DOI: 10.1111/jpn.

12561

ORIGINAL ARTICLE

Performance, metabolic variables and enteric methane

^ s hair lambs fed Orbignya phalerata and
production of Santa Ine
Combretum leprosum
A. L. Abdalla Filho1, D. Dineshkumar1, M. Barreal2, C. McManus1,3, V. R. Vasconcelos4, A. L. Abdalla1 and
H. Louvandini1
1 Centre for Nuclear Energy in Agriculture, University of S~ao Paulo, Piracicaba, Brazil
2 partement Milieux, Productions, Ressources et Syst
De emes, Montpellier SupAgro, Montpellier, France
3 Faculty of Agriculture and Veterinary Medicine, University of Brasilia, Distrito Federal, Brazil, and
4 Federal University of Piauı, Teresina, Brazil

Summary
In this study, the possibility of using Babassu (Orbignya phalerata) and Mofumbo (Combretum leprosum) leaves for
lambs feed was verified. Performance, biochemical and haematological parameters, microbial protein synthesis,
nutrient apparent digestibility and enteric production of methane (CH4) were evaluated. The experimental treat-
ments included diets with forage-to-concentrate ratios of 50:50, with the leaves of the experimental plants
replacing 33% of the Cynodon dactylon (Tifton-85) hay with three treatments: control (no hay replacement) and
substitution with Babassu or Mofumbo. For the performance study, 24 Santa In^ es lambs were used, in a random-
ized experimental design with eight repetitions (5 male and 3 female) for each treatment and 48 days of experi-
mental trial. After this period, for nine days, six animals from each treatment were allocated in metabolic cages
to determine the apparent nutrient digestibility, microbial protein synthesis and nitrogen balance. Simultane-
ously, enteric CH4 was measured in vivo. The control group showed higher (p < 0.05) apparent digestibility of
acid detergent fibre. Enteric CH4 production of lambs fed Mofumbo leaves did not differ from that of the control
group, but was lower (p < 0.05) than in those fed with Babassu. The inclusion of Babassu and Mofumbo leaves
showed no negative effects on animal health and did not compromise performance. Mofumbo also presented
CH4 mitigating potential, indicating that those plants can be used as ingredients in the composition of lamb diets
with the advantage of reducing methane production.
Keywords Babassu, Mofumbo, tannin, tropical legumes

Correspondence A. L. Abdalla Filho, Av. Centen

ario 303, 13400-970, Piracicaba, S~ao Paulo, Brazil. Tel: +55 19 3429 4731; Fax: +55 19 3429 4610;
E-mail: adibefilho@cena.usp.br

Received: 29 March 2016; accepted: 1 June 2016

such as Babassu (Orbignya phalerata) and Mofumbo

Introduction
In particular when there are feed shortages in natural
pastures that grow on arable land used for food pro-
duction, small ruminants can incorporate the leaves
of plants other than grasses into their diets, which do
not necessarily compete with foodstuff. In this scenar-
io, the use of local resources can act as important
alternatives to buying in animal feed such as concen-
trates, contributing to the food security, sustainability
and economic viability of the activity (Schader et al.,
2015).
There is a wide variety of trees and shrubs in Brazil-
ian flora that grow in marginal areas without compet-
ing with food that can be used for feeding ruminants,
(Combretum leprosum). Those plant leaves are con-
sumed by sheep (Meier et al., 2014), but studies on
their possible effects on animal production are still
required.
Commonly, these plants are rich in secondary
metabolites, including tannins, produced by plants in
their intermediary metabolism, which may present
various degrees of condensation and a wide variety of
chemical structures, thus presenting different biologi-
cal properties (Soltan et al., 2013). Even though some
anthelmintic (Cenci et al., 2007) and antioxidative
(Luciano et al., 2011) activities are described in rumi-
nants, tannins can have influence on different physio-
logical aspects such as N metabolism, feed intake and

Journal of Animal Physiology and Animal Nutrition © 2016 Blackwell Verlag GmbH 1
 ^s lambs
Babassu and Mofumbo in diets of Santa Ine A. L. Abdalla Filho et al.

