Effect of synchronization of energy and nitrogen supply on ruminal characteristics

and microbial growth

P. H. Henning, D. G. Steyn and H. H. Meissner

J Anim Sci 1993. 71:2516-2528.

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 Effect of Synchronization of Energy and Nitrogen Supply on
Ruminal Characteristics and Microbial Growthl
P. H. Henning, D. G. Steyn, and H. H. Meissner2
Animal Nutrition Centre, Irene Animal Production Institute, Private Bag X2, Irene, 1675,
Republic of South Africa
ABSTRACT The effect of energy and N synchroni-
zation in the rumen on microbial growth was inves-
tigated. The same daily amount of readily available
energy and N sources ( 19 g of ruminally degradable
N k g of fermentable OM) was supplied intraruminally
to sheep, according t o different patterns, namely both
energy and N as 12-hourly pulse-doses (fast syn-
chronized supply), energy as 12-hourly pulse-doses
and N as a continuous infusion (unsynchronized
supply), energy as a continuous infusion and N as
12-hourly pulse doses (unsynchronized supply), and
both energy and N as continuous infusions (slow
synchronized supply). The study was done near
maintenance (Exp. 1) and at a higher level of
nutrition (Exp. 2). The degree of energy and N
synchronization affected neither microbial flow
nor efficiency of growth ( P > .2 for energy x N interac-
Key Words: Ruminal Fermentation, Energy, Nitrogen, Synchronization
Introduction
Ruminal microbial protein production is generally
considered as a fixed amount per unit of digestible OM
or energy intake, although published values vary
substantially (ARC, 1984). Nocek and Russell (1988)
suggested that efficiency of microbial growth and
microbial protein production may be improved by
balancing the overall daily ratio of ruminally available
energy and N in the diet. The question of concern in
this paper is whether, when feeding the required
balanced amount of ruminally available energy and N,
microbial efficiency and production can be further
lThe authors wish to thank A. Kistner and J. P. Pienaar for
valuable advice and discussions; W. M. Madutlela, W. K. Mashiane,
S. K. Mmola, and J. M. Ramaru for technical assistance; M. Basson,
J. Collier, E. Nel, and H. Smit for chemical analyses; M. F. Smith for
help with statistical analysis; and E. Oberholzer and H. Olivier for
help with the preparation of the manuscript.
2Dept. of Livest. Sci., Univ. of Pretoria, Pretoria, 0001, Republic
of South Africa.
Received August 14, 1992.
Accepted March 11, 1993.
2516
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tion). Continuous infusion of energy resulted in a 17%
( P < .05) and 14% ( P = .18) higher efficiency of
microbial growth than did pulse dosing in Exp. 1 and
2, respectively. This coincided with lower ( P < .01)
ruminal pH and higher ( P < .05) ruminal lactic acid
concentration for energy pulse-dose treatments. The
results suggest that merely improving the degree of
synchronization between energy and N release rates
in the rumen does not increase microbial yield.
Dietary manipulation, rather, should be aimed at first
obtaining the most even ruminal energy supply
pattern, and then at providing the appropriate amount
of ruminally available N. Thereafter some further
advantage may be gained in also ensuring a more
even N supply pattern, particularly avoiding too rapid
a ruminal N release.
J. h i m . Sci. 1993. 71:2516-2528
improved by synchronizing ruminal release of energy
and N over the shorter term (i.e., within a day).
Batch culture studies showed no improvement (Hen-
ning et al., 1991; Newbold and Rust, 1992), whereas
in vivo studies (Casper and Schingoethe, 1989;
Herrera-Saldana et al., 1990; Matras et al., 1991)
suggested a positive response with better synchroniza-
tion. In the in vivo studies different ruminal release
rates of energy and N, to achieve various degrees of
synchronization, were obtained by using different feed
ingredients. This implies that treatment effects may
have been confounded with ingredient effects and with
different total amounts and(or) ratios of nutrients
released in the rumen.
The aim of the present study was to determine to
what extent the degree of synchronization of energy
and N supply to the rumen influence ruminal environ-
ment and microbial growth. This was done by
supplying the same balanced amount of energy-
yielding substrate and N, both in readily available
forms, directly to the rumen of sheep, but according to
different patterns to obtain different degrees of
synchronization.
 ENERGY-NITROGEN SYNCHRONIZATION IN THE RUMEN
Materials and Methods
In Exp. 1 sheep were fed near the maintenance
level because the treatments were expected to cause
ruminal acidosis at higher input levels. It was,
however, realized that the results may not hold true at
higher levels of nutrition. High-producing animals will
have higher levels of intake and will supposedly be
more dependent on increased microbial protein flows;
therefore, the initial study was followed by a second
similar, but independent, study (Exp. 2 ) that used
higher nutrient inputs together with appreciable
amounts of buffer to prevent ruminal acidosis.
Experiment 1
Animals and Management. Sixteen mature South
African Mutton Merino wethers (average BW = 53.5
kg; SD = 8.29) were fitted with ruminal and abomasal
cannulas. Surgical procedures and postoperative care
were done by a qualified veterinarian in accordance
with the National Code for Animal Use in Research,
Education, Diagnostics and Testing of Drugs and
Related Substances in South Africa. Animals were
housed indoors in metabolism cages ( 1.5 m long x 1.0
m wide) under continuous lighting and had free
access, from 14 d before the start of the experiment, t o
the following basal diet, fed in six equal portions at
4-h intervals (gramskilogram of DM): wheat straw
(hammer-milled, 25-mm sieve), 899; fish meal, 56;
molasses, 30; monosodium phosphate, 5 ; salt, 5; urea,
4;and vitamin-mineral premix, 1. Orts were removed
once a day. The basal diet was fed to maintain normal
ruminal function during intraruminal feeding.
Animals had free access to drinking water.
Experimental Treatments. There were four ex-
perimental treatments (i.e., two energy supply pat-
terns and two N supply patterns in a 2 x 2 factorial
arrangement). Treatments consisted of adding the
same daily amount of soluble carbohydrate (CH)
mixture (340 g of 6 maltose:2 dextrose:l maltotriose;
African Products [Pty] Ltd, Johannesburg, South
Africa) as the energy source and N (9.18 g of urea and
10.17 g of sodium caseinate) t o the rumen of each
animal, according t o one of four different patterns:
Treatment EPNP, both the CH mixture and N as two
equal pulse-doses at 0800 and 2000 (representing
rapid synchronized energy and N supply); Treatment
EPNG, the CH mixture as two equal pulse-doses at
0800 and 2000 and the N as a continuous (gradual)
infusion (representing unsynchronized supply);
