1439
INTRODUCTION
Insufficient supply of protein to dairy cows intestines
for digestion and absorption is one of the major problems
for dairy production under tropical conditions (Leng,
1982b). To meet the protein demands of lactating cows, the
flow of microbial protein should be maximized prior to
supplementing the bypass protein. Efficiency of substrate
utilization by rumen microbes involves a strong interaction
between the carbohydrate and protein fractions in the diet
(Oldham, 1984). Rate of starch digestion in the rumen is the
major factor controlling the energy available for microbial
growth (Oldham, 1984), while an adequate supply of
nitrogenous sources (ammonia, peptides and amino acids)
in the rumen will increase microbial growth efficiency
(Leng, 1982b). Therefore the substitution of rumen
undegradable protein (RUP) for rumen degradable protein
(RDP) can reduce the microbial protein entering the host
intestines, which is presumably due to the lack of supply of
peptides and amino acids to the rumen microbes (Clark et
al., 1992).
Ammonia is the primary nitrogen source for rumen
microbes (Bryant and Robinson, 1962). Microbial protein
can be mostly synthesized from the nitrogen of RDP,
passing through the ammonia pool in the rumen (Nolan,
1993). A number of in vivo studies (Redman et al., 1980;
Cruz Soto et al., 1994; Fujimaki et al., 1994) indicate that
peptides and amino acids are not limited for microbial
growth in the rumen. However, the outcomes of the balance
between RDP and RUP are dependent on interactions
among rumen microbes. Changes in availability of
nitrogenous substrates which may influence any change in
interactions among rumen microbes were examined in the
rumen of dairy cows fed TMR containing various levels of
RUP from formalin treated soybean (FSBM).
MATERIALS AND METHODS
Four Holstein×indigenous (93.75×6.25) non-lactating
dairy cows, weighing 460-480 kg with permanent rumen
canulas were held in individual pens and randomly
allocated to dietary treatments according a 4×4 Latin square
design with 21 d periods.
Dietary treatments consisted of 0, 6.1, 12.1 and 18.2 g
as FSBM (37.5 ml of 37% formaldehyde/100 g of soy
protein). Paragrass hay was made from perennial paragrass.
The paragrass was cut, chopped into 2-3 cm length and sun
dried for total mixed rations (TMR). Diets were
isonitrogenous and isocaloric as shown in Table 1.
Experimental diets were offered ad-libitum at 08:00 and
19:00 h and were sampled weekly and bulked for later
analysis of chemical compositions. Within the 21 d
Effects of Formalin Treated Soy Bean as a Source of Rumen Undegradable
Protein on Rumen Functions of Non-lactating Dairy Cows on
Concentrate Based-diets**
J. Kanjanapruthipong*, C. Vajrabukka and S. Sindhuvanich
Department of Animal Sciences, Kasetsart University, Kampaengsaen, Nakornpathom 73140, Thailand
ABSTRACT : An objective of this study was to determine the effects of increasing contents of rumen undegradable protein (RUP)
from formalin treated soy bean (FSBM) on rumen functions. Four rumen canulated non-lactating cows were randomly allocated to total
mixed rations (TMR) containing different proportions of soy bean meal (SBM) and FSBM. Of rumen fermentation characteristics,
concentrations of ruminal fluid ammonia and molar proportions of isoacids decreased with increasing contents of RUP in diets (p<0.01).
The animals on TMR containing only SBM gained less weight and had smaller rumen volume than those on TMR containing RUP from
FSBM (p<0.05). Organic matter and neutral detergent fiber digestibility in sacco were not different (p>0.05). The density of protozoa
particularly small Entodinium sp. in ruminal fluid was higher in animal fed TMR containing SBM:FSBM (34:66) and FSBM than those
fed TMR containing SBM:FSBM (66:34) and SBM (p<0.01). Total viable count, and net microbial protein synthesis as indicated by
purine derivatives in urine increased with increasing contents of RUP from FSBM (p<0.01). It can be concluded that a reduction in net
microbial protein synthesis in the rumen with increasing contents of RUP in the diet can be due to the reduction of preformed protein
available for microbial growth as well as an increased turnover rate of microbial cells by predatory activity of protozoa. (Asian-Aust. J.
Anim. Sci. 2002. Vol 15, No. 10 : 1439-1444)
Key Words : Rumen Undegradable Protein, Rumen Functions
** Supported by National Center for Genetic Engineering and
Biotechnology, Bangkok, Thailand.
