 ng/
mL) and IGF-1 (306 ± 81 ng/mL) were unchanged during the experiment. A curvilinear response
was seen in plasma NH4+ (β1 ± SE, 59.9 ± 16.3; β2 ± SE, -11.2 ± 2.6; Table 1). This suggests that
NH4+ inflow to the liver exceeded hepatic NH4+ removal on the first half of DT. Clinical signs of
hyperammonemia were not observed. PD excretion in urine also showed a curvilinear response (β1
± SE, 81 ± 23; β2 ± SE, -10.5 ± 3.1, Table 1). N intake decreased slightly in DT (β1 ± SE, -0.15 ±
0.07) due to a 1.4 g/d reduction in the urea-N infused into the rumen. N retention (β1 ± SE, -0.96 ±
0.29) decreased 6.8 g N/d during DT, due to an increase in both urinary (β1 ± SE, 0.67 ± 0.25) and
faecal (β1 ± SE, 0.13 ± 0.06) N losses (Table 1).
Table 1. Changes in plasma NH4+, N balance and urinary PD excretion during ruminal infusion of
urea in wethers (Means, N=4).
DT, d Regression model
0a 2b 4c 6d β1, P value β2, P value RMSE
Plasma NH4+, µM 197 235 235 121 0.004 0.001 41.6

N intake, g/d 25.9 24.6 24.8 24.8 0.008 - 0.77
N urine, g/d 9.6 12.5 13.3 14.4 0.02 - 2.55
N faeces, g/d 7.0 7.2 7.7 7.9 0.05 - 0.62
N retention, g/d 9.3 4.9 3.9 2.5 0.007 - 2.98
PD, mgN/d 226 378 364 279 0.005 0.008 69.9
For N balance and urinary PD excretion: a CT7 to CT10; b DT2 to DT3; c DT4 to DT5; d DT6 to DT7.
The results suggest that the reduction of N retention in sheep receiving excess NPN in the rumen
was dependent on the pattern and rate of N infusion rather than on the total amount infused. Lower N
retention was not associated with a decrease in urinary PD excretion (indicative of microbial protein
flow to the duodenum) or changes in the plasma concentration of insulin and IGF-1 (hormones that
participate in the regulation of protein deposition) but was concurrent with a mild and transient
hyperammonemia.
References
Chen, X.B., J. Mathieson, F.D. DeB Hovell and P. Reeds, 1990. Measurement of purine derivatives in urine of ruminants
using automated methods. J. Sci. Food Agr. 53, 23-33.
Choung, J.J. and D.G. Chamberlain, 1995. Effects of intraruminal infusion of propionate on the concentrations of
ammonia and insulin in peripheral blood of cows receiving an intraruminal infusion of urea. J. Dairy Res. 62,
549-557.
Milano, G.D., A. Hotston Moore and G.E. Lobley, 2000. Influence of hepatic ammonia removal on ureagenesis, amino
acid utilization and energy metabolism in the liver. Brit. J. Nut. 83, 307-315.
Ragland-Gray, K.K., H.E. Amos, M.A. McCann, C.C. Williams, J.L. Sartin, C.R. Barb and F.M. Kautz, 1997. Nitrogen
metabolism and hormonal responses of steers fed wheat silage and infused with amino acids or casein. J. Anim.
Sci. 75, 3038-3045.
Sinclair, L.A., P.C. Garnsworthy, J.R. Newbold and P.J. Buttery, 1993. Effect of synchronizing the rate of dietary energy
and nitrogen release on rumen fermentation and microbial protein synthesis in sheep. J. Agr. Sci. (Cambridge)
120, 251-263.

Effects of dietary crude protein and ruminally-degradable protein on
urea recycling and microbial protein production in beef heifers
K. Baker1, T. Mutsvangwa1, J.J. McKinnon1, G. Gozho1 and T.A. McAllister2
1Department of Animal and Poultry Science, University of Saskatchewan, Saskatoon, SK, S7N 5A8
Canada
2Agriculture and Agri-Food Canada, Lethbridge Research Centre, Lethbridge, AB, T1J 4B1
Canada
Introduction
In ruminants, hepatic urea-N output often exceeds apparent digestible N intake; however, animals still
maintain a positive N balance primarily by recycling 40-80% of their hepatic urea-N output to the
gastrointestinal tract (GIT). About 35-55% of this recycled urea-N can be used for microbial protein
production, thereby contributing amino acids to the host animal (Lapierre and Lobley, 2001). The
proportion of hepatic urea-N output that can be returned to the GIT can be influenced by numerous
dietary factors, including the level of dietary N (Lapierre and Lobley, 2001). However, the dietary
level of ruminally-degradable protein (RDP) might also be important since it can influence the
amount of dietary N that is directed towards ruminal ammoniagenesis and, subsequently, hepatic
ureagenesis. Limited information is available on how concomitant changes in dietary content of N
and RDP might influence urea-N kinetics in ruminants. The objective of this study was to determine
how interactions between dietary content of N and RDP alter urea-N transfer to the GIT and the
utilization of this recycled urea-N in growing beef heifers.

Four ruminally-cannulated beef heifers (437 kg BW) were used in a 4×4 Latin square design with
18-d adaptation and 4-d data collection periods. Dietary treatments were arranged as a 2×2 factorial,
with two levels of crude protein (CP: 10 vs. 13%) and two levels of RDP (64.5 vs. 71.0%). Jugular
infusions of [15N15N]-urea (98+ APE; 220 mg/d) were conducted for 4 d (d 19-22) to estimate urea
kinetics (Lobley et al., 2000). During this period, total collection of feces and urine were conducted.
Composite fecal and urine samples were analyzed for N. Proportions of [15N15N]-, [14N15N]- and
[14N14N]-urea in urinary urea, and 15N content in feces were determined by isotope-ratio mass
spectrometry (IRMS). Plateau enrichments of [15N15N]- and [14N15N]-urea in urine, and fecal 15N
were corrected for the 15N natural abundance in urine and fecal samples taken prior to initiation
of isotopic infusions, and were used to calculate urea kinetics (Lobley et al., 2000). Microbial N
production was estimated based on urinary excretion of purine derivatives. Data on DM intake, N
balance, urea kinetics and microbial N production were analyzed as a 4×4 Latin square using the
Proc Mixed procedure of SAS.

The results are presented in Table 1. Increasing dietary CP increased N intake (P=0.01) and urinary
N excretion (P=0.02), but had no effect on fecal N excretion (P=0.50). Retained N tended to increase
(P=0.09) as dietary CP level increased. Altering dietary RDP level had no effect on N balance. Weight
gain ranged from 0.2 to 0.6 kg/d during the experimental period. Urea-N entry rate (UER; endogenous
urea production) increased (P=0.03) with dietary CP level, but the amounts of endogenous urea-N
production partitioned to the GIT (GER; P=0.81) and urea-N utilized for anabolic purposes (UUA;
P=0.72) were unaffected. Urea-N returned to the ornithine cycle (ROC; P=0.07) tended to increase
as dietary CP increased, whereas urea-N transferred to feces (UFE; P=0.06) tended to decrease as
dietary CP increased. Urea-N fluxes were unaffected (P>0.05) by dietary RDP level and interactions

between dietary levels of CP and RDP did not affect (P>0.05) N balance or urea-N kinetics. Fractional
transfers of urea-N were largely unaffected by the diet; however, the proportion of GER that was
voided in feces (GER to feces) decreased (P=0.02) as dietary CP increased. Microbial N supply was
unaffected (P>0.05) by the diet. These results indicate that increasing dietary CP increased N intake
and N retention. The results also indicate that significant amounts of N transit the urea-N pool daily;
however, urea-N transfer to the GIT was similar across diets, and most of the urea-N which entered
the GIT was returned to the ornithine cycle.
Table 1. Effects of dietary CP and RDP levels on N balance, urea-N recycling kinetics and microbial
N production in beef heifers.
10% CP 13% CP SEM P values
Item 64.5% 71.0% 64.5% 71.0% CP RDP CP×RDP

RDP RDP RDP RDP
DMI, kg/d 8.0 7.5 7.6 7.4 0.5 0.61 0.75 0.51
N balance, g/d
N intake 138.5 130.1 171.7 165.3 10.8 0.01 0.51 0.93
Fecal N 28.7 26.9 30.3 28.6 2.4 0.50 0.47 0.98
Urine N 50.7 49.0 69.5 72.9 7.7 0.02 0.92 0.75
Retained N 59.1 54.2 71.9 63.8 6.0 0.09 0.30 0.79
Urea-N fluxes, g/d
UER 135.0 131.7 165.7 166.9 10.8 0.03 0.92 0.84
GER 98.4 93.3 121.1 105.6 11.3 0.81 0.40 0.67
UUA 25.4 20.9 35.1 17.2 7.7 0.72 0.21 0.43
ROC 70.3 69.8 84.0 86.2 6.5 0.07 0.90 0.84
UUE 36.5 43.1 44.6 61.3 5.1 0.04 0.06 0.35
UFE 2.7 2.6 2.0 2.1 0.2 0.06 0.95 0.68
Fractional transfers
UER to urine 0.268 0.311 0.277 0.371 0.04 0.38 0.11 0.50
UER to GIT 0.732 0.689 0.723 0.629 0.04 0.38 0.11 0.50
GER to ROC 0.709 0.753 0.695 0.822 0.06 0.64 0.18 0.49
GER to feces 0.028 0.029 0.019 0.020 0.00 0.02 0.74 0.93
GER to UUA 0.263 0.218 0.286 0.158 0.06 0.76 0.19 0.50
Microbial N, g/d 50.7 55.3 71.0 70.0 15.1 0.27 0.91 0.86
UER, urea-N entry rate; GER, gastrointestinal entry rate; UUA, urea-N utilised for anabolism ROC, return to
ornithine cycle; UUE, urinary urea-N loss; UFE, loss to feces.
References
Lapierre, H. and G. E. Lobley, 2001. Nitrogen recycling in the ruminant: A review. J. Dairy Sci. 84 (Suppl.), E223–
E236.
Lobley, G.E., D.M. Bremner and G. Zuur, 2000. Effects of diet quality on urea fates in sheep as assessed by refined,
non-invasive [15N15N]urea kinetics. Br. J. Nutr. 84, 459-468.

Effect of potato pulp as a dietary carbohydrate source on nitrogen
excretion and urea metabolism in sheep
T. Obitsu, M. Tsunemine, K. Han, T. Sugino and K. Taniguchi
Graduate School of Biosphere Science, Hiroshima University, 1-4-4 Kagamiyama, Higashihiroshima-
shi, 739-8528, Japan
Introduction
Ruminants fed forages containing high crude protein (CP), such as grass silage or legumes, increase
hepatic urea production and urinary nitrogen (N) excretion, when ruminal degradable N is in excess
of the microbial needs and absorbed as ammonia. Supplementation of non-structural carbohydrate
(NSC) sources to such forages facilitates ruminal microbial synthesis to reduce ammonia-N loss
from the rumen. Furthermore, greater ruminal fermentation of NSC may stimulate the entry of
endogenous urea into the rumen (Sutoh et al., 1996). Potato pulp, a by-product of starch extraction,
contains relatively high NSC, and may enhance ruminal microbial activity and urea-N entry into the
rumen. The objectives of this study were to elucidate the effects of potato pulp on N excretion and
urea kinetics in sheep under excess in N intake, by following (i) the responses to different levels of
potato pulp (experiment 1), and (ii) the comparison with other NSC sources (experiment 2).

In experiment 1, 3 ewes fitted with ruminal cannula and jugular vein catheters were used in a 3×3
Latin square design. As a control, ewes were fed alfalfa hay cubes (180 g CP/kg dry matter (DM)) at
maintenance digestible energy (DE). In the other two treatments, the hay cubes were replaced with
dried potato pulp (40 g CP/kg DM) by 25 and 50%, on a DE basis. Dietary urea was added to the
potato pulp diets to match the N intake of the control. Diets were offered hourly in equal portions by
an automatic feeder. Nutrient digestibilities, N balance and urinary excretion of urea and allantoin
were measured by total feces and urine collection for 4 d after a 14 d adjustment period. On the
first d of the collection period, [15N2]urea (99.7 atom%, 0.2 g N) dissolved in sterilised saline was
injected into the jugular vein. Venous blood and rumen contents were collected at intervals over
the 24 h after injection. 15N enrichments of feces and isolated plasma urea in dried eluate obtained
by ion exchange column were analysed by isotope ratio mass spectrometry after combustion by an
element analyser. Urea kinetics was calculated using multi-exponential equations as described by
Nolan and Leng (1974). Fecal 15N excretion was also calculated.
In experiment 2, the 3 ewes were fed alfalfa hay cubes (160 g CP/kg DM) at maintenance intake
plus three different concentrates in a 3×3 Latin square design. The concentrates were comprised
either (i) barley (128 g CP/kg DM) plus corn (73 g CP/ kg DM), (ii) barley plus potato pulp or (iii)
barley plus wheat bran (198 g CP/kg DM), all in a ratio of 1:1 (DM basis), with each supplement
provided at 0.5×maintenance energy. Digestibilities, N balance and urea kinetics were measured
as in experiment 1.

In experiment 1, intakes of DE and digestible N did not differ between treatments. Urinary allantoin
excretion, an indicator of ruminal microbial protein synthesis, increased (P<0.05) in proportion
to the level of potato pulp, probably due to greater starch fermentation in the rumen. However,
production rate, entry rate into the gut and urinary excretion rate of urea were not affected by dietary
treatments (Table 1).

In experiment 2, DE intake for all treatments were similar, but digestible N intake was greater (P<0.05)
for the wheat bran diet than the other two diets due to higher N content of wheat bran. Urinary
allantoin excretion did not differ among treatments even though the amount of digestible starch for
corn diet was greater than that for the other two diets. Unfortunately, significant differences in urea
production rate and urea entry into the gut between treatments were not detected (Table 2), urinary urea
excretion was greater (P<0.05) for the wheat bran diet. Although fecal 15N excretion did not differ,
fecal total N excretion was greater for the potato pulp diet than the corn diet (P<0.05). Compared
with feeding alfalfa alone in experiment 1 (control), additional supplementation of potato pulp to
alfalfa hay cubes (potato pulp diet in experiment 2) may be as effective as isocaloric supplementation
of corn, to reduce urinary urea excretion through the enhancement of urea entry into the gut.
Table 1. Effect of the level of potato pulp on urea kinetics in ewes in experiment 1.
Item Alfalfa hay Replacement with SEM P

cubes potato pulp
Control
25% 50%
Digestible nitrogen intake, g N/kg0.75 1.12 1.07 1.05 0.04 0.162

Urinary allantoin, g/kg0.75 0.053b 0.062ab 0.072a 0.004 0.014
Urea production, g N/kg0.75 1.12 1.12 1.05 0.05 0.623
Urea entry into the gut, g N/kg0.75 0.42 0.43 0.41 0.03 0.943
Urinary urea excretion, g N/kg0.75 0.70 0.69 0.64 0.02 0.148
Urinary urea excretion, g N/kg0.75 0.33 0.30 0.27 0.01 0.115
a,b Means in the same row with different superscript differ significantly (P<0.05).
Table 2. Effect of different carbohydrate sources on urea kinetics in ewes in experiment 2.
Item Corn Potato pulp Wheat bran SEM P
Digestible nitrogen intake, g N/kg0.75 1.05b 1.01b 1.21a 0.04 0.031

Urinary allantoin, g/kg0.75 0.075 0.070 0.082 0.006 0.742
Urea production, g N/kg0.75 1.21 1.15 1.30 0.04 0.349
Urea entry into the gut, g N/kg0.75 0.63 0.63 0.55 0.05 0.602
Urinary urea excretion, g N/kg0.75 0.58b 0.52b 0.75a 0.04 0.016
Fecal nitrogen, g N/kg0.75 0.33b 0.38a 0.36ab 0.01 0.049
a,b Means in the same row with different superscript differ significantly (P<0.05).
References
Nolan, J.V. and R.A. Leng, 1974. Isotope techniques for studying the dynamics of nitrogen metabolism in ruminants.
Proc. Nutr. Soc. 33, 1-8.
Sutoh M., Y. Obara and S. Miyamoto, 1996. The effect of sucrose supplementation on kinetics of nitrogen, ruminal
propionate and plasma glucose in sheep. J. Agric. Sci. Camb. 126, 99-105.

The effect of dietary pectin on concentration of free amino acids and
urea in blood plasma of young pigs
E. Święch, M. Taciak, A. Tuśnio and L. Buraczewska
The Kielanowski Institute of Animal Physiology and Nutrition, Instytucka street 3, 05-110 Jabłonna,
Poland
Introduction
Pectin affects digesta viscosity and passage rate, as well as digesta water-holding capacity and
fermentability and may induce changes in intestinal morphology. In our recent work (Buraczewska
et al., 2007) dietary pectin decreased ileal digestibility of amino acids (AA), increased muscle layer
width of the small intestine and decreased N retention in pigs. The objective of this study, which
was a continuation of research on the effects of pectin on nitrogen metabolism in pigs, was the
assessment of varied levels of pectin on time-related changes in concentrations of free AA and on
post-prandial level of urea in plasma.

The experiment was carried out on three groups, each of 5 male pigs (from 15 to 40 kg BW) fed on
a balanced feed mixture (about 18% protein) composed of wheat, corn, soyabean meal, casein and
crystalline AA and supplemented with 0, 4, or 8% of highly esterified apple pectin. The daily dietary
allowance provided pigs with the same level of nutrients including ileal digestible AA. After 35 d
of feeding diets, pigs were fitted with silicone catheters to the jugular vein to collect blood samples
before and 0.5, 1, 2, 4, 6 h after the morning meal. Blood was collected into heparinised tubes and
immediately centrifuged for 10 min at 3900×g at 4 °C. The plasma proteins were precipitated with
salicylsulfonic acid and centrifuged for 10 min at 10 000×g at 4 °C. Free AA content was measured
using gas chromatography according to the method described by Hušek (1991) with modifications.
Urea concentration was measured in pooled plasma samples, taken at 2 and 4 h after the morning
meal, using a biochemical diagnostic analyser (Vitros DTII). Data were statistically evaluated using
Statistica software with one-way ANOVA or repeated measurements ANOVA. All results were
expressed as means ± SEM of 5 pigs. The level of significance was set at P≤0.05.

Pectin supplementation did not significantly affect free AA concentration in plasma sampled at
different times after the morning meal (Figure 1). Unfortunately, high individual differences were
found among the pigs in each group and only some decreasing tendency was observed in AA level
during the first h after feeding the diet with 8% pectin, as compared to the diet without pectin. This
may result from lower ileal digestibility of AA and higher viscosity of digesta (Buraczewska et al.,
2007). For threonine and branched-chain AA, the highest plasma level was found in most cases
about 1 h after the meal. Literature data of plasma AA in the pig are very variable and affected not
only by analytical but also by animal-related factors (feeding system, age).
The plasma urea concentration was decreased by added pectin, however only the difference between
pigs fed diets containing 0 and 8% pectin was significant (Table 1). This was in agreement with
results obtained by Zhu et al. (2003), who found reduction of plasma urea level by 28% in growing
pigs after infusion of 10% pectin solution to the caecum. Lower plasma urea may suggest that pectin
(fermetable fiber) shifted N excretion from urine to faeces.

Figure 1. Concentration (µmol/mL) of free threonine, isoleucine, leucine and valine in plasma
of young pigs fed diets containing 0, 4, and 8% pectin, determined before and up to 6 h after the
meal.
Table 1. Plasma urea concentration (mmol/l) of young pigs fed diets containing 0, 4, and 8%
pectin.
Pectin, % 0 4 8 P-value
Urea, mmol/L 3.01 ± 0.17 b 2.82 ± 0.29 ab 2.13 ± 0.25 a 0.037
References
Buraczewska, L., E. Święch, A. Tuśnio, M. Ceregrzyn and W. Korczyński, 2007. The effect of pectin on amino acid
digestibility and digesta viscosity, motility and morphology of the small intestine, and on N-balance and performance
of young pigs. Livest. Sci., in press.
Hušek, P., 1991. Amino acid derivatization and analysis in five minutes. FEBS Lett. 280, 354-356.
Zhu, C.L., H. Lapierre, M. Rademacher and C.F.M. de Lange, 2003. Pectin infusion into the caecum reduces the
utilization of threonine intake for body protein deposition and urea flux in growing pigs. In: Ball, R.O. (editor),
Proc. 9th International Symposium on Digestive Physiology in Pigs, Banff, AB, Canada, Vol. 2, 346-348.
Supported by the Ministry of Science and Education, Grant No. 2P06Z 06827.

Effect of different energy supply on microbial protein synthesis and
renal urea handling in Corriedale sheep consuming temperate fresh
forages
I. Tebot, C. Echaides, A. Secchi and A. Cirio
Veterinary Faculty, Physiology Department, Lasplaces 1550, 11600, Montevideo, Uruguay
Introduction
The efficiency of microbial protein synthesis (g of microbial N/kg digestible organic matter apparently
digested in the rumen) in Corriedale ewes is not modified when the animals are fed on a 57% reduced
N diet, and this fact was accompanied by a reduction of the urinary elimination of urea (Tebot et al.,
2002). However, the efficiency of N utilisation could be limited by a low ratio soluble carbohydrates/
N, frequently found in temperate fresh forages consumed by grazing ruminants (Garcia et al., 2000).
For this reason energy supplementation is often practised in the winter time in order to improve the
use of forage N for microbial protein synthesis.
In order to asses the necessity of energy supplementation for microbial protein production in
Corriedale sheep consuming temperate fresh forage, two energy-rich supplements (grain and
molasses) were tested and microbial protein synthesis and renal urea handling were determined.

Eighteen non pregnant, non lactating ewes (43 ± 4 Kg BW), housed in individual pens, were
randomly divided into 3 groups (n=6 each one): F (forage, non supplemented, 44 g DM/kg0.75 BW),
FG (forage (32 g DM/kg0.75 BW) + grain (12 g DM/kg0.75 BW)) and FGM (forage (32 g DM/kg0.75
BW) + grain (6 g DM/kg0.75 BW) + cane molasses (6 g DM/kg0.75 BW)). Forage was fresh oats (DM
14.7%, OM 88.7%, NDF 49.9%, ADF 26.8%, CP 14.4%) and grain was barley grain (DM 86.8%,
CP 10.9%, EM Mcal/kg 3.13, ADF 5%). Ewes were fed 4 times a d, with only the first and the last
meal supplemented. Following a 10-d adaptation period to the diets, different tests were performed
in order to study the urinary allantoin excretion (method of Young and Conway, modified by Fujihara
(Fujihara et al., 1987)), and the renal function and urea handling (urea and creatinine clearances).
Microbial protein synthesis was estimated according to the equation:
y = e (0.830 + 2.089x)
proposed by Puchala and Kulasek (1992), where y = microbial N entering the duodenum and x =
urinary excretion of allantoin N. Statistical significances were evaluated by the ANOVA test.

The daily output of urinary allantoin was 0.64 ± 0.10, 0.60 ± 0.06 and 0.62 ± 0.05 mmol/d/BW0.75
for F, FG and FGM groups respectively. The calculated amount of microbial N entering the small
intestine was 8.6 ± 1.4, 8.1 ± 1.6 and 8.8 ± 1.3 g/d for F, FG and FGM groups respectively. No
statistical differences were found among the groups for both parameters. Renal urea handling is
shown in Table 1.

Table 1. Urea plasma and renal parameters in the three experimental groups.
Parameters F FG FGM
Urea plasma, mg/mL 0.27 ± 0.03 0.25 ± 0.05 0.25 ± 0.04

Urine flow, mL/min 2.21 ± 0.04 1.52 ± 0.03* 1.82 ± 0.03*
Glomerular filtration rate, mL/min 80.2 ± 4.52 79.38 ± 5.76 75.33 ± 3.07
Urea clearance, mL/min 38.44 ± 5.73 38.27 ± 6.18 30.95 ± 4.63
Filtered load of urea, mg/min 21.96 ± 4.26 21.99 ± 2.67 21.44 ± 2.75
Urea eliminated, mg/min 9.0 ± 0.36 8.88 ± 0.07 7.67 ± 0.54
Fractional excretion of urea 0.48 ± 0.09 0.54 ±0.04 0.41 ± 0.07
Data = mean ± SEM * P<0.05 compared with F group, n = 6.
Low plasma urea level in the three groups may be explained, as proposed by Obara et al. (1991),
assuming a high influx of blood urea into the rumen and/or a low rumen ammonia level because of
its higher uptake by energy-rich microbes. The reduced renal urea elimination in low protein (LP)
vs. normal protein (NP) fed sheep, found in a previous work (1.5 vs. 9.3 mg/min respectively), was
mainly due to a decrease in glomerular filtration rate (20.0 vs. 69.5 mL/min in LP and NP groups
respectively, Tebot et al., 2002). A reduced fractional excretion of urea was also associated with
renal urea sparing in sheep: 0.24 (LP) vs. 0.47 (NP) (Cirio and Boivin, 1990); 0.21 (LP) vs. 0.46
(NP) (Tebot et al., 1998). Consequently, the present values of renal parameters do not support a
renal adaptative process for urea sparing.
It seems that local temperate fresh forages provide enough energy for a correct level of microbial
protein synthesis, without requiring energy supplementation.
References
Cirio, A. and R. Boivin, 1990. Urea recycling from the renal pelvis in sheep: a study with [14C]urea. Am. J. Physiol.
258, F1196-F1202.
Fujihara, T., E.R. Orskov, P.J. Reeds and D.J. Kyle, 1987. The effect of protein infusion on urinary excretion of purine
derivatives in ruminants nourished by intragastric nutrition. J. Agri. Sci. Camb. 109, 7-12.
García, S.C., F.J. Santini and J.C. Elizalde, 2000. Sites of digestion and bacterial protein synthesis in dairy heifers fed
fresh oats with or without corn or barley grain. J. Dairy Sci. 83, 746-755.
Obara, Y., D.W. Dellow and J.V. Nolan, 1991. The influence of energy-rich supplements on nitrogen kinetics in ruminants.
In: Tsuda T., Y. Sasaki and R. Kawashima (editors), Physiological aspects of digestion and metabolism in ruminants:
Proc. of the 7th Int. Symposium on Ruminant Physiology. Academic Press, Inc., San Diego, USA, 515-539.
Puchala, R. and G.W. Kulasek, 1992. Estimation of microbal protein flow from the rumen of sheep using microbial
nucleic acid and urinary excretion of purine derivatives. Can. J. Anim. Sci. 72, 821-830.
Tebot, I., A. Britos, J.-M. Godeau and A. Cirio, 2002. Microbial protein production determined by urinary allantoin
and renal urea sparing in normal and low protein fed Corriedale sheep. Vet. Res. 33, 101-106.
Tebot, I., S. Faix, M. Szanyiova, A. Cirio and L. Leng, 1998. Micropuncture study on urea movements in the kidney
cortical tubules of low protein fed sheep. Vet. Res. 29, 99-105.

The effect of potato protein and potato fibre on amino acid digestibility,
small intestinal structure and on N-balance and performance of young pigs
A. Tuśnio, B. Pastuszewska, E. Święch and L. Buraczewska
The Kielanowski Institute of Animal Physiology and Nutrition, Instytucka 3, 05-110 Jabłonna, Poland
Introduction
Potato protein concentrate (PPC) is regarded as a valuable protein supplement for young pigs
(Kerr et al., 1998) but its dietary level is limited by the presence of antinutritional compounds as
protease inhibitors and glycoalkaloids (GA) (Smith et al., 1996) which depress feed intake and
growth performance, and, presumably, health of the gastrointestinal tract. Potato fibre (PF) consists
of fermentable non-starch polysaccharides having functional properties. The objective of the study
was to determine the amino acid availability of PPC and the effects of feeding PPC vs. casein (CAS)
and PF vs. cellulose (CEL) on protein utilisation and morphometry of the small intestine.

Ileal digestibility and nitrogen balance were determined on young male pigs line 990. The animals
were kept in metabolic cages in an environmentally controlled room (22 °C) with ad libitum access
to water. Ileal digestibility was studied in a cross-over design using 6 pigs (about 21 kg initial BW)
per diet. The animals were fed semisynthetic diets with casein or casein and potato protein (1:1),
both with addition of cellulose (6%). The animals were surgically fitted with a post-valve T-shaped
cannula inserted in the caecum and given the experimental diets for seven d. Digesta samples were
collected during 12 h for the last three d of each experimental period.
In a 2×2 factorial nitrogen balance experiment four groups of 6 male pigs (about 15 kg initial BW)
were fed during 21 d on cereal diets containing either CAS or PPC and supplemented with CEL
(6%) or PF (6%). Feeding level ranged from 4.6-4.8% BW. Feed intake and body weight were
controlled. Collection of urine and faeces lasted 6 d. At the end of the experiment, the animals were
sacrificed and samples of the duodenum and mid-jejunum were taken, placed in Bouin solution, cut
and stained with haemotoxylin and eosin. Villus height, crypt depth, thickness of the tunica mucosa
and myenteron were determined. The statistical analyses were carried out using the One-way and
Multifactor ANOVA procedure.

Apparent ileal digestibility (AID) of PPC protein and most essential amino acids was significantly
lower than that of CAS except for AID of threonine, isoleucine and valine which were not statistically
different (Table 1). The effects of PPC and PF on the structure of the small intestine varied between
the segments and parameters under study (Table 2). In the duodenum, PF increased villi length and
decreased crypt depth (only on PPC diet) while PPC increased mucosa thickness. In the mid-jejunum,
feeding the diet with PPC and CEL increased all parameters while decreased or did not affect them with
the PF diet. Feeding PF increased the mid-jejunum myenteron thickness with both protein diets.
Total protein digestibility of the PPC-supplemented diet was lower than that of the CAS diet (Table 3),
the effect being smaller than in the case AID of the proteins fed alone (Table 1). The two indices of
protein utilisation were not significantly affected by the type of protein or fibre, although feeding PF
diets tended to depress protein retention as related to absorbed protein and protein intake.
Average daily body gain was in the range of 660-680 g and was not affected by the treatment. It
may be concluded that potato protein concentrate has lower ileal and total protein digestibility but
similar supplementary protein value compared with casein. Potato fibre does not affect protein

digestibility but tends to depress protein utilisation. Both the nature of protein and type of fibre
affect the structure of the small intestine.
Table 1. Apparent ileal digestibility of protein and amino acids (%).
Diet Protein Lysine Threonine Isoleucine Arginine Histidine Leucine Phenyl- Valine
alanine
CAS 87.0b1 95.9b 86.4 83.8 93.3b 94.9b 94.0b 94.7b 90.0
PPC2 80.4a 87.0a 80.1 84.1 88.3a 84.9a 87.4a 86.7a 86.9
SEM 1.63 1.31 2.56 2.14 1.40 1.40 1.38 1.34 1.42
1Means in the same column with different superscripts are significantly different (P<0.05).
2Calculated by difference method from PPC+CAS diet.
Table 2. Effects of protein and fibre on duodenal and mid-jejunal morphometry (µm).
Item Casein PPC SEM Significance
CEL PF CEL PF Protein Fibre P×F
Duodenum
Villi length 340 292 324 318 9.17 0.59 0.00 0.02
Crypt depth 231 277 257 258 8.99 0.67 0.01 0.01
Mucosa thickness 614 594 648 621 14.33 0.03 0.10 0.82
Myenteron thickness 488 470 466 494 13.47 0.97 0.72 0.09
Mid-jejunum
Villi length 391 462 408 342 11.46 0.00 0.83 0.00
Crypt depth 237 263 275 262 7.44 0.01 0.37 0.01
Mucosa thickness 684 769 740 643 15.27 0.02 0.68 0.00
Myenteron thickness 306 384 358 415 10.01 0.00 0.00 0.28
Table 3. Effects of protein and fibre on nitrogen balance (%).
Item Casein1 PPC2 SEM Significance
CEL PF CEL PF P F P×F
Apparent digestibility 86.6 86.1 84.5 83.8 1.02 0.05 0.57 0.90
N ret. / N absorbed 70.3 66.0 71.2 65.1 2.51 0.99 0.11 0.79
N ret. / N intake 60.8 56.8 60.2 54.3 2.69 0.57 0.08 0.73
1,2 Used as the only protein supplements in cereal diets.
References
Kerr, C.A., R.D. Goodband, J.W. Smith, R.E. Musser, J.R. Bergström, W.B. Jr Nessmith, M.D. Tokach and J.L. Nelssen,
1998. Evaluation of potato proteins on the growth performance of early-weaned pigs. J. Anim. Sci. 76, 3024-3033.
Smith, D.B., J.G. Roddick and J.L. Jones, 1996. Potato glycoalkaloids: Some unanswered questions. Trends in Food
Science and Technology 7, 126-131.
Supported by the Ministry of Science and Education, Grant No. 2P06Z01830.

Part 5. From the parts to the whole of how to use detailed
information to answer applied questions
5. Protein-energy interactions
Protein-energy interactions: horizontal aspects
G.E. Lobley
Obesity and Metabolic Health Division, Rowett Research Institute, Bucksburn, Aberdeen, AB21
9SB, United Kingdom
Abstract
Protein and energy interact at many levels to ensure survival of the organism, productivity in an
agricultural setting and the health of both the animal and consumers of its products. Nutrients interact
through interchange of carbon skeletons via intermediary metabolism and this provides metabolic
flexibility required at the cell, organ and whole body level. This flexibility allows key organs, such
as the digestive tract, muscle and mammary gland, to respond to metabolic demands across a wide
range of nutrient inputs and patterns of supply. Understanding the important factors involved will
aid future strategies to maximise nutrient use for anabolic outputs in farm animals. Furthermore,
recent studies have also indicated how important the interactions between proteins and energy are
in maintaining the metabolic health of humans and animals. Mothers fed adequate energy, but low
protein, place their offspring at risk of late onset diseases e.g. diabetes and cardiovascular disease
linked to insulin insensitivity. In contrast, insulin sensitivity can be improved during post-natal growth
or adulthood by replacement of saturated fat consumptions by polyunsaturated fatty acid intake and
this benefits both amino acid (AA) and glucose metabolism. In other situations, although high rates of
post-natal AA supply can increase hepatic gluconeogenesis this leads to impaired glucose utilisation
in muscle, but not in adipose tissue. Recent advances in the molecular understanding of nutrient/
hormone signal cascades in cells have revealed a key role for the mammalian target of rapomycin
(mTOR) in regulation and integration of AA, glucose and insulin action. Interestingly, the same
pathway may also function in the brain to regulate appetite and food intake, through sensing factors
such as energy (ATP) status and leucine availability. In addition, conversion of AA to glucose by the
small intestine may act as a satiety signal to inhibit intake and provide a means to regulate energy
over-consumption. Therefore, in the areas of both animal production and human health, exciting
challenges exist as we try to understand and manipulate protein-energy interactions at the cell, organ
and whole body level. These different targets may involve manipulation of common mechanisms but
to achieve these future advances necessitates the integration of molecular and modelling approaches
with a clear understanding of the consequences at both the organ and whole animal level.
Keywords: protein, energy, tissues, health, insulin
Introduction
Although it has been traditional to consider ‘protein’ and ‘energy’ metabolism as separate entities in
mammalian metabolism, most scientists recognise this is an artificial divide. Indeed, they should be
considered together as this reflects how nutrients are ingested and utilised as part of normal feeding
patterns during evolution. In the wild, for example, protein is never consumed without co-ingestion
of carbohydrate and/or fat while energy-rich meals that do not contain protein are restricted mainly
to hedonistic selections made by humans. Instead, the organism has to respond to a near-infinite
variety of ingested macronutrient mixtures, while metabolism has adapted to support high rates of
both energy expenditure and protein turnover, even with unbalanced intakes and variations in the
absorptive state. In consequence, animal survival and function has involved the development of a
complex set of intermediary metabolism and hormonal regulatory processes. The sheer complexity
of the signals, controls and responses that arise has required concepts to be refined into smaller,
manageable questions and here scientists have tended to specialise – including within and between

those broad definitions - ‘energy’ and ‘protein’. While it would be foolish to say that our knowledge
is sufficiently advanced to allow these rather artificial barriers to be completely dismantled, we have
reached the stage where serious attempts can be made to integrate metabolism and achieve sensible
predictive responses. Continued advance in this area requires input from those interested in overall
responses, e.g. agriculturists, clinicians, plus metabolists, geneticists, molecular biologists coupled
with mathematicians to combine empirical data and theoretical frameworks into descriptive and
predictive models. Therefore, the recent merging of the separate meetings on Protein Metabolism
and Energy Metabolism, initiated in 2003 and formalised at this Symposium, provides a timely
forum for specialists in both areas to meet and debate within their areas of expertise but also to
discuss the wider integrative issues.
Historical protein-energy interactions

Mammals need to release energy contained in food or stored in endogenous reserves to maintain
body temperature and support homeorhesis and anabolism. They also need to retain dietary energy,
either to deliver a continual supply of specific substrates, such as glucose to support the vital
functions of the brain and red blood cells, or to meet more global needs, as provided by fat and to
a lesser extent protein. Maintenance of these stores of carbohydrate (glycogen), lipid and protein is
not a static process and the required dynamism to allow the necessary metabolic flexibility has an
energy cost. Perhaps this is seen most obviously with protein metabolism, where the synthesis of
peptide units and formation into new protein molecules has a known minimum ATP requirement
(van Milgen, 2002). Classical studies during the 1970 and 1980s linked energy expended to support
protein turnover with whole body heat production (see MacRae and Lobley, 1987) with the minimum
theoretical contribution of 15-25% increased to 33% by practical studies in vitro and in vivo (see
Lobley, 1990). Similarly, energy costs occur to support the continued linkage and cleavage of fatty
acids to glycerol (Dunshea et al., 1990) as well as the dynamic incorporation and release of glucose
from glycogen as part of the diurnal feed-fast cycle of non-ruminants (see van Milgen, 2002). These
dynamic interactions at the protein-energy and energy-energy macronutrient level complemented the
biochemical stoichiometric pathways so elegantly defined during the Twentieth century.
Whole body protein and energy metabolism is a composite of contributions from the various organs
(Lobley, 2003) with the most dynamic in terms of energy expenditure (e.g. digestive tract, liver) often
also having high rates of protein turnover. This led to attempts at the cellular level to identify the
process that contributed to energy expenditure (see Lobley, 1991) and such crude accounting rapidly
became superseded by increasingly sophisticated models to explain the biochemical dynamics of
biological systems (Baldwin et al., 1987; Gerrits et al., 1997; Cherepanov et al., 2000; van Milgen,
2002). Indeed, modelling of biological systems has impacted greatly on our overall thinking in that
inputs and outputs need to be set for all processes – even when these are imprecisely defined or
unknown. Nothing stimulates a scientist more than an area classified as ‘undefined’, especially when
the suspicion arises that this may be important!
So where does such a topic as ‘protein-energy interactions’ go forward? There are many such
directions, with importance to separate areas. Many attendees at this Symposium will come from a
‘traditional’ animal background with a desire to respond to the current pressures that include demands
for a healthy product, sensibly priced but that is profitable for the farmer and to be achieved with
due attention to environmental concerns. Others will have interests in animal health and welfare and
how to minimise stress and imbalances. Human health may also be a key issue, especially with the
ridiculous dichotomies that exist in the modern world: too little food (and of inadequate quality) in
many under-developed regions while, in developed countries, over-consumption is now creating
obesity-linked diseases. In all these areas a clearer understanding of protein-energy interactions will
prove beneficial but in such a short article considerable selectivity is required (Figure 1). The current
article reflects three broad areas of interest. The first discusses examples of the metabolic flexibility

Body composition
Product quality Epigenetics
Appetite
Bioenergetics Protein ↔ Energy
Production Metabolic health

efficiency Metabolic fuels
Figure 1. A selection of the protein-energy interactions that impact on animal productivity and
mammalian health.
Figure 1. A selection of the protein-energy interactions that impact on animal productivity
necessary within and
tissues to face variations in nutrient supply and still meet metabolic demands. The
mammalian health.
second considers how protein and energy interact metabolically, including in appetite regulation,
while the last addresses cellular mechanisms that link amino acid and glucose metabolism.
Metabolic flexibility
In nutrient-based schemes, inputs are often defined under the classical headings of protein or energy but
cells do not adhere to such arbitrary classification and have adapted to perform many inter-conversions.
This provides the all-important metabolic flexibility to respond to and survive to a wide range of
nutritional, environmental and physiological demands and challenges. Such adaptability is illustrated
by consideration of two organs – the gastro-intestinal tract (GIT), where nutrients are first sensed, and
the mammary gland which, for the high yielding cow, exerts the greatest metabolic demand.
Metabolic flexibility of the gastro-intestinal tract
The gut digests food and absorbs nutrients, processes that involve extensive endogenous secretions,
limited re-absorption of which may impact on net availability to other tissues (Ouellet et al., 2002;
Lapierre et al., 2006). At the same time, the tract has to maintain an innate immunity barrier against
pathogenic bacterial invasion, again incurring both energy and protein costs (Smirnov et al., 2004;
Irene et al., 2005; Faure et al., 2006). In both ruminants and non-ruminants, the gastrointestinal
tract contributes between 15-30% towards whole body energy expenditure (Yen and Nienaber, 1993;
Reynolds et al., 1991; Vaugelade et al., 1994) and up to 35% of protein turnover (Lobley, 2003).
Such considerable metabolic activity from a tissue that comprises less than 10% of total protein
mass will inevitably impact on nutrient demands and utilisation.
Besides the general correlations observed at the whole body level between energy expenditure and
protein metabolism there are also specific issues. For example, both processes in the digestive tract are
influenced by the amount and pattern of food intake (Webster et al., 1975; Lobley et al., 1994; Deutz
et al., 1995; Davis et al., 1996). Notably, non-ruminants face the problem of the diurnal feed-fast cycle
and how this impacts on meeting metabolic requirements. Nearly 30 years ago it was realised that
the rat small intestine catabolised considerable amounts of two non-essential amino acids (NEAA),
glutamine and glutamate (Windmueller and Spaeth, 1980), but this was accompanied by an increased
net appearance of other NEAA, particularly alanine, citrulline and proline. So it was unclear exactly
whether the oxidation was complete, thus liberating substantial amounts of energy, or whether the
liberated CO2 was part of the carbon transformations between the various NEAA. Furthermore, it was
realised that the digestive tract could also catabolise essential amino acids (EAA) in most species,
including the branch-chain (BC) AA (Lobley et al., 1995; van der Schoor et al., 2001; Lapierre et al.,

2002), methionine (Lobley et al., 2003; Shoveller et al., 2005) and in pigs, also lysine (van Goudoever
et al., 2000) and phenylalanine (Bush et al., 2003) although not in sheep (Lobley et al., 2003). Such
findings had two important implications, first that both NEAA and EAA may be important energy
providers to the GIT and, second, such catabolism would impact on both the absolute amounts and
relative proportions of AA available to the rest of the animal for anabolic purposes. This raised questions
of the extent of energy contribution from the AA, was this obligate or does it vary with nutritional
and physiological state and, in a wider sense, what purpose does this serve?
Studies in vitro using sections of the rat intestine showed that the amounts of glutamine and
catabolised glucose were reduced if the other were present in substantial amounts (Fleming et al.,
1991). Similarly, enterocytes from dairy cows in early lactation were able to meet all of the energy
demands from either glutamine or glucose alone, but when added together glutamine oxidation
was decreased by approximately 60% (Okine et al., 1995). Furthermore, glutamate oxidation by
duodenal mucosal cells from sheep could be reduced by propionate and glutamine, in a concentration
dependent manner, but not by glucose (Oba et al., 2004). Together, all these data suggest metabolic
flexibility in the biological fuels used and this attractive concept fits with similar findings with other
proliferative cells, such as lymphocytes (Dugan et al., 1994).
Whether such findings in vitro have physiological significance in vivo was addressed with piglets
by Reeds and colleagues. Their studies confirmed that 95% of dietary glutamate but only 13% of
enteral glucose was catabolised by the portal-drained viscera (PDV), with contributions to tissue CO2
production of 36 and 6%, respectively, while a further 15% was derived from catabolism of systemic
glutamine (Stoll et al., 1999; Reeds et al., 2000). While almost all the glutamate catabolism occurred
during absorption from the lumen, i.e. ‘first pass’, most of the glucose use involved removal from
the systemic circulation, i.e. on second (or subsequent) passes (van der Schoor et al., 2001). Most
of the glutamate and glutamine removed by the GIT was oxidised, with only a small conversion to
other products, such as alanine and lactate, whereas extensive conversion to other metabolites did
occur for glucose (Reeds et al., 2000).
So if glutamate and glutamine represent major energy sources for the GIT what happens when supply
of these becomes limited but additional energy sources are given? Based on the enterocyte data in
vitro this should lead to a reduced oxidation of glutamine plus glutamate while glucose catabolism
is increased. When piglets were fed either of two iso-energetic diets with protein reduced from 16
to 10% by substitution with carbohydrate this led to lowered oxidation of leucine, glutamine and
glutamate across the GIT (van der Schoor et al., 2001). Glucose oxidation did increase (by 20%)
but, interestingly, this was derived totally from systemic sources on the higher protein diet but was
divided approximately equally between enteral and systemic sources at the lower protein intake.
This might indicate that villi cells may have a preference for AA as energy sources but, if these
become limiting, then glucose can be utilised instead. Nonetheless, the decrease in oxidation of AA
(glutamate plus leucine) from 50% to only 10% of CO2 production between the HP and LP diets was
not fully compensated by the extra input from glucose and so the contribution from ‘other’ sources
rose from 11 to 40% (van der Schoor et al., 2001). Whether these other sources involve substrates
such as galactose (from the dietary lactose) or lipids remains to be identified and indicates that other
links exist in oxidative metabolism between AA and ‘energy’ metabolites.
So why does the GIT oxidise glutamate and glutamine? The body has extensive needs for both these
NEAA and so reduced absorption from the diet necessitates synthesis de novo by other tissues.
What benefit or detriment does this confer? In fact, conversion of glucose to glutamate in peripheral
tissues costs only 1.25 ATP molecules less than that available from direct catabolism of glucose by
gut tissues (van Milgen, 2002). This is a relatively small cost to pay for metabolic flexibility. The
costs for replacement of glutamine are greater, as an extra ATP molecule is required for amidation.
Although the GIT needs glutamine-N to synthesise purines and pyrimidines to support the nucleic

acid demands of cell proliferation, this represents only 6% of glutamine removal (Gate et al., 1999)
and so is not a main biological drive. A stronger possibility may relate to the fuel used during the
post-absorptive period. Glucose is an essential fuel for the brain and the red blood cells and thus may
be ‘protected’ to support these vital organs. Certainly, supply of carbohydrate leads to increased net
α-amino N retention by the digestive tract (Deutz et al., 1995) but during the post-absorptive phase
only 12% of this appears in the portal vein (van der Schoor et al., 2002) and the remainder may be
used as an energy source, thus sparing glucose and effectively maintaining the neutral GIT balance
for glucose (i.e. no net uptake from the systemic supply). Furthermore, the intestine may also be able
to synthesise glucose under conditions of restricted nutrition (Mithieux et al., 2006).
In forage-fed ruminants, little or no dietary glucose usually reaches the small intestine and any
demand would need to arise from systemic sources and, furthermore, the diurnal feed–fast cycle does
not usually operate. So do these factors impact on protein-energy interactions across the ruminant
GIT? Unfortunately, such data are more limited but casein infusion into the ovine duodenum
increased net portal appearance of many EAA plus proline (El Kadi et al., 2006) but for glutamate
and glutamine only 20-30% of the additional supply was recovered across the PDV. If the other
70-80% were converted to glucose this would have reduced the negative PDV glucose balance, but
this did not occur. As with the pig, this might suggest that glutamate and glutamine interact with
energy metabolites other than glucose.
What happens under circumstances where demands for both energy and protein are increased, as
occurs in the high-yielding dairy cow in early lactation where expansion of the gut plus liver mass
(Gibb et al., 1992) and activity occurs alongside the drive to support mammary gland function? Here,
80% of supplemental glutamine (300 g/d) infusion via the abomasum was recovered in the portal
vein either unchanged or as glutamate (Doepel et al., 2005 and unpublished results), with no impact
on net glucose transfers across the GIT. This was despite low net portal appearance of glutamate
and glutamine with the basal diet. Similar high portal recoveries for supplemented glutamine, but
not glutamate, from casein were observed in late-lactation cows (Hanigan et al., 2004), again with
no change in net glucose appearance. In lactating animals, therefore, the switch between glucose
and AA as intestinal energy sources appears to be relatively restricted. Whether this represents a
limited ability of the digestive tract to catabolise glutamine or because demands elsewhere in the
animal require a ‘sparing’ effect remains to be elucidated.
Metabolic flexibility of the mammary gland
Along with the liver, the mammary gland is one of the most adaptable organs in the body. Indeed,
in energetic terms the high yielding dairy cow is one of the most challenged animals on the planet
(Vandehaar and St-Pierre, 2006) with the relatively small mass of the mammary gland driving large
outputs of both energy (lactose and lipid) and protein (Lobley, 2003). While approximately half of the
lipid and a small component of the protein output is supplied preformed, most of the secreted products
are synthesised within the gland. Such synthesis requires metabolic flexibility, for both energy and
protein metabolites, to cope with changes in nutrient supply that arise from both endogenous stores
(during early lactation) and the wide range of ration formulations offered.
The main sources of nutrient uptake for intermediary metabolism consist of acetate, glucose, ketones
and AA to not only synthesise the required milk fat, lactose and protein but also provide energy
to drive these processes and sustain cellular homeostasis. A number of studies in dairy cows have
shown that either additional protein or AA (see Doepel et al., 2004) or energy supply (Huhtanen et
al., 2002; Rigout et al., 2003; Rulquin et al., 2004) can improve milk output and impact on either
milk protein or milk fat (energy) yield. While the magnitude of responses varies between studies,
determined partly by the basal ration supplied, these data suggest that separately both additional
protein and energy can exert positive anabolic effects. Similar milk anabolic responses to dextrose

(plus insulin) and supplemented protein have been observed in lactating pigs (Dunshea et al.,
2005). The question is do these responses occur via different or similar mechanisms and are there
opportunities for synergism?
A recent study assessed the effects of supplemental protein (casein) and energy (propionate), alone
or in combination, on production parameters and mammary gland metabolism. While milk output
increased more with casein than with propionate, the response was additive when supplied together
(Raggio et al., 2006b). Furthermore, the additional uptake of [13C]leucine by the mammary gland
exceeded the extra secreted in milk protein for the casein treatment but the incremental uptake:output
ratio was close to unity when propionate was present (Raggio et al., 2006a). Correspondingly, the
gland oxidised more leucine with the casein treatment but this was reduced when propionate was
supplemented at the same time. The combined improvement on milk protein output was achieved
without an increase in mammary gland protein synthesis. These data suggest that protein and energy
supply to the mammary gland may act through different mechanisms and provide, at the least, additive
responses. Such additive effects were also observed in lactating pigs (Dunshea et al., 2005).
The ruminant mammary gland extracts more branch chain AA and lysine than needed to support milk
protein output and this uptake:output ratio increases with additional casein supply (e.g. Bequette
et al., 1996b; Raggio et al., 2006a). This helps supply additional N for non-essential AA (NEAA)
synthesis within the mammary gland (Lapierre et al., 2005) but also provides extra energy or act as
precursors for synthesis of other required metabolites. The contributions of these various roles are
not fixed, however, and need to be elucidated from the consideration of C and N balances across the
mammary gland, coupled tracer approaches. For the study discussed above (Raggio et al., 2006a),
uptake of potential precursor-C for lactose exceeded output in the milk and the same occurred for milk
lipids (Lemosquet et al., unpublished results; see Figure 2). This is expected, because the surplus is
needed to fuel the energy demands of the mammary gland, reflected in the CO2 output. In contrast,
the balance of amino acid C uptake:output is close to unity (or just below) but this hides the fact that
some EAA (Group 1; including phenylalanine plus tyrosine, methionine and histidine) are usually
extracted in proportion to secretion in milk, the other essential AA (Group 2; including the branch
chain AA and lysine) are removed in excess while the non-essential AA, combined as Group 3, are
in deficit. A simple accounting would suggest that C from Group 2 is transferred to Group 3. This
would fit with the AA-N balances between the groups (Raggio et al., 2006a; Figure 2) but such a
simplistic approach does not allow for the C losses associated within intermediary metabolism and
Figure 2. C and N balances across the mammary gland of cows fed a basal ration (Raggio et al.,
2006b). Data represent as follows. (a) C movements of uptakes of main nutrients for lactose (lact)
synthesis (glucose uptake), protein (prot) output (AA uptake) and fat synthesis. Oxidation (ox) is
based on net appearance of CO2 across the gland. All data from Lemosquet et al. (unpublished data).
(b) C transfers of Group 1 AA (his, met, phe, trp, tyr), Group 2 AA (leu, lys, ile, val) and Group 3
AA (ala, arg, asp, cys, glu, gln, gly, pro, ser). Data from Raggio et al. (2006a) and Lemosquet et al.
(unpublished data). (c) N transfers of Group 1, 2 and 3 AA. Data from Raggio et al. (2006a).

so other sources must be involved. In media devoid of NEAA, bovine mammary gland explants
synthesised substantial quantities of NEAA-C (e.g. 63-84% of aspartate and 79-95% of glutamate)
from EAA sources (Bequette et al., 2005; Bequette et al., 2006), with the remainder derived from
glucose. Interestingly, this latter contribution was relatively insensitive to the exogenous glucose
concentration, indicating flows and control independent of mass action. In contrast, the proportion
of galactose synthesised from glucose did increase from 3 to 88% as glucose in the medium was
raised from 0.67 to 27.7 mM. Such differential sources of galactose and glucose in milk lactose has
been observed in vivo for goats (Bickerstaffe et al., 1974) and humans (Sunehag et al., 2002). In the
bovine explants, at least 12% of galactose was derived from essential AA catabolism (Bequette et al.,
2006). Most control of glucose metabolism in the mammary gland is via intracellular phosphorylation
(80%) rather than transport into the cells (20%; Xiao and Cant, 2005) and this would allow the
metabolic flexibility to meet changing demands of nutrient supply.
Improved efficiency of EAA use by the mammary gland, and consequently the whole body, has been
demonstrated by studies on reduced supply (Bequette et al., 1996a; Lapierre et al., 2005; Weekes
et al., 2006) and it is attractive to hypothesise that part of this, at least, involves reduced catabolism
within the gland with substitution of either energy availability or intermediary metabolites from non-
AA sources, such as glucose. This needs to be tested directly, as does the impact on bioenergetics.
Does the more efficient use of these EAA reduce or increase the energy costs across the udder?
While this may be quantified experimentally, a first step may be to model the potential interactions
based on present knowledge and determine the consequences for ATP production and utilisation
(van Milgen, 2002).
Protein-energy interactions and health

Although the effects of protein-energy interactions produce obvious and rapid responses in the
quantity and quality of anabolic products, more subtle and longer term implications on the health
of the organism, either animal or human are being increasingly recognised. This is reflected in the
current concerns of consumers about the health effects of what they eat. For example, meat and milk
high in saturated fat has incurred the wrath of human nutritionists (Kuller, 2006) and current trends
are to encourage consumers to seek products with high contents of n-3 and n-6 polyunsaturated
fatty acids (PUFA; Noci et al., 2007) and conjugated linoleic acid (Tricon et al., 2006). Failure to
respond to such dietary advice has wide implications, however, both directly on protein metabolism
and on inter-generational health.
Epigenetics
Imbalanced protein-energy intakes can have more severe long-term effects than previously recognised.
For example, both under- and over-weight offspring tend to develop more late-onset diseases, such as
diabetes, hypertension and cardiovascular problems (Godfrey and Barker, 2000). Such observations
have created an expansion in the study of epigenetics, whereby expression of the foetal genotype is
influenced by maternal nutrition, with consequences for post-natal development and adult health.
Amongst the known epigenetic factors are provision of methyl groups (for example by inadequate
choline, folate or methionine supply), type of fat and level of protein (de Moura and Passos, 2005)
with putative mechanisms including alterations in DNA methylation or the physical state of the
histone-DNA complex during critical periods of development (Feil, 2006).
Diets low in protein (e.g. 8-10% compared with 18-20%), but adequate in energy, given to rat
dams during pregnancy and lactation resulted in offspring with elevated plasma glucose and raised
triglycerides in both plasma and liver (Maloney et al., 2003), a prelude to the development of type
2 diabetes and cardiovascular disease (Kahn et al., 2005). These health problems are exacerbated if
the offspring eat energy-dense diets as they mature (Petry et al., 1997) and may reflect the ‘thrifty

phenotype’ hypothesis (Hales and Barker, 2001), whereby animals subjected to imbalanced or
under-nutrition during development are adapted to conserve nutrients during any subsequent period
of restriction but over-compensate during periods of over-nutrition. Therefore, protein-energy
imbalances in early nutrition impact on subsequent regulation of glucose and fatty metabolism as
well as overall energy balance.
Such deleterious influences of imbalanced maternal protein-energy intakes on the offspring are exerted
during lactation (Bieswal et al., 2004) as well as pregnancy (Zambrano et al., 2006). Surprisingly,
adipocytes from the offspring had more insulin receptors (Bieswal et al., 2004) so the resistance
to the anti-lipolytic effect of insulin must involve defects in the signalling pathway. Nonetheless,
partial alleviation of pregnancy-induced responses can be achieved by feeding high protein diets to
the mother during lactation (Zambrano et al., 2005), probably because the suckling period is critical
for pancreatic development and the subsequent proper functioning of the insulin-glucagon axis.
This relatively new area of research has not yet impacted greatly on agricultural thinking, but clearly
genetic selection is an important factor in animal husbandry. Epigenetic factors, as illustrated by
protein-energy imbalances, can influence whether the so-called ‘genetic potential’ of an animal is
achieved or even detected and lead to faulty selection.
Impacts of polyunsaturated fatty acids on glucose and protein metabolism
Strategies to improve the health qualities of meat and milk (Gonzalez, 2006) include increased
content of mono- and poly-unsaturated fats (e.g. Chilliard and Ferlay, 2004; Ponnampalam et al.,
2006), if necessary even by genetic means (Lai et al., 2006). PUFA impact directly on metabolic
health with the insulin insensitivity induced in rats by diets high in saturated fat reversed by intake
of small amounts of PUFA, especially the n-3 forms (Storlien et al., 1991). This improvement by
n-3 PUFA was associated with a 14-fold increase in insulin binding to muscle membrane (Liu et
al., 1994). This then raised the question - do these effects of n-3 PUFA on insulin also impact on
protein dynamics? The answer appears to be ‘yes’ as growing steers, offered a diet rich in menhaden
(Pacific herring) oil, required more glucose and AA to maintain euglycaemia and euaminoacidaemia
during a hyperinsulinaemic clamp, indicative of increased insulin sensitivity (Gingras et al., 2007).
Furthermore, supplementation with the n-3 PUFA-rich oil decreased whole body leucine oxidation and
this, coupled with lower food intake but improved growth rate, implied increased protein deposition
of absorbed AA. Indeed, muscles from these steers had increased phosphorylation of protein kinase
B (PKB, also known as Akt), mammalian target of rapamycin (mTOR) and ribosomal protein 6
kinase-1 (S6K-1) compared with the control diet. These factors are well established as positive
regulators of protein synthesis (Kimball and Jefferson, 2006).
Effects of amino acids on glucose metabolism during post-natal growth and adulthood
Although AA have the potential to act as gluconeogenic substrates (van Milgen, 2002) quantification
of their exact contribution to glucose synthesis is difficult without consideration of counter-regulatory
mechanisms and use of tracers (Wolff and Bergman, 1972; Darrah et al., 1979). For example, high-
protein moderate-carbohydrate diets in humans show only moderate glycaemia (changes in plasma
glucose) compared with normal-protein diets (Gannon and Nuttall, 2006). Partly this is because
AA stimulate pancreatic secretion of insulin and glucagon (Gabai et al., 2002; Bos et al., 2003) and
together these act to decrease hepatic gluconeogenesis from other substrates (Donkin and Armentano,
1995) and increase peripheral glucose disposal (Rooyackers et al., 2004). Therefore, dynamic effects
on AA-glucose metabolism are not reflected by changes in plasma glucose. Acute AA infusions in
humans result in increased hepatic gluconeogenesis, with glycogenolysis unchanged (Krebs et al.,
2003; Figure 3). In contrast, glucagon increased hepatic glycogenolysis but not gluconeogenesis.
The AA infusion increased glucose production by 33% over basal conditions, only approximately

Figure 3. Impacts of AA on hepatic gluconeogenesis, muscle glucose uptake and glycogen synthesis
in humans. The consequences are separate from the actions of insulin and glucagon also released in
response to elevated AA supply. Based on Krebs et al. (2002 and 2003) and Krebs (2005).
Figure 3. Impacts of AA on hepatic gluconeogenesis, muscle glucose uptake and glycogen
60% of the theoretical
synthesis ingluconeogenic potential
humans. The consequences are of the infusate
separate (fromofKrebs
from the actions et al., 2003; Tremblay
insulin and
glucagons also released in response to elevated AA supply. Based on Krebs et al. (2002 and
et al., 2005; van Milgen, 2002). This
2003) and Krebs (2005).
lower transformation might reflect the basal fasting conditions
when AA may be oxidised to provide ATP.
These effects of AA on hepatic gluconeogenesis in humans concur with earlier findings in farm species
(Wolff and Bergman, 1972) but recent evidence introduces further interactions in peripheral tissues.
For example, a 2-fold rise in plasma AA concentration, induced by infusion, was associated with a
25% decrease in the rate of whole body glucose disposal, Rd (Krebs et al., 2002; Krebs, 2005). Brain,
red blood cells, fat and muscle are amongst the most important tissues for peripheral disposal, with
muscle having the greatest flexibility as this can switch between energy fuels and also store ‘excess’
glucose as glycogen, actions that are regulated in part by insulin (Spriet and Watt, 2003). The AA,
however, impair muscle glucose transport, synthesis of glucose-6-phosphate and glycogenesis (Krebs
et al., 2002; Figure 3). As with the observations with PUFA described earlier, key intermediates in
the inhibition of these processes involve mTOR and S6K-1 phosphorylation with these linked to
phosphorylation of the insulin-receptor substrate-1 (IRS-1) that then interferes with insulin signalling
via the phosphinositol-3 kinase (PI-3K) and suppression of translocation of the GLUT-4 transporter
to the cell membrane (see Figure 4). This is an important pathway for insulin sensitivity because
mice deficient in S6K-1 do not develop peripheral insulin resistance (Um et al., 2004).
What is particularly interesting is that this mechanism appears to be specific for muscle because, in
fresh adipose tissue, AA have a positive effect on insulin-induced glucose transport in adipocytes
of high fat fed mice and this helps normalise the glycaemic response (Hinault et al., 2006a). This
mechanism requires the combined presence of insulin and AA and may involve signalling via PKB
rather than PI-3K (Hinault et al., 2006b). Although rates of glucose disposal are greater in muscle
than fat, the latter may provide a substantial route in obese animals.
Mechanisms that link AA and glucose metabolism

Over the past few years, the rapid growth in molecular knowledge on how AA, energy substrates
and peptide hormones interact through similar signalling pathways has allowed an integrative model
to be constructed for tissue (cellular) responses to nutrients (see simplified version in Figure 4).
As already discussed, the mTOR and S6K signal pathway intermediates become phosphorylated
in response to an anabolic signal (e.g. AA and/or insulin) and these then alter the phosphorylation
status of the eukaryote initiation factors (see Kimball and Jefferson, 2006), and thus regulate protein
synthesis. Interestingly, physiological amounts of leucine also impact on these pathways (see

amino acids glucose insulin
PI3K glycogen PI3K ISR1

ATP
Glut4 synthesis
P
(p)mTOR
(p)S6K1
PKB
S6 eIF4B eEF2
protein synthesis
Figure 4. Simplified scheme of interactions between amino acids, glucose and insulin. Abbreviations:
mTOR, mammalian target of rapomycin; S6, ribosomal protein S6; S6K1, S6 kinase1; eIF, eukaryote
initiation; eEF, eukaryote elongation; IRS1, insulin receptor substrate 1; PI3K, phosphoinositide 3-
Figure 4. Simplified scheme of interactions between amino acids, glucose and insulin.
kinase; PKB (AkT),Abbreviations:kinase
protein B; Glut 4,target
mTOR, mammalian glucose transporter
of rapomycin; 4 (in muscle);
S6, ribosomal protein S6;P,S6K1,
phosphorylation;
S6
p, phosphorylated form.eIF,
kinase1; Amino acid
eukaryote entry or
initiation; eEF,ATP (e.g.elongation;
eukaryote from glucose) leadsreceptor
IRS1, insulin to phosphorylation of
substrate 1; PI3K. phosphoinositide 3-kinase; PKB (AkT), protein kinase B; Glut 4, glucose
mTOR and S6K1transporter
and subsequent activation of eIFand eEF factors to stimulate protein synthesis.
4 (in muscle); P, phosphorylation; p, phosphorylated form. Amino acid entry or
Insulin action is ATP
via IRS1 activation
(e.g. from of PI3K
glucose) leads and this promotes
to phosphorylation of mTOR andPKB (AkT)
S6K1 activity at the membrane
and subsequent
to recruit glucose transporters.
activation When
of eIFand eEF nutrients
factors to stimulateare imbalanced
protein or inaction
synthesis. Insulin excess
is viaover-activation
IRS1 of
S6K1 results in activation of PI3K and this promotes PKB (AkT) activity at the membrane to recruit glucose
phosphorylation of IRS1 and this inhibits the PI3K and PBK action
transporters. When nutrients are imbalanced or in excess over-activation of S6K1 results in
and reduces
nutrient entry and metabolism.
phosphorylation of Positive actions
IRS1 and this inhibits(i.e. stimulation
the PI3K of cell
and PBK action andmetabolism and
reduces nutrient anabolism)
entry
are described byand solid arrowsPositive
metabolism. and inhibitory
actions (i.e. actions
stimulation byofdotted arrows.
cell metabolism andBased on Marshall
anabolism) are (2006),
described by solid arrows and inhibitory actions by dotted arrows. Based on Marshall (2006),
Hinault et al. (2006b) and Um (2006).
Hinault et al. (2006b) and Um (2006).
Kimball and Jefferson, 2006). Insulin also regulates aspects of energy metabolism, notably glucose
transport, with signals though the IRS-1 and this leads to activation of PI3K and PKB (Akt) with
subsequent recruitment of the Glut-4 vesicle to the membrane and transport of glucose. At the same
time, the PKB pathway stimulates mTOR and S6K1 and enhances protein synthesis. This then
provides an integrated system for responses to a meal that contains both energy and protein nutrients.
With nutrient overload, such as excess AA and/or glucose, the mTOR and S6K1 over-activation
phosphorylates membrane-associated IRS1, inhibits complex formation and results in localisation
to the cytosol (for degradation) and prevents the PKB-mediated localisation of the glucose receptor
to the membrane (Um et al., 2006). Thus, imbalanced nutrient supply will alter the capacity of the
cell to utilise specific substrates. In the case of excess AA, for example, the cell can utilise those as
energy sources and the need for oxidation of glucose is reduced, achieved by lowered transport into
the cell and formation of glucose-6-phosphate. In the absence of other routes of glucose disposal
within the body, hyperglycaemia results.
Such a mechanism based on intermediates common to both nutrient and hormone signalling pathways
offers scope for tight integration at the cellular level and thus determines organ responses. These
rely on phosphorylation-based processes and provide rapid, acute responses. Of particular interest,
however, is that responses in glucose transport to AA may differ between tissues, as exemplified by
muscle and adipose tissue (Krebs, 2005: Hinault et al., 2006a and b). Such differences would provide

important flexibility as inhibited (stimulated) pathways in one tissue would increase (decrease)
nutrient concentrations in plasma and create potential counter-regulatory effects unless metabolism
is altered in other organs. While it would be premature to incorporate such data into current
considerations for either animal or human nutrition, these mechanisms may help the understanding
of nutrient diversion between tissues during different physiological states (e.g. growth, lactation),
how imbalances are ‘corrected’ by switches in intermediary metabolism and what underlies metabolic
flexibility during diurnal patterns of food intake.
Regulation of intake
The most influential impact on both protein and energy balance is intake. In terms of growing and
lactating animals although diets can be carefully balanced for macro nutrient supply an important
constraint to total production is intake (Persaud and Simm, 1991; Kerr and Cameron, 1995). In humans,
the current problem in western societies of burgeoning obesity is a consequence of two problems,
increased energy intake and decreased energy expenditure (exercise). For both animal and human
nutritionists, therefore, understanding regulation of intake is of crucial importance. Mammals sense
the major macronutrients to different extents and, in terms of human satiety, lipids provide the weakest
signals, protein the greatest and with carbohydrate intermediate (Batterham et al., 2006). Indeed,
where protein comprises at least 30% of dietary calories, voluntary food intake can be suppressed
below energy maintenance for weeks or months (Skov et al., 1999; Weigle et al., 2005; Johnstone
et al., 2006 and unpublished results). The underlying mechanisms of appetite regulation are still not
fully understood but absorption of glucose in rats induces c-fos activity in brain neurones and this
may signal nutrient sufficiency. Similar elevated c-fos activity is observed even 6 h after a protein
meal (Mithieux et al., 2005) with increased expression in small intestinal tissue of genes related to
gluconeogenesis and associated elevated glucose synthesis (Mithieux et al., 2006). Thus, glucose
synthesised by the intestine from absorbed AA may provide a persistent signal via the vagal nerve
(Mithieux et al., 2005) and suppress appetite. Such a gluconeogenic role for the small intestine has
been challenged recently (Martin et al., 2007) but would fit with the known increase in gut protein
retention during absorption (Deutz et al., 1995) and the partial release and metabolism of AA, coupled
with the net positive or neutral glucose balance, during the early post-absorptive period in pigs (Van
der Schoor et al., 2002). Alternatively, both protein and energy substrates may release GIT peptides,
such as peptide YY, that affect hunger and satiety but with different responsiveness (Batterham et al.,
2006) and with potential interactions to alter hunger responses to mixed meals.
Protein and energy nutrients may also act directly on the brain, rather than through gut-derived
signals, to control intake. The major fuel used by the brain is glucose and subtle mechanisms exist
to sense availability and metabolism of this nutrient, particularly within the hypothalamus. High
glucose supply will inhibit intake (Mobbs et al., 2001; Routh, 2002), while glucose analogues that
inhibit glucose oxidation within the brain result in elevated glycaemia and increase intake (Hudson
and Ritter, 2004). The mTOR pathway has been implicated as a nutrient sensor, partly via intracellular
ATP levels (Dennis et al., 2001) and act on the hunger/satiety mechanisms. When ATP concentrations
are low, as in the fasted state, then levels of pmTOR and pS6K1 are decreased in neurones of the
arcuate nucleus of the hypothalamus but are increased on refeeding (Cota et al., 2006). As in other
tissues, phosphorylation of the mTOR signalling pathway in the brain is enhanced by leucine and
intracerebroventricular injection of this AA increased pmTOR and pS6K1 levels and decreased
food intake (Cota et al., 2006). Interestingly, valine did not produce this effect and this supports
the notion that leucine is a very specific peripheral signal of AA status. Again, however, this is an
example of how common mechanisms that ‘sense’ either energy or specific AA can interact to produce
flexible responses. Such effects are amplified by the sensitivity of the brain mTOR system to peptide
hormones, including insulin and leptin (Niswender et al., 2003), that can act as nutrient sensors or
indicators of body composition and so has the potential to integrate the signals that arise from a
variety of metabolites and hormones that are involved in determining energy balance status.