Treatment EGNP, the CH mixture as a continuous
infusion and N as two equal pulse-doses at 0800 and
2000 (representing unsynchronized supply); and
Treatment EGNG, both CH mixture and N as
continuous infusions (representing slow synchronized
energy and N supply).
The basal diet together with the intraruminally
supplied nutrients were formulated to provide for the
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2517
maintenance requirements of the sheep (NRC, 1985)
and to obtain a ratio of approximately 19 g of
ruminally degradable N ( RDN)/kg of ruminally
fermentable OM ( FOMR) . Nitrogen contents of 5 g k g
of DM and 98 g k g of DM and ruminal degradations of
36 and 40% were allotted t o wheat straw (R. Meeske,
unpublished results) and fish meal (Erasmus et al.,
1988), respectively. The ratio of 19 g of RDN/kg of
FOMR is equivalent t o an average efficiency of
synthesis of microbial N in the rumen (Czerkawski,
1986) and was aimed at ensuring a sufficient but not
excessive N supply to the rumen. The ratio of urea:
sodium caseinate was selected to obtain a ratio of 3:l
for nonprotein N (NPN):amino acid N (Maeng,
1975).
Experimental Protocol. Four sheep were randomly
allocated to each experimental treatment for a first
experimental period of 22 d. After completion of the
first experimental period, intraruminal feeding was
terminated, whereafter all sheep were allowed free
access to the basal diet for the next 7 d. Thereafter,
sheep were again allocated to different treatments for
the second experimental period, which was a repeti-
tion of the first. This allocation was random, except
that no sheep received the same treatment it had
received during the first period. From d 1 of the first
experimental period, each sheep received a continuous
infusion (1,300 mL/d) and two intraruminal pulse-
doses (500 mL at both 0800 and 2000) of water. From
d 1 to 10 the amount of the basal diet offered was
gradually changed from ad libitum to 900 g of air-
dried feed-sheep-1.d-l. Concurrently, the intrarumi-
nally added CH and N were gradually introduced into
the respective continuous infusions or pulse-doses ( t h e
volumes of which were kept constant) until the total
amount was added by d 10.
Two days before the beginning of the experimental
period, and on d 12 of the experimental period, 180
mL of Cr-EDTA solution, prepared according to the
method described by Binnerts et al. (1968), was
mixed into the rumen of each sheep at 0800. Ruminal
fluid samples were subsequently taken at 3, 8, and 24
h after dosing of Cr-EDTA to determine the retention
time of water according t o the procedure of Downes
and McDonald (1964). On d 14, a controlled-release
capsule for continuous uniform release of Cr2O3
(Captec Chrome for Sheep; Captec [NZI Ltd, Auck-
land, New Zealand) was placed into the rumen of each
sheep. Capsules were recovered on d 22, briefly rinsed
under tap water to remove digesta particles, and then
dried in a desiccator. Mass loss of individual capsules
was subsequently determined and used to calculate
the average daily release of Cr203 into the rumen for
each sheep. The Cr203 was used as a digesta flow
marker.
From d 18 to 22, samples of ruminal (150 mL/
collection) and abomasal (140 mL/collection) digesta
were collected from each sheep every 8 h, with a
10-h interval between days to allow a shift in daily
2518 HENNING ET AL.
sampling times. The abovementioned sequence al-
lowed samples to be obtained for every odd hour of the
24-h day (i.e., 1, 3, 5, 7, 9, and 11 h after both the
0800 and 2000 dosing). The corresponding ruminal
samples after the morning and the evening dose were
pooled for each sheep, leaving six ruminal samples ( 1,
3, 5, 7, 9, and 11 h after dosing) per sheep. A
subsample was taken from each ruminal sample and
pooled within sheep to obtain a representative pooled
ruminal sample for every sheep. Abomasal samples
were pooled within sheep to obtain a representative
abomasal sample for every sheep. Immediately after
collection, the pH of the ruminal digesta samples was
measured using a pH electrode, whereafter both
ruminal and abomasal samples were frozen and stored
at -10°C.
Water intake of sheep was monitored daily before
and during the experimental periods. Representative
samples of the basal diet were taken daily from d 18 to
22 and composited.
Sample Preparation and Analysis. The basal diet
samples were analyzed for OM (by ashing at 600°C
for 4 h ) and N (AOAC, 1984).
The pooled ruminal digesta samples were strained
through cheesecloth and the ruminal fluid centrifuged
at 26,000 x g for 15 min at 4°C. The supernatant was
decanted and analyzed for lactic acid (Pryce, 1969).
The time-interval ruminal digesta samples were
treated similarly to the pooled samples and the
supernatant divided into two parts. One part was
analyzed for ammonia N (NH3 N; Technicon Auto
Analyser 11, Industrial Method No. 334-74A, January
1976). The other part was hydrolyzed with 1.0 N
HzSO4 at 121°C for 60 min, to convert all of the added
soluble CH still remaining to glucose. This was
followed by neutralization with 1.0 N NaOH before
determining glucose (GOD-Perid lut, Boehringer
Mannheim GmbH, Mannheim, FRG), as a measure of
added soluble CH still remaining.
A portion of each pooled abomasal digesta sample
was freeze-dried and analyzed for OM and N by the
same methods used for the basal diet samples. The
pooled sample was also analyzed for purine (Zinn and
Owens, 1986) and Cr (determined by atomic absorp-
tion spectrophotometry after processing the samples
according to methods described by Cheong and Salt,
1968). The remaining portion of each sample was
strained through cheesecloth and centrifuged at 1,000
x g for 5 min at 4°C to remove protozoa and solid feed
particles. The resulting supernatant was centrifuged
at 26,000 x g for 15 min and the supernatant thus
obtained was analyzed for NH3 N by the same method
used for ruminal digesta samples, whereas the bac-
terial pellet was resuspended in distilled water and
similarly centrifuged. The supernatant was decanted
and the washed bacterial pellet was freeze-dried
before analysis for OM, N, and purine by the same
methods that were used for the pooled abomasal
digesta samples.
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Flows of dietary components from the rumen were
based on digesta flow, calculated by dividing Cr
release from the capsule in the rumen by Cr concen-
tration in the abomasal sample. Microbial N flow was
calculated as purine N flow divided by the ratio of
purine N:total N in microbial isolates (Zinn and
Owens, 1986).
Statistical Analysis. The number of repetitions
(experimental units) per treatment were eight (i.e.,
four sheep in each of Periods 1 and 2 1. All data except
pH, NH3, and soluble CH were analyzed using
multifactor analysis of variance procedures as im-
plemented in S T A T G W H I C S (Statistical Graphics
