Short communication
Growth performance of faunated and
defaunated Malpura weaner lambs
A. Santra
*, S.A. Karim
Division of Animal Nutrition, Central Sheep and Wool Research Institute, Avikanagar Ð 304 501, Rajasthan, India
Received 1 September 1999; received in revised form 10 April 2000; accepted 7 June 2000
Abstract
An experiment was conducted to study the effect of presence or absence of rumen ciliate protozoa on nutrient intake, its utilization, rate of body weight gain and feed conversion ef®ciency of lambs in post-weaning phase of growth. Forty ®ve weaned Malpura lambs, divided into three equal groups (G1, G2 and G3) were either defaunated by periodic dosing of sodium lauryl sulfate at the rate of 8 g/100 kg body weight (G1) or defaunated and refaunated (G2) or maintained faunated (G3). The lambs were fed for 90 days on 50:50 pala (Zizyphus nummularia) leaf and concentrate based complete feed. With care and segregation the defaunated lambs were maintained free of rumen protozoa for 47 days after which Entodinomorphid appeared while Holotrichs were not detected. The total and differential protozoa count was similar in refaunated and faunated lambs and the residual toxic effect of sodium lauryl sulfate if any was not detected. Even with similar dry matter intake in the three groups, body weight gain and feed conversion ef®ciency were higher in defaunated lambs. The digestibilities of cell and cell wall contents were generally poor in defaunated lambs while intake of digestible protein and energy were similar in the three groups. The lambs in the three groups were in positive nitrogen balance while percent N retention was higher in defaunated than in refaunated and faunated lambs.
It was concluded from the study that with similar dry matter intake and plane of nutrition, the growth performance and feed conversion ef®ciency were better in defaunated than refaunated and faunated lambs. In spite of reduced digestibility of neutral detergent ®bre, acid detergent ®bre and cellulose, the metabolise energy availability for body synthetic activity was probably higher in defaunated lambs as evident from the observed production traits.#2000 Elsevier Science B.V. All rights reserved.
Keywords:Lambs; Defaunation; Nutrient utilization; Plane of nutrition; Growth 86 (2000) 251±260
*Corresponding author. Tel.:91-1437-28143; fax:91-1437-28163. E-mail address: santra@cswri.raj.nic.in (A. Santra).
1. Introduction
Rumen protozoa play important role in utilization of lignocellulosic material by fermentative digestion in ruminants (Bird et al., 1994; Chaudhary et al., 1995; Santra and Jakhmola, 1998). It is now well established by a number of in vivo experiments that elimination of ciliate protozoa from rumen microbial ecosystem (defaunation) increases the intestinal protein ¯ow (Lindsay and Hogan, 1972; Ushida et al., 1984, 1986; Rowe et al., 1985; Meyer et al., 1986; Kayouli et al., 1986) thus improving body weight gain in young ruminants and increasing wool growth in sheep under certain feeding condition (Bird et al., 1979; Ivan et al., 1992; Bird et al., 1994). During last decade many attempts were made in developed countries to generate information regarding role of protozoa on the rumen metabolism and performance of ruminant (Jouany et al., 1988; Demeyer, 1992). However, such studies were almost lacking in tropical environment (Developing countries). Further in developing countries livestock are primarily maintained on low grade roughage (Crop residues) and it is well established that low grade roughage feeding results in more methane production thus leading to wastage of digestible energy in rumen fermentation. In the rumen, protozoa produce hydrogen which is used by the ruminal bacteria to reduce carbon dioxide to methane (Jouany et al., 1988). Hence, defaunation reduces methane production to the tune of 20±30% and increases about 12% metabolizable energy availability to the animals (Whitelaw et al., 1984; Kreuzer et al., 1986; Santra et al., 1994a). Land utilization pattern, agricultural production system and availability of feed resources for livestock in developing countries particularly in India are unlikely to change in near future. Hence, it is felt that 10±12% increment in digestible energy availability to the ruminants simply by defaunation will have sizable contribution in improving their production. The reported experiment was therefore conducted to study the effect of presence or absence of rumen ciliate protozoa on nutrient intake, its utilization, growth and feed conversion ef®ciency of lambs in post-weaning phase of growth.