Summary
The sheer range and complexity of protein-energy interactions presents scientists with a daunting
challenge to even partially understand and quantify the metabolic transformations, let alone understand
the consequences. There is no doubt that the area of most rapid advance relates to understanding the
molecular events within the cell and how metabolites, both ‘energy’ and ‘protein’, are channelled,
interact with hormones and influence metabolic flows. This is an exciting development but the real task
comes when we consider these cells together, first to form tissues and, thus, how do these molecular
events fit with organ function. The organs then interact and integrate to produce the whole animal
response, with this differing dependent on the physiological demands on the animal e.g. growth
or milk production vs. homeostasis at maturity or maintenance. Finally, while the required targets
may appear different, animal production targets meat or milk or human health and well-being, yet
the same underlying principles apply and that involves an understanding of how macronutrients are
sensed and utilised by mammals. The solution requires intellectual flexibility with a willingness to
use information from cells, laboratory species, farm animals and humans.
Acknowledgements
Work at the Rowett Research Institute is funded from a core grant given by the Scottish Executive
Environmental and Rural Affairs Department.
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Vertical integration from ‘omics’ to the whole organism
A. Cornish-Bowden and M.L. Cárdenas
CNRS-BIP, 31 chemin Joseph-Aiguier, B.P. 71, 13402 Marseille Cedex 20, France
Abstract
Although the rapid advance of the various ‘omics’ fields is striking, it consists mainly of accumulating
data in ever-increasing detail at an ever-increasing rate. There is little basis for believing that this
approach is leading to a deeper understanding of living organisms, and the hope of creating life de
novo remains as remote as it has always been. Changing this will require a far greater integration of
systemic ways of thinking into what is loosely called systems biology. At the most profound level
this means incorporating a genuine theory of life, but even at a more superficial level the relatively
simple ideas that have come from metabolic control analysis have yet to be fully incorporated into
biotechnological practice.
Keywords: theory of life, metabolic regulation, control
Introduction
Proteomics, metabolomics, metabonomics, transcriptomics and so on, together with systems biology
as it is currently understood, are essentially products of the 21st century, as one can see from the
distribution of usages of the various terms listed in Table 1, which is based on data from PubMed
(2007). The explosive increase in papers on genomics is, of course, a direct consequence of the vast
amount of information that has come from sequencing genomes, and is a natural development of
the subject. The other ‘omics’ disciplines are fruits of the growing amount of genomic information,
and studies of the proteome and transcriptome have already contributed greatly to understanding of
the net of interactions that connect genes to phenotypes (Cornish-Bowden and Cárdenas, 2001a),
and they have been useful tools for early diagnosis of medical problems. For example, powerful
proteomic technologies now have great potential in cancer research for biomarker discovery, and
for addressing the issue of cancer heterogeneity. Detecting cancer at an early stage, and predicting
how a tumour will develop and how it will respond to therapy, are areas of research that are already
benefiting from proteomics (Celis et al., 2004 and 2006).
Systems biology, however, is more of a new name for an old approach than a genuinely new way
of studying biology. The distinction is important because, as we shall argue in this paper, there is a
real need for an integrated approach to biology in which the components of a biological system are
analysed in terms of their contributions to the organization of the whole system. It is, however, far
from clear that that is what systems biology is in its current form. On the contrary, it appears to be
just as reductionist as less fashionable areas of biochemistry and molecular biology, differing mainly
in being based on an enormously increased body of detailed data available for study.
Expressing the same idea differently, the current obsession with the accumulation of detailed data
is not leading towards a better understanding of organisms: a theory of biological organisation will
not appear spontaneously from beneath a mountain of data, but will need to be actively constructed.
Systems biology in its present form has almost nothing in common with the general systems theory
that Ludwig von Bertalanffy worked to develop, and would not escape his criticism that ‘the only
goal of science appears to be analytical, i.e. the splitting up of reality into ever smaller units and
the isolation of individual causal trains’ (Bertalanffy, 1975). The question therefore arises of what
sort of systemic ideas need to be added to the various ‘omics’ fields to enable them to move away

Table 1. ‘Omics’ terminology in the biological literature.1
Search term Total 2000–2007 (%) First use
‘genome’ 128703 56 1953

‘genomics’ 25675 75 1987
‘systems biology’ 2027 99 1988
‘proteome’ or 15720 98 1995
‘proteomics’
‘transcriptome’ or 2575 99.6 1997
‘transcriptomics’
‘metabolome’ or 612 99.7 1998
‘metabolomics’
‘metabonome’ or 196 99.5 1999
‘metabonomics’
‘fluxome’ or 23 96 1999
‘fluxomics’
‘metagenomics’ or 173 99.6 1998
‘metagenome’ or
‘metagenomic’
‘interactome’ or 161 99.4 1999
‘interactomics’
‘omics’ (whole word) 351 100 2002
1The Table shows information obtained from the PubMed database (PubMed, 2007) as checked in March 2007.
The word ‘genome’ has been in use at least since 1953, but ‘genomics’ as the name of a field of study was invented
by the founding editors of the journal Genomics: ‘For the newly developing discipline of mapping/sequencing
(including analysis of the information) we have adopted the term GENOMICS. We are indebted to T.H. Roderick of
the Jackson Laboratory, Bar Harbor, Maine, for suggesting the term. The new discipline is born from a marriage of
molecular and cell biology with classical genetics and is fostered by computational science’ (McKusick and Ruddle,
1987). Percentages in the third column are approximate, as all of the totals in the second column change rapidly. The
various ‘omics’ terms should not be confused with other terms, such as glycosome, spliceosome, trypanosome, etc.
that contain the unrelated root -some.
from the mere accumulation of data and towards a real contribution to biological understanding. In
a sense, one could hope to move biology away from being a purely descriptive science to becoming
a predictive science.
Eighty years after Heisenberg’s uncertainty principle taught physicists that behaviour at the particle
level cannot be predicted, and 25 years after studies of chaotic dynamics taught them that the long-
term behaviour of many-particle ensembles cannot be predicted either, it may seem futile to try to
make biology something that even modern physics no longer claims to be, but the implied criticism
misses the point. The existence of two areas of physics that are now known to be less amenable to
prediction than they were once hoped to be hardly alters the fact that there is a vast body of theory
underlying physics that does allow the results of many experiments to be predicted. The theory of
biology is far more restricted, being essentially limited to the theory of natural selection. This offers
a very convincing mechanism for evolution and makes some predictions about the characteristics
of a previously unknown species, but has essentially nothing to say about the origin of life, the
moment when organised systems learned how to maintain their organisation, in other words how
to stay alive.

Biology does of course depend on many fragments of theory, such as the understanding of enzyme
mechanisms that comes from studies of organic reaction mechanisms, or, most notably, the whole
area of energy management known as bioenergetics, which depends on the laws of thermodynamics.
However, none of these can be regarded as theories of biology at the same level as the theory of
natural selection, because they apply to non-living systems no less (or more) than they do to living
systems. Indeed, a major part of our understanding of biochemistry came from the overthrow of
vitalism by Buchner (1897) – the recognition that living systems obey the same laws of chemistry
and physics as non-living systems. Volcanic activity depends on the laws of thermodynamics just as
much as a living organism does, but one would not call thermodynamics a theory of volcanoes. The
distinction we are making here (made, of course, by Schrödinger (1944) before us) is that although
no one now doubts that adherence to the laws of physics is necessary for life, it is much less clear
that the currently known laws are sufficient.
Does biology need a theory of life?

The question of how far systemic ideas have influenced systems biology as it is currently understood
can be answered at a fairly simple level, in terms of the ideas of metabolic control analysis developed
from the seminal contributions of Kacser and Burns (1973) and of Heinrich and Rapoport (1974),
or at a much more profound (and difficult) level, in terms of the theory of biological organisation
developed by Rosen (1991). At the simpler level this influence already exists: metabolic control
analysis already forms a significant part of systems biology, though not as large a part as it probably
should. At the more profound level it is probably fair to say that Rosen’s ideas have had no impact
at all on ordinary practice. So far as most biologists are concerned there is no theory of the whole
organism, and for most the lack of one has no importance, as they would agree with Medawar (1977)
that discussing the nature of life represents ‘a low level in biological conversation’.
Medawar’s comment may have had some validity when he made it, as it could be argued that in the
absence of the ‘omics’ technology that we now have there was little that a theory of life or theory
of the whole organism could have contributed. However, that is no longer true, and to advance
significantly further (other than in the accumulation of yet more detailed information) biology will
need to integrate the information that already exists into a whole. It needs what Woese (2004) has
called a guiding vision, because ‘without an adequate technological advance the pathway of progress
is blocked, and without an adequate guiding vision there is no pathway, there is no way ahead.’
A pessimistic view would liken systems biology to cybernetics: in the middle of the 20th century this
was confidently predicted to offer solutions to all problems of organisation and regulation. However,
apart from giving biochemists the idea of feedback inhibition, it has largely vanished from biological
consciousness, after failing to deliver on its early promise. What, then, should the guiding vision of
systems biology be? In the deepest sense, Rosen’s (M,R) systems may provide this (Cornish-Bowden
et al., 2007), but it will be a long time before these have any practical application, except in the
negative sense that recognising that some current objectives are impossible to realise may avoid
some futile effort. In the shorter term, the less profound systemic ideas involved in metabolic control
analysis are already applicable to current biotechnology, and may offer easier and better ways to
success than the brute-force approach that has dominated the field since genetic manipulation became
possible. Analysis of metabolic pathways and networks has a great potential in biotechnology and
medicine, and constitutes a powerful tool in drug research (Eisenthal and Cornish-Bowden, 1998;
Ramos-Montoya et al., 2006).
Machines and organisms

Rosen’s theory of (M,R) systems (Rosen, 1991) treats the fundamental properties of living organisms
as metabolism and repair, though replacement expresses better than repair the intended meaning

(Letelier et al., 2006). It is natural to suppose that ‘repair’ includes the fundamental biological idea
of reproduction, but this is not the case, because Rosen was little concerned with reproduction in the
usual sense or with the other central idea of modern biology, evolution. For most biologists these
will seem to be such crucial omissions that they deprive Rosen’s theory of any interest it might have.
However, the point is that Rosen was interested in life at a more fundamental level: until the early
organisms had succeeded in staying alive, i.e. in maintaining their organization for a significant period,
there was no question of either reproduction or evolution. It follows, therefore, that understanding
how organisms stay alive is more fundamental than understanding how they reproduce or evolve.
The essential property that allows an organism to stay alive is metabolic closure, which allows them
to preserve their integrity of organisation and to be autonomous. We shall discuss later what this
implies, but we note at the outset that no machine has any property equivalent to metabolic closure,
and the fallacy of the machine metaphor for organisms defeats most attempts to understand them
in their entirety. It may have been philosophically tenable for Descartes to hold that organisms are
essentially machines, but it is not tenable today. There is a vast gulf between organisms and machines,
and at the moment we cannot see how it might be bridged, even in principle, let alone in practice. A
sufficiently detailed study of a machine may allow a competent engineer to produce another machine
with the same functionality, but we are totally incapable of designing an organism today, and it may
even be naive to think that it will ever be possible to design an organism ab initio. Everything that
we know about organisms confirms Rosen’s contention that an organism is not a machine.
In January 2007 the popular comic strip Dilbert contained a conversation in which the question ‘Your
sales representative told us that the product heals itself. Is that true?’ received the answer ‘It’s totally
true … that he said that.’ Why was this amusing? It would not have been amusing if the conversation
had been placed at an agricultural fair and the product had been a disease-resistant breed of pig; it
would then have been an uncontroversial claim, because we all know that animals are capable of
recovering from injuries and illnesses without external intervention. No, it was amusing because
the cartoonist knew that his readers know perfectly well that machines cannot recover from damage
without external help. We know this, but we are tempted to ignore it in over-optimistic projections
of where current biological engineering will lead.
Philosophically the difference between organisms and machines lies in the different kinds of causation
described by Aristotle. Machines and organisms both are open to material causation, as both are
constructed from external materials, and both release used materials into their environments. They
differ, however, in final causation, because all machines are made for a purpose, to fulfil particular
functions, whereas organisms have no final causes. More important, they also differ in efficient
causation, because in making a machine the essential decisions about which parts are installed in
which locations are external, whereas in an organism the catalysts that decide how the organism
is to be constructed are themselves products of the same organism; they are not supplied from the
outside, and, in Rosen’s words (Rosen, 1991), an organism is closed to efficient causation. The
essential idea is illustrated in Figure 1, which represents the enzymes that catalyse the metabolic
reactions as being themselves products of the same metabolism. However, there is a serious difficulty
with this representation, because synthesis of the enzymes also requires catalysts, which are not
shown in Figure 1. The problem is illustrated by the more abstract representation of metabolism and
replacement in Figure 2. This illustrates the problem but does not attempt a solution, as this requires
a deeper analysis (Letelier et al., 2006; Cornish-Bowden et al., 2007).
We must not forget, of course, that life must satisfy not only metabolic closure but also bioenergetics,
which implies that a living organism is an open system in the thermodynamic sense, with a flux of
matter and energy. There is no conflict here, however, because the two statements involve different
levels of causation: closure to efficient causation does not imply closure to material causation, which
would be absurd.

Figure 1. A schematic representation of metabolism and replacement. The conventional idea of
metabolism is as a set of chemical reactions, represented here by the steps from S1 to P, catalysed
by a series of specific enzymes, E1 to E4. However, these enzymes are not supplied from outside and
are not indefinitely stable (as represented by the arrows labelled Decay). Accordingly they need
to be synthesised (‘replaced’) by chemical reactions that use products of metabolism as starting
materials.
Figure 2. A more abstract representation of metabolism and replacement. All of the chemical reactions
of metabolism are represented by the single arrow from A to B; thus A (reactants) and B (products)
must be regarded as sets, not as individual metabolites. Catalysis by the set of enzymes needed for
the metabolism is represented by the dashed arrow from f, representing the set of metabolic enzymes,
acting on A. Replacement is represented by the arrow from B to f, and is catalysed by a replacement
system (another set of enzymes) Φ. The diagram raises (but does not answer), the question of how
metabolic closure is achieved, i.e. of how Φ is replaced. Discussion of how the question might be
answered may be found elsewhere (Letelier et al., 2006; Cornish-Bowden et al., 2007).
This excursion into philosophy may appear superfluous in a discussion of biological engineering, but
it is absolutely essential if one is to understand the limitations on what biological engineering can do,
not just at present with present levels of technology, but in principle and for ever. If engineering just
means tinkering with existing organisms then that is, of course, perfectly possible, and that is what
genetic engineering is. But the difference between that and designing an entirely new organism is
more than just a difference of scale; it is absolutely fundamental and probably unbridgeable. If we
accept Rosen’s position it will never be solved; if we think he is mistaken we must still recognise
the magnitude of the gap, and we still need to understand the nature of the difficulty before there
can be any hope of surmounting it. Simply gathering more data about the details of gene products
will not lead to a solution.
This conclusion is pessimistic, and is perhaps wrong. However, if it is wrong it needs to be shown to be
wrong: it will not be sufficient to continue with today’s reductionist approach in a pious hope that the
tremendous advances that this approach brought in the 20th century will be matched by even greater
advances in the 21st. If it is right it has practical consequences, just as much as the understanding
of chaotic dynamics that came from the studies of 25 years ago had practical consequences: it may
have failed to solve the problem of long-range weather forecasting, but it still convinced most
meteorologists not to waste more time and effort in pursuit of an unattainable objective.

Another practical implication of Rosen’s theory comes from the new light that it sheds on the
occurrence and role of multiple functions of proteins. It has been known for many years that some
of the lens proteins of the vertebrate eye are identical to glycolytic enzymes, and examples of such
‘moonlighting’ proteins are being discovered with increasing frequency (Tipton et al., 2003). Until
now they have been regarded as an interesting feature of life, albeit one that can greatly complicate the
diagnosis and treatment of monogenic diseases (Sriram et al., 2005), but without deep significance.
However, analysis of how organisms can be closed to efficient causation indicates that metabolic
closure would be impossible without multifunctionality (Letelier et al., 2006; Cornish-Bowden et
al., 2007). This in turn implies that the instances of multifunctional proteins that are known today
will prove to be a small proportion of those that exist.
The message from metabolic control analysis

Even though the message from the hard systemic approach of Rosen is the rather negative one that
it is useless to continue trying to achieve the impossible, the message from the softer systemic ideas
embodied in metabolic control analysis is more positive. There is little doubt that many of the early
difficulties in interpreting the first genome data were due to failure to incorporate the lessons from
25 years of metabolic control analysis, and little doubt that these lessons will need to be understood
better in the future.
The hope when genome data first became available was that determining the functions of the genes
first identified by examining DNA sequences would be relatively easy: it would be sufficient to
examine what function was lost – or what other deleterious effects appeared – when the gene was
deleted. To the surprise of many, though not of those who had studied metabolic control analysis,
the most common effect of such knockout experiments was that there was no effect: many genes
proved to be ‘silent’ and had no obvious phenotype. In the case of Saccharomyces cerevisiae, the
probability is about 80% that deleting a randomly chosen gene will have no effect on growth or
on any easily measurable metabolic flux (Cornish-Bowden and Cárdenas, 2001b). This should not
have been a surprise, as it follows directly from the summation properties that control coefficients
obey (Kacser and Burns, 1973): these imply, in simple terms, that flux control is shared (unequally)
among all the enzymes in the system, and as the number of enzymes is typically large the share that
each one has is typically small. Although this idea is usually applied to specific metabolic pathways,
it also applies to gross fluxes like rates of growth, where thousands of enzymes may share control.
Eliminating an enzyme entirely may still have an effect – part of the reason why the probability
in yeast is only about 80%, rather than 100% – if the reaction catalysed by the deleted enzyme is
absolutely essential for growth in the culture conditions used; but organisms typically have more
than one way of satisfying an essential function, and eliminating one of the ways will not necessarily
therefore eliminate the function. The simplest way of having more than one way to fulfil a function
is to have multiple enzymes that catalyse the same reaction, or isoenzymes, but more complicated
ways exist as well, and all of them imply that even deleting an enzyme that catalyses an essential
function may be phenotypically silent, at least if its effects are measured solely in terms of fluxes.
The second lesson to be learned from control analysis is that metabolite concentrations are typically
much easier to perturb than fluxes, with the result that a gene that appears to have no phenotype can
often be revealed by studying the effect on metabolite concentrations of deleting it, even though the
deletion has no effect on fluxes, and especially not on a gross flux like growth. When this is done,
even genes for isoenzymes cease to be silent (Cornish-Bowden and Cárdenas, 2001b; Raamsdonk
et al., 2001).

Metabolic regulation
Some of the more hostile attitudes to metabolic control analysis, such as that of Atkinson (1990),
derive from a perception that it has no use for such central concepts in biochemical regulation as
feedback inhibition by end products (Umbarger, 1956; Yates and Pardee, 1956) and cooperative and
allosteric interactions (Monod et al., 1965; Koshland et al., 1966). It is true that these concepts are
not always very evident in discussions of control analysis, but they are not ignored, though their
classical interpretation has required some revision. Contrary to the usual idea, these mechanisms
are almost irrelevant to flux control, but they are crucial for concentration control: fluxes can be
controlled efficiently without using any of the classic mechanisms, but only at the cost of huge
variations in metabolite concentrations (Hofmeyr and Cornish-Bowden, 1991; Cornish-Bowden et
al., 1995; Cornish-Bowden and Cárdenas, 2001c).
The essential points, which apply when the flux through a metabolic pathway is determined by
the need for its product, can be summarised in terms of the law of supply and demand: the classic
mechanisms exist so that when a change in flux occurs in response to a change in demand this is
accompanied by minimal changes in the concentrations of the intermediates in the pathway (Hofmeyr
and Cornish-Bowden, 2000). This law derives, of course, from economic theory, but it works much
more efficiently in biochemistry than it does in economics, as long as one remembers that not all
pathways need to be regulated by demand. The mammalian liver, for example, phosphorylates glucose
not primarily to meet its own relatively modest need for energy, but to prevent hyperglycaemia;
in other words, most glucose phosphorylation in the liver is supply-driven, and hexokinase D
(‘glucokinase’), the principal enzyme involved, shows none of the characteristics that one would
expect an enzyme that responds to demand to have (Cárdenas, 1995; Cornish-Bowden and Cárdenas,
2004; Cornish-Bowden and Nanjundiah, 2006).
Although much of our knowledge of metabolic regulation was originally derived from studies in
bacteria, such as the classic work on aspartate metabolism in Escherichia coli (Stadtman et al.,
1961; Patte et al., 1967), the principles also apply to higher organisms in which different organs
fulfil specialised tasks. This is well illustrated by the hexokinase isoenzymes in the human, where
different isoenzymes predominate in different tissues and have properties appropriate for the needs
of the tissues concerned (Cárdenas, 1995). As we have mentioned, hexokinase D, the characteristic
isoenzyme of the liver, has the properties expected for an enzyme regulated by substrate supply:
lack of saturation at physiological glucose concentrations, a cooperative response to glucose, and
insensitivity to product inhibition by glucose 6-phosphate. In complete contrast, the brain has a need
for glucose that must be satisfied even when other tissues cannot be supplied, and hexokinase A,
which predominates in the brain, is saturated by very low concentrations of glucose, and is inhibited
by glucose 6-phosphate: the rate of glucose phosphorylation thus changes with changes in demand
but not with changes in the availability of glucose. Muscle also has a need for large amounts of
glucose, but it is less crucial to satisfy it than it is for the brain, and hexokinase B, predominant in
the muscle, has similar properties to hexokinase A with the important difference that it is unsaturated
at low glucose concentrations that would be saturating for hexokinase A. This example could be
pursued in greater detail, but the point is already clear, that the principles of metabolic regulation
derived from studies of prokaryotes apply also to higher organisms.
The practical biotechnological importance of this is that before one can modify the metabolic
behaviour of an organism for biotechnological ends one must understand the organism’s regulatory
mechanisms and their functions, which have evolved in order to satisfy its own needs, not those of
biotechnology. This last may seem an obvious point, and indeed it ought to be, but it is sometimes
forgotten. When one reads, for example, that the ability of a virus to resist a drug is due to ‘careless
transcription’ (Smith and Simons, 2004) a line has clearly been transgressed. Whatever intentions
one may meaningfully impute to a virus, a willingness to die in the cause of human health is certainly

not one of them. More generally, many attempts to increase yields of desirable metabolites by
overexpressing supposedly rate-determining enzymes have failed because of failure to recognise
the existence of regulatory mechanisms that have evolved precisely to prevent accumulation of
metabolites that the organism does not need. As a particular example, efforts to increase yields of
ethanol in Saccharomyces cerevisiae have been thoroughly analysed (Niederberger et al., 1992), but
the principle applies widely (Fell, 1997). Only in the unusual case of a pathway regulated by supply,
such as glycogen synthesis in the mammalian liver, does one find a rate-limiting enzyme that one
can overexpress in order to increase the flux (Cárdenas, 1995; Agius et al., 2004; Cornish-Bowden
and Cárdenas, 2004; Cornish-Bowden and Nanjundiah, 2006).
Concluding remarks
Despite the great and rapid advances in biological knowledge that have come from the sequencing
of genomes and from the new fields that have grown out of this, our attitude in this article has not
been entirely positive about the usefulness of the progress in these fields. On the one hand, we cannot
deny our belief that there has been far too much emphasis on the accumulation of data and far too
little on the questions that can be answered by means of the data accumulated, and this belief can
only be strengthened by a recent article (Abecasis et al., 2007) that lays great stress on hopes that
‘new, inexpensive sequencing methods [will] provide even higher-throughput capabilities and allow
more detailed analysis of individual genomes’ but almost none on why this might be desirable. On
the other hand, some suggestions about how systemic ideas should influence how research is actually
done may be helpful. Clearly researchers cannot wait for a complete or even a partial understanding
of life at the sort of level sought by Rosen, because this may not come in this century, if at all.
Nonetheless, realisation of the limitations of the machine analogy may help to avoid some wasted
effort. It is more important also to realise that data accumulation is not useful in itself; it is or may be
useful in relation to long-term questions that the data may help to answer. In other words, research
needs to be hypothesis-driven and not just technique-driven.
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Intravenous administration of lysine and threonine to a deficient diet
results in low nitrogen utilization in preruminant calves
S.J.J. Alferink, J.J.G.C. van den Borne, A. Habets, A.A. Jacobs and W.J.J. Gerrits
Animal Nutrition Group, Wageningen University, P.O. Box 338, 6700 AH, Wageningen, The
Netherlands
Introduction
During our research into the effects of nutrient synchrony on energy and protein metabolism
we performed experiments with heavy preruminant calves (Van den Borne, 2006). One type of
nutrient synchrony concerned the timing of the availability of individual amino acids. Absorption
of supplemented free amino acids occurs shortly after feed intake whereas protein bound amino
acids need to be hydrolysed prior to absorption and are absorbed more slowly. This may result in
an asynchronous availability of the free amino acids and those from dietary protein. Because there
is no real temporary storage of amino acids it was expected that an asynchronous availability would
result in oxidation of the amino acids that can not be used for synthesis because of an imbalanced
amino acid profile. A study was performed in which amino acids were intravenously infused to
create a contrast in the timing of the availability of individual amino acids. The results of this study
indicate large differences in protein metabolism compared to two other experiments (similar in
methodology) in which the administration route of free amino acids, the feeding frequency and/or
protein source were different. The objective of this paper was to identify the role of these factors in
protein metabolism in heavy preruminant calves. For better comparison, the results from a selection
of treatments from the various experiments are presented in this paper.
Animals, material and methods

In experiment 1 (Exp. 1) sixteen male Holstein Friesian calves of 170 kg body weight (BW) were
fed a lysine and threonine deficient milk replacer diet in which 80% of the dietary protein originated
from soluble wheat protein. The diet had an analysed content of 6.1 g lysine, 6.9 g threonine and
19.5 MJ metabolisable energy (ME) per kg and was fed twice daily at 6.00 and 18.00 h at a rate
of 1035 kJ ME/(BW0.7/d). An amino acid supplement containing 15 g L-lysine hydrochloride and
15 g L-threonine per litre was administered during a two h intravenous infusion in the jugular vein.
The intravenous supplemented amount accounted for 9.3 g lysine and 6.0 g threonine per kg of feed
offered. Three 2-h infusion periods were compared, starting at 0, 2 and 8 h after feeding. Since there
were neither significant nor numerical differences between these treatments, the presented results
were averaged across treatments. In experiment 2 (Exp. 2) eight male Holstein Friesian calves of
157 kg BW were fed a diet identical to that of Exp. 1 at the same feeding level. The diets were fed
four times a d (at 6.00, 12.00, 18.00 and 24.00 h). The supplemental L-lysine-HCl (10.4 g/kg feed)
and L-threonine (6.2 g/kg feed) was supplied mixed with the feed.
In experiment 3 (Exp. 3), twelve male Holstein Friesian calves of 175 kg BW were fed a diet in
which all dietary protein originated from whey protein concentrate and sweet whey powder. The
diet had an analyzed content of 16.2 g lysine, 13.2 g threonine and 18.8 MJ ME per kg. Diets were
fed two (at 6.00 and 18.00 h) or four times (at 6.00, 12.00, 18.00 and 24.00 h) daily at a rate of 1150
kJ ME/(BW0.75/d). In all experiments, calves were housed individually in metabolic cages placed
in respiration chambers and nitrogen balance was measured during an experimental period of at
least 7 d.

At similar digestible nitrogen intakes, the efficiency of digestible nitrogen utilization (EDNU)
was 14.3% lower when amino acids were supplemented intravenously (Exp. 1) compared to oral
supplementation (Exp. 2) (Table 1). This comparison was, however, complicated by a difference in
feeding frequency. Comparison of feeding frequencies within Exp. 3 indicates that the difference
in feeding frequency between Exp. 1 and Exp. 2 accounted for only 5.4% of EDNU, leaving the
remainder of the difference between Exp. 1 and Exp. 2 (i.e. 8.9%) unaccounted for. Comparing
Exp. 2 and Exp. 3 at four meals per d shows that the diet based on whey proteins resulted in a 7.7%
higher EDNU compared to the diet based on soluble wheat protein with 60% of lysine and 47% of
threonine intake originating from industrial amino acids.
Table 1. Effects of route of amino acid administration, protein source and feeding frequency on
nitrogen balance. Values are in g/BW0.75 per d unless stated otherwise.
Exp. 1 Exp. 2 Exp. 3
Administration of lysine intravenous oral - -

and threonine
Main protein source soluble wheat soluble wheat whey whey
Meals per d 2 4 2 4
Mean SEM Mean SEM Mean SEM Mean SEM
N intake 1.71 0.05 1.75 0.03 1.83 0.02 1.81 0.02

Faecal N excretion 0.15 0.01 0.17 0.02 0.16 0.01 0.12 0.01
Urinary N excretion 1.05 0.03 0.87 0.03 0.84 0.03 0.76 0.03
N retention 0.51 0.03 0.75 0.04 0.82 0.03 0.95 0.03
EDNU1 32.5% 1.61 46.8% 1.76 49.1% 1.96 54.5% 1.97
1Efficiency of digestible nitrogen utilisation, i.e. nitrogen retention as percentage of digestible nitrogen intake.
Orally supplemented lysine and threonine are transferred across the splanchnic tissues along with
the amino acids originating from dietary protein. When lysine and threonine are supplemented
intravenously they directly enter the systemic circulation. This may have induced oxidation of
amino acids at two sites. First, a deficiency of lysine and threonine may have triggered oxidation of
dietary amino acids in the intestinal tissues. Second, an excess of lysine and threonine in the systemic
circulation may have induced oxidation of these amino acids. Especially the unusually high systemic
threonine supply may have increased hepatic threonine oxidation, because high first-pass extraction
rates of threonine by intestinal tissues (Le Floc’h and Sève, 2005) normally prevent large systemic
excursions of plasma threonine concentrations.
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Wageningen, The Netherlands.

The use of glutamine and glutamate for gluconeogenesis and non-
essential amino acid synthesis in late term chicken embryos
N.E. Sunny, J. Adamany, S.L. Owens and B.J. Bequette
University of Maryland, Animal and Avian Sciences, 20742, College Park, USA
Introduction
On d 0 embryonic, the average broiler egg (60 g) contains only 300 mg of available glucose, yet by
d 18 the embryo accumulates ~1.5 g of glycogen (Hazelwood, 1971). Therefore, the embryo must
rely on gluconeogenesis from amino acids (AA) or triglyceride-glycerol for glycogen synthesis.
Glutamate (Glu) and glutamine (Gln), which together comprise ~14% of egg protein (1.2 g, Ohta
et al., 1999) and whose supply is 2-fold greater than that which is accumulated by the embryo on d
19, could be net contributors to gluconeogenesis. Data also indicates that there is a need for proline
synthesis by the embryo (Ohta et al., 1999), and as the avian lacks a complete urea cycle, Glu and
Gln are the only possible substrates for Pro synthesis. The objective of this study was to determine
the contribution of Glu and Gln to the synthesis of glucose and Pro, and to other non-essential AA.
A further objective was to determine whether the metabolism of Glu and Gln towards these synthetic
pathways is compromised in small, compared to large, eggs where in-ovo nutrient supply is less and
where embryonic weight on d 20 is lower (31.8 vs. 36.8 g; N.E. Sunny, unpublished results).

Fertilized small (54 to 58 g; n=10) and large (66 to 70 g; n=10) eggs from a broiler flock (Ross
strain) were incubated at 37 °C and 65% relative humidity. Beginning on d 16 of incubation, half
of the small and the large eggs were injected into the amniotic fluid with [13C5]Glu or [13C5]Gln
(3.5 mg in 90 µL H2O) for three consecutive d prior to tissue and blood collection on d 19. For all
blood and tissue (intestines and liver) samples, Glu and aspartate were separated from basic AA
using sequential anion and cation exchange resins. 13C-Mass isotopomer enrichments of AA were
determined by gas chromatography-mass spectrometry (Bequette et al., 2006). Blood glucose was
isolated and 13C-mass isotopomer enrichments determined as above. The contribution of Glu and
Gln to glucose and non-essential AA synthesis was determined by precursor-product relationships.
Here, the M+n (where n is the number of carbon atoms) isotopomer enrichment for each AA was
used for calculations. Data were analysed using the MIXED procedure of SAS (2006), with [13C5]Glu
and [13C5]Gln injected eggs as blocks.

There were no differences in enrichment of either the Glu [M+5] or Gln [M+5] isotopomers within
blood, liver and intestines when comparing small and large egg embryos, however, for each of
these AA their enrichments did differ between blood, liver and intestinal pools. Thus, enrichments
of tissue free Glu [M+5] and Gln [M+5] were both higher in intestines compared to liver, indicating
that a greater proportion of Glu and Gln flux in the intestines derived from the tracer. Blood Glu
contributed <5% to Glu flux in the liver and intestines. By contrast, blood Gln contributed to 34%
of intracellular Gln flux in the intestines which was greater (P=0.05) than that in the liver (14%).
Because there were no differences between small and large egg embryos in their metabolism of
Glu and Gln towards glucose and non-essential AA synthesis, all fluxes are reported as the mean of
small and large eggs (Table 1).
Despite our expectations, there was no contribution of either Glu or Gln to blood glucose synthesis,
nor did these two AA contribute to the synthesis of aspartate and arginine in the blood, liver and

intestines. Thus, even though the availability of Glu and Gln in the egg is 2-fold greater than that
required for net tissue growth of the embryo, these AA are not the source of carbon to account for the
accumulation of glycogen by d 18 (Hazelwood, 1971). In contrast, both Glu and Gln contributed to
tissue intracellular fluxes of alanine and proline. Further, Glu and Gln both made a larger contribution
to intracellular proline flux in the liver compared to intestines.
Table 1. Glu and Gln enrichments and their contributions to liver and intestinal alanine and proline
intracellular fluxes1.
Liver Intestine SED P-value
Isotopic enrichments
Glu [M+5] (13C5 Glu tracer) 0.18 0.32 0.07 0.066
Gln [M+5] (13C5 Glu tracer) 0.19 0.35 0.08 0.084
Glu [M+5] (13C5 Gln tracer) 0.16 0.39 0.12 0.02
Gln [M+5] (13C5 Gln tracer) 0.20 0.59 0.12 0.003
Proportion of intracellular flux from Glu or Gln
Ala flux from Glu 0.22 0.13 0.07 NS
Ala flux from Gln 0.12 0.10 0.03 NS
Pro flux from Glu 0.27 0.09 0.07 0.05
Pro flux from Gln 0.24 0.03 0.05 0.004
SEM, Standard error of the difference between means. NS, not significantly different (P>0.10).
1Enrichments expressed as moles tracer/100 moles tracee.
In summary, embryos from the small and large eggs, despite a 5 g difference in body weight by d 19
embryonic (N.E. Sunny, unpublished results), maintain similar rates of Glu and Gln metabolism with
virtually no contribution of these AA towards gluconeogenesis, and thus glycogen synthesis. The
results also indicate that metabolism of Glu and Gln to proline probably accounts for the shortage
of proline in the developing chick embryo (Ohta et al., 1999).
References
Bequette, B.J., N.E. Sunny, S.W. El-Kadi and S.L. Owens, 2006. Application of stable isotopes and mass isotopomer
distribution analysis to the study of intermediary metabolism of nutrients. J. Anim. Sci. 84 (E. Suppl.), E50-
E59.
Hazelwood, R.L, 1971. Endocrine regulation of avian carbohydrate metabolism. Poult. Sci. 50, 9-18.
Ohta, Y., N. Tsushima, K. Koide, M. T. Kidd and T. Ishibashi, 1999. Effect of amino acid injection in broiler breeder
eggs on embryonic growth and hatchability of chicks. Poult Sci. 78, 1493-1498.
SAS, 2006. Statistical Analysis Software (Release 8.02). SAS Inst. Inc., Cary, NC.

Metabolic flexibility of lactating mink (Mustela vison) is not reflected at
transcriptional level but by changes in functional liver mass
R. Fink, P.D. Thomsen and A.-H. Tauson
Department of Basic Animal and Veterinary Sciences, Faculty of Life Sciences University of
Copenhagen, Groennegaardsvej 3, DK-1870 Frederiksberg C, Denmark
Introduction
Among strict carnivores the generally accepted view has been that the capacity to adapt to a
varied dietary nutrient supply is very limited, with animals constantly having a high rate of hepatic
gluconeogenesis and consequently, high obligatory N losses (Morris, 2002). However, the authors
have previously shown that lactating mink are able to regulate protein oxidation rate, and that milk
yield was improved when protein supply was reduced and replaced by readily available carbohydrates.
The objective of this study was to test whether the indicated metabolic flexibility in the mink is
reflected at the transcriptional level of key metabolic enzymes or if it may be caused by changes in
functional liver mass.

Twelve lactating mink dams were fed ad libitum, from parturition until ten wk post partum, with
diets either high (H), medium (M) or low (L) in protein content. Quantitative energy metabolism
traits, including substrate oxidation, measured by means of balance studies in combination with
indirect calorimetry, and protein turnover determined by means of 15N-glycine end-product
technique, were performed in lactation wk one to four. Ten wk post partum the dams were killed
and blood and liver samples were collected for determination of plasma amino acid profiles by
micelle electrokinetic capillary chromatography, and abundance of mRNA for phosphoenolpyruvate
carboxykinase (PEPCK), fructose 1,6-biphosphatase (Fru 1,6-P2ase), pyruvate kinase (PK) and
glucose-6-phosphatase (G-6-Pase) by quantitative RT-PCR analyses.

The live weights of the kits indicated that dams fed the L diet had the highest milk yield, corresponding
to earlier results (Fink et al., 2004, 2006). Metabolisable energy (ME) intake did not differ (P>0.05)
between dams. However, dams fed the H diet had a higher (P<0.05) nitrogen (N) intake and
consequently a higher (P<0.05) N excretion than dams fed the L diet. Diet did not affect (P>0.05)
total heat production (HE), though the oxidation of protein (OXP) accounted for much higher
(P<0.01) proportion of the total heat production (HE) in H dams than in L dams (Table 1). The
protein flux increased (P<0.05) with progressing stage of lactation, but was not affected (P>0.05)
by diet. Similarly, there were no differences (P>0.05) in protein synthesis and breakdown between
dams. However, dams fed the H and M diets had a higher (P<0.001) net protein synthesis (NPS) than
dams fed the dams fed the L diet, whereas L dams had the most (P=0.003) efficient re-utilisation of
amino acids (Table 1). This was the contrary to results in cats, where a decrease in dietary N content
decreased the protein synthesis (Russell et al., 2003). However, the cat data were calculated based on
label excreted in urea and ammonia, whereas our data were calculated based on total urinary N.
Ten wk post partum dietary treatment was not reflected (P>0.05) in the plasma amino acid profiles
(data not shown), and there were no differences (P>0.05) in the relative abundance for any of the
studied mRNA among dams. However, the liver mass and kidneys were larger (P<0.05) in H dams
than in L dams (Table 2), and an increase in the size of the liver will increase the rate of amino
acid catabolism, despite unchanged activity of the amino acid degradative enzyme per gram liver

(Morris, 2002). Thus, the capacity to adapt to a wide range of protein supplies in mink seems to be
caused by adaptive changes in functional liver mass rather than adjustments of the transcriptional
level of key enzymes.
Table 1. ME, N intake and excretion, oxidation of nutrients, and protein turnover traits.
Protein supply P-value; effect of
H H M L RR* Diet Wk D×Wk
ME intake, kJ/d 1174 1320 1260 145 0.15 <0.01 0.50

N intake, g/d 8.0a 6.7b 3.9c 0.76 <0.01 <0.01 0.04
N excretion, g/d 6.6a 5.2b 3.1c 0.72 <0.01 <0.01 0.02
OXP, % of HE 40a 33b 18c 4.23 <0.01 0.55 0.75
OXF, % of HE 55 48 56 10.57 0.29 0.86 0.56
OXCHO, % of HE 5a 19b 26c 6.91 <0.01 0.36 0.80
Flux, g N/d 9.67 9.99 8.66 2.86 0.50 0.02 0.86
Synthesis, g N/d 4.64 5.33 6.13 2.51 0.72 0.03 0.91
Breakdown, g N/d 1.13 1.78 4.05 2.42 0.19 0.11 0.86
NPS, g N/d 3.52a 3.54a 2.08b 0.49 <0.001 <0.001 0.14
R, % 46.5a 49.6a 69.7b 9.42 <0.001 0.18 0.86
a, b values that share no common superscript differ significantly (P<0.05); effect of diet.
* Residual error.
Table 2. Relative abundance of the study’s mRNA, and liver and kidney mass.
Protein supply R-MSE* P-value
H M L
G-6-Pase 1.08 1.19 1.02 0.46 0.87

Fru 1.6-P2ase 2.02 2.26 1.91 1.05 0.89
PEPCK 1.24 1.66 1.46 0.73 0.74
PK 2.01 1.38 1.61 1.67 0.73
Liver mass, % of kg0.75 4.01a 3.47b 3.36b 0.33 0.01
Kidney mass, % of kg0.75 0.80a 0.67b 0.64b 0.04 0.01
a, b valuesthat share no common superscript differ significantly (P<0.05); effect of diet.

* Root mean square error.
References
Fink, R., A-H. Tauson, A. Chwalibog, N.E. Hansen, N.B. Kristensen and S. Wamberg, 2004. Effects of substitution
of dietary protein with carbohydrate on lactation performance in the mink (Mustela vison). J. Anim. Feed Sci.
13, 647-664.
Fink, R., A-H. Tauson, A. Chwalibog and N.E. Hansen, 2006. A first estimate of the amino acid requirement for milk
production of the high-producing female mink (Mustela vison). J. Anim. Physiol. Nutr. 90, 60-69.
Morris, J.G., 2002. Idiosyncratic nutrient requirements of cats appear to be diet-induced evolutionary adaptations.
Nutrition Research Reviews 15, 153-168.
Russell, K., G.E. Lobley and D.J. Millward, 2003. Whole-body protein turnover of a carnivore, Felis sivestris catus.
Br. J. Nutr. 89, 29-37.

Heat production in broilers is not affected by dietary crude protein
J. Noblet1, S. Dubois1, J. van Milgen1, M. Warpechowski2 and B. Carré3
1INRA, UMR1079 SENAH, F35590 Saint Gilles, France
2Universidade Federal do Paraná, 88035 Curitiba, Brazil
3INRA, UR83 URA, F37380 Nouzilly, France
Introduction
The energy value of poultry feeds is usually estimated according to their metabolisable energy (ME)
contents. However, literature indicates that the ‘true’ energy value of a feed is better estimated by
its net energy (NE) content, at least in pigs or polygastric species (Noblet et al., 1994). In the case
of poultry, the advantage of NE over ME is less clear and uncertainty remains with controversial
conclusions. The objective of the studies reported here was to quantify the efficiency of utilization
of ME for NE according to the dietary crude protein (CP) content in order to evaluate the possible
interest of proposing a NE system for poultry; complementary studies have been carried out in order
to evaluate the effects of fat and dietary fibre on energy utilisation. Indirect calorimetry was used
to estimate energy values.

Experimental designs
In trial 1, 8 groups of male broilers were offered a low (LP) or a normal (NP) CP diet (4 groups per
diet). Measurements were carried out twice at 3 wk (stage 1) and 6 wk (stage 2) of age. The dietary
CP and amino acid levels were adapted according to requirements at each age and levels of limiting
essential amino acids were identical in both diets at each stage. Birds were offered feed ad libitum;
2 h of darkness was imposed daily. In trial 2, 12 groups of male broilers were offered a NP or a high
(HP) CP diet. Measurements were carried out once (6 groups per diet), either at 4 or 5 wk age; the
average age was equivalent for both dietary treatments. Feed was offered for 6 periods of 30 min
each at 3 h intervals; 6 h of darkness was imposed daily. Due to the high CP level of the HP diet,
only the sulphur amino acid content was equivalent in both diets.
The difference in CP level between diets within each trial was about 4.7%. The increase in CP content
was achieved by replacing corn starch and free amino acids by soybean protein concentrate. Diets
were based on wheat, corn and soybean meal. Birds had ad libitum access to water and were kept at
24 °C. Measurements in the open-circuit respiration chamber were conducted for 7 d for groups of
broilers kept in a metabolic cage; no feed was provided on the last day to measure the fasting heat
production (FHP). Measurements were carried out after at least 7 d of adaptation to the diet and
housing conditions. Since the metabolic cage and chamber had a fixed size, the number of birds per
group (6 to 15) depended on their age.
Measurements and calculations
Feed intake, BW gain and excreta were measured during 6 d during the fed period. The O2 and
CO2 gas concentrations in the chamber were used to calculate total daily heat production (HP). Its
partitioning between activity-related heat production (AHP; estimated from force sensors on which
the metabolic cage was mounted), thermic effect of feeding (TEF) and FHP was obtained using
modelling techniques (van Milgen et al., 1997). The ME value of the diet and energy, protein and
fat balances were calculated according to standard procedures; balance data were expressed per
unit of metabolic body weight (kg BW0.70; unpublished data). The NE intake was calculated as:
ME intake – TEF – AHP.

There was no interaction between dietary CP and age for the response criteria. Although essential
amino acid supplies were similar for both diets within each trial, BW gain and protein deposition
were improved for birds receiving the higher CP diet. Unlike what has been observed in growing
pigs (Le Bellego et al., 2001), none of the criteria related to energy utilisation was affected by
dietary CP (Table 1). The energy used for physical activity represented about 10% of ME intake.
No clear explanations can be given for the higher HP at a lower ME intake in trial 2. Differences in
diet characteristics (more dietary fibre, less starch and less fat with subsequent lower ME value in
trial 2) could contribute to this difference. In addition, these studies were conducted at a 3 yr interval
with genetically different birds. It is unlikely that the difference in feeding techniques (ad libitum vs.
meals) would generate this difference (unpublished data). In conclusion, the partial replacement of
starch by dietary protein in broiler feeds is not associated with changes in energy utilisation.
Table 1. Effect of dietary protein level on energy utilisation in broilers.1
Trial 1 Trial 2
LP NP RSD3 Stat.3 NP HP RSD3 Stat.3
Dietary crude protein2, % 18.0 22.7 - - 22.5 27.3 - -

Average BW, kg 1.46 1.47 0.10 NS 1.35 1.34 0.03 NS
Feed intake2, g/d 142 142 7 NS 142 138 7 NS
Body weight gain, g/d 76 85 8 0.05 78 83 8 NS
Energy balance4, kJ/kg0.70
ME intake 1609 1609 - - 1457 1457 - -
Total HP2 853 846 21 NS 892 872 18 0.08
As FHP 450 439 17 NS 419 416 15 NS
As AHP 146 153 21 NS 173 168 12 NS
As TEF 257 256 35 NS 299 288 17 NS
Total energy gain 756 763 21 NS 565 585 18 NS
As protein 331 369 22 0.01 332 369 12 0.01
ME content2, MJ/kg 13.49 13.52 0.40 NS 12.72 12.64 0.15 NS
NE/ME, % 75.1 74.8 1.7 NS 67.7 68.6 1.3 NS
1Abbreviations: ME: metabolisable energy, NE: net energy, HP: heat production, FHP: Fasting heat production,
AHP: Activity heat production, TEF: Thermic effect of feed.
2Values adjusted for 89% feed dry matter; in trial 1, only values for stage 2 are given (see text).
3RSD: Residual standard deviation, Stat.: level of significance (within trial).
4Data adjusted for the same ME intake.
References
Le Bellego, L., J. van Milgen and J. Noblet, 2001. Energy utilization of low protein diets in growing pigs. J. Anim.
Sci. 79, 1259-1271.
Noblet, J., H. Fortune, X.S. Shi and S. Dubois, 1994. Prediction of net energy value of feeds for growing pigs. J.
Anim. Sci. 72, 344-354.
Van Milgen, J., J. Noblet, S. Dubois and J.F. Bernier, 1997. Dynamic aspects of oxygen consumption and carbon
dioxide production in swine. Br. J. Nutr. 78, 397-410.

Diet-induced thermogenesis and feed intake regulation in the chicken:
effect of diet and genotype
Q. Swennen1, A. Collin2, G.P.J. Janssens3, E. Le Bihan-Duval2, A. Bordas4, E. Decuypere1 and J.
Buyse1
1Department Biosystems, K.U.Leuven, Kasteelpark Arenberg 30, 3001 Heverlee, Belgium
3Laboratory of Animal Nutrition, Ghent University, Heidestraat 19, 9820 Merelbeke, Belgium
4INRA, UMR1236 Genetique et Diversite Animales, F-78350 Jouy en Josas, France
Introduction
The regulation of voluntary feed intake is a complex mechanism involving several levels of control.
In mammals, many models have been proposed to understand the mechanisms that match energy and
nutrient balance with food intake and energy expenditure to maintain body homeostasis. Diet-induced
thermogenesis (DIT) plays a role in the regulation of feed intake, as defined in the hierarchic oxidation
theory (Stubbs et al., 1997). Protein is preferentially oxidised, then carbohydrates and finally fat,
corresponding with their satiating capacity, suggesting that obligatory oxidative disposal plays a role
in feed intake regulation. In addition, genotype has a profound effect on appetite (Rolls, 1995).
A series of studies was initiated to study the role of DIT in the regulation of feed intake in chickens
using diet composition, genotype and the combination of these factors as a model.

To assess a nutritional model, commercial broilers were reared on diets with an isoenergetic
substitution between fat and protein and similar carbohydrate level (low protein (LP): 126 g protein/
kg, 106 g fat/kg; low fat (LF): 242 g protein/kg, 43 g fat/kg; 16.6 MJ gross energy/kg). From 21
d of age, repeated during 4 wk, 3 LP and 3 LF broilers were placed into respiratory cells of the
indirect calorimeter unit to measure DIT and feed intake (FI). To evaluate DIT, chickens were feed-
deprived during 24 h and the average heat production during the last 8 h was calculated. During the
next 7 h, the animals were given their diet and O2 and CO2 exchanges and FI were recorded. The
difference between the average feed-deprived heat production and heat production during refeeding
was calculated. The DIT was then calculated as the area under the resulting curve, expressed as a
proportion of FI (Swennen et al., 2004). In addition, genetically fat and lean broilers (Leclercq et
al., 1980) were reared on the same isoenergetic diets. From 21 d, repeated during 5 wk, 3 chickens
per group were monitored for DIT and FI (Swennen et al., 2006). In the following experiments,
poultry genotypes differing in feed efficiency (on the same diet) were studied. Cockerels from a
laying strain, selected for egg production were compared to age-matched broiler chickens, selected
for body weight gain. In addition, cockerels selected for high (R+) or low (R-) residual feed intake
were compared (Bordas and Mérat, 1984). From 21 d or 30 wk of age, respectively, repeated during
4 wk, DIT and FI were measured for 3 animals per genotype (Swennen et al., 2007; Swennen et
al., unpublished results).

In adult mammals, a hierarchy exists in the satiating capacity of the macronutrients: protein >
carbohydrates ≥ fat, as well as a positive relation between DIT and satiety (Crovetti et al., 1998).
In spite of a 13% higher DIT and a 24% lower FI per metabolic body weight (MBW; kg0.75) in LP
broilers compared to their LF fed counterparts, this difference was not statistically verifiable. Also, no
combined effect of diet composition and genotype on DIT or FI was observed between broilers of a

genetically fat and lean line reared on the same isoenergetic diets. In contrast to the lack of significant
effect of diet on DIT and FI, genotype significantly affected both parameters. Layer cockerels had a
significantly higher DIT per MBW compared to age-matched broilers (Figure 1A). However, layer
cockerels also had a significantly elevated FI per MBW (Figure 1B). Similarly, cockerels of the R+
genotype had a significantly higher DIT and FI per MBW compared to R- cockerels. In other words,
in spite of the effects of genotype on DIT and FI, no feedback effect of DIT on FI was observed.
100 1.2
* A B
1.0
DIT/FI (kJ/MBW*g feed*7h)

80 *
Feed intake (g/MBW*7h)
0.8
60 *
*
0.6 * *
*
*
40
0.4
20 0.2
0.0
0
Week 4 Week 5 Week 6 Week 7 Week 4 Week 5 Week 6 Week 7
Figure 1. Feed intake (FI) (A; g/metabolic body weight (MBW)) and diet-induced thermogenesis
(DIT) per FI (B; kJ/kg MBW per g FI) of broilers (filled bars) and layer cockerels (open bars).
Significant genotype effects are indicated by * P<0.05, ***P<0.0001.
Taken together, these results strongly suggest that, in contrast to mammals, DIT is not directly
involved in the control and regulation of feed intake in the broad range of models used to study the
effects of diet composition, genotype and the interaction between these variables.
References
Bordas, A. and P. Mérat, 1984. Correlated responses in a selection experiment on residual feed intake in Rhode Island
Red chickens. Br. Poult. Sci. 44, 233-237.
Crovetti, R., M. Porrini, A. Santangelo and G. Testolin, 1998. The influence of thermic effect of food on satiety. Eur.
J. Clin. Nutr. 52, 482-488.
Leclercq, B., J.C. Blum and J.P. Boyer, 1980. Selecting broilers for low or high abdominal fat: initial observations.
Br. Poult. Sci. 21, 107-113.
Rolls, B.J., 1995. Carbohydrates, fats and satiety. Am. J. Clin. Nutr. 61, 960S-967S.
Stubbs, R.J., A.M. Prentice and W.P.T. James, 1997. Carbohydrates and energy balance. Ann. NY Acad. Sci. 819,
44-69.
Swennen, Q., G.P.J. Janssens, E. Decuypere and J. Buyse, 2004. Effects of substitution between fat and protein on
food intake and its regulatory mechanisms in broiler chickens: Energy and protein metabolism and diet-induced
thermogenesis. Poult. Sci. 83, 1997-2004.
Swennen, Q., G.P.J. Janssens, A. Collin, E. Le Bihan-Duval, K. Verbeke, E. Decuypere and J. Buyse, 2006. Diet-
induced thermogenesis and glucose oxidation in broiler chickens: influence of genotype and diet composition.
Poult. Sci. 85, 731-742.
Swennen, Q., E. Delezie, A. Collin, E. Decuypere and J. Buyse, 2007. Further investigations on the role of diet-
induced thermogenesis in the regulation of feed intake in chickens: comparison of age-matched broiler versus
layer cockerels. Poult. Sci., in press.

Protein intake but not feed intake may affect dietary net energy for
finishing pigs
S. Moehn and R.O. Ball
Swine Research and Technology Centre, 4-10 Agriculture/Forestry Centre, University of Alberta,
Edmonton AB, T6G 2P5 Canada
Introduction
Dietary net energy (NE) is regarded as independent of feed intake level and dietary protein contents
(Noblet et al., 2003). However, theoretical calculations (Susenbeth, 2005) indicate that the energetic
value of protein should vary with its content in a diet. Changing feeding level may alter the ratio of
protein to lipid deposition, and hence the efficiency of growth, and thus NE. Our objective was to
study the effects of protein and feeding level on net energy in finishing pigs.

Twelve castrated male pigs, in 2 blocks, were fed either ad libitum (AL) or restrictively (RF), at twice
the metabolizable energy (ME) requirement for maintenance (458 kJ/kg0.75). Within feeding levels,
pigs received a low-protein (LP) and a high-protein (HP) diet in a cross-over design. Pigs were adapted
to the feeding level for 6 wk prior to measurements. Body weight (BW) during the experimental
period was between 50 and 95 kg, with a mean of 75.4 kg (SE 2.8) for AL and 63.2 kg (SE 1.4) for
RF. Measurements started at 50 kg for a period of 4 wk. Each measurement period consisted of 7 d
adaptation to each diet, a 7-d nitrogen balance and 24-h indirect calorimetry. The LP diet consisted
of barley plus free amino acids and contained 11.5% crude protein and 0.61% lysine. The HP diet,
formulated for the same ME (13.6 MJ/kg) and lysine content, was based on barley, soybean-, canola-
and corn gluten meal without free amino acids and contained 19.9% crude protein.
Heat production was calculated using indirect calorimetry. NE was calculated as ME intake minus
heat increment, calculated as heat production minus NE for maintenance (NEm). Because feeding
level can affect NEm (de Lange et al., 2006), NEm was taken as 644 kJ/BW0.60 for RF and as 729
kJ/BW0.60 for AL. Predicted dietary NE was calculated according to Noblet et al. (2003) from the
digestible protein, lipid, organic residue (OR), NDF and ADF contents.
Mixed model analysis (SAS, 2002) was used to assess the impact of feeding level, protein level and
body weight on dependent variables. The interaction ‘feeding level × protein level’ was retained in
the model if P<0.1. The model included ‘pig’, ‘block’ and ‘period’ as random variables. Significance
was taken as P<0.05, P<0.1 as a tendency.
Results
AL pigs gained weight approximately 30% faster than RF pigs at almost twice the feed intake
(Table 1). Digestibility of OR was greater for RF (P=0.05). Digestibility of protein, OR and ADF
was greater for HP (P<0.01), while the digestibility of gross energy, lipid and NDF was lower
(P<0.1) for HP vs. LP, due to differences in ingredient composition. HP had greater gross energy
content than LP, which balanced the difference in gross energy digestibility and resulted in similar
digestible energy contents. ME was lower (P=0.01) by 0.65 MJ/kg (SE 0.17) for HP than for LP,
and tended to increase with BW (P=0.06). Heat production (Table 1) was not affected by feeding or
protein level but increased with BW. Retained protein and lipid energy were greater for AL than for
RF. Retained protein energy was greater for HP probably caused by greater than anticipated lysine
contents in HP, while retained lipid energy was lower. Predicted NE content was not affected by

treatment. Measured NE content tended (P=0.07) to be lower for HP. This effect was small (-0.08
MJ per 1% increase in dietary protein content) and not likely to affect the estimate of dietary NE
under conditions of commercial pig production. Measured NE content was not affected by feeding
level or BW. The estimated change in energetic efficiency due to changing protein to lipid retention
was approximately 5%; this change may be too small to affect practical application of NE in diet
formulation.
Table 1. Means for performance and energy measurements1 in pigs fed high protein (HP) or low
protein (LP) diets ad libitum or restrictively.
Ad libitum Restricted SEM Significance of effects, P=
HP LP HP LP Feeding Protein Body

level level weight
Daily gain, g/d 1056 1058 720 660 35 0.02 0.58 0.01
Daily feed2, g 2961 3222 1619 1621 118 0.02 0.01 0.01
RPE, MJ/d 5.94 5.28 3.10 2.49 0.24 0.01 0.01 0.02
RLE2, MJ/d 11.57 18.04 2.17 3.54 1.14 0.04 0.01 0.01
Heat, MJ/d 21.80 22.03 16.3 16.26 0.65 0.12 0.68 0.01
NE, MJ/kg 9.22 10.17 8.08 8.49 0.26 0.33 0.07 0.32
NEp, MJ/kg 11.95 11.93 11.94 11.85 0.03 0.99 0.38 0.20
1RPE: retained protein energy, RLE: retained lipid energy, NE: net energy, NEp: net energy predicted from digestible
nutrient content.
2Interaction ‘protein level × feeding level’: P<0.05.
Conclusions
Dietary net energy may be affected by the pigs’ protein intake, although the effect was rather small.
There was no evidence that feed intake or body weight affected dietary net energy.
References
De Lange, K., J. van Milgen, J. Noblet, S. Dubois and S. Birkett, 2006. Previous feeding level influences plateau heat
production following a 24 h fast in growing pigs. Br. J. Nutr. 95, 1082-1087.
Noblet, J., V. Bontems and G. Tran, 2003. Estimation de la valeur energetique des aliments pour le porc. INRA Prod.
Anim. 16, 197-210.
SAS (Statistical Analysis System), 2002. Release 9.1 Edition. SAS Institute Inc., Cary, NC, USA.
Susenbeth, A., 2005. Bestimmung des energetischen Futterwerts aus den verdaulichen Nährstoffen beim Schwein.
Űbers. Tierernährg. 33, 1-16.

Influence of dietary protein/energy ratio on growth, body composition,
protein, lipid and energy retentions as well as amino acid metabolism of
Nile tilapia
J. Gaye-Siessegger, S.M. Mamun and U. Focken
Department of Aquaculture Systems and Animal Nutrition in the Tropics and Subtropics, University
of Hohenheim (480B), 70593 Stuttgart, Germany
Introduction
The present study was conducted to investigate the effect of different dietary protein contents and
feeding levels on growth performance, proximate composition, protein, lipid and energy retentions,
apparent crude protein digestibility as well as activities of enzymes involved in amino acid metabolism
of Nile tilapia, Oreochromis niloticus (L.) under controlled laboratory conditions.

Fifty-five tilapia were reared individually in aquaria connected to a recirculation system. The water
temperature was maintained at 27 ± 0.2 °C. After an initial phase to standardise their body mass,
seven fish were sacrificed to determine the initial body composition. The remaining fish (22.3 ± 3.7
g) were assigned randomly to eight groups and were fed two purified diets differing in their protein
contents (CP 20.9 and 41.2%, hereafter referred to as P21 and P41 respectively) at four different
levels (4, 8, 12 and 16 g/kg0.8/d, hereafter referred to as F4, F8, F12 and F16 respectively). The
experiment lasted ten wk. The diets were formulated to be isolipidic and isoenergetic (on the basis of
metabolisable energy) and were made from defatted fish meal, wheat starch, sunflower oil, fish oil,
vitamin and mineral premixes, carboxymethylcellulose, cellulose and titanium dioxide. Faeces were
collected during the last wk of the experiment. At the end of the experiment, the livers of fish were
removed on ice, weighed and stored at -80 °C until the measurement of enzyme activities (aspartate
aminotransferase, alanine aminotransferase and glutamate dehydrogenase). Feed and individual fish
were analysed for their proximate composition, gross energy content and δ13C and δ15N values.

Body mass gain, growth rates and HSI varied significantly according to both feeding level and
dietary protein content (Table 1). There were significant differences in body composition between the
treatments (data not shown). Protein and energy gain of fish were significantly affected by feeding
level and dietary protein content with the highest values in fish fed diet P41 at the highest level
and the lowest values in fish fed diet P21 at the lowest level (Table 2). Lipid gain was significantly
affected only by feeding level. Utilisation of protein and energy did not differ significantly between
the treatments while lipid utilisation was significantly affected by dietary protein content. Apparent
protein digestibility of the high and low protein diet was 98% and 93%, respectively, for fish fed
at 4 g/kg0.8/d. Apparent protein digestibility decreased with increasing feeding level to 73% (high
protein diet) and 84% (low protein diet) at the highest feeding level. The decrease in apparent protein
digestibility with increasing feeding level was more pronounced in the high protein diet.
It can be summarised that feeding level significantly affected growth, body composition, HSI as well
as protein, lipid and energy gain but did not affect utilisation parameters whereas dietary protein
content significantly affected growth, body composition, HSI, protein and energy gain as well as
lipid retention. To find whether the low growth rate of fish fed Diet P21 at the highest level was
due to oxidation of feed, any factor so far unknown or just by incidence needs to be investigated
in a following study using a respirometric system for fish in order to determine complete energy
budgets of individual fish.

Table 1. Initial and final body mass as well as body mass gain, metabolic growth rate (MGR) and
hepatosomatic index (HSI) of tilapia fed the high and low protein diet at different levels. Arithmetic
means (n=6) and probability of parameter effects.
Diet F, Initial body Final body Body mass MGR, HSI,

g/kg0.8/d mass, g mass, g gain, g g/kg0.8/d %
P21 4 22.3 25.8 6.4 1.0 2.5

8 22.1 32.2 12.6 2.6 3.0
12 22.1 39.3 19.8 4.0 3.6
16 22.3 37.0 17.4 3.4 3.5
P41 4 22.3 27.4 7.9 1.4 1.3
8 22.4 37.9 18.1 3.6 1.9
12 22.2 50.8 31.2 5.6 2.2
16 22.4 66.3 46.7 7.5 1.9
S.E.D. 4.67 9.92 9.71 1.15 0.85
Parameter df
Diet 1 >0.05 <0.001 <0.001 <0.001 <0.001
F 3 >0.05 <0.001 <0.001 <0.001 <0.05
Diet × FL 3 >0.05 <0.01 <0.01 <0.001 >0.05
F = feeding level, S.E.D.=standard error of the difference.

MGR = body mass gain/average metabolic live weight (kg0.8)/d.
HSI = liver mass (g) × 100/(body mass (g) – liver mass (g).
Table 2. Protein, lipid and energy gain as well as utilisation parameters of tilapia fed the high and
low protein diet at different levels. Arithmetic means (n=6) and probability of parameter effects.
Diet F, Protein PPV, Lipid ALC, Energy ktot,

g/kg0.8/d gain, g % Gain, g % gain, kJ %
P21 4 0.6 26.0 0.8 66.3 48.0 20.7

8 1.7 32.3 1.7 64.1 104.3 20.9
12 2.9 33.8 2.8 62.7 174.8 21.6
16 2.6 22.1 2.7 45.9 163.6 15.0
P41 4 1.3 24.8 0.5 29.1 45.6 18.1
8 2.9 25.0 1.5 44.8 121.4 21.9
12 4.4 22.4 2.5 43.5 192.1 20.4
16 7.2 24.4 4.4 51.0 333.9 23.4
S.E.D. 1.65 10.82 0.99 19.60 72.89 7.40
Parameter df
Diet 1 <0.001 >0.05 >0.05 <0.001 <0.01 >0.05
F 3 <0.001 >0.05 <0.001 >0.05 <0.001 >0.05
Diet × FL 3 <0.01 >0.05 <0.05 <0.05 <0.01 >0.05
F = feeding level, S.E.D.=standard error of the difference.

PPV = (final fish body protein (g) – initial fish body protein (g)) × 100 / crude protein intake (g).
ALC = (final fish body lipid (g) – initial fish body lipid (g)) × 100 / crude lipid intake (g).
ktot = (final gross energy (kJ) – initial gross energy (kJ)) × 100 / gross energy intake (kJ).

Efficiency of energy and protein deposition in swine during compensatory
growth measured by dual energy X-ray absorptiometry (DXA)
A.D. Mitchell1 and A.M. Scholz2
1USDA-ARS, Growth Biology Laboratory, Building 200, 20715 Beltsville, MD, USA
2University Munich, Livestock Center, Hubertusstr. 12, 85764 Oberschleissheim, Germany
Introduction
Following a period of restricted dietary intake, young pigs exhibit compensatory growth that is
characterized by an accelerated growth rate that usually includes more fat and less muscle than in
pigs continuously fed ad libitum (Mersmann et al., 1987). Fat and lean deposition in growing pigs
can also be influenced by the level of protein in the diet and by feeding the β-adrenergic agonist,
ractopamine (Mitchell et al., 1990; Carr et al., 2005). The purpose of this study was to examine the
effects of controlled intake, dietary protein (CP) level, and ractopamine supplementation on growth,
body composition, and the efficiency of energy and protein deposition in pigs during uninterrupted
or compensatory growth from 60 to 100 kg.

Seven groups of pigs (58 pigs in total) were scanned by DXA for body composition analysis at a
starting weight of 61.4 ± 1.9 kg and at a final weight of 100.9 ± 2.9 kg. Three groups of pigs were fed
at continuous intake levels from 60 kg: ad libitum (A) the basal diet (186 g/kg CP and 13.58 MJ/kg
ME), ad libitum plus 20 mg/kg ractopamine (Rac), and limited at calculated (C) energy intake level
(NRC, 1988). Four groups of pigs were maintained at 60 kg for 56 d, scanned by DXA again, and
then fed: 200 g/kg CP (13.73 MJ/kg ME) diet at C intake (M-HP), 120 g/kg CP (13.79 MJ/kg ME)
diet at C intake (M-LP), ad libitum intake (M-A), and ad libitum intake plus R (M-Rac).
Total body protein was calculated from the DXA lean values using the following equation:
protein (g) = -1.062 + (0.2·DXA lean).
Feed intake was measured for each pig individually. Total body fat and protein deposition were
based on the differences between the 60-kg (2nd measurement for M-groups) and 100-kg DXA
measurements of fat and lean. Energy deposition was calculated as the sum of fat deposition (39.6
MJ/kg) and protein deposition (23.7 MJ/kg).