digestibility affecting the animals’ performance (Patra and the proportion of ingredients used in the
and Saxena, 2011). experimental diet of each treatment is presented in
Also, tannins, provided in the form of supplements Table 1.
or present in plants in the diet, often, but not always Chemical analyses of plants and diets (Tables 2 and
(Beauchemin et al., 2007), show the potential for the 3) were based on the content of dry matter (DM).
reduction in CH4 emissions (Abdalla et al., 2012; Concentrations of organic matter (OM; ID number
Staerfl et al., 2012; Moreira et al., 2013). Methane is 934.01), crude protein (CP; ID number 2001.11) and
one of the by-products of carbohydrate ruminal fer- ether extract (EE; ID number 2003.5) were deter-
mentation and, to a lesser extent, of amino acids in mined according to AOAC (2011). As recommended
the rumen and hindgut of ruminants. Depending on by Uden et al. (2005), neutral detergent fibre content
their sizes and the consumption of dry matter, sheep (NDF; ID number 2002.04) was analysed according to
and goats produce 10 to 16 kg CH4/yr (Hristov et al., Mertens (2002), and acid detergent fibre (ADF; ID
2013). In addition to contributing to the intensifica- number 973.18) and lignin (LIG; ID number 973.18)
tion of climate change, these emissions represent a were determined sequentially following the method-
loss of approximately 5 to 7% of dietary energy (Hris- ology proposed by Van Soest et al. (1991). Hemicellu-
tov et al., 2013). lose (HEMI) and cellulose (CEL) were calculated by
The aim of this study was to examine the effects of the difference between NDF, ADF and LIG. Total phe-
including leaves of Babassu and Mofumbo in the diet nols (TP), total tannins (TT) and condensed tannins
for lambs. (CT) of plants and diets were evaluated using the
methodology proposed by Makkar (2003).
Materials and methods
Location Table 1 Proportion of ingredients (DM) used in each experimental
diet (%)
The experiment was approved by the Ethics Commit-
tee on Use of Animals of the Escola Superior de Agri- Treatments
cultura ‘Luiz de Queiroz’ – CEUA-ESALQ/USP
Ingredients Control Babassu Mofumbo
(Protocol 2013-7) and conducted in the Animal Nutri-
tion Laboratory of Centre for Nuclear Energy in Agri- Tifton-85 51 34 34
culture, University of S~
ao Paulo, located in the City of Plant leaves 0 16 16
Piracicaba, State of S~ao Paulo, Brazil (LANA-CENA/ Corn grain 34 34 34
USP). Soya bean meal 13 14 14
Mineral mix 2 2 2

Experimental treatments
The experimental treatments were diets based on the Table 2 Chemical analyses, total phenolic (TP), total tannin (TT) and
condensed tannins (CT) content of the plants used
use of the leaves of two taniniferous plants as rough-
age hays: Mofumbo or Babassu. The leaves were Plants
obtained from the experimental farm of the Federal
Parameters Tifton-85 Babassu Mofumbo
University of Piaui (UFPI), dried in the shade and
chopped to a maximum length of 1 cm. Dry matter (g/kg) 945.7 945.0 946.2
Tifton 85-hay (Cynodon dactylon) was used as a con- Organic matter* 943.2 943.8 933.2
trol treatment. The diets consisted of 33% replace- Crude protein* 62.4 82.6 82.8
Neutral detergent fibre* 826.7 755.7 664.3
ment of Tifton-85 by the plant to be evaluated, on a
Acid detergent fibre* 519.9 568.6 505.7
dry matter (DM) basis, resulting in three treatments Hemicellulose* 306.9 187.1 158.5
(T): control, Babassu and Mofumbo. In addition, each Cellulose* 262.4 314.0 144.1
animal received approximately 335 g/day of concen- Lignin* 245.4 231.8 349.7
trate mixture composed of 69% crushed corn, 29% Ether extract* 16.0 20.4 50.6
soya bean meal and 2% mineral mixture, to meet the Total phenols† 5.09 76.06 217.55
requirements of weaned lambs weighing 20 kg and Total tannins† 2.71 60.74 171.91
Condensed tannins‡ 0.14 78.38 245.18
with daily gain of 100–150 g/day, according to the
NRC (2007). To achieve the similar protein values *Expressed in g/kg DM.
between the treatments, a 50:50 roughage/concen- †Expressed in grams of tannic acid/kg DM.
trate ratio was used. Water was provided ad libitum, ‡Expressed in equivalent gram leucocyanidin/kg DM.