2. Material and methods
2.1. Animals and defaunation protocol
Forty ®ve Malpura weaner (90 days) lambs with average body weight of 10.6 kg were randomly divided into three equal groups (G1, G2and G3) of 15 lambs each. All the lambs
of G1 and G2 were defaunated by fasting them for 24 h followed by administration of 8 g sodium lauryl sulfate/100 kg body weight for 2 consecutive days as 10% solution directly into rumen through a stomach tube. On the ®rst day of defaunation the lambs received only 200 g air dried and conserved Pala (Zizyphus numularia) leaves in the evening. After 6 days of defaunation (sodium lauryl sulfate treatment), all the lambs of G2 were
refaunated by inoculating 100 ml/lamb rumen content drawn from normal faunated and ®stulated rams maintained on similar diet. The lambs in G1were maintained defaunated
throughout the study by periodic dosing of sodium lauryl sulfate when required. The status of defaunation in G1animals was checked in 7 days intervals by collecting rumen
¯uid from these intact animals using a stomach tube. The collected rumen ¯uid was strained through one layer of muslin cloth.
2.2. Experimental feeding and metabolism trial
All the animals in the three groups were individually fed for 90 days on a composite feed containing 50:50 Pala (Zizyphus nummularia) leaf and concentrate ingredients (ground pala 50, crushed barley grain 37, groundnut cake 20, mineral mixture 2 and common salt 1% along with Vitablend120 g/100 kg feed) in mash form. The chemical composition of the experimental feed and the Pala leaf is presented in Table 1. Feed was offered once daily at 0900 h after discarding previous day's residue and record of daily feed intake was maintained throughout the experimental period. All the lambs were weighed in weekly intervals for 2 consecutive days during the entire course of the study. After 80 days of the experimental feeding a metabolism trial of 7 days duration was conducted on nine lambs from each group in metabolic cages with provision for quantitative collection of faeces and urine. Composited samples of feed offered, residue, faeces and urine from the metabolic trial were subjected to preliminary processing and preserved for subsequent chemical analysis. Blood samples were also collected from all the animals in the morning hours before feed offer by jugular vein puncture at 0, 45 and 90 days of the experimental feeding.
2.3. Chemical analysis
The feed offered, residue left and faeces and urine voided were analyzed for dry matter (DM) by drying at 1008C for 24 h, organic matter (OM) by ashing at 5508C for 4 h and crude protein (CP) by Kjeldahl technique (AOAC, 1980). Neutral detergent ®ber (NDF), acid detergent ®ber (ADF) and acid detergent lignin (ADL) were estimated by the method of Van Soest et al. (1991). Cellulose was calculated by the difference of ADFÿADL. Blood glucose was estimated in whole blood (Cooper and Mc Daniel, 1970) and urea nitrogen (Rahamatullah and Boyde, 1980), total protein and albumin (Annino, 1976) in separated serum. The counting of rumen protozoa was carried out as per the method described by Kamra et al. (1991).
Table 1
Chemical composition (on % DM) of the diet
Particulars Composite ration Pala (Zizyphus numularia) leaves
Organic matter 87.7 83.8
Crude protein 14.2 11.8
Neutral detergent ®bre 50.3 58.7
Acid detergent ®bre 30.2 33.5
Cellulose 23.9 18.7
Lignin 6.1 6.9
2.4. Statistical analysis
The data on feed intake, nutrient utilization and feed conversion ef®ciency were subjected to test of signi®cance between the treatments using least squares analysis of variance (Snedecor and Cochran, 1989) and signi®cant treatment means were compared by Duncan's multiple range test (Duncan, 1955). Weekly body weight changes of individual lamb was traced by ®tting polynomial curves and the generated constants were subjected to analysis of variance to assess treatment differences. The pooled constants of the three groups are presented in Fig. 1 to show their growth pro®le.
3. Results and discussion
3.1. Nutrient intake and growth performance
The lambs were periodically screened for presence of rumen protozoa by drawing rumen liquor from all the defaunated lambs using stomach tube. It was evident from the results that the defaunating agent sodium lauryl sulfate used in the study was fully effective in removing protozoa from the rumen and successfully defaunated the lambs. With little extra care in management and segregation such defaunated lambs could be maintained free of rumen ciliate protozoa till 47 days. After 47 days of defaunation and experimental feeding, Entodinomorphid protozoa appeared in defaunated animals whereas holotrichs were not detected. The results indicate that as compared to holotrichs, the Entodinomorphid protozoa were more resistant to defaunating agent used in the study.