During the 56 d period of maintenance feeding, the pigs on that dietary regime lost an average
of 2.2 ± 0.4 kg of fat, but gained 2.6 ± 0.7 kg of lean and 0.13 ± 0.02 kg of bone mineral, as the
percentage of body fat dropped from 13.3 to 9.7. During the final phase, there was no difference in
the growth rate (0.95 ± 0.09 kg/d) for pigs fed at the calculated energy intake level (C), however the
M-LP pigs deposited more fat and less lean (protein) than the C or M-HP pigs (Table 1). For pigs
fed ad libitum, the addition of Rac resulted in a 13% increase in the rate of weight gain compared
to A (1.15 vs. 1.02 kg/d, P<0.05), consisting of a 29% increase in the rate of lean tissue deposition
(0.86 vs. 0.67 kg/d, P<0.05) and an 18% reduction in the rate of fat deposition (0.27 vs. 0.34 kg/d,
P<0.05). A similar response to ractopamine was observed in pigs fed continuously (Rac) and those
that had been previously fed at maintenance intake (M-Rac). Also for pigs fed ad libitum, those that
had been previously fed at maintenance intake exhibited compensatory growth ([M-A + M-Rac]/2:
1.23 vs. [A + Rac]/2: 0.94 kg/d, P<0.05), consisting primarily of lean tissue (0.90 vs. 0.62 kg/d,

P<0.05). During maintenance feeding the pigs continued to deposit bone mineral, consequently during
the final phase those fed at the calculated intake exhibited a lower rate of bone mineral deposition
compared to those fed on a continuous basis or refed ad libitum.
The efficiencies of energy (kg) and protein (PE) deposition are shown in Table 1. For pigs fed at the
calculated intake level, the M-LP group had higher efficiency for both energy and protein deposition
compared to the C or M-HP groups. These differences were consistent with the effects of a low
protein diet on nitrogen excretion and heat production as reported by Le Bellego et al. (2001). For
pigs fed ad libitum, Rac resulted in an improvement in PE in both groups (Rac, M-Rac), but not in
kg. Similarly, compensatory growth in the ad libitum intake pigs had no effect on kg, but resulted in
a significant improvement in PE ([M-A + M-Rac]/2: 0.26 vs. [A + Rac]/2: 0.20, P<0.05).
Table 1. Least squares means for intake, growth, and the efficiency of energy (kg) and protein (PE)
deposition in pigs during continuous or compensatory growth from 60 to 100 kg body weight.
Calculated intake2 Ad libitum intake SEM
Group1 (n) C (8) M-HP (8) M-LP (8) A (8) Rac (9) M-A (9) M-Rac (8)
Intake
DE3, MJ/d 38.3a 4 38.3a 38.7a 46.3b 46.2b 52.1c 50.7c 0.43
CP3, g/d 526b 560b 337a 637c 635c 716d 696d 5.6
Growth
Fat, g/d 249a 246a 309bc 338c 266ab 333c 284ab 6.2
Prot3, g/d 145b 139b 116a 109a 139b 155b 208c 2.7
BMC3, g/d 20.1b 15.7a 15.7a 17.8ab 17.3ab 19.4b 19.0ab 0.4
kg 0.348b 0.341b 0.390c 0.345b 0.301a 0.325ab 0.319ab 0.005
PE 0.276c 0.253bc 0.350d 0.171a 0.221b 0.217ab 0.301c 0.006
1Groups: C, 186 g/kg CP (13.58 MJ/kg ME); M-HP, fed at maintenance (M) for 56 d followed by 200 g/kg CP
(13.73 MJ/kg ME); M-LP, fed at M followed by 120 g/kg CP (13.79 MJ/kg ME); A, same diet as C; Rac, A plus 20
mg/kg ractopamine; M-A, M followed by A; M-Rac, M followed by Rac.
2Calculated DE intake, MJ/d=55.07·(1-e-0.0176·BW), (NRC, 1988).
3Abbreviations: DE, digestible energy; CP, crude protein; Prot, total body protein; BMC, bone mineral.
4Means followed by different letters were significantly different at P<0.05.
References
Carr, S.N., D.J. Ivers, D.B. Anderson, D.J. Jones, D.H. Mowrey, M.B. England, J. Killefer, P.J. Rincker and F.K.
McKeith, 2005. The effects of ractopamine hydrochloride on lean carcass yields and pork quality characteristics.
J. Anim. Sci. 83, 2886-2893.
Le Bellego, L., J. van Milgen, S. Dubois and J. Noblet, 2001. Energy utilization of low-protein diets in growing pigs.
J. Anim. Sci. 79, 1259-1271.
McKeith, 2005. The effects of ractopamine hydrochloride on lean carcass yields and pork quality characteristics. J.
Anim. Sci. 83, 2886-2893.
Mersmann, H.J., M.D. MacNeil, S.C. Seideman and W.G. Pond, 1987. Compensatory growth in finishing pigs after
feed restriction. J. Anim. Sci. 64, 752-764.
Mitchell, A.D., M.B. Solomon and N.C. Steele, 1990. Response of low and high protein select lines of pigs to the
feeding of the beta-adrenergic agonist ractopamine (phenethanolamine). J. Anim. Sci. 68, 3226-3232.
NRC, Nutrient Requirements of Swine, 1988. National Research Council, National Academy Press, Washington, DC,
USA, 8 pp.

Dynamic integration of biological processes into models: contribution to
prediction of cattle growth and body composition
F. Garcia1, R.D. Sainz2, J. Agabriel1 and J.W. Oltjen2
1INRA, UR1213 Herbivores, Site de Theix, F-63122 Saint-Genès-Champanelle, France
2Department of Animal Sciences, University of California, Davis, CA 95616, USA
Introduction
Feeding systems use empirical relationships to predict energy requirements and composition of
growing beef cattle (INRA, 1989; NRC, 1996). Whereas their estimations are often satisfactory,
they do not take into account the effects of previous nutrition. Mechanistic and dynamic models
of cattle growth and body composition cope with various genotypes, growth trajectories and types
of diets. The Davis Growth Model (DGM, Oltjen et al., 1986) simulates biological mechanisms of
hyperplasia and hypertrophy. It predicts empty body protein, estimates the heat production, and then
deduces accretion in the body fat pool. The INRA Growth Model (IGM, Hoch and Agabriel, 2004)
predicts carcass and non-carcass pools of protein and lipids, integrating the processes of synthesis
and degradation in each. Both models work with metabolizable energy intake (MEI) as input and
mature (frame) size as a driver of protein deposition. This paper presents a comparative evaluation
of these models in order to identify strengths and weaknesses and improve our knowledge of the
underlying processes.

Two datasets involving Salers heifers (n1=23) or Angus-Hereford steers (n2=120), various types
of diets and growth trajectories were used. Salers heifers were fed (from 8 to 34 mo) forage diets
of different energy concentrations to obtain continuous and discontinuous growth trajectories. The
Angus-Hereford steers were fed (from 8 to 16 mo) a high-energy diet ad libitum (CA), a high-energy
diet in a limited quantity (CL) or a low-energy diet ad libitum (FA). We investigated the ability of
both models to predict the quantities of body protein and lipids in these experiments. Parameterization
and evaluation were performed with the same data. A method for empirical validation based on the
deviations between observed and model predicted protein and lipid (Mitchell, 1997), was adopted.
The models were also evaluated as to mean bias (MB) and mean square error of prediction (MSEP)
(Tedeschi, 2006). The significances of MB and MSEP were tested using a linear mixed model that
included the model and the treatment effects and their interactions with the individual as random
effects.

Mean bias and MSEP for body protein were always similar with both models and much lower than
for body lipids. For body lipids, MSEP were 382 kg² for IGM and 967 kg² for DGM (versus 39
kg² and 39 kg² for body protein) with the Salers heifers dataset; 352 kg² for IGM and 171 kg² for
DGM (versus 17 kg² and 19 kg² for body protein) with the Angus-Hereford steers dataset. Both
models performed very well for body protein whatever the growth trajectory, suggesting that the
mathematical formulation based on frame size (FS) is generic for fat free matter growth. Indeed, FS
‘pulls’ the growth rate and represents the homeorrhetic control of growth.
DGM tended to underestimate body fat accretion for the growth restricted Salers heifers (Figure 1)
which suggests that DGM overestimated heat production (HE) in those animals. DGM estimates HE
as a function of empty body weight, consequently overestimation during feed restriction suggests
that changes in maintenance energy expenditures with varying diet quantity and quality are not well

represented. Estimation of HE as a function of viscera protein should improve the DGM’s biological
realism and predictive ability. Finally, IGM underestimated lipid quantities for high energy diets and
overestimated lipid quantities for low energy diets (Figure 1), indicating that IGM is not sensitive
enough to MEI. Indeed lipid accretion in IGM is related to a maximum quantity of lipids at a given
physiological stage, which means that the homeorrhetic control of fat accretion in IGM is stronger
than what is observed. Dynamic integration of biological processes improves predictive ability.
Evaluation of the models investigates the hypotheses built into the models and thus contributes to
the knowledge of the underlying processes.
Figure 1. Lipid mean bias with DGM (¢) and IGM (£) for i) Angus-Hereford steers fed a CA
diet, CL diet or a FA diet; and ii) for Salers heifers in continuous growth (CG) or submitted to a
feeding restriction (DG). Test of difference with 0 can be NS (non significant), ** (P<0.01) or ***
(P<0.001).
References
Hoch, T. and J. Agabriel, 2004. A mechanistic dynamic model to estimate beef cattle growth and body composition:
1. Model description. Agric. Syst. 81, 1-15.
INRA, 1989. Ruminant nutrition: recommended allowances and feed tables. R. Jarrige (Ed.) John Libbey, Eurotext,
Montrouge, France.
Mitchell, P.L., 1997. Misuse of regression for empirical validation of models. Agric. Syst. 54, 313-326.
NRC, 1996. Nutrient requirements of Beef Cattle. 7th revised edition. National Academy Press, Washington, DC,
242 pp.
Oltjen, J.W., A.C. Bywater, R.L. Baldwin and W.N. Garrett, 1986. Development of a dynamic model of beef cattle
growth and composition. J. Anim. Sci. 62, 86-97.
Tedeschi, L.O., 2006. Assessment of the Adequacy of mathematical models. Agric. Syst. 89, 225-247.

5A. Whole organism energy metabolism

The efficiency of utilisation of metabolisable energy of diets rich in
saturated or polyunsaturated fats in broiler chickens
G. Ferrini1, M. Lachica2, A.C. Barroeta1, J.F. Aguilera2 and J. Gasa1
1Dept. de Ciencia Animal y de los Alimentos, Edif. V, Campus UAB, 08193 Barcelona, Spain
2Estación Experimental del Zaidín (CSIC), Camino del jueves s/n, 18100 Granada, Spain
Introduction
It is well established that the lipid depot of chicken may change according to fatty acids in dietary fat.
Chickens fed ad libitum a diet rich in polyunsaturated fatty acids (PUFA) show lower total carcass
fat content than chickens fed a diet rich in saturated fatty acids (SFA) (Sanz et al., 1999; Crespo and
Esteve-Garcia, 2001). Assuming similar digestibility coefficients among dietary fats, this increased
fat deposition can be due to differences in their metabolic use, a fact that may involve differences in
energetic efficiency. As a result, diets of similar proximal composition that differ in their fatty-acid
profile (PUFA/SFA) should differ in energetic efficiency. Consequently, the objective of this study
was to determine in broiler chickens whether the metabolisable energy from a diet containing linseed
oil fat is used with a lower efficiency than that including an equal percentage of tallow fat.

Three groups of female broiler chickens (n=24; 8 chickens per treatment) were fed ad libitum at
1 wk of age a control diet based on maize and soybean meal (diet C: without additional fat; 2.6%
EE) or each of two diets containing 9% of tallow (diet T: rich in SFA; 11.1% EE) or 9% of linseed
oil (diet LO: rich in PUFA; 11.0% EE). The contents of crude protein were 197.8, 198.9 and 202.9
g/kg as-fed, for diets C, T and LO, respectively. Corresponding ME values (13.20, 14.35 and 14.75
MJ/kg as-fed) were determined using the classical total collection method (on d 25 to 27 d of age; 8
chickens per treatment). Between d 39 and 52 of age heat production (HP) of each treatment group
was measured twice at two levels of feeding (ad libitum or 0.5 × ad libitum, one meal), after 2 d of
adaptation, by placing 4 chickens in each of two confinement chambers (Lachica et al., 1995) for
24 h. Retained energy (RE) was calculated as ME intake (MEI) minus HP. At the end of calorimetry
measurements, chickens were slaughtered by electrical stunning and the abdominal fat pad (from the
proventriculus surrounding the gizzard (down to the cloaca) was removed from the hot carcass, and
weighed. The experimental protocol was approved by the Bioethical Committee of the CSIC. The
SAS GLM procedure (SAS Institute, 2000) was used for statistical analysis of lipid depots data.

Table 1 shows the average values observed for MEI and HP. A lower percentage of abdominal fat
pad, considered as the main index of fat deposition, was observed in the animals fed diet C (0.725
g/100 g of BW; P<0.01) with respect to T and LO diets (1.257 and 1.013 g/100 g BW), which differed
significantly (P<0.05), in agreement with previous observations of Sanz et al. (1999) and Crespo
and Esteve-Garcia (2001) who found that chickens fed tallow diet showed a significantly higher
abdominal fat pad than birds on the linseed oil dietary treatment. Sanz et al. (2000) suggested that
PUFA decrease hepatic lipogenesis and enhance fatty acid β-oxidation rate.

Table 1. Means of metabolic energy intake (MEI, kJ/kg0.75/d) and heat production (HP, kJ/kg0.75/d)
according to dietary treatment and level of feeding.
MEI HP
0.5 × ad libitum ad libitum 0.5 × ad libitum ad libitum
Item
C 525 ± 37.9 1044 ± 81.4 240 ± 67.0 437 ± 119.1
T 604 ± 64.1 1220 ± 136.3 267 ± 72.5 425 ± 116.8
LO 582 ± 86.3 1118 ± 136.8 269 ± 68.8 421 ± 105.6
The following linear regression equations were calculated relating RE and MEI, both expressed as
kJ/kg0.75/d:
Diet C: RE = -22.9 (s.e. 109.7) + 0.598 (s.e. 0.132) × MEI; P<0.01; R2 = 0.773
Diet T: RE = -54.1 (s.e. 84.4) + 0.680 (s.e. 0.087) × MEI; P<0.001; R2 = 0.910
Diet LO: RE = -46.9 (s.e. 72.7) + 0.650 (s.e. 0.081) × MEI; P<0.001; R2 = 0.915
The regression coefficient of equation from diet C tends to indicate the well-known lower efficiency
of utilisation for growth (kg) of ME from carbohydrates in comparison with fats. Moreover, although
the kg values of the regressions calculated with data from the T and LO treatments do not differ
statistically, due to their relatively high standard errors, they suggest that the efficiency of utilisation
of ME for growth may vary according to the fatty acid profile. Since the amount of fat absorbed from
treatments T and LO is similar, it may be argued that the energy from unsaturated fat might have
been preferentially used for processes other than lipogenesis, which would result in a tendency to
reduce kg. Previous research by Baillie et al. (1999) performed in rats supports the hypothesis that
dietary PUFA reduce fat deposition by increasing the expression of mitochondrial uncoupling proteins
and enhancing fatty acid β-oxidation. Nevertheless, these mechanisms remain to be conclusively
demonstrated.
References
Crespo, N. and E. Esteve-Garcia, 2001. Dietary fatty acid profile modifies abdominal fat deposition in broiler chickens.
Poult. Sci. 80, 71-78.
Baillie, R.A., R. Takada, M. Nakamura and S.D. Clarke, 1999. Coordinate induction of peroxisomal acyl-CoA oxidase
and UCP-3 by dietary fish oil: a mechanism for decreased body fat deposition. Prostaglandins Leukot Essent.
Fatty Acids 60, 351-356.
Lachica, M., J.F. Aguilera and C. Prieto, 1995. A confinement respiration chamber for short gaseous exchange
measurements. Arch. Anim. Nutr. 48, 329-336.
Sanz, M., A. Flores, P. De Ayala and C.J. Lopez-Bote, 1999. Higher lipid accumulation in broilers fed on saturated fats
than in those fed on unsaturated fats. Br. Poult. Sci. 40, 95-101.
Sanz, M., C.J. Lopez-Bote, D. Menoyo and J.M. Bautista, 2000. Abdominal fat deposition and fatty acid synthesis
are lower and β-oxidation is higher in broiler chickens fed diets containing unsaturated rather than saturated fat.
J. Nutr. 130, 3034-3037.

Energy utilization and growth responses of broilers to supplementation
of enzyme cocktails
O.A. Olukosi1, A.J. Cowieson2 and O. Adeola1
1Department of Animal Sciences, Purdue University, West Lafayette, IN 47907, USA
2Danisco Animal Nutrition, Marlborough, Wiltshire, SN8 1XN, United Kingdom
Introduction
Phytate and non-starch polysaccharides (NSP) are anti-nutrients present in cereals and to a lesser
extent in legumes. Phytate can complex with starch and reduce their digestibility, whereas NSP could
increase digesta viscosity thus reducing metabolizable energy (ME). Therefore, it could be reasoned
that the use of phytase and carbohydrases would improve energy utilization. However, the use of these
enzymes has not consistently led to improvement in ME. Whereas some have reported improvement
in ME with phytase (Driver et al., 2006) or carbohydrase (Villamide et al., 1997) others reported no
effect of the enzymes on ME (Onyango et al., 2004). Hence, it is likely that ME may not be sensitive
enough to detect improvement in energy utilization in response to enzyme supplementation and net
energy for production (NEp) may be a more sensitive measure. The objective of this study was to
examine the use of NEp and ME in assessing energy utilization.
Materials and methods

A total of 480 one-d old broiler chicks were used for the experiment. At d 1, the chicks were divided
into 4 slaughter groups. All the groups had equal average body weight (BW). Thirty chicks were
used as the initial slaughter group at d 1 whereas the remaining 450 chicks were used for the growth
trial and as final slaughter groups. The 450 broiler chicks were allocated to 5 dietary treatments with
each diet having similar initial BW. Each dietary treatment had 6 replicate cages with 5 birds per
replicate cage for each of weeks (wk) 1, 2 or 3 final slaughter age (group). The treatments were the
following: (1) positive control (PC) with adequate P and ME; (2) negative control (NC) marginal
deficient in P and ME; (3) NC plus phytase at 1000 FTU/kg; (4) NC plus an enzyme cocktail added to
supply 650, 1650 and 4000 U/kg of xylanase, amylase and protease (XAP); and (5) NC plus phytase
and XAP at levels in 3 and 4, respectively. The diets were corn-soyabean meal based with wheat
as a source of NSP and were fed throughout the experiment. The chickens used for final slaughter
groups in the comparative slaughter were asphyxiated with CO2 at the end of wk 1, 2 and 3. Excreta
was collected in the last three d of each wk to determine ME and data on growth performance was
collected every wk. All the data were subjected to the GLM procedure of SAS and means were
separated by orthogonal contrasts.

Chickens receiving the PC diet and diets supplemented with phytase alone or combined with XAP
gained more (P<0.05) than those on NC diet at all ages. Chickens receiving diets supplemented
with XAP alone gained more (P<0.05) than those on NC diet only in wk 2. There were no effects
of any of the enzymes on gain:feed in wk 1 but in wk 2, gain:feed was higher (P<0.05) in all the
treatments compared to the NC treatment. However, in wk 3 supplementation of XAP alone had
no effect on gain:feed whereas other treatments improved (P<0.05) this response compared to the
NC treatment. There were no effects of the enzyme on ME of the diets in wk 1 of the study, phytase
supplementation improved ME in wk 2 whereas supplementation of phytase alone or in combination
with XAP improved ME in wk 3. The enzymes had no effect on NEp in wk 1, however in wk 2
and 3, supplementation of phytase alone or combined with XAP improved NEp. Heat production

was higher in chickens receiving phytase alone in wk 2, but in wk 3 heat production was higher in
chickens receiving phytase alone or in combination with XAP.
The results of the experiment show that energy utilization response to dietary enzyme supplementation
for chickens as determined by ME and NEp is not the same. For example, data for wk 2 show
that in the treatment receiving combination of the enzymes there was no improvement in ME but
improvement in NEp was observed. Very few studies have examined the use of NE as a measure
for energy utilization in response to enzyme supplementation. Daskiran et al. (2004) used the
carbon-nitrogen method to assess the effect of endo-β-D-mannanase and reported improvement
in NE for gain as a result of enzyme supplementation of a corn-soyabean meal diet whereas ME
did not change. However, subsequent addition of guar gum with enzyme supplementation did not
improve net energy for gain. In the current study, phytase supplementation alone or combined with
XAP improved weight gain and NEp in wk 2 and 3. This improvement in NEp could be attributed
to improvement in BW gain due to enzyme supplementation because the gain in BW represents
a deposition of energy in the form of fat and protein in the tissues gained. Both ME and NEp are
positively correlated with BW gain, but NEp has higher correlation coefficient (r = 0.99) compared
with ME (r = 0.67). Intuitively, NEp would be expected to correlate highly with BW, however
NEp is a product of BW and gross energy content of the carcass and as the data from the current
experiment show improvement in weight gain in wk 2 due to XAP supplementation did not result
in improvement in NEp in that treatment and hence NEp will not necessarily follow the same
trend as BW. Expressing energy utilization as NEp affords the partitioning energy deposition into
that deposited as fat and protein. This also enables the determination of efficiency of diet energy
utilization. In the current study, both energy deposited as fat and as protein were higher (P<0.01) in
diets containing phytase alone or in combination with XAP. Significantly also, phytase promoted
higher deposition of protein than of fat; an indication that the enzyme promoted eficiency of dietary
energy usage because the efficacy of using dietary energy for lean deposition is greater than for fat
deposition. In conclusion, phytase alone or combined with XAP improved NEp in wk 2 and 3 and
NEp can be used as a more sensitive criterion to measure energy utilization by chickens in response
to enzyme supplementation.
References
Daskiran, M., R.G. Teeter, D. Fodge and H.Y. Hsiao, 2004. An evaluation of endo-(beta)-D-mannanase (hemicell) effects
on broiler performance and energy use in diets varying in (beta)-mannan content. Poult. Sci. 83, 662-668.
Driver, J.P., A. Atencio, H.M. Edwards and G.M. Pesti, 2006. Improvement in nitrogen-corrected apparent metabolizable
energy of peanut meal in response to phytase supplementation. Poult. Sci. 85, 96-99.
Onyango, E.M., M.R. Bedford and O. Adeola, 2004. The yeast production system in which Escherichia coli phytase is
expressed may affect growth performance, bone ash, and nutrient use in broiler chicks. Poult. Sci. 83, 421-427.
Villamide, M.J., J.M. Funete, P.P. de Ayala and A. Flores, 1997. Energy evaluation of eight barley cvs for poultry:
effect of dietary enzyme addition. Poult. Sci. 76, 834-840.

Phytase reverses negative effect of dietary phosphorus reduction on
energy metabolism in growing pigs fed restrictively
Y. Zhang, S. Moehn and R.O. Ball
Swine Research and Technology Centre, 4-10 Agriculture/Forestry Centre, University of Alberta,
Edmonton AB, T6G 2P5 Canada
Introduction
In ad libitum fed growing pigs, reducing dietary crude protein (CP) while adding limiting amino
acids improved protein retention and dietary net energy (NE) (Moehn et al., 2007). In that study,
a negative effect of dietary phosphorus (P) reduction on energy metabolism was found, which
could be corrected by supplementing phytase. Phytase and xylanase in pig diets may improve pig
performance (Kim et al., 2005). Our objective was to study the effects of dietary CP and P reduction
with phytase and/or xylanase inclusion on performance, ileal and total tract nutrient digestibility and
energy metabolism in finishing pigs fed restrictively.

Six gilts (35.0 kg initial weight, W) were surgically fitted with a T-canula at the terminal ileum, and
allowed to recover for 1 wk. Each pig received each of the 6 diets in a Latin square. Each measurement
period consisted of 7 d adaptation, 5-d nitrogen balance, 2 12-h periods of ileal digesta collection
and 24-h indirect calorimetry. Six diets were formulated for equal metabolizable energy (ME, 13.2
MJ/kg) and based on wheat and 10% wheat millrun, containing 0.5% TiO2 as indigestible marker.
Amino acids, vitamins and minerals – except P – were supplied at least at requirements (NRC, 1998).
The control diet contained 19.2% protein without free amino acids. In the other five diets, the protein
content was reduced to 16.0% by reduction of soybean meal and inclusion of L-lysine-HCl and L-
threonine. The low protein diet ‘LP+’ contained di-calcium-phosphate to elevate total P contents
to 0.54%. In diet LP-, only limestone was added to achieve P and Ca concentrations of 0.45% and
0.70%, respectively. The remaining diets were identical to LP-, except for the inclusion of 500 units
phytase (Phyzyme XP, Danisco Animal Nutrition, Marlborough, UK; diet H), 4000 units xylanase
(Porzyme 9300, Danisco Animal Nutrition, Marlborough, UK; Diet X) or 500 units phytase plus
4000 units xylanase (diet HX). Diets were offered at 1.15 MJ ME/kg0.75 W.
Heat production was calculated using indirect calorimetry. Retained energy (RE) was calculated as
ME intake minus heat production. Retained lipid energy (RLE) was calculated as RE minus retained
protein energy (RPE). Net energy (NE) was calculated as RE plus maintenance energy requirement
(664 kJ/W0.60, de Lange et al., 2006).
Mixed model analysis (SAS, 2002) was used to assess the impact of the factors ‘protein level’,
phosphorus level’, ‘phytase’ and ‘xylanase’ and W on dependent variables and ‘pig’ and ‘period’ as
random variables. The interaction phytase x xylanase was retained if P<0.1. Significance was taken
as P<0.05, P<0.1 as a tendency.
Results
Body weight (59.0 kg, SE 2.2), feed intake (1761 g/d, SE 48) and daily gain (516 g/d, SE 27) did not
differ (P>0.1) among treatments. CP level did not affect parameters of energy metabolism (P>0.1,
Table 1). P reduction reduced RE and NE, but had no effect on digestible energy (DE) and ME. P
reduction reduced (P<0.05) the hindgut disappearance (% of intake) of gross energy and organic
matter, indicating that P limited hindgut fermentation. Phytase addition increased RPE (P=0.05);

its beneficial effects on RE, DE, ME and NE did not reach significance. Xylanase increased RLE
and RE (P<0.1) but did not improve other parameters of energy metabolism significantly. RLE, RE,
NE and heat production increased with body weight (P<0.1). The current results obtained under
restricted feeding conditions confirmed the negative effects of P reduction and indicate that phytase
and xylanase addition may have beneficial effects on energy metabolism.
Table 1. Effect of dietary protein and phosphorus level, and phytase and/or xylanase addition on
performance and parameters of energy metabolism1 in pigs fed restrictively.
C2 LP+ LP- H X HX SEM Prot2 Phos2 Phyt2 Xyl W
RE, MJ/d 6.1 6.5 4.7 5.9 7.7 6.1 0.5 0.68 0.07 0.14 0.08 0.02
DE, MJ/kg 14.5 14.4 14.3 14.2 14.3 14.2 0.04 0.17 0.19 0.68 0.52 0.40
ME, MJ/kg 13.9 13.7 13.6 13.4 13.7 13.6 0.06 0.45 0.51 0.69 0.21 0.85
NE, MJ/kg 8.0 8.2 7.0 7.7 8.3 7.3 0.27 0.79 0.04 0.14 0.25 0.02
Heat, MJ/d 16.8 16.3 18.5 17.7 17.6 18.0 0.54 0.92 0.08 0.62 0.35 0.06
1RE: retained energy, DE: digestible energy, ME: metabolizable energy, NE: net energy.
2Significance of effects:Prot: protein level, Phos: phosphorus level, Phyt: phytase addition, Xyl: xylanase addition,
W: body weight. Protein level. Interaction of phytase × xylanase was not significant (P>0.1).
3C: control, LP+: low protein, high phosphorus, LP-: low protein, low phosphorus, H: LP- plus phytase, HX: LP-
plus phytase and xylanase, X: LP- plus xylanase.
Conclusions
Reduction of dietary phosphorus impaired energy metabolism, while phytase and xylanase addition
exerted positive, non-significant effects. In this experiment, phytase and xylanase addition to diets
were not additive for the specific ingredients tested and at the levels included.
References
Lange, K. de, J. van Milgen, J. Noblet, S. Dubois and S. Birkett, 2006. Previous feeding level influences plateau heat
production following a 24 h fast in growing pigs. Br. J. Nutr. 95, 1082-1087.
Kim, J.C., P.H. Simmins, B.P. Mullan and J.R. Pluske, 2005. The digestible energy value of wheat for pigs, with special
reference to the post-weaned animal [Review]. Anim. Feed Sci. Technol. 122, 257–287.
Moehn, S., J.K.A. Atakora, J. Sands and R.O. Ball., 2007. Effect of phytase-xylanase supplementation to wheat-based
diets on energy metabolism in growing–finishing pigs fed ad libitum. Livest. Prod. Sci. In press.
National Research Council (NRC), 1998. Nutrient Requirements of Swine. National Academic Press, Washington,
DC.
SAS (Statistical Analysis System), 2002. Release 9.1 edition. SAS Institute Inc., Cary, NC.

Effects of feed intake in the rate of protein deposition in heavy Iberian
pigs
R. García-Valverde, R. Barea, R. Nieto and J.F. Aguilera
Department of Animal Nutrition, Estación Experimental del Zaidín, Spanish Council for Scientific
Research (CSIC), Camino del Jueves s/n, 18100 Armilla, Granada, Spain
Introduction
In some areas of the Mediterranean basin (Southwest of Spain, Portugal, Corsica) heavy pig
production relies on local breeds (Iberian, Corsican). Intensification of the management during the
first stages of growth allows the Iberian pig to attain the slaughter weight (150-160 kg) at 14-18
mo of age, after a final fattening extensive period at the Mediterranean forest. Top quality products
derive from this rearing system. Nevertheless, there is also an increasing demand of both fresh and
dry-cured products from Iberian pigs reared intensively due to their high quality attributes at lower
prices. Previous studies of this department carried out in Iberian pigs at two stages of growth (15-
50 kg, Nieto et al., 2002; 50-100 kg, Barea et al., 2007) fed diets that differed widely in protein:
energy ratio have revealed a low capacity of this pig breed for lean tissue deposition. To extend
these observations to the final phase of production, we determined the effects of the level of feed
intake (FL) on growth performance, partition of energy retention and body composition in Iberian
pigs growing from 100 to 150 kg live weight fed a diet with a protein:energy ratio assumed to be
well-balanced.

Eighteen purebred castrated male Iberian pigs of the Silvela strain of 89 ± 3.0 kg BW were used.
They were placed in individual 5-m2 pens, in a thermo-neutral environment. The experimental diet,
supplying a digestible ideal protein:ME ratio of 4.82 g/MJ, was then introduced and 4.0 kg feed were
given daily in two equal meals, until they reached 100 ± 0.5 kg when six pigs randomly selected were
slaughtered. The twelve additional pigs were randomly allocated to one of the two feeding levels
tested: 0.70 or 0.95 × ad libitum, with six pigs per treatment (ad libitum intake defined as in Barea et
al. (2007). Water was freely available. During the trial, the pigs were weighed weekly before feeding
and the daily food allowance for each pig for the next wk was adjusted accordingly. Two classical
digestibility trials were conducted at approximately 110 and 140 kg BW. At 150 ± 0.5 kg BW the
pigs were electrically stunned and bled. After emptying the gut, collected blood, carcass and non-
carcass parts were weighed separately. The right half of the carcass and the rest of the components
were ground separately, homogenised and sub-samples were taken for freeze-drying and subsequent
analysis. The experimental protocol was approved by the Bioethical Committee of the CSIC.
The effects of feeding level on pig performance and comparative slaughter parameters were assessed
by a one-way analysis of variance with six pigs per treatment group, by means of SAS (SAS Inst.,
Inc., Cary, NC, 1990).

Average ME intakes were 1.29 and 1.70 MJ/kg BW0.75 per d, respectively, for 0.70 and 0.95 × ad
libitum and provided 3.3 and 4.3 times MEm. Average daily gain increased with the increase in FL
(691 and 918 g/d, respectively). Nevertheless, the gain:ME intake ratio was not significantly affected.
An energy cost of 66.4 kJ ME/g gain was calculated. The whole-body rate of protein deposition
(PD) was not significantly altered by FL. On average, it attained 80 g/d. On the contrary, the pigs
fed at the lowest level exhibited lower fat deposition (389 vs. 515 g/d, P<0.01) and total energy

retention (17.26 vs. 22.40 MJ/d, P<0.01) than the pigs from the group subjected to a slight feed
restriction. The present results and previous data (Nieto et al., 2002; Barea et al., 2007) suggest
a relatively constant rate for maximum PD (71-80 g/d) for the growing Iberian pig in the 15-150
kg BW range. Our results were in agreement with those reported by Whittemore et al. (1988) and
Möhn and de Lange (1998), who observed in lean pigs no effect of the stage of growth on PDmax.
In our study, a significant increase in the daily rate of lipid deposition was observed on increasing
energy intake (P<0.01). This increase in fat deposition attained a mean value of 8.84 g/MJ ME, far
below the average value of 12.20 g/MJ ME that we have observed in the Iberian pig growing from
50-100 kg BW (Barea et al., 2007). On average, 0.903 of the energy retained was deposited as fat,
of which a considerable portion may have been synthesised from dietary amino acids, with a low
energetic efficiency. The gross efficiency of utilisation of the ME for energy gain (ER:ME intake)
remained at 0.364, irrespective of the feeding level imposed. Nieto et al. (2002) and Barea et al.
(2007) found MEm requirements of 422 and 396 kJ/kg BW0.75 per d, for the 15-50 and 50-100 kg
BW pigs. Assuming the lowest MEm value for the heavy (more fattened) pig, then an average net
efficiency of energy gain (kg) of 0.481 was obtained. kg values of 0.582 and 0.545 were estimated
for the growing and fattening stages (Nieto et al., 2002; Barea et al., 2007), suggesting an increased
energetic cost of growth as the pig ages. Deposition of fat, expressed as a proportion of retained
energy, increased from 0.838 (Nieto et al., 2002) to 0.926 (Barea et al., 2007) or 0.903, on average,
for the 15-50, 50-100 and 100-150 kg stage of growth, respectively. Compared with observations
in the growing Iberian pig, kg shows a trend to decline in the heavier pig, in spite of the increased
fat deposition found in both the fattening and finishing pig, probably as a result, unless in part, of
an increased contribution of hind gut fermentation to ME supply to tissues, as reported by Noblet
et al. (1994). Further studies are needed to throw light on this subject.
The dietary treatments imposed did not provoke differences in whole-body or carcass composition,
except for the significant increase in percentage of lean tissues in the carcass (P<0.05) and the
strong tendency in whole-body protein content to increase on raising FL. Therefore, a degree of
feed restriction may result in a discernable improvement of carcass quality and deserves attention
in the management of the finishing Iberian pig reared indoors.
References
Barea, R., R. Nieto and J.F. Aguilera, 2007. Effects of the dietary protein content and the feeding level on protein and
energy metabolism in Iberian pigs growing from 50 to 100 kg body weight. Animal (in press).
Nieto, R., A. Miranda, M.A. García and J.F. Aguilera, 2002. The effect of dietary protein content and feeding level
on the rate of protein deposition and energy utilization in growing Iberian pigs from 15 to 50 kg body weight.
Br. J. Nutr. 88, 39-49.
Noblet, J., H. Fortune, X.S. Shi and S. Dubois, 1994. Effect of body weight on net energy value of feeds for growing
pigs. J. Anim. Sci. 72, 648-657.
Möhn, S. and C.F.M. de Lange, 1998. The effect of body weight on the upper limit to protein deposition in a defined
population of growing gilts. J. Anim. Sci. 76, 124-133.
Whittemore C.T., J.B. Tullis and G.C. Emmans, 1988. Protein growth in pigs. Anim. Prod. 46, 437-445.

Feeding frequency alters protein and energy metabolism of sows fed 1 ×
and 2 × the energy requirement for maintenance
R.S. Samuel1, S. Moehn1, L.J. Wykes2, P.B. Pencharz3 and R.O. Ball1
1Swine Research and Technology Centre, 4-10 Agriculture/Forestry Centre, University of Alberta,
Edmonton, AB, T6G 2P5 Canada
2School of Dietetics and Human Nutrition, McGill University, Montreal, PQ, H9X 3V9 Canada
3Research Institute, Hospital for Sick Children, Toronto, ON, M5G 1X8 Canada
Introduction
Energy and protein intake recommendations may need reassessment for modern very high producing
sows. Protein and energy metabolism can be studied simultaneously by combining indirect calorimetry
with amino acid oxidation techniques using stable isotopes. To develop this methodology for a long-
term research program, energy and protein metabolism of sows was studied at two feeding levels
using the planned feeding regiment for future experiments.

Animals, diets, housing, feeding, and indigestible marker
Non-pregnant sows (n=5, 174 kg ± 11 kg) were selected from the University of Alberta’s Swine
Research and Technology Centre. Diets were formulated for 13 MJ DE/kg and 0.65% lysine content
and composed of barley, wheat, and canola and soybean meals. Sows were housed individually and
fed one-half of their daily feed allowance twice daily, except on study days, when they received half
their daily feed allowance in 16 ½-hourly meals, followed by the remaining half in one meal. Thus,
physiological ‘states’ were identified for sows: 8-h ‘nibbling’ when eating the ½-hourly meals, 2-h
at ‘full feeding’ when eating the large meal, 8-h ‘post-prandial’ after the last meal and the last 6-h of
the study ‘fasted’. The sows rapidly consumed all meals. Daily feed allowances were calculated to
deliver 458 kJ ME/BW0.75 (1 × MEm) and 916 kJ ME/BW0.75 (2 × MEm) (ARC, 1981). Individual
nipple drinkers provided free access to water. An indigestible marker was mixed into individual
batches of complete diet at 10 g/kg of diet and analyzed as acid insoluble ash (AIA) for determination
of energy and nutrient digestibility.
Indirect calorimetry, isotope dilution, and sample collection
Sows were individually housed in respiration chambers for 24-h measurements of energy (open-
circuit indirect calorimetry) and protein metabolism (infusion of L-[1-13C]leucine). The 24-h gas
exchange was recorded for O2, CO2, and CH4 in 1 min intervals. Expired CO2 and blood plasma
were collected in 30 min intervals for determination of 13C enrichment.
Statistics
The effects of ‘state’ and ‘feeding level’ were assessed using mixed model analysis (SAS, 2002)
using ‘sow’ as a random variable. The ratio of short-term to 24-h values for oxidation and heat
production (HP) was compared between feeding levels using the Student t-test. Significance was
taken at P<0.05, a tendency at P<0.1.

Results
Sows (175.7 ± 3.3 kg BW) fed 1 × MEm lost weight at the rate of –198 ± 96 g/d (over 7 d). Sows
(174.1 ± 3.2 kg BW) fed 2 × MEm gained weight up to 186.6 ± 3.3 kg at the rate of 1292 ± 166 g/d
(over 7 d). Daily feed intake was 1.84±0.03 kg and 3.69 ± 0.05 kg for sows fed 1 × and 2 × MEm.
The digestible energy (DE) content of the diet was determined to be 80.9 ± 0.6% of gross energy
(GE) or 13.0 MJ DE/kg. Digestibility of nitrogen (81.2 ± 0.5%), carbon (81.8 ± 0.3%), fat (86.7
± 0.7%), and organic matter (84.3 ± 0.3%) was not different between feeding levels. NDF (53.9 ±
1.4% vs. 42.3 ± 2.4%) and ADF (61.4% vs. 40.7%, SEM=6.0) digestibility was greater (P<0.01)
for 2 × MEm than 1 × MEm fed sows. Calculated (Brouwer, 1965) average daily heat production
(HP) of sows fed 2 × MEm was greater (P<0.001) than for sows fed 1 × MEm (631 ± 10 kJ/BW0.75
vs. 523 ± 11 kJ/BW0.75). Actual energy intakes were 470 ± 6 kJ/BW0.75 and 934 ± 6 kJ/BW0.75.
Calculated (Lodge et al., 1979) maintenance energy requirement (MEm) was 515 ± 8 kJ/BW0.75 for
sows fed 1 × MEm and 495 ± 13 kJ/BW0.75 for sows fed 2 × MEm. Sows had greater (P<0.05) HP
during nibbling than during fasting or following a large meal.
Average 24-h tracer appearance as labeled CO2 in breath (F13CO2) tended to be lower (31.8 µmol/kg
vs. 44.5 µmol/kg) for 1 × MEm than 2 × MEm fed sows. F13CO2 was lower when sows were nibbling
(P<0.05) than during fasting or following a large meal. Leucine oxidation (% of dose) was greater
(P=0.001, SEM=2.8) for 2 × MEm (17.6%) than for 1 × MEm (11.4%). Leucine oxidation tended to
be reduced during ‘nibbling’ compared to ‘post-prandial’.
Discussion
Heat production (523 ± 11 kJ/BW0.75) of sows fed 470 ± 6 kJ ME/BW0.75 per d was greater than
intake, leading to an average weight loss of –198 ± 96 g/d. A new MEm value of 505 ± 8 kJ ME/BW0.75
was calculated (Lodge et al., 1979). Oxidation was not twice as great in 2 × MEm vs. 1 × MEm fed
sows. Only a portion of the additional protein intake was utilized for lean tissue deposition. In fact,
lean tissue deposition was limited by dietary lysine intake (24 g/d). Significant growth (1292 ± 166
g/d) occurred in sows fed 2 × MEm.
Energy and protein metabolism were impacted oppositely by frequent feeding; when nibbling, sows
had greater HP but lower leucine oxidation than during fasting or following a large meal. Greater
HP was partially due to increased physical activity during nibbling. However, feeding level had no
significant effect upon the ratio of ‘nibbling’ to 24-h values for HP or oxidation. This indicates that
measurements during the ‘nibbling’ phase accurately predict the effects of protein and energy intake
on 24-h values. Future studies will be conducted during the ‘nibbling’ state.
References
Agricultural Research Council, 1981. The nutrient requirements of pigs: technical review, revised edition. Commonwealth
Agricultural Bureaux, Slough, England.
Brouwer, E., 1965. Report of the sub-committee of the European Association for Animal Production on constants and
factors. In: K. L. Blaxter (editor), Energy metabolism. Academic Press, New York, NY, 441–443.
Lodge, G.A., D.W. Friend and M.S. Wolynetz, 1979. Effect of pregnancy on body composition and energy balance of
the gilt. Can. J. Anim. Sci. 59, 51-61.
SAS (Statistical Analysis System), 2002. Release 9.1 Edition. SAS Institute, Inc., Cary, NC.

Influences of feeding intensity on protein and energy deposition in calves
H. Janssen, U. Meyer and G. Flachowsky
Federal Agricultural Research Centre (FAL), Institute of Animal Nutrition, Bundesallee 50, D38116
Braunschweig, Germany
Introduction
There exists inconsistent literature data on the effect of an intensive rearing practice on body
composition of calves and heifers as well as the later potential for milk performance of cows (e.g.
Capuco et al., 1995; Macdonald et al., 2005; Meyer et al., 2004; Serjsen et al., 2000). Therefore,
the objective of the study was to consider the effect of different rearing intensities of female calves
on feed consumption, body development and on the body composition of growing cattle.

Eighty-four female calves (initial body weight: 44.8 kg/animal) of the German Holstein breed were
divided into two groups (L, H) and fed with low (~ 25 kg milk replacer over 43 d) and high (~ 85
kg milk replacer over 92 d) intensity (Period 1). From the 15th to the 25th wk (Period 2) the animals
of both groups were fed on the same level (2 kg calf-starter as concentrate per d, grass silage ad
libitum). Feed intake and live weight development were registered individually during the whole
course of all experimental periods.
At the start of the experiment, four calves were slaughtered for whole carcass analysis and additionally
four animals per group after each period (altogether 20 animals).
Results
Some results of the experiment are shown in Table 1. The high drinking intensity in Period 1 resulted
in a higher average daily dry matter intake (1.74 vs. 1.55 kg) and a significantly higher daily weight
gain (838 vs. 766 g). The ME-intake amounted to 24.1 (group H) and 19.5 MJ/d and was likewise
significantly different.
During Period 2, the groups H and L indicated almost equal daily dry matter intake (3.79 vs. 3.73
kg) and ME-intake (41.7 vs. 41.0 MJ/d). The daily live weight gain averaged 994 and 971 g and
the difference in daily live weight gain between treatments declined by more than half compared
to Period 1.
Considering the complete experimental time (wk 0 to 25), the group with high feeding intensity (H)
showed significant higher average daily dry matter intake (2.66 vs. 2.52 kg), ME-intake (32.0 vs.
29.0 MJ/d) and daily weight gain (909 vs. 857 g).
The empty body composition is influenced by the energy content respectively the energy intake of
animals (Table 1).
The deposition of protein, fat and energy during Period 1 was significantly influenced by drinking
intensity (87 and 114 g protein, 39 and 67 g fat; 3.5 and 5.2 MJ/d respectively for the rearing intensities
L and H). The deposition during Periods 1 and 2 averaged 112 and 122 g protein as well as 70 and
75 g fat, the mean of daily energy retention amounted to 5.2 (L) and 5.7 MJ (H). These results did
not differ between treatments.

Table 1. Influence of low (L) and high (H) rearing intensity on dry matter intake (DMI), live weight
(LW), live weight gain (LWG) and empty body composition (EBC) of female calves and heifers
(n=4).
Period Rearing DMI, LW, EBC, /kg

intensity kg/d g/d
(n) Dry matter, Protein, Fat, Energy,
g g g MJ
Start - - - 284 190 47 6.1
1 L (40) 1.55 766a 292a 184 63a 6.6a

(wk 0 to 14) H (40) 1.74 838b 307b 183 82b 7.3b
2 L (36) 3.73 971 320 180 93 7.7

(wk 15 to 25) H (36) 3.79 994 318 179 92 7.6
a,b Different superscripts in one period and item show significant differences (P<0.05).
The utilisation of metabolisable energy for energy gain (kpf) over the whole experimental period
averaged 0.45 (L) and 0.37 (H) and showed a statistically significant difference.
Conclusion
Feeding practice of calves and heifers significantly influences growth and body composition of
animals. Presently the potential for milk production of variously fed animals will be tested.
References
Capuco, A.V., J.J. Smith, D.R. Waldo and C.E. Rexroad, 1995. Influence of prepubertal dietary regimen on mammary
growth of Holstein heifers. J. Dairy Sci. 78, 2709-2725.
Macdonald, K.A., J.W. Penno, A.M. Bryant and J.R. Roche, 2005. Effect of feeding level pre- and post-puberty and
body weight at first calving on growth, milk production, and fertility in grazing dairy cows. J. Dairy Sci. 88,
3363-3375.
Meyer, M.J., A.V. Capuco and M.E. Van Amburgh, 2004. Effects of energy intake and time to puberty on mammary
growth of prepubertal Holstein heifers. J. Dairy Sci. 87 (Suppl. 1), 275.
Sejrsen, K., S. Purup, M. Vestergaard and J. Foldager, 2000. High body weight gain and reduced bovine mammary
growth: physiological basis and implications for milk yield potential. Domest. Anim. Endocrinol. 19, 93-104.

Energy metabolism and energy requirement for maintenance of
Brahman steers in tropical conditions
A. Chaokaur1, T. Nishida2, I. Phaowphaisal3, P. Pholsen3, R. Chaithiang3 and K. Sommart1
1Department of Animal Science, Khon Kaen University, 40002, Thailand
2Japan International Research Center for Agricultural Sciences, Japan
3Khon Kaen Animal Nutrition Research and Development Center, Department of Livestock
Development, 40260, Khon Kaen, Thailand
Introduction
Feed resources for ruminants in the humid tropical countries of Southeast Asia are usually scarce and
of poor quality. There are also apparent differences between temperate Bos taurus and the tropical
Bos indicus breeds of cattle. The National Research Council (1996) suggested that growing Bos
indicus cattle require about 10% less metabolisable energy requirements for maintenance (MEm)
than Bos taurus cattle. However, there is very limited research on the metabolisable energy (ME)
requirement of Brahman cattle. Therefore, this energy balance study was emphasised on energy
metabolism and requirement in Brahman cattle steer using an open-circuit indirect gas exchange
respiration calorimetry method in tropical conditions.

Four steer, Brahman cattle, average body weight 385±37.9 kg, were housed in individual stalls. They
were halter-trained and adapted to the respiration calorimetry with a head hood. Dietary treatments
were applied in a 4×4 Latin square design and consisted of total mixed diets containing 100% of
Cavalcade (Centrosema pubescens) hay (T1), 23.41% of Pangola grass (Digitaria eriantha) hay with
76.59% of Cavalcade hay (T2), 63.59% of Pangola grass hay with 36.41% of coconut meal (T3)
and 47.65% of Pangola grass hay with 52.35% of palm kernel cake (T4). The daily amount of dry
matter feed offered was fixed at a rate of 1.5% of body weight. The experimental period consisted
of4 periods of 20-d feeding periods (14-d preliminary and 6-d sampling). They were standing or
lying in an individual respiration head hood for heat production measurements. After the last feeding
period, the animals were fasted (post-absorption, very low methane production) for 5 d and samples
were collected on the last 2 d. The weight of feed offered and refused, feces, and urine were recorded
and sampled daily. The samples were analysed by using standard methods. Heat production was
calculated according to Brouwer (1965). Data were subjected to analysis of variance.

The results indicate that energy excretion in feces and urine and methane production in T1 and T2
were higher (P<0.01) than in T3 and T4. Heat production was not different among the treatments (see
also Table 1). MEm was determined from the regression of energy retention (ER) on metabolisable
energy intake (MEI); a significant linear relationship was obtained ER = (0.578 (SE=0.022) ×
MEI) – 265 (SE=10.4), [R2=0.976; P<0.001; RSD=4.903; n=19] (see also in Figure 1). Thus, the
equation estimate and a value for fasting heat production (FHP) of 265 kJ/MBW/d, gave a value
for efficiency of utilisation of ME for maintenance (km; km=FHP/MEm) of 0.578 and the estimated
MEm requirement of 458 kJ/metabolic body weight (MBW, kg BW0.75)/d was lower than the values
reported by Ferrell and Jenkins (1998) for growing Brahman crossbred steer (501 kJ/MBW/d).

Table 1. Energy partition of Brahman cattle given dietary treatments.
Treatment T1 T2 T3 T4 Fasted P-value SE
Number animal, head n=4 n=4 n=3 n=4 n=4 - -

Average body weight, kg 386 390 386 379 385 - -
Energy partition1, kJ/MBW/d

GE intake 1152 1152 1130 1152 - 0.19 5.70
DE intake 603b 615bc 682a 709a - 0.01 14.46
ME intake 471b 484b 571a 601a - <0.01 12.08
Feces excretion 548a 537a 448b 444b 143 <0.01 12.92
Urine excretion 34a 29ab 22b 23b 13 0.05 2.30
Methane production 99a 103a 89b 85b 6 0.01 2.41
Heat production 477 481 499 504 259 0.35 10.69
Energy retention, kJ/MBW/d -7b 2b 71a 97a -259 <0.01 13.33
1GE, Gross energy; DE, Digestible energy; ME, Metabolizable energy.

a, b, c
Least squares means with different superscripts among treatments significantly differ (P<0.05).
SE, standard errors; MBW, metabolic body weight (kg BW0.75).
350
Energy retention, kJ/MBW/day
250
150
50
-50 0 100 200 300 400 500 600 700

-150
-250
-350 Metabolizable energy intake, kJ/MBW/day
Figure 1. TheFigure
relationship between metabolisable
1. The relationship energy intake
between metabolizable andintake
energy energyand
retention.
energy retention.
References
Brouwer, E., 1965. Report of subcommittee on constants and factors in energy metabolism. In: Blaxter, K.L. (editor),
Proceedings of the 3rd Symposium on Energy Metabolism. Academic Press, New York, European Association of
Animal Production Publication no. 11, 441-443.
Ferrell, C.L. and T.G. Jenkins, 1998. Body composition and energy utilization by steers of diverse genotypes fed a
high concentrate diet during the finishing period. II. Angus, Boran, Brahma, Hereford and Tuli sires. J. Anim.
Sci. 76, 647–657.
National Research Council, 1996. Nutrient Requirements of beef cattle. 7th edition. National Academy Press,
Washington, DC.
The authors wish to express their thanks to Khon Kaen University, Japan International Research
Center for Agricultural Sciences and Department of Livestock Development for this research project
supported.

Effects of dairy cow genotype and plane of nutrition on energy
partitioning between milk and body tissue
T. Yan, R.E. Agnew and C.S. Mayne
Agri-Food and Biosciences Institute, Hillsborough, Co. Down BT26 6DR, United Kingdom
Introduction
Breeding programmes for the Holstein-Friesian dairy cow have historically focused on improved
milk production with little emphasis on functional traits such as fertility or disease resistance. In
contrast, Norwegian dairy cattle have been bred using a multi-trait selection procedure. Differences
in selection procedures for the two breeds may have major effects on the efficiency of food use and
partitioning of nutrients that may offset the potential advantages of improvements in functional traits.
The objectives of the present study were to examine possible differences in energy partitioning and
the efficiency of energy utilisation between Holstein-Friesian and Norwegian dairy cows.

Sixteen first lactation dairy cows (8 Holstein Friesian and 8 Norwegian) were used in a 2 (breed)
× 2 (plane of nutrition) factorial design study. Each breed was offered two levels of concentrates
(proportion of total diet, g/kg DM) for d 1-100 (600 vs. 300), 101-200 (500 vs. 200) and 201-308
(400 vs. 100) of lactation. The concentrates consisted of 230, 225, 300 and 245 g/kg fresh weight of
barley, wheat, sugar beet pulp and soyabean meal respectively. The remainder of the diet was primary
growth grass silage. Energy metabolism studies were undertaken on each animal at d 80, 160 and 240
of lactation. Each animal was placed in open-circuit indirect respiration calorimeters for 72 h with
measurements of gaseous exchange during the final 48 h. Animals were then transferred to digestibility
units for a 6-d total collection of faeces and urine. Data were analysed by ANOVA to examine the
effects of cow genotype and plane of nutrition using the experiment period as a block.
Results
The mean energy utilisation data averaged over 3 periods are presented in Table 1. There was no
significant interaction between cow genotype and plane of nutrition on any variable, except for milk
energy output (El), for which the interaction was significant (P<0.01). Neither genotype nor plane of
nutrition significantly affected Eg/MEI (energy balance/ME intake) or the efficiency of utilisation of
ME for lactation (kl) when ME requirement for maintenance was calculated from Thomas (2004).
However, Holstein-Friesian cows partitioned more consumed ME into milk than Norwegian cows
(P<0.001), irrespective of plane of nutrition. The El/MEI was higher with Holstein-Friesian than
Norwegian cows (P<0.01) using mean data of both planes of nutrition. When examining individual
treatment effects, this improvement in energy partitioning into milk with Holstein-Friesian cattle was
mainly derived from the high plane of nutrition. With the low plane of nutrition, both genotypes had
a similar ratio of El/MEI. When examining cow genotype effects in individual periods (Figure 1),
Holstein-Friesian cows partitioned more ME into milk in early and mid lactation. In late lactation
there was no difference in El/MEI between genotypes. However, Holstein-Friesian cows partitioned
more ME into body tissue than Norwegian cows in late lactation.

Table 1. Effects of cow genotype and plane of nutrition on energy utilisation.
Holstein Norwegian s.e. Significance1
Plane of nutrition High Low High Low Genotype Plane G×P
Energy utilisation
MEI, MJ/d 207c 156a 193b 145a 4.4 ** ***
El, MJ/d 76d 53b 63c 46a 1.2 *** *** **
Eg, MJ/d 7 -2 5 0 3.3 *
El/MEI 0.37b 0.33a 0.33a 0.32a 0.008 ** **
Eg /MEI 0.03 -0.01 0.02 -0.01 0.018
kl 0.59 0.55 0.54 0.57 0.021
1* = P<0.05; ** = P<0.01; *** = P<0.001.
0.45
0.35
Energy partitioning
El /ME intake Holstein

Norwegian
0.25
0.15
Eg /ME intake
0.05
-0.05
0 1
Early 2
Mid 3
Late
Lactation stage
Figure 1. Effects of cow genotype and plane of nutrition on energy partitioning.
Conclusions
Figure
Neither 1. Effects nor
cow genotype of cow genotype
plane and plane
of nutrition of nutrition
affected on energy
kl. However, partitioning.cows partitioned
Holstein-Friesian
more consumed ME into milk than Norwegian cows. This effect was mainly observed in early
lactation and with animals offered a high plane of nutrition.
References
Thomas, C. (editor), 2004. Feed into milk: A new applied feeding system for dairy cows. Nottingham University
Press, Nottingham, UK.

Comparison of energy consumption and respiratory quotient in chicken
embryos with different growth rate
M. Sato1, K. Noda2, K. Kino2, A. Nakamura2 and M. Furuse1
1Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan
2Aichi-ken Agricultural Research Center, Nagakute-cho, Aichi 480-1193, Japan
Introduction
Broiler chickens have been selected for rapid growth and high meat yield. As a result, at 6 wk
of age, the body weight of broilers is five times that of layer chickens, which are selected for
egg production (Zhao et al., 2004). Mechanisms explaining the difference in growth rate are not
completely elucidated. Food intake is considerably higher in broilers than in layers (Hocking et al.,
1997), and the difference in growth of the two types of chickens is largely due to differences in food
intake. However, embryonic growth of broilers is also faster than that of layers (Ohta et al., 2004).
By focussing on embryonic growth, it is possible to elucidate the genetic factors causing growth
differences because the embryo is enclosed by eggshell and they are largely uninfluenced by external
factors. In the present study, we investigated the difference in energy consumption, which partially
relates to protein synthesis, between broilers and layers during embryonic development. In addition,
the Nagoya breed, known as Japanese native chickens improved as a dual-purpose chicken, i.e., egg
and meat production, and the meat-type has been improved to increase body size and rapid growth.
As a result, body weight of the meat-type female has increased about 1.3 times compared to that of
egg-type females at 150 d of age (Kino et al., 1991 and 1999). The physiological differences within
the Nagoya breed of these two types have not been investigated during embryonic stages. Therefore,
we also investigated the difference in energy consumption between meat- and egg-types in Nagoya
breeds during embryonic development.

To investigate energy consumption and respiratory quotient (RQ), oxygen (VO2) consumption and
carbon dioxide (VCO2) production were measured using an open-circuit calorimeter system (MK-
5000RQ, Muromachi Kikai Co. Ltd., Tokyo, Japan). An acrylic chamber (150 mm × 150 mm × 150
mm) was put into an incubator kept at 37.6 °C and a relative humidity of 58-68%. Atomospheric
air in the incubator was drawn at a rate of 300 mL/min and then passed through VO2 and VCO2
detectors (MM202R, Muromachi Kikai Co., Ltd.). The analyzer was calibrated using primary gas
standards of high purity at every h. VO2 consumption and VCO2 production were measured from 30
min to 1 h after introduction to the chamber because VO2 and VCO2 concentrations became stable
from 30 min after introduction. VO2 consumption and VCO2 production were measured every 3
min for 30 min and mean values of ten measurements were used to calculate energy consumption
and RQ. Energy consumption was calculated by the formula of Romijn and Lockhorst (1961). The
formula is as follows: Energy consumption (J) = (the volume of VO2 consumption (mL) × 3.871
+ the volume of VCO2 production (mL) × 1.194 × 1000) × 4.184. The eggs used were opened and
embryo weights were also measured after the experiment. The values obtained were divided by
embryo weights. Embryos used for measurement were 12, 14, 16 and 18 d after incubation (E12,
E14, E16 and E18, respectively).

Energy consumption was significantly lower in broilers than in layers. The RQ values were around
0.7, implying that lipids are mainly used as an energy source. In addition, the RQ values were similar
in the two types. In Nagoya breeds, energy consumption was significantly lower in meat-type than

in egg-type chickens. The RQ values were around 0.7 and were similar to the values obtained in
broilers and layers. In the present study, we found that energy consumption in faster growing chicken
embryos was lower than that in slower growing chicken embryos.
(A) (B)
Energy consumption (J/min/g embryo weight)
Type P<0.05 Type P<0.05
0.8 Broiler 0.8 Stage P<0.0001 Meat-type
Stage P<0.0001
Type x Stage P>0.05 Layer Type x Stage P>0.05 Egg-type
0.7 0.7
0.6 0.6
0.5 0.5
0.4 0.4
0.3 0.3
0.2 0.2
0.1 0.1
0 0
E12 E14 E16 E18 E12 E14 E16 E18
Embryonic development (day)
Figure 1. Energy consumption (J/min/g embryo weight) in broilers and layers (A), and meat- and
egg-types in Nagoya breeds (B) during embryonic development. Abbreviations were as follows;
E12, embryonic d 12; E14, embryonic d 14; E16, embryonic d 16; E18, embryonic d 18. Data are
expressed as means ± SEM.
References
Hocking, P.M., B.O. Hughes and S. Keer-Keer, 1997. Comparison of food intake, rate of consumption, pecking activity
and behavior in layer and broiler breeder males. Br. Poult. Sci. 38, 237-240.
Kino, K., K. Noda, H. Miyakawa, H. Bamba and H. Murayama, 1999. Production of a laying-type strain of Nagoya
breed. Res. Bull. Aichi Agric. Res. Ctr. 31, 281-288.
Kino, K., M. Yamada, T. Ohyabu, K. Otsuka, K. Noda, H. Murayama, K. Hirose and M. Ohta, 1991. Improvement in
efficiency of meat production in Nagoya breed. Res. Bull. Aichi Agric. Res. Ctr. 23, 443-452.
Ohta, Y., T. Yoshida and N. Tsushima, 2004. Comparison between broilers and layers for growth and protein use by
embryos. Poult. Sci. 83, 783-787.
Romijn, C and W. Lokhorst, 1961. Some aspects of energy metabolism in birds. Proceedings of 2nd Symposium on
Energy Metabolism. European Association for Animal Production 10, 49-58.
Zhao, R., E. Muehlbauer, E. Decuypere and R. Grossmann, 2004. Effect of genotype-nutrition interaction on growth
and somatotropic gene expression in the chicken. Gen. Comp. Endocrinol. 136, 2-11.

Incubation circumstances affect energy metabolism in avian embryos
H. van den Brand1, A. Lourens2, R. Meijerhof3, M.J.W. Heetkamp1 and B. Kemp1
1Adaptation Physiology Group, Wageningen Institute of Animal Sciences, Wageningen University,
P.O. Box 338, 6700 AH, Wageningen, The Netherlands
2Applied Research of the Animal Sciences Group of Wageningen University and Research Centre,
P.O. Box 65, 8200 AB, Lelystad, The Netherlands
3Hybro B.V., P.O. Box 30, 5830 AA, Boxmeer, The Netherlands
Introduction
During the incubation process, energy in the egg (both albumen and yolk) is metabolised and
deposited into the embryo or lost as heat (Romanoff, 1972). A part of the yolk remains unused in the
hatchling (residual yolk). It has been demonstrated that chick quality and post hatch performance is
improved when yolk free body weight at hatch is increased (Lourens et al., 2005). The amount of
residual yolk, and consequently the quality of the hatchling, can probably be affected by incubation
circumstances, such as temperature and oxygen supply. The aim of this study was to investigate
which factors affect incubation quality from an energy metabolism point of view. The effects of
incubation temperature and oxygen supply on energy metabolism were investigated in large and
small eggs in two experiments.

Eggs from Hybro G+ grandparents were used. Eggs were incubated in small climate respiration
chambers, at which chamber temperature was adapted, based on required egg shell temperature (EST).
EST was determined with Pt100 sensors attached to the shell of five individual eggs and the median
EST was used to regulate chamber temperature (Lourens et al., 2006). Oxygen consumption and
carbon dioxide production were determined every 9 min and HP was calculated using the formula
of Romijn and Lokhorst (1961). Additionally, albumen and yolk from fresh eggs and yolk free body
(YFB) and residual yolk (RY) of hatchlings were determined to calculate energy partitioning.
In experiment 1, small (54.0-56.0 g) or large (70.0-72.0 g) eggs were incubated at 37.8 °C EST
throughout incubation. Thirty eggs per chamber were incubated and repeated 4 times. In experiment
2, average sized eggs (60-65 g) were incubated at 37.8 or 38.9 °C EST and at 17, 21, or 25% oxygen
from d 8 of incubation onwards, using a 2 × 3 factorial design. Each combination of factors was
repeated twice.

In experiment 1, HP was higher in large eggs, only from 15 onwards. At d 19, the difference in HP
was 15 mW/egg, meaning that more energy was lost in the large eggs. On the contrary, large eggs
deposited more energy in the YFB and more energy remained in the RY. In total, the energy efficiency
[YFB (kJ)/(albumen + yolk – RY (kJ))] was similar for small and large eggs (Table 1), meaning that
energy partitioning from the egg to the hatchling is not affected by egg size.
Experiment 2 demonstrated that a higher EST increased HP from d 8 till 15. From d 14 till 17 a
higher oxygen supply also increased HP and on d 17 and 18 an interaction between EST and oxygen
supply was found, with the higher values for high EST and high O2 and the lowest for high EST
and low O2 (Figure 1).

Table 1. Total energy partitioning from small (54.0 – 56.0 g) and large eggs (70.0 – 72.0 g) to
hatchlings.
Hatching egg, kJ Hatchling, kJ Lost, kJ Efficiency,

%
Albumen Yolk YFB RY
Small eggs 68.8a 258.7b 155.4b 26.5b 145.5a 51.7

Large eggs 89.5a 320.2a 178.0a 50.6a 181.1b 49.6
180
160
37.8C x 17%
140 37.8C x 21%
37.8C x 25%
120 38.9C x 17%
38.9C x 21%
HP (mW/egg)
100
38.9C x 25%
80
60
40
20
0
6 8 10 12 14 16 18 20
day of incubation
Figure 1. Heat production of embryos during incubation affected by temperature and oxygen
supply.
We concluded that energy metabolism (both HP and energy allocation) is largely influenced by
incubation circumstances and consequently affects hatchling quality and growth out performance.
References
Lourens, A., H. van den Brand, R. Meijerhof and B. Kemp, 2005. Effect of eggshell temperature during incubation on
embryo development, hatchability, and posthatch development. Poult. Sci. 84, 914-920.
Lourens, A., R. Molenaar, H. van den Brand, M.J.W. Heetkamp, R. Meijerhof and B. Kemp, 2006. Effect of egg size
on heat production and the transition of energy from egg to hatchling. Poult. Sci. 85, 770-776.
Romijn, C. and M.W. Lokhorst, 1961. Some aspects of energy metabolism in birds. In: Brouwer, E. and A.J.H. van Es
(editors), Proc. Second Symposium on Energy Metabolism. Wageningen Academic Publisher, Wageningen, The
Netherlands, EAAP Series 10, 49-59.
Romanoff, A.L., 1972. Assimiliation of avian yolk and albumen under normal and extreme incubation temperatures.
Pathogenesis of the avian embryo. Wiley Interscience, New York.

Heat production of two lineages of broilers fed diets of different physical
form
L.J.C. Lara, W.E. Campos, N.C. Baião, N.M. Rodríguez, M.V. Triginelli and R.S. Leite
Veterinary School of Universidade Federal de Minas Gerais, MG, Brazil
Introduction
The use of pelleted rations for broilers is usual practice in the Brazilian poultry industry. Pelleting
seems to be responsible for improving weight gain and feed conversion. The benefits of pelleting
are related to higher feed consumption and modification of bird behavior reducing eating time,
expending less energy in feed apprehension and having therefore more energy available for growth
(Jensen et al., 1962). This change in behavior may depend on the lineage of the broilers (Skinner-
Noble et al., 2005).

To guarantee a uniform flock, 320 one d-old male chicks were selected among 510, of Cobb and
Ross lineages, with 10 birds used for every repetition. The birds were raised in metal cages with 1
m2, with adequate tray for excreta collection. The assay was divided into four periods of 12 d each,
and the birds were 24 d old. Excreta collections were made during four d (24 to 27 d old) and the
respirometric data were obtained on subsequent d.
Two types of rations were used according to growing period: initial - from d one to d 21; and growth
- from d 22 to d 36. In each phase, the rations were the same (Table 1) differing only in physical
form.
Table 1. Calculated nutrition levels of the diets.
Nutrional levels Initial Growth
Crude protein, % 22.5 21.0

Apparent Metabolizable energy, kcal/kg 3050 3210
Total lysine, % 1.27 1.21
Total Methionine, % 0.54 0.48
Total Methinine + Cystine, % 0.92 0.89
The ingredients utilized were: maize, soybeam meal 46%, full fat treated soybean, poultry fat, bone and meat meal,
feather meal, gut meal, sodium chloride, DL-Methionine, L-Lysine, Vitamin mix, Mineral mix, Ronozyme, Enradin
F80, Surmax 100, Lerbec, Coxistac 12%, limestone.
The treatments were defined by line, physical format of the rations and by feed intake, being: treatment
A – mash ration; treatment B – pelleted ration and treatment C – pelleted ration with same feed intake
of the mash ration. To control feed consumption of the pelleted ration in treatment C, 30 birds of
each lineage were used one d before the beginning of the trial, and the daily mash feed consumed
was noted and this value was given to the birds of treatment C in pelleted form.
The determination of bird heat production was made by indirect calorimetry. The oxygen consumed
and carbon dioxide produced were determined by an open respiratory chamber system, and heat
production (HP) was calculated using the Brouwer equation (Brouwer, 1965). The values of nitrogen
urinary excretion and basal metabolism were obtained in 40 birds of each lineage after 72 h of

fasting (Chwalibog, 1991). The heat increment (HI) was calculated as the difference between heat
production obtained in fed birds minus heat production obtained in fasting birds. The requirement of
metabolizable energy (MEm) for maintenance was obtained by dividing the heat production of the
fasting bird by the efficiency of use of the metabolizable energy for maintenance (Km); a value of
0.80 was adopted for this variable (Chawlibog, 1991). The experimental design was a randomized
block in a 3 × 2 factorial (3 diets and 2 lineages) in four replicates (blocks); the results were compared
by the Student-Newman-Keuls (SNK) test.

The broiler dry matter intakes between d 24 and 27 were 130a, 134a and 120b g/animal/d for animals
fed mash, pelleted and restricted pelleted diets, respectively.
Table 2 shows the results of heat production and heat increment of broilers in the fed phase. Heat
production in fasting birds of the Cobb line was 312 kj/kg BW/d and the Ross line 347 kj/kg BW/d.
This is very important because the lower energy need for maintenance may represent higher weight
gain due to a better energy partition toward gain.
Table 2. Bird heat production (kJ/kg BW/d).
Treatment BW, g ME intake Heat Heat

production increment
Physical form Mash 1045 1667a 691a 362a

Pelleted 1161 1587ab 658a 329a
Restricted 1079 1524b 663a 334a
pellet
Line Cobb 1127 1550b 655a 343a
Ross 1063 1635a 687a 340a
CV - 4.9 7.8 15.3
Values with different letters within each treatment were different SNK (P<0.05).
The calculated values of maintenance metabolizable energy needs (MEm) were 390 kj/kg BW/d for
the Cobb line and 434 kj/kg BW/d for the Ross line. There was no interaction between the physical
form of the rations and lineages, and the physical form and line did not change the heat production
and heat increment of the birds.
References
Brouwer, E., 1965. Report of Sub-Committee on Constants and Factors. Proc. 3rd Symp. on Energy Metabolism, 11,
441-443.
Chwalibog, A., 1991. Energetics of Animal Production. Acta Agric. Scand. 41, 147-160.
Jensen, L.S., L. Merril, C.V Reddy and J. McGinnis, 1962. Observations on eating patterns and rate of food passage
of birds fed pelleted and unpelleted diets. Poult. Sci. 41, 1414-1419.
Skinner-Noble, D.O., L.J. McKinney and R.G. Teeter, 2005. Predicting effective caloric value of nonnutritive factors:
III. Feed form affects broiler performance by modifying behavior patterns. Poult. Sci., 84, 403-411.

Energy metabolism of high productive laying hens in daily light and
dark periods
A. Chudy
formerly Research Institute for the Biology of Farm Animals, Dummerstorf, OT Warsow 11, D-17154
Neukalen, Germany
Introduction
The aim of this investigation was to determine the energy and nutrient metabolism of two high
productive strains of laying hens (Lohmann Brown (LB) and Lohmann White (LSL)) under different
ambient and feeding conditions. In this paper the heat production in the light and dark periods were
studied.

