Proceedings Buffalo Workshop December  2001

http://www.mekarn.org/procbuf/wanapat.htm

 

Swamp buffalo rumen ecology and its manipulation

M Wanapat 

Department of Animal Science

Faculty of Agriculture

Khon Kaen University, Khon Kaen  40002, Thailand
metha@kku1.kku.ac.th

http://web.kku.ac.th/~metha 

Abstract

Rumen ecology has been playing an important role in fermentation process and providing end-products for ruminants.  These studies were carried out to investigate on rumen factors namely pH, NH₃-N, microorganisms in cattle and swamp buffaloes raised on traditional system.  Furthermore, study on diurnal pattern of rumen fermentation and effect of rumen digesta transfer from buffalo to cattle was conducted.  Based on these studies, it was found that buffalo and cattle raised under similar condition exhibited a diverse rumen ecology. Diurnal fermentation patterns in both cattle and buffaloes were revealed.  It was found that rumen NH₃-N appeared to be a limiting factor.  Rumen digesta transfer from buffalo to cattle was achievable. Monitoring of digesta transfer up to 14 days resulted in normal rumen ecology as compared to that of original buffalo.  However, further research should be undertaken in these regards in order to improve rumen ecology especially buffalo-based rumen. 

Key words : Swamp buffalo, rumen ecology, manipulation, digesta transfer

Introduction

The rumen has been well recognized as an essential fermentation vat that is capable of preparing end-products particularly volatile fatty acids (VFAs) and microbial proteins as major energy and protein for the ruminant host. The more efficient the rumen is the better the fermentation end-products being synthesized.  In recent years, there has bee increasing research directed to rumen ecology and rumen manipulation (Orskov and Flint, 1989; Martin, 1998; Weimer, 1998).  However, most of these papers have dealt with ruminants raised in temperate areas and fed on good-quality roughages and with high levels of concentrate supplementation.  However in the tropics, most ruminants have been fed on low-quality roughages, agricultural crop-residues, industrial by-products which basically contained high levels of ligno-cellulosic materials, a low level of fermentable carbohydrate and a low level of good-quality protein.  In addition, long dry seasons, a prevailing harsh environment, especially high temperature, low soil fertility and lees feeds available throughout the year, all influence rumen fermentation.  Recently, Wanapat (2000) reported on rumen fermentation to increase the efficient use of local feed resources and productivity of ruminants in the topics.

Traditionally, swamp buffaloes (Bubalus bubalis) have been kept by small-holder farmers in Southeast Asia, as multi-purpose animals. According to De Haan et al (no date) there are 148 million buffaloes in the world and 99.9 % are  raised in the developing countries. The present numbers of swamp buffaloes in Thailand are about 2 million heads and more than 90% are in the Northeast which are serving as a vital component in the mixed farming  systems along with cattle and/or small ruminants.  It has previously been reported that when cattle and buffalo were kept under similar conditions, buffalo utilize feed more efficiently with the digestibility of feeds being typically 2-3 percentage units higher (Wanapat 1989;  Wanapat et al 1994; Kennedy and Hogan 1994). Nitrogen utilization in swamp buffalo was found more efficient than that in Malaysian cattle (Devendra 1985). The superiority is particularly noticeable in situations where the feed supply is of low quantity and/or quality. The reasons for the superior digestive capacity of buffalo over cattle have not been fully elucidated.  However it is likely that much of the superiority may be explained by differences in the nature of rumen microbial population which would affect the type of fermentation occurring and the end-products resulting from fermentation. Thus, any variations between cattle and buffalo in the proportions and numbers of ruminal bacteria, protozoa and fungi might contribute to the explanation of differences in digestive  capability due to fermentation end-products available for absorption and utilization by ruminants.

The objectives of these experiments were to study on the rumen ecology of buffalo and cattle raised on traditional system and to identify the rumen fermentation pattern in buffalo and cattle fed on untreated and urea-treated rice straw. 

Materials and methods

Experiment 1

The rumen samples for subsequent study of microbial populations were obtained from animals kept under traditional village conditions in the Northeast of Thailand. The experiment was conducted in September to April when swamp buffalo and crossbred cattle (Brahman x Native) were both grazing seasonally on available native grasses, rice stubble and rice straw. No concentrates were given to the animals at this time. A total of 40 animals which were being  kept under similar conditions were identified, brought from a local marker and slaughtered.  The animals (20 cattle and 20 buffaloes) were of both sexes and were between 2-4 years of age.