* Address reprint request to J. Kanjanapruthipong. Dairy
Research and Development Center, Kasetsart University,
Kampaengsaen, Nakornpathom 73140, Thailand. Tel: +66-34-
351033, Fax: +66-34-351892, E-mail: agrjck@nontri.ku.ac.th
Received January 10, 2002; Accepted May 13, 20021440 KANJANAPRUTHIPONG ET AL.
experimental periods, the first 16 d was regarded as an
adaptation period and within the last 5 d, intensive sampling
was undertaken. On day 17, ruminal fluid was sampled via
a probe covered with a double layers of nylon stocking
material for total viable and cellulolytic bacteria counts
prior to incubation of nylon bags. Digestibilities of organic
matter (OM), neutral detergent fiber (NDF) and crude
protein (CP) of the diet was assessed. A nylon bag
containing 10-12 pieces of rice straw samples was
suspended for 24 h in the rumen to allow fungi to colonize
the rice straw blades and produce sporangia. On day 18, CrEDTA (I mg Cr/kg BW) used as a ruminal fluid marker was
injected into the rumen at 05:00 h. From 3-27 h after
injection, 10 samples of ruminal fluid were taken
periodically via the rumen canula with a probe covered with
the stocking material. Prior to acidification, samples were
removed to a vial for enumeration of protozoa and for
measurement of pH. The rest was acidified for later analysis
of Cr, VFA and NH3-N. On days 19-21, daily urine voided
by individual animal was collected into a container with
2 litres of 2% (V/V) CH3COOH and 1% (V/V) H2SO4. The
urine sample was diluted by 3-4 times just after collection
and then stored at -20°C. Equal portions of the daily urine
samples from each animal from each day were pooled prior
to analysis of purine derivatives.
Crude protein, ether extract (EE), DM, OM and ash
contents of the experimental diets were determined
according to the AOAC (1980). Neutral detergent fiber
(NDF) was measured following the method of Van Soest et
al. (1991).
Fresh ruminal fluid (0.1 ml) was used as inoculum and
serially diluted in a bicarbonate buffer containing L-a
cystine –HCL as a reducing agent. Medium 98-5 agar roll
tubes (Bryant and Robinson, 1961) and cellulose broth
tubes (Halliwell and Bryant, 1963) were used for the
determination of the densities of total viable and cellulolytic
bacteria (most probable numbers) in ruminal fluid.
Enumeration of fungal sporangia followed the technique
that recommended by Bauchop (1979). The rice straw
leaves were removed from the nylon bags after 24 h
incubation in the rumen and then dipped in 4% formal
saline. The leaves were randomly sampled and stained with
lactophenol cotton blue (Gurr, 1965) for 2 min and then
washed with deionized water to remove the excess stain.
Sporangial counts were made under a light microscope. The
technique of enumeration of protozoa was similar to that
recommended by Bird and Leng (1984). Protozoa were
counted and also classified into three groups, namely small
and large Entodinium sp. and Holotrich sp. under a light
microscope.
Prior to analysis of Cr, VFA and NH3-N, the acidified
sample was thawed and centrifuged at 3,000 g and 4°C for
10 min. The supernatant was kept for analysis. The
concentration of Cr was measured according to the method
of Downes and McDonald (1964). The concentration and
molar proportions of VFA were determined according the
method of Erwin et al. (1961). Iso-caproic acid was used as
an internal standard (Geissler et al., 1976). The
concentration of NH3-N was analyzed according the method
of Weatherburn (1967).
Allantoin in urine was analyzed according to the method
of Borchers (1977) and uric acid was measured following
the method described by Fujihara et al. (1987).
The statistical significance of the data was analyzed by
SAS (1989). The difference between treatments means was
assessed by the Least squares means.
RESULTS
Dietary treatments were isonitrogenous and isocaloric
and contained similar levels of nutrients. Values of nutrient
composition on a DM basis for TMR were as follows: CP,
15.6%, EE, 2.3%; NDF, 30%, TNFC, 39.4% and ash, 9.9%.