Respiration experiments (climatically controlled respiration chamber with 4 cages per 3 hens (n=12),
27 measuring periods of 4–18 d) were carried out in two series with two groups, in each 12 or 8 laying
hens – LB (BW 1840 ± 106 g) and LSL (BW 1530 ± 82 g) in series 1 with 6 different treatments and
in series 2 with LB (BW 1888 ± 43 g) only with 10 different treatments; both series in the course
of laying period from the 18th to 77th wk of age. The hens were fed ad libitum. The variations of
exogenous factors were the following: Feed composition: Protein 16.1–26.9%, fat 2.7–9.1% and
starch+sugar 32.2–49.8% of DM; temperature 5–30 °C; two light programs. The two investigated
light/dark programs (LD) were LD 1: 12 h light from 6.00–18.00 and 12 h dark from 18.00–6.00 h,
respectively. LD 2: 11 h light from 7.00–15.00 and from 1.00-4.00 in the night and 13 h dark from
15.00-1.00 and from 4.00–7.00 in the morning. In all we got complete N-, C-, energy, protein and
amino acid as well as nutrient balances.
The measurements of CO2 production and O2 consumption were carried out continuously over 23
h and 40 min daily. The air input and the chamber gas O2- and CO2- concentrations were measured
and automatically recorded every 20 s (Chudy, 2003). Therewith it was possible to calculate the gas
exchange and heat production according to Brouwer (1965) in time segments as required; in these
analyses calculated for each h over 22 h totally, from 11.00 to 9.00 h the next day.

The ME-intake and the dependence of HP on light programs are given in Table 1. The course of
heat production (HP) is demonstrated in Figures 1A (LB) and 1B (LSL). The figures illustrate the
enormous dependence of the HP levels on the light and dark periods. After a short smooth transition
HP changes to the dark or light level at any time. This is equal in LB and LSL. The differences in
HP between LB and LSL are not significant in LD 1 and LD 2, whereas HP is 10–15% higher in
LD 2 versus LD1. The differences between light and dark conditions are very high; ≈60% in LB
and ≈40-50% in LSL respectively. The O2-consumption is in relation to CO2-production higher in
LD 2 (RQ). The absolute values are directly proportional to the daily HP (estimated by means of
N-, C-balance) in dependence on exogenous factors, as environmental temperature or performance,
which influence feed intake. Consequently, the differences between light and dark are caused by the
influence of different physical activities (feed intake and moving).
In conclusion, all in all the results over 24 h lighting programs are without influence, if the totalized
duration of the light respectively dark periods per day are equal. The ratio of the duration between
light and dark periods may be economically important.

Table 1. Heat production in dependence on light programs.
Light program LB LSL
ME kJ/hen×d RQ HP kJ/hen×h ME kJ/hen×d RQ HP kJ/hen×h
LD 1 1361±119 1.04 35.9±8.4 1291±181 1.00 34.7±6.0

LD 2 1250± 92 0.87 39.6±8.4 1284± 55 0.94 39.9±7.9
LD 1 light 45.5±2.3 41.7±1.2
dark 28.3±0.7 29.2±0.8
rel. (dark=100) 161 143
LD 2 light 50.2±1.5 49.6±1.8
dark 31.9±1.0 32.6±1.2
rel. (dark=100) 157 152
Figure 1A. LB - Heat production (HP) - absolute(kJ/hen×h) and relative to average (%).
Figure 1B. LSL - Heat production (HP) - absolute(kJ/hen×h) and relative to average (%).
References
Brouwer, E., 1965. Report of sub-committee on constants and factors. In: Blaxter K.L. (editor), Energy metabolism.
Academic Press, London, 441-443.
Chudy, A., 2003. Two different methods for measuring of gas exchange and heat production in farm animals. EAAP
publication No. 109, 481-484.

Studies on energy needs and nitrogen metabolism of cats during
pregnancy and lactation
B. Wichert1, L. Schade1, B. Bucher2, C. Wenk2 and M. Wanner1
1Institute of Animal Nutrition, Vetsuisse Faculty University Zurich, Winterthurerstr. 260, 8057
Zurich, Switzerland
2Institute of Animal Sciences, Swiss Federal Institute of Technology Zurich Center, 8092 Zurich,
Switzerland
Introduction
The NRC (2006) energy requirements for pregnant and lactating cats have been based on very few
data up to now. The aim of the present study was to collect new experimental data on the energy and
nitrogen intake and balance of pregnant and lactating cats. For lactating cats energy requirements
should be calculated.

Ten queens (2 domestic short hair and 8 European short hair) were measured in the 4th and 7th wk
of pregnancy in respiration chambers for 4 d each. During measurements of O2 consumption and
CO2 production, the cats were kept in the respiration chambers for 22.5 h per d (Hadorn, 1994).
Faeces and urine were collected completely in special cat toilets allowing cooling of urine. In the
2nd and 6th wk of lactation the total urine and faeces of 6 respective 5 queens were collected for 7
d. During the whole experiment, the cats were fed a commercial dry food (Biomill® kitten) with an
addition of 2.1% of celite (HCl-unsoluble ash) as an internal marker ad libitum. Food (dried) and
faeces (lyophylised) were analysed for crude nutrients (proximate analyse) and HCL insoluble ash.
In food, faeces and urine (lyophylised) gross energy was determined by bomb calorimetry (IKA
calorimeter C2000 basic). Further defrosted urine, lyophylised faeces and dried food were analysed
for nitrogen and carbon with a CN-analyser (Leco CN-2000). From these data respectively energy
and nitrogen protein balances were calculated. Energy balances for lactating cats were calculated
with data from the literature for maintenance requirements (Schade, 2006) milk yield depending on
litter size, milk energy content (Zottmann, 1997) and the assumed utilisation of metabolisable energy
for milk production (NRC, 2006). The difference of the number of kittens is taken into account by
using data per kg BW and the calculations depend on litter size.

The energy intake of the cats in the 4th and 7th wk of pregnancy as well as in the 2nd and 6th wk of
lactation is shown in Table 1. The number of kittens differed between 2 kittens (one cat), 4 kittens
(2 cats) and 5 kittens (3 cats).
The data on energy intake shown in Table 1, led to an average total energy intake per cat in the 4th
wk of pregnancy of 1205 ± 177 kJ ME/d. That was about 1.8 times as much as the energy intake of
the adult cat (704 ± 82 kJ ME/d). In the 7th wk of pregnancy the total energy intake of the cats with
1437 ± 257 kJ ME/d was double the maintenance requirement of the adult cats. This high energy
intake seems to be necessary to prepare the cats for normal weight loss during lactation (NRC, 2006).
From the present data it cannot be concluded, that the queens had an adequate energy intake or a
higher one than they would have needed.

Table 1. Average energy intake in kJ/kg BW/d and retained protein in g/kg BW/d during the 4th and
7th wk of pregnancy and during the 2nd and 6th wk of lactation, retained energy in kJ/kg BW/d in
the 4th and 7th wk of pregnancy.
Pregnancy Lactation
4th wk (n=10) 7th wk (n=10) 2nd wk (n=6) 6th wk (n=5)
Energy intake 378 ± 57 366 ± 44 482 ± 102 659 ± 191

Energy retained 140 ± 48 91 ± 83 549 ± 1151 748 ± 2181
Protein retained 1.8 ± 0.8 1.7 ± 0.7 2.3 ± 1.01 4.2 ± 2.51
1before subtraction of milk yield and without regard to number of kitten.
The calculated energy balances in the 2nd wk of lactation were negative independent of the litter size
(between -646 kJ/cat with 5 kitten and -66 kJ/cat with 4 kitten). Nevertheless only 4 from 6 cats lost
weight during this time (weight loss 10-19g/d). The other 2 cats gained weight. This shows that at
least individually the data from the literature about maintenance energy requirements and milk yield
are overestimated or the utilisation of metabolisable energy for milk production in the present study
was higher than the 60% stated by the NRC (2006). Also the energy intake of the queens during the
2nd wk of lactation was higher than their calculated energy requirement (NRC, 2006).
In the 6th wk of lactation, all cats were gaining weight again and showed a positive energy balance
(between +754 kJ/cat with 2 kitten and +1907 kJ/cat with 5 kitten). The cats were heavier than before
pregnancy. Therefore, it can be concluded that their energy intake exceeded their energy needs. To
get more specific information about the energy requirement of pregnant and lactating cats, more
data about body composition during pregnancy and the factor for utilisation of metabolisable energy
for milk production is needed.
References
Hadorn, R., 1994. Einfluss unterschiedlicher Nahrungsfaserträger (Soja- und Hirseschalen) im Vergleich zu
Weizenquellstärke auf die Nährstoff- und Energieverwertung von wachsenden Schweinen und Broilern. Diss.
ETH Zürich, Nr. 10946.
National Research Council, 2006. Nutrient requirements of dogs and cats. The National Academies Press, Washington,
DC, USA.
Schade, L., 2006. Untersuchungen zum Energiestoffwechsel von trächtigen Katzen. Diss Vet. Med. Zürich.
Zottmann, B. 1997. Untersuchungen zur Milchleistung und Milchzusammensetzung der Katze (Felis catus). Diss.
Vet. Med. München.

Changes in energy metabolism during gestation and lactation in sows
R.S. Samuel1, S. Moehn1, L.J. Wykes2, P.B. Pencharz3 and R.O. Ball1
1Swine Research and Technology Centre, 4-10 Agriculture/Forestry Centre, University of Alberta,
Edmonton, AB, T6G 2P5 Canada
2School of Dietetics and Human Nutrition, McGill University, Montreal, PQ, H9X 3V9 Canada
3Research Institute, Hospital for Sick Children, Toronto, ON, M5G 1X8 Canada
Introduction
Energy metabolism of sows changes as a result of pregnancy and lactation. Diets should be adjusted
accordingly to optimize sow nutrition. However, data on energy metabolism in sows are scarce and
recommendations may not be reflective of current high producing animals. Energy metabolism in
gestating and lactating sows was investigated as part of a long-term research program to redefine
the energy and protein requirements of breeding sows.

Animals, diets, housing, and feeding
Sows (n=5, 192 ± 2 kg) were selected from the University of Alberta’s Swine Research and
Technology Centre. Gestation diets were formulated for 13 MJ DE/kg and 0.65% lysine content.
Lactation diets were formulated for 14 MJ DE/kg and 1.02% lysine content. Diets were composed
of barley, wheat, and canola and soybean meals. Sows were housed individually and fed one-half of
their daily feed allowance twice daily during gestation and ad libitum throughout lactation. Gestation
feed allowances were determined by measurement of P2 backfat depth post-breeding. Individual
nipple drinkers provided free access to water.
Indirect calorimetry and respiratory quotient
Sows were individually housed in respiration chambers for 24-h measurements of energy (open-
circuit indirect calorimetry) on d 30, 45, and 105 of gestation and with their litters on d 7 and 19
of lactation. The 24-h gas exchange was recorded for O2, CO2, and CH4 in 1 min intervals. The
respiratory quotient (RQ) was calculated to determine nutrient utilization.
Body composition measurements
Real-time ultrasound measurements of backfat and loin thickness and loin area were collected at
each of the three d of gestation and the two d of lactation.
Statistics
The effect of ‘d’ was assessed using mixed model analysis (SAS 2002) using ‘sow’ as a random
variable. Significance was taken at P<0.05, a tendency at P<0.1.
Results
Daily weight gain of sows (232 g/d) was the lowest (P<0.01, SEM=64) from breeding to d 30 of
gestation compared to d 30 to d 45 (532 g/d) or d 45 to d 105 (574 g/d). Sow body weight increased
(P<0.01, SEM=4) from 183.8 kg at breeding to 190.6 kg on d 30 up to 198.8 kg on d 45 and finally
to 229.1 kg on d 105 of gestation. Sows were fed 2.4 ± 0.1 kg of gestation diet daily resulting in

565, 553, and 524 kJ/BW0.75 (SEM=14) of energy intake for the periods between measurement
d. Measured heat production (HP) on d 45 (510 kJ/BW0.75) tended to be lower than on d 30 (554
kJ/BW0.75) and was lower (P<0.05) than on d 105 of gestation (570 kJ/BW0.75). Measured backfat
thickness increased (P<0.05) from breeding (18.8 mm) to late gestation (21.5 mm) and tended to
decrease from early (19.1 mm) to late lactation (17.1 mm). The RQ for sows at d 45 was greater than
1, indicating lipogenesis. Lipid gain in gestation was 6.4 kg or 26% of maternal gain. Loin area was
not different (P=0.29) from breeding to late gestation or from early to late lactation.
Sow plus litter heat production (63.5 vs. 83.6 MJ/d), milk production per piglet (656 g/d vs. 940 g/d),
and total milk production (7.1 vs. 9.4 kg/d) increased (P<0.05) from d 7 to d 19 of lactation. Calculated
(Jakobsen et al. 2005) sow only heat production (734 vs. 829 kJ/BW0.75) increased (P<0.05) from
early to late lactation. Energy intake from early to late lactation (1146 vs. 1495 kJ/BW0.75) was not
different (P=0.14, SEM=244).
Discussion
Energy expenditure (Brouwer, 1965) at d 105 of gestation (570 kJ/BW0.75) was greater than intake
(524 kJ/BW0.75). As a result, sows in late gestation were in negative energy balance. Calculated
(Lodge et al., 1979) maintenance energy requirements (MEm) were 541, 482, and 572 kJ/BW0.75 for
sows between breeding to d 30, d 30 to 45, and d 45 to 105 of gestation, respectively. Energy intake
for lactating sows was not different (P=0.14) between early and late lactation, indicating maximum
feed intake was achieved. Sow only heat production was not greater than energy intake, but coupled
with the additional energy required for milk production, energy intake was not sufficient. Backfat
thickness indicated excess energy intake during gestation up to d 105 (gain) and insufficient energy
intake during lactation (loss). Ultrasonic measurements also indicated sufficient protein intake since
sows neither gained nor lost loin area during pregnancy.
Dietary energy intakes for late gestation and lactation were insufficient. Feeding a diet greater in
energy (at least 5% greater) to sows in late gestation is required to ensure sufficient energy intake.
Lactating sows required 30% more energy than was consumed.
References
Brouwer, E, 1965. Report of the sub-committee of the European Association for Animal Production on constants and
factors. In: K. L. Blaxter (editor), Energy metabolism. Academic Press, New York, NY, 441–443.
Lodge, G.A., D.W. Friend and M.S. Wolynetz, 1979. Effect of pregnancy on body composition and energy balance of
the gilt. Can. J. Anim. Sci. 59, 51-61.
Jakobsen, K., P.K. Theil and H. Jørgensen, 2005. Methodological considerations as to quantify nutrient and energy
metabolism in lactating sows. J. Anim. Feed Sci. 14 (Supp 1), 31-47.
SAS (Statistical Analysis System), 2002. Release 9.1 Edition. SAS Institute, Inc., Cary, NC.

Determination of the energy cost of physical activity in veal calves
E. Labussière1,2, J. van Milgen1, S. Dubois1, G. Bertrand2 and J. Noblet1
1INRA, UMR Systèmes d’Elevage, Nutrition Animale et Humaine, 35590 Saint-Gilles, France
2Institut de l’Elevage, Monvoisin, BP 85225, 35652 Le Rheu Cedex, France
Introduction
The energy cost of physical activity in veal calves has been little studied and mainly in young animals
(Ortigues et al., 1994; Schrama et al., 1995). Nevertheless, the European legislation by promoting
group-housing and banning the tying-up of animals has created conditions in which the energy
expenditure for activity increases. Indeed, group-housed calves spend more time standing (Bertrand
and Martineau, personal communication) and agitation may also be increased. The objective of the
present paper was to propose a method for estimating the energy cost of physical activity during the
whole fattening period of veal calves.

Animals and management
The data used in this paper were obtained during three experiments implying energy balance
measurements on veal calves in respiration chambers. In total, 62 prim’ Holstein male calves,
weighing from 58 to 250 kg and ranging in age between 5 and 26 wk, were housed in individual
metabolism cages with wooden slatted floors for a total of 89 measurement periods. After two wk of
adaptation to the diet and the housing system, the calf in its metabolism cage was placed during six d
in a 12 m3 open-circuit respiration chamber. The cage was mounted on force sensors that produced
an electrical signal proportional to the physical activity of the calf. Infrared beams detected the
number and duration of standing periods. The temperature and the relative humidity in the chamber
were maintained constant at 18 °C and 70% respectively. A 12 h lighting scheme (from 07.30 h to
19.30 h) was used. The calves were fed at 80 to 100% of their assumed ad libitum intake with a milk
replacer that was distributed twice daily using an automatic feeder. Calves had no access to solid
feed or water. The distribution recipient was mounted on a weight sensor to determine the beginning
of the milk replacer intake. The meal was assumed to last four min.
Heat production calculations
Gas concentrations (CO2, O2) of outgoing air and ventilation rate were continuously recorded
according to van Milgen et al. (1997). Gas concentrations, the signals of the force sensors and infrared
beams, the weight of the distribution recipient and physical characteristics of gas in the chamber
were measured 60 times per s, averaged over ten s intervals, and recorded for further calculations.
The variations in O2 and CO2 concentrations in the chamber were related to events occurring in the
chamber and resulting in gas exchanges due to feed intake and physical activity in addition to the gas
exchanges corresponding to basal metabolic rate. A modelling approach was used for partitioning
total gas exchanges between these three components (van Milgen et al., 1997). Heat production
(HP) values due to physical activity while standing or lying were then calculated for each d from
respective O2 consumption and CO2 production; signals of the force sensors during standing were
corrected for basal intensity measured during lying. The HP was calculated using the simplified
formula of Brouwer (1965) excluding urinary N excretion and methane production. Data of HP
during standing were expressed per h of standing and regressed against (daily) body weight (BW)
with a non-linear regression model: HPstanding, kJ/h = aBWb (n=482).

On average, calves stood up seventeen times (from 2 to 36, SD=5) per d and spent 5.5 h (from 2.7
to 9.8 h, SD=1.3) per d standing. These results are in close agreement with those of Ortigues et al.
(1994) and Schrama et al. (1995) in young calves. Total daily ME intake, HP and HP related to
standing activity averaged 38.5, 25.0 and 1.65 MJ per d respectively. This latter value is equivalent
to 6.6% (from 2.8 to 17.3%, SD=1.7) of the total HP or 4.2% (from 1.6 to 12.0%, SD=1.2) of ME
intake. The HP due to standing, expressed per kg BW0.75, was approximately 30% higher than the
value calculated by Schrama et al. (1995) for young calves (from 41 to 47 kg of initial BW) housed
in similar conditions while standing duration was only 20% higher. The non-linear regression model
indicates that the b exponent in the HPstanding prediction equation was 0.645 (SD=0.027, P<0.001)
over the 60 to 250 kg BW range. If expressed per kg BW0.65, HP due to standing activity was 12.5
kJ/kg BW0.65/h (P<0.001). This means that for a 100-kg calf spending one additional h standing,
its ME requirement for maintenance (considered to be 418 kJ/kg BW0.75/d) increases by about
1.8%. Our method of calculation of the energy cost of standing differed considerably from previous
experiments where the energy cost of standing was assumed to be the difference between average
HP values during standing and lying and therefore ignoring the impact of feed intake on variations
in HP (Ortigues et al., 1994). In addition, our results have been obtained for a much wider BW range
than in all previous studies.
Conclusion
This study provides a method for estimating the energy requirements of veal calves for standing on
wooden slatted floors. The result, obtained with isolated animals and expressed per h of standing,
can be implemented in calves reared in groups according to indicators of their standing duration.
Complementary studies might be necessary to estimate the energy cost of locomotion.
References
Brouwer, E., 1965. Report of sub-committee on constants and factors. In: Blaxter, K.L. (editor), Energy metabolism.
Academic Press, London, New York, EAAP 2, 441-443.
Ortigues, I., C. Martin, M. Vermorel and Y. Anglaret, 1994. Energy-cost of standing and circadian changes in energy-
expenditure in the preruminant calf. J. Anim. Sci. 72, 2131-2140.
Schrama, J.W., J.P. Roefs, J. Gorssen, M.J. Heetkamp and M.W. Verstegen, 1995. Alteration of heat production in
young calves in relation to posture. J. Anim. Sci. 73, 2254-2262.
Van Milgen, J., J. Noblet, S. Dubois and J.F. Bernier, 1997. Dynamic aspects of oxygen consumption and carbon
dioxide production in swine. Br. J. Nutr. 78, 397-410.

The relationships between fasting energy expenditure and intermediary
metabolites in growing lambs
A. Kiani1,2, M.O. Nielsen1 and A. Chwalibog1
1Department of Basic Animal and Veterinary Sciences, Faculty of Life Sciences, Copenhagen
University, Groennegaardsvej 7, DK-1870 Frederiksberg C, Denmark
2Animal Sciences Group, Faculty of Agricultural Sciences, Lorestan University, Iran
Introduction
Feed deprivation causes negative energy balance which subsequently leads to changes in metabolites
and hormones. It also decreases the whole body energy expenditure. Although it is known that
some hormones are up-regulated and some down-regulated with feed deprivation, the relationships
between circulating hormones and metabolites and fasting energy expenditure (FEE) have received
little attention.
The objective of this study was to determine the effects of 72 h fasting and re-feeding on changes
in plasma concentrations of leptin, insulin-like growth factor-1 (IGF-1), insulin, glucose, and
nonesterified fatty acids (NEFA), 3-β-hydroxybutyrate (BOHB), urea in growing lambs and also to
determine the relationships between these metabolites and fasting energy expenditure (FEE).

Seven-mo old lambs (n=14) were fasted for 72 h. Blood samples were taken at the fed state, after 48
and 72 h of fasting and 2 h after re-feeding. Plasma concentrations of leptin, IGF-I, insulin, glucose,
BOHB, NEFA and urea were determined. Additionally, during the last 22 h of fasting, FEE was
calculated from respiratory gaseous exchange measurements (O2, CO2 and CH4) in combination
with nitrogen excretion in urine according to Brouwer (1965). Furthermore, oxidation of fat (OXF),
carbohydrate (OXCHO) and protein (OXP) were calculated according to Chwalibog et al. (1992).
The Pearson correlation coefficient was used to determine the relationships between FEE, OXCHO,
OXF and OXP with circulating metabolites and hormones at 72 h of fasting.
Results
Plasma glucose concentration dropped sharply from the fed to fasted state and reached the lowest
level at 48 h thereafter gradually increasing towards fed state values by 72 h of fasting. Circulating
insulin was the highest at the fed state after which it decreased rapidly to reach the lowest level at 48
h of fasting and thereafter increased towards a basal level at 72 h of fasting. Plasma concentration
of NEFA increased sharply from the fed state to the fasted upon 72 h of fasting. Circulating urea
reached a peak level by 48 h of fasting, thereafter returning to a lower level which was maintained
2 h after re-feeding. Concentrations of IGF-1 and leptin significantly decreased after 48 h of fasting.
Plasma leptin concentration remained low with prolonged fasting for 72 h and 2 h after re-feeding,
whereas IGF-1 progressively decreased with fasting prolongation.

Table 1. Plasma concentrations of metabolites and hormone concentrations in growing lambs at the
fed state (00H), after 48 and 72 h of fasting (48H and 72H) and 2 h after re-feeding (+2H).
Fasting h (Time) SE P-values
00H 48H 72H +2H Time Gender
Glucose, mM/L 3.6a 2.7c 3.1b 3.3ab 0.14 <0.001 NS

Insulin 1,1000×µg/mL 6.0a 3.9d 5.4b 4.6c 0.14 <0.001 NS
NEFA, ng/mL] 0.1d 1.3b 1.9a 1.2b 0.05 <0.001 0.03
BOHB1, 1000×ng/mL 6.6a 6.4ab 6.5ab 6.3b 0.11 0.04 NS
Urea, Mm/L 6.6c 11.9a 9.8b 9.9b 0.54 <0.001 NS
Leptin 1, 1000×ng/mL 7.4a 6.7b 6.7b 6.7b 0.09 <0.001 NS
IGF-1, ng/mL 46.0a 19.6b 12.8bc 10.4c 4.49 <0.001 NS
Values are least squares means ± standard error. Means in the same row with different letters are statistically
different (P<0.05). 1 Data are transformed to a natural logarithm. NEFA, nonesterified fatty acids; BOHB, β-
hydroxybutyrate; IGF-1, insulin-like growth factor-1; NS, non-significant.
Table 2. Pearson correlations of body weight, fasting energy expenditure (FEE) and oxidation of fat
(OXF), carbohydrate (OXCHO) and protein (OXP) in proportion to FEE with circulating metabolite
concentrations in growing lambs after 72 h of fasting.
n Body weight FEE, kJ/W0.75 OXCHO, % OXF, % OXP, %
Body weight 14 - -0.51† 0.19 -0.32 0.33

Glucose 13 0.01 0.21 -0.02 0.25 -0.38
Insulin 12 0.10 -0.56† 0. 00 -0.06 0.10
BOHB 13 -0.25 0.34 0.06 -0.29 0.41
NEFA 11 0.22 -0.28 0.33 -0.41 0.26
Urea 13 -0.20 0.29 0.26 -0.53† 0.58*
NEFA, nonesterified fatty acids; BOHB, β-hydroxybutyrate; †: P<0.10; *: P<0.05; **: P<0.01.
Conclusion
Feed deprivation results in a reduction in plasma concentrations of insulin, glucose, leptin, IGF-1
and elevation in NEFA, β-hydroxybutyrate and urea. When fasting was prolonged from 48 to 72 h,
NEFA concentration increased even further, whereas glucose, insulin and urea concentration were
normalised and stabilised at an intermediate level between 48 h and pre-fasting values. This may be
associated with development of insulin resistance during a prolonged fasting period. Oxidation of
protein was positively correlated to urea concentration. However, FEE (kJ/W0.75) did not show any
significant correlation with intermediary metabolite and hormones except a tendency for a negative
correlation with circulating insulin.
References
Brouwer, E., 1965. Report of sub-committee on constants and factors. In: Blaxter, K.L. (editor), Energy metabolism
of farm animals. Academic Press, London, 441-443.
Chwalibog A., K. Jakobsen., S. Henckel and G. Thorbek, 1992. Estimation of quantitative oxidation and fat retention
from carbohydrate, protein and fat in growing pigs. J. Anim. Physio. Anim. Nutr. 68, 123-135.

Energy requirements of gestating Santa Inês ewes
W.E. Campos1, G.L. Macedo Jr.1, M.I.C. Ferreira1, I. Borges1, N.M. Rodríguez1 and M.L.
Lachica2
1Escola de Veterinária da Universidade Federal de Minas Gerais (EV-UFMG), Brazil
2Animal Nutrition Department, Estación Experimental del Zaidın, Granada, Spain
Introduction
Fasting heat production (FHP) provides an estimate of the basal metabolic rate of an animal because
it excludes most of the energy required for digestion, related tissue deposition, and activity. Thus,
FHP measures the differences in maintenance between different types of animals, assuming constant
efficiency in the use of energy for maintenance.
The present experiment was designed to measure FHP of Santa Inês breed sheep at two gestation
stages and previously fed at two feeding levels.

The experimental data was obtained with 31 first gestating Santa Inês breeding ewes housed in
metabolic cages and fed with Tifton hay (Cynodon spp.), soybean meal and corn grain. Diets were
formulated according to NRC (1985) with one group being fed at maintenance (M) and the other
at 85% of the maintenance requirements (0.85M) from 70 to 130 d of gestation. The ewes’ average
daily heat production was measured at d 100 and 130 of gestation in a respirometry chamber after
60 h fasting. The same ewes were measured at both stages of pregnancy.
An open respirometry system was used at a controlled flow rate of 65 L/min. Inside chamber air
was continuously sent to the gas analysis equipment, which was composed of O2, CO2 and CH4
analysers set in serial connection and the results were automatically recorded by specific software
every three to six min. For every sample analysed from the chamber a simultaneous determination
of the outside air composition was made, assuring a reliable base line. Heat production (kJ) was
calculated according to the Brower equation (Brower, 1965) (1) for O2 consumption (L), CO2 (L),
CH4 (L) and urine-N (Nur, g) production.
HP (kJ) = 16.18 O2 + 5.02 CO2 – 2.17 CH4 – 5.99 Nur (1)
To uniform the animals, all uteri were scanned by ultrasound to check for a presence of one or two
foetuses, with only animals that had one fetus being selected.
The experimental design was a randomised blocks with the results compared by the Student-
Newman-Keuls (SNK) test.

Ewe heat productions are shown in Table 1.

Table 1. Nuliparous ewe heat production at d 100 and 130 of gestation fed at maintenance (M) and
85% of maintenance (0.85M) after 60 h fasting.
Gestation Weight kj/d Kj/BW/d Kj/kg0.75/d RQ
M 100 34.4a 6222a 210a 487a 0.73a

0.85M 100 35.4a 6163a 183a 438a 0.76a
M 130 34.5a 5903a 176a 423a 0.74a
0.85M 130 38.1a 7490b 210a 512a 0.74a
Average 35.6 6445 195 465 0.74
CV 18.7 19.0 30.0 25.0 10.8
BW = Body Weight; kg0.75 = metabolic weight; RQ = respiratory quotient; Numbers with different letters are
different at P=0.09.
At 100 d of gestation, feed supply did not change the ewe’s daily heat production, but at 130 d, the
animals fed with 85% of maintenance requirements produced 21% less heat after 60 h fasting. This
shows that previous food restriction resulted in lower maintenance requirements (P=0.09). When
total daily heat production from 130 d gestation animals was isolated, contrasts between the groups
M and 0.85M showed differences at 3% probability (P=0.03) besides the lower degrees of freedom
which supports the difference shown in Table 1. However, when the heat production as a function
of body weight and metabolic size was evaluated, the values were similar.
The interaction between gestation and feed restriction was evaluated and showed that at late gestation
(130 d), nutrition has a higher impact on animal metabolism (P=0.06) than at earlier stages (100 d).
These results shows that animals fed at 85% of maintenance in the last third of gestation reduced
the net energy requirement by 11%. It seems that in response to a feed restriction, an animal’s
metabolism adaptation occurs.
Considering diet formulations, the metabolisable energy supplied to the animals was 8.54 MJ/d (85%
of maintenance) and 10.04 MJ/d (maintenance). If heat increment is not taken into account and those
energy values are related to averages reported in Table 1, the efficiencies of use of metabolisable
energy were 71% and 69% for animals fed at 85% of maintenance and maintenance, respectively.
The discrepancy between these values (71 × 69) could be higher if heat increment was measured
because the animals fed at maintenance probably had shown higher heat loss than those fed at 85%
maintenance due to higher dry mater intake.
It was concluded that feed restriction had a higher impact on animal metabolism at 130 d of gestation
than at 100 d.
References
Brower, E. 1965. Report of sub-committee on constants and factors. In: Blaxter, K.L. (editor), Energy Metabolism.
Academic Press, London, 442-443.
National Research Council (NRC), 1985. Nutrient requirements of sheep. 6th edition. National Academy Press,
Washington, DC, USA.

Adaptational response of energy metabolism to varying environmental
conditions in Hereford oxen
M. Derno1, W. Jentsch1, M. Schweigel1, H.-D. Matthes1 and E. Mohr2
1Research Institute for the Biology of Farm Animals (FBN), 18196, Dummerstorf, Germany
2University of Rostock, Justus-von-Liebig-Weg 6, 18052, Rostock, Germany
Introduction
In their natural habitat, animals are often exposed to daily changes in weather conditions and/or
feed availability. Thus, short-term adaptations of tissue metabolism and energy expenditure are
essentially needed to maintain homeostasis. In this study, we investigated the short-term response
of heat production (HP), heart rate (HR) and physical activity to a reduction of nutrition level (NL)
and altered ambient temperature (AT). By analysing the variability of HP and HR, closer insight
into the activity patterns of involved regulatory systems were achieved.

An indirect calorimetry study was conducted with 7 Hereford oxen, 20 mo of age weighing 316
± 7 kg. Before starting the experiment the animals were halter trained, adapted to handling and to
the respiration chambers. The experimental design is illustrated by Figure 1. The entire experiment
lasted 15 d and was divided into three 5-d periods. The oxen were fed dried chopped grass (ME
= 11.56 MJ/kg DM) twice daily at levels of 1.5, 1.0 or 0.5 × MEm.(450 kJ/kg0.75/d). During each
feeding period the ambient temperature was changed daily as indicated in Figure 1. Measurements
of O2-consumption, CO2- and CH4-production, heart rate and activity were performed as described
previously (Derno et al., 2005).
Statistical evaluation was done using SigmaStat 3.5 (Systat Software Inc.). The differences and
interactions between various feeding levels or temperatures were analysed by two way repeated
measurements ANOVA allowing comparing repeated measurements for the same individuals.
The level for significant differences was set to P<0.05. Pre-test for normal distribution and equal
variance were performed, post hoc multiple comparison test was done using the Holm-Sidak method.
Correlations were calculated using the Pearson product-moment-correlation procedure. Changes in
the underlying regulatory mechanisms were inspected by analysing the long-term (variations of the
standard deviation) and the short-term (root mean square of successive differences) variability.

Lowering the nutrition level was accompanied by a decrease in 24-h HP and HR but no significant
changes in physical activity occurred. To exclude the food intake-related effects on the investigated
parameters only data taken during the nocturnal period (0.00 to 6.00 h) were analysed further. The
results are summarised in Figure 1.
The resting metabolic rate showed an immediate response to changes in energy supply. In this context,
it has been shown that a fast reduction of HP results mainly from a decreased O2-consumption of the
splanchnic organs related to a strong and rapid decrease in blood flow (Lomax and Baird, 1983).
At NL 1.5 and 1.0 × MEm the time course of HP and HR is determined by AT. While HR and HP are
strictly correlated to AT at NL 1.5 × MEm, the time courses at NL 1.0 × MEm are more characterised
by a parabolic shape with a minimum level at 18 °C (entering the thermoneutral zone). In contrast
to this, at NL of 0.5 × MEm no correlation of either HP or HR to AT could be observed. This effect

NL 1.5 x MEm 1.0 x MEm 0.5 x MEm
T (°C) 6 12 18 24 30 30 24 18 12 6 6 12 18 24 30
d )
-1
80 500
1
Heart rate (beats min )
-1
-0.75
75 c d 480
Heat production (kJ kg

460
70 b e 4
2 440
65 a
3 420
g
60
f 400
55 heart rate
380
heat production 5 h
50 360
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
day
Figure 1. Experimental design and the related time courses of HP and HR. Significant differences
(P<0.05) are indicated by different letters (HR) or numbers (HP). NL = nutrition level.
could be explained by the long-lasting inadequate food supply, an assumption also supported by the
observed decrease in RQ reflecting an elevation in fat oxidation.
The rise in HR and HP at the change from NL 1.0 × MEm to 0.5 × MEm was accompanied by a
significant rise of long-term and short-term variability, a first hint that the process is the outcome of
a change in the dynamics of the underlying regulatory mechanisms.
References
Derno, M., W. Jentsch, M. Schweigel, S. Kuhla, C.C. Metges and H.D. Matthes, 2005. Measurements of heat production
for estimation of maintenance energy requirements of Hereford steers. J. Anim. Sci. 83, 2590-2597.
Lomax, M.A. and G.D. Baird, 1983. Blood flow and nutrient exchange across the liver and gut of the dairy cow. Effects
of lactation and fasting. Br. J. Nutr. 49, 481-496.

5B. Whole organism protein metabolism

Changing dietary lysine level from a deficient to a sufficient level greatly
enhances growth rate of growing rats
A. Ishida, K. Nakashima and M. Katsumata
NILGS, Tsukuba, Ibaraki 305-0901, Japan
Introduction
Since amino acids are the main components of body protein, a number of studies have been carried
out to elucidate the effects of dietary amino acid levels on growth of animals. In these previous
studies, they focussed only on the effects of an excess or deficiency of dietary amino acids (Cree
and Schalch, 1985; Cieslak and Benevenga, 1985). However, our knowledge of the response of
animals to a change of dietary amino acids from a deficient to a sufficient level is limited. The aim
of this study was, therefore, to elucidate the response of animals to the change of a single amino
acid level in the diet. Focus was on lysine because it is a major limiting amino acid in diets both
for humans and animals.

Twenty-four male 4-wk-old rats were randomly assigned to one of two diets: a control diet and a low
lysine (LL) diet. The control diet contained all essential amino acids in the recommended amounts,
including 13 g lysine/kg while the low lysine diet was similar but contained only 4.5 g lysine/kg. The
animals were subjected to these diets during the first 14 d. From d 14, the rats fed the LL diet were
given the control diet until the end of the experiment (d 21) while the rats in the control group were
continuously fed the control diet throughout the 21 d experimental period. Rats were allowed free
access to the diets and water. Samples of urine were taken based on the following schedule: from
d 12 to 14, from d 15 to 17 and from d 19 to 21. The fractional degradation rate of muscle protein
(FDR) was calculated from the rate of excretion of Nτ-methylhistidine. The details of the method
are described elsewhere (Nishizawa et al., 1977). The fractional growth rate (FGR) was defined as
the mean increment in body weight. The fractional synthesis rate (FSR) was calculated as the sum of
FDR and FGR. This calculation is a modification of the calculation used by Millward et al.(1975).
On d 14 and 21, liver and gastrocnemius muscle samples were taken from six rats from each group
in order to determine the expression of proteolytic-related genes, m-calpain, proteasome, cathepsin
L and caspase, by Real-time PCR method.

Growth rate and feed efficiency from d 0 to 14 of rats fed the LL diet were lower than those of
the control rats (Table1; P<0.01). A change of dietary lysine level at d 14 induced enhancement
of growth rate and feed efficiency of rats of the LL group at 70 and 80%, respectively (Table1;
P<0.01). As a result, growth rate of rats of the LL group during the 3rd wk was higher than those
of the control group (Table 1; P<0.01). Although rate of muscle protein synthesis from d 15 to 17
was not different between the two groups, rate of muscle protein degradation was lower in the LL
group (Figure 1; P<0.01). Abundances of mRNA of m-calpain in the liver and muscle of the rats
fed the LL diet were approximately 20 and 40% lower than those of the control group, respectively
(liver; P<0.01, muscle; P=0.14).
Lower degradation of muscle protein in the LL group may be partly explained by concurrent down-
regulation of proteolytic-related genes. Perhaps rats fed the LL diet adapted to a shortage of a single
amino acid in the diet in order to maintain the amount of body protein through a lowering protein
degradation. We infer this lower degradation of protein contributed to considerable enhancement of
growth rate induced by changing dietary lysine level from a deficient to a sufficient level.

Table 1. Effect of low lysine (LL) diet and a changed dietary lysine level on growth performance
of rats.
Term (T) Root Significance

MSE
1 wk 2 wk 3 wk
Feed (F) Control LL Control LL Control LL F T F×T
Feed intake, g/d 12.0d 12.5cd 13.9b 13.6b 14.8a 12.9c 1.21 *** *** **
Growth rate, g/d 6.16b 3.96c 5.84b 3.71c 5.69b 6.67a 1.21 *** *
Feed efficiency, 0.51a 0.32d 0.42c 0.27e 0.40c 0.46b 0.09 *** *** ***
g gain/g feed
*P<0.05, ** P<0.01 and *** P<0.001.
Figure 1. Effect of low lysine diet and a changed dietary lysine level on protein synthesis and
degradation of rats. A,B Values of each dietary group with different superscripts differ at P<0.05,
*P<0.05 and ** P<0.01.
References
Cree T.C. and D.S. Schalch, 1985. Protein utilization in growth: effect of lysine deficiency on serum growth hormone,
somatomedins, insulin, total thyroxine (T4) and triiodothyronine, free T4 index, and total corticosterone.
Endocrinology 117, 667-673.
Cieslak D. C. and N.J. Benevenga, 1984. The effect of amino acid excess on utilization by the rat of the limiting amino
acid-lysine. J. Nutr. 114, 1863-1870.
Nishizawa N., M. Shimbo and S. Hareyama, 1977. Fractional catabolic rates of myosin and actin estimated by urinary
excretion of Nτ-methylhistidine: the effect of dietary level on catabolic rates under conditions of restricted food
intake. Br. J. Nutr. 37, 345-353.
Millward D.J., P.J. Garlick, R.J. Stewart, D.O. Nnanyelugo and J.C. Waterlow, 1975. Skeletal-muscle growth and
protein turnover. Biochem. J. 150, 235-243.

The effect of dietary methionine concentrations on the efficiency of
energy utilisation in broiler chickens
N. Priyankarage1, S.P. Rose1, S.S.P. Silva2 and V.R. Pirgozliev3
1The National Institute of Poultry Husbandry, Harper Adams University College, Newport,
Shropshire,TF10 8NB, United Kingdom
2Veterinary Research Institute, Peradeniya, Sri Lanka
3ASRC, SAC, West Mains Road, Edinburgh, EH9 3JG, Scotland, United Kingdom
Introduction
Dietary protein has a lower efficiency of utilisation of metabolisable energy than other dietary
nutrients in poultry feeds. The utilisation of a dietary protein supply is dependent upon its amino
acid composition and the effects of amino acid imbalances on the growth of poultry have been well
established (Morris et al., 1999). The possibility of amino acid balance on the efficiency of energy
utilisation is understood (MacLeod, 1994) but these effects have not been quantified. Imbalances of
dietary lysine within the protein supply have significant effects on the efficiency of energy utilisation
(Priyankarage et al., 2006) in poultry. The efficiency of energy retention of broiler chickens was
increased with a deficiency of dietary lysine, however, the efficiency of energy retention was markedly
reduced with the excess levels. There is no further evidence of the effects of other essential amino
acids on the efficiency of energy utilisation. Methionine is also an important limiting amino acid
in poultry feeding therefore an experiment was conducted to examine its effects on efficiency of
energy utilisation. Carcass energy deposition and efficiency of energy utilisation in broiler chickens
were observed with six different methionine levels (27, 34, 42, 49, 74 and 98 g methionine/kg crude
protein).

Forty-eight male Ross 308 broiler chicks were randomly allocated to metabolism cages on d 7 of age
and fed a proprietary starter diet. Maize-based practical diets were formulated that had six different
methionine levels. Methionine concentrations were changed by substitution of L-glutamic acid with
DL-methionine. The bioassay was started at 10 d of age and each chicken was given a 12 d feeding
period. Daily feed intakes were limited to 85% of ad libitum so that all birds had the same total feed
intake. Droppings were collected over the final four d of the bioassay and at the end of the bioassay
birds were slaughtered and their carcasses were ground, freeze-dried and the dry matter, nitrogen,
fat contents were determined. Energy from protein and fat were calculated using the energy values
of 23.68 MJ/kg for protein and 39.12 MJ/kg for fat. Eight replicate chickens were fed each of the
experimental diets. Data were compared in a randomised block analysis of variance, and treatments
were compared by partitioning the sums of squares into their linear and quadratic effects.

The growth response was asymptotic with different dietary methionine concentrations and this agrees
with Morris et al. (1992). The lowest (P<0.001) growth was observed with 27 g methionine/kg
CP (Table 1) and this indicates that methionine was limiting in the basal diet. There was relatively
small, albeit significant difference (P<0.05) in determined apparent metabolisable energy (AME)
values between the diets. Carcass nitrogen deposition was affected (P<0.001) by dietary methionine
concentrations and the lowest nitrogen deposition occurred with the 27 g methionine/kg CP. However,
there was no significant differences (P>0.05) in carcass fat deposition. Energy gain and efficiency
of energy retention were also not affected (P>0.05) by dietary methionine concentration.

A previous study (Priyankarage et al., 2006) showed that deficiency and excess of dietary lysine
increased and decreased (P<0.001) respectively the efficiency of energy utilisation in broiler chickens.
There was no effect observed with deficiency or excess dietary methionine in the present experiment.
The difference in response to methionine imbalance compared to lysine imbalance is difficult to
explain although there are differences in the catabolic pathways between these two amino acids.
Table 1. Effect of different methionine concentrations on growth, metabolisable energy, nitrogen,

fat and energy gains and efficiency of energy retention in broiler chicks.
Methionine Body weight Metabolisable Nitrogen Fat gain, Energy gain, Efficiency
concentration, g/ gain, g energy, MJ/kg gain, g/bird g/bird MJ/bird of energy
kg crude protein retention†
27 302.7 12.94 9.67 23.38 2.35 0.301

34 334.1 13.12 10.97 18.42 2.34 0.296
42 336.7 12.99 10.58 18.62 2.29 0.293
49 348.7 13.12 11.17 20.26 2.45 0.309
74 344.6 13.06 11.38 15.92 2.30 0.293
98 350.4 12.96 11.09 18.21 2.35 0.303
SED 7.45 0.071 0.346 2.409 0.098 0.0117
Probabilities of statistical differences
Methionine <0.001 0.014 <0.001 0.105 0.817 0.805
concentration
Linear <0.001 0.551 0.001 0.084 0.952 0.798
Quadratic <0.001 0.011 0.002 0.080 0.958 0.710
† The ratio of carcass energy gain (MJ) to the AME (MJ) intake.
References
MacLeod, M.G., 1994. Dietary energy utilisation: prediction by computer simulation of energy metabolism. Proceedings
of the 13th symposium on energy metabolism of farm animals, Mojacar, Spain, 237-240.
Morris,T.R., R.M. Gous and S. Abebe, 1992. Effects of dietary protein concentration on the response of growing chicks
to methionine. Br. Poult. Sci. 33, 795-803.
Morris, T.R., R.M. Gous and C. Fisher, 1999. An analysis of the hypothesis that amino acid requirements for chicks
should be stated as a proportion of dietary protein. Worlds Poult. Sci. J. 55, 7-22.
Priyankarage, N., S.P. Rose, S.S.P. Silva and V.R. Pirgozliev, 2006. Dietary lysine concentration and the efficiency of
metabolisable energy utilisation in broiler chickens and turkeys. Br. Poult. Abstr. 2, 8-10.

Nitrogen metabolites and enzymatic activity during the weaning period
in goat kids
D. Magistrelli1, L. Pinotti2 and F. Rosi1
1Department of Animal Science, Agricultural Faculty, University of Milan, Via G. Celoria 2, 20133
Milan, Italy
2Department of Veterinary Sciences and Technology for Food Safety, Veterinary Faculty, University
of Milan, Via G. Celoria 10, 20133 Milan, Italy
Introduction
In mammals weaning is the transitional stage of life from milk to other sources of nutrients. It is
characterised by several changes that can deeply influence growth, maturation of gastro-intestinal and
reproductive apparatus (Garnsworthy, 2005) and even the quality of the end products (Schoonmaker
et al., 2002). In light of this, the aim of the present study was to evaluate the variations in protein
metabolism and in the liver and pancreas enzymatic activity, involved in the adaptation process of
young animals to the solid diet.

At birth, 11 Saanen goat kids were assigned to one of the two experimental groups: MILK (6 animals)
and WMIX (5 animals). All kids were fed colostrum for 3 d and then goat milk (3.22% crude protein,
3.61% fat, 4.73% lactose, as fed) for the first 4 wk of life. Starting from the 5th wk, MILK group
continued to receive goat milk, while the WMIX group was fed milk in decreasing quantity plus a
weaning feed mixture (15.6% crude protein, 5.36% ether extract, 16.7% starch, as fed) ad libitum;
on d 47 of age, the WMIX group was completely weaned. Weaning feed mixture was constituted
by grass hay (30%), dehydrated alfalfa (10%), steam-flaked maize (19%), maize gluten meal (3%),
dried sugar beet pulp (8%), soybean meal (15%), sunflower seed (4%), sugar cane molasses (4%)
and mineral/vitamin supplement (7%). During the experimental period, mean feed intake of the
two groups was recorded every d and individual body weight was recorded weekly. On wk 3, 4,
5, 6 and 7 of age, blood samples were taken before the first meal of the d. Blood was immediately
centrifuged at 2000×g for 15 min and then stored at -20 °C until analysis for total protein, urea,
creatinine (Giesse Diagnostics Snc, Roma, Italy), albumin, globulin (Boehringer Mannheim GmbH,
Mannheim, Germany) and free aminoacid (Goodwin, 1968). On d 50 of age, all kids were slaughtered
and carcass and liver weight recorded. Liver and pancreas samples were stored at -80 °C and then
analysed for DNA and RNA (Munro, 1969). Phospholipid (Munro, 1969) and soluble protein level
(Pierce, Rockford, IL, USA) and alanine aminotransferase (ALT) and aspartate aminotransferase
(AST) activity (Boehringer Mannheim GmbH, Mannheim, Germany) were analysed in the liver.
Zymogen content and α-amylase activity (Giesse Diagnostics Snc, Roma, Italy) were determined
in the pancreas. Data obtained were evaluated by analysis of variance.

Dry matter intake began to differ (P<0.01) on the 6th wk of age (328 vs. 407 g, for WMIX and
MILK group respectively), when WMIX kids progressively began to ingest the weaning mixture.
This difference can be explained by the difficulty of the WMIX group to accept the new diet. By
contrast, no difference was observed in body weight, during the entire period, but the weight of
WMIX kids could be overestimated by the presence of feed in the rumen. The weaning protocol
significantly decreased (P<0.01) the overall mean of plasma free aminoacid and increased (P<0.05)
plasma creatinine. Major differences were observed on d 50 of the experiment (Table 1), when free
aminoacid and urea were lower (P<0.01) in the WMIX group than in the MILK one, as a possible
consequence of the lower availability of nutrients in the solid diet. As shown in Table 1, plasma

creatinine level was higher (P<0.01) in WMIX group, although no difference was observed in body
and carcass weight, neither in plasma protein.
Table 1. Slaughtering parameters, plasma, liver and pancreas analysis on d 50 of age.
Slaughtering parameters Body weight, kg Carcass weight, kg Liver weight, g Liver weight, % BW
WMIX 15.3 9.54 330 2.17*

MILK 16.2 10.7 377 2.76*
Plasma Total protein, g/L Albumin, g/L Aminoacid, mM Urea, mM Creatinine, mM
WMIX 53.1 38.9 4.21** 5.67** 78.9**

MILK 55.4 39.9 5.58** 7.66** 69.3**
Liver DNA, mg/g RNA, mg/g Phospholipid, mg/g Soluble prot., mg/g ALT, U/g AST, U/g
WMIX 2.96 5.47 3.80 223 7.71 200

MILK 2.54 5.29 3.87 238 8.22 229
Pancreas DNA, mg/g RNA, mg/g Zymogen, mg/g α-amylase, U/g α-amylase, U/mg Z
WMIX 3.36 13.3 10.8 8.23* 0.70**

MILK 3.16 11.8 9.65 29.5* 2.79**
* P<0.05; ** P<0.01.
On d 50 of age, liver weight, hepatic DNA, RNA and phospholipid content and ALT and AST activity
were not different between the two experimental groups (Table 1). However, liver weight expressed
as a percentage of body weight (P<0.05) (Table 1), as well as liver RNA expressed on kg of body
weight (118 vs. 146 mg/kg BW, P<0.01) and liver content of soluble protein on mg of RNA (40.4
vs. 44.9 mg/mg RNA, P<0.05) were lower in WMIX animals than in the MILK group. On d 50
of age, pancreatic zymogen, DNA and RNA content was not different between groups (Table 1).
By contrast, pancreatic α-amylase activity expressed as unit on g of fresh tissue, as well as when
expressed on zymogen (Table 1), on DNA (2.94 vs. 8.87 U/mg DNA), and on RNA (0.57 vs. 2.34
U/mg RNA), was more than three times lower (P<0.05) in the WMIX group than in the MILK one.
Pancreatic amylase activity was also correlated with plasma free aminoacid concentration (r=+0.80;
P<0.01). The data here reported suggest that weaning can affect protein metabolism and pancreatic
amylase activity. However, it is surprising that the MILK group had a higher level of pancreatic α-
amylase, even if there was no starch in the milk diet. So, more extensive experiments are required
in order to establish what could be the stimulator of pancreatic α-amylase synthesis and secretion,
at the time of weaning.
References
Garnsworthy, P.C., 2005. Calf and heifer rearing: principles of rearing the modern dairy heifer from calf to calving.
Nottingham University Press, Nottingham, UK.
Goodwin, J.F., 1968. The colorimetric estimation of plasma amino nitrogen with DNFB. Clinic. Chem. 14 (11),
1080-1090.
Munro, H.N., 1969. Mammalian Protein Metabolism, vol. III. Academic Press, London, UK.
Schoonmaker, J.P., S.C. Loerch, F.L. Fluharty, T.B. Turner, S.J. Moeller, J.E. Rossi, W.R. Dayton, M.R. Hathaway and
D.M. Wulf, 2002. Effect of an accelerated finishing program on performance, carcass characteristics and circulating
insulin-like growth factor I concentration of early-weaned bulls and steers. J. Anim. Sci. 80, 900-910.

Influence of dietary tryptophan concentration on performance and
dietary selection by starting pigs
T. Ettle1, J. Bartelt2, C. Relandeau3 and F.X. Roth4
1BOKU University Vienna, Gregor Mendel Str. 33, 1180 Vienna, Austria
2Lohmann Animal Health GmbH & Co.KG, 27472 Cuxhaven, Germany
3Ajinomoto Eurolysine S.A.S., 153 rue de Courcelles, 75817, Paris, France
4TU Munich-Weihenstephan, Hochfeldweg 6, 85350 Freising, Germany
Introduction
Tryptophan (Trp) may be a growth limiting factor in pigs especially when diets are mainly based on
corn products. Given that requirement derivations from different studies show a high variability, a
dose response trial was conducted to estimate the starting pigs Trp requirement. Besides effects on
growth, Trp is considered to be involved in feed intake regulation. A former study (Ettle and Roth,
2004) demonstrated that piglets prefer diets adequate in Trp over Trp deficient diets. In the second
experiment of the present study, those effects of Trp on dietary selection were proven using a broader
range of Trp concentrations of diets on offer.

In experiment (exp) 1, 48 ad libitum fed, individually housed crossbred piglets (7.2±1.0 kg) were
subdivided into 5 treatments (treat; n=10 for treat 1-3, n=9 for treat 4 and 5) and fed diets with varying
Trp concentrations. Diets were similar for all treatments (1.13 and 1.06% lysine for d 1 to 25 and for d
26 to 47, respectively) except varying inclusion rates of crystalline L-Trp. Dietary Trp concentrations
for treatments 1 to 5 were by analysis 0.18, 0.20, 0.22, 0.23 and 0.26% for d 1 to 25 and 0.17, 0.19,
0.21, 0.23 and 0.26% for d 26 to 47, respectively. In exp 2, 48 individually housed crossbred piglets
(7.5 ± 0.7 kg) were used to investigate preferences of piglets for Trp in a 6-wk choice study. Piglets
were randomly subdivided into six treatment groups of eight animals each. Animals of group 1 to 3
were used as reference groups whereas animals of group 4 to 6 were given the choice of two diets
with similar amino acid levels (1.10% lysine) but various Trp concentrations. Trp concentrations in
diets given to animals of groups 1 to 3 were by analysis 0.09, 0.16 and 0.24%, respectively. Animals
of group 4 to 6 were offered simultaneously pairs of diets with Trp concentrations of 0.09 or 0.16%
(group 4), 0.09 or 0.24% (group 5) or 0.16 or 0.24% (group 6), respectively.

In exp 1, performance responded in a linear manner to the increasing Trp supply during the first 25
experimental d (Table 1) leading to a requirement estimate of at least 0.26% Trp (highest Trp level
used). In the second half of the experiment performance was again significantly (P<0.05, SNK-test)
improved by the increasing dietary Trp supply. Non-linear regression analysis (exponential model)
revealed a Trp requirement of 0.25% (R2=98.7) to optimise performance. Even if the exponential
model may lead to slightly higher requirements compared to the broken-line model, these data clearly
indicate higher dietary Trp requirements (23% Trp:Lys ratio) compared to current recommendations
(NRC, 1998).
In exp 2, the animals of groups 4 and 5 met on average 8 and 12% (P<0.05, Student t-test) of total
feed intake by consumption of the low Trp diet (0.09% Trp) offered. Contrarily, animals of group 6
showed no preference for the diet higher in Trp (0.24% Trp) but met 58% (N.S.) of total feed intake
by consumption of the 0.16% Trp diet. The animals of groups 4 and 5 increased their preference for
the diets higher in Trp from about 50% at the onset of the experiment to more than 90% in the last

experimental week, whereas preferences for the diets on offer in group 6 remained almost constant
(Figure 1). As a mean of the experiment, chosen total diets in groups 4 to 6 had Trp concentrations
of 0.16, 0.22 and 0.19%, respectively. Dietary Trp concentration did highly influence performance
(Table 2). However, on the contrary to exp 1 dietary Trp concentration above a level of 0.16% did
not increase performance. This effect may be due to a one-sided diet composition (high levels of corn
products) resulting in a generally poor performance. In accordance to a former study (Ettle and Roth,
2004), the results of exp 2 indicate that piglets are able to prefer a diet higher in Trp concentration
over a low Trp diet in order to avoid deficiency. Piglets of group 6 selected both diets to a similar
extent. Thus, it can be concluded that piglets were able to balance the intake of the two diets on test,
however, they did not select for the highest possible Trp concentration in the total diet.
Table 1. Growth and Feed intake in experiment 1.
Treat 1 Treat 2 Treat 3 Treat 4 Treat 5 SE
d 1-25 Gain, g/d 172c 230b 289a 304a 346a 52

Feed intake, g/d 297d 364c 424b 447ab 491a 56
d 26-47 Gain, g/d 308c 444b 543a 592a 588a 88
Feed intake, g/d 596c 803b 952a 1073a 1069a 154
100
Proportion of total feed intake, %
75
50
25
0.16 % Trp diet (treat 4)
0
1 2 3 4 5 6
Week
Figure 1. Selection of 0.16 or 0.24% Trp diet by piglets given the choice of the 0.09 or 0.16% Trp
diet (group 4), the choice of the 0.09 or 0.24% Trp diet (group 5) or the choice of the 0.16 or 0.24%
Trp diet (group 6).
Table 2. Growth and Feed intake in experiment 2.
Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 SE
Gain, g/d 50c 341ab 342ab 286b 301ab 363a 48

Feed intake, g/d 214b 584a 573a 501a 527a 606a 72
References
Ettle, T. and F.X. Roth, 2004. Specific dietary selection for tryptophan by the piglet. J. Anim. Sci. 82, 1115-1121.
NRC, 1998. Nutrient requirements of swine. 10th revised edition. Natl. Acad. Press, Washington, DC, USA.

Histidine maintenance requirement and the efficiency of its utilisation
for protein accretion in pigs
P. Patráš, S. Nitrayová and J. Heger
Slovak Agricultural Research Centre, Research Institute of Animal Production, Hlohovská 2, 949
92 Nitra, Slovakia
Introduction
Very little is known on the pig maintenance requirement (MR) for histidine (His). Also, the essentiality
of His under maintenance conditions is still a matter of some controversy. In our preceding study using
a classical balance technique (Heger et al., 2003), His MR was estimated to be 14 mg/kg BW0.75.
However, the precision of the estimate might be questioned because of the confounding effect of
endogenous His reserves or a presumed His synthesis de novo. Therefore, the objective of the present
study was to estimate His MR in a prolonged time period using the carcass analysis technique.

Twenty-four pigs weaned at 30 d were used. During a 5 d preliminary period, they were fed a low-
protein adaptation diet to deplete labile protein reserves. Six animals were killed at the beginning of
the experiment (mean BW 9.4 kg, 3.01 g His/16 g N) to estimate the initial body composition. The
remaining pigs were fed experimental diets for 21 d and their mean final BW was 11.7 kg. Based
on carcass analysis data, daily retention of His and of total N was calculated and converted to units
per kg BW0.75. Three purified diets were formulated: (i) diet devoid of His, (ii) diet in which His
corresponded to its MR (14 mg/kg0.75) and (iii) diet providing 56 mg His/kg0.75. The pigs were fed
at a daily rate of 100 g/kg BW0.75. Linear regression equations were fitted to experimental data with
total N retention and His retention regressed on His intake. His MR was calculated as His intake
at zero N or His retention. His retention was also regressed on N retention to determine whether N
equilibrium was attained at zero His retention.

The regression equation relating His retention to His intake (Figure 1) indicates that 15.5 mg dietary
His per kg BW0.75 was required to maintain His equilibrium. The slope of the regression line (0.94)
was not significantly different from 1.0 (P<0.05). Regressing total N retention to His intake (Figure
2) showed that His intake required for zero N retention was 4.1 mg/kg BW0.75. The regression
equation relating His retention (y) to N retention (x) was y = -8.56(SE 3.30) + 0.105(SE 0.019)x (R2
= 0.65; P<0.001). The equation indicates that for each gram of N retained, His retention increased
by 105 mg and that at zero His retention, the pigs retained daily 82 mg N/kg BW0.75. The positive
N retention at zero His retention suggests that at His deficiency, a substantial quantity of this amino
acid may be released from its labile tissue stores such as carnosine and/or haemoglobin. Another
explanation may be de novo His synthesis. However, based on the present results, this eventuality
is unlikely in pigs. The slope of the regression line relating His retention to N retention (105 mg/g
N) was much lower than His concentration found in body protein (179 mg/g N). It therefore seems
that the positive N balance at His deficiency might be due to the synthesis of proteins with lower His
content at the expense of compounds containing higher concentrations of His. Taking the above into
consideration, His should be classified essential both for maintenance and growth. The slope of the
regression line relating His retention to His intake (0.94) suggests that His is efficiently conserved
when its dietary supply is limited. It is concluded that His intake corresponding to zero His retention
is a better criterion of its MR than His intake corresponding to zero N retention.

50
40
30
)
0.75
Daily histidine retention (mg/kg
20
10
-10
-20
-30
-40
0 10 20 30 40 50
Daily histidine intake (mg/kg0.75)
Figure 1. His retained as a function of His intake. Plotted from the equation y = -14.61(SE 3.42) +
0.94(SE 0.14)x (R2 = 0.73; P<0.001).
Figure 1. His
500
retained as a function of His intake. Plotted from the equation y = -14.61(SE 3.42)
+ 0.94(SE 0.14)x (R2 = 0.73; P<0.001).
400
300
Daily N retention (mg/kg0.75)
200
100
-100
-200
0 10 20 30 40 50
0.75
Daily histidine intake (mg/kg )
Figure 2. N retained as a function of His intake. Plotted from the equation y = - 30.59(SE 24.2) +
7.47(SE1.00)x (R2 = 0.78; P<0.001).
References
Heger, J., T.V. Phung, L. Křížová, M. Šustala and K. Šimeček, 2003. Efficiency of amino acid utilization in the growing
pig at suboptimal levels of intake: branched-chain amino acids, histidine and phenylalanine + tyrosine. J. Anim.
Physiol. Anim. Nutr. 87, 52-65.

Protein oxidation measured by breath test in mink fed bacterial protein
meal
A.L.F. Hellwing1, A.-H. Tauson1,3 and A. Skrede2,3
1Department of Animal and Veterinary Basic Sciences, Faculty of Life Sciences, University of
Copenhagen, Grønnegaardsvej 3, DK-1870 Frederiksberg C, Denmark
2Department of Animal and Aquacultural Sciences, Norwegian University of Life Sciences, P.O.
Box 5003, N-1432 Ås, Norway
3Aquaculture Protein Centre, Centre of Excellence, P.O. Box 5003, N-1432 Ås, Norway
Introduction
Bacterial protein meal (BPM) is a protein source produced from natural gas by fermentation. The
protein content is approximately 70% and the amino acid composition is comparable with fish meal
except that lysine levels are lower, and tryptophan and threonine levels are higher (Skrede et al.,
1998). RNA and DNA make up 10% of the dry matter, which is higher than in fish meal but similar
to other sources of BPM (Kiessling and Askbrandt, 1993). Protein and energy metabolism studies
have shown that up to 40% of dietary nitrogen can be derived from BPM without negative effects
(Hellwing et al., 2005). The objective of this study was to determine whether the oxidation of glycine,
measured by the 13C/12C breath test, as an indicator of protein oxidation, in growing mink kits was
affected by replacing fish meal with increasing levels of BPM.

Sixteen male mink kits of the standard brown genotype were randomly allocated to four dietary
treatments: A control diet (M1) was based on high-quality fish meal which was replaced with
increasing levels of BPM on the basis of digestible nitrogen so that 20% (M2), 40% (M3) and 60%
(M4) of the digestible nitrogen was derived from BPM. The calculated contents of glycine and
threonine were 9.6/5.9, 9.3/6.4, 8.9/6.9, and 8.5/7.5 g/kg on M1, M2, M3 and M4 respectively. For
details on dietary composition see Hellwing et al. (2005). The animals were fed ad libitum, and
because minks are intermittent eaters food was available throughout the measurement period. The
oxidation of glycine was measured when the mink kits were about 9.5 (period 1), 17.5 (period 2),
23.5 (period 3) and 28.5 (period 4) wk old. The breath tests were performed simultaneous with 22-h
respiration experiments and measurements of protein turnover by 15N-glycine end-point technique,
hence the choice of glycine as an indicator amino acid. A baseline measurement of the 13C/12C ratio
in expired air drawn from the respiration chambers was made 10 min before the mink were given
an intraperitoneal injection of 1 mL/kg body weight of a solution containing 5 mg of [15N][1-13C]
glycine dissolved in 1 mL of isotonic saline. The 13C/12C ratio in the breath air was then measured
every 10 min for the next four h. Data was analysed with the MIXED procedure of SAS as repeated
measurements and the autoregressive order 1 (AR1) covariance structure was fitted (Littell et al.,
1996). Diet, period, time of measurement and the interaction effects between these were treated as
fixed effects and animal as random effect.

The amino acid composition (g/16 g N) of BPM and fish meal is very similar and there were no
differences between diets in the cumulative recovery of 13C (Figure 1a), but it differed significantly
between periods, the lowest recovery being recorded when the minks were 28.5 wk old (Figure
1b), thus suggesting a lower rate of glycine oxidation in this period. Measurements of the protein
oxidation by indirect calorimetry indicated the same differences (Hellwing et al., 2005) as observed
by the breath test. However, because the dietary protein concentration was the same throughout the

experiment and the protein requirement decreases when animals approach maturity, an increased rate
of oxidation would have been expected. The lower rate of glycine oxidation might reflect a decrease
in protein turnover with age (Hellwing et al., 2007), and could also be a result of winter fur growth,
a process involving a high rate of nitrogen retention. In conclusion, glycine oxidation as an indicator
of protein utilisation was not affected by diet, but by the age of the animals.
Figure 1. Cumulative excretion of 13C in expired air from mink fed increasing levels of bacterial
protein meal during the growth period a) Effect of diet in animals fed diets with approximately 0%
(M1), 20% (M2), 40% (M3) and 60% (M4) of N derived from BPM b) Effect of period when the
animals were approximately 9.5 (period 1), 17.5 (period 2), 23.5 (period 3) and 28.5 (period 4) wk
old. P-values: diet P=0.65, period P=0.003, time P<0.001, diet×period P=0.74, diet×time P=0.13,
period×time P<0.001, diet×period×time P=0.32.
References
Hellwing, A.L.F., A.-H. Tauson, Ø. Ahlstrøm and A. Skrede, 2005. Nitrogen and energy balance in growing mink
(Mustela vison) fed different levels of bacterial protein meal produced with natural gas. Arch. Anim. Nutr. 59,
335-352.
Hellwing, A.L.F., A.-H. Tauson, A. Skrede, N.P. Kjos and Ø. Ahlstrøm, 2007. Bacterial protein meal in diets for pigs
and minks - Comparative studies on protein turnover rate and urinary excretion of purine base derivatives. Arch.
Anim. Nutr., in press.
Kiessling, A. and S. Askbrandt, 1993. Nutritive-value of two bacterial strains of single-cell protein for rainbow-trout
(Oncorhynchus-Mykiss). Aquaculture 109, 119-130.
Littell, R.C., G.A. Milliken, W.W. Stroup and R.D. Wolfinger, 1996. SAS® System for Mixed Models. SAS Institute
Inc., Cary, NC.
Skrede, A., G.M. Berge, T. Storebakken, O. Herstad, K.G. Aarstad and F. Sundstøl, 1998. Digestibility of bacterial
protein grown on natural gas in mink, pigs, chicken and Atlantic salmon. Anim. Feed Sci. Technol. 76, 103-116.

Response of pigs in the weight ranges 35 to 60 kg and 80 to 100 kg to
increasing ileal digestible Threonine: ileal digestible Lysine ratios in the
diet
M.K. O’Connell1, C. Relandeau2, M. Overend3 and P.B. Lynch1
1Pig Production Development Unit, Teagasc, Moorepark, Fermoy, Co. Cork, Ireland
2Ajinomoto Eurolysine S.A.S., 153 rue de Courcelles, 75017 Paris, France
3Forum Products Ltd., 41-51 Brighton Road, Redhill, Surrey, RH16YS, England
Introduction
Optimum dietary threonine: lysine ratio for pigs increases with bodyweight (Tuitoek et al., 1997;
Pedersen et al., 2003), and can be affected by growth rate. At higher weights, boars have higher growth
rates than gilts (O’Connell et al., 2005). The objective of this study was to determine the response of
boars and gilts in the weight ranges of 35 to 60 kg and 80 to 100 kg to increasing standardised ileal
digestible threonine:standardised ileal digestible lysine (idTHR:idLYS) ratio in the diet.