Immediately, after slaughtering, samples of fresh digesta (500g) were taken from the rumen of each animal. Rumen digesta were squeezed through 4 layers of cheesecloth to ensure a sample which contained microbial population from both the liquid and solid phases. The subsequent rumen fluid was immediately fixed with 10% formalin solution ( Galyean 1989). The total direct count of bacteria, protozoa and fungal zoospores were made using the methods of Galyean (1989) based on the use of a haemacytometer (Boeco) and total numbers were studied for bacterial and protozoal shapes under microscope. Differentiation of rumen  fungal zoospores from small protozoa was based on  characteristics  having flagellae while protozoa had ciliates around them. Rumen fluid was diluted  using autoclaved distilled water (121^oC  for 15 minutes) as a medium, by 100, 10 , and 10 times for bacteria, protozoa and fungal zoospores counting using 10x40, 10x10 and 10x40 ocular x objective of haemacytometer, respectively. For further identifying different shapes of bacteria rumen fluid was made at 10 times and counted at 10x40 ocular x objective of haemacytometer.  Scanning electron microscopy was used for further determinations of bacteria, protozoa and fungal zoospores. Samples were fixed in buffered glutaraldehyde dehydrated in a graduated ethanol series and dried in a criteria-point dryer. They were mounted on aluminum stubs and sputter coated with gold prior to viewing. The data were subjected to statistical analysis using students t- test. 

Experiment 2

Digestion trial

Six rumen-fistulated buffaloes and cattle (3 each) were randomly assigned according to a 3 x 3 Latin square design to receive three roughage sources and the treatments were as follows:

•    T1 = untreated rice straw (URS)

•    T2 = urea-treated (5%) rice straw (UTRS)

•    T3 = URS and UTRS (1:1) (MX)

All animals received the roughage on ad libitum basis and in addition rice bran was supplemented at 0.5% of body weight. Digestion trial lasted for 21 days each.  Feed intakes were measured during the first two weeks and followed by a 24-h rumen fluid sampling for every hour.  Samples were measured for pH immediately and prepared for later analyses of NH₃-N, VFAs, total viable counts of cellulolytic, proteolytic and amylolytic bacteria.  During the last 5 days the animals were put onto metabolism crates when 90% of previous feed intakes were given and for total collection of feed, feces and urine.

Rumen fluid was collected at 0, 4 h-post feeding and measured for pH immediately and samples were prepared for later analysis of NH₃-N (Bromner and Keeney  1965), volatile fatty acids (VFAs) using HPLC (Samuel et al 1997), total viable cellulolytic, proteolytic and amylolytic bacteria were measured using roll tube technique (Hungate  1969).  Digestibilities of nutrients were calculated.  All data were subjected to ANOVA and treatment means comparisons were conducted by Duncan’s New Multiple Range Test using Proc GLM (SAS 1985) 

Digesta transfer study

All rumen fistulated buffaloes and cattle (3 each) were fed with three kinds of roughage treatments using a 3 x 3 Latin square design: untreated rice straw (URS), urea-treated (5%) rice straw (UTRS) and URS and UTRS (1:1) (MX).  They were fed for two weeks and then rumen fluid samples were collected at 0 and 4 h post feeding.  Measurements of pH were taken immediately while other rumen fluid samples were treated and prepared for later analyses of NH₃-N (Bromner and Keeney 1965), volatile fatty acids (VFAs) using HPLC as the above. Total viable cellulolytic, proteolytic and amylolytic bacteria were counted using roll tube technique (Hungate 1969).

After the initial sampling period (3 weeks), the rumen digesta from each buffalo fed on each respective roughage were transferred to each respective cattle for each roughage after rumen digesta of the cattle were removed completely.  These transfer were done as quickly as possible to avoid longer exposion of digesta to the air.  After complete transfers, all lids of fistulae were closed.  Samplings of rumen fluid were take at 0, 4 h post feeding, before transfer, and 7 and 14 days after rumen digesta transfer to be measured for rumen pH, NH₃-N, VFAs and total viable counts of cellulolytic, proteolytic and amylolytic bacteria using standard methods as indicated above. All data were subjected to ANOVA and treatment means were compared using Duncan’s New Multiple Range Test (Proc. GLM) (SAS  1985). 