2 Journal of Animal Physiology and Animal Nutrition © 2016 Blackwell Verlag GmbH
A. L. Abdalla Filho et al. ^s lambs
Babassu and Mofumbo in diets of Santa Ine

Table 3 Chemical analyses, total phenolic content (TF), total tannin (TT) blood with EDTA were used to determine haematocrit
and condensed tannins (CT) of the different diets according to Coles (1984), and haemoglobin, using
Treatments the colorimetric cyanomethaemoglobin method
(Thrall, 2006). The samples without EDTA were cen-
Parameters Control Babassu Mofumbo trifuged (10 min at 1310 g and 4 °C) for the separa-
Dry matter (g/kg) 944.3 944.1 944.3 tion of blood serum. The samples were stored at
Organic matter* 945.1 945.2 943.5 20 °C for further analyses of the parameters includ-
Crude protein* 119.7 124.2 124.2 ing aspartate aminotransferase (AST), creatinine, total
Neutral detergent fibre* 701.8 687.9 673.3 protein, glucose, urea and serum albumin. These
Acid detergent fibre* 364.6 369.2 359.2
parameters were evaluated using commercial Labtestâ
Hemicellulose* 337.2 318.7 314.1
kits and a spectrophotometer (PerkinElmer-UV/Vis
Cellulose* 176.5 183.0 155.8
Lignin* 179.6 176.0 194.9 Spectrophotometer EZ150 Lambda, S~ ao Paulo, Brazil).
Ether extract* 33.4 34.9 39.4
Total phenols† 2.20 10.50 26.80
Apparent digestibility of nutrients
Total tannins† 1.20 8.00 20.80
Condensed tannins‡ 0.10 9.30 28.50 After the performance of trial period, six animals of
*Expressed in g/kg DM. each treatment (four males and two females) were
†Expressed in grams of tannic acid/kg DM. placed in metabolic cages for nine days for daily diet
‡Expressed in equivalent gram leucocyanidin/kg DM. sample collection of feed offered and total orts, as well
as for the collection of faeces and urine excreted, in
order to determine the nutrient apparent digestibility,
Performance evaluation
microbial protein synthesis using urine purine deriva-
Twenty-four Santa In^ es lambs (9 females and 15 tives and nitrogen balance. The diet, orts and faeces
males), 149  4.7 (mean  standard deviation) days were weighed and a representative sample of 10% of
old and average live weight of 20.4  5.31 kg, were each material was stored under refrigeration at 4 °C.
used in a randomized experimental design with eight Urine samples were collected in buckets previously
repetitions. The animals were maintained in individ- acidified with 100 ml of 10% H2SO4, the volume was
ual pens (1.0 m x 1.5 m) fitted with a feeder, water determined and the sample was stored in a graduated
trough and recycled rubber flooring. For five days, the cylinder. The diet offered, orts and faeces were dried
animals underwent adaptation to the diet, fed twice a in a forced ventilation oven at 60 °C for 48 h, milled
day, in the morning (08:00) and in the afternoon to 1 mm in a Willey-type mill and subjected to chemi-
(15:00). The amount of food provided was adjusted to cal analysis as previously described.
give 20% orts daily. The trial period lasted 48 days
including the adaptation days.
Microbial protein synthesis by purine derivatives
Daily, before the provision of the diet, the orts from
the previous day were weighed and daily dry matter For the estimation of microbial protein synthesis
intake (dDMI) was determined. Weekly, the animals in vivo, purine derivatives (PD) were determined in
were weighed before the morning feeding, to deter- the samples of urine using high-performance liquid
mine the average daily weight gain (ADG), calculated chromatography (HPLC), under the protocol adapted
by linear regression of individual animal weights over from Balcells et al. (1992) and Makkar and Chen
time and the total gain in weight (TGW) calculated (2003).
throughout the experimental period. Feed conversion After thawing at room temperature, the acidified
(FC) was calculated as the ratio between the total con- urine samples were homogenized in a sonicator (ul-
sumption and the total weight gain of the animals. trasound) for 5 min due to the presence of particulate
material deposited at the bottom of tubes. Then, 5 ml
of these samples was centrifuged for 20 min at 582 g
Biochemical and haematological parameters
and 4 °C. Two millilitres of the supernatant fraction
Animal health was evaluated by monitoring the bio- was sampled and transferred to another container
chemical and haematological parameters. On days 1, where 0.25 ml of oxypurinol and 2.75 ml of ammo-
24 and 45, blood samples were collected through nium monobasic phosphate 0.0025 M were added.
jugular puncture using vacuum tubes, with and with- The samples were then homogenized in a vortex.
out anticoagulant (ethylenediamine tetraacetic acid – With the aid of a syringe, 1 ml of the samples was fil-
EDTA). Immediately after the collection, samples of tered using a Millexâ 13-mm filter (0.45-lm),