Two separate trials were conducted. In experiment 1 (Exp. 1) 72 single-gender pairs of pigs (36
boars, 36 gilts) were assigned to one of five idTHR:idLYS ratios: 0.56, 0.58, 0.62, 0.65 and 0.69 in
a randomised block design. Initial weight was 34.4 ± 2.64 kg, final weight was 61.5 ± 5.66 kg and
diets were fed ad libitum for 38.2 ± 3.31 d. In a second experiment (Exp. 2), 66 single gender pairs
(31 boars, 35 gilts) were assigned to one of five idTHR:idLYS ratios: 0.55, 0.61, 0.65, 0.70 and 0.73,
in a randomised block design. Initial weight was 77.9 ± 5.88 kg, final live weight was 100.3 ± 7.29
kg. Diets were fed ad libitum for 28.4 ± 7.94 d. Ten pelleted diets were used in each experiment (5
each for boars and gilts). Ileal digestible (id) amino acids were calculated from the analysed amino
acid contents and digestibility coefficients from CVB. In Exp. 1 crude protein, idlysine, and net
energy concentrations of diets were 162 g/kg, 8.1 g/kg and 9.8 MJ/kg and 150 g/kg, 7.0 g/kg and 9.8
MJ/kg for boars and gilts, respectively. In Exp. 2, corresponding values were 160 g/kg, 7.9 g/kg, 9.8
MJ/kg (boars) and 132 g/kg, 6.2 g/kg and 10.0 MJ/kg (gilts). Lysine concentrations were set below
requirements (O’Connell et al., 2005) to ensure that performance response was due to increasing
threonine in the diet. Methionine plus cystine and tryptophan were at ratios of 0.66 and 0.19 to
lysine. Increasing idTHR:idLYS ratios were achieved by incremental addition of L-threonine, at the
expense of wheat. Diets were composed of barley, wheat and soyabean meal, with added soya oil,
amino acids, vitamins and minerals. Pigs were weighed at the beginning and end of experiments,
and feed offered was recorded. Pigs in Exp. 2 were slaughtered at a commercial facility. Carcass
data including cold weight (CW, kg), muscle (MU, mm) and backfat depth (BF, mm at 3rd/4th last
rib), estimated lean content (LM, g/kg) and kill out proportion (KO, g/kg) were collected. Data were
analysed using the PROC GLM procedure of SAS version 9.1 (SAS Institute Inc., Cary, NC), to
determine effects of treatment, gender and interactions on response variables. Initial weight was a
covariate in growth performance analysis and slaughter weight in analysis of carcass data. Quadratic
regression models were developed separately for boars and gilts using PROC REG (SAS 9.1). idTHR:
idLYS ratios for highest predicted average daily gain (ADG) or best predicted feed conversion ratio
(FCR) were determined.
Results
Table 1 shows the least squares means values per treatment for boars and gilts in each experiment.
In Exp. 1, idTHR:idLYS ratios for predicted max ADG were 0.65 for boars (R2=0.98, P<0.05,
RSD=9.82, max ADG=795 g/d)and 0.62 for gilts (R2=0.96, P<0.05, RSD=4.23, max ADG=677

g/d). In Exp. 2, idTHR:idLYS ratios for predicted min FCR were 0.68 for boars (R2=0.99, P<0.01,
RSD=0.01, min FCR=2.52 kg/kg) and 0.74 for gilts (R2=0.99, P<0.01, RSD=0.05, min FCR=3.39
kg/kg).
Table 1. Least squares means values for boars and gilts in each experiment.
Boars Gilts seg1 set1
Experiment 1
Ratio 0.56 0.58 0.62 0.65 0.69 0.56 0.58 0.62 0.65 0.69
ADGc 679 739 775 788 774 641 666 675 669 642 17 26
FI 1726 1768 1784 1730 1753 1687 1813 1864 1816 1668 41 64
FCRc 2.60 2.41 2.31 2.30 2.26 2.65 2.72 2.63 2.73 2.86 0.1 0.1
Experiment 2
Ratio 0.55 0.61 0.65 0.70 0.73 0.55 0.61 0.65 0.70 0.73
ADG2b 864 852 834 852 922 591 697 735 770 766 31 49
FI 2460 2143 2305 2470 2318 2453 2484 2226 2674 2292 73 11
FCR2c 2.87 2.62 2.55 2.52 2.59 4.20 3.73 3.62 3.41 3.40 0.2 0.2
CWb 75.5 75.2 74.9 75.2 75.5 76.4 76.4 77.1 76.9 76.8 0.3 0.5
KOc 750 748 748 751 752 763 765 770 765 768 3.2 5.0
MUa 47.1 47.4 48.4 46.9 47.7 48.4 50.2 48.8 48.2 52.0 0.5 0.8
LCa 574 582 584 579 568 581 591 582 581 594 2.4 0.4
BFd 11.5 10.7 10.6 11.0 12.5 10.9 10.2 11.0 10.9 10.1 0.3 0.4
1standard error of means for gender (g) and treatment (t); 2Linear treatment effect P=0.08; Gender effect: aP<0.1,
bP<0.05, cP<0.01, dP<0.001.
Conclusion
idTHR:idLYS ratio for maximum performance was influenced by both gender and weight range. At
the higher weight range, idTHR:idLYS for gilts was marginally greater than for boars. Further work
is needed in this area to determine if this will influence future dietary formulations for maximum
performance.
References
O’Connell, M.K., P.B. Lynch and J.V. O’Doherty, 2005. Determination of the optimum dietary lysine concentration for
boars and gilts penned in pairs and in groups in the weight range 60 to 100 kg. Anim. Sci. 82, 65-73.
Pedersen, C., J.E. Lindberg and S. Boisen, 2003. Determination of the optimal dietary threonine: lysine ratio for
finishing pigs using three different methods. Livest. Prod. Sci. 82, 233-243.
Tuitoek, K., L.G. Young, C.F.M. de Lange and B.J. Kerr, 1997. The effect of reducing excess dietary amino acids on
growing-finishing pig performance: an evaluation of the ideal protein concept. J. Anim. Sci. 75, 1575-1583.

Lysine requirement for maintenance in growing pigs
J. Ringel and A. Susenbeth
Institute of Animal Nutrition and Physiology, Christian-Albrechts-University Kiel, 24098 Kiel,
Germany
Introduction
Due to methodological problems, precise information about the requirements of amino acid (AA) for
maintenance in pigs is limited. Roth et al. (2003) determined the AA requirement for maintenance
of adult sows as the x-intercept of the linear regression equation between N retention and AA intake.
This approach requires a feeding strategy close to the expected value for maintenance raising the
question of the applicability of those values to intensively fed animals when lactating or growing.
The approach used by Fuller et al. (1989) is based on the response of protein accretion at different AA
intake levels in growing animals. The regression coefficient reflects the dietary AA requirement for
the accretion of 1 g body protein whereas the daily amount of the respective AA required to maintain
N equilibrium is estimated by extrapolating the linear regression to the x-intercept. The advantage
of the latter approach is that values for maintenance are derived from animals in a physiologically
normal situation, however the far extrapolation to N equilibrium reduces the accuracy of the estimate.
The present study used a third approach to determine the lysine requirement for maintenance in
growing pigs. Based on the assumption of a constant marginal efficiency of lysine utilisation for
protein retention (Susenbeth, 1995), the decrease in protein retention with increasing body weight
(BW) reflects the increasing requirement of lysine for maintenance when the amount of lysine intake
is kept constant for all animals irrespective of their BW and is below the required level necessary
for achieving protein retention capacity.

Twelve castrated male pigs were used from 23 to 147 kg of body weight (BW). All animals received
throughout their growing period 1.25 kg/d of a basal diet which consisted (g/kg) of 365 g barley,
365 g wheat, 120 g wheat gluten, 100 g soybean meal, 20 g soybean oil, and 30 g vitamin-mineral-
premix, and contained 215 g crude protein and 4.3 g lysine/16 g N resulting in a daily lysine intake
of 11.5 g. Appropriate amounts of wheat starch were fed separately from the remainder of the diet
to meet the increasing demand for energy with BW and to maintain an energy intake level of 1.3 MJ
ME/kg BW0.75. Additional amounts of the mineral-vitamin-premix were given separately to ensure a
proportion of 30 g/kg of the total diet. Six out of the 12 pigs were randomly selected which received
2 g free lysine to the diet resulting in a total intake of 13.5 g lysine/d. This lysine supplementation
to the basal diet was necessary to prove whether the intake of 11.5 g lysine/d is below the level for
a maximum response. In addition, lysine is the fist-limiting AA in the diet which is a prerequisite of
the approach applied here. N-balances were performed at approximately 35, 55, 80, 105, and 140
kg of BW. Periods for total feces and urine collection lasted seven d.

Two animals receiving 11.5 g lysine/d did not ingest 17% of the wheat starch offered during the
last N-balance period; they were not excluded from statistical analysis, since all other components
of the diet were completely ingested. The effects of lysine intake and BW on protein retention are
presented in Table 1. Protein retention showed a numerical increase by additional lysine supply
within each BW group, therefore confirming that N retention of the lysine un-supplemented groups
was limited by lysine intake. A further prerequisite of the approach used here to determine lysine
maintenance requirement is that marginal efficiency of lysine utilisation is not affected by BW;

this was shown earlier by Dourmad et al. (1996) and Möhn et al. (2000). For the animals receiving
11.5 g lysine/d the relationships between protein retention (RP, g/d) and body weight (BW, kg) or
metabolic body weight (BW0.75, kg) are described by the following equations: RP = 135.5 – 0.1735
BW (r2=0.29; n=30) and RP = 139.6 – 0.6848 BW0.75 (r2=0.28; n=30), respectively. The negative
regression coefficients reflect the increasing demand for lysine with increasing BW. Assuming that
(i) protein retention is overestimated by the N balance method by 11% (Susenbeth et al., 1999),
(ii) retained protein contains 7.2% lysine (Susenbeth, 1995), and total dietary lysine is utilised for
retention with a gross efficiency of 0.63 (Bikker, 1994; Susenbeth et al., 1999), lysine requirement
for maintenance can be calculated as follows: regression coefficients of 0.1735 or 0.6848 divided
by 1.11, multiplied by 0.072, and divided by 0.63 result in 18 mg/kg BW and 71 mg/kg BW0.75,
respectively. The estimate of the lysine requirement for maintenance by 71 mg/kg BW0.75 is higher
than that found in other studies (e.g. 36 mg; Fuller et al., 1989). This may be caused by the high
feed intake level in the present study, since according to Jansmann et al. (2002) it can be assumed
that the largest fraction of AA maintenance requirements are related to the amount of endogenous
gut protein losses which are affected by the amount of feed ingested.
Table 1. The effect of body weight and lysine intake on protein retention (mean1 ± SEM).
Lysine intake, g/d 11.5 15.5 11.5 13.5 11.5 13.5 11.5 13.5 11.5 13.5
Body weight, kg 35 35 52 55 79 77 107 108 137 142
IME2, MJ/kg 1.32 1.31 1.31 1.28 1.26 1.28 1.27 1.23 1.12 1.25
BW0.75
Protein retained3, 127 ± 7 145 ± 7 131 ± 4 141 ± 4 116 ± 4 127 ± 4 123 ± 4 126 ± 4 109 ± 13 123 ± 13
g/d
1Six observations per treatment.

2Intake of metabolisable energy.
3Effect of body weight (P<0.05).
References
Bikker, P., 1994. Protein and lipid accretion in body components of growing pigs: Effects of body weight and nutrient
intake. Ph.D. thesis, Univ. of Wageningen, The Netherlands.
Dourmad, J.Y., D. Guillou, B. Seve and Y. Henry, 1996. Response to dietary lysine supply during the finishing period
in pigs. Livest. Prod. Sci. 45, 179-186.
Fuller, M.F., R. McWilliam, T.C. Wang and L.R. Giles, 1989. The optimum dietary amino acid pattern for growing
pigs. 2. Requirements for maintenance and for tissue protein accretion. Br. J. Nutr. 62, 255-267.
Jansmann, A.J.M., W. Smink, P. van Leeuwen and M. Rademacher, 2002. Evaluation through literature data of the
amount and amino acid composition of basal endogenous crude protein at the terminal ileum of pigs. Anim. Feed
Sci. Technol. 98, 49-60.
Möhn, S., A.M. Gillis, P.J. Moughan and C.F.M. de Lange, 2000. Influence of dietary lysine and energy intakes on
body protein deposition and lysine utilization in growing pigs. J. Anim. Sci. 78, 1510-1519.
Roth, F.X., B.M Jahn and W. Schönberger, 2003. Essential amino acid requirements for maintenance in adult sows.
In: Souffrant, W.B. and C. C. Metges (editors). Wageningen Academic Publishers. Wageningen, The Netherlands,
EAAP Publication No. 109, 717-720.
Susenbeth, A., 1995. Factors affecting lysine utilization in growing pigs: An analysis of literature data. Livest. Prod.
Sci. 43, 193-204.
Susenbeth, A., T. Dickel, A. Diekenhorst and D. Höhler, 1999. The effect of energy intake, genotype, and body weight
on protein retention in pigs when dietary lysine is the first-limiting factor. J. Anim. Sci. 77, 2985-2989.

Maintenance protein requirement and efficiency of utilization in poultry
N.K. Sakomura, J.B.K. Fernandes, R. Neme, C.B.V. Rabelo and F.A. Longo
Universidade Estadual Paulista, Faculdade de Ciências Agrárias e Veterinárias, Via de Acesso Prof.
Paulo Donato Castelane, s/n, Jaboticabal, São Paul, Brazil
Introduction
Protein requirement can be established based on the factorial method, which takes into account
maintenance and protein deposition requirements. Maintenance protein requirements can be estimated
as the amount of protein needed to maintain the bird in nitrogen balance (Scott et al., 1982).
Nitrogen requirement for maintenance (Nm) can be determined by the relationship of N intake with N
retention estimated by the slaughter comparative method or N balance measured in metabolic trials.
However, the studies carried out at UNESP, based on N retention determined by the comparative
slaughter technique, provided Nm higher than those reported in the literature.
Thus, several metabolic trials were conducted at UNESP to determine maintenance protein
requirements by the Nitrogen Balance Technique and the efficiency of nitrogen utilization by the
Slaughter Comparative Method.

Five metabolic trials were conducted to access maintenance protein requirements for broiler breeders,
laying hens and broilers by the nitrogen balance technique.
The trials were conducted in metabolic cages during 8 d, 4 d for adaptation and 4 for excreta collection.
The birds were distributed in 4 dietary treatments and 6 replicates.
The diets were formulated to be isocaloric with four graded levels of CP (3, 6, 8, and 15% CP) to
promote positive, zero and negative nitrogen balance (NB). Total feed intake and excreta production
by total excreta collection were measured. Diets and excreta were analyzed for N and DM. NB was
determined by the difference between N intake and N excreted.
A linear regression of NB as a function of nitrogen intake (NI), provides maintenance nitrogen

requirement (Nm) as the intercept of the x-axis, as being the N intake at zero NB. Endogenous and
metabolic losses represent the negative intercept on the y-axis; it is the N excreted at zero of the N
intake. The protein requirement was calculated by the factor (6.25).
Five feeding trials were conducted to determine efficiencies of N utilization in broiler breeders,
laying hens and broilers by the Slaughter Comparative Method.
The birds were fed four feeding levels: ad libitum, and about 70%, 50% and 30% of the ad libitum
intake. Body nitrogen retained (NR) was determined by the Slaughter Comparative Method. At the
beginning and the end of the assay, the birds were slaughtered in order to determine nitrogen in
carcass and feathers. The NR was obtained by the difference of body N at the end and body N of
birds at the beginning of the trials. A linear regression of NR as a function of NI was adjusted, and
the slope provided the efficiency of N utilization for protein deposition.

Results
The maintenance nitrogen (Nm) and nitrogen endogenous losses (Ne) (mg/kg0.75/d) were calculated
according to regression equations of NB as a function of NI (Table 1). We can observe that the
requirements did not vary for poultries. However, the Pm and Ne for broiler chicks were lower than
those found for other poultry. The results found in our studies were lower than those reported by
Scott et al. (1982) (410 mg N/ kg/d) Albino et al. (1994) (540 mg N/kg/d) for growing chickens.
McLeod (1990) also found higher Nm for broilers chicks (900 and 1110 mg N/kg/d).
The efficiencies of N utilization obtained from linear regressions between NR and NI, were higher
for growing poultry, laying type pullets, broiler breeder pullets, and broilers, compared to laying hens
and broiler breeder hens. This difference could be due to higher requirements for protein accretion
in growing poultry. Our results of efficiency of N utilization were in accordance to those found by
Scott et al. (1982) 55%, Albino et al. (1994) 62% and MacLeod (1991) 46%.
The coefficients for Pm (mg/kg0.75/d) and the efficiencies of N utilization can be used to calculate
the protein requirements by a factorial method that takes into account maintenance and growth
requirements.
Table 1. Nitrogen (Nm) and protein (Pm) maintenance requirements, endogenous loss of nitrogen
(Ne) and efficiencies of N utilization (kg) for poultry.
Regressions Nm1 Pm1 Ne1 kg2

mg/kg0.75/d g/kg0.75/d mg/kg0.75/d %
Laying-type NB = -0.257 + 0.672.NI 383 2.37 258 67

pullets (R2=0.96)
Laying hens NB = -0.1812 + 0.589.NI 307 1.92 181 38
(R2=0.97)
Broiler breeder NB = -0.1789 + 0.554.NI 323 2.02 179 54
pullets (R2=0.97)
Broiler breeder NB = -0.2216 + 0.607.NI 365 2.28 222 40
hens (R2=0.76)
Broiler chickens NB =-0.172.NI2 + 0.974.NI 212 1.32 198 72
- male – 0.198 (R2=0.97)
Broiler chickens NB =-0.1803.NI2 + 0.972.NI 280 1.75 258 72
- female – 0.257 (R2=0.97)
1Determined by N balance trials, according to regressions of NB as a function of NI.

2Determined by the Slaughter comparative technique, according to regressions of NR as a function of NI.
References
Albino, L.F.T., F.B. Fialho, C. Bellaver, C. Hara and G.J. Paiva, 1994. Estimativas das exigências de energia e proteína
para frangas de postura em recria. Pesq. Agropec. Bras. 29, 1625-1629.
Macleod, M.G., 1990. Energy and nitrogen intake, expenditure and retention at 20 °C in growing fowl given diets with
range of energy and protein contents. Br. J. Nutr. 64, 625-637.
Scott, M. L., M.C. Nesheim and R.J. Young, 1982. Nutrition of the chicken. 3rd edition. Scott & Associates, Ithaca,
NY, 562 pp.

Oral and intravenous phenylalanine kinetics in adult mixed hounds
A.K. Shoveller1, G.M. Davenport2, J.P. Cant1, S. Robinson1 and J.L. Atkinson1
1Dept. of Animal and Poultry Science, University of Guelph, Guelph, ON, N1G 2W1 Canada
2P&G Pet Care, Lewisburg, Ohio, 45338 USA
Introduction
There are few estimates of single amino acid (AA) kinetics in adult mammals and none in adult
dogs. Knowledge of the differences in AA kinetics between healthy adult dogs and those in different
physiological stages or disease states could help to elucidate potential improvements to diets
recommended for those populations. The objectives of these studies were the following: i) to validate
the use of oral (O) isotope studies, ii) to define phenylalanine (PHE) kinetics when the tracer was
given intravenously (IV) and orally, and iii) to compare kinetics from IV and O studies to enable an
estimate of the effects of gastric emptying and splanchnic metabolism on PHE kinetics in 5 adult,
spayed, mixed hound female dogs.

Five spayed, female, mixed hound dogs (~3 yr, ~22 kg) were fed diets that met or exceeded AAFCO
guidelines for individual nutrients. On the d of study, dogs were weighed and a catheter was inserted in
the cephalic vein for blood sampling and isotope infusion. Dogs received 25 ½ hourly meals providing
a daily intake of 13 g/kg BW. After two feedings, blood samples (1 mL) were taken immediately
before each feeding for the remainder of the study. After an additional 5 feedings, dogs received either
an O or IV bolus of L- [1-13C] PHE (12 mg/kg BW). Oral dosing was accomplished by adding the
isotope to the ½ hourly feeding. All dogs received both O and IV dosing on separate d. Enrichment
and concentrations of plasma PHE were measured using liquid chromatography-MSMS.
Curves of plasma enrichment following IV dosing (PV(t)) were fit with the one-compartment
model:
PV(t) = dose/(a×BW)e-kt where a = PHE pool size (1)
Curves of enrichment following oral dosing (PG(t)) were fit with the plasma model modified according
to Cant et al. (2006) to include a first-order gastric emptying rate constant (kG), 100% absorption of
the oral dose, and constant percentage extraction (ex) of tracer on first pass through the splanchnic
bed, as:
PG(t) = dose×kG(1 - ex)/(a×BW(kG - k))(e-kt - e-kGt) (2)
Effects of route of administration were analyzed using a 2-tailed unpaired t-test, because one dog
was removed from the O data set. Differences were considered significant when P<0.05.

Data were normally distributed for both IV and O routes of isotope dosing. There were no differences
in plasma PHE concentrations (34 µmol/L ± 0.61) between groups (P>0.05). Typical decay curves for
PHE are presented in Figure 1. R2 values for the model fits ranged from 0.98-0.99 for IV and 0.99-
1.00 for oral dosing. The square root of mean square prediction errors, as a percentage of the mean,
ranged from 15-33 for IV and 15-25 for O dosing. Oral dosing resulted in a greater PHE turnover
time (1.403 vs. 0.724 h, P<0.05) and half life (0.972 vs. 0.502 h, P<0.05) than IV dosing. Using the

plasma elimination kinetics determined from IV dosing of each dog, the half-life for gastric emptying
was determined from the O dose to be 0.402 h, on average, and first-pass extraction by the splanchnic
bed was 49.4%. This figure is slightly higher than the 35% first-pass intestinal metabolism reported
in milk-fed piglets (Stoll et al., 1998), but the present results also account for the effects of hepatic
metabolism. When a single-exponential equation was fit to decay curves from both IV and O doses,
there were no differences in PHE pool size (9.97 µmol/kg ± 0.36) between groups (P>0.05). This
lack of difference indicates that O dosing is a useful and a comparable technique to IV dosing.
30
20
10
0
0 100 200 300 400 500 600 700
Time (minutes)
Figure 1. Typical plasma enrichment decay curves after a single bolus injection of 13C-PHE either
administered IV (n) or orally (*) to an Adult Spayed Female Hound Dog.
Implications 13
Figure 1. Typical plasma enrichment decay curves after a single bolus injection of C-PHE
either administered
Together, these data demonstrate IV () ordosing
that O isotope orally (*)
cantobeanused
Adult
in Spayed
dogs to Female Hound
determine AA Dog.
kinetics,
avoiding the use of the more invasive IV dosing. Furthermore, PHE kinetics have been defined in
the healthy, adult, female dog and we found that gastric emptying is 0.402 h and the splanchnic bed
accounts for ~ 49% of PHE turnover.
References
Cant, J.P., V.N. Walsh and R.J. Geor, 2006. Obtaining information on gastric emptying patterns in horses from appearance
of an oral acetaminophen dose in blood plasma. In: Kebreab, E., J. Dijkstra, A. Bannink, W.J.J. Gerrits and J.
France (editors), Nutrient Digestion and Utilization in Farm Animals: Modelling Approaches. CABI Publishing,
Stoll, B., J. Henry, P.J. Reeds, H. Yu, F. Jahoor and D.G. Burrin, 1998. Catabolism dominates the first-pass intestinal
metabolism of dietary essential amino acids in milk protein-fed piglets. J. Nutr. 128, 606-614.

Effect of feeding carefully dried and ensiled tanniniferous sainfoin
(Onobrychis viciifolia) on protein metabolism of lambs
A. Scharenberg1, Y. Arrigo1, A. Gutzwiller1, H.D. Hess1, U. Wyss1, M. Kreuzer2 and F. Dohme1
1Agroscope Liebefeld-Posieux Research Station ALP, Tioleyre 4, 1725 Posieux, Switzerland
2ETH Zurich, Institute of Animal Science, 8092 Zurich, Switzerland
Introduction
Condensed tannins (CT) are plant polyphenols with the ability to precipitate proteins. Furthermore,
they are not being degraded or absorbed in the gastrointestinal tract. These properties enable CT to
reduce ruminal protein degradability thus resulting in an increased flow of undegraded dietary protein
to the small intestine. The CT-protein complexes may dissociate at low pH in the abomasum. This
could change the quality of protein supplied to the host animal and eventually result in an improved
metabolic supply with essential amino acids (Min et al., 2003). Because of the diversity of CT and
feed proteins, it is difficult to accurately predict the effects of CT on protein utilisation in ruminants
and to give general recommendations for all diet types. Furthermore, controversial information is
available on the effects of feed conservation on the fate and effectiveness of CT in forages. While
Ott et al. (2005) observed a decline in CT by ensiling, Salawu et al. (1999) reported effects of CT
on the ensiling process but not on ensiled forages. The present study compared intake as well as
urinary and faecal excretions of nitrogen (N) of lambs fed dried or ensiled Onobrychis viciifolia
(sainfoin) compared both to sainfoin treated with polyethylene-glycol (PEG, a synthetic polymer
that neutralises CT) and to a conventional grass-clover mixture with low CT content.

The experiment was carried out with 12 lambs of the Swiss White Hill breed in a cross-over design,
with each lamb being consecutively fed 3 different diets (n=6). Sainfoin, either carefully dried or
ensiled, was tested against the same diets supplemented with PEG and against dried or ensiled
grass-clover-mixtures. Careful air drying at 35 °C was used to obtain conserved forage almost equal
in its nutritive value to fresh material. Daily, 66 g of forage organic matter were offered per kg of
metabolic bodyweight (BW0.75). The animals had free access to fresh water and received 20 g/d of
a commercial mineralised salt mixture for growing sheep. The PEG added to the feed amounted to
100 g per animal per d (>1 g PEG/g CT). Each experimental period consisted of a 14-d adaptation
period and a 7-d balance period. During the balance periods, daily forage intake and faecal and
urinary excretions were determined quantitatively. Samples were pooled across each balance period
and the N-contents were analysed. To analyse blood urea and rumen fluid ammonia, samples were
taken 2 h after feeding on the d before and after the balance periods. The butanol-HCl method as
described by Terrill et al. (1992) was used to analyse CT content in forages. To investigate the effect
of CT, a contrast model of SAS (Mixed) was used. This included comparing differences between
sainfoin with and without PEG and between sainfoin without PEG and the grass-clover mixture.
Besides, differences between dried and ensiled forages were evaluated.

As a consequence of the equal dry matter intake and the differences in N content between forages, N
intake was higher with sainfoin than with grass-clover (2.32 vs. 2.05 g/d and BW0.75 P<0.001). The
addition of PEG affected N utilisation in lambs fed sainfoin, which was consistent with results of
previous studies with tanniniferous feeds (Min et al., 2003). In particular, concentrations of ruminal
ammonia (14.8 vs. 11.3 mmol/L) and, blood urea (9.17 vs. 7.74 mmol/L), and urinary N excretion
(0.91 vs. 0.74 g/d and BW0.75) were increased (P<0.01) in lambs fed PEG-treated sainfoin. This

indicates that CT contained in sainfoin suppressed ruminal protein degradation. When expressed as
a proportion of total dietary N intake, N excretion did not differ (P>0.05) between the grass-clover
mixture and the PEG-treated sainfoin. Therefore, it seems that the differences observed between
sainfoin with and without PEG were exclusively due to the CT-neutralising effect of PEG and not
due to PEG effects on other dietary components. The addition of PEG did not affect (P>0.05) body
N retention, since the higher urinary N excretion was fully compensated for by a decreased faecal N
excretion. These findings confirm that CT increase N losses through faeces which could be related
to an incomplete dissociation of the CT-protein complexes in the small intestine and, therefore,
negatively affects animal performance. On the contrary, the lower ruminal ammonia and blood
urea levels indicate a lower level of liver strain, which could result in lower energy requirements
for ammonia detoxification. Condensed tannins in sainfoin increased) total plasma concentration
of free essential amino acids (1146 vs. 992 μmol/L, P<0.05), including the branched-chain amino
acids, but also of some non-essential amino-acids. This could be due to the increased intestinal flow
and absorption of ruminally undegraded feed protein (Waghorn et al., 1994). The fact that the serum
concentration of methionine, which is probably the first limiting amino acid, was not increased, might
explain the lack of effect on N balance. If this was the case, strategic methionine supplementation
might improve N balance in sainfoin-fed ruminants. Ensiling had no effect on N and fibre content of
the forages. However, it decreased the proportion of extractable CT and increased those of protein-
and fibre-bound CT in sainfoin, but it did not affect total CT content.Ensiling increased faecal N
excretion (0.96 vs. 0.75 g/d and BW0.75; P<0.001) and decreased N retention (0.54 vs. 0.65 g/d and
BW0.75; P<0.05), independent of the forage species.
In conclusion, CT in sainfoin obviously decreased ruminal protein degradation and urinary N losses,
increased faecal N losses, but did not affect N retention. However, the reduced energy requirements
for ammonia detoxification and the increased plasma concentration of essential amino acids could
be beneficial in animals fed diets containing sufficient methionine. Ensiling seems to increase the
CT effects of sainfoin to an extent that was undesired.
References
Min B.R., T.N. Barry, G.T. Attwood and W.C. McNabb, 2003. The effect of condensed tannins on the nutrition and
health of ruminants fed fresh temperate forages: a review. Anim. Feed Sci. Technol. 106, 3-19.
Ott E.M., A. Aragón and M. Gabel, 2005. Ensiling of tannin-containing sorghum grain. In: Park, R.S. and M.D. Stronge
(editors), Silage production and utilisation: Proceedings of the 14th International Silage Conference, a satellite
workshop of the 20th International Grassland Congress, Belfast, Northern Ireland, S.178.
Salawu M.B., T. Acamovic, C.S. Stewart, T. Hvelplund and M.R. Weisbjerg, 1999. The use of tannins as silage
additives: effects on silage composition and mobile bag disappearance of dry matter and protein. Anim. Feed Sci.
Technol. 82, 243-259.
Terrill T.H., A.M. Rowan, G.B. Douglas and T.N. Barry, 1992. Determination of extractable and bound condensed tannin
concentrations in forage plants, protein-concentrate meals and cereal-grains. J. Sci. Food Agri. 58, 321-329.
Waghorn G., I.D. Shelton, W.C. McNabb and S.N. McCutcheon, 1994. Effects of condensed tannins in Lotus
pedunculatus on its nutritive value for sheep. 2. Nitrogenous aspects. J. Agr. Sci. 123, 109-119.

Intake of fermentable fibre and body protein deposition in pigs fed
methionine or tryptophan limiting diets
C.L. Zhu1, M. Rademacher2 and C.F.M. de Lange1
1Department of Animal and Poultry Science, University of Guelph, Guelph, ON, Canada
2Degussa AG, Hanau, Germany
Introduction
It was demonstrated previously that dietary pectin (purified fermentable fibre) induces endogenous
protein secretions into the hindgut and reduces the efficiency of using ileal digestible threonine intake
for body protein deposition (PD) in pigs (Zhu et al., 2003 and 2005). Because of the relatively high
cysteine (CYS) content in endogenous gut protein, intake of fermentable fibre may also reduce the
efficiency of utilising ileal digestible intake of methionine (MET), or MET plus CYS, for PD. In
contrast, the content of tryptophan (TRP) in endogenous gut protein secretions is low and microbial
protein production in the upper gut may contribute to a net increase in TRP supply to the pig. Nitrogen
(N)-balance experiments were conducted to determine the impact of feeding additional pectin on
utilisation of ileal digestible intake of MET, MET plus CYS, and TRP for PD in growing pigs.

Two N-balance experiments were conducted to determine the impact of dietary pectin level and
dietary cellulose on PD in pigs fed cornstarch and soybean meal-based diets that were first limiting
in either MET (exp. 1) or TRP (exp. 2). In each experiment, 8 pigs were assigned to one of 5 diets
according to a partly replicated Latin Square design. Diets varied in added dietary pectin level (0,
4, 8, 12%) or contained 8% purified cellulose (a source of non-fermentable fibre; another control;
Zhu et al., 2005), with fibre sources replacing cornstarch. Pigs were adjusted to the experimental
diets for 9 d followed by a 5 d N-balance period (Zhu et al., 2005). Apparent and standardised
ileal digestible (SID) amino acid (AA) intake was calculated from analysed dietary AA contents
and previously determined ileal digestibility values (Zhu et al., 2005). The efficiency of utilising
SID intake of MET, CYS, and TRP for PD (K) was calculated (Zhu et al., 2005), assuming that the
content of these AA in PD was 1.8, 1.4, and 1.1% and that maintenance requirements were 10, 34,
and 9 mg/kg BW0.75/d, respectively.

The inclusion of cellulose in the diet did not influence PD and the efficiency of utilising SID MET,
MET plus CYS, or TRP intake for PD (P>0.10). With increasing dietary pectin level the utilisation
of SID intake of MET, and MET plus CYS, for PD was reduced linearly (P<0.05), while there was a
clear trend towards increased utilisation of SID TRP intake tryptophan for PD (P=0.08; linear). The
utilisation of SID intake of CYS for PD was not affected by dietary pectin level (P>0.10), suggesting
that the conversion of MET to CYS was increased with fermentable fibre intake. These observations
were consistent with previously threonine utilisation studies and increased endogenous protein
losses into the hindgut with increased intake of fermentable fibre (Zhu et al., 2003 and 2005). In the
current study, feeding fermentable fibre results in a net increase in supply of tryptophan to the pig;
this may be attributed to microbial production of tryptophan in the upper gut of pigs (Torrallardona
et al., 2003). The lack of response to intake of non-fermentable fibre suggests that diet effects on
AA utilisation are mediated via effects on enteric fermentation.

Table 1. The efficiency of using ileal digestible methionine or tryptophan intake for body protein
deposition in growing pigs fed soybean meal and cornstarch-based diets that are limiting in either
methionine or tryptophan with added pectin or cellulose.
Item Diets SEM Probability1
Added dietary pectin Cellulose Cellulose L Q
0% 4% 8% 12% 8%
Methionine intake, g/d

Apparent digestible 1.89 1.87 1.89 1.82 1.82 0.02 <0.01 0.01 0.15
Standardised digestible 1.95 1.94 1.95 1.88 1.88 0.02 <0.01 0.02 0.17
PD2, g/d 95.7 90.5 89.2 84.0 89.0 2.63 0.09 0.01 0.99
Kmethionine3, % 93.6 87.1 85.7 84.2 91.2 3.02 0.58 0.04 0.46
Tryptophan intake, g/d
Apparent digestible 1.43 1.43 1.37 1.28 1.38 0.01 0.04 <0.01 <0.01
Standardised digestible 1.55 1.56 1.50 1.40 1.51 0.01 0.06 <0.01 <0.01
PD2, g/d 89.9 89.3 94.0 88.8 82.9 2.85 0.14 0.92 0.47
Ktryptophan3, % 65.0 63.6 72.7 70.9 62.3 2.96 0.57 0.08 0.95
18% cellulose versus 0% pectin diet (Cellulose), linear (L) and quadratic (Q) effects of dietary pectin level; 2Whole
body protein deposition (N retention×6.25); 3Efficiency of using standardised ileal digestible AA intake above
maintenance requirements for PD.

Inclusion of non-fermentable fibre in the diet does not influence the use of SID intake of MET, MET
plus CYS and TRP for PD in growing pigs. In contrast, increasing the dietary level of fermentable
fibre reduces the efficiency of using SID intake of MET, and MET plus CYS, for PD and increases the
efficiency of using SID intake of TRP for PD. Feeding additional fermentable fibre to pigs increases
the dietary requirements for MET, or MET plus CYS, and appears to reduce the dietary requirements
of TRP. The impact of enteric fermentation on AA utilisation in pigs should be explored further.
References
Torrallardona, D., C.I. Harris and M.F. Fuller, 2003. Lysine synthesized by the gastrointestinal microflora of pigs is
absorbed mostly in the small intestine. Am. J. Physiol. Endocrinol. Metab. 284, E1177-1180.
Zhu, C.L., H. Lapierre, M. Rademacher and C.F.M. de Lange, 2003. Pectin infusion into the hindgut reduces the
utilization of threonine intake for body protein deposition and urea flux in growing pigs. In: Ball, R. (editor),
Proceedings 9th International Symposium on digestive Physiology in the Pig. Department of Agricultural, Food
and Nutritional Science, University of Alberta, Edmonton, Canada. Vol. 2, 346-348.
Zhu, C.L., M. Rademacher and C.F.M. de Lange, 2005. Increasing dietary pectin level reduces utilization of digestible
threonine intake, but not lysine intake, for body protein deposition in growing pigs. J. Anim. Sci. 83, 1044-
1053.

Influence of dietary benzoic acid on nitrogen metabolism in growing/
finishing pigs
K. Bühler, S. Gebert and C. Wenk
Institute of Animal Sciences, ETH Zurich, Universitätstrasse 2, 8092 Zurich, Switzerland
Introduction
Organic acids naturally occur in different organisms and are widely distributed as metabolic
byproducts in plants and animals. As feed additives in pig diets, organic acids are known to enhance
health and performance, which is induced by positive influences on gut microflora (Canibe et al.,
2001). Thus, organic acids are proven alternatives to antimicrobial growth promoters. One of these
acids is benzoic acid (approved 2003, EC877/2003) which is also a strong acidity regulator for urine
in pigs since it is excreted as hippuric acid.
The aim of this study (Bühler et al., 2006) was to determine whether dietary benzoic acid influences
N-metabolism, especially N-balance, in pigs and how this depends on dietary protein level. To gain
more information on this topic a balance study with growing/finishing pigs fed a low and a high
protein diet was conducted.

A total of 24 crossbred healthy barrows were individually housed in six series with an initial and final
body weight (BW) of 26 ± 1.2 kg and 105.6 ± 2.8 kg. The animals were fed restrictively one of four
cereal based diets according to a BW-based feeding scale (190 g feed*BW0.569): low protein diet
without and with 1% benzoic acid added (LP-/LP+), high protein diet without and with 1% benzoic
acid added (HP-/HP+). Water was available ad libitum. Benzoic acid was added as VevoVitall®,
DSM Nutritional Products, Basel. Calculated crude protein (Lys/DE) content in the grower period
(25 kg to 55 kg BW) was 165 g/kg (0.74 g/MJ) in the LP diets and 189 g/kg (0.94 g/MJ) in the HP
diets. The corresponding numbers in the finisher period (55 kg to 105 kg BW) were 134 g/kg (0.55
g/MJ) and 156 g/kg (0.71 g/MJ), respectively.
To estimate overall N-metabolism urine and faeces had to be collected separately. Therefore, the
pigs were kept in metabolic cages for four consecutive d every third wk. This rhythm guaranteed
two sampling periods in the grower and finisher period each.

Benzoic acid did not influence daily weight gain significantly (P=0.06) although it was about 40 g/d
higher in LP+ and HP+ diets than in LP- and HP- diets. In both feeding periods N intake and digested
N were only influenced by the protein level of the diet (P<0.01) but remained unaffected (P>0.2)
by dietary benzoic acid (Table 1). Neither protein level nor addition of benzoic acid affected the N
balance of the pigs (P>0.2). These results were consistent in both feeding periods. In the grower
period the addition of dietary benzoic acid increased N digestibility by almost 4% (P<0.01). No
effect on N digestibility was observed in the finisher period (P=0.30).
Although performance was similarly increased as it was in the study of van der Peet-Schwering
et al. (1999), the improvement was not significant. This may be due to the low number of animals
used in this experiment. N retention was unaffected by the addition of benzoic acid, too. Lysine
levels in diets HP- and HP+ were slightly higher than recommended whereas lysine levels in diets
LP- and LP+ were according to recommendation (Agroscope Liebefeld-Posieux, 2004). It seems

possible that the excess of proteins in the HP diets prevented an increase in N retention despite the
addition of benzoic acid. However, it is not clear, whether the increase in N digestibility indicates
a better N utilisation – and thus an additional nutritional value – by the pig. It may also point to a
reduction in excreted microbial N caused by effects of benzoic acid on the gastrointestinal microflora.
However, the scale of increase of microbial N excretion remains unclear, since the microflora and
its metabolites were not parts of this study.
Table 1. Weight gain (g/d), N metabolism (g/kg/BW0.75/d) and N digestibility in growing/finishing

pigs.
Diet1 SEM P-value2
LP- LP+ HP- HP+ P B P×B
Grower period
Weight gain 662 754 703 746 50 0.64 0.06 0.47
N intake 2.63a 2.74a 3.12b 3.03b 0.09 <0.01 0.83 0.08
N digested 2.46a 2.58a 2.94b 2.89b 0.08 <0.01 0.44 0.11
N balance 1.65 1.80 1.83 1.82 0.09 0.22 0.37 0.32
N digestibility 0.830a 0.856ab 0.843ab 0.879b 0.01 0.08 <0.01 0.61
Finisher period
Weight gain 807 838 839 874 40 0.17 0.17 0.94
N intake 1.99a 1.98a 2.28b 2.24b 0.03 <0.01 0.24 0.51
N digested 1.89a 1.88a 2.17b 2.15b 0.03 <0.01 0.43 0.61
N balance 1.18 1.19 1.20 1.15 0.05 0.94 0.72 0.58
N digestibility 0.865 0.872 0.876 0.888 0.01 0.16 0.30 0.75
1LP: Low protein diet; HP: High protein diet; -: without benzoic acid; +: with 1% benzoic acid.
2Effects of protein level (P), benzoic acid (B) and their interaction (P×B). Different superscripts in a row indicate
significant differences (P<0.05) among diets. SEM: Maximal Standard error of the means.
References
Agroscope Liebefeld-Posieux, 2004. Fütterungsempfehlungen und Nährwerttabellen für Schweine. LmZ, Zollikofen,
242S.
Bühler, K., C. Wenk, J. Broz and S. Gebert, 2006. Influence of benzoic acid and dietary protein level on performance,
nitrogen metabolism and urinary pH in growing-finishing pigs. Arch. Anim. Nutr. 60, 382-389.
Canibe, N., R.M. Engberg and B.B. Jensen, 2001. An overview of the effect of organic acids on gut flora and gut
health. Available: http://www-afac.slu.se/Workshop%20Norge/organic_acids_canibe_et_al.pdf. Accessed 10
April 2007.
Van der Peet-Schwering, C.M.C., N. Verdoes and J.G. Plagge, 1999. Influence of benzoic acid in the diet on performance
and urine pH of growing and finishing pigs. Report P 5.8, Englisch translation of Report P1 212, 1-24.
This study was supported by DSM Nutritional Products Ltd, CH – 4002 Basel.

Growth response of pigs to dietary threonine:lysine ratio is affected by
the withdrawal of anti microbial growth promoters
P. Bikker1, J. Fledderus1, L. le Bellego2 and M. Rovers3
1Schothorst Feed Research, PO Box 533, 8200 AM Lelystad, The Netherlands
2Ajinomoto Eurolysine s.a.s, 153 rue de Courcelles, 75817 Paris Cedex 17, France
3Orffa, Vierlinghstraat 51, 4251 LC Werkendam, The Netherlands
Introduction
Increasingly, worldwide animal feeds are produced without inclusion of anti-microbial growth
promoters (AGP). In previous studies we showed that the withdrawal of AGP may increase the
amino acid requirements for maximum growth performance in growing pigs (Bikker and Dirkzwager,
2003). That study did not allow us to conclude whether requirements of specific amino acids and
hence the optimal balance between essential amino acids is influenced by the usage of AGP. The
withdrawal of AGP may allow higher microbial growth in the digestive tract, increase endogenous
protein losses (mucus, enzymes, bacteria) and reduce ileal digestibility of amino acids. Because of
differences in the amino acid pattern for protein retention and endogenous protein, an increase in
endogenous losses may alter the optimal dietary amino acid pattern. This could especially increase
the optimal threonine:lysine ratio in the diet because of the high threonine content of endogenous
protein (e.g. Wang and Fuller, 1989). Therefore our hypothesis was that withdrawal of AGP from
the diet increases the threonine:lysine ratio for optimal animal performance. This hypothesis was
tested in growing-finishing pigs.
Methods
This study was conducted with 288 pigs of normal health status, in a 2×4 factorial experiment with
AGP (0 and 60/30 ppm salinomycin) and dietary threonine content as respective factors. The ratio
of standardised ileal digestible (SID) threonine to lysine, based on analysed amino acid content,
increased in four equidistant steps, 55, 60, 65 and 71%, by inclusion of graduated amounts of L-
threonine. Pens with six pigs, castrates and females, all mixed, were the experimental unit. The pigs
had free access to feed and water in a wet and dry feeder. The pigs received grower diets (9.5 MJ
NE, 7.9 g SID lysine/kg) and finisher diets (9.5 MJ NE, 6.7 g SID lysine/kg) from 25-45 and 45-
110 kg body weight, respectively. Dietary SID lysine was 10% below the estimated requirements.
At 110 kg body weight, the pigs were slaughtered and carcass composition was determined by an
optical probe (HGP). Linear and quadratic effects of threonine content and the interaction with AGP
were determined.
Results
The main effects of AGP and threonine content are presented in Table 1. In the entire period,
withdrawal of AGP reduced daily gain by 32 g/d and increased FCR by 0.06. Moreover, the dressing
percentage was lower in pigs receiving diets without AGP indicating a higher weight of the digestive
organs including gut contents. The main effects of threonine were most pronounced in the grower
phase with a linear increase in daily gain and a quadratic decrease in FCR. In the finisher phase
and the entire period, the main effects of dietary threonine were small. However, in these periods
we found a significant interaction between AGP and dietary threonine content. In diets including
AGP, threonine content did not significantly affect daily gain and FCR. However, in diets without
AGP increasing the threonine:lysine ratio linearly increased daily gain and quadratically decreased
the FCR (Figure 1).

Table 1. Main linear (L) and quadratic (Q) effects of threonine:lysine ratio and AGP in the diet on
growth performance from 25-110 kg body weight.
SID threonine:lysine, % AGP Effects1
55 60 65 71 yes no THR AGP INT
25-45 kg
Body gain, g/d 773 798 837 829 835 784 L*** *** ns
FCR 2.21 2.12 2.05 2.08 2.07 2.16 L***Q* ** ns
45-110 kg
Body gain, g/d 964 953 969 959 972 950 ns † L*
FCR 2.64 2.66 2.64 2.60 2.61 2.66 ns * Q†
25-110 kg
Body gain, g/d 905 908 934 923 934 902 L† *** L*
FCR 2.52 2.52 2.49 2.47 2.47 2.53 L† ** L†Q*
Dressing, % 78.6 78.6 78.6 78.3 78.9 78.1 ns ** ns
HGP lean, % 54.8 54.8 55.3 55.3 55.0 55.1 ns ns ns
1Statististical effects: † tendency, P<0.1, * P<0,05, ** P< 0,01, *** P<0.001; INT = interaction THR×AGP.
a b
gain + AGP gain - AGP FCR + AGP FCR - AGP
1000 2,7
Gain (25-110 kg), g/d
950
FCR (25-110 kg)
2,6
900
2,5
850
800 2,4
50 55 60 65 70 75 50 55 60 65 70 75
Stand. dig. threonine / lysine, % Stand. dig. threonine / lysine, %
Figure 1. Response relationship between dietary threonine:lysine ratio and (a) growth rate and (b)
feed conversion ratio, in growing pigs fed diets with and without AGP.
We concluded that the optimal dietary threonine:lysine ratio is higher in diets without AGP. The
results do not allow an exact quantification of this effect. Increased usage of amino acid in the
digestive tract may explain the increased response to dietary threonine.
References
Bikker, P. and A. Dirkzwager, 2003. Withdrawal of anti microbial growth promoters from pig diets increases the amino
acid requirements for growth performance. In: Souffrant, W.B. and C.C. Metges (editor) Progress in research on
energy and protein metabolism. Wageningen Academic Publishers, The Netherlands, EAAP 109, 593-596.
Wang, T.C. and M.F. Fuller, 1989. The optimum dietary amino acid pattern for growing pigs. 1. Experiments by amino
acid deletion. Br. J. Nutr. 62, 77-89.

5C. Multicriteria evolution of nutritional recommendations

Empirical modelling by meta-analysis of digestive interactions and CH4
production in ruminants
D. Sauvant and S. Giger-Reverdin
UMR INRA-AgroParisTech, Physiology of Nutrition and Feeding, 16 rue Claude Bernard, 75005
Paris, France.
Introduction
In ruminants, digestive interactions may alter the energy value of a mixture of forage (for.) and
concentrate (CO) (Vermorel and Coulon, 1998). They are mainly caused by feeding level (FL, in
DMI%LW) or CO proportion in the diet DM (0<CO<1). The target was to study the respective
influences of FL and CO and of their interactions, on energy digestibility (ED%GE), methane
energy (ECH4%GE), urinary energy (EU%GE) and metabolisable energy (ME%GE). All values
were expressed in % of gross energy (GE).

Two databases of energy balances performed either on growing or on lactating cattle, sheep and goats
were built. The first base pooled experiments focussed on the influences of FL (101 experiments,
290 treatments, FL=1.61±0.64, min=0.56, Max=4.01), the 2nd base pooled experiments focussed
on the impact of CO (87 experiments, 260 treatments, CO=0.39±0.26, min=0.0, Max=0.87). Meta-
analyses were performed to split among and within experiment influences, within relationships
were only considered.
Results and conclusion

Regressions are presented in Table 1 (standard deviation in brackets).
Table 1. Regression results.
Items Constant FL effect CO effect CO2 FL×CO rmse
ED%GE 72.6 –2.62 (0.19) 1.7

ECH4%GE 9.2 –1.36 (0.09) 0.8
EU%GE 5.7 –0.71(0.04) 0.4
ME%GE 57.6 –0.53 (0.21) 1.9
ED%GE 59.3 +21.22 (0.80) 2.7
ECH4%GE 6.4 +5.42 (0.80) –7.64 (1.10) 1.0
EU%GE 4.4 –1.12 (0.20) 0.6
EM%GE 48.6 +16.64 (2.30) +8.39 (2.9) 2.6
ED%GE 67.2 –2.88 (0.30) +20.83 (0.80) 2.1
ECH4%GE 7.78 –0.67 (0.20) +8.38 (8.38) –8.14 (0.80) –1.29 (0.30) 0.5
EU%GE 6.4 –0.80 (0.10) –2.96 (0.40) +0.62 (0.20) 0.6
EM%GE 48.7 +18.80 (2.7) +6.25 (3.4) 2.4
Influence of feeding level

FL presented a linear and negative influence on ED. The coefficient of regression was close to –2.9
which was obtained with another data base from experiments dealing with the FL influence on organic
matter digestibility in cattle (Sauvant, 2003). There was a compensation of almost half of the FL

influence on ED through its negative influence on CH4. The depressive effect on methanogenesis
likely resulted from a lower residence time of digesta in the rumen when FL increased (Michalet-
Doreau et al., 1997). There was also a compensatory influence of FL on EU. As a consequence, the
influence of FL on ME content was only of about 20% of that observed for ED. If maintenance is
assumed to be approximately achieved for FL=1 (frequent standard values for feed evaluation), it
is possible to calculate the influences of FL variations on the different predicted values above from
the ‘a’ coefficients: ∆FL = a (FL – 1). For instance, ED%GE was equal to 70.0 when FL=1, and to
64.8 when FL=3, the decrease of ED was of 5.2, or 7.4% of the standard value. For ME%GE the
corresponding values were 57.1, 56.0, 1.1 and 1.9%, showing that the influences of FL were much
less marked for ME than ED.
Influence of concentrate proportion

CO presented a positive and linear influence on ED. The linearity suggested that there was no
detectable interaction between the mean for. (EDf=59.3%) and CO (EDc=80.5%) of this relation. In
contrast, ECH4 varied curvilinearly in function of CO revealing interactions between for. and CO.
The maximum value (ECH4%GE=7.36) was achieved for CO=0.35. The range of variation among
experiments was more important for rations rich in CO For EU, the regression was negative and
linear. As a result of the above relationship on CH4, there was a positive and curvilinear influence
of CO on ME content. Thus, for a given level of COi the effect of interaction on ME was ∆COi=8.39
COi2. For instance, if COi=0.5, the theoretical value of ME%GE without interaction was of 56.8,
and with interaction it was of 58.9% because ∆COi=2.1% of GE, or 3.7% of this ME value.
Simultaneous influences of feeding level and concentrate proportion

In a third step, the two sub-bases were pooled. FL and CO influences were additive on ED. In contrast,
for CH4, there was a negative interaction between FL and CO and a confirmation of the curvilinear
influence of CO. Thus, the depressive effect of FL was more marked when diets were rich in CO.
Contrasting with ECH4, there was a positive interaction between FL and CO for EU. For ME, the
influence of FL and the interaction FL×CO were not significant. Thus, when all data were pooled,
there was no interaction between FL and CO at the metabolisable energy level and only the influence
of CO remained. This result on ME was the outcome of the compensations between the interactions
on ECH4 and EU. When CH4 production was expressed as L/kgDMI, the coefficients did not differ
from those previously obtained by Giger-Reverdin et al. (2000) on lactating ruminants.
Conclusion
This approach updates equations published on digestive interactions and simultaneous CH4 production
under the influences of FL and CO. CH4 production efficiently compensated for the influence of FL
on diet digestibility. When CO varied, the relationship between ED and CH4 appeared to be more
complicated with a high level of compensation for diets rich in CO When all data were pooled only
interactions due to CO level remained.
References
Giger-Reverdin, S., Sauvant D., Vermorel M. and Jouany, J.P., 2000. Empirical modelling of methane losses from
ruminants. Renc. Rech. Ruminants 7, 187-190.
Michalet-Doreau, B., C. Martin and M. Doreau, 1997. Optimization of fiber ruminal digestion: interactions of fiber
digestion with other dietary components. Renc. Rech. Ruminants 4, 103-112.
Sauvant, D., 2003. Modelling the effects of digestive and metabolic interactions on cattle. Renc. Rech. Ruminants
10, 151-158.
Vermorel, M. and J.B. Coulon, 1998. Comparison of the National Research Council energy system for lactating cows
with four European systems. J. Dairy Sci. 81, 846-855.

Evaluation of the German net energy system and estimation of the
energy requirement of cows on the basis of an extensive data set from
feeding trials
L. Gruber1, A. Susenbeth2, F.J. Schwarz3, B. Fischer4, H. Spiekers5, H. Steingass6, U. Meyer7, A.
Chassot8, T. Jilg9 and A. Obermaier10
1HBLFA Raumberg-Gumpenstein, Inst. for Livestock Res., Gumpenstein, AT-8952, Irdning,
Germany
2University of Kiel, Inst. of Anim. Nutr. and Physiology, Olshausenstr. 40, D-24118, Kiel,
Germany
3TUM München, Department for Anim. Sciences, Hochfeldweg 4-6, D-85350, Freising, Germany
4LVA Iden, Inst. for Agriculture, Forestry and Gardening, Lindenstrasse 18, D-39606, Iden,
Germany
5Landwirtschaftskammer Nordrhein-Westfalen, Endenicher Allee 60, 53115, D-Bonn, Germany
6University of Hohenheim, Inst. for Anim. Nutr., Emil-Wolff-Str. 8, D-70599, Stuttgart, Germany
7FAL Braunschweig, Inst. for Anim. Nutr., Bundesallee 50, D-38116, Braunschweig, Germany
8ALP Agroscope, Liebefeld-Posieux Res. Station, Rte de la Tioleyre 4, CH-1725, Posieux, France
9LVVG Aulendorf, Department for Anim. Production, Atzenberger Weg, 88326, D-Aulendorf,
Germany
10LfL Grub, Inst. for Anim. Nutr., Prof.-Dürrwaechter-Platz 3, D-85586, Poing, Germany
Introduction
The maintenance energy requirement (NEm) of the dairy cow is assumed to be 0.300–0.350 MJ net
energy (NEL)/kg metabolic live weight (LW0.75) in the systems established in Europe and the USA
(INRA, 1989; AFRC 1993; GfE 2001; NRC 2001). The efficiency of utilisation of metabolisable
energy (ME) for milk production (kl) is in the range of 0.60–0.63 in these systems. Recent results
of Agnew et al. (2003) indicate both a higher NEm and a higher kl.

A comprehensive data set (n=24 583; means of 2 lactation wk of individual cow measurements)
obtained from long term feeding experiments with lactating dairy cows carried out in 9 research
institutes in Germany, Austria and Switzerland (Gruber et al., 2005) was used to evaluate the current
German feeding standards (GfE, 2001). The experiments were carried out with Holstein Friesian,
Brown Swiss and Simmental cows and the data showed a wide variation in animal parameters
[(mean±SD, range); d in milk (138±78, 2–459), milk yield (24.3±8.1, 2.2–60.6 kg/d), feed intake
(18.5±3.5, 5.4–31.6 kg dry matter (DM)/d)] as well as nutritional factors [NEL content (5.9±0.5,
4.1–7.4 MJ/kg DM), proportion of concentrate in the diet (25.6±17.9, 0.0–81.1% of DM)]. The NEL
system was validated by regressing NEL requirement, calculated on the basis of its assumptions
[0.293 MJ NEL/kg LW0.75 for maintenance, NE in milk=0.38×fat+0.21×protein+0.95 (Tyrrell and
Reid, 1965), 25.5 MJ NEL for gain and 20.5 MJ NEL for loss of 1 kg LW, NEL pregnancy = (0.044×
exp(0.0165×d of gestation/0.175×0.6)], on actual NEL intake (MJ), considering the decrease of
dietary energy content with feeding level in the requirements (GfE, 2001). ME requirement in this
study was estimated using multiple regression analysis with LW0.75 (kg), milk energy output (LE,
MJ/d) and live weight change (LWC, kg/d) as independent variables.

The results of the validation of the NEL system are shown in Figure 1A. The regression equation
shows a bias of 3.7% of mean squared prediction error (MSPE) (mean NE requirement=124.5

MJ, mean NE intake=121.1 MJ) and an even higher error caused by a systematic deviation of the
regression line from 1 (10.9% of MSPE). In addition, there was a high prediction error (MPE=17.8
MJ NEL (=14.7%), 85.4% of MSPE). In order to find reasons for the relatively low correlation, a
multiple regression analysis was carried out relating ME intake (MJ/d) to LW0.75, LE and LWC.
Requirement for pregnancy was calculated according to GfE (2001) and subtracted from total ME
intake: MEintake= 0.652×LW0.75+1.41×LE+16.6×LWC; R²=0.711, RSD=24.1 MJ (Eq 1). The
results reveal a considerably higher maintenance energy requirement (MEm=0.652 MJ/kg LW0.75)
than in the other energy systems (ca. 0.500 MJ ME/kg LW0.75; INRA, 1989; AFRC, 1993; GfE,
2001; NRC, 2001), but they are in line with recent observations in Northern Ireland (0.600–0.660
MJ ME/kg LW0.75; Agnew and Yan, 2000; Agnew et al., 2003; FiM, 2004); kl is also higher than in
current systems (1/1.41=0.71). Agnew and Yan (2000) and Agnew et al. (2003) reported values of
kl of 0.64–0.69, based on literature data since 1976 and their own experimental results. The higher
kl value could be due to an increased proportion of ruminally undegraded nutrients in the actual
diets, resulting in decreased microbial fermentation losses and a relatively lower chewing activity
per kg DM (Susenbeth et al., 2004). Eq 1 gives a much lower estimate for the energy content for
mobilisation/retention of body reserves (ELWC=16.6×0.71=11.8 MJ/kg) than usually expected.
Furthermore, the relationship between LWC and calculated net energy balance is not significant
(Figure 1B). Actually, LWC cannot be regarded as a useful predictor of energy balance. In their
review, Agnew and Yan (2000) concluded that a fixed energy value for LWC as used in NRC and
European systems is incorrect since it varies with body condition (BCS) and change of lactation
(Tamminga et al., 1997). Unfortunately, BCS is not available in these data. Agnew and Yan (2000)
assumed that both an increased internal organ mass associated with higher feed intake and a higher
protein content of the body (lower body condition score) of high yielding dairy cows are possible
reasons for the enhanced maintenance energy requirement. This is supported by the present results
when considering an interaction term of [live weight×milk yield] in the following equation: ME
intake=(0.637+(0.0088×milk yield))×LW0.75+1.09×LE+ 16.7×LWC; R²=0.722, RSD=23.8 MJ (Eq
2). It is concluded that recent data from feeding trials provide evidence that current NE systems
for lactating dairy cows underestimate the energy requirement for maintenance and overestimate
the requirement for lactation, leading to a higher total energy requirement at lower milk yields and
a lower total requirement at higher yields (>30 kg/d). These findings should lead to more accurate
diet formulation for lactating dairy cows.
Calc. NEL requ. = 24.1 + 0.83 × NEL intake (MJ)
NEL balance (MJ) = 1.4 + 11.3 × LWC (kg)
R2 = 0.660, RSD = 17.8 MJ NEL (14.7%)
R2 = 0.114, RSD = 15.5 MJ NEL
MSPE: 3.7% bias, 10.9% line, 85.4 random
250 90
[calculated acc. to GfE 2001]
GfE (2001)
[calculated acc. to GfE 2001]
NEL requirement (MJ/d)
60
NEL balance (MJ/d)
200
30
150
0
100
-30
50
-60
0 -90
0 50 100 150 200 250 -1,5 -1 -0,5 0 0,5 1 1,5
NEL intake (MJ/d) Live weight change (kg/d)
Figure 1. Observed (A) NEL intake and calculated NEL requirement (based on GfE, 2001) and (B)
live weight change and calculated NEL balance.
For reference list, contact Dr L. Gruber at leonhard.gruber@raumberg-gumpenstein.at.

Development of a simple nutrient based feed evaluation model: net
energy versus glycogenic nutrient supply in predicting milk output
H. van Laar1, A. van Vugt1,2, T. van de Broek1,2, C. Soulet de Brugiere1 and J. Dijkstra2
1Nutreco Ruminant Research Centre, Veerstraat 32, P.O. Box 220, 5830 AE, Boxmeer, The
Netherlands
2Wageningen Institute of Animal Sciences, Animal Nutrition Group, Wageningen University, P.O.
Box 338, 6700 AH Wageningen, The Netherlands
Introduction
Current feed evaluation systems for dairy cows predict milk output based on metabolisable or net
energy (NE) supply to the animal. However, the amount of milk produced is mainly governed by the
synthesis of lactose for which glucose is the sole precursor. This paper focuses on the development of
a simple, static nutrient based feed evaluation model that predicts the supply of glycogenic nutrients
(GN) and its potential to predict milk lactose production. The main goal of this model was a broad
applicability in practical diet optimisation.

A dataset of more than 8000 weekly averages of individual intake and production for 2 yr for all
the cows at the Nutreco ruminant research farm was used to calculate NE with the Dutch energy
system (VEM, CVB, 2005), and GN supply (g/d) according to a previously described nutrient
based model (Van Laar et al., 2005). This model calculates GN from three sources, (i) the supply
of propionic acid, estimated with a simple degradation and passage rate based rumen model for
fermentable carbohydrates and protein, (ii) intestinal digestible bypass starch, with starch digestibility
depending on the bypass level of starch, and (iii) intestinal digestible protein that is not used for
milk protein synthesis. To obtain NE available for production (NEp), supply of NE was corrected
for body weight (BW) and pregnancy according to CVB (2005). GN for production (GNp) was
calculated by correcting for maintenance (4 g/kg BW0.75/d; Dijkstra et al., 1996) and pregnancy.
GN requirements for pregnancy were assumed to be negligible up to 125 d of pregnancy, linearly
increasing to 718 g/d at 270 d, modified from Overton (2003). GN available for the production of
lactose (GNpl) was calculated by correcting GNp with the GN requirement for milk fat, assuming
0.12 g GN for glycerol and 0.19 g GN for liponeogenesis per g of milk fat (Dijkstra et al., 1996).
The energy balance (EB) was calculated by correcting NE supply with NE requirement for milk
production, pregnancy and BW. The supply of NE, GN, NEp, GNp and GNpl were regressed to milk,
fat and protein corrected milk (FPCM) and lactose production using linear regression, furthermore
the additional effect of EB was investigated.

Table 1 displays the results of the linear regression between milk lactose production and NE or GN
supply. The R2 of the relationship between NE and lactose production was numerically higher than
for GN. These R2 values were higher than that between DMI and lactose production (0.30). The R2
of regressions between NE or GN and FPCM were numerically lower than with lactose production
(results not shown). BW and pregnancy correction raised the R2 of the regression of NEp or GNp
with lactose production to similar levels. Correcting GNp for glucose required to synthesise milk
fat (GNpl) decreased the R2 value, indicating that a fixed requirement per unit milk fat produced is
not valid.

Table 1. Intercept, slope and explained variation (R2) for the linear regression of milk lactose (g/d)
production with net energy (NE; VEM1/d), glucogenic nutrients (GN; g/d) and energy balance (EB;
VEM/d) either as such or corrected for production (NEp, GNp) or milk fat (GNpl).
Intercept NE GN NEp GNp GNpl EB R2
Lactose -180 0.0722 0.39

-55 0.511 0.35
190 0.0745 0.43
212 0.52 0.43
510 0.476 0.29
-2 0.66 -0.080 0.80
69 0.79 -0.101 0.80
1VEM, Dutch energy unit 1 VEM = 6.9 kJ net energy for lactation.
When the data were split into different stages of lactation, R2 for both NE and GN were the highest
for early lactation (around 0.50) and the lowest for end lactation (around 0.25). These results
indicate that when working with individual cow wk data, EB, with body mobilisation in early and
body accretion in late lactation, has a major impact on the prediction of production from intake. The
regression of GNp or GNpl and EB on lactose production gives an estimate of the supply and cost of
GN per energy unit of body mobilisation or accretion. According to this regression, 1 gram of GNp
or GNpl provides respectively 0.66 and 0.79 gram of milk lactose. Depending on the model, 1000
VEM units (6.9 MJ NE) of EB is associated with an increase (negative EB) or decrease (positive
EB) of 80 to 101 g of milk lactose. Using the regression coefficients from GNp or GNpl this would
equal 120 to 127 g of GN.
Conclusions
GNp, but not GN, rivals NE for the prediction of lactose production. Further improvements in
the description of nylon bag rumen degradation characteristics of individual feed ingredients and
an improved description of the fate of small intestinal digestible starch, may improve the model.
Furthermore, the results indicate that EB can represent a cost or source of GN.
References
CVB, 2005. Veevoedertabel 2005, gegevens over chemische samenstelling, verteerbaarheid en voederwaarde van
voedermiddelen.[in dutch]. CVB, Lelystad, The Netherlands.
Dijkstra, J., France, J., Neal, H.D.St.C., Assis, A.G., Aroeira, L.J.M. and O.F. Campos, 1996. Simulation of digestion
in cattle fed sugarcane: model development. J. Agric. Sci. Camb. 127, 231-246.
Overton, T.R., 2003. Managing the metabolism of transition cows. Proceedings of the 6th Western Dairy Management
Conference, Reno, NV, USA.
Van Laar, H., R. Meijer, K. Mulder, W. Burema and M. Brok, 2005. Development of a simple nutrient based feed
evaluation model for dairy cows. In: Garnsworhty, P.C. and J. Wiseman (editors), Recent Advances in Animal
Nutrition 2004. Nottingham University Press, Nottingham, UK, 277-297.

A new practical feed evaluation system for pigs
S. Boisen1 and P. Tybirk2
1Danish Institute of Agricultural Sciences, Department of Animal Nutrition and Physiology, Research
Centre Foulum, P.O. Box 50, DK-8830 Tjele, Denmark
2Danish Agricultural Advisory Centre, DK 8200 Aarhus, Denmark
Introduction
A new feed evaluation system for pigs has been implemented in practical pig production in Denmark
in 2006 (Tybirk et al., 2006). The annual production of slaughter pigs is about 25 million, i.e. almost
five times the number of inhabitants. Correct feeding of pigs is, therefore, essential for a sustainable
production in Denmark.
The purpose of this short paper was to give a brief description of the new basic principles for feed
evaluation as well as the implementation of the system in the practical pig production.
Basic principles for the Danish feed evaluation system

The energy value of feedstuffs and diets is based on the potential physiologically available energy
(PPE) from the relevant nutrient fractions, i.e. starch, crude protein, crude fat, and fermentable
carbohydrates. PPE is based on the production of ATP, which is the fundamental energy source for
all living organisms. ATP production from all nutrient fractions is precisely described (Boisen and
Verstegen, 2000).
The protein value of feedstuffs and diets is based on standardised digestible amino acids (SDAA)
and are, furthermore, related to their contribution to the ideal protein for the specific pig category
(Boisen, 1998).
Actual batches of feedstuffs are analysed for organic matter digestibility (OMD) corresponding to
ileal and faecal level, respectively. The analyses are based on two laboratory methods that simulate
digestion using digestive enzymes corresponding to feed degradation in the stomach, small intestine
and hindgut, respectively (Boisen and Fernandez, 1995 and 1997). The difference between the results
for the two analysis methods corresponds to the fermentable fraction of carbohydrates. The PPE
of the different fractions are compared to energy values corresponding to different national feed
evaluation systems based on net energy (NE) in Table 1.
In practical feed evaluation, the relative energy values for the different nutrient fractions are central for
optimisation of diets for the actual purpose. Although the Danish system is based on new principles
the relative energy values are quite similar to those based on NE.
However, while energy factors in NE systems are based on regression analyses between feed
composition and production results, obtained under specific experimental conditions, energy factors
in the new PPE system is based directly on the properties in the actual feed samples. Furthermore,
the system includes a new fraction, i.e. EIDMi, which has a negative energy factor caused by an
increased energy cost from digestion processes (see Table 1).
It follows, that feed evaluation in the new Danish system is based on a very simple system, which
includes two new analysis methods for analysing the digestibility of the actual samples. Furthermore,
the defined feed value is only related to the feed itself. Finally, the recommendations for the feed
composition are directly related to the actual and specific pig production.

Table 1. Energy value (in MJ/kg and relative to starch, respectively) of main nutrient fractions
in different feed evaluation systems for growing pigs based on net energy (NE), ATP energy from
potentially retained nutrients (NER), and potentially available physiological energy (PPE).
NE1 NER PPE

Former Dutch (1993) French Rostock General New Danish3
Danish (2000) (2003)2 (2000)1
Starch 10.7 (100) 13.5 (100) 14.4 (100) 12.7 (100) 11.7 (100) 11.7 (100)
FMC4 10.7 (100) 9.5 (70) 12.1 (84) -5 7.0 (60) 7.0 (60)
Crude 13.2 (123) 10.8 (80) 11.3 (78) 11.0 (87) 10.4 (89) 9.9 (85)
protein
Crude fat 23.4 (219) 36.1 (267) 35.0 (243) 27.0 (213) 26.1 (223) 31.7 (271)
EIDMi6 - - - - - - 2.8 (-24)
1Boisen and Verstegen (2000).

2Jentsch et al. (2003).
3Tybirk et al. (2006).
4Fermentable carbohydrates.
5Corresponding fraction calculated from the equation: (12 – 0.14 (80 – dE)) dNFR; dE: faecal digestibility of
energy; dNFR: digestible N-free residue, g (dNFR = digestible organic matter – (dCP + dCF + dST + dSU).
6Enzyme indigestible dry matter at the ileal level.
Consequently, the new Danish feed evaluation system is a basic and flexible system. It can be further
improved when more knowledge is obtained on specific properties of actual batches of individual
feedstuffs, e.g. specific effects of dietary fibre, anti-nutritional compounds, as well as of specific
effects of diet composition on animal health, behaviour and activity, which may influence nutrient
requirements and production results.
An international agreement on the basic principles for feed evaluation, as well as of a common
practical feed evaluation system, would offer the optimal conditions for future concerted actions
for further developments within pig research.
References
Boisen, S., 1998. A new protein evaluation system for pig feeds and its practical application. Acta Agric. Scand., Sect.
A, Anim. Sci. 48, 1-11.
Boisen, S. and J.A. Fernandez, 1995. Prediction of the apparent ileal digestibility of protein and amino acids in feedstuffs
and feed mixtures for pigs by in vitro analyses. Anim. Feed Sci. Technol. 51, 29-43.
Boisen, S. and J.A. Fernandez, 1997. Prediction of the total tract digestibility of energy in feedstuffs and pig diets by
in vitro analyses. Anim. Feed Sci. Technol. 68, 277-286.
Boisen, S. and M.W.A. Verstegen, 2000. Developments in the measurement of the energy contents of feed and energy
utilisation in animals. In: Moughan, P.J., M.W.A. Verstegen and M. Visser (editors), Feed evaluation. Principle
and practice. Wageningen Pers, Wageningen, The Netherlands, 57-76.
Jentsch, W., A. Chudy and M. Beyer, 2003. Rostock feed evaluation system. Plexus Verlag, Miltenberg-Frankfurt,
392 pp.
Tybirk, P., A.B. Strathe, E. Vils, N.M. Sloth and S. Boisen, 2006. The new Danish feed evaluation system for pig feeds
(in Danish). Danish Pig Production, 76 pp.