Corporation, STSC, Rockville, MD). Energy and N
supply pattern and experimental period were specified
as main effects. Residual mean square was used as the
error term. Individual treatment means were com-
pared using the multiple-comparison method of Tukey.
Data for pH, NH3, and soluble CH were analyzed
using GENSTAT (Lawes Agricultural Trust, 1987).
Energy and N supply pattern and experimental period
were regarded as whole plots and time after dosing as
subplots in a split-plot analysis. Residual mean square
was used as the error term.
Experiment 2
Animals and Management. A group of 16 South
African Mutton Merino wethers not previously used in
Exp. 1 (average BW = 56.1 kg; SD = 13.2) were
managed similarly to those in Exp. 1 except for diet
composition. The diet consisted of (gramskilogram of
DM) wheat straw (hammer-milled, 25-mm sieve),
881.5; fish meal 56; molasses, 50; dicalcium phos-
phate, 6; urea, 4; magnesium sulphate, 1.5; and
vitamin-mineral premix, 1. Salt was excluded, because
sufficient levels of sodium and chloride were consumed
via NaHC03 and NH&1 used to stabilize ruminal pH.
Magnesium sulphate was added to prevent any
induced magnesium deficiency that may have resulted
from high input of potassium (NRC, 1985) and was
also used to stabilize ruminal pH.
Experimental TYeatments. There were four ex-
perimental treatments (i.e., two energy supply pat-
terns and two N supply patterns in a 2 x 2 factorial ar-
rangement). Treatments consisted of adding the same
daily amount of soluble CH mixture (680 g of 6
maltose:2 dextrose:l maltotriose; African Products) as
energy source and N (32.6 g of NH4C1 and 22.8 g of
sodium caseinate) into the rumen of each animal,
according to one of four different patterns: Treatment
EPNP, one-half of the CH mixture plus all of the N as
two equal pulse-doses at 0800 and 2000, with the
remaining half of the CH mixture as a continuous
infusion (representing rapid synchronized energy and
N supply); Treatment EPNG, one-half of the CH
mixture as two equal pulse-doses at 0800 and 2000,
and the remaining half of the CH mixture plus the N
as a continuous infusion (representing unsyn-
 ENERGY-NITROGEN SYNCHRONIZATION IN THE RUMEN
chronized supply); Treatment EGNP, the CH mixture
as a continuous infusion and N as two equal pulse-
doses at 0800 and 2000 (representing unsynchronized
supply); and Treatment EGNG, both CH mixture and
N as continuous infusions (representing slow syn-
chronized energy and N supply).
A 4:3 (wt/wt) mixture of NaHC03:KHC03 was
supplied together with the CH mixture at a rate of 76
g/d and according to the same regimen as the CH
mixture t o help prevent the expected decline in
ruminal pH with high inputs of CH. Ammonium
chloride instead of urea was used as the NPN source
t o facilitate preventing metabolic alkalosis due to
feeding of high sodium bicarbonate and potassium
bicarbonate levels (Kronfeld, 1979). It was pointed
out by Belasco (1954) and Acord et al. (1967) that
NH4Cl was equivalent to urea as a source of NH3 N in
the rumen.
Initially, the protocol of this study was planned to
duplicate exactly that of Exp. 1, except for the higher
total nutrient input. However, it was decided to
modify the pulse-dosing by splitting the EP treat-
ments half and half as pulse-doses and continuous
infusions. The reason was to minimize the rapid
decline in pH associated with pulse-dosing. It was
evident from preliminary trials that higher levels of
buffer may result in metabolic disorders, but the
present level was insufficient to prevent ruminal pH
from declining to well below 6.0 when 680 g of soluble
CH was supplied as two, equally distributed, daily
pulse-doses.
As with Exp. 1 and based on the same assumptions,
diet and intraruminally supplied nutrients were
formulated to obtain a ratio of approximately 19 g of
RDN/kg of FOMR and a ratio of 3:l for NPN:amino
acid N.
Experimental Protocol. With a few exceptions, the
same protocol used in Exp. 1 was followed. Pulse-dose
volumes were 700 mL instead of 500 mL and rate of
infusion was 1,800 m u d instead of 1,300 m u d . The
amount of basal diet offered changed from an ad
libitum amount before intraruminal feeding to 1,200 g
of air-dried feed per day during intraruminal feeding.
Total feces and urine were collected daily from d 18 to
22 and bulked for each sheep. Feces samples were
dried to constant weight in a forced-draught oven at
55°C.
Sample Preparation and Analysis. All procedures
were similar to those described for Exp. 1. In addition
NDF (Robertson and Van Soest, 19811, ADF and ADL
(Goering and Van Soest, 1970) were determined in
feed, abomasal, and fecal samples. Flow of dietary
components from the rumen was calculated using ADL
as flow marker. Chromium oxide was originally mixed
with the feed as a digesta flow marker. However, Cr
values resulted in unacceptable flow data, as evi-
denced by higher ruminal than total tract digestibili-
ties of NDF and ADF for some treatments. Owens and
Hanson ( 1992) suggested that such illogical differ-
Downloaded from jas.fass.org by on March 31, 2011.
2519
'r
-3 -1 1 3 5 7
Time relative to dosing, h
Figure 1. Ruminal pH values in sheep receiving
similar amounts of soluble carbohydrates and nitrogen
intraruminally according to different patterns E P N P
(A),E P N G (e), E G N P ( + ) and E G N G (m) (Exp. 1).The
SE was .05 when comparing treatment means at a
specific time and .03 when comparing means at
different times within the same treatment. E = energy;
N = nitrogen; P = pulse; G = gradual (even).
ences in digestibility of fiber fractions are indicative of
errors made with the use of the digesta flow markers.
The nature of the problem in the present study was
not apparent, although it may be related to incomplete
mixing of the Cr with the feed. In this respect ADL
has the advantage that it forms an integral part of the
feed, and hence digesta, and was present in the basal
diet in relatively large quantities. Flow was calculated
by dividing ADL intake by ADL concentration of
abomasal digesta.
Statistical Analysis. Data were analyzed as for Exp.
1.
Results
Experiment 1
Ruminal pH and Lactic Acid. There was an
interaction between energy supply pattern, N supply
pattern, and time ( P < .001; Figure 1).Ruminal pH
for both EG treatments was relatively constant over
time (mean value 6.41, whereas that for the EP
treatments declined sharply after pulse-dosing to < 6.0
by 1 h and returned to near predosing levels by 3 h
after dosing. Ruminal lactic acid concentration was
higher ( P < .05) for EP than for EG treatments
(Table 1).
Ruminal Ammonia Nitrogen Concentration. Energy
and N supply patterns and time all had an effect ( P <
2520 HENNING ET AL.
Table 1. Ruminal lactic acid concentration and retention time of water in sheep receiving similar amounts of
soluble carbohydrates (energy) and nitrogen intraruminally according to different patterns (Exp. 1 and 2)