Journal of Animal Physiology and Animal Nutrition © 2016 Blackwell Verlag GmbH 3
 ^s lambs
Babassu and Mofumbo in diets of Santa Ine
transferred to vials (Agilent 1.5 ml) and subjected to
HPLC analysis (Agilent 1100). The HPLC system was
equipped with a quaternary pump, degasser, auto-
matic gun samples, thermostat, photodiode array
(UV–Vis) and Zorbax ODS C18 column (250 9
4.6 mm, 5 lm particles). After the quantification of
allantoin, uric acid, xanthine and hypoxanthine,
microbial nitrogen (MN) absorbed in the small intes-
tine was calculated according to Makkar and Chen
(2003).
Nitrogen balance
For the nitrogen balance calculation, the total N con-
centration (N) of urine and faeces samples was deter-
mined by the micro-Kjeldahl method (AOAC, 2011),
using steam distillation with sodium hydroxide solu-
tion 12 M (NaOH), collecting this in the solution of
boric acid (H3BO3) and titration with a solution of
0.025M H2SO4.
The N retained was calculated as the difference
between the total N consumed and the total N
excreted: N retained = (N consumed) (N excreted),
where N consumed = N ingested from the diet offered
and N excreted = N excreted in the faeces and urine.
Enteric methane production
Simultaneously with the apparent digestibility assay,
enteric CH4 production was quantified in vivo. In each
test, six different animals were housed individually in
open circuit chambers for gas emission measurements
following the methodology described by Abdalla et al.
(2012).
The animals were kept in the chambers for two con-
secutive days, with an average flow of indoor air
removal of 125 L/min (measured by anemometer-EC
AD Instrutherm-250, S~ ao Paulo, Brazil); the internal
temperature (24.2  0.72 °C) and humidity
(66.5  2.21%) of the chambers were measured reg-
ularly at 2-h intervals. The quantity of eliminated
gases and CH4 concentration was measured daily for
each animal in each experimental treatment.
The CH4 concentration was assessed in a gas chro-
matograph (Shimadzu GC2010TM, Tokyo, Japan)
equipped with a flame ionization detector (FID) and a
capillary HP-Molesieve column (GC 30 m 9 0.53 mm
9 25 lm). The operation conditions of the chro-
matograph were column temperature (isothermal) at
60 °C, injector at 200 °C, detector at 240 °C and car-
rier gas (He) in constant flux at 10 ml/min. The con-
centration of CH4 was determined using a calibration
curve (0, 30, 90 and 120 ml/l) prepared with a
4 Journal of Animal Physiology and Animal Nutrition © 2016 Blackwell Verlag GmbH
A. L. Abdalla Filho et al.
commercial CH4 standard (Praxair Industrial Gases,
Osasco, SP, Brazil; 995 ml/l purity).
The production of CH4 was expressed in g/day, g/kg
of dry matter intake [g/kg DMI, considering the
changes in DMI as the lambs enter the chambers
(Moate et al., 2012)], g/kg of live weight (g/kg LW),
and the CH4 emission intensity, calculated according
to Hegarty et al. (2010), was expressed as g/kg in
average daily weight gain (g/kg ADG).
Statistical analysis
Statistical analyses of the data were performed
using SASâ v.9.2 (Statistical Analysis System Insti-
tute, Cary NC, USA). The variables obtained were
submitted to analysis of variance using PROC GLM.
For biochemical and hematological repeated mea-
sures, PROC MIXED was used fixed effects of treat-
ment (T), sex (S) and their interaction (T*S) were
tested. PROC REG was used to analyse the effects
of concentrations of CT of diets on all the variables
studied. A 5% significance level was used.
Results
No differences were found between the treatments
(p > 0.05) in terms of average body weight at each
weighing (Fig. 1). Daily dry matter intake, average
daily gain, the total weight gain and feed conversion
also did not show significant differences between the
treatments (p > 0.05) (Table 4). An effect of lamb sex
(p < 0.05) was observed for ADG, TGW and FC where
male animals showed higher average daily weight
gain (147  10.0 g/day) and the total gain in
weight (6.63  0.456 kg) compared to females
28
Control
27
Babassu
26
Body weight (kg)
Mofumbo
25
24
23
22
21
20
0 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45
Days
Fig. 1 Average body weight at each weighing day of Santa In^es sheep
fed Tifton hay (control), Babassu or Mofumbo according to the experi-
mental treatments.
A. L. Abdalla Filho et al. ^s lambs
Babassu and Mofumbo in diets of Santa Ine