Prediction of the metabolizable energy intake and energy balance of
goats with the Small Ruminant Nutrition System model
A. Cannas1, L.O. Tedeschi2 and D.G. Fox3
1Dipartimento di Scienze Zootecniche, University of Sassari, 07100 Sassari, Italy
2Department of Animal Science, Texas A&M University, College Station, TX 77843, USA
3Department of Animal Science, Morrison Hall, Cornell University, Ithaca, NY 14853, USA
Introduction
A computer model (Small Ruminant Nutrition System, SRNS) to predict site-specific nutrient
requirements and feed biological values for sheep was developed, based on the structure of the
Cornell Net Carbohydrate and Protein System for Sheep (Cannas et al., 2004 and 2006). The SRNS
model uses animal and environmental factors to predict energy, protein, Ca and P requirements.
Feed biological values are predicted based on carbohydrate and protein fractions and digestion
rates, forage, concentrate and liquid passage rates, and microbial growth. In the SRNS for goats,
energy requirements are predicted based on the equations developed for the SRNS for sheep,
modified to account for specific requirements of goats. In this paper, the predictions for adult goats
of metabolisable energy intake (MEI) and energy balance (EB) by the SRNS were evaluated.
Model description and evaluation

In the SRNS, the energy requirement for basal metabolism, expressed as MEm, is adjusted for age,
physiological state, environmental effects, activity, urea excretion, acclimatization and cold stress
in order to estimate total NEm and MEm as shown in Equation 1.
MEm = ((SBW0.75 × a1 × a2 × exp(-0.03 × AGE)) + (0.09 × MEI × km) + ACT + NEmcs + UREA) / km (1)
where MEm is in Mcal/d; and SBW0.75 is metabolic shrunk body weight, kg. The factor a1 is the
thermal neutral maintenance requirement per kg of metabolic weight for fasting metabolism; it is
assumed to be 0.325 and 0.273 MJ of NEm/kg0.75 for dairy goats and for other breeds, respectively.
The term (0.09 × MEI × km) accounts for the increase in the size of the visceral organs as nutrient
intake increases. The factors a2, AGE, ACT, NEmcs and UREA are adjustments described by Cannas
et al. (2004). The efficiency coefficient km is fixed at 0.644. Metabolisable energy requirements
for milk production and pregnancy are estimated as described by Cannas et al. (2004). The energy
available for growth or for body reserve changes depends on the EB after maintenance, lactation, and
pregnancy requirements are satisfied. The predictions of the SRNS on MEI and EB were evaluated
using 5 published studies in which balance experiments including indirect calorimetric measurements
(2 to 8 d) on lactating does (15 treatment means: Aguilera et al., 1990. Brit. J. Nutr. 63, 165-175.;
Rapetti et al., 1997. Zoot. Nutr. Anim. 23, 317-328; Rapetti et al., 2002. Ital. J. Anim. Sci. 1, 43-
53; Rapetti et al., 2005. Ital. J. Anim. Sci. 4, 71-83) and wethers (6 treatment means: Ngwa et al.,
2007. Small Rumin. Res., in press.) were performed. The evaluations were carried out using the
mean BW, dry matter intake (DMI) and diet composition, and milk yield and composition reported
in these publications as inputs in the SRNS. The assessment of the adequacy of the models was
carried out with the Model Evaluation System, which is based on statistical techniques as discussed
by Tedeschi (2006).
Results
The publications used to evaluate the SRNS reported only part of the information on feed composition
required by the model; therefore, many values had to be estimated. The SRNS predictions were

highly accurate for daily MEI when DMI was an input (Table 1). Milk NE also had high accuracy
(Table 1). The ME balance, which was calculated as the difference between MEI and ME requirements
for maintenance and lactation, was converted by the SRNS to NE using the kg value of 0.6 both
for lactating and dry adult goats. The SRNS under-predicted the NE balance (Table 1). A similar
problem was observed by Cannas et al. (2006) evaluating the SRNS ability to predict the growth
rate of lambs. They observed that the main cause of the under-prediction was that the SRNS over-
estimated the maintenance requirements because of the term (0.09 × MEI × km) in Eq. 1. For this
reason, the EB of adult goats was estimated without the production adjustment factor. Indeed, this
markedly improved the prediction of the EB (Table 1). The RMPSE was almost reduced by half
and the CCC reached 0.90.
Table 1. Comparison of observed (O) and predicted (P) daily MEI (n=21), milk NE (n=15), and
net EB (NEB; n= 21)1.
Variables (MJ/d) P P–O MSEP partition, % RMSEP r2 Cb ρc
Ub Us Ur
MEI 16.757 -0.167 2.8 7.1 90.1 0.987 0.99 1.00 0.99
Milk NE 6.962 0.343 65.4 11.3 23.3 0.427 0.99 0.99 0.99
NEB 0.213 -1.297 61.0 7.6 31.4 1.669 0.79 0.72 0.64
NEB, no MEI adjustment 1.197 -0.314 13.8 7.8 78.4 0.841 0.87 0.97 0.90
1MSEP = mean squared error of prediction; RMSEP = root of the MSEP; r2 = coefficient of determination of the best
fit regression line not forced through the origin; Cb is accuracy of the model; ρc is concordance correlation coefficient
(CCC); Ub = mean bias; Us = systematic bias; and Ur = random error (Tedeschi, 2006).
Conclusions
The SRNS for goats was able to accurately and precisely predict both the MEI and the EB of lactating
and non-lactating adult goats.
References
Cannas A., L.O. Tedeschi, D.G. Fox, A.N. Pell and P.J. Van Soest, 2004. A mechanistic model to predict nutrient
requirements and feed biological values for sheep. J. Anim. Sci. 82, 149-169.
Cannas, A., L.O. Tedeschi, A.S. Atzori and D.G. Fox, 2006. Prediction of energy requirement for growing sheep with
the Cornell net carbohydrate and protein system. In: Kebreab, E., J. Dijkstra, A. Bannink, W.J.J. Gerrits and J.
France (editors), Nutrient digestion and utilization in farm animals: modelling approaches. CAB International,
Tedeschi, L.O., 2006. Assessment of adequacy of mathematical models. Agric. Syst. 89, 225-247.

Inter- and intraindividual variation of feed intake and metabolic
parameters of dairy cows related to energy supply
U. Meyer1, K. Horstmann2, M. Kaske3 and G. Flachowsky1
1Federal Agricultural Research Centre (FAL), Institute of Animal Nutrition, Bundesallee 50, D-
2Clinic for Cattle, University of Veterinary Medicine Hannover, Bischofsholer Damm 15, D-30173
Hannover, Germany
3Physiology Weihenstephan, Technical University Munich, Weihenstephaner Berg 3, D-85354
Freising, Germany
Introduction
Early lactation represents the period with the highest risk for production diseases such as hypocalcemia,
retained placenta, endometritis, ketosis, mastitis, and claw diseases, throughout the reproductive cycle
of dairy cows (Fleischer et al., 2001). Metabolic disturbances have in particular been attributed to
insufficient energy supply during the first wk post partum (p. p.) because of the delayed increase of
feed and energy intake in the light of high milk production. The subsequent mobilisation of body
adipose tissue is usually associated with a distinct reduction of body weight (BW) and body condition
score (BCS). The objective of this study was to demonstrate the inter- and intraindividual variation
of feed and energy intake, BW, BCS as well as the variation of some metabolic indicator parameters
of dairy cows during early lactation.

The experiment was carried out with 20 clinically healthy multiparous Holstein cows in a free stall
barn with cubicles at the FAL Experimental Station (Braunschweig). The cows had an initial BW of
607 ± 44 kg and a milk yield of 9338 ± 1089 kg fat corrected milk (FCM) in the previous lactation.
They were fed a mixture of corn silage and alfalfa silage (65:35 on dry matter basis) for ad libitum
consumption. To meet the energy and protein requirements (GfE, 2001) concentrates were offered
according to milk yield by automatic concentrate feeders. The concentrates mainly consisted of
soybean meal, barley, wheat, sugar beet pulp, soybean oil, and a mineral premix. Animals had free
access to water.
Feed intake was recorded by a computerised feeding system (Insentec, Marknesse, The Netherlands).
The cows were milked twice daily. In the first 12 wk p. p. milk yield, feed intake and body weight
were assessed daily, venous blood samples were taken weekly, liver biopsies were obtained two wk
prior to calving and on the d 1, 15, 29 and 43 p. p., respectively. For each animal, intake characteristics
were calculated as means for each wk of lactation. The BW was measured after each milking, BCS
(1 to 5 scale) was assessed weekly.
In order to calculate the energy intake of the cows, the nutrient digestibility of the silages was
determined in balance experiments with wethers (GfE, 1991), and the energy concentration of the
diet was calculated on the basis of the digested nutrients according to the regression equations as
given in the GfE guidelines (2001).
Results
Beginning from 9.5 kg dry matter (DM)/d in the first wk the average silage intake reached a plateau
at about 11.5 kg DM/d already four wk after parturition. The concentrate intake increased until wk
6 of lactation and a steady level of about 11 to 12 kg DM. Mean DM intake increased gradually up

to about 22 kg/d during the first four wk of lactation. The interindividual variation of DM intake was
impressive during the first wk of lactation with individual values ranging from 11 to 31 kg DM/d.
The coefficient of variation (CV) of silage intake was found to be larger in the first wk of lactation
(40%) than in the following wk (27%). The interindividual CV of concentrate intake was distinctly
lower and averaged out at 20%. The cows reached maximum energy intake in wk 8 p. p. (170 MJ
NEL/d). The distinctive negative energy balance in the first wk of lactation (on average -40 MJ NEL/d
in wk 2 p. p.) was balanced in wk 7 p. p. The CV for energy intake was not influenced by the wk of
lactation and amounted to 18%. High correlations were found between energy and DM intake in wk
1 to 4 p. p. and energy and DM intake in wk 5 to 8 and 9 to 12 p. p. (P<0.001), respectively.
Average milk yield peaked at wk 5 of lactation and reached 40.5 kg FCM/d. After 12 wk the daily
milk yield declined to 36 kg FCM. The CV of milk yield amounted to 14% and remained unchanged
during the whole experimental period. Due to the post partum delay in feed intake, the average BW
and BCS declined slightly from 625 to 590 kg and from 3.0 to 2.75, respectively. The nadir of BW
and BCS was reached in the third and seventh wk p. p..
The changes of blood parameter and liver triglyceride content exposed only a moderate mobilisation
of fat during the first wk of lactation. The mean results for the measured variables during the first 12
wk p. p. were as follows: 240 μmol/L non-esterified fatty acids (NEFA), 5 μU/L insulin, 0.6 mmol/L
beta-hydroxybutyrate (BHBA) and 45 mg/g liver triglyceride content. No correlations were found
between blood concentrations of NEFA, insulin and BHBA on the one hand and the extent of energy
intake or –balance on the other.
Conclusions
Under the conditions of the present experiment with multiparous Holstein cows, different blood
parameters (NEFA, insulin, BHBA) did not refer to the energy intake or the energy balance of the
animals. The loss of body condition was linked to lower body weight. For this reason the determination
of BCS might be an easy way to estimate the energy status of the cows.
References
Fleischer, P., M. Metzner, M. Beyerbach, M. Hoedemaker and W. Klee, 2001. The relationship between milk yield and
the influence of some production diseases in dairy cows. J. Dairy Sci. 84, 2025-2035.
GfE (Gesellschaft für Ernährungsphysiologie), 1991. Leitlinien für die Bestimmung der Verdaulichkeit von Rohnährstoffen
an Wiederkäuern; herausgegeben vom Ausschuss für Bedarfsnormen der Gesellschaft für Ernährungsphysiologie,
Frankfurt. J. Anim. Physiol. a. Anim. Nutr. 65, 229-234.
GfE (Gesellschaft für Ernährungsphysiologie), 2001. Energie- und Nährstoffbedarf landwirtschaftlicher Nutztiere,
Nr. 8, Empfehlungen zur Energie- und Nährstoffversorgung der Milchkühe und Aufzuchtrinder. DLG-Verlag,
Frankfurt (Main), 136 pp.

5D. Modelling and meta-analysis

Assessment of duodenal starch as a predictor of portal absorption of
glucose in ruminants
C. Loncke1, I. Ortigues-Marty1, J. Vernet1, H. Lapierre2, D. Sauvant3 and P. Nozière1
1INRA UR 1213 Herbivores, Site de Theix, 63122 St Genès Champanelle, France
2Dairy and Swine Research and Development Centre, Agri & Agri Food Canada, Lennoxville,
Quebec JIMIZ3 Canada
3INRA UMR 791, Physiologie de la Nutrition et Alimentation, INAPG, 75231 Paris, France
Introduction
To adapt the feeding strategies for ruminants to new demands of animal production, important and
systematic advances have been made in the characterisation of feed chemical composition. A further
advance will be to predict the amount and nature of absorbed nutrients. In 2007, Bermingham et al.
studied by meta-analysis the responses to increasing digestible organic matter intake of the net portal
appearance (NPA) of energetic nutrients. Significant response equations were obtained for the NPA
of volatile fatty acids but not for glucose. Glucose appearance in the portal vein is highly dependent
on dietary duodenal starch and of portal drained viscera metabolism (PDV). The objectives of this
work were to establish the responses of the NPA of glucose to calculated duodenal starch escaping
from rumen fermentation.

The FLORA (Vernet and Ortigues-Marty, 2006) database, which is an exhaustive data base from
the 150 international publications presenting results of net splanchnic nutrient fluxes in ruminants,
was used. For the present work, six datasets were selected out of FLORA. For the first four
datasets, experimental diet groups were coded according respectively to changes in dry matter intake
(‘DMI’), in percentage in concentrates (‘conc’), in percentage of concentrates and dry matter intake
(‘concDMI’) and in the nature of concentrates (‘nat’). Experiments which presented an abosamal
or duodenal infusion of glucose (‘glucose inf’) or starch (‘starch inf’) were also coded. Quantity of
duodenal starch (or glucose) was defined as the sum of the amount of starch (or glucose) infused
plus the amount of dietary starch escaping rumen degradation, estimated according to INRA-AFZ
Feed Tables (Sauvant et al., 2004), as starch intake x (1- in sacco starch degradability), assuming a
particulate passage rate of 0.06h-1. In each data set, changes in duodenal starch (or glucose) were
defined as a variation between control and experimental treatments within an experiment greater
than 0.5 g/d/kg BW. The data were submitted to meta analyses (Sauvant et al., 2005). In order to
study relationships between net portal flux of glucose (Y) and duodenal starch (or glucose), a nested
covariate model on species was used: Y = α + βX + species + study (species) + (species x X) + e
(where e=error). The interaction (species x X) was not significant and thus removed from the model.
All statistical analyses were carried out using the GLM model (Minitab, Version 14).
Results
The number of experimental groups selected ranked from 4 to 8 for the different datasets (Table 1),
except for dataset ‘nat’, where the 2 publications available were considered insufficient. Duodenal
glucose (or starch) in control treatments averaged 1.35, 0.72, 0.60, 0.47 and 0.59 g/d/kg BW, and
were increased by 0.75, 2.93, 1.74, 2.28, and 2.47 g/d/kg BW in experimental treatments, for ‘DMI’,
‘conc’, ‘concDMI’, ‘glucose inf’ and ‘starch inf’ data sets, respectively. An increment of duodenal
starch due to changes in DMI decreased NPA of glucose (Table 1). The model obtained was not
robust with a R2 equal to 49.5% and despite a low residual error (S=0.04). In contrast, an increase
in duodenal starch (or glucose) due to changes in percentage of concentrates, glucose or starch

infused, significantly increased NPA of glucose. The models obtained were robust. Interestingly, the
response slopes were similar when duodenal starch increased thanks to ‘conc’, ‘concDMI’ ‘starch
inf’ and lower than with ‘glucose inf’.
Table 1.Linear (β) response equations for the relationships between the duodenal starch (or
glucose)(g/d/kg BW) and the net portal appearance of glucose (mmol/h/kg BW).
Dataset n gp n tr n bv α se β se S R2(%)
DMI 7 15 11 0.084* 0.036 -0.054* 0.021 0.04 49.5

conc 8 17 10 -0.081*** 0.021 0.043*** 0.007 0.05 82.8
concDMI 8 19 8 -0.142** 0.036 0.050* 0.019 0.08 44.7
glucose inf 6 15 12 -0.153** 0.045 0.137*** 0.021 0.10 81.2
starch inf 4 10 10 -0.125** 0.021 0.057*** 0.009 0.04 91.7
Probability of significance: *P<0.05; **P<0.01; ***P<0.001; gp: experimental group; tr: treatments; bv: bovine.
Discussion
The results show that the variation in glucose NPA averaged -23%, 19%, 22%, 59%, 25% of the
variations in duodenal starch (or glucose) with ‘DMI’, ‘conc’, ‘concDMI’, ‘glucose inf’, and ‘starch
inf’, respectively. The similarity of response between ‘conc’, ‘concDMI’ and ‘starch inf’ datasets
indicates that INRA-AFZ feed tables allow to predict duodenal starch and NPA of glucose. The slope
for ‘glucose inf’ is higher than for the ‘starch inf’ dataset, probably because incomplete hydrolysis
of starch may limit intestinal absorption of glucose. The negative slope for ‘DMI’ may reflect the
increase in glucose utilisation by PDV induced by the increase in digesta mass, and may explain
the lower precision (lower R2) for ‘concDMI’ than for ‘conc’. The surprising positive intercept
with ‘DMI’ was related to the high level of duodenal starch with control treatments in this group.
In conclusion, duodenal starch, as estimated from INRA Feed Tables, appears as a reliable tool for
prediction of glucose reaching the liver.
References
Bermingham, E.N., I. Ortigues-Marty, J. Vernet, H. Lapierre, S. Léger, D. Sauvant and P. Nozière, 2007. The
relationships between intake and portal fluxes of energy metabolites in ruminants: a meta analysis. Anim. Feed.
Sci. Technol., in press.
Vernet, J. and I. Ortigues-Marty, 2006. Flux of nutrients through organs and tissues in Rruminant animals. Reprod.
Nutr. Dev. 5, 257-546.
Sauvant, D., J.M Perez., G. Tran (editors), 2004. Tables de composition et de valeur nutritive des matières premières
à destination des animaux d’élevage, INRA Edition,Versailles, 304 pp.
Sauvant, D., P. Schmidely and J.J Daudin, 2005. Les méta-analyses des données expérimentales: applications en
nutrition animale. INRA Prod. Anim. 18, 63-73.
Financial support from LIMAGRAIN and INZO is acknowledged.

Energy and protein requirements of purebred and crossbred Nellore
bulls, steers, and heifers: a meta-analysis evaluation
M.L. Chizzotti1,2, L.O. Tedeschi2, S.C. Valadares Filho1, P.V.R. Paulino1 and F.H.M. Chizzotti1,2
1Universidade Federal de Viçosa, Departamento de Zootecnia, 36571,Viçosa, MG, Brazil
2Texas A&M University, Department of Animal Science, 77843, College Station, TX, USA
Introduction
Several studies have been independently conducted to determine energy and protein requirements of
B. indicus purebreds and their crossbreds with B. taurus. A meta-analysis of this data is necessary to
provide an overall summary of current results and directions for future experiments. Therefore, the
objective of this study was to perform a meta-analysis of this data to determine energy and protein
requirements for maintenance and growth of bulls, steers, and heifers of Nellore and Nellore×B.
taurus crossbreds from independent studies, which used a comparative slaughter technique to
measure energy and protein balances.

A database of 16 comparative slaughter studies (n=389 animals) was gathered to provide enough
information to develop equations to predict net energy and protein requirements for maintenance
(NEm and NPm) and growth (NEg and NPg). Animals from all studies were individually fed and no
implant was used. The data were analyzed using a random coefficients model (Littell et al., 1999),
considering studies as random effects, and genders (bulls, steers, and heifers; n=262, 103 and 24,
respectively) and breeds as fixed effects. Breeds were coded as Nellore purebreds (n=271) and
crossbreds (with Angus, Red Angus, Simmental, Limousin, or Brangus; n=118).
The antilog of the linear regression between the log of heat production (HP) on metabolizable energy
intake (MEI) was used to estimate the NEm (Lofgreen and Garrett, 1968). The ME required for
maintenance (MEm) was calculated by iteration, assuming that the maintenance requirement is the
value at which HP is equal to MEI according to this equation: HP = β0 × e(β1 × MEI). The slope of the
regression of retained energy (RE) on MEI was assumed to be the efficiency of energy utilization
for growth (Kg). Alternatively, the intercept divided by the slope (Kg) was used to compute MEm.
The NPm was assumed to be the intercept of the linear regression of the retained N on N intake.
The NEg was calculated as a × EBW0.75 × EWGb, where EBW is empty BW; a and b are the
antilog of the intercept and the slope of the linear regression of the log of the RE on the log of the
empty body gain (EWG). The NPg was calculated as c + (d × EBW) + (e × RE) where c, d, and e
are the intercept and slopes of the multiple regression of the retained protein on the EBW and RE,
respectively. The general statistical model used was: Υij = β0 + β1Xij + β2Si + β3SiXij + εij; where
Υij = the dependent variable Y at level j of the independent variable X in the study i, β0 = overall
intercept with fixed effect, β1 = overall slope that result from regressing Y on X across all studies
with fixed effect, Xij = observed value j of the independent variable X in the study i, β2 = effect of
study i (Si) on the intercept, β3 effect of study i on the slope of the regression of Y on X in study i,
and εij = the random, unexplained error.
An initial analysis was conducted assuming random slope and intercept effects, assuming a covariance
between the slope and intercept using an unstructured variance-(co)variance matrix. In instances
in which covariance parameters were not different from zero (P>0.10), a variance components
structure of the variance-(co)variance matrix was used. Outliers were identified and removed if the
studentized residue were outside of the range -2.5 and 2.5.

The nonlinear regression indicated that HP increased exponentially MEI increased. There were no
differences in NEm requirements among genders (P=0.73) and breeds (P=0.82). The combined data
indicated a NEm requirement of 75.0 (± 1.03) kcal/kg0.75 EBW, which is slightly lower than the NEm
of 77 kcal/kg0.75 EBW reported by Lofgreen and Garrett (1968). Similarly, the estimate of the MEm
based on the relationship between RE and MEI indicated no differences in the MEm requirement
among breeds and genders. Nonetheless, the overall estimate of MEm was smaller than the exponential
relationship between HP and MEI (107 ± 20.2 vs. 112 ± 1.54 kcal/kg0.75 EBW, respectively). The
partial efficiency of conversion of MEm to NEm was similar among genders and breeds with an
average value of 0.67. The regression of the log of RE on the log of EWG indicated a similar
slope (P=0.92) but a different intercept (P=0.01) among genders. The NEg requirement calculated
assuming a common slope among genders were the following: NEg = 0.0514 × EBW0.75 × EWG1.070
(R2=0.76) for bulls; NEg = 0.0700 × EBW0.75 × EWG1.070 (R2=0.78) for steers and NEg = 0.0771 ×
EBW0.75 × EWG1.070 (R2=0.64) for heifers. The Kg was not different among genders (P=0.33) and
breeds (P=0.20), and averaged 0.44 (± 0.03). There were no differences in NPm requirement; The
overall NPm was 1.74 (± 0.41) g of NPm/kg EBW0.75/d. The overall metabolizable protein (MP)
requirement for maintenance was 2.59 g of MPm/kg EBW0.75/d. The NPg was not different among
genders (P>0.59) and breeds (P>0.14); the overall equation was NPg (g/d) = EWG × (217 - 12.8 ×
RE/EWG) (R2=0.63). The percentage of RE deposited as protein (%REp) decreased exponentially
as the content of RE in the gain (REc, Mcal/kg of EWG) increased. Because no study effect was
observed, we pooled the data across studies and the overall equation for %REp was 0.1012 + 1.6673
× e(-0.6605 × REc) (R2=0.63).
Conclusion
Our results do not support the hypothesis that bulls have greater NEm requirements than steers and
heifers. Similarly, no differences in the NPm among bulls, steers, and heifers were detected. The
NEg of steers might be greater than that for bulls and lesser than that for heifers. Although the %REp
was negatively correlated with the concentration of energy in the EWG, our results indicated no
differences in NPg among bulls, steers, and heifers.
References
Littell, R.C., G.A. Milliken, W.W. Stroup and R.D. Wolfinger, 1999. SAS System for Mixed Models. SAS Institute,
Cary, NC.
Lofgreen, G.P. and W.N. Garrett, 1968. A system for expressing net energy requirements and feed values for growing
and finishing beef cattle. J. Anim. Sci. 27, 793–806.

Portal absorption of N: partition between amino acids and ammonia in
relation with nitrogen intake in ruminants
H. Lapierre1, J. Vernet2, R. Martineau1, D. Sauvant3, P. Nozière2 and I. Ortigues-Marty2
1Agriculture and Agri-Food Canada, Stn Lennoxville, Sherbrooke, QC, J1M 1Z3 Canada
2Institut National de la Recherche Agronomique, Theix, 63122 St Genès Champanelle, France
3Institut National de la Recherche Agronomique, 75231 Paris Cedex, France
Introduction
Reducing protein intake and N excretion without a detrimental impact on productivity requires a
better understanding of the relationship between intake, nutrient supply and utilization for metabolic
functions. In ruminants, the first challenge is to adequately predict the supply of nutrients to the
animal, after dietary ingredients have traversed the complex rumen ecosystem. The nitrogen (N)
digested is mainly absorbed as free amino acids (AA), used by the animal for protein synthesis, or
ammonia, which needs to be detoxified by the liver into urea. The forms under which N are absorbed
will therefore be critical in determining the proportion of N digested available to support protein
synthesis. The objective of this study was to examine the relationship between net portal absorption
of AA-N (NPA-AAN) or ammonia (NPA-NH3) and N intake (NI) from studies in ruminants where
NI was increased.

The original database used was the FLORA database (FLux of nutrients through Organs and tissues
in Ruminant Animals), including data from publications reporting splanchnic fluxes of nutrients
in ruminants (Vernet and Ortigues-Marty, 2006). Seventeen publications, for a total of 28 group
comparisons (called below ‘study’: 15 in cattle, n=31; and 13 in sheep, n=30), were selected based on
variation in daily NI with a standard deviation greater than 0.04 g/kg BW and a forage to concentrate
ratio varying by less than 5%. To allow comparison between species, all data were expressed in g
of N per d per kg body weight (Vernet et al., 2005). Linear and curvilinear relationships between
net NPA-AAN, NPA-NH3, the ratio of NPA-NH3 relative to NPA-AA and NI were tested through
meta-analysis (St Pierre, 2001; Sauvant et al., 2005) using the statistical model: Y = a × NI2 + b ×
NI + c + species + αi(species) + species × NI + e, where αi=study effect (fixed) and e=error. With
a significant interaction between species and NI, the following model (Y = b × NI + c + αi + error)
was rerun within species.
Table 1. Characteristics of the data used in the meta-analysis.
Variables, g N/d/kg Cattle Sheep

BW
Mean STD Min. Max. Mean STD Min. Max.
N ingested 0.481 0.158 0.147 0.845 0.386 0.176 0.127 0.754

NPA-AAN 0.130 0.064 0.001 0.308 0.178 0.082 0.021 0.319
NPA-NH3 0.208 0.096 0.076 0.500 0.128 0.087 0.042 0.327
NPA-NH3 / AAN 1.75 0.77 0.68 4.18 0.87 0.59 0.22 2.67
Cattle: Bruckental et al., 1997; Eiseman et al., 1996; Guerino et al., 1991; Huntingon et al., 1988; Lapierre et al.,
2000; Maltby et al., 1992; Reynolds et al., 1988, 1991 and 1992. Sheep: Burrin et al., 1991; Ferrel et al., 1999 and
2001; Freetly et al., 1998; Fukuma et al., 2005; Goetsh et al., 1994; Ortigues et al., 1994; Patil et al., 1996.

For the three variables, more than 95% of the variation was explained by the linear model, with no
improvement with the inclusion of the quadratic component of NI; the effect of the study was always
significant (P<0.001). There was a linear relationship between NPA-AAN and NI, with an effect of
species on the intercept (P=0.003: lower absorption of AA-N for a same NI in cattle than in sheep)
but no interaction between species and NI (P=0.94: no effect of the species on the slope). The pattern
of NPA-NH3 was different, with an interaction (P<0.001) between species and NI: NPA-NH3 was
linearly related to NI, but with a smaller slope in sheep than in cattle. As a result of these differences,
the ratio of NPA-NH3/NPA-AAN was higher in cattle than in sheep (Table 1) and negatively linearly
related with NI, with a steeper slope for cattle (P<0.001). The analysis of the average quantitative
interfering factors (forage to concentrate ratio, metabolizable energy and digestible organic matter
concentration) did not show any correlation with NI, NPA-AAN and NPA-NH3, but NPA-NH3 and
NPA-NH3/NPA-AAN were correlated with the ratio of diet concentrations of N/digestible organic
matter or N/metabolizable energy. These latter parameters were higher in cattle than in sheep.
Table 2. Relationships between net portal absorption of AA-N (NPA-AAN), ammonia (NPA-NH3),
NPA-NH3 /NPA-AAN and N intake (NI), expressed in g N/d kg body weight.
Y Equation P int. P slope Syx
NPA-AAN Cattle -0.092(0.026) + 0.459(0.053)NI + αi 0.003 <0.001 0.023

Sheep 0.011(0.018) + 0.453(0.050)NI + αi 0.58 <0.001 0.022
NPA-NH3 Cattle 0.013(0.026) + 0.406(0.052)NI + αi 0.60 <0.001 0.022
Sheep 0.056(0.014) + 0.163(0.037)NI + αi 0.001 <0.001 0.017
NPA-NH3 / AAN Cattle 3.23(0.41) – 3.02(0.82)NI + αi 0.002 <0.001 0.33
Sheep 1.23(0.14) – 1.13(0.39)NI + αi <0.001 0.01 0.17

This study is a first step to relating NPA-AAN and NPA-NH3 to NI: in situations where NI is
increased through increments of dry matter intake or through protein supplementation, for both
sheep and cattle, 0.46 of the increment of ingested N is recovered in the portal vein as AA, but with
higher absolute values for sheep. In contrast, the increment in NPA-NH3 relative to NI occurs at a
faster rate in cattle than in sheep. The ratio of diet concentrations of N on energy seems to affect
NPA-NH3 but not NPA-AAN.
References
Sauvant, D., P. Schmidely and J.J. Daudin, 2005. Les méta-analyses des données expérimentales: applications en
St-Pierre, N.R., 2001. Integrating quantitative findings from multiple studies using mixed model methodology. J.
Dairy. Sci. 84, 741-755.
Vernet, J., H. Lapierre, P. Nozière, S. Léger, D. Sauvant and I. Ortigues-Marty, 2005. Prediction of blood nutrient
supply to tissues of economical interest in Ruminants: a first step with the prediction of portal blood flow. In: Hill,
D.R.C., V. Barra and M.K. Traore (editors), Oicms. Blaise Pascal University, France, 163-173.
Vernet, J. and I. Ortigues-Marty, 2006. Conception and development of a bibliographic database of blood nutrient
fluxes across organs and tissues in ruminants: data gathering and management prior to meta-analysis. Reprod.
Nutr. Dev. 46, 527-546.

Effects of protein supply on whole body glucose rate of appearance and
mammary gland metabolism of energy nutrients in ruminants
S. Lemosquet1, G. Raggio2, H. Lapierre2, J. Guinard-Flament1 and H. Rulquin1
1INRA, Agrocampus Rennes, UMR 1080, Dairy Production, F-35590 St-Gilles, France
2Agriculture and Agri-Food Canada, Lennoxville STN, Sherbrooke, J1M 1Z3, QC Canada
Introduction
Increasing intestinal supply of protein through diet supplementation or post-ruminal infusions
usually leads to increased yields of milk and lactose in lactating dairy cows. Glucose (GLC) is the
main precursor of lactose synthesis in the mammary gland so by which mechanism does increased
intestinal amino acid supply increase lactose yield? Is it through elevated gluconeogenesis coupled
with increased mammary GLC uptake or through a change in mammary metabolism? Furthermore,
what is the effect of increasing protein supply on milk fat yield and metabolism of other energy
nutrients? These questions were addressed through a meta-analysis of the effects of increasing
intestinal supply of protein on whole body GLC rate of appearance (Ra) and mammary metabolism
of GLC and of other energy nutrients.

The effect of increasing protein digested in the intestine (PDI, g/d) in ruminants, mainly through
post-ruminal infusions of casein, was analysed using two databases. The first database was used to
analyse whole body GLC Ra measured with labelled glucose in 7 trials (Clark et al., 1977. J. Nutr.
107, 631; El-Kadi et al., 2006. J. Nutr. 136, 1261; König et al., 1984. Br. J. Nutr. 52, 319; Oldham
et al., 1980. 3rd EAAP, 460; Putmam et al., 1999. J. Dairy Sci. 82, 1274; Ranawana et al., 1977.
Br. J. Nutr. 37, 395 and personal data from Raggio et al., 2006. J. Dairy Sci. 89, 4340) in ruminants
(sheep, goats and cows). The effect on mammary nutrient metabolism was analysed using 11 trials in
lactating dairy cows (second database: Guinard and Rulquin, 1994. J. Dairy Sci. 77, 222; Korhonen
et al., 2002. J. Dairy Sci. 85, 3336; Mackle et al., 2000. J. Dairy Sci. 83, 93; Metcalf et al., 1991.
J. Dairy Sci. 74, 3412; Metcalf et al., 1994. J. Dairy Sci. 77, 1816; Metcalf et al., 1996. J. Dairy
Sci. 79, 603; Miettinen et al., 1997. J. Sci. Food Agric. 74, 459; Rulquin et al., 1983. Reprod. Nutr.
Dévelop. 23, 1029; Vanhatalo et al., 2003a and b. J. Dairy Sci. 86, 3246 and 3260; personal data from
Raggio et al., 2006). Yields of milk, lactose and fat were analysed as were arterial concentrations
(A), mammary plasma flow, arterio-venous differences (AV), extractions, and uptakes (U) of GLC,
β-hydroxybutyrate (BHBA), acetate, non esterified fatty acids (NEFA), total glycerol and Ala. Plasma
flow was either determined by ultrasound probe (1 trial), p-amino hippurate dilution (2 trials) or
by the Fick principle (Phe + Tyr; 8 trials). Data were corrected for trial effect using the statistical
model: Y = trial + a + b × PDI + e; where Y = the variable studied and e = error.

All the significant relationships were linear (Table 1). In the first database, PDI supply averaged 12.9
g/d/kg BW0.75 (from 4.5 to 22.4). Ra of GLC increased with PDI supply expressed as kg BW0.75
across species but with a high gradient.
In the second database, PDI supply averaged 1787 g/d (from 1469 to 2079). Increasing PDI supply
decreased lactose concentration but increased milk and lactose yields, insulin A but did not affect
GLC A. The GLC AV increased with PDI supply as did GLC extraction (not shown). This suggested
an improved efficiency of mammary gland to extract GLC but, in practice, no clear response was
obtained for mammary GLC U because although 8 of the trials showed a linear increase with increased

PDI supply the other 3 trials showed a decrease. This variable response may be linked to problems
in estimation of plasma flows. In fact plasma flow exhibited no clear relationship with increased PDI
supply. Most plasma flow calculations involved the Fick principle, which has been questioned when
high protein supplements are given (Rulquin et al., 2007). The absence of increased GLC U may also
be linked to a change in mammary metabolism, with a greater proportion of GLC used for lactose
synthesis. The ratio of lactose yield to GLC U exhibited no clear relationship with increased PDI
supply, however. This ratio averaged 0.73 (0.47 to 1.01). Although GLC U almost always covered
lactose yield, it has been suggested that other nutrients contribute to galactose synthesis since only
80% of lactose carbons originated from GLC (Bickerstaffe et al., 1974). Ala, whose carbons could
be used for galactose synthesis, is probably not a good candidate since its AV and U decreased with
increasing PDI supply. Increasing PDI supply increased milk fat yield, AV and extractions of BHBA
and NEFA but no clear relationship was obtained for their uptakes, as for GLC. In addition, neither
acetate nor total glycerol parameters were modified. In conclusion, increasing PDI supply increased
whole body GLC Ra, yields of milk, lactose and fat and mammary AV of GLC and BHBA. Further
research is required, however, to clarify the response of net uptakes of GLC and energy nutrients
to support these increased yields and to determine how mammary gland metabolism is altered to
allow increased lactose synthesis.
Table 1. Relation between energetic nutrients and protein digested in the intestine (PDI1).
Y² Mean Range Equation P slope R2 Syx N
First database3
Ra4, g/d/kg BW0.75 19.2 11.1-26.3 8.7 + 0.8 PDI/BW0.75 <0.001 0.94 1.04 24
Second database3
Milk, kg/d 25.2 20.1-31.4 17.1 + 4.53 10-3 PDI <0.001 0.92 0.76 36
Lactose, g/kg 48.2 44.7-52.1 52.9 - 2.67 10-4 PDI <0.001 0.90 0.50 33
Lactose, g/d 1222 981-1510 847 + 0.21 PDI <0.001 0.93 33.0 33
Fat, g/kg 41.3 36.7-45.7 47.2 - 3.3 10-3 PDI <0.001 0.73 1.14 33
Fat, g/d 1034 851-1236 752 + 0.159 PDI <0.001 0.89 31.3 33
Insulin A5, ng/mL 0.653 0.549-0.759 0.420 + 1.3 10-4 PDI 0.002 0.44 0.04 20
GLC AV6, mM 0.763 0.565-1.033 0.426 + 1.88 10-4 PDI <0.001 0.93 0.03 36
Ala U7, mmol/h 7.74 0.46-11.5 15.9 - 4.7 10-3 PDI <0.001 0.54 7.22 30
BHBA AV6, µM 0.288 0.234-0.365 0.182 + 6.7 10-5 PDI <0.001 0.54 0.02 26
NEFA AV6, µM -0.26 -24.4-25.6 -36.5 + 2.27 10-2 PDI <0.001 0.52 8.1 25
1g/d; 2Y:corrected for trial effect; 3References for databases 1 and 2 are listed in Material and methods; 4Whole body
glucose rate of appearance; 5Arterial concentration; 6Mammary arterio-venous difference; 7Uptake.
References
Bickerstaffe, R., E.L. Annison and J.L. Linzell, 1974. Metabolism of glucose, acetate, lipids and amino acids in lactating
dairy cows. J. Agric. Sci. (Camb.) 82, 74-85.
Rulquin, H., G. Raggio, H. Lapierre and S. Lemosquet, 2007. Relationship between intestinal supply of essential
amino acids and their mammary metabolism in the lactating dairy cow. Int. Symp. on Energy and Protein Metab.
and Nutr., Vichy, France, in press.

Nitrogen transactions along the gastrointestinal tract in cattle: a meta-
analytical approach
J. Marini1, D.G. Fox2 and M. Murphy3
1Baylor College of Medicine, 1100 Bates Street, Houston, TX 77030, USA
2Cornell University, 130 Morrison Hall, Ithaca, NY 14850, USA
3University of Illinois, 1207 Gregory Dr., Urbana, IL 61801, USA
Introduction
Endogenous nitrogen (EN) secretions occur along the whole gastrointestinal (GI) tract of animals
constituting a loss of amino acids for the animal, but a supply of nitrogen (N) for the microbial
population of the foregut and hindgut of ruminants. The quantification of these transactions is not
only challenging, but also expensive and consequently very little data is available in cattle. To
determine the N transactions along the GI tract of cattle, we utilized a statistical approach on data
obtained from the literature.

A database on N flow along the GI tract in cattle was compiled from published data on intestinally
cannulated cattle, by systematically searching two leading animal science journals (J. Anim. Sci.
and J. Dairy Sci.) between January 1990 and December 2006. A multilevel analysis was performed
on the 455 treatment diets (from 108 studies) included in the database utilizing a mixed model,
with study as a random component of the model (St-Pierre, 2001). Model selection was done by
utilizing the corrected Aikake information criterion. Observed values of the dependent variables (Yis)
come from a multidimensional space, and because it is of interest to represent the data solely as a
function of the main variable of interest (N entering a particular segment of the GI tract), the value
of the observations were adjusted (Y’is) for the lost dimensions (St-Pierre, 2001). This was done by
adjusting the Yis values to a reference diet (32% NDF, carbohydrates of medium fermentability rate,
ingested at an intake level of 2% body weight).
Y’is = µ + βN • XN(is) + eis (1)
where,
Y’is = adjusted value of the dependent variable for the i treatment in the s study,
βN = regression coefficient,
XN = the value of the continuous variable N entering the GI segment in study s, treatment i.

The EN entering the foregut, small intestine and hindgut, as well as metabolic fecal N, were estimated
from the intercept of the regressions (Table 1). These values were consistent with the ones reported
previously in the literature. True digestibility of N for each segment, as well as for the whole GI
tract, was derived from the slope of the regressions (Table 1).
Utilizing the coefficients reported in Table 1, a model for the N transactions along the whole GI tract
was constructed for the reference diet containing 24.2 g N/kg OM (Figure 1). The contribution of
EN to ruminal microbial production (16%) and to duodenal flow (22%) was similar to the values
obtained utilizing isotopic labeling methods (Ouellet et al., 2002).

Table 1. Regression coefficients on adjusted data for N digested in different segments of the GI
tract.
Intercept1 ± (SEM) Slope ± (SEM) r2
Total tract digested N -4.3 (0.15) 0.84 (0.006) 0.979

Ruminal apparently digested N -13.4 (0.29) 0.51 (0.011) 0.819
Ruminal truly digested N -3.2 (0.29) 0.68 (0.011) 0.895
Bacterial N yield 10.5 (0.22) 0.15 (0.008) 0.407
Small intestinal digested N -5.6 (0.53) 0.75 (0.036) 0.974
Large intestinal digested N -15.6 (0.80) 0.49 (0.088) 0.727
1All values in g nitrogen/kg OM entering the GI tract segment.
Figure 1. Nitrogen transactions along the GI tract of cattle. Values are expressed in g N/kg OM
intake. It was assumed that 0.56 and 0.26 kg OM/kg OMI entered the duodenum and hindgut,
respectively.
Conclusion
EN is an important contributor to total duodenal N flow. The statistical approach followed can
be utilized to determine the N transactions along the GI tract of cattle under a variety of feeding
conditions.
References
St-Pierre, N.R, 2001. Invited review: Integrating quantitative findings from multiple studies using mixed model
methodology. J. Dairy Sci. 84, 741-755.

Predicting in vivo production of volatile fatty acids in the rumen from
dietary characteristics by meta-analysis: description of available data
P. Nozière1, F. Glasser1, C. Martin1, D. Sauvant2
1INRA, UR1213 Herbivores, Site de Theix, F-63122 Saint-Genès-Champanelle, France
2INRA, UMR791 Physiologie de la nutrition et alimentation, INAPG, 16 rue Claude Bernard, F-
75231 Paris, France
Introduction
Volatile fatty acids (VFA) produced in the rumen represent 40% to 70% of digestible energy intake
in ruminants (France and Dijkstra, 2005), and yet their composition is poorly described by existing
rumen models (Offner and Sauvant, 2004). The estimation of the quantitative supply of individual
VFA to intermediary metabolism thus remains a key question in ruminant nutrition. Several authors
measured in vivo production rate of individual VFA in the rumen, using mainly isotope dilution,
which is recognised as the more reliable method (France and Dijkstra, 2005). By applying a meta-
analysis approach to these data, we aim to produce quantitative information on (i) the effects of
intake and dietary composition on the individual VFA production rates in the rumen (VFA-PR); (ii)
the partition of fermented energy between VFA, heat and gas productions, and their relation with
microbial synthesis. The present paper describes the available data and presents preliminary results on
the effects of the amount of organic matter digested in the rumen (OMDR) and concentrate level.

All the available publications measuring in vivo VFA-PR by isotope dilution techniques were included
in a database. All data related to animals (species, body weight, age, physiological stage), intake,
diets (feedstuffs, chemical composition), ruminal and total tract digestibility of dietary components,
VFA-PR (methods, results), microbial synthesis (methods, results) and ruminal fermentations (pH,
ammonia) were included. Preliminary relationships were determined by meta-analysis (Sauvant et al.,
2005), with statistical models including an experiment effect: (i) between the amount of OMDR and
VFA-PR, and (ii) between the percentage of concentrate and the acetate/propionate ratio (C2/C3).

We identified 66 publications (Table 1) reporting results of VFA-PR determined by isotope dilution
techniques on ovine (N=30), growing buffalo or cattle (N=29) and cows (N=7). This corresponded
to 203 dietary treatments, among which 96 on ovine, 89 on growing buffalo or cattle and 18 on
cows. The percentage of concentrates in the diet was much lower in ovines and growing buffalo
than in dairy cow, with growing cattle being intermediary. However, a wide diversity of diets was
available within each group. The main variation factors studied in the experiments were the nature of
forage (N=14 publications), the effect of additives or preservatives (N=14), the nature of concentrate
(N=11), the forage:concentrate ratio (10), and the level of intake (N=6). More than 70% of the
measurements were performed under steady state conditions (i.e. more than 8 meals/d). Except in
3 publications using 13C, all measurements were performed using 14C isotopes. More than 80% of
the publications reported data on individual VFA, although in more than 60% of these, only one
isotope (mostly 1 or 2-14Cacetate) was administrated, assuming total VFA was a homogenous pool.
In the other publications (40%), 2 or more often 3 isotopes were administrated. Interconversions
between VFA, mainly between acetate and butyrate, were quantified on 12 publications, using two
or three-pool schemes.

Table 1. Description of available data of VFA production rate in the rumen (VFA-PR) determined
by isotope dilution techniques. Values are means ± SD.
Ovine Growing Growing Dry cow Dairy cow

buffalo cattle
No. of publications 30 10 19 2 5
with individual VFA 25 5 17 2 4
No. of treatments 96 33 56 6 12
DM intake, g/kg BW 19 ± 5 19 ± 5 20 ± 6 13 ± 1 29 ± 7
% Concentrate 6 ± 20 13 ± 19 37 ± 29 2±2 74 ± 16
Range 0-90 0-50 0-100 0-4 45-91
VFA-PR, mmol/kg BW/d 105 ± 36 71 ± 40 80 ± 48 60 ± 29 130 ± 23
VFA-PR, mol/kg DMI 5.7 ±1.6 3.6 ± 1.7 4.4 ± 3.0 4.6 ± 2.6 4.7 ± 1.4
Acetate/propionate ratio 3.4 ± 0.6 3.6 ± 1.0 3.1 ±1.0 3.8 ± 0.6 2.2 ± 1.0
Publications with variations in OMDR higher than 1 g OMDR/kg BW/d were selected for the 1st
analysis. They comprised 11 experiments and 38 treatments, all on ovines fed only forages. The
adjusted model indicated a linear increase in VFA-PR of 9.15 ± 1.07 mol per kg OMDR, which was
highly consistent with what is classically admitted: VFA-PR (mol/d) = 1.54 (SE=0.46, P<0.01) +
9.15 (SE=1.07, P<0.001) × OMDR (kg/d); (Syx=0.58; R2=0.84).
Publications with variations in % concentrate higher than 15% were selected for the 2nd analysis.
They comprised 11 experiments and 26 treatments on sheep (1 exp), cattle (6 exp), and dairy cows
(4 exp). The adjusted model indicated a quadratic decrease in C2/C3 with increasing % concentrate
(%CONC): C2/C3 = 3.40 (SE=0.19, P<0.0001) – 2.13⋅10-4 (SE=0.48⋅10-4, P=0.001) × %CONC²;
(Syx=0.57; R2=0.62). This within-experiment quadratic coefficient was similar to the one obtained
by global regression (–1.95⋅10-4, SE=0.44⋅10-4).
This overview of available data emphasizes the lack of measurements of VFA-PR in dairy cows
and in non steady state conditions. However, considering the amount of available data, the range
of intake levels and the diversity of dietary conditions, this database constitutes a helpful tool for
empirical modelling, by a meta-analysis approach, of the VFA-PR from dietary characteristics.
These preliminary results appear promising. Analyses are in progress concerning the VFA-PR, the
relationships between VFA concentrations and their PR, their relations with microbial synthesis
and ruminal fermentations. This work will contribute to subsequent developments of nutrient-based
energetic feeding systems.
References
France, J. and J. Dijkstra, 2005. Volatile fatty acid production. In: Dijkstra, J., J.M. Forbes and J. France (editors),
Quantitative aspects of ruminant digestion and metabolism, 2nd edition. CAB International, Wallingford, UK,
157-175.
Offner, A. and D. Sauvant, 2004. Comparative evaluation of the Molly, CNCPS, and LES rumen models. Anim. Feed
Sci. Technol. 112 (1-4), 107-130.
Sauvant, D., P. Schmidely and J.J. Daudin, 2005. Les méta-analyses des données expérimentales: applications en

Relationship between intestinal supply of essential amino acids and their
mammary metabolism in the lactating dairy cow
H. Rulquin1, G. Raggio2, H. Lapierre3 and S. Lemosquet1
1UMRPL INRA 35590 SaintGilles, France
2Université Laval, Québec, G1K7P4 Canada
3Agriculture and Agri-Food Canada, Sherbrooke, J1M 1Z3 Canada
Introduction
Prediction of uptake and utilisation of amino acids (AA) by the mammary gland is one of the main
challenges in modelling mammary gland metabolism. Mepham (1982) defined 2 groups: Group 1
(Phe and Tyr, Met and His) including AA always taken up in accordance with their output in milk
protein; Group 2 (Lys, Leu, Ile, Val and Arg) taken up in excess to their output, with this especially
large for Arg (2-3 fold). Thr was not allocated to a specific group. Dietary conditions of animals
(goats and cows) used to constitute these groups were not described, however, and it is possible that
the intra-mammary behaviour of AA may differ, for example, dependent on whether the animals
are in protein deficit or in protein excess. The present meta-analysis was performed to address this
point.

From experiments where altered protein supply was studied, a database was constructed based
on essential AA mammary arterio-venous difference or uptake, milk protein yield, and mammary
blood or plasma flow. The data were obtained from 11 publications in which supplement(s) of
protein were fed or infused post-ruminally. These reported 38 treatments and were regrouped into
17 experimental treatments for the meta-analysis (Raggio, 2006). The supplements consisted of
casein (10), casein + branched-chain AA (2), fish meal or fish meal and blood meal (3) and soybean
meal (2). Intestinal supply of individual digestible AA (AADI) was estimated according to Rulquin
et al. (1998). Requirements of individual AA were based on PDI requirements according to INRA
(1989) and ideal protein composition: Lys: 7.30%, Met: 2.50% (Rulquin et al., 1993), Phe: 4.60%
(Rulquin and Pisulewski, 2000), Leu: 8.90% (Rulquin and Pisulewski, 2006b), Thr: 4.02% (Rulquin
and Pisulewski, 2006a), His: 3.03% (Rulquin and Delaby, 2006). Ideal compositions for Val (5.33%),
Ile (4.45%) and Arg (3.14%) were calculated by applying the relative ratio to Lys (0.73, 0.61, 0.43)
obtained by Fraser et al. (1991) to the value of Lys. Free AA concentrations were determined after
deproteinisation (sulphosalicylic acid) on blood (1) and on plasma (16). Blood or plasma flows
were measured by an ultrasound probe (1), p-amino hippurate dilution (2) or estimated by the Fick
principle using Phe + Tyr as AA markers (14). Data were corrected for an experiment effect by
applying the statistical model: Y = experiment + a + b × (intestinal AA supply / AA requirement)
× 100 + e, where Y = the ratio of mammary uptake relative to milk output (U/O) in protein for the
AA considered; experiment = fixed experiment effect; e = error.
Results
Cows produced between 20.7 and 36.2 kg/d of milk. Protein supply covered 83 to 184% of predicted
requirements. For His, Phe, Leu, Lys, Ile, Val, the ratio U/O was linearly related to the supply
(P<0.05; Table 1). For Met this ratio did not differ significantly from 1. For Arg and Thr the ratio
was greater than 1 but was not related to intestinal supply. Similar conclusions, but with lower R²,
were obtained when the supply was expressed in absolute values (g/d).

Table 1. Relationships between uptake/output (Y) and intestinal supply (X) of essential AA.
Y X1 X2 Equation P slope R² Syx n
His 1.15 82 58-148 Y = 0.77 + 4.64 × 10-3X 0.0085 0.70 0.07 37

Phe 1.09 126 93-217 Y = 0.66 + 3.47 × 10-3X 0.0123 0.79 0.05 37
Lys 1.45 106 80-181 Y = 0.52 + 8.68 × 10-3X 0.0010 0.85 0.08 37
Met 1.01 89 64-149 Y = 1.10 – 1.09 × 10-3X 0.5733 NS 0.11 35
Thr 1.24 142 110-234 Y = 0.89 + 2.52 × 10-3X 0.2850 NS 0.11 35
Arg 2.65 169 129-297 Y = 2.99 – 1.94 × 10-3X 0.7106 NS 0.38 32
Ile 1.51 135 101-259 Y = 0.48 + 7.70 × 10-3X 0.0004 0.89 0.09 37
Leu 1.33 111 78-218 Y = 0.41 + 8.35 × 10-3X 0.0006 0.90 0.09 37
Val 1.57 125 95-210 Y = -0.19 + 1.43 × 10-2X 0.0002 0.91 0.12 35
Y = uptake/output corrected for experiment effect; X = AADI % requirements.

1mean.
2range.
Conclusion
Only Met respected the rule decreed for Group 1. For His, the rule was true only when supply was
close to the requirements. The ratio for Phe needs to be analysed with caution though because, for
14 of the studies, the Fick principle was used, based on a 1:1 uptake/output ratio for Phe+Tyr. The
increment in the U/O ratio of His, however, questions the stoechiometric transfer of Group 1 AA
and the use of the Fick principle to estimate mammary blood flow irrespective of protein supply.
Other essential AA behave as expected for Group 2. Arg and Thr, however, were different because
their U/O ratio was not related to intestinal supply.
References
Fraser, C., E.R. Ørskov, F.G. Whitelaw and M.F. Franlin, 1991. Limiting amino acids in dairy cows given casein as
the sole source of protein. Livest. Prod. Sci. 28, 235-252.
INRA, 1989. Ruminant Nutrition: recommended allowances and feed tables. J. Libbey Eurotext, Paris, France.
Mepham, T.B, 1982. Amino acid utilization by lactating mammary gland. J. Dairy Sci. 65, 287-298.
Raggio, G., 2006. Effets des apports protéiques et énergétiques sur le métabolisme protéique chez la vache laitière.
Thèse Doctorat, Fac. Sci. Agri. Alim. Univ. Laval, Québec, Canada.
Rulquin, H. and P. Pisulewski, 2000. Effects of duodenal infusions of graded amounts of Phe on mammary uptake and
metabolism in dairy cows. J. Dairy Sci. 83 (Suppl. 1), 267-268.
Rulquin, H. and P. Pisulewski, 2006a. Effects of duodenal infusion of graded amounts of threonine on lactational
performances of dairy cows. J. Dairy Sci. 89 (Suppl. 1), 401.
Rulquin, H. and P. Pisulewski, 2006b. Effects of graded levels of duodenal infusions of leucine on mammary uptake
and output in lactating dairy cows. J. Dairy Res. 73, 328-339.
Rulquin, H. and L. Delaby, 2006. Estimation du besoin en Histidine (His) des vaches laitières. Renc. Rech. Ruminants
13, 100.
Rulquin, H., P.M. Pisulewski, R. Vérité and J. Guinard, 1993. Milk production and composition as a function of
postruminal lysine and methionine supply: a nutrient-response approach. Livest. Prod. Sci. 37, 69-90.
Rulquin, H., J. Guinard and R. Vérité, 1998. Variation of amino acid content in the small intestine digesta of cattle:
development of a prediction model. Livest. Prod. Sci. 53, 1-13.

Effect of species (ovine, bovine) and feeding level on portal blood flows
and net volatile fatty acid (VFA) fluxes: a meta-analysis
J. Vernet1, P. Nozière1, S. Léger2, H. Lapierre3, D. Sauvant4 and I. Ortigues-Marty1
1UR 1213, Herbivores, INRA, Theix, 63122 Saint Genès Champanelle, France
2Laboratoire de Mathématiques, Université de Clermont II, 63177 Aubière Cedex, France
3Agriculture and Agri-Food Canada, Stn Lennoxville, Sherbrooke, QC, J1M 1Z3 Canada
4UMR INRA-INAPG, Physiol. Nutr. et Alimentation, INRA, 75231 Paris, France
Introduction
In studies measuring net nutrient flux across tissues in ruminants, ovines have often been used as
an animal model because of lower experimental cost and work load. It is then often assumed that
conclusions can be extrapolated to bovines. Still, the results obtained by Bermingham et al. (2007)
by meta-analysis suggested that the incremental response of the net portal appearance (NPA) of
energetic nutrients to variations in energy intake (between 0 and 371 kJ MEI/kg BW/d) is higher
in bovines than in ovines. The objective of the present work was to further explore the difference
previously observed between species by describing and comparing published NPA of volatile fatty
acids (VFA) within narrower ranges of energy intake.

The exhaustive FLORA (FLux of nutrients across Organs and tissues of ruminant Animals)
bibliographical database (Vernet and Ortigues-Marty, 2006) was used. All results published in fed
animals, without fasting or infusions were selected, i.e. 193 treatments in ovines and 205 in bovines
for a total of 129 experiments. Ovines were mainly non-productive adults (n=137) and growing
(n=23). Bovines were mainly growing (n=121), adults (n=35) and lactating (n=33). Three ranges
of metabolisable energy intake were expressed on a body-weight basis for a better overlap of data
between species (Vernet et al., 2005; MEI, kJ/kg BW/d): low (50 to 150), medium (150 to 250)
and high (250 to 350) ranges. Within each MEI range, diet composition was characterised by the
proportion of concentrate (%conc), the ME content and organic matter digestibility (OMd). The
influence of species and MEI range was tested by analysing dietary characteristics and results of
portal blood flows and NPA of VFA (acetate, C2; propionate, C3 and butyrate, C4) available in the
selected dataset by ANOVA using the following model: Y= species + MEI range + (species × MEI
range) + e and by discriminant analysis.

The number of observations obtained from ovines and bovines within each MEI range was relatively
well balanced (Table 1). Significant differences were noted between species in the nature and
composition of diets, with bovines being fed diets with higher proportions of concentrate, organic
matter digestibility (OMd) and ME content, especially at the lowest MEI ranges. Portal blood flow
was lower (P<0.001) in bovines than in ovines, independently of the MEI range. NPA of C2 was
higher in ovines at high MEI (P<0.001) but not different at low and medium MEI. NPA of C3 was
higher in bovines at low and medium MEI, and higher in ovines at high MEI (P<0.05). NPA of
C4 was always higher in bovines (P<0.001). The discriminant analysis showed a good separation
between the 2 species (the eigenvalue of the discriminant axis was 0.892). The variables that
allowed to differentiate species were %conc, MOd, EM content and portal blood flow. NPA of VFA
did not allow this differentiation. This description of published data shows that it is not possible
to compare the variables between species at similar MEI range, except at high MEI range. At the
latter, for which diet chemical composition was similar between the 2 species except for ME content

(P<0.01), portal blood flow and NPA of C2 and C3 were higher in ovines (P<0.05), while NPA of
C4 was not different.
Table 1. Comparison of MEI, diet composition, portal blood flow and net portal absorption (NPA)
of volatile fatty acid (VFA) between ovines and bovines in published A-V studies.
n Low MEI Medium MEI High MEI SEM Treatment effect
Ov Bo Ov Bo Ov Bo Sp MEI-R Sp×MEI-R
n 46 67 99 82 48 56
MEI1 398 110 124 198 195 283 284 3.19 NS *** *
Diet composition
ME content2 398 9.1 9.9 9.7 11.3 10.6 11.2 0.2 *** *** *
% Conc3 393 9.3 32.7 20.9 54.3 53.4 53.4 4.2 *** *** ***
OMd (%) 392 61.8 68.1 68.6 76.3 73.5 75.5 0.9 *** *** **
Portal blood flow4 340 2.02 1.77 2.42 2.08 3.05 2.54 0.07 *** *** NS
VFA NPA5
acetate 202 1.14 1.57 1.58 1.74 3.07 2.51 0.13 NS *** **
propionate 215 0.36 0.52 0.58 0.69 1.20 1.05 0.06 NS *** *
butyrate 197 0.04 0.11 0.09 0.13 0.18 0.22 0.01 *** *** NS
Sp: species; MEI-R: MEI range; Ov: ovine; Bo: bovine; Sp: species; MEI-R: MEI range; 1kJ/kg BW/d; 2MJ/kg DM;
3DM basis; 4L/kg BW/h; 5mmol/kg BW/h; * P<0.05, ** P<0.01, *** P<0.001.
A characteristic of the published studies reporting NPA of VFA is that diet composition is different
between species. In the current state of knowledge, it is not clear if differences observed between
species are a true species difference or more related to difference in the diets fed to the different
species. Furthermore, the selection of a sub-sample with completely similar dietary characteristics
in the 2 species is not possible, even at high MEI. Gaps of knowledge are thus clearly identified
for ovines fed concentrate based diets and for bovines fed with forages alone. In the mean time,
relationships relating NPA of VFA and other energetic nutrients to MEI and diet characteristics need
to be established for ovines and bovines.
References
Bermingham, E., I. Ortigues-Marty, J. Vernet, H. Lapierre, S. Léger, D. Sauvant and P. Nozière, 2007. The relationship
between intake and portal fluxes of energy metabolites in ruminants: a meta-analysis. Anim. Feed Sci. Technol.,
in press.
Vernet, J., H. Lapierre, P. Nozière, S. Léger, D. Sauvant, I. Ortigues-Marty. 2005. Prediction of blood nutrient supply to
tissues of economical interest in Ruminants: a first step with the prediction of portal blood flow. In: Hill, D.R.C.,
Barra V. and Traore M.K. (editors), 1 st Open Intern. Conf. on model. and simul. OICMS, Clermont-Ferrand,
France, 163-173.
Nutr. Dev. 5, 527-546.

Bibliographical database applied to ruminant nutrition
J. Vernet, J.P. Brun and I. Ortigues-Marty
UR1213, Unité de Recherche sur les Herbivores, INRA, Theix, 63122 Saint Genès Champanelle,
France
Introduction
Animal nutrition calls upon various fields of knowledge (animal production, nutrition, metabolism).
In these various fields, researchers need references and increasingly need to integrate the available
knowledge. It is thus necessary to gather the published and available data in databases before
analyzing them by meta-analyses or using them in mathematical modelling. To date, an operational
database exists under Access which gathers the published results on nutrient exchanges between
organs and tissues in Ruminants (FLORA, Fluxes of nutrients through ORgans and tissues in
Ruminant Animals, (Vernet and Ortigues-Marty, 2006). The objectives of the present work were
to develop a new database tool to face new stakes such as a generalisation and widening to other
fields of knowledge, data sharing between more numerous and distant users, easiness of use for an
exploitation on a larger scale. Consequently, the present paper presents the specifications and the
preliminary steps of the construction of the new database.

The objectives for the new database were to create a general structure that would accept a wide
range of published results obtained on ruminant nutrition research, excluding those obtained via
high, throughput techniques. The database should therefore encompass various fields of knowledge,
e.g. zootechnical results, digestive fluxes of nutrients, metabolic regulations and also the physico-
chemical composition of animal products (e.g. muscle).
At a practical level, several objectives were set for the new database. It had to enable the management
of numerous data (currently Flora contains more than 50 000 lines of information) in a robust way.
In particular, to facilitate the data management, operations such as input, calculations, data retrieval
had to be made directly in the database. To increase easiness of use, a man-machine interface was
required. Finally, to be used as a research tool in different research projects, the database had to be
installed on Internet with a management of access rights in order to be shared in real time between
the various users. An APACHE, MySQL, PHP (AMP) environment was chosen for the development
of the new database, because of its capacities for data sharing on Internet, data management and
reliability.
In addition, meta-analyses on published results clearly showed that when combining data from
experiments with animals, two major difficulties appeared (Loncke et al., 2007). First, it was
difficult to define and describe feeds and diets homogenously. Usually, the chemical composition
and the nutritional values of the feeds are either reported in publications or estimated from the feed
composition tables of the various countries. However, differences exist among Feeding Systems
(e.g. INRA, NRC) and thus in the characterisation and expression mode of the values of feeds. It
was thus necessary to build a structure that made it possible to connect the various feeds used in
a publication with their feeding values expressed according to several Feeding Systems. Another
important difficulty when gathering data was to evaluate the potential impact of measurement
methods on data. These methods had thus to be described and coded in the database, to be used when
necessary to explain the observed differences at the intra- or inter-experiment levels.

Results
The relational model was defined with only 9 data tables and several reference tables (with free
or administered input). The publications, experiments, animals and treatments were described and
organized in 4 tables in a hierarchical way. All the various types of results obtained on the animals
(e.g. animal growth and body composition, digestive fluxes, blood fluxes of nutrients) were gathered
in one table. The corresponding variables were identified in 4 ways: (i) according to their nature (e.g.
dry matter intake, milk production, glucose), (ii) the anatomical or physical site of measurement (e.g.
whole body, liver, portal vein), (iii) the matrix of the sample (e.g. blood, plasma, digesta, tissue) and
(iv) the type of result (e.g. concentration, flux, quantity).
The description of diets and rations was subdivided into 2 tables: (i) the description of the nature of
feeds, diets and infused substrates which are reported in the publication and (ii) the description of
the physico-chemical composition of the feeds and diets as reported in the publication, or estimated
from different feed composition tables. The source of estimation was traced, so it was possible to
input several values from different Feeding Systems for each feed/diet and parameter of chemical
composition. The methods were described thanks to 2 distinct elements of structure. The experimental
measurement methods (e.g. infusion of markers, sampling methods) appeared in a separate table that
was associated to the publication table. The methods of determination (e.g. chemical determination
methods) were associated to the measured value or the result to indicate the conditions of their
acquisition. When meta-analyses are carried out, many codes are created to identify the various
eligible groups of treatments for the study one or more of variation factor (e.g. variation on intake,
variation on diet composition). For sake of traceability and in order to re-use them in other analyses,
the codes were gathered in a table.
For reasons of efficiency, the data management which was carried out previously under Excel was
now done under the database. For example, specific SQL queries based on the relational model to
cross information were created to estimate the chemical composition of the diets in FLORA from
the different Feeding Systems and to calculate new variables from existing ones. The data extraction
for meta-analyses was facilitated by means of a multi-criteria querior and a ‘dynamic crossing table’
tool. An interface towards the statistical treatment of data was sketched (R, SAS). In conclusion, the
developed database answers criteria to be used as a research tool for analysis of literature in ruminant
nutrition, by different researchers and on different independent projects.
References
Loncke, C., I. Ortigues-Marty, J. Vernet, H. Lapierre, D. Sauvant and P. Nozière, 2007. Assessment of duodenal starch
as a predictor of portal absorption of glucose in ruminants. Int. Symp. on Energy and Protein Metab. and Nutr.,
Vichy, France, in press.
Nutr. Dev. 5, 527-546.

Alternative regression approaches when modelling energy components
M.S. Dhanoa1, R. Sanderson1, S. Lopez2, J. Dijkstra3, E. Kebreab4 and J. France4
1Institute of Grassland and Environmental Research, Plas Gogerddan, Aberystwyth, SY23 3EB,
United Kingdom
2Department of Animal Production, University of León, E-24071 León, Spain
3Wageningen Institute of Animal Sciences, Animal Nutrition Group, Wageningen Agricultural
University, Marijkeweg 40, 6709 PG Wageningen, The Netherlands
4Centre for Nutrition Modelling, Department of Animal and Poultry Science, University of Guelph,
Guelph, ON, N1G 2W1 Canada
Introduction
Components of energy balance data in animals are generally inter-related and they are also subject
to measurement errors. Reduction or attenuation of the estimated regression slope when using
ordinary least squares linear regression is a well known effect (Dhanoa et al., 2000). This leads to
bias in the derived quantities such as the estimate of metabolisable energy intake for maintenance
(MEm) which is a function of the estimates of regression intercept and slope. Here we illustrate
this problem and suggest some alternative regression models, such as type II regression models, to
minimise the size of the slope-bias.