Results and Discussion:

Experiment 1

The methods used proved successful at estimating the numbers of micro-organisms in the samples. Data on the numbers of bacteria, protozoa and fungal  zoospores and shown in Table 1.  

Based on this experiment, it was found that the rumen pH was similar for both species, but significant differences were found in the numbers of micro-organisms. There was a trend for a higher population of bacteria, a lower population of protozoa and significantly more fungal zoospores in the ruminal fluid of buffaloes as compared with those of cattle.

The results obtained show microbial counts which are broadly in agreement with other studies reported by other workers for cattle and buffalo (Langar et al 1968). Wattanachant et al (1990) found higher total bacterial count and cellulolytic bacteria in swamp buffalo than those in cattle. Interestingly, in the present study where animals were subjected to similar feeding, there were strong trends for a higher bacteria and lower protozoa counts but significantly more fungal zoospore counts in buffaloes than those in cattle. As clearly known, rumen bacteria are far most important among protozoa and fungi, but moreover, their close association and balances in forming optimal rumen ecology are paramount in providing useful fermentation end-products for the ruminants.

The presence and role of fungi in the rumen have until recently been a contentious issue. In earlier years, Orpin (1975), Akin et al (1983), Akin and Benner (1988) and Ho and Abdullah (1999) reported the findings of rumen fungi and their roles in degrading ligno-cellulosic materials which stimulated further studies to be conducted in these regards. Studies by Ho et al (1988) indicated the presence of fungi in buffalo and in cattle and it was found that the rate and method of colonization by rumen fungi in swamp buffalo and native cattle of Malaysia were similar when fed on guinea grass. In addition, Ho and Barr (1995) offered detailed information regarding classification of rumen fungi from Malaysia. The present study clearly illustrates the presence of fungi in the rumen digesta of both cattle and buffaloes. It is interesting that, on similar diets, fungal zoospores in swamp buffalo were found twice as many as those found in cattle. Using the method of Ho and Barr (1995), in swamp buffaloes the fungi predominantly found were Anaeromyces sp.  having sporangia with acuminate apex. Previous workers ( Williams and Coleman 1992) have suggested that there is a negative relationship between the number of protozoa and fungi in ruminal fluid. This is supported by the present work where protozoal numbers were lower in buffalo and fungal zoospores were higher than in cattle.  As Williams and Coleman (1992) found that rumen protozoa predated on fungal zoospores therefore the lower numbers of protozoa present, the higher fungal zoospores would be.  Rumen fungi would possibly act to control protozoal population as well.

Figures 1, 2, 3, 4, 5, 6 and 7  illustrate pictures of rumen bacteria attachment, protozoa  distribution, fungal sporangia, rumen fungus, Anaeromyces sp., having sporangium with acuminate apex and rhizoid forming “appressorium” in swamp buffaloes.

 

Figure 1.  Attachment of rumen bacteria on rice straw of swamp buffalo, Bar = 16 mm	Figure 2. Rumen protozoa of cattle fed rice straw, Bar = 16 mm
Figure 3. Rumen protozoa, Entodiniomorph sp. of swamp buffalo, Bar = 16 mm	Figure 4. Sporangia of rumen fungi of swamp buffalo burst out from the surface of rice straw, Bar = 16 mm
Figure 5. Rumen fungus of swamp buffalo, Anaeromyces sp. With acuminate apex, Bar = 16 mm	Figure 6. Rumen fungal rhizoid with penetrated appressorium of swamp buffaloes, Bar = 16 mm

 

Figure 7.  Rumen fungal sporangium with flagellae, Bar = 16 mm

 Based on this study, interesting findings revealed differences in rumen baterial, protozoal population and fungal zoospore counts in swamp buffaloes and cattle raised under similar conditions. Since rumen microorganisms have impact on fermentation pattern and fermentation end-products particularly volatile fatty acids and NH₃-N, it therefore could be speculated that higher bacteria, lower protozoa and higher fungal zoospores in swamp buffaloes would result in greater fermentation efficiency and productivity than in cattle. However, further studies in this regard as well as classification of rumen microbes of swamp buffaloes should be looked at more closely and warrant immediate research undertakings. 

Experiment 2

The diurnal patterns during 24 h of rumen fermentation characteristics in beef cattle and swamp buffaloes fed on untreated and urea-treated rice straw. 