The ®nding is in agreement with observations of Veira et al. (1983) and Santra et al. (1994b). After 47 days, on reappearance of protozoa in rumen, the lambs of G1 group were again defaunated as per earlier protocol. Total and differential counts of the rumen protozoa were similar in refaunated and normal faunated lambs (Table 2) indicating that SDS treatment of the lambs did not have visible carry over harmful effect on the rumen environment.
The average daily dry matter intake of the lambs during the entire period of growth study was similar in the three groups amounting to 761.1, 754.4 and 757.8 g/day in defaunated, faunated and refaunated groups, respectively. The trends remained similar on its expression in terms of unit weight or metabolic body size. The dry matter intake (DMI) was also similar in the three groups during metabolic trial period. It is evident from the results of DMI that the chemical agent employed for defaunation in the study did not have adverse effect on animal's voluntary feed intake.
Initial body weight was similar in the three groups while ®nishing weight was signi®cantly (p<0.01) higher in defaunated than refaunated and faunated groups (20.8 kg). Similarly total body weight gain during the study and average daily gain (ADG) were also higher (p<0.01) in defaunated than refaunated and faunated lambs Table 2
Changes in body weight and nutrient intake in experimental lambs
Particulars Defaunated
Initial body weight (kg) 10.6 10.7 10.5 0.32 NSc Final body weight (kg) 23.0 bb 20.9 a 20.7 a 0.35 p<0.01 Total body weight gain (kg) 12.4 b 10.2 a 10.2 a 0.26 p<0.01 Average daily gain (g) 138 b 113 a 113 a 2.8 p<0.01 Total dry-matter intake (kg) 68.5 67.9 68.2 1.95 NS
Dry-matter intake (g/day) 761 754 758 27.6 NS
Total DCP intake (kg) 6.3 6.4 6.4 0.19 NS
DCP intake (g/day) 70 71 71 4.1 NS
Total DE intake (Mcal) 202 207 209 5.8 NS
DE intake (Mcal/day) 2.2 2.3 2.3 0.78 NS
Per cent feed conversion ef®ciency 18.1 b 15.1 a 15.1 a 0.42 p<0.01
Nutrient intake/kg gaind
DM (kg) 5.5 a 6.7 b 6.7 b 0.15 p<0.01
DCP (kg) 0.51 a 0.63 b 0.63 b 0.01 p<0.05
DE (Mcal) 16.3 a 20.3 b 20.5 b 0.44 p<0.01
Rumen ciliate protozoal population (104/ml SRLe)
Holotrich 00 4.9 4.8 0.15 ±
Entodinomorphid 00 28.4 28.1 0.31 ±
Total 00 33.3 32.9 0.79 ±
aStandard error mean.
bValues bearing different letters in a row differ signi®cantly. cNon-signi®cant.
(Table 2). On an average the defaunated lambs showed 21% growth improvement over faunated and refaunated lambs. Similar studies conducted elsewhere on growing lambs (Bird and Leng, 1985) and calves (Bird et al., 1979) have also indicated on an average 15±20% improvement in growth of defaunated animals. The changes in the weekly body weight of the experimental lambs in the three groups was charted by polynomial curve ®tting and pooled constants of the groups are presented in Fig. 1. The pattern of body weight gain was similar in the three groups till 15 days of the study. Thereafter, the defaunated lambs had consistently better (p<0.01) growth response compared to other two groups which was also re¯ected by improved nitrogen retention in them.
With similar total feed intake in the three groups during the entire period of study, higher gain in defaunated lambs was re¯ected in improved (p<0.01) feed conversion ef®ciency. Further the conversion ef®ciency in terms of digestible protein and energy intake also followed similar trend. Higher growth rate and feed conversion ef®ciency in defaunated compared to faunated and refaunated lambs was probably due to reduced methanogenesis (Kreuzer et al., 1986; Santra et al., 1996) as well as increase protein ¯ow to the duodenum of defaunated lambs (Demeyer et al., 1982; Van Nevel et al., 1985). As a result, more metabolisable energy was available for body's synthetic activity. Moreover, the observed improved growth rate and feed conversion ef®ciency in defaunated lambs could also be due to improved microbial and dietary protein ¯ow to duodenum in defaunated compared to faunated lambs (Bird and Leng, 1985).