Carbohydrate-nitrogen supply patterna Significance of effect‘

Item EPNP EPNG EGNP EGNG SEM Energy Nitrogen

Exp. 1
Lactic acid concentration, mM 6.6 6.7 4.4 5.0 .86 <.05 NSC
Retention time of water, h 12.3 12.1 11.6 12.1 .81 NS NS
Exp. 2
Lactic acid concentration, mM 7.7x 7.OXY 5.OXY 3.9y .a1 <.001 NS
Retention time of water, h 13.9 12.4 12.8 12.5 .73 NS .15
aE = energy; N = nitrogen; P = pulse; G = gradual (even).
bEffect of energy x nitrogen interaction: P > .2 for all items.
‘NS = P > .2.
XJMeans within a row that do not have a common superscript differ ( P < ,051.

.001) on ruminal NH3 N concentration (Figure 2 ) . to just c 6.0 by 1 h after dosing but returned to > 6.0
There was also interaction between N supply pattern by 3 h after dosing. Nitrogen pulse-dosing resulted in
and time. Ruminal NH3 N levels remained relatively a greater decline ( P < .O 1) in pH than did the
constant and mostly higher when N source was corresponding NG treatments for some time after the
infused than when it was pulse-dosed. Supplying N as pulse. Lactic acid concentration was higher ( P < . O O l )
a pulse-dose resulted in a sharp increase in ruminal for E P than for EG treatments (Table 1).
NH3 N directly after dosing, followed by a rapid Ruminal Ammonia Nitrogen Concentration. There
decrease t o predosing levels by 5 h after dosing. was a n interaction ( P < .001) between N supply
Ruminally Soluble Carbohydrate Concentrations. pattern and time (Figure 5). The NG treatments
Energy supply pattern affected soluble CH concentra- resulted in a relatively constant ruminal NH3 N
tions in the rumen (Figure 3); a n interaction occurred concentration throughout the day. The NP treatments,
between energy supply pattern and time ( P < . O O l ) . conversely, caused a sudden fivefold increase in
For the EG treatments, appreciable levels of soluble
CH were present in the rumen at all times, whereas
for the E P treatments these were much lower (mostly
zero), except 1 h after dosing, when they were higher
( P < ,051 than for the EG treatments.
Intake, Flow, and Digestibility o f Organic Matter.
Microbial OM flow was higher ( P < .05) for EG than
for E P treatments (Table 2). Ruminal outflow of
dietary OM tended ( P = . 0 7 ) to be lower for the NP
than for the NG treatments. In accordance, true
digestibility of OM in the rumen was higher ( P < ,051
for NP than for NG treatments. There was no effect ( P
> . 2 ) of treatments on the ruminal retention time of
water (Table 1).Retention time of water was shorter
( P < .001) during than before the addition of water to
the rumen (Table 3 ) .
Microbia7 Growth Efficiency and Intake, Flow, and
Digestibility of Nitrogen. Total N, nonammonia N, and
microbial N outflow from the rumen ( P < .001), as
well as both apparent and true microbial growth
Time relative to dosing, h
efficiency (MOEFF; P < .05), were higher for EG
treatments than for E P treatments (Table 4). Neither
N supply pattern nor energy x N interaction had a n Figure 2. Ruminal ammonia N concentrations in
effect ( P > . 2 ) on these traits. sheep receiving similar amounts of soluble carbohy-
drates and nitrogen intraruminally according to differ-
Experiment 2 ent patterns EPNP (A), EPNG (.), EGNP ( + ) and EGNG
(H) (Exp. 1).The SE was .99 when comparing treatment
Rumina7 pH and Lactic Acid. Both energy and N means at a specific time and .81 when comparing
supply patterns showed interactions with time ( P < means at different times within the same treatment. E
.001; Figure 3 ) . After a pulse-dose of CH, pH declined = energy; N = nitrogen; P = pulse; G = gradual [even).