Table 4 Means of daily dry matter intake (dDMI), dDMI in percentage of body weight, average daily gain (ADG), total weight gain (TWG) and feed
^s lambs fed Tifton hay (control), Babassu or Mofumbo according to the experimental treatments
conversion (FC) presented by Santa Ine

Treatments (T) p value

Parameters Control Babassu Mofumbo SEM T S T*S REG

Daily dry matter intake (g DM) 663.06 738.73 728.56 67.362 0.6948 0.4260 0.9521 0.6363
Daily dry matter intake (% LW) 2.94 3.12 3.21 0.144 0.4284 0.8182 0.9969 0.3611
Average daily gain (g/day) 98.45 103.31 110.78 12.063 0.4145 0.0267 0.4979 0.5084
Total weight gain (kg) 5.01 6.11 6.06 0.645 0.4145 0.0267 0.8653 0.5084
Feed conversion 6.49 5.52 5.46 0.422 0.5906 0.0389 0.7338 0.3272

T, treatment effect; S, sex effect; T*S, interaction between T and S; REG, linear regression; SEM, standard error of the means.

(107  13.1 g/day and 4.83  0.589 kg). Females
presented higher FC values (6.43  0.386) than males
(5.22  0.299).
No differences were found between the treatments
(p > 0.05), but an effect of sex was found for haemat-
ocrit (p < 0.05), where female animals showed higher
values (32.7  0.54%) than males (30.8  0.42%).
The biochemical parameters such as total protein
(g/dl), albumin (g/dl), glucose (mg/dl), urea (mg/dl),
aspartate aminotransferase (U/l) and creatinine (mg/
dl) of animals during the experiment did not show the
differences (Table 5) between the treatments
(p > 0.05). Female animals showed greater (p < 0.05)
creatinine (0.94  0.040 mg/dl) and urea
(36.5  1.50 mg/dl) levels than males (0.78  0.031
and 30.8  1.16 mg/dl).
The control diet had a greater apparent digestibility
of ADF (p < 0.05) than the other diets. There were no
differences between the diets in the other nutritional
characteristics (Table 6).
Purine derivatives concentration (mmol/day and
lmol/day/LW0.75) excreted in the urine, the estima-
tion of MN (g N/day) absorbed and the amount of
nitrogen consumed (g/day), excreted (g/day) and
retained (g/day) by lambs of the treatments did not
show differences (p > 0.05) between the treatments
(Table 7).
When the enteric methane production was
expressed in grams of CH4/kg of live weight and CH4
intensity (g/kg ADG), animals on Mofumbo treatment
showed lower values than animals on Babassu
(p < 0.05), but neither was different to the control
(Table 8). Also, sex affected CH4 production, where
female animals showed higher (p < 0.05) values
(144.5  15.57 g/kg ADG) than males
(95.4  11.01 g/kg ADG).
Discussion
Lambs fed Babassu or Mofumbo grew faster than
those fed control diet, but the variation between indi-
vidual lambs meant that the differences were not sta-
tistically significant. The inclusion of Babassu and
Mofumbo leaves in the diet of lambs was well
accepted by the animals because no significant differ-
ence was observed in the daily dry matter intakes and
were in accordance with NRC (2007).