3.2. Digestibility of nutrients and plane of nutrition
Apparent digestibility of DM, OM, NDF, ADF and cellulose were signi®cantly (p<0.01) lower in defaunated than refaunated and faunated lambs while the digestibility of CP was similar in the three groups (Table 3). Reduced digestibility of ®ber fraction in defaunated lambs could be attributed to elimination of speci®c cellulytic activity of largeEntodino morphid ciliate. Further, better digestibility of cell wall constituents in faunated than defaunated lambs was probably due to increased retention time of feed particle in their rumen (Kayouli et al., 1983±1984; Ushida and Jouany, 1990), stabilization of rumen environ-ment favoring developenviron-ment of cellulytic ¯ora (Hegarty et al., 1991) and stimulatory effect of protozoa over bacteria (Onodera et al., 1988). The nutritive value of the test diet was similar in the three groups which is in agreement with previous report of Chaudhary et al. (1995). In spite of lower digestibility of DM, cell and cell wall constituents in defaunated lambs, the plane of nutrition was similar in the three groups because of similar DMI. Santra et al. (1994b) also reported similar ®ndings in faunated and defaunated animals.
3.3. Nitrogen balance and blood pro®le
The data on nitrogen intake, its loss through faeces and urine and balance are presented in Table 4. The daily N intake and its loss through faeces and urine was similar in the three groups while N retention was signi®cantly (p<0.05) higher in defaunated than refunated and faunated lambs. Accordingly the N retention was 41% in defaunated whereas it was 37% in refaunated and faunated lambs. Higher N retention in defaunated lambs was possibly due to greater microbial protein synthesis in their rumen (Bird et al.,
Plane of nutrition and nutrient digestibility in experimental lambs
g/day 779 760 753 41.1 NS
g/kg body weight/day 37.7 39.2 39.3 1.30 NS
g/kg W0.75/day 80.4 82.3 82.2 3.10 NS
Nutrient intake (g/day) and digestibility (%) Dry-matter
Intake 779 760 753 41.1 NS
Digestibility 59.4 ab 61.8 b 61.5 b 0.39 p<0.05
Organic matter
Intake 682 665 660 36.0 NS
Digestibility 62.7 a 65.3 b 65.1 b 0.45 p<0.01
Crude protein
Intake 110 108 106 5.8 NS
Digestibility 65.4 66.5 66.5 0.47 NS
Neutral detergent ®bre
Intake 392 382 379 20.7 NS
Digestibility 49.2 a 52.6 b 52.9 b 0.50 p<0.01
Acid detergent ®bre
Intake 235 229 227 12.4 NS
Digestibility 41.1 a 43.0 b 42.9 b 0.30 p<0.05
Cellulose
Intake 186 181 180 9.8 NS
Digestibility 43.6 a 46.3 b 46.9 b 0.41 p<0.01
Energy
Intake (Mcal) 3.4 3.3 3.3 1.80 NS
Digestibility 68.7 69.4 69.5 0.34 NS
Nutritive value of rationd
DCP (%) 9.26 9.41 9.40 0.06 NS
DE (Mcal/kg feed) 3.0 3.1 3.1 0.01 NS
Nutrient intake DCP
g/day 72.0 71.6 70.8 3.70 NS
g/kg body weight/day 3.5 3.7 3.7 0.12 NS
g/kg W0.75/day 7.4 7.7 7.7 0.28 NS
DE
Kcal/day 2300 2324 2304 127.8 NS
Kcal/kg body weight/day 111 120 120 4.1 NS
Kcal/kg W0.75/day 237 251 251 9.7 NS
aStandard error mean.
bValues bearing different letters in a row differ signi®cantly. cNon-signi®cant.
1994). It is now generally accepted that in absence of rumen ciliate, the ef®ciency of rumen microbial growth is enhanced and more microbial and dietary protein ¯ows from reticulo-rumen to duodenum (Bird and Leng, 1985).
Blood glucose level was signi®cantly (p<0.01) higher in defaunated (60.3 mg%) than refaunated and faunated lambs (55.0 mg%) while serum urea N, total protein and albumin were similar in the three groups. Higher blood glucose level in defaunated lambs was possibly due to higher availability of propionate in their rumen.