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 ENERGY-NITROGEN SYNCHRONIZATION IN THE RUMEN
a 5%
I
I Ruminally Soluble Carbohydrate Concentrations.
I
-3 -1 1 3
Time relative to dosing, h
Figure 3. Total soluble carbohydrate concentrations
in the rumen of sheep receiving similar amounts of
soluble carbohydrates and nitrogen intraruminally ac-
cording to different patterns EPNP (A),EPNG (e),
EGNP ( + ) and EGNG (B) (Exp. 1). The SE was 55.1
when comparing treatment means at a specific time and
37.7 when comparing means at different times within
the same treatment. E = energy; N = nitrogen; P =
pulse; G = gradual (even).
5.51
.I.
>-3 -1
I I
1
I
3 5
Tire relative to dosing, h
Figure 4. Ruminal pH values in sheep receiving
similar amounts of soluble carbohydrates and nitrogen
intraruminally according to different patterns EPNP
(A), EPNG (@I, EGNP ( + ) and EGNG (m) (Exp. 2). The
SE was .05 when comparing treatment means at a
specific time and .03 when comparing means at
different times within the same treatment. E = energy;
N = nitrogen; P = pulse; G = gradual (even).
Downloaded from jas.fass.org by on March 31, 2011.
252 1
ruminal NH3 N by 1 h after dosing, whereafter levels
gradually declined back to predosing levels, which
were reached by 9 h after dosing.
There was a n interaction ( P e .001) between the
effects of energy supply pattern and time on soluble
CH concentrations (Figure 6 ) . The EG treatments
resulted in relatively constant soluble CH levels,
whereas E P treatments produced comparatively lower
soluble CH levels, which increased drastically after
pulse dosing and then declined to prepulse levels by 7
h after pulse-dosing.
Intake, Flow, and Digestibility of Organic Matter.
Organic matter infused was slightly ( 5 9% ) higher for
EP than for EG treatments (Table 5 ) . This difference
5 7 was unintentional and was a n artifact of the infusion
apparatus. Microbial OM flow was 12% higher ( P =
.07) for NG than for NP treatments. Total tract OM
digestibility was 3% higher ( P < .05) for E P than for
EG treatments. True and apparent ruminal OM
digestibility showed the same tendency ( P > .2).
There were no differences ( P > . 2 ) in retention time of
water between treatments (Table 3). Treatments
resulted in larger increases in total water intake ( P <
.001) during the experimental period than before it
began.
Microbial Growth Efficiency and Intake, Flow, and
Digestibility of Nitrogen. Apparent and true MOE ?F
were 20% ( P = .16) and 14% ( P = . I S ) higher,
respectively, for EG than for E P treatments (Table 6 ) .
I L
7 -3 -1 1 3 5 7
Tile relative to dosing, h
Figure 5. Ruminal ammonia N concentrations in
sheep receiving similar amounts of soluble carbohy-
drates and nitrogen intraruminally according to differ-
ent patterns EPNP (A), EPNG ( @ ) , EGNP ( + ) and EGNG
[B) (Exp. 2). The SE was .67 when comparing treatment
means at a specific time and .48 when comparing
means at different times within the same treatment. E
= energy; N = nitrogen; P = pulse; G = gradual (even).
2522 HENNING ET AL.
Table 2. Intake, flow, and digestibility of organic matter in sheep receiving similar amounts of soluble
carbohydrates (energy) and nitrogen intraruminally according to different patterns (Exp. 1)

Carbohydrate-nitrogen supply patterna Significance of effectb

Item EPNP EPNG EGNP EGNG SEM Energy Nitrogen
Intake, gid
Feed 676 650 691 710 20.4 .10 NSC
Infusion 318 3 18 3 18 318 - - -
Total 994 968 1,009 1,028 - - -

Ruminal outflow, gid

Total 494x 541'y 539Xy 600y 22.3 .12 .10
Microbial 87'y 83x 94Y 93xy 2.9 <.05 NS
Dietary (apparent) 408 458 445 507 29.5 .16 .07
Ruminal digestibility, %
Apparent 50.4 44.5 46.4 41.7 1.99 NS .06
True 59.2' 53.0q 55.8'y 50.7y 1.89 NS <.05
aE = energy; N = nitrogen; P = pulse; G = gradual (even).
bEffect of energy x nitrogen interaction: P > .2 for all items.
'NS = P > .2.
XJMeans within a row that do not have a common superscript differ ( P c .05).

The NP treatments resulted in a 25% higher ( P = .2)
urinary N excretion, a 54% higher ( P < .001) ruminal
outflow of NH3 N, a 47% lower ( P = .09) N retention,
an 11%lower ( P= . 0 7 ) microbial N flow, and a 10%
lower ( P = ,19) true MOEFF than the NG treatments.
Intake, FZow, and DigestibiZity o f CeZZ WuIZ Consti-
tuents. There were no differences ( P > .2) between
treatments in any of these traits (Table 7). Ruminal
digestion of NDF and ADF, expressed as a percentage
of total tract digestion, averaged 72 and 7996, respec-
tively.

Discussion
.rl

"r
a
rl
W
Effectiveness o f Buffer

From the pH data presented in Figure 4,it seems

200 - that the use of a buffer, in conjunction with giving
-3 -1 1 3 5 7
Time relative to dosing, h
Figure 6. Total soluble carbohydrate concentrations
in the rumen of sheep receiving similar amounts of
soluble carbohydrates and nitrogen intraruminally ac-
cording to different patterns EPNP (A),EPNG (a),
EGNP ( + ) and EGNG (m) (Exp. 2). The SE was 23.4
when comparing treatment means at a specific time and
17.9 when comparing means at different times within
the same treatment. E = energy; N = nitrogen; P =
pulse; G = gradual (even).
only half the soluble CH in the EP treatments as a
pulse-dose, was successful in preventing a major
decline in ruminal pH in Exp. 2. The decline in pH for
EP treatments to between 5.6 and 5.9 at 1 h after
dosing is in agreement with a pH of 5.8 observed by
Chamberlain et al. (1985) after they dosed silage-fed
sheep with 190 g of sucrose plus 50 g of NaHC03. The
present data, furthermore, suggest that adding high
levels of buffer to fast-fermenting diets may not
prevent pH from falling to < 6.0 for at least part of the
time. This is confirmed by the results of Kovacik et al.
(19861, who allowed sheep ad libitum access to a 50:
50 c0ncentrate:roughage mixture but with only one
90-min period per day in which to consume it. They
found that even with 4.5% NaHC03 included in the
diet, pH remained < 6.0 for up to 10 h.
The lower ( P < .01) pH values for the N P than for
the NG in Exp. 2 reflect the fact that NP treatments
included the addition of a 2.3% (wt/wt) NH4C1
solution, with a pH of approximately 5.5, as a pulse-
dose to the rumen.