In both cattle and buffaloes, rumen pH and temperature were maintained constant and the values were 6.5-6.7; 38-39 °C, respectively.  However, VFA production patterns were fluctuating as well as C₂ concentration while C₃ and C₄ were similar which indicated an active role of rumen microbes and on-going fiber fermentation of cellulolytic bacteria.  It was also found that rumen NH₃-N was very consistent and relatively low (<5 mg/100ml) throughout the period.  However, all of the fermentation aspects except rumen pH and temperature were notably enhanced by feeding urea-treated rice straw. Rumen fermentation end-products were significantly different as a result of feeding different types of roughages.  As shown in table 2 that rumen NH₃-N, C₂, C₄ were increased as a result of using urea-treated rice straw and were also higher in buffalo than in cattle. Ratios of (C₂+C₄₎/C₃ and TVFA/NH₃-N were also narrower. Based on this study, low rumen NH₃-N could be a limiting factor on rumen fermentation and would ultimately affect on rumen ecology.

In ruminants fed on low-quality roughages, critical rumen NH₃-N levels for microbial activities were found a 5-20 mg/100ml (Boniface et al 1986; Perdok and Leng  1989).  While Chanthai et al (1987) demonstrated that rumen NH₃-N in cattle and buffaloes fed on untreated rice straw were less than 2 mg/100ml and were increased to 9 mg/100ml with urea-treated rice straw. Perdok and Leng (1989) further showed that higher level of rumen NH₃-N (15-30 mg/100ml) improved intake and digestibility.  Increasing rumen NH₃-N level up to 30 mg/100ml significantly decreased C₂+C₄/C₃, increasing rumen fungal zoospores as well as increasing microbial protein synthesis (17-47%) (Kanjanapruthipong and Leng  1998).  In a most recent experiment in swamp buffaloes fed on untreated rice straw, Wanapat and Pimpa (1999) also found similar results that rumen NH₃-N levels of 13.6-34.4 mg/100 ml  improved rumen fermentation by increasing digestibility and intake of straw.  As rumen NH₃-N increased, rumen bacteria and protozoa, as well as urinary purines were also increased.  It was suggested that optimum rumen NH₃-N level would be higher than 15 mg/100ml. Nguyen Van Thu and Preston (1999) also found rumen NH₃-N (5-6 mg/100ml) of swamp buffaloes fed on rice straw or grass and were significantly increased to 8-18 mg/dl by adding urea-treated rice straw, urea-molasses cake and Sesbania leaf.  The increases of rumen bacteria, protozoal population as well as DMI were also concomitantly found with increases in rumen.

Effect of buffalo rumen digesta transfer 

Among diets, urea-treated rice straw (UTRS) digestibility was highest (P<.05) and digestibility of nutrients particularly those of organic matter and crude protein were higher in buffalo than in cattle (Table 3).  Several factors have been claimed to attribute to these values.

Intakes of roughages were highest in both cattle and buffaloes fed on UTRS in terms of kg/d, % BW, g/kgW^.75. In general, intakes of these roughages before and after buffalo digesta transfer were similar at 7 and 14 days after transfer (Table 4). 

Rumen pH in all treatments and animals were similar and were in normal range of rumen ecology (pH 6.2-6.7). Digesta transfer did not show any effects (Table 5).   

Rumen NH₃-N concentrations were lowest in animals fed on untreated rice straw (URS) and highest in UTRS fed groups.  These NH₃-N values remained low in URS fed group after buffalo digesta transfer of 7 and 14 d, respectively and were lower than those reported as optimum (20-30 mg%) (Boniface et al 1989; Perdok and Leng 1989; Wanapat and Pimpa 1999). Values in cattle and buffalo fed on UTRS and mixture of URS+UTRS (MX) were found higher and were maintained after digesta transfer for 14 d.  Values after 4 h-post feeding were slightly increased in some treatments (Table 5). 

Total volatile fatty acids (TVFAs) at 0 h post-feeding were highest in UTRS and in buffaloes, while at 4 h-post feeding in group fed on UTRS and MX were increased.  After 7 and 14 d transfer, TVFAs of cattle were comparable to those of buffaloes.  This could be an attributing factor from digesta transfer.  For C₂, C₃ and C4 all values were similar from, before and after 7, 14 d digesta transfer for both cattle and buffaloes. It is noticeable that C₃ concentrations were relatively high in all fed groups (Table 6, 7, 8, 9).  