Effects of TMR containing formalin treated soy bean
(FSBM) on dry matter intake (DMI) and average daily gain
(ADG) were shown in Table 2. Voluntary intake was not
influenced by the contents of rumen undegradable protein
(RUP) from FSBM in the diet (p>0.05). A significant
increase in ADG was observed in the animals fed TMR
containing RUP from FSBM compared with those fed TMR
containing only SBM (p<0.05).
Effects of TMR containing FSBM on ruminal
Table 1. Feed ingredients of total mixed rations (TMR)
TMR containing
Ingredients
SBM FSBM-6.1 FSBM-12.1 FSBM-18.2
Soy bean meal (SBM) 18.2 12.1 6.1 -
Formalin treated soy bean meal (FSBM) - 6.1 12.1 18.2
Extracted rice bran 14.7 14.7 14.7 14.7
Cassava chips 28 28 28 28
Molasses 5.6 5.6 5.6 5.6
Urea 1.1 1.1 1.1 1.1
Minerals+vitamins 2.4 2.4 2.4 2.4
Paragrass hay 30 30 30 30
Total 100 100 100 100 UNDEGRADABLE PROTEIN AND RUMEN FUNCTIONS 1441
fermentation characteristics were shown in Table 3. The pH
of ruminal fluid was not influenced by dietary treatments
(p>0.05). Concentrations of ruminal fluid ammonia
(NH3-N) were decreased with increasing contents of RUP
form FSBM in the diet (p<0.01). Of the pattern of VFA in
ruminal fluid, only isoacids were decreasing with increasing
contents of RUP from FSBM in the diet (p<0.01).
Effects of TMR containing FSBM on the kinetics of
ruminal fluid and 24 h in sacco OM, NDF and CP
digestibilities were shown in Table 4. Rumen volume in the
animals fed TMR containing RUP from FSBM was larger
than those fed TMR containing only SBM (p<0.05).
Outflow rate and turnover rate of ruminal fluid were
affected by the different contents of RUP from FSBM in the
diet (p<0.01). The 24 h in sacco (OM) and (NDF)
digestibility was not influenced by dietary treatments
(p>0.05). The 24 h in sacco CP digestibility decreased with
increasing contents of RUP from FSBM (p<0.01).
Effects of TMR containing FSBM on microbial
ecosystem in the rumen and net microbial protein synthesis
as indicated by purine derivatives in urine were shown in
Table 5. Of microbial ecosystem in the rumen, the
populations of total viable bacteria (p<0.05) and protozoa
particularly Entodinium sp. (p<0.01) were affected by the
contents of RUP from FSBM in the diet. Net microbial
protein synthesis in the rumen as indicated by purine
derivatives in urine was decreasing with increasing contents
of RUP from FSBM in the diet (p<0.05 and p<0.01).
DISCUSSION
Rumen undegradable protein (RUP) is one of the
limiting factors affecting milk yield and its composition and
ovarian functions during early lactation of dairy cows in the
tropics (Kanjanapruthipong and Buatong, 2002). Changes
in availability of nitrogenous substrates will result in a
change in microbial ecology in the rumen due primarily to
differences in the capabilities of substrate assimilation
among microbial species. The outcomes of the level and
balance of RDP and RUP in the rumen are dependent on
interaction between bacteria, protozoa and fungi in the
rumen. Changes in these interactions can result in changes
in either the specific growth rate of microbes or the turnover
of microbial cells within the rumen as implied by a
changing protozoal density in ruminal fluid. The
contribution of the microbial cells that leave the rumen was
assumed to follow closely the relative changes in purine
derivatives excreted in urine (Chen and Gomes, 1992).
Table 2. Effects of total mixed rations (TMR) containing
formalin treated soy bean (FSBM) on dry matter intake (DMI)
and average daily gain (ADG)
TMR containing
Item
SBM FSBM-6.1 FSBM-12.1 FSBM-18.2
S.E
DMI
- kg/d 13.4 13.0 13.8 13.3 0.46
- %BW 3.42 3.38 3.52 3.38 0.12
ADG, kg 0.643
1
0.875
2
0.964
2
0.946
2
0.19
1,2
Mean within a row without a common superscript letter differ (p<0.05).