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 ENERGY-NITROGEN SYNCHRONIZATION IN THE RUMEN 2523
Table 3. Mean intake and ruminal retention time of water in sheep before and during intraruminal supply
of carbohydrate-nitrogen solutions (Exp. 1 and 2)

Before During
Item infusioddosing infusioddosing SEAM Significance
Exp. 1
Water intake, gld
Voluntary 3,230 2,002 124.9 <.001
Infused/dosed 0 2,300 - -
Total 3,230 4,302 - -
Retention time of water, h 17.1 12.0 .77 <.001
Exp. 2
Water intake, gld
Voluntary 2,187 2,275 283.3 NSa
Infuseddosed 0 3,200 - -
Total 2,187 5,475 - -
Retention time of water. h 16.6 12.8 .93 <.001

Effect of Energy and Nitrogen Synchronization
on Rumen Characteristics and
Microbial Growth
Experiment 1 . Degree of energy N synchronization
had no significant effect on microbial N flow or
MOEFF, as is evidenced by the absence of any energy
x N interactions ( P > . 2j for these traits (Table 4 j.
This is in agreement with results obtained in vitro
(Henning et al., 1991; Newbold and Rust, 1992). It
may be argued that, although the time pattern of
ruminal N H 3 N concentration differed between treat-
ments, the actual concentration was never limiting
relative to the microbial growth resulting from the
various energy supply patterns. From Figure 2, it can
be seen that, whereas NH3 N for the NG treatments
remained >7 mM at all times, that for treatments
EPNP and EGNP, respectively, declined to between
4.6 and 5.2 mM and 3.3 and 4.3 mM for 65% of the
time. These values are, however, still at or above the
mean concentration of 3.5 mM generally considered
sufficient for maximum microbial growth (Satter and
Slyter, 1974; ARC, 1984). The argument is further
supported by the fact that microbial flow and MOEFF
did not differ between the NG and NP treatments
(Table 4).
The data presented in Figure 2 suggest that the N
supplied as pulse-doses in the NP treatments was
"depleted" by 5 h after dosing. Ruminal NH3 N
concentration nevertheless remained above approxi-
mately 3.5 mM until the next dosing (Figure 2).
Although N released from the basal diet would have

Table 4. Intake and flow of nitrogen and microbial growth efficiency in sheep receiving similar amounts of
soluble carbohydrate (energy) and nitrogen intraruminally according to different patterns (Exp. 1)

Carbohydrate-nitrogen supply patterna Significance of effect'

Item EPNP EPNG EGNP EGNG SEM Energy Nitrogen
Intake, gld
Feed 8.5 8.2 8.6 8.9 .25 .ll NSC
Infusion 5.7 5.7 5.7 5.7 - - -
Total 14.2 13.9 14.3 14.6 - __ -

Ruminal outflow, g/d

Total 16.2x 15.gX 19.5y 19.0s .74 <.001 NS
Ammonia 1.4 1.2 1.3 1.1 .10 .14 .17
Nonammonia 14.BX 14.6' 18.2Y 17.9y .69 <.001 NS
Microbial 7.3xy 7.1' 8.2xy 8.g .33 <.001 NS
Dietary (apparent 1 7.5x 7.5x 10.1y 9.5xy .65 <.01 NS
Microbial growth efficiency, g of microbial Nlkg of organic matter fermented
Apparent 15.0 17.3 18.7 20.3 1.77 <.05 NS
True 12.6 14.2 15.0 16.3 1.12 <.05 NS
___ ~ ~ ~

aE = energy; N = nitrogen; P = pulse; G = gradual (even).

'Effect of energy x nitrogen interaction: P > .2 for all items.
'NS = P > .2.
XJ'Means within a row that do not have a common superscript differ ( P < ,051.

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2524 HENNING ET AL.
Table 5. Intake, flow, and digestibility of organic matter in sheep receiving similar amounts of soluble
carbohydrates (energy) and nitrogen intraruminally according to different patterns (Exp. 2)

Carbohydrate-nitrogen supply patterna Significance of effect

E x N
Item EPNP EPNG EGNP EGNG SEM Energy Nitrogen interaction
Intake, gld
Feed 718 838 783 786 48.1
Infusion 590 590 560 560 -
Total 1,308 1,428 1,343 1,346 -

Ruminal outflow, gid

Total 642 730 696 758 50.4 NS .11 NS
Microbial 159 182 169 186 12.4 NS .07 NS
Dietary ( a p p a r e n t ) 483 548 527 572 41.3 NS .18 NS
Ruminal digestibility, %
Apparent 50.8 48.9 48.1 44.5 2.87 NS NS NS
True 62.9 61.7 60.8 58.2 2.37 NS NS NS
Fecal output, gid 402 465 463 452 27.1 NS NS .18
Total tract apparent digestibility, % 69.4 67.4 65.6 66.7 1.13 <.05 NS .19
aE = energy; N nitrogen; P = pulse; G = gradual (even).
bNS = P > .2.

made some contribution in this respect, it is likely
that recycling of N from blood urea, via saliva or
through the ruminal epithelium, played a n important
role (Preston and Leng, 1987). This recycling can be
considerable, as shown by Potthast et al. (19771, who
calculated that 9.5 g of urea N daily entered the
rumen of sheep given a N-free basal diet plus 300 g of
sucrose per day. Recycling of urea to the rumen seems
to be positively related to the amount of soluble
carbohydrate entering the rumen or to the rate of OM
fermentation in the rumen (Houpt, 1970; Kennedy
and Milligan, 1980).
The plasma urea pool may be considered as a
“reserve N pool.” During periods of excessive N
availability in the rumen, NH3 N is absorbed and
appears as urea in the plasma urea pool. This N may
then be recycled back to the rumen during subsequent
periods of N shortage. Nitrogen recycling may explain
in the present study the lack of response to better
synchronization of energy and N supply t o the rumen.

Table 6. Intake, flow, and retention of nitrogen and microbial growth efficiency in sheep receiving similar
amounts of soluble carbohydrate (energy) and nitrogen intraruminally according to different patterns (Exp. 2)

Carbohydrate-nitrogen supply patterna Significance of effect

Item EPNP EPNG EGNP EGNG SEM Energy Nitrogen E x N interaction
Intake, gld
Feed 9.5 11.0 10.3 10.3 .56 NSb NS .14
Infusion 10.8 10.8 10.3 10.3 - - - -
Total 20.3 21.8 20.6 20.6 .56 NS NS .14
Ruminal outflow, gld
Total 25.2 27.1 27.4 28.0 1.99 NS NS NS
Ammonia .64x .55x .88Y .44x ,061 NS <.001 <.01
Nonammonia 24.5 26.6 26.5 27.5 1.95 NS NS NS
Microbial 14.1 16.2 15.0 16.5 1.10 NS .07 NS
Dietary (apparent) 10.4 10.4 11.4 11.0 1.07 NS NS NS
Microbial growth efficiency, g of microbial N k g of OM fermented
Apparent 22.1 23.3 24.7 29.7 2.90 .16 .20 NS
True 17.4 18.4 19.0 21.8 1.72 .18 .19 NS
Fecal output, gid 8.1X 9.1XY 9.7y 9.1XY .41 .07 NS <.05
Urinary output, g/d 8.4 7.1 9.3 7.1 1.13 NS .20 NS
Retention, gid 3.7 5.6 1.7 4.5 1.16 .20 .09 NS
aE = energy; N = nitrogen; P = pulse; G = gradual (even).
bNS = P > .2.
xJ’Means within a row that do not have a common superscript differ ( P < ,051.