Table 5 Haematocrit, haemoglobin and biochemical means values of Santa In^es lambs fed Tifton hay (control), Babassu or Mofumbo according to the
experimental treatments

Treatments (T) p value

Parameters Control Babassu Mofumbo SEM Ref T S T*S REG

Haematocrit (%) 32.18 31.91 31.38 0.600 24–50 0.6438 0.0135 0.3106 0.4752
Haemoglobin (g/dl) 11.74 11.89 11.63 0.383 8–16 0.8905 0.3235 0.4816 0.9691
Total protein (g/dl) 6.74 7.29 6.94 0.217 6–7.9 0.1136 0.7333 0.1975 0.7747
Albumin (g/dl) 2.63 2.69 2.49 0.129 2.4–3 0.5546 0.2374 0.3115 0.3896
Glucose (mg/dl) 47.36 48.45 47.65 3.165 50–80 0.9690 0.7498 0.3375 0.9412
Urea (mg/dl) 35.06 33.64 32.31 1.650 17.1–42.8 0.5128 0.0080 0.9829 0.3064
AST (U/l) 91.56 84.79 86.27 5.970 60–280 0.7062 0.5708 0.4387 0.6404
Creatinine (mg/dl) 0.84 0.84 0.90 0.044 1.2–1.9 0.4870 0.0051 0.9214 0.3280

AST, aspartate aminotransferase; T, treatment effect; S, sex effect; T*S, interaction between T and S; REG, linear regression; Ref, reference values
according to Meyer and Harvey (2004); SEM, standard error of the means.

Journal of Animal Physiology and Animal Nutrition © 2016 Blackwell Verlag GmbH 5
 ^s lambs
Babassu and Mofumbo in diets of Santa Ine A. L. Abdalla Filho et al.

Table 6 Apparent nutrient digestibility (%) of Tifton hay (control), Babassu and Mofumbo diets fed to Santa In^es lambs

Treatments (T) p value

Variables (%) Control Babassu Mofumbo SEM T S T*S REG

DM apparent digestibility 63.4 59.5 61.8 2.46 0.5465 0.6617 0.7988 0.7897
OM apparent digestibility 64.8 60.7 62.8 2.41 0.4918 0.6749 0.8127 0.6744
CP apparent digestibility 67.9 61.9 64.6 2.92 0.3732 0.3727 0.5297 0.4828
NDF apparent digestibility 59.7 54.3 55.8 2.67 0.3634 0.9343 0.6145 0.3184
ADF apparent digestibility 54.6a 44.0b 45.7b 2.54 0.0258 0.2138 0.8575 0.0605
HEMI apparent digestibility 65.9 67.3 68.1 3.62 0.9088 0.3298 0.5428 0.7322
CEL apparent digestibility 41.2 31.6 36.9 7.37 0.2442 0.1460 0.0702 0.7074

DM, dry matter; OM, organic matter; CP, crude protein; NDF, neutral detergent fibre; ADF, acid detergent fibre; HEMI, hemicellulose; CEL, cellulose. T,
treatment effect; S, sex effect; T*S, interaction between T and S; REG, linear regression; SEM, standard error of the means.
a,b
Different letters in the same row indicate statistical difference (p < 0.05).

^s lambs fed Tifton

Table 7 Purine derivatives (PD) excreted, estimation of microbial nitrogen absorption (MN) and nitrogen (N) balance of Santa Ine
hay (control), Babassu and Mofumbo according to the experimental treatments

Treatments (T) p value

Parameters Control Babassu Mofumbo SEM T S T*S REG

PD (mmol/day) 6.13 4.70 6.00 1.276 0.6915 0.5479 0.6345 0.6418

PD (lmol/day/kg0.75) 568.46 437.19 516.97 112.537 0.7147 0.5202 0.6976 0.9380
MN (g N/day) 2.82 2.85 2.87 0.114 0.9648 0.5197 0.6766 0.6987
N consumed (g) 14.45 16.30 15.95 1.391 0.6964 0.3313 0.9946 0.5418
N excreted (Faeces) (g) 4.78 6.03 5.80 0.615 0.3290 0.2293 0.7942 0.3611
N excreted (Urine) (g) 4.56 4.29 3.31 0.575 0.5447 0.4876 0.6726 0.1200
N retained (g) 5.11 5.98 6.83 0.827 0.5652 0.7692 0.7178 0.1594

T, treatment effect; S, sex effect; T*S, interaction between T and S; REG, linear regression; SEM, standard error of the means.