Table 3. Effects of total mixed rations (TMR) containing formalin treated soy bean (FSBM) on ruminal fermentation characteristics
TMR containing
Item
SBM FSBM-6.1 FSBM-12.1 FSBM-18.2
S.E
pH 6.38 6.29 6.15 6.25 0.19
HN3
-N, mgN/l 221.4
a
165.4
b
154.0
c
129.3
d
7.61
Acetate, % 56.0 56.1 56.5 57.0 0.79
Propionate, % 28.0 28.6 28.4 28.7 0.62
Butyrate, % 10.7 10.6 11.0 10.8 0.15
Isobutyrate, % 1.8
a
1.6
b
1.3
c
0.9
d
0.06
Valerate, % 1.4 1.2 1.3 1.4 0.03
Isovalerate, % 2.1
a
1.9
b
1.5
c
1.3
d
0.04
Total VFA, µm/ml 139.0 137.8 138.2 136.9 3.2
a,b
Mean within a row without a common superscript number and letter differ (p<0.01).
Table 4. Effects of total mixed rations (TMR) containing formalin treated soy bean (FSBM) on the kinetics of ruminal fluid and 24 h
in sacco digestibility
TMR containing
Item
SBM FSBM-6.1 FSBM-12.1 FSBM-18.2
S.E
Kinetics of ruminal fluid
Rumen volume, l 60.0
1
62.2
2
63.9
2
63.9
2
1.6
Outflow rate, l/d 101.1
3,c
95.1
2,a,b
93.7
1,2,a,b
91.1
1,a
3.7
Turnover rate, /d 1.58
2,b
1.59
2,b
1.50
1,2,a,b
1.44
1,a
0.12
24 h In sacco digestibility, %
Organic matter 82.2 81.6 80.1 79.8 3.01
Neutral detergent fiber 48.2 48.5 47.8 47.5 0.05
Crude protein 75.5
a
71.6
b
66.2
c
62.7
d
2.90
1,2, a,b
Mean within a row without a common superscript number and letter differ. p<0.05 and p<0.01. 1442 KANJANAPRUTHIPONG ET AL.
These changes were examined in the rumen of dairy cows
fed on concentrate based-diets containing various contents
of RUP from FSBM.
Santos et al. (1998) reported that there were no
statistically significant differences in DMI when SBM was
replaced by the high RUP sources in any of the comparisons.
A similar result was also observed in this study.
When the rate of protein proteolysis exceeds the rate of
small peptide and amino acid assimilation, excessive RDP
undergoes hydrolysis and deamination to form ammonia,
VFA and CO2 (Annison, 1956). In this study, there was a
decrease in ruminal ammonia concentrations with
increasing contents of FSBM in diets. The lower
concentrations of ruminal ammonia can be reflective of the
less rapidly degradable protein in FSBM compared with
that in SBM.
Isoacids appear to be essential and required by
cellulolytic bacteria in the rumen (Van Gylswyk, 1970).
However, a decrease in isoacids with increasing contents of
FSBM in diets reported in this study did not appear to
influence populations of cellulolytic bacteria in the rumen.
This indicates that isoacids did not limit cellulolytic
bacteria growth in the rumen on these diets.
Feed intake, rumen digestion, salivary secretion and
retention time of the feed in the rumen, all affect the
kinetics of rumen digesta, An increase in rumen volume is
generally accompanied by an increase in digesta in the
rumen. Increasing feed intake results in increasing pools of
rumen digesta (Owen et al., 1984) and, at constant feed
intake, the opposite would be expected with increasing the
digestibility in the rumen (Colucci et al., 1982). Organic
matter digested in the rumen reported here tended to
decrease with increasing levels of RUP in the diets and thus
it may be assumed that decreasing degradability of protein
did not influence digestion in the rumen. However,
increasing degradability of protein in the rumen will
increase concentrations of ruminal fluid ammonia
(Tamminga, 1983). Hume et al. (1970) reported that
ruminal fluid ammonia concentrations can influence rumen
volume. An increase in concentrations of ruminal fluid
ammonia above 200 mgN/l were associated with a decrease
in volume of ruminal fluid (Kanjanapruthipong and Leng,
1998). The smaller volume of ruminal fluid in the animal on
the diet containing SBM as RDP, reported in this study, is
likely influenced by the concentration of ruminal fluid
ammonia.
Digestibility primarily determines the amounts of
nutrients extracted from the feed and also the level of intake
and therefore the amounts available for absorption by the
host animal. The availability of microbial protein and VFA
for absorption is dependent on the availability of the
monomers required for microbial cell synthesis in the
rumen. Regardless of diet, it has been reported that peptides
and amino acids added to the rumen appear to give no
benefit of OM digested in the rumen over urea (Redman et
al., 1980; Cruz Soto et al., 1994; Fujimaki et al., 1994). A
decrease in OM digested in the rumen with increasing
FSBM reported in this study was partly due to an increase
in contents of RUP in diets.