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 ENERGY-NITROGEN SYNCHRONIZATION IN THE RUMEN 2525
Table 7. Intake, flow, and digestibility of neutral detergent fiber (NDF) and acid detergent fiber (ADF) in
sheep receiving similar amounts of soluble carbohydrates (energy) and nitrogen intraruminally
according to different patterns (Exp. 2)

Carbohydrate-nitrogen supply patterna

Significance of
Item EPNP EPNG EGNP EGNG SEM effect. nitroeen’
Intake, gld
NDF 555 647 605 608 37.2 .20
ADF 330 383 362 359 22.1 NSC
Ruminal outflow, g/d
NDF 381 427 405 429 35.3 NS
ADF 208 243 2 18 238 17.8 .13
Ruminal digestibility, r0
NDF 29.9 34.1 32.8 31.1 4.19 NS
ADF 35.9 36.6 39.4 35.1 3.16 NS
Fecal output, gid
NDF 307 345 345 342 22.3 NS
ADF 177 189 196 199 13.2 NS
Total tract digestibility, %
NDF 44.5 46.5 42.4 43.9 2.17 NS
ADF 46.0 50.4 45.4 44.8 2.07 .14
aE = energy; N = nitrogen; P = pulse; G = gradual (even).
bEffect of energy and energy x nitrogen interaction: P > .2 for all items.
ws = P > .2.

This may not hold true at higher levels of energy and
N input, at which point excessive absorbed N may be
excreted in urine and recirculation of N to the rumen
at a later stage may be insufficient to meet the needs
dictated by the higher energy supply. Cocimano and
Leng (1967) showed that high NPN input into the
rumen, without a concomitant utilization of the
resulting N H 3 N, led t o more NH3 N being absorbed
from the rumen into the blood, resulting in increased
plasma urea concentrations and subsequent increased
urinary excretion of urea. Inefficient ruminal utiliza-
tion of NH3 N would thus be reflected in increased
urinary N excretion.
Experiment 2. Similar to Exp. 1,better synchroniza-
tion of energy and N supply to the rumen at the
higher level of nutrition gave no significant improve-
ment ( P > .2 for energy x N interaction) in microbial
protein flow or MOEFF (Table 6). The higher N H 3 N
outflow and urinary N excretion and the lower N
retention, microbial N flow, and MOEFF for NP than
for NG treatments (Table 6 ) support the abovemen-
tioned notion that high inputs of readily available N
into the rumen may result in inefficient NH3 N
utilization for microbial growth. A more synchronized
energy supply did not seem to improve the situation,
as is evidenced by a comparison of microbial N flow
and MOEFF values between EPNP and EGNP treat-
ments in Table 6. In contrast, the lower ( P< ..05) NH3
N outflow from the rumen for treatment EPNP than
for treatment EGNP does suggest some advantage to
better energy and N synchronization with a fast N
supply.
Ruminal N H 3 N concentration for treatment EGNP
was approximately 2.5 mM for approximately 30% of
the time (Figure 4). This is lower than the mean
concentration of 3.5 mM generally considered to be
sufficient for maximum microbial growth (Satter and
Slyter, 1974; ARC, 1984) and suggests the possibility
of insufficient recirculation of N H 3 to the rumen
during the later stages of fermentation. This notion is
further supported by the lower microbial N flow,
MOEFF, and N retention for treatment EGNP than
for EGNG.
The average ratio of N:energy for all treatments in
the present study was approximately 19 g of RDNkg
of FOMR. This corresponds to the value suggested as
ideal for ruminal microbial growth by Newbold and
Rust (1990), based on their own results and those
presented by Czerkawski (1986). The need to balance
daily inputs of ruminally available energy and N is
well recognized (Stern, 1986; Nocek and Russell,
1988). From the foregoing discussion, it may be
postulated that, provided the overall balance between
ruminally available N and ruminally fermentable OM
in the daily intake is sufficient, there is no further
advantage in synchronizing the release of energy and
N in the rumen over the shorter term.
Salter et al. (1983) added tapioca or glucose ( a s
energy source) and urea ( a s N source) t o the rumens
of straw-fed steers according to various degrees of
synchronization. Their results support our conclusion
that no advantage is gained from diets and feeding
regimens that aim at a high degree of energy and N
synchronization in the rumen. Studies with silage-
based diets apparently yielded contradictory results.
The synchronization of N and energy release from
silage is often considered to be poor (Rooke et al.,