^s lambs fed Tifton hay (control), Babassu or Mofumbo according to the experimental
Table 8 Enteric methane (CH4) production of Santa Ine
treatments

Treatments (T) p value

Enteric CH4 production Control Babassu Mofumbo SEM T S T*S REG

g/day 9.7 14.9 8.7 1.76 0.0608 0.3553 0.9210 0.4769

g/kg DMI 14.3 19.0 10.9 2.16 0.0625 0.7822 0.8403 0.2275
g/kg LW 0.36ab 0.54a 0.30b 0.061 0.0489 0.4765 0.9238 0.3256
g/kg ADG 121.6ab 163.7 a 74.6b 16.52 0.0085 0.0244 0.0941 0.1476

DMI, dry matter intake; LW, live weight; ADG, average daily gain; T, treatment effect; S, sex effect; T*S, interaction between T and S; REG, linear regres-
sion; SEM, standard error of the means.
Different letters in the same row indicate statistical difference (p < 0.05).

Studies have shown that when the concentration of
tannins in the diet exceeds 50 g CT/kg DM, feed con-
sumption by ruminants was reduced, due mainly to
the reduction in acceptability and conditioned aver-
sion (Frutos et al., 2004; Mueller-Harvey, 2006). As
the level of CT observed here was well below this
value, no negative effect was seen on the consump-
tion of the diet, as other authors have shown when
using diets with similar CT content, irrespective of the
plants used (Barry and Duncan, 1984; Waghorn et al.,
1994; Aerts et al., 1999).
The Babassu and Mofumbo leaves provided the
nutritional requirements for growing lambs, allowing
the animals to perform adequately. There were no sig-
nificant differences between the diets in ADG and FC,
suggesting that the inclusion of these plants as
replacement for Tifton-85 hay may be useful in the
feeding of lambs. Although not statistically different,