Data were used to estimate the efficiency of energy utilisation for milk production (kl). The database
used is a subset of that used by Kebreab et al. (2003) consisting of 433 metabolisable energy intake
(MEI) and net milk energy observations from 37 calorimetry studies. MEI was adjusted for tissue
gain with kg=0.60 and milk energy for tissue depletion with kt=0.84. All data used were as MJ/kg
body size0.75.
^
In ordinary least squares (OLS), simple linear model: yi = α^ + βxi + εi is fitted to a set of paired
^
sample values (xi, yi) to estimate parameters α^ and β which minimise Σε2i . The slope (kl) is given
^ ^ ^
by β = Σ(yi-y)(xi-x)/Σ(xi-x) , α = y - β x and MEm = -α^ / β . The problem arises because in OLS we
2 ^
minimise differences between the model and the observations in a direction that is parallel to the
y-axis whereas in the case of ‘errors in both variables’ we need to minimise these differences in
both directions simultaneously. Various options in model II regression provide alternatives in energy
balance modelling. In addition, energy data are also collinear.
If both error variances are unknown then Bartlett’s 3-group method (Bartlett, 1949) is appropriate
^
with slope given by β = (y3-y1) / (x3-x1) (Method II-1). Reduced major axis regression is appropriate
when both variables are measured on the same scale and the correlation among them is >0.6. The
slope is given by
–––––––
β = √ βyx / βxy
^ ^ ^
(Method II-2). Major principal axis regression (Sokal and Rohlf, 1995) is also a suitable option
with slope
––––––––––––––––––––
β = s12 / (λ - s12), λ = 0.5 [s12 + s22 + √ (s12 + s22) – 4(s12s22 - s12
^ 2 2 )]
where and are variances for milk energy and MEI respectively and is their covariance (Method
II-3). Inverse Gaussian regression (Folks and Chhikara, 1978; model C) may also be used as milk

^
energy as a function of MEI. Here β = (Σ (yi-y)2 / x2i ) / (Σ (xi-x)2 / x2i (Method II-4). A good estimate
of MEm is given by the intercept from OLS regression of MEI on (say) net milk energy (Method
II-5; W.M. Patefield, personal communication).
Estimates of kl and MEm were derived using the various type II regression methods and the 95%
confidence intervals were calculated using Bootstrap (Efron and Tibshirani, 1993; 1000 repetitions)
(Table 1).
Table 1. Estimates of kl and MEm from OLS and type II methods.
Method Estimate 95% Confidence interval
OLS kl 0.5554 (0.5341 - 0.5754)

MEm 0.5063 (0.4592 - 0.5465)
II-1 kl 0.5603 (0.5328 - 0.5856)
MEm 0.5159 (0.4589 - 0.5681)
II-2 kl 0.6130 (0.5927 - 0.6356)
MEm 0.6102 (0.5703 - 0.6504)
II-3 kl 0.5857 (0.5649 - 0.6081)
MEm 0.5636 (0.5211 - 0.6062)
II-4 kl 0.5554 (0.5331 - 0.5758)
MEm 0.5064 (0.4579 - 0.5471)
II-5 MEm (OLS) 0.7023 (0.6575 - 0.7527)
MEm (HGLM)1 0.7221 (0.6716 - 0.7761)
1Intercept corrected for multiple trials (normal-normal mixed model).
Conclusions
In energy balance modelling OLS is generally a poor choice since it is inappropriate when expressing
a relationship between two random variables (Kendall and Stuart, 1960) such as milk energy and
MEI. Method II-2 appears to give sensible estimates of kl and MEm and method II-5 seems to have
potential although further research is needed to arrive at firm recommendation.
References
Bartlett, M.S., 1949. Fitting a straight line when both variables are subject to error. Biometrics 5, 207–212.
Dhanoa, M.S., R. Sanderson and J. France, 2000. Dependence of kf and maintenance estimates on the choice of
regression model: model II regression. In: Chwalibog, A., Jakobsen, K. (editors), Energy Metabolism in Animals.
Wageningen Academic Publishers, Wageningen, The Netherlands, EAAP Publ. 103, 43-46.
Efron, B. and R.J. Tibshirani, 1993. An Introduction to the Bootstrap. Chapman & Hall, London.
Folks, J.L and R.S. Chhikara, 1978. The Inverse Gaussian Distribution and its Applications – A Review. J. R. Statist.
Soc. B, 40, 263-289.
Kebreab, E., J. France, R.E. Agnew, T. Yan, M.S. Dhanoa, J. Dijkstra, D.E. Beever and C.K. Reynolds, 2003. Alternatives
to linear analysis of energy balance data from lactating dairy cows. J. Dairy Sci. 86, 2904-2913.
Kendall, M.G. and A. Stuart, 1960. The Advanced Theory of Statistics. Charles Griffin and Company Limited,
London.
Sokal, R.R. and F.J. Rohlf, 1995. Biometry. 3rd edition. W.H. Freeman & Company, New York.

Comparison of mathematical models to evaluate various in situ
ruminant feed crude protein degradation kinetics
M. Danesh Mesgaran, T. Tashakkori, A.R. Vakili and A.R. Heravi Mousavi
Dept. of Animal Science, Excellence Center for Animal Science, Faculty of Agriculture, Ferdowsi
University of Mashhad, P O Box 91775-1163, Mashhad, Iran
Introduction
The in situ technique is a direct method of measuring the rumen degradation kinetics of a feed. It
was suggested to describe the data obtained by this technique by an exponential curve (Ørskov and
McDonald, 1979). This first order exponential model is a most common procedure for determining
crude protein and dry matter degradation coefficients. However, very low attention has been paid
to the choice of mathematical model to fit the curves and goodness-of-fit of the model. Different
mathematical models were evaluated to describe ruminal dry matter (DM) and crude protein (CP)
degradation kinetics of various feeds (Lopez et al., 1999; Fathi et al., 2006). In the present study,
two different mathematical models of a straight line or a negative exponential (Fathi et al., 2006)
were selected to evaluate crude protein (CP) degradation kinetics of various ruminant feeds including
alfalfa hay, corn and whole barley silages, barley and corn grains, cottonseed and soybean meals
using data obtained from the in situ technique.

Samples of various ruminant feeds including alfalfa hay, corn and whole barley silages, barley and
corn grains, cottonseed and soybean meals were incubated in the rumen of four Holstein steers (330
± 15 kg body weight) for 0.0, 2, 4, 8, 16, 24, 48, 72 and 96 h (8 replicates) using polyester nylon
bags (10×20 cm, 50 µm pore size). Animals were fed to maintenance body weight with a medium
quality alfalfa hay (35%), corn silage (15%), wheat straw (15%) and concentrate (25%) twice a
d, at 9:00 and 16:00 h, individually. Data of CP degradation were further adjusted to a segmented
linear model [model I, P = a + ct, Fathi et al., 2006] or a negative exponential model [model II, P
=a + b(1-e-ct), Ørskov and McDonald, 1979]; where P = fraction degraded in the time t, a = rapidly
degradable fraction, b = slowly degradable fraction, c = fractional degradation rate and t = incubation
time. Several statistics, including mean square prediction error (MSPE), root of MSPE (rMSPE)
and coefficient of determination (R-square) were used to evaluate goodness-of-fit of each model.
After fitting each model to each disappearance curve, statistics were calculated to detect significant
differences between models in MSPE, rMSPE and R-square using GLM of SAS (Y= mean + model
+ parameter + residual).

The results of CP degradation kinetics of each sample using models I and II are shown in Table 1.
In addition, for each model, the MSPE, rMSPE and R-square have been presented. The R-square
and MSPE, as indicators of model accuracy, show that model II gave significantly (P<0.05) better
fits to the feed CP degradation kinetics than model I. However, based on rMSPE, models I and II
showed the same fit to the data on CP disappearance. R-square showed that the variation was high
for models I compared with model II. Model I is a segmented model and needs a sufficient number
of observations in each segment to obtain a consistent solution, and model II is an exponential
model which is sometimes inadequate for describing ruminal disappearance curves (Fathi et al.,
2006). Lopez et al. (1999) pointed out that the disappearance of some feed components, particularly
structural carbohydrates, exhibits a larger variety of forms than does CP. In addition, the ruminal CP
degradation does not follow a zero order. The results of the present experiment showed that, based on

various statistical tests, the negative exponential model is well suited to describing the degradability
patterns obtained for CP of the feed samples.
Table 1. In situ dry matter degradation parameters estimated for various ruminants feed crude
protein using model I (P = a+ ct) and model II (P = a + b(1-e-ct)).
Model I Model II
Forages a b c MSPE rMSPE2 R2 a b c MSPE rMSPE2 R2
Alfalfa hay 0.35 0.47 0.06 0.02 4.12 0.90 0.43 0.49 0.06 0.01 3.99 0.93
Corn silage 0.35 0.33 0.04 0.04 5.71 0.81 0.28 0.33 0.05 0.03 5.50 0.88
Whole barley silage 0.33 0.42 0.04 0.03 5.2 0.89 0.43 0.33 0.02 0.02 4.84 0.94
Barley grain 0.29 0.63 0.07 0.03 4.84 0.90 0.34 0.44 0.06 0.02 4.92 0.97
Corn grain 0.31 0.50 0.06 0.03 4.99 0.81 0.29 0.41 0.05 0.02 5.02 0.91
Cottonseed meal 0.31 0.45 0.07 0.03 5.01 0.79 0.31 0.32 0.04 0.03 5.11 0.94
Soybeanmeal 0.42 0.49 0.09 0.02 4.16 0.89 0.44 0.46 0.09 0.01 3.78 0.97
a = rapidly degradable fraction, b = slowly degradable fraction, c = fractional degradation rate, MSPE = mean square
prediction error, rMSPE = root of MSPE expressed as a percentage of the observed mean and R2 = coefficient of
determination.
References
Fathi Nasri M.H., M. Danesh Mesgaran, J. France, J.P. Cant and E. Kebreab, 2006. Evaluation of models to describe
ruminal degradation kinetics from in situ ruminal incubation of whole soybeans. J. Dairy Sci. 89, 3087-3095.
Lopez, L., J. France, M.S. Dhanoa, F. Mould and J. Dijkstra, 1999. Comparison of mathematical models to describe
disappearance curves obtained using the polyester bag technique for incubating feeds in the rumen. J. Anim. Sci.
77, 1875-1888.
Ørskov, E.R. and I. McDonald, 1979. The estimation of protein degradability in the rumen from incubation measurements
weighted according to rates of passage. J. Agric. Sci. (Camb.) 92, 449-503.
This research was supported by a grant from Ferdowsi University of Mashhad and Excellence
Center for Animal Science.

Modelling energy partition in growing pigs
A. Strathe, A. Danfær, G. Thorbek and A. Chwalibog
Department of Basic Animal and Veterinary Sciences, Faculty of Life Sciences, University of
Copenhagen, Grønnegårdsvej 7, 1870 Frederiksberg C, Denmark
Introduction
Animal variation in partial energetic efficiencies of protein and fat retention are seldom estimated
when data from balance and respiration studies are analysed. The aim of this study was to reanalyse
previous measurements and model energy partitioning in growing pigs by means of random
regression by applying a model that estimates animal variation in energetic efficiencies of protein
and fat retention.

Forty-eight barrows were assigned to six different dietary treatments (i=1, 2,…, 6) with twelve pigs
in treatments 1 and 2 and six pigs per treatment for the rest of the groups. The pigs were fed close
to ad libitum. Eight repeated balance and respiration measurements were made on each pig from
20 to 90 kg of body weight (BW). For details see Thorbek (1975).
The population’s metabolisable energy (ME) requirement for maintenance (MEM) was determined
by the function MEM, MJ/d = 7033 + 33.9 × BW (Thorbek, 1975). Then the following relationship
between ME intake (MEI) and ME for production (MEP) (MEP, MJ/d = MEI – MEM) was applied.
Finally, the partial efficiencies α1i (MEP, MJ/RP, MJ) and α2i (MEP, MJ/RF, MJ) for retaining protein
(RP) and fat (RF) for the i’th treatment were derived from the following linear model (1):
MEPij = α1i × RPij + α2i × RFij +eij (1)
where refers j=1, 2,…, (8 x ni) – e.g. measurements from the same pig are independent.
Model (1) ignores the structure of the data set because the effect of animal was not taken into account
although it ought to be modelled as a random effect. This was corrected in the random regression
model (2):
MEPijk = Aij + (βi + βij) × RPijk + (γi + cij) × RFijk +eijk (2)
Where: i = 1, 2, ..., 6; j = 1, 2, ..., ni; k = 1, 2, ..., 8
 Aij   0 σA2  
 Bij  ~N  0; σAB σB2 2   with eijk ~ N (0, σ2)
 Cij   0 σAC σBC σC  
The coefficients (βi and γi) vary systematically with treatment and the coefficients (Aij, Bij and Cij)
vary among individuals within treatment according to a three dimensional normal distribution. The
deviation from the linear equation is modelled by eijk which has a constant variance. Model (2) thus
separates variation within individuals, in terms of eijk, from variation between individuals, in terms of
(Aij, Bij and Cij). Furthermore, Aij can be regarded as the random residual maintenance requirement for
the jth pig within the ith treatment because MEP was calculated at the population level. Appropriate
tests of both fixed and random effects were based on the likelihood ratio test and parametric bootstrap
if P-values were close to the significance limit (P<0.05) (Pinheiro and Bates, 2000). All computations
were done in R (R project, 2004) utilising the nlme package by Pinheiro and Bates (2000).

The data was fitted to model (2) and the following model reductions were attempted. The first
simplification of the model (2) was to set the non-diagonal elements in the covariance matrix equal
to zero (P≤0.001). It is clear that variance components (Aij, Bij and Cij) are not independent due to
the correlation between MEP, RP and RF. The effect of treatment on utilisation of MEP for protein
and lipid deposition was (P=0.442 and P=0.036), respectively. Thus, the utilisation of MEP for fat
gain was affected by treatment whereas protein gain was not. The parameters of the final model are
presented in Table 1 for both fixed and random effects.
Table 1. Estimated parameters in the random regression model.
Solutions for fixed effects
β γ1 γ2 γ3 γ4 γ5 γ2
Estimate 1.854 1.391 1.391 1.271 1.307 1.398 1.424

Std. error 0.047 0.034 0.034 0.044 0.046 0.046 0.046
Solution for random effects
σ2A σAB σAC σ2B σBC σ2C σ2
Estimate 9.758 -4.527 0.316 0.203 -0.144 0.018 8.700
A wide range of values for the energy cost of both protein (1.30 to 2.82, MEP, MJ/RP, MJ) and lipid
(1.01 to 1.71, MEP, MJ/RF, MJ) deposition in pigs are reported by Noblet et al. (1999). The present
analysis presented estimates that were all in the range of those previously reported. The random
effect of animal was presented in a simulation study where 1000 samples are drawn from the three
dimensional normal distribution based on the estimated covariance matrix. From the simulation study
and utilising the estimates (β=1.854 and γ1=1.391) the 5% quantile, mean and 95% quantile of the
sampled animal variation in protein and lipid retention were estimated. For protein these amounted to
1.35, 1.85 and 2.34 (MEP, MJ/RP, MJ) and lipid 1.29, 1.39 and 1.51 (MEP, MJ/RF, MJ), respectively.
However, the present analysis also reveals that the random covariance between (Aij, Bij and Cij) was
strong and that the present analysis does not present solutions to circumvent this problem.
In conclusion, it was demonstrated that the data was successfully fitted to a random regression model
and thus it was possible to extract information about animal variation in the efficiency of protein
and lipid accretion.
References
Noblet, J., C. Karege, S. Dubois and J. van Milgen, 1999. Metabolic utilization of energy and maintenance requirements
in growing pigs: effects of sex and genotype. J. Anim. Sci. 77, 1208-1216.
Pinheiro, J.C. and M.B. Bates, 2000. Mixed effects models in S and S-PLUS. Statistics and Computing. Springer,
New York, 528 pp.
R Development Core Team, 2004. A language and environment for statistical computing. R Foundation for Statistical
Computing, Vienna, Austria. http://www.Rproject.org.
Thorbek, G., 1975. Studies on energy metabolism in growing pigs II. Protein- and fat gain in growing pigs fed different
feed compounds. Efficiency of utilization of metabolizable energy for growth. Beret. Statens Husdyrbrugsfor.
424, 1-159.

Using meta-analysis to study residual feed intake and CVDS model
predictions of feed intake and efficiency in growing and finishing cattle
B.M. Bourg, L.O. Tedeschi, G.E. Carstens and P.A. Lancaster
Texas A&M University, College Station, TX 77843, USA
Introduction
The conversion of feed into animal products during the post-weaning growth phase has a large
influence on the cost of producing beef (Herd et al., 2003). The Cattle Value Discovery System
(CVDS; http://nutritionmodels.tamu.edu/cvds.htm) was developed to predict growth and feed
requirements of individual cattle fed in groups based on animal, diet, and environment information
(Tedeschi et al., 2006). CVDS utilizes body weight (BW), average daily gain (ADG), carcass
measurements, environmental conditions, and diet metabolizable energy (ME) to predict BW at
28% empty body fat, feed dry matter required (DM) for maintenance (FFM), and feed DMR for gain
(FFG). Model predicted intake difference (PID) is computed as observed DM intake (DMI) minus
DMR, partial efficiency of growth (PEG) is computed as ADG/FFG, and DMR to ADG ratio (R:
G). Residual feed intake (RFI) is the residual error of the linear regression between observed DMI
on metabolic BW and ADG.
The objectives of this study were (i) to evaluate the effectiveness of the CVDS in predicting DMR
from observed animal ADG and (ii) to examine phenotypic correlations between predicted and
observed DMI and feed efficiency traits from eight studies using meta-analysis.

Database description
Two databases were compiled based on growing or finishing diets. The growing database contained
4 studies of steers and heifers (n=514; initial and final BW of 274 to 352 kg) fed high-roughage diets
(2.06 to 2.14 Mcal ME/kg). The finishing database contained 4 studies of steers (n=321; initial and
final BW of 358 to 521 kg) fed high-concentrate diets (2.73 to 2.99 Mcal ME/kg). Within studies
cattle were individually fed and managed in a similar manner.
Statistical analysis
The MIXED procedure of SAS (SAS Institute Inc., Cary, NC) was used to compute the mixed RFI
(RFIx) for growing and finishing databases assuming studies within databases as random effects
and variance components for the variance-(co)variance matrix (Ψ) using Equation [1]. A simpler
random coefficient model (RCM) was used to adjust the Y-variate to the effects of studies. The fixed
effects plus the residual error from the RCM were combined and Pearson correlation coefficients
were obtained. In these two RCM, both intercept and slopes were adjusted for studies. Similarly, a
third RCM was used to compare RFIx and other variables, but no adjustment on the intercept for
studies was allowed.
RFIxij = DMIij - DMIij and DMIij = ai + bi × ADGij + ci × BW0.75 (1)
Where a, b, c ~ N((β0, β1, β2), Ψ) and RFIxij ~ N(0, σ2) for ith study and jth level of X-variate.

The CVDS was able to account for 56 and 66% of the variation in DMI for growing and finishing
calves; respectively. Accordingly, PID was able to account for 61% and 34% of the variation in
RFIx in growing and finishing calves; respectively. Moderate to strong correlations were observed
in both databases between R:G and actual feed efficiency traits (FCR, PEG), but no correlation
was observed with RFIx. With growing calves, random study effects accounted for up to 27% of
variation, whereas, fixed effects accounted for up to 97%. Random study effects accounted for up
to 58% of the variation in the finishing database, indicating a greater variation among studies in
the finishing database. Overall, correlations in this meta-analysis tended to be stronger than those
reported by Arthur et al. (2001). However, a weaker correlation between DMI and FCR was noted
in this analysis. Correlations between PID and RFI were 0.58 and 0.78 for finishing and growing
calves, respectively, and were similar to the correlation reported by Arthur et al. (2001) in which
PID was computed using French feeding standards. As was noted in Arthur et al. (2001), PID was
not independent of BW and ADG.
Table 1. Pearson correlation of variables adjusted for study variation of growing (above diagonal)
and finishing (below diagonal) cattle databases.
DMI ADG BW0.75 FCR PEG PID FFM FFG DMR RFIx R:G
DMI - 0.68 0.72 0.12 -0.46 0.19 0.67 0.69 0.75 0.56 -0.15
ADG 0.76 - 0.43 -0.74 0.47 -0.66 0.43 0.98 0.95 0.0a -0.77
BW0.75 0.73 0.47 - 0.13 -0.21 -0.18 1.0 0.58 0.73 0.0a 0.33
FCR 0.10b -0.77 0.17 - -0.91 0.90 0.12 -0.65 -0.55 0.60 0.84
PEG -0.37 0.68 -0.27 -0.95 - -0.92 -0.19 0.38 0.28 -0.85 -0.59
PID 0.42 -0.34 0.06a 0.75 -0.88 - -0.15 -0.64 -0.60 0.78 0.47
FFM 0.63 0.35 0.90 0.15 -0.19 -0.08a - 0.56 0.70 0.0a 0.30
FFG 0.78 0.95 0.61 -0.63 0.50 -0.55 0.60 - 0.99 0.01a -0.61
DMR 0.81 0.92 0.73 -0.54 0.38 -0.51 0.79 0.99 - 0.01a -0.48
RFIx 0.57 0.0a 0.0a 0.66 -0.78 0.58 0.03a 0.03a 0.03a - 0.03a
R:G -0.25 -0.80 0.24 0.83 -0.72 -0.11 0.39 -0.32 -0.08a 0.02a -
aNot different from zero at P>0.05.

bCorrelations tended to differ from zero at P<0.10.
Conclusion
Models can account for up to 66% of the variation in observed DMI, but more research is needed to
account for more individual variation beyond BW0.75 and ADG alone. PID and RFIx had medium
to high correlations, suggesting some similarities in these variables.
References
Arthur, P.F., G. Renand and D. Krauss, 2001. Genetic and phenotypic relationships among different measures of growth
and feed efficiency in young Charolais bulls. Livest. Prod. Sci. 68, 131-139.
Herd, R.M., J.A. Archer and P.F. Arthur, 2003. Reducing the cost of beef production through genetic improvement in
residual feed intake: Opportunity and challenges to application. J. Anim. Sci. 81 (E. Suppl. 1), E9-E17.
Tedeschi, L.O., D.G. Fox, M.J. Baker and D.P. Kirschten, 2006. Identifying differences in feed efficiency among
group-fed cattle. J. Anim. Sci. 84, 767-776.

Net partial efficiencies of metabolizable energy utilization for protein
and fat gain in Nellore cattle
P.V.R. Paulino1, S.C. Valadares Filho1, M.L. Chizzotti1, E. Detmann1, M.A. Fonseca1, M.I. Marcondes1
and R.D. Sainz2
1Universidade Federal de Viçosa, Departamento de Zootecnia, Av. P.H. Rolfs, sn, Campus
Universitário, 36570-000, Viçosa, MG, Brazil
2University of California, One Shields Avenue, Davis, CA, 95616-8521, USA
Introduction
Nellore, a Bos indicus breed, is the main breed used in Brazil for beef production. Currently, it
accounts for more than 80% of the Brazilian bovine herd, which is estimated to have over 200
million heads. It is a very adapted breed and the animals can deal pretty well in harsh environments.
A great amount of the Brazilian beef production systems are located in the Mid-West of the country,
where the animals have to deal with high temperatures and humidity, high load of parasites and
remarkable fluctuation on availability and quality of the feed throughout the year, since the beef
production systems are based on pasture, whose quality and amount decreases from the rainy to
the dry season of the year. The Nellore cattle can survive and be productive under these conditions
due to particular adaptations of their metabolism. It has been shown that the energy requirement
for maintenance of Nellore cattle is lower than that of Bos taurus breeds as a result of differences
in the body composition, protein turnover rate and gut size (Sainz et al., 2006). In spite of that, few
studies have been conducted on the energetic metabolism of Nellore cattle in Brazil and there is no
evidence of any study that estimated the net partial efficiencies of metabolizable energy utilization
for protein and fat gain in Nellore cattle, raised under the Brazilian conditions. Hence, that was the
objective of this work.

Data of eleven trials involving measurements of energetic metabolism in Nellore cattle in Brazil
were gathered and analyzed. After the removal of outliers, the data set consisted of 185 individual
observations, with complete information available on initial and final empty body weight (EBW),
d on feed, initial and final body composition, retained energy (RE), retained protein (RP) and
metabolizable energy intake (MEI). The descriptive statistics of the data is shown in Table 1. The
MEI ranged from maintenance to ad libitum level. A multiple regression analysis to partition MEI
(Mcal/kg EBW0.75/d) to protein and fat deposition was done as suggested by Williams and Jenkins
(2003), according to the model:
MEI = b0 + b1 × REprot + b2 × REfat (1)
where REprot and REfat stand for recovery energy as protein and fat, respectively (Mcal/kg EBW0.75/
d). The intercept (b0) of this equation is interpreted as an estimate of the maintenance requirement
(MEm) and the partial regression coefficients b1 and b2 represent the amounts of ME required for the
deposition of 1 Mcal of ME as protein or fat, respectively. Since the metabolizable energy required
for maintenance is not used to deposit protein or fat, the estimate of MEm (b0) obtained was used to
discount on the MEI of each animal and the equation was fitted again.

The equation developed was:

MEI = 0.00 + 4.66 REprot + 1.30 REfat, r2=0.739; Sxy=0.026 (2)
The intercept of the equation was not significantly different from zero (P>0.01), suggesting that
the maintenance requirement was completely accounted for and the remainder of the MEI was then
partitioned between protein and fat deposition. Before discounting, the b0 value obtained was 136
kcal/EBW0.75 that represents the energy requirements for maintenance. The efficiencies of protein
and fat deposition were 0.21 (1/4.66) and 0.77 (1/1.30), respectively. These values are within the
range considered adequate for ME partial efficiencies in bovines, which are 0.10 to 0.40 for protein
gain and 0.60 to 0.80 for fat gain (Baldwin, 1995). So, since the nutritional requirement guidelines
of Nellore cattle are still being established in Brazil, the results provided herein can be effectively
incorporated into any empirical feeding system for Nellore cattle that could be built in the near
future, using only Brazilian data.
Table 1. Descriptive statistics for the data used to estimate the partial efficiencies of metabolizable
energy utilization for protein and fat gain in Nellore cattle.
Items1 Mean Minimum Maximum Std
iBW, kg 297.8 151.1 433.2 61.09

fBW, kg 392.5 191.0 520.0 69.83
iEBW, kg 256.0 130.4 355.5 49.70
fEBW, kg 350.0 169.3 452.2 61.41
MMEBW, kg0,75 72.41 44.50 89.07 9.36
ADG, kg/d 0.86 -0.12 1.72 0.40
EBWG, kg/d 0.86 0.03 1.67 0.38
dProt., kg/d 0.13 -0.05 0.34 0.08
dFat, kg/d 0.32 -0.11 0.75 0.19
MEI, Mcal/d 17.32 6.43 28.79 4.91
RE, Mcal/d 3.80 0.00 8.09 1.98
REprot., Mcal/d 0.76 -0.28 1.91 0.44
REfat, Mcal/d 3.05 -1.03 7.04 1.79
1iBW = initial body weight; fBW = final body weight; iEBW = initial empty body weight; fEBW = final empty body
weight; MMEBW = mid-point metabolic body weight; ADG = average daily gain; EBWG = empty body weight
gain; dProt = change in weight of protein in EBW; dFAT = change in weight of ether-extractable lipid in EBW; MEI
= metabolizable energy intake; RE = retained energy; REprot. = retained energy as protein; REFat = retained energy
as fat.
References
Baldwin, R.L., 1995. Energy requirements for maintenance and production. In: Baldwin, R.L. (editor), Modeling
ruminant digestion and metabolism. Chapman and Hall, London, UK, 148-188.
Sainz, R.D., L.G. Barioni, P.V. Paulino, S.C. Valadares Filho and J.W. Oltjen, 2006. Growth patterns of Nellore vs.
British beef cattle breeds assessed using a dynamic, mechanistic model of cattle growth and composition. In:
Kebreab, E., J. Dijkstra, A. Bannink, W.J.J. Gerrits and J. France (editors), Nutrient digestion and utilization in
farm animals. CABI Publishing, Oxfordshire, UK, 160-170.
Williams, C.B. and T.G. Jenkins, 2003. A dynamic model of metabolizable energy utilization in growing and mature
cattle. III. Model evaluation. J. Anim. Sci. 81, 1390-1398.

Residual feed intake, energy and protein metabolism in beef steers
R.D. Sainz1, F.C.P. Castro Bulle1,2, P.V.R. Paulino1,3 and J.F. Medrano1
1University of California-Davis, 95616 Davis, CA USA
2Bolsista CNPq - Brazil
3Universidade Federal de Viçosa, Viçosa-MG, Brazil
Introduction
Residual feed intake (RFI), the difference between actual and expected intake (Koch et al., 1963),
is moderately heritable (Arthur et al., 2001). Possible mechanisms underlying differences in RFI
include differences in digestive efficiency, maintenance energy requirement, net energetic efficiency,
and composition of gain. Changes in protein degradation may affect both maintenance and feed
conversion efficiency due to altered protein:fat in the gain. The present study was aimed at examining
the relationships among feed intake, efficiency of gain, carcass composition, energy metabolism
and myofibrillar protein turnover.

Twenty-four beef steers, predominantly Angus×Hereford and approximately 14 to 18 mo of age
with a mean weight of 403 (± 3) kg were housed and fed ad libitum in individual pens for 112 to
135 d (mean=122 d). The diet was based on cracked corn and alfalfa, with 3.06 Mcal ME/kg and
13.6% CP on a DM basis, respectively. DMI was regressed against the average metabolic BW and
ADG (Archer et al., 1997):
DMI (kg/d) = β0 + β1 BW0.75 + β2 ADG + ε
where ε represents the RFI. Steers with RFI≥0.5 SD above or below the mean were classified as High
or Low residual feed intake (n=8/class), respectively. Myofibrillar protein breakdown rate (FDR)
was estimated from the urinary excretion of 3-methylhistidine (3MH; Harris and Milne, 1981) in
12 steers. Energy and protein retention and maintenance energy requirement (MEm) were estimated
by comparative slaughter after calculation of net energetic efficiency of growth (kg) from the gains
of protein and fat (Williams and Jenkins, 2003).

By definition, average weights and daily gains were similar between groups although DM intakes (kg/
d) were greater in High (7.52) than in Low (6.61 kg/d) RFI steers. Average RFI for the two groups were
-0.367 and 0.380 kg/d, respectively. Energy retention (Mcal/kg0.75/d) tended to be greater (P=0.08)
in High (0.0773) than in Low (0.0585) RFI steers. There were no significant differences in net
energetic efficiency, maintenance energy or fractional rate of myofibrillar protein degradation (FDR).
There was, however, a significant correlation between FDR and MEm (r=0.76). This relationship is
shown in Figure 1. Several DNA markers were examined; for example, polymorphisms in calpain
and DGAT1 were related to differences in growth, post-mortem proteolysis and meat tenderness,
and possibly energy and protein metabolism as well.

Figure 1. Regression between ME requirement for maintenance (MEm) and fractional degradation
rate (FDR) of myofibrillar proteins; ■, High RFI steers; □, Low RFI steers; ▲, steers with RFI
within 0.5 standard deviation of the mean (zero).

This preliminary study included a small number of animals, and found no significant differences in
MEm or protein degradation between low- and high-RFI steers. However, the positive relationship
between these variables across all groups confirms the contribution and importance of protein
turnover to maintenance energy expenditures. Therefore, it is impossible to separate energy and
protein metabolism. Improvements in energetic efficiency will likely involve alterations in protein
metabolism, which may have implications for post-mortem product quality development.
References
Archer, J.A., P.F. Arthur, R.M. Herd, P.F. Parnell and W.S. Pitchford, 1997. Optimum postweaning test for measurement
of growth rate, feed intake, and feed efficiency in British breed cattle. J. Anim. Sci. 75, 2024–2032.
Arthur, P.F., J.A. Archer, D.J. Johnston, R.M. Herd, E.C. Richardson and P.F. Parnell, 2001. Genetic and phenotypic
variance and covariance components for feed intake, feed efficiency, and other postweaning traits in Angus cattle.
J. Anim. Sci. 79, 2805-2811.
Harris, C.I. and G. Milne, 1981. The urinary excretion of Nτ-methylhistidine by cattle: validation as an index of muscle
protein breakdown. Br. J. Nutr. 45, 411-429.
Koch, R.M., L.A. Swiger, D. Chambers and K.E. Gregory, 1963. Efficiency of feed use in beef cattle. J. Anim. Sci.
22, 486-494.
Williams, C.B. and T.G. Jenkins, 2003. A dynamic model of metabolizable energy utilization in growing and mature
cattle. II. Metabolizable energy utilization for gain. J. Anim. Sci. 81, 1382–1389.

Body nutrient composition of two broiler chicken strains
N.K. Sakomura, S.M. Marcato, J.B.K. Fernandes and I.A.M.A. Teixeira
Universidade Estadual Paulista, Faculdade de Ciências Agrárias e Veterinárias, Via de Acesso Prof.
Paulo Donato Castelane, s/n - Jaboticabal 14884-900, São Paulo, Brazil
Introduction
The adjustment in the nutritional levels for broilers and the resulting increase in animal performance
and profits require the knowledge of body composition as well as the genetic potential of bird
growth (Hruby et al., 1996). In this sense, growth curves of a given strain may help to define
specific nutritional programs and the optimum age for slaughtering (Knízetová et al., 1991). It is
important to know the body nutrient composition of broiler strains to achieve successful adjustments
on the nutritional levels, aiming to increase performance and profits. The growth of body chemical
components can be predicted by growth parameter estimation of nonlinear function, and the chemical
component weights can be predicted from allometric relationships between body nutrient and
body protein weights. The aim of this work was to estimate growth parameters of body chemical
components and allometric relationships between body chemical components and body protein
weight of 2 broiler strains.

A total of 1920 male and female Cobb 500 and Ross 308 chicks were distributed randomly in a
factorial arrangement (2 strains×2 sex), 4 replicate pens of 120 birds from 1 to 56 d of age. The birds
were fed a corn/soybean-based diet formulated to supply the EM and CP requirements according
to phase: pre-starter 3010 EM and 22% PB, starter 3150 EM and 21.50% CP, grower 3200 EM and
20% CP, final 3245 EM and 18% CP. The birds were weighed weekly (1 to 56 d) to determine body
weight and 4 birds were sampled in each replicate pen, a total of 16 birds were slaughtered weekly.
After 24 h of fasting they were slaughtered, grinded and sampled. The samples were freeze-dried
to be analyzed for dry matter, protein, ash and fat. The parameters of 4 replicates of weight (water,
protein, fat and ash) obtained weekly were adjusted by the Gompertz equation in order to estimate
the growth parameters Wt = Wm exp(-exp(-b(t-t*))). The body nutrient weight at time (Wt) were
described in terms of the mature weight (Wm, g), their rates of maturing (b) and the time to reach
the maximum rate of growth of each component (t*,d). Allometric relationships between the
body chemical components and body protein weight were determined by using logarithmic linear
regressions. The data were adjusted by using PROCNLIN procedure of SAS software.
Results
As demonstrated in Table 1, there was no significant interaction of strains and sex (P>0.05) for the
parameters of the Gompertz equation for protein and fat body weight. The Ross showed lower b and
consequently higher t and Wm compared to Cobb for protein weight. For fat weight, just a difference
in the b value was observed between strains. For water and ash weight, there was an interaction of
strains and sexes. For water weight, the male Ross had higher b and consequently lower t and Wm
than those of Cobb. However, those parameters were similar for Ross and Cobb females. For ash
weight, male and female Ross showed lower b, but t and Wm were superior to those of Cobb. Not
considering the strain effect, males showed higher Wm and t* and lower b than those of females
for all nutrients. These results indicate that the Cobb was more precocious than the Ross strain. The
allometric equations of body fat, ash and water by protein weight are shown in Table 2. According
to allometric coefficients of equations, higher ratios of fat per body protein than ash and water per
body protein were observed. Males had lower allometric coefficients for fat, showing that females

had higher fat deposition by protein weight than males. Cobb males had lower allometric coefficients
than Ross males, however, the coefficients for females were similar for both strains. The coefficients
for ash and water by protein weight were similar for both sexes and strains.
Table 1. Parameter estimates of the Gompertz equation for protein, fat, water and ash body weight
of Ross and Cobb male and female broilers.
Wm, g b, per d t*, d
Strains Males Females Means Males Females Means Males Females Means
Protein
Ross 1308.6 865.7 1087.2a 0.037 0.044 0.040b 44.0 37.9 40.9a
Cobb 1041.9 666.7 854.3b 0.047 0.056 0.051a 37.2 31.0 34.1b
Means 1175.3A 766.2B 0.041B 0.049A 40.6A 34.5B
Fat
Ross 907.2 810.9 859.0 0.039 0.041 0.039b 46.8 44.5 45.6
Cobb 930.7 780.8 855.7 0.041 0.043 0.042a 46.5 42.9 44.7
Means 918.9A 795.85B 0.040 0.041 46.6A 43.8B
Water
Ross 3215.7Ab 2269.9Ba 2742.8 0.052Ba 0.057Aa 0.054 32.7Ab 28.9Ba 30.8
Cobb 4027.9Aa 2342.5Ba 3185.3 0.045Bb 0.054Aa 0.049 37.3Aa 29.2Ba 33.3
Means 3621.8 2306.2 0.048 0.055 35.0 29.1
Ash
Ross 360.3Aa 115.1Ba 237.70 0.038Bb 0.061Ab 0.049 52.6Aa 29.2Ba 40.9
Cobb 173.8Ab 87.4Ba 130.59 0.051Ba 0.082Aa 0.067 34.1Ab 23.9Ba 28.9
Means 267.1 101.2 0.045 0.071 43.4 26.5
Wm = weight at mature in d; b = mature rate per d; t* = Time at maximum mature rate in d; ab Means with different
small letters in the same column differ significantly (P<0.05); AB Means with different capital letters in the same row
differ significantly (P<0.05).
Table 2. Logarithmic linear regression of body weight expressed as fat (F), water (W) and ash (A)
to body protein weight (PW), of Ross and Cobb male and female broilers.
Ross Cobb
Males Females Males Females
Fat F = -0.77 + 1.21PW F = -0.79 + 1.26PW F =-0.64 + 1.16PW F = -0.77 + 1.24PW

(R2 =0.99) (R2 =0.99) (R2 =0.99) (R2 =0.99)
Water W = -0.77 + 0.92PW W = -0.81 + 0.91PW W = -0.78 + 0.92PW W = -0.75 + 0.93PW
(R2 =0.99) (R2 =0.99) (R2 =0.99) (R2 =0.99)
Ash A = -0.52 + 0.92PW A = -0.53 + 0.92PW A = -0.50 + 0.91PW A = -0.43 + 0.86PW
(R2 =0.98) (R2 =0.98) (R2 =0.98) (R2 =0.98)
References
Hruby, M., M.L. Hamre and N. Coon, 1996. Non-linear and linear functions in body protein growth. J. Appl. Poult.
Res. 5, 109-115.
Knízetová, H., J. Hyánek, B. Knize and J. Roubícek, 1991. Analysis of growth curves of fowl. I. Chickens. Br. Poult.
Sci. 32, 1039-1053.

5E. Feed evaluation and methanogenesis

Rumen methanogenesis of dairy cows in response to increasing levels of
dietary extruded linseeds
C. Martin, A. Ferlay, Y. Chilliard and M. Doreau
INRA, UR1213 Herbivores, Site de Theix, F-63122 Saint-Genès-Champanelle, France
Introduction
A major concern of citizens in many countries is the increase in greenhouse gas production and
their impact on climate changes. Methane (CH4) is a greenhouse gas for which the release into
the atmosphere is directly linked with animal agriculture, particularly ruminant production. CH4
emissions from ruminants also represent a loss in productive energy for the animal. Dietary fat seems
to be the most promising dietary alternative to depress ruminal methanogenesis (Martin et al., 2006).
It has been shown in vivo that fatty acids (FA) from linseeds may decrease methane production in
growing lambs (Machmüller et al., 2000) or in sheep (Czerkawski et al., 1966). But to our knowledge,
this effect has never been confirmed on dairy cows. Moreover, the effects of extruded linseeds, and
of the level of linseeds FA supplementation in the diet on methanogenesis are unknown, whereas
little is known on changes in milk fatty acid composition (Chilliard and Ferlay, 2004). The present
study was aimed at evaluating the changes in rumen methanogenesis of dairy cows to increasing
levels of extruded linseeds (ELS) in the diet.

Four Holstein dairy cows (average initial milk yield and BW of 32.5 ± 5.1 kg/d and 627 ± 54 kg,
respectively) were randomly assigned to four dietary treatments in a 4 × 4 Latin square design. Diets
were a control diet (50% natural grassland hay and 50% concentrate on a DM basis) without extruded
linseeds (ELS0) or supplemented with 5% (ELS5), 10% (ELS10), or 15% (ELS15) of extruded
linseeds and corresponding to oil levels of 2, 4, and 6% of dietary DM, respectively. Cows had ad
libitum access to the diets (10% refusals). The desired forage:concentrate ratio was maintained by
daily adjustment of offered amounts of forage and concentrate, depending on the composition of
the refusals of the previous day.
Each experimental period lasted 4 wk, measurements occurring the last wk of each period. Feed
offered and refused as well as total collection of faeces were quantified daily on 6 consecutive d
to determine intake and total tract digestibility. Milk yield was determined on 5 consecutive d and
individual CH4 productions on 4 consecutive d using the sulphur hexafluoride (SF6) tracer technique
as described by Pinares-Patiño et al. (2003).
Data on DM intake, milk production, diet digestibility and CH4 production were averaged over the
d of measurements before statistical analysis. All data from experiment were analysed as a 4 × 4
Latin square using the MIXED procedure of SAS (2000). The statistical model included cow, period,
treatment and residual error. Fixed effects included period and treatment. Cow was the random effect.
Overall differences between treatment means were declared significant at P<0.05. Trends towards
significance were considered at P<0.10.
Results
Feed intake (20.0 kg/d at mean; P=0.29) and milk yield (26.6 kg/d at mean; P=0.57) were similar
among treatments as well as total-tract DM digestibility (70.3% at mean; P=0.26) (Table 1). The
amount of CH4 emitted daily by dairy cows decreased with the level of extruded linseed in the diet
(P<0.01): -15% with ELS5 (P=0.09), -19% with ELS10 (P<0.05) and -40% with ELS15 (P<0.01)

compared to ELS0. The same significant response to increasing levels of extruded linseeds was
observed for CH4 output expressed in proportion to a gross energy intake (P<0.01) or in L/kg of
DM digested (P<0.05). This evolution was less marked for CH4 production expressed in L/kg of
milk (P=0.09).
Table 1. Intake, milk production, digestibility and methane emissions from dairy cows in response
to increasing levels of dietary extruded linseeds.
Diet1 SE Diet effect

P-value
ELS0 ELS5 ELS10 ELS15
DM intake, kg/d 20.9 18.9 20.3 19.9 0.79 0.29

Milk, kg/d 26.1 25.8 28.3 26.4 1.68 0.57
DM digestibility, % 71.0 68.9 71.5 69.8 0.88 0.26
Methane
L/d 679.8a 575.7ab 552.4b 405.3c 41.86 <0.01
% gross energy intake 6.8a 5.8ab 5.5b 4.0c 0.34 <0.01
L/kg DM digested 45.8a 44.2a 38.3ab 29.1b 2.99 <0.05
L/kg milk 26 23.6 19.9 16.4 2.25 0.09
1ELS0 = control diet without extruded linseeds; ELS5, ELS10 and ELS15 = diets including 5, 10 and 15% of
extruded linseeds, respectively; a, b Means within rows with same superscript letters are not significantly different
(P>0.05).
Conclusion
This study shows that CH4 production of dairy cows decreased in response to increasing levels of
ELS. The addition of ELS up to 15% of dietary DM (i.e. 6% of oil) decreases rumen methanogenesis
without altering cow performance (DM intake, milk yield) and total-tract digestibility. Further work
is in progress to evaluate the effect of ELS on milk nutritional quality, and their suitability to reduce
CH4 emissions across a range of diets.
References
and sensory properties. Reprod. Nutr. Dev. 44, 467-492.
Czerkawski, J.W., K.L. Blaxter and F.W. Wainman, 1966. The metabolism of oleic, linoleic and linolenic acids by
sheep with reference to their effects on methane production. Br. J. Nutr. 20, 349-362.
Machmüller, A., D.A. Ossowski and M. Kreuzer, 2000. Comparative evaluation of the effects of coconut oil, oilseeds and
crystalline fat on methane release, digestion and energy balance in lambs. Anim. Feed Sci. Technol. 85, 41-60.
Martin, C., D.P. Morgavi, M. Doreau and J.P. Jouany, 2006. Comment réduire la production de méthane chez les
ruminants? Fourrages 187, 283-300.
Pinares-Patiño, C.S., R. Baumont and C. Martin, 2003. Methane emissions by Charolais cows grazing a monospecific
pasture of timothy at four stages of maturity. Can. J. Anim. Sci. 83, 769–777.
Statistical Analysis Systems Institute, 2000. SAS Version 8. SAS Inst. Inc., Cary, NC.
The authors thank experimental cowshed workers and staff of the laboratory.

Energy utilisation and methane conversion rate in Indonesian
indigenous sheep fed Napier grass supplemented with pollard
A. Purnomoadi1, F.Y. Devi1, R. Adiwinarti1, E. Rianto1, O. Enishi2 and M. Kurihara2
1Faculty of Animal Agriculture, Diponegoro University, Semarang 50275, Indonesia
2National Institute of Livestock and Grassland Science, Tsukuba, Ibaraki 305-0901, Japan
Introduction
Sheep production in Indonesia is low since most sheep are raised in small-holder farms that allow
the sheep to grass only without any supplements. In fact, supplementation may increase feed quality
and animal productivity (Leng, 1993; Purnomoadi et al., 2003), but consequently it will increase
the production cost. Therefore, supplementation with cheap feedstuff such as by-products should
be explored for this purpose. Pollard, the by-product from the noodle industry is a very promising
feedstuff due to its high protein, carbohydrate and energy content. The type of carbohydrate influences
methane production since fermentation of soluble carbohydrate is less methanogenic than that of
cell wall carbohydrates (Moe and Tyrrell, 1979). Energy losses in the form of methane vary from
approximately 2 to nearly 12% of Gross Energy (GE) intake (Johnson and Johnson, 1995). This
loss of energy from feed reduces retained energy and in turn reduces productivity. Moreover, this
loss of methane contributes to greenhouse gas and to global warming. Hence, reducing methane
production from ruminants must be achieved for production efficiency and environmental issues.
In this study, the use of pollard to improve animal productivity and reduce methane production was
investigated.

Twelve indigenous sheep, aged 12 mo and weighing 23 kg were used in this study. The sheep
were allocated into a completely randomised design (CRD) with 3 treatments and 4 replicates. The
sheep were fed Napier grass ad libitum as a basal diet. The treatments applied were supplemented
with pollard, namely NP0 (Napier grass and pollard supplemented at 0% body weight, or no
supplementation), NP1 (pollard supplemented at 1% BW) and NP2 (pollard supplemented at 2%
BW). The sheep were adapted to the diets for a mo prior to the data collection period. The chemical
composition (dry matter basis) of Napier grass was 13.1% Crude Protein (CP), 31.1% Crude Fibre
(CF), 33.7% Nitrogen Free Extract (NFE) and GE 16.75 kJ/g, while the composition of pollard was
18.7% CP, 6.9% CF, 65.9% NFE and GE 19.73 kJ/g, respectively. Daily dry matter intake (DMI),
gross energy intake (GEI), methane production and average daily gain (ADG) were measured. Energy
balance trial was done by 7-d total collection, while methane was measured by the Facemask method
that was done for 10 min with 3-h intervals for 2 × 24 h after total collection d (Purnomoadi et al.,
2003). Daily gain was measured after the sheep were raised for 12 wk. The data were analysed with
the F-test (noticed by P value of 5%), and was followed by the Duncan Multiple Range Test if there
was a significant difference.

The results showed that pollard supplementation (NP1 and NP2) increased GEI as well as DMI
(P<0.05) compared to the diet without supplementation (NP0), but it did not increase energy loss in
feces and urine. However, energy loss in methane was increased due to increasing DMI, and therefore
increased digestible and metabolisable energy (P<0.05). Moreover, there were no differences between
NP1 and NP2 in energy utilisation.

Table 1. Energy utilisation and methane production of sheep fed pollard in this study.
NP0 NP1 NP2 P
Initial body weight, kg 22.9 24.2 21.0

Average daily gain, g/d 14.6a 68.5b 94.1c 0.0001
Dry matter intake, g/d 729a 1013b 908b 0.0129
Gross energy intake, MJ/d 12.21a 17.76b 16.65b 0.0010
Energy loss, MJ/d
Feces 4.70 5.18 5.15 0.2466
Urine 0.18 0.37 0.18 0.1115
Methane 1.55a 1.98b 1.98b 0.0225
Available energy
Digestible, MJ/d 7.51a 12.58b 11.50b 0.0408
Metabolisable, MJ/d 5.78a 10.23b 9.34b 0.0106
Methane conversion rate* 12.9 11.8 12.0 0.2926
Methane per gain, g CH4/kg 2865a 522b 390c 0.0001
ADG
* Methane conversion rate = (MCR; CH4 MJ/100 MJ Gross energy intake); a,b,c different superscript in same row
showed significance (P<0.05).
This study showed that the methane conversion rate (MCR) of diets with pollard (NP1 and NP2) was
similar with that of the diet without pollards (NP0). Pollard increased the productivity of the sheep
as shown by increasing ADG since pollard supplementation increased (P<0.05) due to its nutrient
content in the diet. The protein content of NP0, NP1 and NP2 in the present study was 13.1, 14.6 and
16.1%, respectively. That condition resulted in that sheep fed pollard produced fewer methane per
unit gain (NP1: 522; NP2: 390 g CH4/kg ADG; P<0.05) than that of the sheep fed without pollard
(NP0: 2865 g CH4/kg ADG). The value of methane per unit gain of sheep fed pollard in this study
is still higher than that of cattle fed Lucerne hay and high grain ad libitum (127 g CH4/kg ADG),
but similar or lower to that of cattle fed Rhodes grass (500 g CH4/kg ADG) (Kurihara et al., 1999).
This study, however, suggested that pollard supplementation could lead to a significant productivity
in addition to mitigate methane production from sheep.
References
Johnson, K. A. and D. E. Johnson, 1995. Methane emissions from cattle. J. Anim. Sci. 73, 2483-2492.
Kurihara, M., T. Magner, R.A. Hunter and G.J. McCrabb, 1999. Methane production and energy partition of cattle in
tropics. Brit. J. Nut. 81: 227-234.
Leng, R.A., 1993. Quantitative ruminant nutrition - A green science. Aust. J. Agri. Res. 44, 363-380.
Moe, P.W. and H.F. Tyrell, 1979. Methane production by dairy cows. J. Dairy Sci. 62, 1583-1586.
Purnomoadi, A., E. Rianto, F. Terada and M. Kurihara, 2003. Reduction of methane production from buffalo heifers
fed soy-sauce by-product in South East Asia. Proceedings of the 3rd International Methane and Nitrous Oxide
Mitigation Conference (II), Beijing, 135-142.

Methane production in lactating dairy cows on fat or corn silage rich
diets compared to Intergovernmental Panel on Climate Change (IPCC)
estimates
W.M. van Straalen1, H. van Laar1,2 and H. van den Brand3
1Schothorst Feed Research, P.O. Box 533, NL-8200 AM, Lelystad, The Netherlands
2Current address: Nutreco Ruminant Research Centre, P.O. Box 220, NL-5830, Boxmeer, The
Netherlands
3Adaptation Physiology Group, Wageningen University, P.O. Box 338, NL-6700 AH, Wageningen,
The Netherlands
Introduction
Methane produced by ruminants contributes for a large part to greenhouse gas emission in The
Netherlands. Methane production can be estimated according to the Intergovernmental Panel on
Climate Change-guidelines (6% of gross energy intake) (IPCC, 1996), but this model ignores the
opportunity to reduce methane production by selecting the type of energy (fat or starch) in the
diet.
The objective of this study was to determine the effect of diets rich in either fat or corn silage on
methane production by dairy cows and to compare methane production with estimates according
to IPCC-guidelines.

Twelve lactating dairy cows (120-200 d in milk) were used in the experiment. Animals were subjected
to three treatments: 1: control (CON) (grass silage and standard concentrates with 44 g fat/kg); 2:
fat rich (FAT) (grass silage and concentrates with 62 g fat/kg from soya and palmoil, resulting in
20 g C18:2+C18:3/kg) and 3: corn silage (CS) (grass and corn silage, 50/50 DM, and concentrates
high in protein (208 g CP/kg)). Animals were milked and fed (roughage ad libitum and concentrates
according to production level) twice a d (6.00 h and 17.00 h).
Methane production was measured in two respiration chambers at Wageningen University during
three periods of four weeks. Each chamber housed two animals and per period two treatments were
compared. A period consisted of three wk adaptation period followed by one wk of measurements.
During the measuring period the methane production of each chamber was measured each 9 min.
Milk production and feed intake were registered daily. Milk samples were taken 4 times a wk and
feed samples were taken daily. Animals were weighed before and after each period.
Methane concentration in outflowing air was analysed with a spectrophotometer (Hartman & Baum,
URAS 10E) by non dispersive infrared. Samples of feeds were analysed for moisture, ash, crude
protein, crude fat, NDF, ADF, ADL, starch and sugar. Milk samples were analysed by NIRS for
fat, protein and lactose. Based on feed analyses gross energy (GE) was calculated according to the
VEM-system (CVB, 2005). Calculated GE was used to predict methane production according to
ICCP (6% of GE). The effect of treatment on response parameters (methane production, feed intake
and milk parameters) was tested with the following model: Y = µ + Treatment-effect + ε

Effects of treatments on intake, milk production parameters and methane production are presented in
Table 1. The composition of the CON, FAT and CS diets was respectively 209, 180 and 185 g CP/kg

DM; 419, 416 and 373 g NDF/kg DM and 58, 60 and 154 g starch/kg DM. Concentrate intake was
40, 43 and 40% of total DM intake. Treatments did not influence feed intake or milk composition.
The feed intake on the FAT diet was numerically lower than on the other diets, which is observed
frequently (Chilliard, 1993). Milk production was higher on the CS diet, which could probably be
explained by the higher starch intake on this diet.
Table 1. Effect of treatments on intake, milk production parameters and methane production.
Control Fat Corn silage L.s.d.2
Total DM intake, kg DM/d 20.3 18.8 20.7 2.2

Milk production, kg/d 29.7a 29.8a 32.7b 0.6
Fat, % 4.24 4.10 3.95 0.67
Protein, % 3.29 3.14 3.31 0.39
Measured methane production
g/d 405 378 395 81
% of GE intake 6.0 6.0 5.8 -
Predicted methane production1
g/d 404 377 412 -
1According to ICCP; 2 L.s.d. = least significant difference (P<0.05); a,b Means with different superscripts within a
row are significantly different (P<0.05).
The methane production was in the range as observed in other studies (Wilkerson et al., 1995).
Methane production on all diets was close to the estimated values by the IPCC model (6.0% of
GE). Methane production per animal per d tended to be lower on the FAT and CS diets compared
to the CON diet, but due to large variation between replicates in the CON treatment not statistically
different. The depressive effect of these treatments on methane production is also predicted in other
studies (Plöchl and Berg, 2002).
In conclusion, FAT and CS diets tended to reduce methane production in dairy cows compared to
the CON diet, but due to large variation between replicates in the CON diet these differences were
not significant. Measured methane production was close to the prediction of ICCP irrespective of
the diet (6% of GE).
References
Chilliard, Y., 1993. Dietary fat and adipose tissue metabolism in ruminants, pigs and rodents: a review. J. Dairy Sci.
76, 3897-3931.
Centraal Veevoederbureau (CVB), 2005. Veevoedertabel. Gegevens over chemische samenstelling, verteerbaarheid
en voederwaarde van voedermiddelen. CVB, Lelystad, The Netherlands.
Intergovernmental Panel on Climate Change, 1996. Revised 1996 IPCC guidelines for national greenhouse gas
inventories: reference manual, vol. 3. IPCC, Bracknell UK.
Plöchl, M., W. Berg and R. Brunsch, 2002. Modelling the emissions of methane and nitrous oxide from dairy cattle.
In: Van Ham et al. (editor), Non-CO2 Greenhous gasses: Scientific understanding, control options and policy
aspects. Millpress, Rotterdam, 45-46.
Wilkerson, V.A., D.P. Casper and D.R. Mertens, 1995. The prediction of methane production of Holstein cows by
several equations. J. Dairy. Sci. 78, 2402-2414.

Manipulation of rumen methanogenesis with saponin-containing plant
extracts
J. Takahashi1, B. Pen2 and R. Asa1
1Graduate School of Animal Food Hygiene, Obihiro University of Agriculture and Veterinary
Medicine, Obihiro, Hokkaido, Japan
2The United Graduate School of Agricultural Sciences, Iwate University, Obihiro, Hokkaido, 080-
8555 Japan
Introduction
Ruminal microbial activity is essential for the use of structural carbohydrates and synthesis of
high quality protein in ruminants. However, microbial fermentation in the rumen may result in
considerable energy and protein losses such as methane and ammonia. A growing concern about
global warming and N pollution has increased attention on ways to abate ruminal methanogenesis
and to improve nitrogen utilisation. Recently, there has been increased interest in saponin-containing
plants for modifying ruminal fermentation. The objectives of the study were to evaluate the effects
of saponin-rich extracts of Quillaja saponaria (QE) and Yucca schidigera (YE), and their additive
effects with galacto-oligosaccharides (GO) on methane emission and nitrogen utilisation in ruminant
livestock.

An in vitro continuous methane quantification system was used to evaluate the effects of QE and
YE addition at 0, 2, 4 and 6 mL/L on ruminal fermentation and methanogenesis. Strained rumen
fluid obtained from non–lactating Holstein cows was used as inoculums (Exp 1). The effects of QE
and YE with or without GO on methanogenesis and N utilisation were evaluated in a 4×6 Youden
square design experiment with four sheep (Exp 2). Sheep were fed a basal diet comprising 40% of
concentrate and 60% of Italian ryegrass hay at 55 g/kg BW0.75 daily. Dietary treatments were the
following: (1) control; (2) 14 mL of QE; (3) 14 mL of YE; (4) 20 g of GO; (5) 14 mL QE + 20 g
GO and (6) 14 mL YE + 20 g GO per d. The CH4 production was measured by using four paralleled
and fully-automated head cage respiration systems.

Addition of YE and QE up to 6 mL/L in vitro, linearly increased (P<0.05) propionate concentrations
and decreased (P<0.05) protozoa numbers (Table 1), which was consistent with Pen et al. (2006a).
An antiprotozoal effect of saponin compounds has been reported from an aspect of nutritional
potential by Cheeke (1999). Methane production decreased with YE in a dose dependent manner,
which was consistent with Takahashi et al. (2000). The NDF digestibility increased (P<0.05) in
sheep treated with QE, QE+GO and YE+GO (Table 2), which was in agreement with Pen et al.
(2006b). Protozoa tended to decrease in sheep treated with YE (P<0.04). Nitrogen retention tended
to increase in sheep treated with QE (P=0.19). Methane production tended to decrease in sheep
treated with QE, YE (P=0.13). The results suggest that saponin-rich extracts of QE and YE did not
depress fibre digestibility and tended to mitigate methane emission in vivo.

Table 1. Effect of YE and QE addition on ruminal fermentation characteristics.
YE, mL/L QE, mL/L
0 2 4 6 0 2 4 6
Total VFA, mM 66.5 64.2 68.0 69.2 62.4 66.6 66.6 66.5
Acetate, mM 47.7 44.1 45.5 46.3 44.8 47.1 46.9 46.3 Li
Propionate, mM 12.4 14.2 17.1 19.0 Li 11.5 13.2 13.3 13.7
Butyrate, mM 5.7 5.5 4.9 3.6 Li 5.4 5.4 5.7 5.7
A:P ratio 4.4 3.9 3.6 3.5 Li 4.4 4.3 4.2 4.1
Protozoa, ×104/mL 13.2 9.4 5.9 5.8 Li 10.1 6.6 5.9 6.1 Li
CH4, mL/min 0.24 0.20 0.17 0.14 Li 0.24 0.23 0.24 0.25
Li = Linear effect.
Table 2. Effect of YE and QE with or without GO on fibre digestibility, methane emission and
nitrogen utilisation.
Control QE YE GO Q+G Y+G SEM
NDF digestibility 0.55b 0.58a 0.57ab 0.56ab 0.59a 0.57a 0.033

ADF digestibility 0.53 0.56 0.54 0.54 0.56 0.56 0.029
Protozoa, ×104/mL 16.0 15.0 14.0 16.5 15.1 12.65 4.11
CH4, L/kg BW0.75 1.86 1.53 1.64 1.78 1.76 1.60 0.10
N retention, % N 24.5 27.5 23.4 29.2 24.1 24.8 0.47
intake
a,b,c Mean values within a row with different superscript letters differ (P<0.05).
References
Cheeke, P.R., 1999. Actual and potential application of Yucca schidigera and Quillaja saponaria saponins in human
and animal nutrition. Proc. Am. Soc. Anim. Sci. 1-10.
Pen, B., C. Sar, B. Mwenya, K. Kuwaki, R. Morikawa and J. Takahashi, 2006a. Effects of Yucca schidigera and
Quillaja saponaria extracts on in vitro ruminal fermentation and methane emission. Anim. Feed Sci. Technol.
129, 175-186.
Pen, B., K. Takaura, S. Yamaguchi, R. Asa and J. Takahashi, 2006b. Effects of Yucca schidigera and Quillaja saponaria
with or without β 1-4 galacto-oligosaccharides on ruminal fermentation, methane production and nitrogen utilization
in sheep. Anim. Feed Sci. Technol., in press. doi:10.1016/j.anifeedsci.2006.11.018.
Takahashi, J., T. Miyagawa, Y. Kojima and K. Umetsu, 2000. Effects of Yucca schidigera extract, probiotics, monensin
and L-cysteine on rumen methanogenesis. Asian-Aust. J. Anim. Sci. 13, 499-501.

Influence of tanniniferous shrubs (Calliandra calothyrsus and Flemingia
macrophylla) in tropical diets on energy metabolism and methane
emission of lambs
T. Tiemann1, H.-R. Wettstein1, A.C. Mayer1, M. Kreuzer1, C.E. Lascano2 and H.D. Hess3
1ETH Zurich, Institute of Animal Sciences, CH-8092 Zurich, Switzerland
2Tropical Grass and Legume Project, Centro Internacional de Agricultura Tropical, CIAT, Cali,
Colombia
3Agroscope Liebefeld-Posieux (ALP), Tioleyre 4, CH-1725 Posieux, Switzerland
Introduction
Tropical fodder shrub legumes are often characterised by high contents of condensed tannins (CT).
While the influence of CT on protein metabolism is well studied (e.g. Makkar, 2003) few data are
available on their effects on metabolic energy turnover and the existing indications for methane-
suppressing effects of CT (Waghorn et al., 2002) require further confirmation. The aim of the present
study was to measure the effect of partly replacing an expensive, CT free high-quality legume
(Vigna unguiculata) by promising tanniniferous shrub legumes (Calliandra calothyrsus, Flemingia
macrophylla) to a tropical grass diet (Brachiaria brizantha) on digestion, energy metabolism and
methane emission of sheep.

Six complete tropical diets were formulated. Five of them represented mixtures of a tropical grass and
legume foliage (0.55:0.45), a grass-only diet served as a negative control. The legume supplements
consisted either of V. unguiculata alone (high quality diet) or of mixtures of V. unguiculata with
leaves of C. calothyrsus or F. macrophylla in ratios of 2:1 and 1:2. All forages were dried and
shipped from Colombia to Switzerland. Diets were tested in six lambs of the Swiss White Mountain
breed (22.7 ± 3.8 kg) for their effect on energy turnover and methane release using dual open circuit
respiratory chambers and applying a Latin square design (n=6). Dry matter allocation was 60 g/kg of
metabolic bodyweight (BW0.75). Animals were adapted to the experimental diets for 2 wk, followed
by 1 wk of quantitative recording of intake and excretion of feces and urine. During 2 d, the gaseous
exchange of the animals was determined. In addition, blood and rumen fluid samples were taken.
Diets, refusals, feces and urine were analysed for contents of nitrogen and carbon, feed, refusals and
feces also for energy and fibre. Data were subjected to analysis of variance with SAS considering
diet, animal and experimental run as factors. Multiple comparisons among means were performed
with Ryan-Einot-Gabriel-Welsch range test (REGWQ).

Calliandra and Flemingia contained about 103 and 30 g soluble CT/kg dry matter and 79 and 189
g/kg of insoluble CT, respectively. The level of consumption of the tanniniferous legumes was
comparably high (>85% of the amounts offered). Rumen fluid ammonia and plasma urea (PUN) N
concentration (mmol/L) were the lowest (P<0.05) with high proportions of Calliandra and Brachiaria
only (Table 1). PUN and apparent digestibility and metabolisability of energy were in general lower
(P<0.05) with tanniniferous supplements than with Brachiaria/Vigna. Heat energy expenditure in
MJ/kg BW0.75 was lower (P<0.01) for the high tannin diets than for the high quality diet. Fat tissue
retention did not differ between treatments. Fecal N excretion was the highest for the diets high
in Calliandra and Flemingia. Body protein retention did only show a tendency to be lower in the
high Calliandra diet than in the Brachiaria/Vigna diet (P<0.1). Despite higher overall CT contents,
Flemingia macrophylla supplementation was less detrimental than that of Calliandra in terms of

metabolic protein supply while adverse effects on energy supply were similar. Also, the profile of
volatile fatty acids was similar (P>0.1) among treatments. Methane emission (L/kg BW0.75) was
reduced (P<0.05) by including tanniniferous legumes. Reductions compared to the high quality diet
were 8.4% and 25.7% for low and high Calliandra and 9.1% and 21.8% for low and high Flemingia,
respectively. When related to intake of gross energy this effect was significant only for the high-
tannin diets. There was no effect (P>0.1) on methane emission per unit of either digested energy
or digested NDF. This indicates that the measured suppression when using tanniniferous legumes
seems to have been mainly mediated by associated reductions in ruminal fibre and organic matter
degradation and less by a direct adverse action against the methanogenic microbes what coincides
with the findings of Carulla et al. (2005).
Table1. Effect of feed supplementation with tanniniferous legumes.
NH3 PUN ME Heat Fecal N Prot. ret. CH4 CH4

Mmol/L Mmol/L g/kg it kJ/BW0.75 g/kg it g/d L/BW0.75/ mL/g
d NDF
B 3.89b 2.44c 420ab 408a 562bc 6.85a 1.68b 86.4a

B/V 6.54a 6.28a 446a 396ab 476c 1.08a 1.82a 97.0a
C 15% 6.05ab 4.77b 391abc 393ab 609b 1.45a 1.67b 104a
F 15% 5.62ab 5.41ab 380bc 383ab 563bc 2.05a 1.65b 97.6a
C 30% 3.83b 3.38c 370bc 345c 727a -5.36a 1.42c 95.9a
F 30% 5.32ab 4.57b 352c 369bc 626b 3.33a 1.35c 91.0a
B, Brachiaria; V, Vigna; C, Calliandra; F, Flemingia; NH3, ruminal ammonia; PUN, plasma urea nitrogen; ME,
metabolisable energy; Heat, heat energy expenditure; Prot. ret., protein retention; CH4, methane emission; CH4/
NDF, methane emission/neutral detergent fibre intake; it, intake. Means followed by unequal letters within column
are different at P<0.05.
In conclusion, the results demonstrate that the use of tanniniferous tropical shrub legumes, in
replacement of a high-quality legume, results in a reduced metabolic energy and protein supply.
However, as an alternative on poor acidic soils, characterised by low-protein grass species, part of
the care-intensive CT-free Vigna could be replaced by Flemingia macrophylla in improving such
tropical grass-only diets.
References
Carulla, J.E., M. Kreuzer, A. Machmüller and H.D. Hess, 2005. Supplementation of Acacia mearnsii tannins decreases
methanogenensis and urinary nitrogen in forage-fed sheep. Aust. J. Agric. Res. 56, 961-970.
Makkar, H.P.S., 2003. Effects and fate of tannins in ruminant animals, adaptation to tannins, and strategies to overcome
detrimental effects of feeding tannin-rich feeds. Small Rum. Res. 49, 241-256.
Waghorn, G.C., M.H. Tavendale and D.R. Woodﬁeld, 2002. Methanogenesis from forages fed to sheep. Proc. N. Z.
Grasslands Assoc. 64, 167–171.

Enteric methane emission of Japanese native goats
O. Enishi, N. Takusari, K. Higuchi, I. Nonaka, M. Kurihara and F. Terada
National Institute of Livestock and Grassland Science, Ikenodai 2, 305-0901,Tsukuba, Japan
Introduction
Global warming results from increases in atmospheric concentrations of gases such as carbon dioxide
(CO2) and methane (CH4), which are potential greenhouse gas that are directly linked with livestock
industry. The microbes in the rumen convert the plant material into nutrients that ruminants can
use, such as volatile fatty acids. Microbial fermentation of feed in the rumen results in enteric CH4
production that represents a loss in productive energy for ruminants. In ruminants, CH4 production
represents a loss of 3 to 13% of the feed’s energy (McCaughey et al., 1997). To help guide policies
and strategies, greenhouse gas inventories that can provide accurate information of these emissions
and trends are critically important. National inventories for enteric CH4 emissions are calculated
according to the Intergovernmental Panel on Climate Change (IPCC) guidelines. The Tier 1 method
involves multiplying the number of livestock in various classes by a fixed emission factor (EF),
whereas the Tier 2 method calculates emissions from the number of livestock in various classes, their
gross energy intake (GEI) and CH4 conversion rate (MCR: % of GEI). However, there is no MCR
for the goat. Therefore, given the importance of MCR in calculating national CH4 inventories, this
study was performed to examine the enteric methane production of Japanese native goats.

The data were obtained from 33 total energy balance trials with four mature Japanese native goats
(body weight 28.3 ± 2.8 kg: mean ± standard error; aged from 25 to 33 mo). Goats were given
roughage (Timothy hay and Alfalfa hay) and concentrate mainly made up of corn, soybean meal
and by-products at maintenance level (NRC, 1981). The ratio of roughage and concentrate is from
55:45 to 30:70. The experiment was designed as a one-way layout design assigned four goats to
each feed. CH4 production was measured for 3 consecutive d in open circuit respiration chambers
during each energy balance trial.