Various species of rumen microbes are all responsible
for proteolysis of RDP (Nolan, 1993). Proteolytic activity of
bacteria in the rumen may be more active for soluble
protein (Nugent and Mangan, 1981) while that of protozoa
and fungi is apparent for insoluble particulate protein
(Wallace and Munro, 1986). In this study, a decrease in
ruminal fluid bacteria as indicated by total viable count with
increasing contents of RUP from FSBM in diets would be
possibly due to lower soluble peptides and amino acids
available for assimilation (Annison, 1956). Michalowski
(1989) reported that numbers of protozoa in ruminal fluid
appeared to increase with increasing RUP. A similar result
was also observed in this study.
Any excess of peptides and amino acids from RDP over
that are required for microbial protein synthesis can be
utilized as ATP-yielding substrates. Amino acids
catabolized anaerobically are low ATP-yielding substrates
and could contributed to energetic-spilling reactions
occurring under the condition that the rate of ATP
Table 5. Effects of total mixed rations (TMR) containing formalin treated soy bean (FSBM) on microbial ecosystem of in the rumen and
net microbial protein synthesis in the rumen indicated by purine derivatives in urine
TMR containing
Item
SBM FSBM-6.1 FSBM-12.1 FSBM-18.2
S.E
Total viable count, 10
-11
/ml 9.41
1,a
9.35
1,a
9.17
2,a,b
9.02
2,b
0.58
Cellulolytic bacteria count, 10
-7
/ml 0.94 0.91 0.89 0.90 0.05
Sporangia, No/mm
2
19 19 18 17 2.02
Large entodinium, 10
-4
/ml 1.15
a
0.47
b
0.57
b
0.42
b
0.29
Small entodinium, 10
-4
/ml 7.29
a
8.80
a
14.79
b
12.83
b
0.41
Dasytricha sp., 10
-4
/ml 0.47 2.63 0.83 0.84 0.09
Isotricha sp., 10
-4
/ml 0.52 0.63 0.58 0.42 0.08
Protozoa, 10
-4
/ml 9.43
a
10.53
a
16.77
b
14.51
b
0.65
Purine derivatives, mmol/d 270.9
1,a
269.1
1,a
264.2
2,a,b
262.3
2,b
8.9
Microbial N supply, g/d 196.9
1,a
195.6
1,a
192.1
2,a,b
190.7
2,b
4.2
1,2 and a,b
Mean within a row without a common superscript number and letter differ p<0.05 and p<0.01. UNDEGRADABLE PROTEIN AND RUMEN FUNCTIONS 1443
production by catabolism is in excess of the rate of ATP
utilization by anabolism (Stouthamer, 1979). On the other
hand, a supply of a small amount of peptides and amino
acids in addition to ammonia to a sugar or starch but not
cellulose diet in vitro studies resulted in a substantial
increase in net bacterial protein synthesis (Maeng and
Baldwin, 1976). A lack of response in the majority of in
vivo studies (Redman et al., 1980; Cruz Soto et al., 1994;
Fujimaki et al., 1994) can be due primarily to a large
diversity of rumen microbes and their interactions. In this
study, a decrease in net microbial protein synthesis in the
rumen (as indicated by purine derivatives in urine) with
increasing contents of RUP from FSBM was associated
with increasing numbers of protozoa in ruminal fluid. The
possible explanation is that the predatory activity of
protozoa appeared to be the main factor causing
considerable turnover of bacterial and fungal cells within
the rumen (Wallace and McPherson, 1987). Whereas the
majority of protozoa lyse and are degraded within the
rumen and only 10-30% enter the intestine (Leng, 1982a).
It was likely that reduction in net microbial protein
synthesis in the rumen with increasing contents of RUP in
diets may be due to the reduction of preformed protein
available for microbial growth as well as an increased
turnover rate of bacterial cells by predatory activity of
protozoa. However, a substantial increase in average daily
gain in dairy cows fed TMR containing RUP from FSBM
reported in this study appeared to be due to on increase in
dietary protein available for digestion and absorption by the
animals.
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