4. Conclusions
It is concluded from the study that with similar dry matter intake and plane of nutrition the growth performance and feed conversion ef®ciency was better in defunated than refaunated and faunated lambs. In spite of reduced digestibility of neutral detergent ®bre, acid detergent ®bre and cellulose in defaunated lambs their ME availability for body synthetic activity was probably higher as evident from the observed better growth rate.
Acknowledgements
The authors are grateful to the Director of the Institute for providing necessary facilities for conducting the study and technical assistance rendered by Mr. M. Asgar is acknowledged.
Table 4
Nitrogen balance and blood biochemical constituents in experimental lambs
Particulars Defaunated
g/kg body weight/day 0.85 0.89 0.89 0.03 NS
g/kg W0.75/day 1.32 1.86 1.86 0.13 NS
Faecal nitrogen (g/day) 6.1 5.8 5.7 0.35 NS
Urinary nitrogen (g/day) 4.3 5.1 5.0 0.32 NS
Nitrogen retained
g/day 7.2 6.4 6.3 0.01 NS
g/kg body weight/day 0.35 0.33 0.33 0.01 NS
g/kg W0.75/day 0.75 0.68 0.69 0.02 NS
As% nitrogen intake 41.0 bc 37.1 a 37.1 a 0.44 p<0.01 Blood glucose (mg/dl) 60.3 b 55.0 a 54.8 a 0.55 p<0.01
Serum urea (mg/dl) 19.2 19.1 19.3 0.30 NS
Serum total protein (mg/dl) 7.2 7.1 7.0 0.15 NS
Serum albumin (mg/dl) 4.1 4.0 4.1 0.11 NS
aStandard error mean. bNon-signi®cant.
cValues bearing different letters in a row differ signi®cantly.
References
Annino, J.S., 1976. Determination of total proteins and albumins in serum. Clinical Chemistry: Principles and Procedures, 4th Edition. Little Braken and Company, Bogoton, pp. 184±188.
AOAC, 1980. Of®cial Methods of Analysis, 13th Edition. Association of Of®cial Analytical Chemists, Washington, DC, pp. 125±139.
Bird, S., Hill, M.K., Leng, R.A., 1979. The effects of defaunation of the rumen on the growth of cattle on low-protein high-energy diets. Br. J. Nutr. 42, 81±87.
Bird, S.H., Leng, R.A., 1985. Productivity response to eliminating protozoa from the rumen of sheep. Rev. Rur. Sci. 6, 109±117.
Bird, S.H., Romulo, B., Leng, R.A., 1994. Effects of lucerne supplementation and defaunation on feed intake, digestibility, N retention and productivity of sheep fed straw based diets. Anim. Feed Sci. Technol. 45, 119± 129.
Chaudhary, L.C., Srivastava, A., Singh, K.K., 1995. Rumen fermentation pattern and digestion of structural carbohydrates in buffalo (Bubalus bubalis) calves as affected by ciliate protozoa. Anim. Feed Sci. Technol. 56, 111±117.
Cooper, G.R., Mc Daniel, V., 1970. Determination of glucose by orthotoludine method. In : Standard Method of Clinical Chemistry. Academic Press, New York, 159 pp.
Demeyer, D.I., Van Nevel, C.J., Van de Voorde, G., 1982. The effect of defaunation on the growth of lambs fed three urea containing diets. Arch. Tierernahr. 32, 595±605.
Demeyer, D.I., 1992. Biotechnology and the quality of animal products in sustainable agriculture. J. Appl. Anim. Res. 1, 65±80.
Duncan, B.B., 1955. Multiple range and multipleF-test. Biometrics. 11, 1±42.
Hegarty, R.S., Nolan, J.V., Leng, R.A., 1991. Sulphur availability and microbial fermentation in fauna free rumen. Arch. Anim. Nutr. 41, 725±736.
Ivan, M., Dayrell, de M., Mahadevan, S., Hidiroglou, M., 1992. Effect of bentonite on wool growth and nitrogen metabolism in fuana free and faunated sheep. J. Anim. Sci. 70, 3194±3202.
Jouany, J.P., Demeyer, D.I., Grain, J., 1988. Effect of defaunating the rumen. Anim. Feed Sci. Technol. 21, 229± 265.