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2526 HENNING ET AL.
1987). Dietary treatments aimed at rectifying this
problem gave positive results, which were interpreted
as indicating a response in microbial growth to energy
and N synchronization in the rumen (Rooke et al.,
1987; Dawson et al., 1988; Rooke and Armstrong,
1989). None of these studies was, however, designed
to critically test the “energy and N synchronization”
idea per se and other factors may have played a role.
There are a number of recent studies specifically
aimed at determining the effect of synchronizing the
supply of protein and energy to the rumen on
microbial protein synthesis and animal production
(Casper et al., 1990; Herrera-Saldana et al., 1990;
Matras et al., 1991). In all these studies, ingredients
with different rates of starch and N fermentation in
the rumen were used t o formulate diets supposedly
varying in degree of synchronization of energy and N
released in the rumen. Some studies did show
increased microbial flow and efficiency of growth for
“better-synchronized” treatments (Herrera-Saldana et
al., 1990), whereas others showed no response
(Casper et al., 1990). Treatments in these studies,
however, differed in ingredient composition, so degree
of energy and N synchronization may have been
confounded by other ruminal effects inherent to
specific ingredients. Furthermore, using different in-
gredients, each with a different fermentation rate,
would also cause differences between treatments in
respect to the ratios and total amounts of energy and
N released into the rumen. It is quite likely that
responses in microbial growth ascribed to improved
energy and N synchronization may instead have been
the result of an improvement in overall balance of
energy and N supply t o the rumen.
Effect o f Energy Supply Pattern on Ruminal
Characteristics and Microbial Growth
The EP treatments in Exp. 1 resulted in lower
microbial N flows ( P < .OO 1) and MOEFF ( P < . 0 5 )
than the respective EG treatments (Table 4). Effi-
ciency of microbial growth in Exp. 2 showed a similar
trend ( P = .16) (Table 6). This may be ascribed t o the
abundance of rapidly fermentable CH in the rumen
directly after dosing for the EP treatments (Figures 3
and 61, which in turn would have caused the observed
increase in ruminal lactic acid concentration (Table
1) (Dawson and Allison, 1989). Although lactic acid
as such is not detrimental to MOEFF (Jaakkola and
Huhtanen, 19891, lactate production implies a lower
energy yield per unit of carbohydrate fermented
(Strobel and Russell, 1986). Energetic uncoupling of
microbial growth was also found to occur at a pH of 5
6.0 (Russell and Dombrowski, 1980; Strobel and
Russell, 1986). The combined effect of these events
would be a decrease in MOEFF and microbial flow.
The situation may have been further aggravated by
the relatively lower soluble CH availabilities that
coincide with the return of ruminal pH to levels
similar to those before the pulse-dose was given.
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(Compare Figures 1 and 4 with 3 and 6, respectively.)
The fact that in the present in vivo study energy
gradually supplied to the rumen resulted in higher,
more efficient microbial production than the same
amount of energy supplied according to a rapid
pattern seems t o conflict with results obtained using
similar treatments in vitro (Henning et al., 1991).
This, however, may be explained by the fact that in
the in vitro study, pH remained well above 6.0 for
most of the incubation time for all treatments under
consideration and never declined below 6.0. Further-
more, the MOEFF values referred t o were only
calculated up to the point at which the energy-yielding
substrate was depleted. This is in contrast to the
periods of low pH and low levels of readily fermentable
substrate encountered in the present study. The
present results are also in contrast to those of Salter
et al. (19831, who added starch or glucose to the
rumens of straw-fed steers, either as single doses or as
three, equally divided doses at 2-h intervals. They
found no significant differences in MOEFF between
the various supply patterns, in spite of the fact that
the pulse-dose resulted in the decline of ruminal pH to
approximately 5.5, whereas pH remained well above
6.0 for treatments in which CH was more evenly
supplied. Results of Casper and Schingoethe ( 1989)
and Casper et al. (1990) with diets that differed in
starch degradation rates also suggest that there is no
difference between faster or slower release of energy
in the rumen. In contrast to these findings, Herrera-
Saldana et al. (1990) and Matras et al. (1991), using
complete diets varying in the ruminal degradation
rate of ingredients, found improved microbial protein
flow and efficiency of growth with the faster-ferment-
ing diets. Again, it must be emphasized that treat-
ments in these studies differed not only in energy
supply pattern but also in ingredient composition.
Thus, the effect of energy supply pattern may have
been confounded with other ruminal effects inherent
to the specific ingredients. The use of different
ingredients with different fermentation rates also
implies treatment differences in the total amount of
energy released in the rumen.
Fiber Digestion (Experiment 2)
The absence of any significant treatment effects on
ruminal or whole-tract fiber digestibility (Table 7 ) is
similar to the findings of Herrera-Saldana and Huber
(1989) when they fed diets that varied in protein and
starch degradation to dairy cows. The NDF and ADF
digestibility values are in the expected range for high-
concentrate diets, in which the presence of CH and the
lower ruminal pH may have had a negative effect on
fiber digestibility (McCarthy et al., 1989). Ruminal
digestibility of NDF and ADF was on average 72 and
79% of the respective total-tract digestibilities. This
compares well with corresponding values of 72 and
76% found by Cecava et al. (1990).
Fahey and Jung (1983) reviewed the use of lignin
as a digesta flow marker. They pointed out that many
 ENERGY-NITROGEN SYNCHRONIZATION IN THE RUMEN
investigators reported lignin to be indigestible; it is for
this reason that it is considered a suitable marker.
However, they also cite several reports indicating that
various amounts of lignin might be digested in the
ruminant digestive tract. Fahey and Jung (1983)
stressed that, in the light of data suggesting possible
degradation and solubilization of lignin in the diges-
tive tract, lignin should be used as a marker with
caution, because incomplete recovery will result in
underestimation of nutrient digestibility. There was,
however, no net loss of ADL between intake and feces
in the present study. Taking into account our discus-
sion of NDF and ADF digestibilities in the present and
other studies, as well as the data that are summarized
by Church (1975) on the ratio of hindgut:foregut
fermentation of fiber, we suggest that there was no
serious over- or underprediction of flow at the
abomasum.
Intake and Retention Time of Water
In Exp. 1 sheep drank less water to compensate for
that being supplied intraruminally (Table 3). In Exp.
2 voluntary water intake remained essentially the
same during the daily infusion of 3.2 L of water as
before (Table 3 ) . The fact that infusion of water did
not cause voluntary water consumption t o decline, as
was the case in Exp. 1, probably reflects an increased
water requirement due to the relatively high levels of
buffer fed in Exp. 2. The total water intake of between
4.3 and 5.5 Wd during experimental periods could be
considered to be within physiological limits.
The shorter retention time of water during infusion
reflects increased water intake, as well as the effect of
buffers (Stokes, 1983) in Exp. 2. Retention time of
ruminal fluid is known to affect MOEFF (Harrison
and McAllan, 1980). This would not have confounded
matters in the present study because there were no
significant differences in retention time of water
between treatments (Table 1).
Implications
The present results suggest that merely improving
the degree of synchronization between energy and N
release rates in the rumen does not increase microbial
yield. It seems that manipulation of energy and N
fermentation in the rumen to increase microbial yield
should first be aimed at obtaining the most even
ruminal energy supply pattern possible with a particu-
lar dietary regimen. Second, it should be ensured that
the total daily amount of ruminally available N is
sufficient for the total amount of energy expected to be
released in the rumen per day. There may then be
some further advantage in also ensuring a more even
N supply pattern.
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2527
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