6 Journal of Animal Physiology and Animal Nutrition © 2016 Blackwell Verlag GmbH
A. L. Abdalla Filho et al.
lambs fed Babassu and Mofumbo had an ADG of
4.9% and 12.5% greater than those fed the control
diet, suggesting the possible benefits to be investi-
gated, because these differences may have been signif-
icant if more animals per treatment had been used.
Health of lambs was not compromised by the
feeding of Babassu or Mofumbo. In addition to the
presence of CT, the plants tested may also present a
wide variety of compounds that require further study,
including toxicity, which could affect the animal
health. Hence, haematological and biochemical
parameters were monitored and the results observed
showed that there were no intoxication symptoms in
lambs fed with 33% of Babassu or Mofumbo replacing
Tifton hay for 57 days.
The haematocrit and haemoglobin were within the
reference values for the species, as well as total pro-
teins, albumin, urea and AST. Glucose and creatinine
values were below, but close to, the reference values
(Meyer and Harvey, 2004).
Glucose is the only source of energy for the central
nervous system, and the regulation of its concentration
in the blood is related to glycogenesis in the liver (Val-
adares Filho and Pina, 2011). The values found in this
study did not differ between the treatments and are
slightly lower than the reference values, ranging from
50 to 80 mg/dl (Meyer and Harvey, 2004), but do not
necessarily characterize hypoglycaemia, because no
clinical symptoms or metabolic disorders were
observed. In addition, small differences regarding the
reference values may be observed between the breeds,
because Santa In^es hair sheep is a tropical breed.
Creatinine is formed from condensation and sponta-
neous muscle creatinine dehydration, and its elimina-
tion depends on the individual’s muscle mass, age and
sex (Fettman and Rebar, 2006). The concentration of
creatinine did not differ between the treatments and
was slightly below the reference values ranging from
1.2 to 1.9 mg/dl (Meyer and Harvey, 2004), which
may be related to the young age of animals
(149.4  4.7 days) and, consequently, lower muscle
mass in relation to adult animals on which the refer-
ence values are based.
With regard to the digestibility of nutrients, signifi-
cant differences were observed in ADF: the control
treatment showed the values higher than Babassu and
Mofumbo treatments, suggesting that specific parame-
ters such as the CT content of plants included in the
diets with Babassu and Mofumbo interfered with the
digestibility of this fibre fraction. Although tannins
have their effects primarily on proteins, they also have
effects on carbohydrates (Schofield et al., 2001). Sev-
eral studies have shown that the digestibility of fibre
Journal of Animal Physiology and Animal Nutrition © 2016 Blackwell Verlag GmbH
^s lambs
Babassu and Mofumbo in diets of Santa Ine
fractions in the rumen of animals fed with tannins can
be dramatically reduced (McSweeney et al., 2001;
Herv as et al., 2003).
As an estimate of microbial protein synthesis,
excreted PD were measured and were not affected by
the inclusion of Babassu or Mofumbo leaves in the
diets. Several studies have reported the effects of tan-
nins reducing the growth of several species of ruminal
micro-organisms (Jones et al., 1994; Nelson et al.,
1997; Min et al., 2002; Sivakumaran et al., 2004).
According to Patra and Saxena (2011), the sensitivity
of micro-organisms to tannins can vary substantially,
which is dependent on the microbial species as well as
the type or source of tannins used. According to these
authors, the effects of tannins on microbial growth are
attributed to their interactions with extracellular
enzymes and bacterial cell walls, causing morphologi-
cal changes in these structures, in addition to acting
directly on the microbial metabolism; these can form
complexes with proteins, preventing them from being
degraded in the rumen.
Generally, the improved animal performance
observed when tannins are at moderate levels in the
diet is attributed to these complexes, increasing the
flow of essential amino acids to the intestine and
increasing its absorption in the blood (Makkar, 2003),
which is dependent on the quantity and quality of
dietary protein, in conjunction with the protein of
microbial origin that flows from the rumen to the abo-
masum (Patra and Saxena, 2011). Nitrogen balance
was not affected by feeding Babassu or Mofumbo,
without the changes in the route of dietary nitrogen
excretion (urine/faeces), which were observed by
other authors using CT aiming CH4 mitigation (Soltan
et al., 2013).
The effect of tannins on the reduction in enteric
CH4 production is usually related to its direct action,
by inhibiting the activity of methanogenic micro-
organisms and/or reducing the digestibility of rumen
fibre fractions (Patra and Saxena, 2011). In the treat-
ment with highest content of CT (Mofumbo), this rel-
ative decrease in fibre digestibility and the reduction
in enteric CH4 emission were observed. However, ani-
mals on Babassu, despite the decrease in fibre
digestibility, did not show lower CH4 production,
probably because the content and activity of CT in this
treatment affected the digestibility without affecting
the population of methanogenic micro-organisms.
According to Hristov et al. (2013), it is important
that the benefits of the reduction in the emission of
CH4 do not hide the possible harmful effects of tannins
on nutrient digestibility and production parameters.
In this work, despite the lower digestibility of a fibre
7
 ^s lambs
Babassu and Mofumbo in diets of Santa Ine A. L. Abdalla Filho et al.

fraction in Babassu and Mofumbo treatments, there ADF, did not compromise animal performance, indi-
was no productivity reduction in the lambs. cating that those plants can be used as ingredients in
Numerically, CH4 yield (g/kg DMI) and intensity the composition of lambs’ diets. Also, although there
(g/kg ADG) from the lambs fed Mofumbo diet were is no statistical evidence, Mofumbo may have CH4
23.4 and 38.6% lower in relation to the lambs fed mitigation potential, contributing to the sustainability
control diet. This difference may be not statistically of the sheep production activity.
significant due to the small number of animals used
and a large variation between individual lambs. The
Acknowledgements
reduced CH4 emission, without compromising the
performance of animals, shows that this plant has The authors thank Comiss~ ao Nacional de Energia
the potential to be used as a CH4 mitigation agent. Nuclear (CNEN) and Conselho Nacional de Desen-
volvimento Cientıfico e Tecnol
ogico (CNPq) for pro-
viding financial support.
Conclusion
The inclusion of Babassu and Mofumbo leaves in the
Conflict of interest
diet showed no deleterious effects on animals’ health
and, despite having reduced apparent digestibility of The authors declare no conflict of interest.

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