Kamra, D.N., Sawal, R.K., Pathak, N.N., Kewalramani, N., Agarwal, N., 1991. Diurnal variation in ciliate pro-tozoa in the rumen of black buck (Antilope cervicapra) fed green forage. Lett. Appl. Microbiol. 13, 165±167. Kayouli, C., Demeyer, D.I., Van Nevel, C.J., Dendooven, R., 1983\|±\|1984. Effect of defaunation on straw
digestion in sacco and on particle retention in rumen. Anim. Feed Sci. Technol. 10, 165±172.
Kayouli, C., Demeyer, D.I., Van Nevel, C.J., Dendooven, R., 1986. Effect of defaunation and refaunation of the rumen on rumen fermentation and N-¯ow in the duodenum of sheep. Arch. Anim. Nutr. 36, 827±837. Kreuzer, M., Kirchgessner, M., Muller, H.L., 1986. Effect of defaunation on the loss of energy in wethers fed
different quantities of cellulose and normal or steam ¯aked maize starch. Anim. Feed Sci. Technol. 16, 233± 241.
Lindsay, J.R., Hogan, J.P., 1972. Digestion of two legumes and rumen bacterial growth in defaunated sheep. Aust. J. Agr. Res. 23, 321±330.
Meyer, J.H.F., van der Walt, S.I., Schwatrtz, H.M., 1986. In¯uence of diet and protozoal numbers on the breakdown and synthesis of protein in the rumen of sheep. J. Anim. Sci. 62, 509±520.
Onodera, R., Yamasaki, N., Murakami, K., 1988. Effect on inhabitation by ciliate protozoa on the digestion of ®brous materials in vivo in the rumen of goats and in vitro rumen microbial ecosystem. Agric. Biol. Chem. 52, 2635±2637.
Rahamatullah, M., Boyde, T.R.C., 1980. Improvements in the determination of urea using diacetyl monoxime methods with and without deproteinization. Clin. Chem. Acta. 107, 3±9.
Rowe, J.B., Davies, A., Broome, A.W.S., 1985. Quantitative effects of defaunation on rumen fermentation and digestion in sheep. Br. J. Nutr. 54, 105±119.
Santra, A., Kamra, D.N., Pathak, N.N., Khan, M.Y., 1994a. Effect of protozoa on the loss of energy in Murrah buffalo (Bubalua bubalis) calves. Buff. J. 3, 249±253.
Santra, A., Kamra, D.N., Pathak, N.N., Khan, M.Y., 1996. In¯uence of ciliate protozoa on biochemical changes and hydrolytic enzymes in the rumen of Murrah buffalo (Bubalus bubalis). Buffalo J. 1, 95±100. Santra, A., Jakhmola, R.C., 1998. Effect of defaunation on animal productivity. J. Appl. Anim. Res. 14, 103±
116.
Snedecor, G.W., Cochran, W.G., 1989. Statistical Methods, 8th Edition. Iowa State University Press, Ames, IA. Ushida, K., Jouany, J.P., Lassalas, B., Thivend, P., 1984. Prtozoal contribution to nitrogen digestion in sheep.
Can. J. Anim. Sci. 65 (Suppl.), 20±21.
Ushida, K., Jouany, J.P., Thivend, P., 1986. Role of rumen protozoa on nitrogen digestion in sheep given two isonitrogenous diets. Br. J. Nutr. 56, 407±419.
Ushida, K., Jouany, J.P., 1990. Effect of defaunation on ®bre digestion in sheep given two isonitrogenous diets. Anim. Feed Sci. Technol. 29, 153±158.
Van Nevel, C.J., Demeyer, D.I., Van de Voorde, G., 1985. Effect of defaunating the rumen on growth and carcass composition of lambs. Archiv fur Tierernahrung 35, 331±337.
Van Soest, P.J., Robertson, J.B., Lewis, B.A., 1991. Methods for dietary ®bre, neutral detergent ®bre and non-starch polysaccharides in relation to animal nutrition. J. Dairy Sci. 74, 3584±3597.
Veira, D.M.., Ivan, M., Jui, P.Y., 1983. Rumen ciliate protozoa: effects on digestion in the stomach of sheep. J. Dairy Sci. 66, 1015±1022.
Whitelaw, F.G., Eadie, J.M., Bruce, L.A., Shand, W.J., 1984. Methane production in faunated and ciliate free cattle and its relationship with rumen volatile fatty acid production. Br. J. Nutr. 52, 261±275.