 

 

 
Effect of digesta transfer on rumen microoganisms

Total viable bacteria counts were found to be similar among treatments and sampling times. Cellulolytic, proteolytic and amylolytic bacterial counts of cattle were increased after 7, 14 d digesta transfer.  The most pronounced values were obtained in buffaloes fed on UTRS and particularly at 7 d after digesta transfer. This could mean that after removal of digesta, buffalo rumen could still have functionally higher rumen turn over rate and while in cattle the digesta transfer could be sustainable as seen on 14 d after transfer (Table 10, 11, 12, 13).

Other means of manipulating the rumen could be used (e.g. feeding of condensed tannins).  Condensed tannins contained in cassava hay has been shown to improve rumen microorganisms and fermentation and to enhance rumen by-pass of the dietary protein (Wanapat 2000; Wanapat et al 1999, 2000a,b). 

  

  

 

 

Conclusions and recommendations

Experiment 1

Cattle and buffaloes raised under traditional raising system could result in having different rumen microorganisms.  Counts of bacteria and fungal zoospores were higher and protozoa were lower in the rumen of buffaloes than in cattle. These could attribute to higher ability to utilize feeds and hence higher digestibility of feed in buffaloes.

Experiment 2

Rumen NH₃-N appeared to be the limiting factor on untreated straw. UTRS resulted in higher nutritive value than URS and MX.  The transfer of rumen digesta transfer from buffalo to cattle indicated that intake, digestibility and rumen ecological parameters criteria from the buffaloes could be sustained in cattle at least for 14 days. 

Acknowledgements

The author wishes to express gratitude to the National Center for Bio-Engineering and Biotechnology of Thailand for the financial support of this research. Special thanks to SIDA-SAREC for providing an opportunity for the author to participate in the MEKARN workshop at the National Institute of Animal Husbandry in Hanoi, Vietnam. 

 

References

Akin, D.E., G.L. Gordon and J.P. Hogan, 1983. Rumen bacterial and fungal degradation of Digitaria pentzii grown with or without sulfur. Appl. Environ. Microbiol. 46:738-748.

Akin, D.E. and R.Benner. 1988. Degradation of polysaccharides and lignin by ruminal bacteria and fungi. Appl. Environ. Microbiol. 54:1117-1125.

Boniface, A.N., R.M. Murray and J.P. Hogan.  1986.  Optimum level of ammonia in the rumen liquor of cattle fed tropical pasture hay. In:Proc. Aust. Soc. Anim. Proc. 16:151-154.

Bromner, J.M., and D.R. Keeney.  1965.  Steam distillation methods of determination of ammonium, nitrate and nitrite. Anal. Chem. Acta.  32:485.

Chanthai, S., M. Wanapat and C. Wachirapakorn.  1989. Rumen ammonia-N and Volatile fatty acid concentrations in cattle and buffalo given rice straw-based diets. In:Proc. 7^th AFAR Int. Workshop. (Eds. R. Dixon), IDPD, Canberra, Australia.

De Haan, H. Steinfeld and H. Blackburn. No date. Livestock and the Environment: Finding a balance. European Commission Directorate-General for Development and Natural Resources, EU Publication 115 pp.

Devendra, C. 1985. Comparative nitrogen utilization in Malaysia Swamp buffaloes and Kedah-Kelanton cattle. In : Proc. The 3 rd AAAP Animal science Congress, Seoulk, Korea, Vol 2:873-875.

Galyean, M. 1989. Laboratory procedure in animal nutrition research. Department of Animal and Range Sciences. New Mexico State University, U.S.A.

Hungate, R.E.  1969.  A roll tube method for cultivation of strict anaerobes. In:Methods in Microbiology, edited by J.R. Norris and D.W. Ribbons. New York:Academic. 313:117.

Ho, Y.W. and D.J.S. Barr. 1995. Classification of anaerobic gut fungi  from herbivores with emphasis on rumen fungi from Malaysia. Mycologia, 87:655-677.

Ho, Y.W. and N. Abdullah. 1999. The role of rumen fungi in fiber digestion. Asian-Aus. J. Anim. Sci. 12:104-112.

Ho, Y.W., N.Abdullah and S. Jalaludin. 1988. Colonization of guinea  grass by anaerobic rumen fungi in swamp buffalo and cattle. Anim. Feed Sci. Technol. 22: 161-171.