The mean, standard deviation and range (min.-max.) were 517.9, 128.4, 331.7-740.6 g/head/d in
dry matter intake; 8576.9, 1496.8, 6396.0-11383.3 kJ/head/d in GEI; 16.3, 3.5, 8.9-24.3 L/head/d
in CH4 production, respectively. Table 1 shows the mean, standard deviation and range (min.-max.)
of chemical composition, nutritive value and digestibility of feeds. On the whole, the nutritive value
of feeds used in this study was high. The relationship between GEI and MCR is shown in Figure 1.
MCR was negatively related to GEI (r=-0.76; P<0.05). As the CP and EE content of feeds increased,
the MCR increased (r=0.98, 0.95; P<0.01). Meanwhile, when ADF content of feeds increased, the
MCR decreased (r=0.62; P<0.05). When highly available carbohydrates are fed at limited intakes,
high fractional methane loss occurs (Johnson and Johnson, 1995). Therefore it is thought that MCR
is low in the feed when ADF content is high because available carbohydrate is low.
From the result of the present study, the observed CH4 emission of Japanese native goats averaged
10.9 g/head/d and 3.99 kg/head/yr. According to the IPCC Tier 1 estimates, expected CH4 production
from the goat is 5.0 kg/head/yr. The Japanese native goat in this study produced 3.99 kg which was
lower than the value of IPCC (5.0 kg/head/yr; IPCC, 2006). Animal sizes are important factors when
estimating CH4 production from the ruminant. For this reason, it was thought that body weight (28.3
kg) and feed intake of a goat of this study were different from IPCC (body weight 40 kg). This study

suggests that MCR (7.21%) of the Japanese native goat was higher than the mature sheep value of
IPCC (6.5%; IPCC, 2006).
Table 1. Chemical composition, nutritive value, methane conversion rate and digestibility (n=33).
Chemical composition, dry matter basis % MCR, Digestibility, %

%
OM CP EE ADF TDN OM CP EE ADF
Mean 92.8 15.3 3.8 23.9 73.2 7.21 76.2 74.6 75.2 58.2
Max. 95.9 18.7 6.1 35.7 83.1 10.35 84.4 83.2 87.6 72.2
Min. 88.0 11.0 1.5 12.1 55.1 5.02 61.9 62.2 29.9 46.9
Std. 2.4 2.5 1.1 6.2 8.0 1.33 5.7 5.8 14.2 7.1
OM: Organic matter, CP: Crude protein, EE: Ether extract, ADF: Acid detergent fiber, TDN: Total digestible
nutrients, MCR: Methane conversion rate = CH4 kJ/Gross Energy Intake kJ.
Methane conversion rate (% GEI)

Fig. 1
15
10
r=-0.63, P<0.05
0
5000 10000 15000
Figure 1. Relationship between gross

Grossenergy
energyintake and(GEI,
intake methane
kJ) conversion rate in Japanese native
goats.
References
Jhonson, K. and D.E. Jhonson, 1995. Methane emissions from cattle. J. Anim. Sci. 73, 2483-2492.
IPCC, 2006. Guidelines for national greenhouse gas inventories. Agriculture, forestry and other land use, vol. 4.
IGES, Japan.
National Reseach Council, 1981. Nutrient requirements of goats. National Academy Press, Washington, DC.
McCaughey, W.P., K. Witterberg and D. Corrigan, 1997. Methane production by steers on pasture. Can. J. Anim. Sci.
77, 519-524.

Energy value of wheat bran and dried beet pulp in finishing Italian
heavy pigs
G.M. Crovetto, G. Galassi, L. Rapetti and S. Colombini
Istituto di Zootecnia Generale, Università degli Studi di Milano, via Celoria 2, Milan, Italy
Introduction
Despite being a monogastric, mature pigs and pig approaching maturity can digest unlignified fibre
to a certain extent (Noblet and Le Goff, 2001; Noblet and Bourdon, 1997; Galassi et al., 2005). In
pigs, fibre plays a role primarily as dietary factor, enhancing peristalsis and guaranteeing a normal
functioning of the gastro-intestinal tract, but its contribution to energy balance is not negligible in
mature/heavy animals. Italy maize is by far the most important cereal used in pig diet formulation,
but its lack in fibre must be balanced with the inclusion of limited amounts of fibrous feed, primarily
wheat bran, in the diet. Previous works (Rijnen et al., 2001; Galassi et al., 2004) indicate that beet
pulp is utilised more efficiently than wheat bran. The aim of the present experiment was to determine
the nutritive value of wheat bran and beet pulp for the heavy fattening pig (>100 kg BW).

Thirty castrated male pigs Large White×Landrace (initial bodyweight 130 kg, final bodyweight 181
kg) were divided into 5 groups of 6 pigs each and fed 5 different diets: a traditional control diet (C,
with 51% maize, 36% barley and 8% soybean meal), two diets with 12 and 24% wheat bran (WB12
and WB24) and two other diets with 12 and 24% dried beet pulp (BP12 and BP24). The two fibrous
ingredients substituted equivalent amounts of barley. Analyses (% on DM) of the diets C, WB12,
WB24, BP12, BP24, respectively are as follows: CP 13.5, 14.0, 14.6, 12.7, 12.8; NDF 11.8, 14.4,
17.2, 13.3, 16.1; Starch 54.4, 50.9, 47.4, 49.0, 43.0.
The trial included 5 consecutive periods (7 d of adaptation + 7 d of excreta collection) for the
determination of digestibility and energy balance (pigs in metabolic cages individually confined
into open circuit respiration chambers).
The data were analysed by the GLM procedure (SAS, 2001) considering the effect of treatment.

Table 1 reports the daily energy balance. Energy digestibility was significantly higher for diet C
(P<0.01) in comparison with the other diets. Among the fibrous diets, BP diets were better digested
than WB diets, for equivalent levels of inclusion. Significant decreases of energy digestibility were
also registered for increasing dietary fibre contents both for WB (P<0.01) and BP (P<0.05).
Methane energy losses were significantly higher for diet BP24. Consistently with a previous
experiment (Galassi et al., 2004), wheat bran did not increase methane emission in comparison with
the control, despite its high fibre content. Energy metabolisability shows the same trend of d(E)
for the different treatments, whilst heat production was similar for all the diets. RE was similar for
diets C, WB12 and BP12; on the contrary, WB24 and BP24 had significantly lower RE (% IE) as
compared to diets C and BP12.
The efficiency of utilisation of ME for growth (k=NE/ME) of the five diets altogether was calculated
considering NE = RE + NEm, with NEm assumed as 261 kJ/kg BW0,75 (Noblet et al., 1993). As

expected, k increased with increasing BW0.75 (x), as follows: k = 0.00425x + 0.54977 (P<0.001).
This was consistent with the higher fat deposition of heavier pigs.
The regression equations to determine NE (y = MJ NE/kg DM) of wheat bran (WB) and beet pulp
(BP) from their content (%) in the diet (x) resulted in the following:
y (WB) = -0.04157x + 11.758 (P<0.001); y (BP) = -0.03748x + 11.819 (P=0.013)
Therefore, NE of WB and BP for the finishing heavy pig resulted in being 7.601 and 8.071 MJ
NE/kg DM, respectively.
Table 1. Daily energy utilisation.
Diet C WB12 WB24 BP12 BP24 SEM
Intake energy (IE) MJ 48.79 49.40 49.02 47.32 48.04 0.82

kJ/kg BW0.75 1083 1134 1131 1165 1075 32.3
Faecal energy kJ/kg BW0.75 100C 152B 181A 136B 147B 7.4
% IE 9.4C 13.4Ba 16.0A 11.6Bb 13.7Ba 0.58
DE kJ/kg BW0.75 982 982 950 1029 928 29.9
% IE 90.6A 86.6Bb 84.0C 88.4Ba 86.3Bb 0.58
Urinary energy kJ/kg BW0.75 29.6 29.5 33.5 28.9 30.3 1.31
% IE 2.8ab 2.6b 3.0a 2.5b 2.8ab 0.11
Methane energy kJ/kg BW0.75 7.3B 7.1B 6.7B 9.1B 14.6A 1.22
% IE 0.7B 0.6B 0.6B 0.8B 1.4A 0.11
ME kJ/kg BW0.75 945 945 910 991 883 29.3
% IE 87.2Aa 83.4BCc 80.5Dd 85.1ABb 82.1CDc 0.55
Heat production kJ/kg BW0.75 506 518 499 526 497 15.9
% IE 46.8 45.7 44.2 45.1 46.3 0.79
RE kJ/kg BW0.75 439 427 411 465 386 19.2
% IE 40.4a 37.7ab 36.3b 40.0a 35.8b 1.13
a, b, c, d P<0.05; A, B, C, D P<0.01.
References
Galassi G., G.M. Crovetto, L. Rapetti and A.Tamburini, 2004. Energy and nitrogen balance in heavy pigs fed different
fibre sources. Livest. Prod. Sci. 85, 253-262.
Galassi G., G.M. Crovetto and L. Rapetti, 2005. Trend of energy and nitrogen utilization of high fibre diets in pigs
from 100 to 160 kg bodyweight. Ital. J. Anim. Sci. 4, 149-157.
Noblet, J. and D. Bourdon, 1997. Valeur énergétique comparée de onze matiéres premiéres chez le porc en croissance
et la truie adulte. Journées Rech. Porcine en France 29, 221-226.
Noblet, J. and G. Le Goff, 2001. Effect of dietary fibre on the enegy value of feeds for pigs. Anim. Feed Sci. Technol.
90, 35-52.
Noblet, J., X.S. Shi and S. Dubois, 1993. Metabolic utilization of dietary energy and nutrients for maintenance energy
requirements in sows: basis for a net energy system. Br. J. Nutr. 70, 407-419.
Rijnen, M.M.J.A., M.W.A. Verstegen, M.J.W. Heetkamp, J. Haaksma and J.W. Schrama, 2001. Effect of dietary
fermentable carbohydrates on energy metabolism in group-housed sows. J. Anim. Sci. 79, 148-154.
SAS, 2001. Release 8.01. SAS Inst. Inc., Cary, NC, USA.

Energy value of crude glycerol in 11 and 110 kg pigs
P. Lammers1, B. Kerr2, T. Weber2, W. Dozier3, M. Kidd4, K. Bregenhahl1 and M. Honeyman1
1Iowa State University, Ames, IA, 50011, USA
2USDA Agricultural Research Service, Ames, IA, 50011, USA
3USDA-Agricultural Research Service, Mississippi State, MS, 39762, USA
4Mississippi State University, Mississippi State, MS, 39762, USA
Introduction
Production of biofuels is increasing due to rising energy prices and recognition of the environmental
impacts of using fossil fuel. Biodiesel is alternative to diesel fuel consisting of the monoalkyl esters
formed by a catalyzed reaction of the triacylglycerides in oils or fats with an alcohol with glycerol the
chief co-product of biodiesel production (Van Gerpen, 2005; Thompson and He, 2006). Widespread
processing of crude glycerol to pure glycerol may become uneconomical given continued growth
in biodiesel production (NBB, 2006) such that crude glycerol may become available for use as an
energy feedstuff for livestock.
Multiple reviews of the metabolic effects of glycerol have been presented (Lin, 1977; Brisson et
al., 2001). Glycerol is absorbed by the gastrointestinal tract of monogastrics and is utilized as a
source of dietary energy. Glycerol is gluconeogenic with gluconeogenisis appearing to be limited
only by the availability of glycerol (Cryer and Bartley, 1973; Baba et al., 1995). Studies examining
the effects of feeding crude glycerol to monogastric food animals have been reported (Mourot et
al., 1994; Kiljora et al., 1995; Simon et al., 1996) but no studies have reported the ME of crude
glycerol fed to pigs.

Starter pigs (24 barrows, 11.0 ±0.5 kg initial BW) were randomly assigned to individual metabolism
crates (0.53×0.71 m) equipped with screens and trays that allowed for total but separate collection
of feces and urine. Dietary regimen consisted of a common corn-soybean meal-12.5% dried whey
basal diet or the basal diet mixed with an additional of 5, 10, or 20% crude glycerol (86.95% glycerol,
9.63% water, 3.13% NaCl, 0.028% methanol, and 0.029% free fatty acid; 3625 kcal GE/kg, as-is).
Pigs fed the basal diet received 188 g/meal while pigs assigned to the 5, 10, or 20% glycerol addition
treatment received 197, 207, and 226 g/meal, respectively. In a second experiment, finisher pigs (24
gilts, 109.6 ±5.5 kg initial BW) were randomly assigned to similarly designed, but larger (0.8×2.1
m) metabolism crates. Dietary regime consisted of a common corn-soybean meal basal diet or the
basal diet mixed with an additional 5, 10, or 20% crude glycerol. Pigs fed the basal diet received 1146
g/meal while pigs assigned to the 5, 10, or 20% glycerol addition treatment received 1204, 1260,
and 1375 g/meal, respectively. In each experiment, a 10 d adjustment period was used to determine
appropriate meal size and to ensure that all animals were adapted to the diet and twice-a-day feeding
regimen. Afterwards, a 5 d total fecal and urine collection period was conducted. For each experiment,
the increase in feed intake above the basal represented an increase in glycerol consumption, since
all pigs (within each experiment) were offered the same amount of basal feed.
Feed samples were oven dried at 70 °C for 48 h, while fecal samples were thawed, dried at 70 °C
for 48 h, weighed, both of which were ground through a 1-mm screen in preparation for energy
analysis. For urine energy determination, cellulose was dried at 100 °C for 24 h after which 2 mL
of urine added to 0.5 g of dried cellulose. The combined urine and cellulose samples were weighed
and dried at 50 °C for 24 h, prior to energy determination. Apparent ME of the diet was calculated

as the difference between feed GE intake less fecal and urinary energy losses. Data were analyzed
using linear regression with ME regressed against total feed intake (Adeola, 2002).
Results
Starter pigs fed 0 to 20% crude glycerol exhibited a quadratic (P<0.01) response to crude glycerol
suggesting glycerol having an apparent ME of 3601, 3239, and 2579 kcal/kg for pigs consuming 5,
10, and 20% glycerol, respectively. This was not noted in finishing pigs where the response remained
linear (P<0.01), providing an apparent ME estimate of 3081 kcal/kg (± 122). With little change in
plasma glycerol or excretion of glycerol in starting pigs fed 20% glycerol, we have no explanation
for the quadratic effect noted in starting pigs. With the reduction of ME most noted in starting pigs
fed 20% glycerol, data from starter pigs fed up to 10% glycerol was reanalyzed. Upon reanalysis,
the response was linear (P<0.01), providing an apparent ME estimate for glycerol of 3463 kcal/kg
(± 480). In summary, this data suggests that up to 10% glycerol can be supplemented to starting
pigs, with an ME of approximately 96% of glycerol’s GE. In finishing pigs, higher levels may be
utilized, with an ME of approximately 85% of glycerol’s GE. Statistically, ME estimates between
starting and finishing pigs are similar, averaging 3272 kcal/kg.
References
Adeola, 2002. Digestion and balance techniques in pigs. In: Lewis, A.J. and L.L. Southern (editors), Swine Nutrition.
CRC Press, Boca Raton, FL, 903-916.
Baba, H., X.-J. Zhang and R.R. Wolfe, 1995. Glycerol gluconeogenesis in fasting humans. Nutr. 11, 149-153.
Brisson, D., M.-C. Vohl, J. St-Pierre, T. J. Hudson and D. Gaudet, 2001. Glycerol: A neglected variable in metabolic
processes? BioEssays 23, 534-542.
Cryer, A. and W. Bartley, 1973. Studies on the adaptation of rats to a diet high in glycerol. Int. J. Biochem. 4, 293-
308.
Kiljora, C., H. Bergner, R.-D. Kupsch and L. Hageman, 1995. Glycerol as feed component in diets of fattening pigs.
Arch. Anim. Nutr. 47, 345-360.
Lin, E.C.C., 1977. Glycerol utilization and its regulation in mammals. Ann. Rev. Biochem. 46, 765-795.
Mourot, J., A. Aumaitre, A. Mounier, P. Peiniau and A.C. Fracois, 1994. Nutritional and physiological effects of dietary
glycerol in the growing pig. Consequences on fatty tissues and post mortem muscular parameters. Livest. Prod.
Sci. 38, 237-244.
NBB, 2006. Biodiesel production in US. National Biodiesel Board. Available: http://www.nbb.org/buyingbiodiesel/
guide/guide_findbio.shtm. Accessed 10 December 2006.
Simon, A., H. Bergener and M. Schwabe, 1996. Glycerol-feed ingredient for broiler chickens. Arch. Anim. Nutr. 49,
103-112.
Thompson, J.C. and B.B. He, 2006. Characterization of crude glycerol from biodiesel production from multiple
feedstocks. Appl. Eng. Agric. 22, 261-265.
Van Gerpen, J., 2005. Biodiesel processing and production. J. Fu. Proc. 86, 1097-1107.

Quantitative and qualitative analyses of seed storage proteins from toxic
and non-toxic varieties of Jatropha curcas L
N. Selje-Assmann, H.P.S. Makkar, E.M. Hoffmann, G. Francis and K. Becker
University of Hohenheim, Institute for Animal Production in the Tropics and Subtropics, Department
of Aquaculture Systems and Animal Nutrition, Fruwirthstr. 12, D-70599 Stuttgart, Germany
Introduction
Interest in Jatropha curcas has increased noticeably due to the multifunctional characteristics of
the plant. Of particular interest is the high oil content of the seeds which can be used for biodiesel
production. The extraction of the oil from the seeds leaves a meal highly enriched in protein (crude
protein ~ 60%), providing a potential protein source for animal nutrition. In order to characterise
the seed proteins, protein fractions were extracted from defatted kernel meals of three varieties of J.
curcas, a toxic and a non-toxic variety from Mexico, and a toxic variety from Cape Verde, and analysed
with respect to their amino acid composition, rumen degradability and gastric digestibility.

Seeds of J. curcas were de-shelled, and the kernel ground and defatted, termed as J. curcas kernel
meal. Albumins, globulins, prolamins and glutelins from all varieties were extracted sequentially
based on the method of Osborne (1924), dialysed and lyophilised. A highly enriched protein
concentrate was prepared only from the defatted kernel meal of the toxic Mexican variety by alkaline
extraction, isoelectric precipitation, and dialysis.
Crude protein content was determined by the Kjeldahl method (N×6.25), true protein content was
determined by dot-blot (Hoffmann et al., 2002). Protein patterns were investigated after separation
by PAGE. Amino acids were analysed according to the EC directive 98/64/EC (1998). In vitro
degradable nitrogen was determined according to the method of Raab et al. (1983). Impacts on
overall rumen fermentation were investigated in batch cultures using a substrate mix of maize silage
and barley grain (Selje et al., 2007) either supplemented by soybean meal or by J. curcas kernel
meal. Two-step digestibility by gastrointestinal enzymes, pepsin and pancreatin, was performed as
described by Calsamiglia and Stern (1995).
Results
Recovery of protein after sequential extraction, dialysis and lyophilisation was 89.3% including
protein fractions plus protein that remained in the pellet. Average proportions of albumins, globulins,
prolamins, glutelins, and non-extracted residue amounted to 10.8, 27.4, 0.6, 56.9, and 4.3% of total
protein recovered. The yield for the protein concentrate of the toxic Mexican variety was 26.5% of
initial sample weight of a defatted kernel meal.
PAGE gels revealed characteristic bands for each of the protein fractions, while banding patterns of
the respective fractions did not differ amongst the varieties. In accordance, amino acid profiles of
the three meals were similar, whereas in the three major fractions, albumins, globulins and glutelins,
proportions of individual amino acids deviated. Total sum of essential amino acids (EAA) of J. curcas
kernel meals, of the protein concentrate as well as of individual protein fractions exceeded the EAA
requirement of preschool children (FAO/WHO 1990). Except for lysine, individual essential amino
acids in J. curcas meals exceeded the requirements for preschool children. In the albumin fraction,
lysine was enriched to 7.9% and by far exceeding the FAO recommendations (5.8%).

In 24 h, the in vitro batch incubations with bovine rumen fluid, the isonitrogenous exchange of
soybean meal by J. curcas meals affected neither the production of gas and short chain fatty acids
nor the in vitro apparent or true degradability. Low solubility of J. curcas protein (~20%), however,
led to a reduction in protein degradation as indicated by lower concentrations of end products of
proteolysis, ammonia and branched short chain fatty acids.
Rumen in vitro degradable nitrogen determined for J. curcas kernel meal (Mexico, toxic) and for the
protein concentrate in comparison to soybean meal and purified soya protein confirmed the lower
N degradability of J. curcas protein compared to soya protein.
Gastric digestion by pepsin and pancreatin yielded protein digestibilities of albumins and globulins
of 64% and 61%, respectively, while a higher value of 95% was observed for glutelins. Protein
digestibility of the meal and the protein concentrate from the toxic Mexican variety was 76%
and 90%.
Discussion and conclusion

The seeds of J. curcas contain a high proportion of high quality protein, which provides all essential
amino acids in sufficient amounts with the exception of lysine. The glutelin fraction amounts to more
than 50% of the protein and may be the reason for the low solubility of the protein. Low solubility
led to decreased rumen degradability when compared to soybean protein, whereas digestibility by
pepsin and pancreatin was high. The results further indicate a high potential of J. curcas kernel
meal in ruminant nutrition, providing a high amount of rumen undegradable protein that will be
available post-ruminally. The high gastric digestibility of the meal and the protein extract suggest
a high potential for protein utilization in monogastric animals as well, if the non-toxic variety or
meal after detoxification is used.
References
Calsamiglia, S. and M.D. Stern, 1995. A three-step in vitro procedure for estimating intestinal digestion of protein in
ruminants. J. Anim. Sci. 73, 1459-1465.
EC, 1998. Establishing Community methods of analysis for the determination of amino-acids, crude oils and fats, and
olaquindox in feedingstuffs, and amending. Directive 71/393/EEC. Commission Directive 89/64/EC, Off. J. Eur.
Communities L257, 14-23.
FAO/WHO/Expert Consultation, 1990. Protein Quality Evaluation. FAO/WHO Nutrition Meetings, Report Series
51, Rome.
Hoffmann, E.M., S. Muetzel and K. Becker, 2002. A modified dot-blot method of protein determination applied in
the tannin-protein precipitation assay to facilitate the evaluation of tannin activity in animal feeds. Br. J. Nutr.
87, 421-426.
Osborne, T.B., 1924. The vegetable proteins. 2nd edition. Longmans, Green, New York.
Raab, L., B. Cafantaris, T. Jilg and K.H. Menke, 1983. Rumen protein degradation and biosynthesis. I. A new method
for determination of protein degradation in rumen fluid in vitro. Br. J. Nutr. 50, 569-582.
Selje, N., E.M. Hoffmann, S. Muetzel, R. Ningrat, R.J. Wallace and K. Becker, 2007. Results of a screening programme
to identify plants or plant extracts that inhibit ruminal protein degradation. Br. J. Nutr., in press.

Potato protein concentrate – nutritional value and effects on gut
morphology, ileal digestibility, and caecal fermentation in rats
M. Taciak and B. Pastuszewska
The Kielanowski Institute of Animal Physiology and Nutrition, Instytucka 3, 05-110 Jabłonna,
Poland
Introduction
Potato protein concentrate (PPC) has a balanced amino acid composition and high nutritional value
as determined in rats (Nestares et al., 1993). However, in the studies of Morita et al. (2004), PPC
was used as a model of protein resistant to enzymatic digestion and intensively fermented in the
caecum, but its real ileal digestibility was not determined. PPC contains protease inhibitors and
glycoalkaloids which induce gastrointestinal disorders (Smith et al.,1996) and may, presumably,
affect gut structure. The objective of the present study was to determine the nutritional value, rate
of digestion in the small intestine and caecal fermentation of PPC as compared with casein (CAS)
and soybean oil meal (SBOM), and to assess its effects on gut morphology.

Three experiments were performed. The diets contained PPC, CAS or SBOM as the only sources
of protein at the 9.5% level in exp. 1 and 2, and 20% level in exp. 3, cellulose, sucrose, soya oil,
minerals and vitamins, and corn starch at 100%. 0.3% Cr2O3 as a marker and 5% of pectin were
added to the diets used in exp. 3. In exp. 1, true digestibility (TD) and biological value (BV) of
protein were determined according to Thomas-Mitchel on 28-d old male rats during 10 d, while in
exp. 2 growth performance was assessed on 24-d old males fed ad libitum during 28 d. The animals
were killed by CO2 inhalation and the weights of the small intestine, caecum, pancreas and liver
were recorded. In exp. 3, the male rats of 395 g mean body weight, 30 animals per treatment, were
meal-fed during 10 d, and sacrificed 4 h after the last meal. The duodenum and mid-jejunum were
sampled and histological measurements were performed using a light microscope. Digesta from the
last segment of the ileum was collected, freeze dried, pooled from 4-5 animals and analysed for N,
while in digesta collected from caecum pH, NH3, and short chain fatty acids (SCFA) were determined.
Statistical analysis was performed using the one-way ANOVA procedure (Duncan test).

Protein TD of PPC and SBOM was lower than that of CAS while BV of all three protein sources
was uniform (Table 1). The nutritional value of PPC protein was very close to that reported by
Nestares et al. (1993). In spite of similar protein value, growth performance of rats fed on PPC was
significantly poorer than on CAS and SBOM mainly due to considerably lower feed intake (Table 1).
Feeding PPC induced overgrowth of the small intestine as compared with CAS and SBOM and of
the pancreas and liver as compared with SBOM (Table 2). The hypertrophic effect of PPC on the
small intestine was confirmed by significantly greater thickness of mucosa and myenteron of the
duodenum and mid-jejunum as well as greater height of villi and depth of crypts.
Ileal digestibility of the PPC protein was significantly lower than those of SBOM and CAS (Table 3),
the difference between PPC and CAS was far greater than in the whole gut. This finding may confirm
suggestions of Morita et al. (2004) on the resistance of PPC to enzymatic digestion but may also
indicate a stimulation of greater endogenous secretion by antinutritional factors present in PPC. Due
to different ileal digestibility, the amount of protein entering the caecum differed among the protein
sources under study and was the greatest on PPC while it did not differ between CAS and SBOM.

SBOM lowered caecal pH and increased acetate and propionate concentrations (Table 3), most
probably due to fermentation of some amounts of fibre present in this product. PPC enhanced
caecal butyrate and ammonia concentrations, probably due to a greater amount of protein entering
the caecum. It may be concluded that potato protein concentrate in spite of its high protein value
depresses feed intake, affects the rate of digestion and morphology of the small intestine, and
modifies caecal fermentation.
Table 1. Protein value (exp. 1) and growth performance (exp. 2), (n=8).
Diet TD BV NPU2 Feed intake, g Weight gain, g FCR3, g/g
PPC 92.3a1 82.2a 75.9a 332a 90.7a 3.68c

CAS 96.9b 82.3a 79.8b 369b 122.7b 3.02a
SBOM 91.4a 80.8a 73.9a 387b 115.3b 3.37b
1Differentletters in columns denote significant differences (P≤0.05).

2Netprotein utilisation NPU=TDxBV/100.
3Feed conversion ratio.
Table 2. Weight of internal organs, g/100 g body mass (exp. 2), (n=8).
Diet Small intestine Pancreas Caecum Liver
PPC 3.32b1 0.32b 0.30a 5.50b

CAS 2.71a 0.31b 0.29a 5.48b
SBOM 2.76a 0.25a 0.38b 4.93a
1Different letters in columns denote significant differences (P≤0.05).
Table 3. Ileal protein digestibility (%), SCFA (mM/100 g) and NH3 (mg/100 g) concentration and
pH in caecal contents (exp. 3).
Diet Ileal digestibility Caecal parameters
acetate propionate butyrate NH3 pH
PPC 71.3a1 6.04ab 1.20a 1.27b 34.5b 6.66b

CAS 83.3c 4.91a 1.26a 0.33a 16.4a 6.82b
SBOM 75.7b 7.74b 1.64b 0.59a 13.2a 6.26a
1Different letters in columns denote significant differences (P≤0.05).
References
Morita, T., S. Kasaoka and S. Kiriyama, 2004. Physiological functions of resistant proteins: proteins and peptides
regulating large bowel fermentation of indigestible polysaccharide. J. AOAC Int. 87 (3), 792-796.
Nestares, T., M. Lopez-Jurado, A. Sanz and M. Lopez-Fria, 1993. Nutritional assessment of two vegetable protein
concentrates in growing rats. J. Agric. Food Chem. 41, 1282-1286.
Smith, D.B., J.G. Roddick and J.L. Jones, 1996. Potato glycoalkaloids: Some unanswered questions. Trends Food
Sci. Technol. 7, 126-131.
Partly supported by the Ministry of Science and Education, Grant No 2P06Z01830 and
2P06Z03128.

Effect of mannan oligosaccharides on protein metabolism in broiler
chickens depending on the dietary fibre content of the feed
B. Prause1, R. Messikommer1, P. Spring2 and C. Wenk1
1Institute of Animal Sciences, ETH Zurich, 8092 Zurich, Switzerland
2Swiss College of Agriculture (SHL), 3052 Zollikofen, Switzerland
Introduction
Mannan oligosaccharides (MOS) derived from the cell wall of Saccharomyces cerevisiae, have shown
promising results in broilers for health and in some case for enhancing performance and feed efficiency
(Rosen, 2003). Its effect is probably due to the benefit of eubiosis in the gastrointestinal tract. They
might selectively enhance desired microorganisms like bifidobacteria or lactobacilli by serving them
as a fermentation substance. Above all, MOS are able to reduce undesired microorganisms like
coliforms and salmonellae via competitive inhibition and using specific-mannose-Type1-fimbriae
for cell adhesion (Spring, 1996). Furthermore, according to Newman (1994) and Spring (1996)
MOS are able to modulate immune response. This is why a MOS effect depends on consistent
housing conditions and feed composition as reported by Wenk and Messikommer (2002). At optimal
husbandry conditions, moderate doses of MOS (100 ppm and 1000 ppm) had no positive influence
on the performance of broilers. Furthermore, according to the authors, an overdose of MOS (10 000
ppm) tends to have a negative impact on growth and metabolism probably caused by an increased
digesta viscosity.
The current experimental series are conducted linked to consistent questions about the interaction
between the dietary fibre content in broiler diets and the effect of MOS.

In three series a total of 216 d-old male chicks (PM3 Ross) were fattened over a period of five
wk. They were kept in cages of six birds each and assigned to four treatments. A basic diet was
supplemented with 20% of a low (18% wheat starch and 2% potato protein) or a high dietary fibre
component (20% soy hulls) and either added with 1000 ppm MOS or not (LF+, LF- and HF+,
HF-). Performance parameters (body weight, feed intake and feed to gain ratio) were monitored
weekly. In addition during two periods (wk 3 and wk 5) excreta were collected for estimating the
metabolisability of gross energy (GE), the utilisation of crude protein (CP) and the degradability
of neutral detergent fibre (NDF) and acid detergent fibre (ADF). After slaughtering the birds on d
35, the weight of carcass and organs were acquired per individual as well as intestinal samples for
microbial and histological analysis. Differences within LF and HF due to addition of MOS were
investigated. All data were interpreted statistically by disposal of SYSTAT 7.0® for Windows® with
a multi-attribute analysis of variance (ANOVA).
Results
Analysed nutrient contents were 197 g CP; 16.9 MJ GE, 79 g NDF and 33 g ADF per kg feed in LF
and 205 g CP; 16.9 MJ GE, 193 g NDF and 120 g ADF per kg feed in HF treatments. Daily weight
gain (55.9 g/d (LF) and 51.6 g/d (HF)), daily feed intake (91.9 g/d (LF) and 95.9 g/d (HF)) and
feed conversion ratio (1.58 (LF) and 1.78 (HF)) were not influenced by MOS. MOS in the HF diet
improved the utilisation of protein in wk 3 by about 2.6% (P<0.001) and decreased degradability of
NDF and ADF in wk 5 (P<0.05). MOS in the LF diet had no effect on these parameters (Table 1).

Table 1. Effect of MOS on the nutrient utilisation of broilers in subject to dietary fibre content.
Low fibre (LF)1 High fibre (HF)1
LF- LF+ P-Value HF- HF+ P-Value
Wk 3 GE 0.812 0.808 0.322 0.649 0.654 0.453

CP 0.618 0.610 0.400 0.543 0.569 <0.001
NDF 0.459 0.472 0.434 0.252 0.228 0.153
ADF 0.316 0.292 0.559 0.117 0.080 0.097
Wk 5 GE 0.805 0.809 0.432 0.663 0.663 0.969

CP 0.591 0.602 0.616 0.546 0.562 0.390
NDF 0.450 0.465 0.522 0.263 0.219 0.032
ADF 0.326 0.320 0.835 0.150 0.083 <0.001
1LF- and HF- were fed without MOS additive and LF+ and HF+ were supplemented with 1000 ppm MOS.
Discussion and conclusion

The results of this study indicate that the dietary fibre content in broiler diets makes a contribution
to a MOS effect. MOS in the HF diet enhanced protein utilisation and decreased the degradability
of detergent fibres whereas it did not have an impact on the LF diet. These effects might be caused
by modifying eubiosis.
To get more information about this effect further analysis, trials with other dietary fibre substrates
are in progress.
References
Newman, K., 1994. Mannan-Oligosaccharides: Natural polymers with significant impact on the gastrointestinal
microflora and the immune system. In: Lyons, T. P. and K.A. Jacques (editors), Biotechnology in the feed industry.
Proceedings of Alltech’s 10th Annual Symposium. Nottingham University Press, Nottingham, UK, 167-174.
Rosen, G., 2003. Setting standards for the efficient replacement of pronutrient antibiotics in poultry and pig nutrition.
Schriftenreihe aus dem Institut für Nutztierwissenschaften, Ernährung- Produkte- Umwelt, ETH-Zürich, 15 May
2003. Gesunde Nutztiere - Heutiger Stellenwert der Futterzusatzstoffe in der Tierernährung, 72-88.
Spring, P., 1996. Effects of Mannanoligosaccharide on different caecal concentrations of enteric pathogens in poultry,
Diss. am Institut für Nutztierwissenschaften, ETH-Zürich.
Wenk, C. and R. Messikommer, 2002. Bedeutung von Mannan-Oligosacchariden bei Broilern unter günstigen
Haltungsbedingungen. Schriftenreihe aus dem Institut für Nutztier-wissenschaften, Ernährung- Produkte- Umwelt,
ETH-Zürich, 15 May 2002. Optimale Nutzung der Futterressourcen im Zusammenspiel von Berg- und Talgebiet,
126-129.

Lowered feed consumption and utilisation of diets containing Acacia
villosa leaves in experimental female rats
E. Harlina1, B.P. Priosoeryanto1, B. Tangendjaja2, L.K. Darusman3 and D. Sastradipradja4
1Dept. of Vet. Clinic, Repro. & Pathology, Institut Pertanian Bogor, Bogor 16680, Indonesia
2Research Institute for Animal Production, AARD Indonesia, P.O. Box 221, Bogor 16002,
Indonesia
3Dept. of Chemistry, Institut Pertanian Bogor, Jalan Pajajaran, Bogor 16151, Indonesia
4Retired Professor, Institut Pertanian Bogor
Introduction
Red acacia (Acacia villosa) (AV), a leguminous tree of Southeast Asia has potential usage as ruminant
feed due to its high protein content (22-28% of DM), but anti-nutritional and toxic contents of non-
protein aminoacids (NPAA) and polyphenols have hindered its use. Wina and Tangendjaja (2000)
reported 3 out of 4 sheep consuming AV as 75% of total feed resulted in death displaying neural
disturbances with hemorrhages of all organs and damages of the liver and kidney. The toxic compound
might be 2,4-diamino butyric acid possibly arising from non-toxic 4-N-acetyl-2,4-diamino butyric
acid. A sequential study using rats as models was conducted to reveal the detrimental effect of AV
and whether steamed AV leaves or leaves incubated with AV adapted rumen fluid microflora would
improve the end results.

Experiment 1 was aimed at determining the accepted safe level of AV in the diet of female rats. A
complete randomly designed 4 wk trial was conducted consisting of 15 female Spraque-Dawley
rats, 4 wk old, divided into 5 test groups, i.e. receiving rat standard pellets containing respectively
0, 9, 15, 21 and 27% of powdered AV (dietary composition: 3800-3900 kcal, protein 15%, fat 5.9%,
ash 4.0%). Daily feed intakes (FI) and bodyweights (BW) were noted. Rats were sacrificed at the
end of the trial, necropsied and liver and kidney specimens were collected. Each histological slide
was scanned for totally 20 different microscopic fields (176 µm2 each) and scored for lesions. The
Duncan Multiple Range Test after ANOVA was used. Histopathological score (HPS) analysis used
Kruskall-Wallis non-parametric statistics. SPSS software Windows version 13.0 was used. The same
dealings apply for other trials.
Experiment 2 studied the effect of steaming (70 °C) AV leaves prior to its addition into the test diet
using 18 female 4 wk rats divided into a control (no AV addition), a group receiving 21% steamed and
another 21% non-steamed AV. An increase of body performance parameters indicates no conversion
of the non-toxic to toxic compound, or detoxification of the toxic compound in leaves.
Experiment 3 is similar to Experiment 2, except that instead of steaming, added AV was previously
incubated in vitro in rumen fluid taken from an AV adapted or a non-adapted goat.

The results of Experiment 1 on daily BW gains and HPS are presented in Table 1 and those, plus
regression equations of FI and BW growth, of Exp. 2 and 3 in Table 2. Daily weight gain and HPS
shown in Table 1 indicate that the safe level of AV in the diet is 21% for female rats. A separate
study on acute toxicity of AV by Harlina et al. (2007) revealed that AV has definite neuro-, cardio-,
hepato- and nephro-toxic qualities due to NPAA/polyphenols.

Table 1. Daily weight gains, and liver and kidney histopathological scores of initial 4 wk old female
rats consuming diets with different levels of Acasia villosa for 4 wk (Exp 1).
AV diet1, % 0 9 15 21 27
DWG2, g/d 2.75d 2.45c 2.27c 2.09b 1.53a

HPS3
liver4 7.70b 7.00b 9.50b 10.00ab 18.50a
kidney4,5 0.11±0.03bb 0.12±0.01ab 0.16±0.03ab 0.15±0.03ab 0.19±0.07a
1Acacia villosa level in diet; 2DWG = daily weight gain; 3HPS = Score of histopathological lesions; 4Different letter
superscripts within the same row denote significant difference at P<0.05; 5Mean ± SD.
Table 2. Weekly feed intakes, Body weights and Feed Conversion Ratios (M ± SD) of female rats
receiving diets with 21% steamed and non-steamed Acasia villosa and those receiving diets with
21% in vitro fermented Acasia villosa with rumen fluid from an AV adapted and AV non-adapted
donor goat (Exp 2 and 3).
Treatment reg.coeff. b of1 FCR2 Treatment reg.coeff. b of1 FCR2
Control, no AV FI 6.99 4.46 ± 0.51a Control, no AV FI 5.613 3.44 ± 0.54a

BW 20.157 BW 22.295
Steamed AV FI 8.34 6.46 ± 0.74b Rumen adapted FI 4.383 5.24 ± 0.60b
BW 13.397 AV BW 11.927
Non-steamed AV FI 7.36 5.45 ± 1.65b Rumen non- FI 2.808 4.75 ± 0.28b
BW 15.122 adapt.AV BW 11.403
1Regr. coeff. b of Y = bX + a; Y=feed intake (FI, g) or body weight (BW, g), X=week.
2FCR=feed conversion ratio.
The data suggest no improvements arise by steaming AV, nor by its prior in vitro incubation in rumen
fluid from the AV adapted goat, but regression analysis of feed intakes and growth curves of the
rumen fluid incubation trials (Table 2) indicate beneficial NPAA biotransformation. Better results
require more factors to be measured e.g. blood constituents.
Conclusions
(i) Female rats are good model animals for AV studies, though extrapolation of rat data into ruminants
need further research; (ii) AV lowers the intake and use of the diet; 21% AV is a safe level for female
rats; (iii) bioconversion of toxic AV into its non-toxic form occurs in the rumen.
References
Harlina, E, D.R. Agungpriyono and M. Rahminiwati, 2007. Kajian toksikopatologi fraksi asam amino daun Lamtoro
Merah (Acacia villosa) pada tikus. (Toxicopathological studies of the aminoacid fraction of Acasia villosa leaves
in rats). J. Med. Vet. Indon., in press.
Wina E and B. Tangendjaja, 2000. The possibility of toxic compounds present in Acacia villosa. Bull. Pet. 24 (1),
34-42.

Estimating forage digestibility from faecal crude protein concentration
in grazing sheep
C.J. Wang1, B.M. Tas1, T. Glindemann1, G. Rave2, L. Schmidt3, F. Weißbach3and A. Susenbeth1
1Institute of Animal Nutrition and Physiology, Christian Albrechts University Kiel, Hermann-
Rodewald-Str. 9, D-24118 Kiel, Germany
2Variationsstatistik, Christian Albrechts University Kiel, Hermann-Rodewald-Str. 9, D-24118 Kiel,
Germany
3Federal Research Centre of Agriculture, D-38116 Braunschweig, Germany
Introduction
Organic matter digestibility (dOM) is the basal information required for feed evaluation. In grazing
animals a direct estimate of dOM by measuring feed intake and faecal excretion is not possible.
Therefore, indigestible external and internal markers are used to indirectly estimate feed intake and
faecal excretion, and hence dOM. Most of these marker techniques depend on samples that represent
the herbage ingested by the grazing animal, which may be difficult or impossible to receive in
heterogeneous pastures. The faecal crude protein (CP) concentration was proposed as a measure of
diet digestibility. It depends, besides on the excretion of indigestible feed protein and endogenous
protein, on indigestible microbial protein, which is positively related to dOM (Schmidt, 1993; Lukas
et al., 2005; Schlecht and Susenbeth, 2006). Regression equations have been developed for sheep and
cattle, and can be used to estimate the dOM of herbage ingested by grazing animals. However, the
relationship may depend on the type of diet, and a general regression equation may not be adequate.
Moreover, indigestible feed protein occurring in the feces may differ between forages. Therefore,
equations have been developed describing the relationship between faecal acid detergent soluble
CP (ADSCP) concentration and dOM (Lukas et al., 2005). The first objective of this study was to
develop a regression equation between dOM and faecal CP based on a large data set with sheep fed
different forages with a large range in dOM, and to test the effect of type of diet. The second objective
was to test whether faecal ADSCP is a superior predictor of dOM than faecal CP.

Data were obtained from in vivo digestibility trials (153 diets, including fresh grass, grass silage, hay
with soy bean meal (18-39% of diet), red clover, lotus silage, alfalfa silage and galega silage, total of
676 observations) with sheep conducted at the Federal Agricultural Research Centre, Braunschweig,
Germany. The dataset was enlarged with digestibility trials (6 diets, including locally produced hay
without or with soy bean meal (33% of diet), total of 45 observations) conducted in Inner Mongolia,
China. OM digestibility ranged between 41 and 83%. In all faecal samples CP was analysed, whereas
ADSCP was analysed in a subset of 161 samples.
Regression equations were determined using PROC NLMIXED in SAS (v8.2) with the following
model:
yij = a – (b + ui) × e (-c × xij /100) + eij (1)
where yij represents the jth dOM (%) of the ith diet type; xij is the corresponding CP or ADSCP
in faecal OM (g/kg); a, b and c are fixed effect parameters; ui is random effect parameter of diet
type, and eij is the residual error. The regression error was assessed by calculation of the mean and
mean squared difference between estimated and observed values of dOM (mean bias and MSPE,
respectively). The size of the error was calculated as the root MSPE and expressed as a percentage
of the mean observed values (RMSPE). MSPE was decomposed into error due to the overall bias

of prediction, error due to deviation of the regression slope from unity, and error due to random
variation (Bibby and Toutenburg, 1977).

Using the total dataset, the following regression equation was developed:
dOM (%) = 89.9 – 64.4 × e (-0.5774 × faecal CP [g/kg OM]/100) (2)
The parameters in the equation were significant (P<0.05), and random effect of type of diet was
not observed. The error due to overall bias and RMSPE were small, and a large proportion of the
prediction error was due to random variation (Table 1). The dOM was slightly under-predicted in
Braunschweig data (mean bias of -1.6), whereas slightly over-predicted in Inner Mongolia data
(mean bias of 2.2). Using the subset of data, the regression equation with ADSCP: dOM (%) =
84.9 – 58.9 × e (-0.8802 × faecal ADSCP [g/kg OM]/100), showed a slightly lower under-prediction and a
slightly higher accuracy than with CP (Table 1). Faecal CP from forages - except lotus silage - had a
relatively high and constant fraction of ADSCP (82.1 ± 0.04% of CP), indicating a low indigestible
feed protein concentration. Since laboratory analyses for ADSCP compared to CP are more laborious
and expensive, predicting the dOM from faecal CP concentration can be taken as the preferential
method for forages with a relatively low and constant indigestible feed protein concentration. It is
concluded that dOM of ingested roughages can be predicted from faecal CP concentration in sheep
grazing on heterogeneous pasture.
Table 1. Accuracy of regression equations predicting dOM (%; estimated (est) – observed (obs))
based on faecal CP or ADSCP concentration in sheep.
Dependent No of dOMest dOMobs Mean bias1 RMSPE2 Proportion (%) of MSPE3

variable observations
overall slope random
CP 721 65.1 66.5 -1.4 7.7 7.6 15.9 76.5

ADSCP 161 66.0 67.1 -1.1 7.3 5.1 18.2 76.7
CP 161 65.2 67.1 -1.9 8.2 11.8 17.2 71.0
1Meanbias = dOM estimated – dOM observed.

2 RMSPE = root mean square prediction error.
3 MSPE = mean square prediction error.
References
Bibby, J. and H. Toutenburg, 1977. Prediction and improved estimation in linear models. John Wiley and Sons,
London.
Lukas, M., K.H. Südekum, G. Rave, K. Friedel and A. Susenbeth, 2005. Relationship between fecal crude protein
concentration and diet organic matter digestibility in cattle. J. Anim. Sci. 83, 1332-1344.
Schlecht, E. and A. Susenbeth, 2006. Estimating the digestibility of Sahelian roughages from faecal crude protein
concentration of cattle and small ruminants. J. Anim. Physiol. Anim. Nutr. 90, 369-379.
Schmidt, L., 1993. Die Schätzung des Futterwertes nach der Kotstickstoff-Methode (methodische Aspekte). VDLUFA-
Schriftenreihe 37, 681–684.

Effects of feeding different levels of lauric acid on ruminal protozoa
kinetics and fermentation pattern in dairy cows
A.P. Faciola1, G.A. Broderick2 and A.N. Hristov3
1University of Wisconsin, Department of Dairy Science, 1925 Linden Drive West, 53706, Madison
WI, USA
2Agricultural Research Service, USDA US Dairy Forage Research Center, 1925 Linden Drive West,
53706, Madison WI, USA
3University of Idaho, 219 Ag Science, 83844, Moscow ID, USA
Introduction
Reducing ruminal protozoa (RP) may improve N utilization in the rumen. In our previous study
(Faciola et al., 2004) lauric acid (LA), a saturated medium chain fatty acid (C12:0) showed high
anti-protozoal activity when a single dose of 160 g/d was given via ruminal cannulae, reducing the
RP by 90% within two d of treatment. In the same study, ruminal ammonia and total free amino
acid (TAA) concentrations were reduced by 60 and 40% respectively. In a second trial (Faciola et
al., 2005), LA fed at 160 and 240 g/d in the diet of dairy cows reduced RP by only 25 and 30%,
respectively, and no changes in ruminal fermentation were observed. These results suggested that
these levels of LA in the diet were not sufficient to achieve a ruminal concentration that promoted
the anti-protozoal effect. Therefore, the objectives of the current experiment were as follows: (i) to
determine the level of LA in the diet that would effectively suppress RP; (ii) to assess the changes
in ruminal fermentation patterns associated with partial suppression of RP; and (iii) to determine
the time needed to re-establish RP after LA treatment.

Two ruminally cannulated Holstein cows averaging 697 kg of body weight, 164 d in milk, and 35
kg/d of milk were used in this trial. Cows were fed a 16.5% CP diet of 40% grain mix (dry ground
corn, soybean meal, minerals and vitamins), 30% corn silage, and 30% alfalfa silage. Cows were
fed the same level of LA for a 7-d period, starting with 0 g/d, and increasing stepwise to 160, 320,
and 480 g/d mixed into the diet. RP numbers, dry matter intake (DMI), ruminal pH, individual and
total volatile fatty acids (VFA), ruminal ammonia, and TAA concentrations were determined at each
level of LA feeding. After, LA was withdrawn from the diet, daily counts were done in order to
determine the time needed to re-establish RP. Data were analyzed using Proc Mixed in SAS (1999),
RP numbers were analyzed in a repeated measures ANOVA with autoregressive covariance structure
within cow, and ruminal traits were analyzed in a two-way repeated measures ANOVA with time
nested within periods and compound symmetry correlation structure.
Results
In this trial, LA level was confounded with time; however, there was no evidence that RP would
normally change in the course of the experiment, so all change in RP was attributed to LA treatments.
Feeding LA at 160, 320, and 480 g/d in the diet of dairy cows reduced (P<0.01) the RP population
by 37, 67, and 80%, respectively (Figure 1). Lauric acid fed at 320 g/d reduced ruminal ammonia
and TAA concentrations by 48 and 51%, respectively, suggesting decreased intraruminal turnover
of bacterial protein which may improve N utilization in the rumen. In addition, LA fed at 320 g/d
increased ruminal propionate concentration by 55%; however, due to the low animal numbers,
these results were not statistically significant. Beyond 320 g/d no change in fermentation pattern
was observed. In this study, there were no changes in DMI, ruminal pH, or other VFA. After LA
was removed from the diet, the RP population re-established itself within 12.07 ± 1.26 d (95%

Confidence interval) showing a fast recovery rate. However, VFA, ammonia, and TAA were not
measured after RP numbers were re-established. The effects of feeding LA on milk production and
milk components are currently being tested in a trial with 48 lactating dairy cows.
70 5.5
NH3 5.0
60
TAA
Protozoa
Protozoa, x 105 cells per ml
4.5
Ruminal NH3 and TAA, mM

50
4.0
40
3.5
30
3.0
20
2.5
10 2.0
0 1.5
0 160 320 480
Lauric acid intake, g/d
Figure 1. Least squares means of ruminal protozoa numbers and ruminal concentration of ammonia,
and TAA at different LA intakes.
Conclusions
Under the conditions of this experiment a dose of 320 g/d of LA added to the total mixed ration
significantly suppressed RP and may increase N utilization in the rumen. After withdrawing LA
from the diet, RP numbers were re-established in about 12 d.
References
Faciola, A.P., G. Broderick, A.N. Hristov and M.I. Leao, 2004. Effect of two forms of lauric acid on ruminal protozoa
and fermentation pattern in dairy cows. J. Dairy Sci. 87, (Suppl. 1), 53.
Faciola, A.P., G. Broderick, A.N. Hristov and M.I. Leao, 2005. Effect of feeding different levels of lauric acid on
ruminal protozoa, and milk production in dairy cows. J. Dairy Sci. 88, (Suppl. 1), 178.
SAS, 1999–2000. SAS/STAT User’s Guide. Release 8.1. SAS Institute, Inc., Cary, NC, USA.

Effect of different energy supply on rumen fermentation of Corriedale
sheep consuming temperate fresh forage
I. Tebot1, C. Cajarville2, A. Pereira1, J.L. Repetto2, V. Elizondo1, A.L. Falero1 and A. Cirio1
1Physiology Department, Veterinary Faculty, Lasplaces 1550, 11600 Montevideo, Uruguay
2Nutrition Department, Veterinary Faculty, Lasplaces 1550, 11600 Montevideo, Uruguay
Introduction
Energy supplementation is often practised when energy availability is low in grazed forage. Ruminants
fed on energy-supplemented diets based on fresh forage are able to keep their ruminal pH between
6.2 and 6.8, avoiding ruminal acidosis (Church, 1993). This range of values is coincident with data
of assays made in Uruguay (Repetto et al., 2000), working with high quality forage and low level of
supplementation. However, Grant and Mertens (1992) showed that supplements containing excessive
amounts or inappropriate types of carbohydrates may negatively affect ruminal fermentation, specially
inducing an important decrease in ruminal pH affecting animal health (acidosis).
The aim of this work was to test the impact of two energy-rich supplements (grain and molasses)
on ruminal pH of Corriedale sheep consuming temperate fresh forage.

Eighteen non-pregnant, non-lactating ewes (43 ± 4 kg BW), housing in individual pens, were
randomly divided into 3 groups (n=6 each one). F (forage, non-supplemented, 44 g DM/kg0.75 BW),
FG (forage (32 g DM/kg0.75 BW)+grain (12 g DM/kg0.75 BW)) and FGM (forage (32 g DM/kg0.75
BW)+grain (6 g DM/kg0.75 BW)+cane molasses (6 g DM/kg0.75 BW)). Forage was fresh oat plants
(DM 14.7%, OM 88.7%, NDF 49.9%, ADF 26.8%, CP 14.4%) and grain was barley grain (DM
86.8%, CP 10.9%, ME 3.13 Mcal/kg, ADF 5%). Weighed food was distributed 4 times a d (at 9.00,
13.00, 18.00 and 22.00 h), being only the first and the last meal supplemented. Following a 20 d
adaptation period to diets, a ruminal cannula was placed in all animals. Ten d after surgery, pH was
measured hourly with an electronic pHmeter (Cole Parmer) during 24 h. Significance was evaluated
by a 3 way (sheep, h, treatment) ANOVA test. P-values were adjusted by a Bonferroni test.

The results are shown in Table 1. Ruminal pH variations during the experimental d were 6.12 to 6.59
(F), 5.92 to 6.53 (FG) and 6.31 to 6.73 (FGM). Statistical differences were found for treatments and
h between FGM group and the others (P<0.05). The pattern of variation in ruminal pH along the d
showed a decrease one h after feeding and an increase one h before feeding. The same pattern was
reported by Chamberlain et al. (1993) in sheep.
Unexpectedly, the highest pH was observed in the group supplemented with readily fermentable
carbohydrates (FGM) and the forage diets (F) showed intermediate values. At the moment, we have
no satisfactory explanation for these findings. Molasses is a concentrated source of simple sugars,
with a high proportion of sucrose. Chamberlain et al. (1993) found that sheep supplemented with
sucrose or lactose (200 g/d) did not depress their ruminal pH when compared with a basal diet of grass
silage. Trevaskis et al. (2001) showed a significant decrease in ruminal pH when sucrose (40 g/d) or
barley grain (150 g DM/d) were added to fresh forage fed sheep. But unfortunately, these authors did
not statistically compare these data with those of basal diets without supplementation. Longer-term
studies are needed to further understand the relationship between simple sugar supplementation
and ruminal pH.

Table 1. Hourly pH values for F, FG and FGM groups during the experimental d.
pH
Daytime F FG FGM
13.00 6.39 ± 0.14 6.34 ± 0.19 6.60 ± 0.14

14.00 6.29 ± 0.11 6.18 ± 0.28 6.58 ± 0.19
15.00 6.31 ± 0.17 6.06 ± 0.27 6.76 ± 0.13
16.00 6.59 ± 0.26 6.22 ± 0.17 6.73 ± 0.15
17.00 6.53 ± 0.37 6.36 ± 0.31 6.75 ± 0.28
18.00 6.50 ± 0.22 6.33 ± 0.26 6.74 ± 0.22
19.00 6.37 ± 0.26 6.26 ± 0.36 6.68 ± 0.18
20.00 6.21 ± 0.25 5.95 ± 0.42 6.53 ± 0.28
21.00 6.23 ± 0.17 6.00 ± 0.45 6.64 ± 0.26
22.00 6.43 ± 0.21 5.92 ± 0.31 6.57 ± 0.23
23.00 6.39 ± 0.25 5.92 ± 0.36 6.54 ± 0.15
0.00 6.07 ± 0.25 6.00 ± 0.26 6.37 ± 0.13
1.00 6.12 ± 0.33 6.07 ± 0.16 6.33 ± 0.41
2.00 6.24 ± 0.32 5.97 ± 0.15 6.24 ± 0.45
3.00 6.29 ± 0.33 5.98 ± 0.30 6.48 ± 0.39
4.00 6.31 ± 0.41 6.15 ± 0.35 6.31 ± 0.40
5.00 6.17 ± 0.41 6.26 ± 0.50 6.48 ± 0.42
6.00 6.43 ± 0.39 6.31 ± 0.37 6.51 ± 0.38
7.00 6.47 ± 0.29 6.41 ± 0.32 6.51 ± 0.45
8.00 6.48 ± 0.25 6.53 ± 0.23 6.51 ± 0.37
For abbreviations and statistical significance see text (n=6).
References
Chamberlain, D.G., S. Robertson and J-J. Choung, 1993. Sugars versus starch as supplements to grass silage. Effects
on ruminal fermentation and the supply of microbial protein to the small intestine, estimated from the urinary
excretion of purine derivatives, in sheep. J. Sci. Food. Agric. 63, 189-194.
Church D.C., 1993. El rumiante. Fisiología digestiva y nutrición. Ed. Acribia, Zaragoza, 641 pp.
Grant, R.J. and D.R. Mertens, 1992. Influence of buffer pH and raw corn starch addition on in vitro fiber digestion
kinetics. J. Dairy Sci. 75, 2762-2768.
Repetto, J.L., M. Mota, P. Marinho, L. Vega and C. Cajarville, 2000. pH ruminal y cinéticas de degradación del forraje
en bovinos que pastorean praderas suplementadas o no con concentrados energéticos. Acta de la XVI Reunión
Latinoamericana de Producción Animal, Montevideo, Uruguay.
Trevaskis, L.M., W.J. Fulkerson and J.M. Gooden, 2001. Provision of certain carbohydrates-based supplements to
pasture-fed sheep, as well as time of harvesting of the pasture, influences pH, ammonia concentration and microbial
protein synthesis in the rumen. Aust. J. Exp. Agric. 41, 21-27.

Author index
A Béchet, D. 173, 303, 365
Abbas, M. 183 Beck, P.A. 425
Abe, H. 185 Becker, K. 625
Abreu, D.C. 165 Beckers, Y. 419
Adamany, J. 475 Bellmann, O. 103
Adeola, O. 495 Bellof, G. 113
Adin, G. 219 Ben Chedly, M.H. 249
Adiwinarti, R. 611 Bequette, B.J. 293, 475
Agabriel, J. 489 Berk, A. 117, 375
Agnew, R.E. 507 Bermingham, E.N. 333
Aguer, C. 121 Bernard, C. 299
Aguilera, J.F. 97, 383, 385, 493, 499 Bernard, L. 285
Aharoni, Y. 219, 221, 251 Berri, C. 63, 199
Akiba, Y. 55 Berthiaume, R. 241
Albertí, P. 109, 111 Bertoni, G. 395
Albrecht, D. 287, 305 Bertrand, G. 521
Albrecht, E. 357 Berwid, S.J. 393
Alferink, S.J.J. 473 Bes, S. 285
Alliot, J. 303 Besson, C. 307, 309
Amarger, V. 291 Beyer, M. 195
Archibeque, S. 337 Bikker, P. 203, 557
Arrigo, Y. 407, 551 Blache, D. 201
Asa, R. 615 Blum, J.W. 233, 361, 367
Atkinson, J.L. 549 Blum, S. 187
Attaix, D. 173, 365 Boirie, Y. 59, 225
Aubry, L. 99 Boisen, S. 567
Auclair, S. 309 Bonekamp, P.R.T. 247
Audouin, E. 179, 181, 183 Bonnet, M. 295
Bordas, A. 481
B Bordeau, T. 63, 199
Back, P. 143 Bordenave, S. 121
Bahelka, I. 115 Borges, I. 525
Baião, N.C. 513 Borthaire, M. 127
Baker, K. 433 Bourg, B.M. 599
Balage, M. 169 Bouritius, H. 247
Bałasińska, B. 159 Boutinaud, M. 249
Baldi, A. 373 Bregenhahl, K. 623
Baldwin VI, R.L. 293 Brennan, K.M. 57, 67
Ball, R.O. 243, 483, 497, 501, 519 Breuillé, D. 187
Barea, R. 97, 499 Briclot, G. 65
Barendse, W. 75 Broadhurst, R. 297
Baris, O. 301 Brocht, D.M. 369
Barletta, A. 37 Broderick, G.A. 635
Barnett, M.P.G. 279, 297, 315 Brömmel, C. 157
Barroeta, A.C. 493 Brosh, A. 219, 221, 223, 251
Bartelt, J. 239, 537 Brossard, L. 93
Bartl, K. 163 Brun, J.P. 591
Bauchart, D. 137, 389 Bucher, B. 517
Baumgartner, M. 397, 399 Buffière, C. 353
Beauvieux, M.-C. 51 Bugeon, J. 229

Bühler, K. 555 Conde-Aguilera, J.A. 383
Bujko, J. 95, 237 Connor, E.E. 293
Buraczewska, L. 437, 441 Cookson, A.L. 297
Busson, M. 301 Coon, C.N. 369
Buyse, J. 65, 397, 399, 481 Cornish-Bowden, A. 463
Coudray, C. 391
C Couet, C. 217
Cabello, G. 301, 391 Cowieson, A.J. 495
Cajarville, C. 637 Coyral-Castel, S. 377
Callesen, J. 405 Cristol, J.-P. 391
Campagnoli, A. 373 Crochet, S. 63, 65, 179, 181, 183
Campos, W.E. 215, 513, 525 Crovetto, G.M. 621
Cannas, A. 569
Cant, J.P. 415, 549 D
Caraty, A. 377 D’Ambrosio, F. 373
Cárdenas, M.L. 463 Damon, M. 63, 123, 289
Carré, B. 479 Danesh Mesgaran, M. 409, 595
Carstens, G.E. 57, 599 Danesh Mesgaran, S. 409
Casas, F. 301 Danfær, A. 597
Cassar-Malek, I. 295, 299 Dardevet, D. 169, 173, 175
Cassy, S. 91 Darusman, L.K. 631
Castro, P.F.C. 165 Davenel, A. 229
Castro Bulle, F.C.P. 603 Davenport, G.M. 549
Caton, J. 101 Davis, M.E. 57
Causeur, D. 161 Davis, S. 143
Chabrolle, C. 377 Davy, M. 297
Chaithiang, R. 505 De Beer, M. 369
Chamoux, A. 225 De Brabander, D.L. 149
Chanséaume, E. 59 De Campeneere, S. 149
Chaokaur, A. 505 Decuypere, E. 65, 481
Chardigny, J.M. 53 Deighton, D.L. 345
Charkhkar, S. 401 Deighton, M.H. 281
Chassot, A. 563 Delamaire, E. 151, 161, 249
Cherel, P. 289 De Lange, C.F.M. 171, 329, 415, 553
Chevalet, C. 311 Dell’Orto, V. 373
Chilliard, Y. 153, 285, 609 Derno, M. 195, 207, 527
Chizzotti, F.H.M. 577 Derouet, M. 179
Chizzotti, M.L. 577, 601 Detmann, E. 601
Christensen, M. 109, 111 Deutz, N.E.P. 319
Christopherson, R.J. 69 Devi, F.Y. 611
Chudobinska, E. 95 Dhanoa, M.S. 593
Chudy, A. 515 Dietrich, N. 207
Chwalibog, A. 61, 205, 213, 403, 523, 597 Dijkstra, J. 565, 593
Ciani, E. 291 Dixon, W.T. 69
Cirio, A. 439, 637 Djakovi, N. 133
Claustre, A. 173, 365 Doepel, L. 145
Codran, A. 173, 365 Dohme, F. 407, 551
Collewet, G. 229 Dokoupilová, A. 135
Collin, A. 63, 65, 181, 481 Dolev, A. 221
Colombani, P.C. 411 Dommels, Y.E.M. 279
Colombini, S. 621 Doran, O. 125
Combaret, L. 173, 175, 187, 365 Doreau, M. 609

Dozier, W. 623 Freetly, H. 337
Dubois, S. 217, 479, 521 Fujihara, T. 423
Duchene, S. 183 Fuller, M.F. 415
Duclos, M.J. 199 Fumita, T. 197
Dupont, J. 181, 377 Furuse, M. 509
Durand, D. 137, 389 Furuta, H. 381
Furuya, Y. 381
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Echaides, C. 439 G
Ecolan, P. 63, 289 Gabillard, J.C. 91
Effertz, C. 101 Gaddini, A. 109
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El-Kadi, S.W. 293, 343 Garcia, F. 489
Elia, C. 107 García-Valverde, R. 499
Elizondo, V. 637 Gas, M. 95
Ender, K. 115 Gasa, J. 493
Enishi, O. 611, 619 Gatellier, Ph. 99
Erez, A. 245 Gaye-Siessegger, J. 485
Ertbjerg, P. 109, 111 Gebert, S. 555
Etoh, T. 197 Geelen, M.J.H. 203
Ettle, T. 537 Geraert, P.A. 179
Even, P. 217 Gerrits, W.J.J. 211, 233, 235, 361, 367, 473
Giger-Reverdin, S. 561
F Gigli, S. 109, 111
Faciola, A.P. 635 Gin, H. 51
Fadaei, M. 401 Giraudet, C. 53
Failla, S. 109, 111 Gjøen, T. 131
Falero, A.L. 637 Gladine, C. 389
Fandrejewski, H. 89, 191 Glasser, F. 153, 585
Fardet, A. 307 Glindemann, T. 633
Feillet-Coudray, C. 391 Goddeeris, B.M. 397, 399
Ferlay, A. 609 Godet, E. 63, 199
Fernandes, J.B.K. 547, 605 Goetsch, A.L. 227
Fernández-Fígares, I. 97, 383, 385 Goglia, F. 37
Ferreira, G. 377 Gomez, C.A. 163
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Ferrell, C. 337 Gotoh, T. 197
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Fischer, B. 563 Grandemange, S. 301
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Fonseca, M.A. 601 Gruber, L. 563
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Fox, D.G. 569, 583 Gubbiotti, A. 395
France, J. 593 Guinard-Flament, J. 151, 161, 249, 581
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Gunter, S.A. 425 J
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Jahreis, G. 157
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Haj Hattab, N. 199 Janssens, G.P.J. 397, 481
Hallett, K.G. 125 Jatkauskas, J. 139
Hamard, A. 331 Jeanpierre, E. 377
Hammer, C. 101 Jentsch, W. 195, 527
Hammon, H.M. 103 Jilg, T. 563
Hampson, D.J. 405 Johnson, K.A. 57, 67
Han, K. 435 Joubert, R. 63, 65
Hansen, C.F. 405 Jourdan, A. 283
Harlina, E. 631 Junghans, P. 103, 195, 239
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Harris, P. 143
Hayashi, K. 197 K
Hayashi, M. 371 Kadanga, A.K. 295
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Heger, J. 539 Kaushik, S. 91
Heipertz, K.S. 357 Kebreab, E. 593
Hellwing, A.L.F. 541 Kemp, B. 511
Henkin, Z. 221 Kerr, B. 623
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Heo, J.M. 405 Kidd, M. 623
Heravi Mousavi, A.R. 409, 595 Kido, K. 371
Herpin, P. 63 Kikusato, M. 55
Hertzberg, H. 407 Kim, E.J. 427
Hess, H.D. 163, 407, 551, 617 Kim, J.C. 405
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Hodate, K. 371 Kluess, J. 203
Hoffmann, E.M. 625 Knoch, B. 279, 297
Honeyman, M. 623 Knowles, S.O. 279
Hoppen, H.-O. 375 Kolditz, C. 127
Horstmann, K. 571 Kolisek, M. 357
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Iwamoto, H. 197 Kucia, M. 207
Izhaki, I. 251 Kuhla, B. 287
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Lopez, S. 593 Milenkovic, D. 259, 309
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Luiking, Y.C. 319 Mohr, E. 527
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Energy and Protein Metabolism and Nutrition: EAAP Publication No. 124, 2007 Vichy, France 9-13 September, 2007

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Energy and Protein Metabolism and Nutrition: EAAP Publication No. 124, 2007 Vichy, France 9-13 September, 2007

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Energy and protein

EAAP publication No. 124, 2007

INRA – Institut National de la Recherche Agronomique

EAAP publication No. 124

The individual contributions in this

ISBN: 978-90-8686-041-8 The designations employed and the

The publisher is not responsible for

The scientific programme was organised by the National Scientific Committee:

In interaction with the International Scientific Committee:

All communications were reviewed by a Reviewing Board:

6 Energy and protein metabolism and nutrition

Locally, the Symposium was organised by the National Organising Committee:

The Coordinators in charge of the plenary sessions and workshops were:

With the contribution of the General Discussion Leaders:

Energy and protein metabolism and nutrition  7

Part 1. Nutrition and mitochondrial functions

On-going and prospective research on nutrition and mitochondrial functions 37

Insulin modulates the decrease of oxidative phosphorylation induced by ingested butyrate.

Up-regulation or activation of avian UCP attenuates mitochondrial ROS production and

Influence of protein-free diet on morphology of rat hepatocytes’ mitochondrium 61

Expression of uncoupling proteins and mitochondrial activity are dependent on muscular

Regulation of mitochondrial and tissue oxidations by thyroid hormones in chicken muscle 65

Inter-muscular variability of metabolic properties of bovine muscle fibres 71

Energy and protein metabolism and nutrition  9

2. Body composition and/or product quality

Study, in model system, of the effect of oxidation on myofibrillar protein digestibility 99

Glucose metabolism and its relation to milk production in F2 offspring of Charolais ×

Expression of beta-adrenergic receptors during lactation in Holstein dairy cattle 105

10  Energy and protein metabolism and nutrition

Influence of various Iodine supply on growing-finishing performance and on Iodine status

2A. Regulation of lipid deposition and intramuscular lipids

Intramyocellular lipid accumulation and insulin resistance development 121

Regional specifities in transcriptomic profiles of adipocytes isolated from skeletal muscle

Effect of a reduced protein diet on expression of lipogenic enzymes in relation to

Energy and protein metabolism and nutrition  11

2B. Regulation of milk production and composition

Does a dietary arginine deficiency limit milk protein yield? 145

Feeding measures to improve nitrogen efficiency in dairy cattle 147

Estimation of the intra-mammary metabolic fate of glucose and acetate in response to

Effect of rumen-protected conjugated linoleic acid in combination with propylene glycol

Variations in mammary extraction of nutrients under the effect of a 36-h milk

12  Energy and protein metabolism and nutrition

Development of low grade inflammation during aging impaired postprandial muscle

Changes in the expression of selected proteins elucidate skeletal muscle type-specific

Leucine suppresses myofibrillar proteolysis by down-regulating ubiquitin-proteasome

Skeletal muscle response to an endotoxin injection followed by malnutrition is similar in

Estrogenic and isoflavonic actions on differentiation and protein metabolism in porcine

Energy and protein metabolism and nutrition  13

Temporary consumption of a soy-based diet induces persisting alterations in protein

Effect of in utero metabolic programming on postnatal growth of mink kits 201

Heat production in chicken embryos 205

Comparing techniques to estimate energy expenditure on physical activity in individually

Quantitative partitioning of energy expenditure in pregnant ewes 213

A calorimetry system for metabolism trials 215

Determining cattle production efficiency without measuring the intake 219

14  Energy and protein metabolism and nutrition

Heart rate measurements as an index of energy expenditure and energy balance in

Characterisation of lipid distribution in rainbow trout by magnetic resonance, colour

Effect of graded dietary tryptophan levels on [1-13C]leucine oxidation and nitrogen

A multi-isotope approach to studying arginine metabolism in neonatal piglets 243

Gut function of pigs in relation to weaning 247

Energy and protein metabolism and nutrition  15

Part 3. ‘Omics’ in metabolism and nutrition studies

6 Energy and protein metabolism and nutrition

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10 Energy and protein metabolism and nutrition

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12 Energy and protein metabolism and nutrition

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14 Energy and protein metabolism and nutrition

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16 Energy and protein metabolism and nutrition

Part 4. Coordination between tissues for the metabolic utilisation of nutrients

4. Splanchnic metabolism and interorgan exchanges of nutrients

Energy and protein metabolism and nutrition 17

18 Energy and protein metabolism and nutrition

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20 Energy and protein metabolism and nutrition

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22 Energy and protein metabolism and nutrition

Energy and protein metabolism and nutrition 23