Kanjanapruthipong and R.A. Leng.  1998. The effects of dietary urea on microbial populations in the rumen of sheep. Asian-Aus. J. Anim. Sci. 11:661-667.

Kennedy, P.M. and J.P. Hogan.  1994. Digestion and metabolism in buffaloes and cattle:are there consistent diffences. In: Proc. The 1^st Asian buffalo Association Congress. (Eds. M. Wanapat and K. Sommart), Khon Kaen University, Khon Kaen, Thailand.

Langar, P.N., G.S. Sidhu and I.S. Bhatia.  1968. A study of the microbial population in the ruminal of buffalo (Bos Bubalis) and Zebu (Bos indicus) on a feeding regimen deficient in carbohydrates. Indian. J. Vet. Sci. 38:333-336.

Martin, S.A.  1998.  Manipulation of ruminal fermentation with organic acids: A reviews.  J. Anim. Sci. 76:3123-3132.

Nguyen Van Thu and T.R. Preston.  1999. Rumen environment and feed degradability in swamp buffaloes fed different supplements. Livestock Res for Rural Dev. 11(3): http://www.cipav.org.co/lrrd/lrrd 11/3/thu 113.htm.

Orpin, C.G. 1975. Studies on the rumen flagellate Neocallimastix frontalis. J. Gen. Microbiol. 91:249-262.

Orskov, E.R. and H.J. Flint.  1989.  Manipulation of rumen microbes or feed resources as methods of improving feed utilization. In:Proc. The Biotechnology in Livestock in Developing Countries. (Ed. A.G. Hunter). Rkitchie of Edinburgh Ltd. United Kingdom.

Perdok, H.B., and R.A. Leng.  1989.  Effect of supplementation with protein meal on the growth of cattle given a basal diet of untreated ammoniated rice straw. Asian-Aus. J. Anim. Sci.  3:269.

Samuel, M., S. Sagathewan, J. Thomas and G. Mathen. 1997. An HPLC method for estimation of vollatile fatty acids of ruminal fluid. Indian. J. Anim. Sci. 67:805. 

SAS.  1985.  User’s Guide : Statistics, Version 5 Edition.  SAS. Inst. Cary, N.C.

Wanapat, M. 1989, Comparative aspects of digestive physiology and nutrition in buffaloes and cattle. In:Proc. Ruminant Physiology and Nutrition in Asia. (Eds. C. Devendra and E. Imaizumi) Jap. Soc. Zootech. Sci. Sendai, pp.27-43.

Wanapat, M. 2000.  Rumen Manipulation to Increase the Efficient use Local Feed Resources and Productivity of Ruminants in the Tropics. In:Proc. Of the 9^th AAAP Congress. (Eds. G.M. Stone),  July 3-7, 2000. University of New South Wales, Sydney, Australia.  13:59-67.

Wanapat, M. and O. Pimpa. 1999. Effect of ruminal NH3-N levels on ruminal fermentation purine derivatives, digestibility and rice straw intake in swamp buffaloes. Asian-Aus. J. Anim. Sci. 12:904-907.

Wanapat,M., K Sommart.,C. Wachirapakorn, S. Uriyapongson,  and C.Wattanachant. 1994. Recent advances in swamp buffalo nutrition and feeding. In:Proc.The 1^st Asian Buffalo Association Congress.(Eds. M. Wanapat and K.Sommart),Khon Kaen University, Khon Kaen, January 17-21, 1994, Thailand.

Wattanachant, C.,M. Wanapat, S. Sarangbin, S. Chanthai and C. Wachirap;akorn. 1990. A Comparative stydy on rumen cellulolytic bacteria  in  swamp buffaloes and cattlel. In Proc. 28^th  Annual Conference Kasetsart University, Bangkok.

Wanapat, M., O. Pimpa, W. Sripuek, T. Puramongkol, A. Petlum, U. Boontao, C. Wachirapakorn and K. Sommart.  1999.  Cassava Hay:an Important On-Farm Feed for Ruminants. In:Tannins in Livestock and Human Nutrition. (Eds. J.D. Brooker), May 31 June – 2 July, 1999, Adelaide, Australia.

Wanapat, M., O. Pimpa, A. Petlum and C. Wachirapakorn.  2000a. Participation scheme of small holder dairy farmers in the northeast Thailand on improveing feeding systems.  Asian-Aus. J. Anim. Sci. 13:600-604.

Wanapat, M., T. Puramongkon and W. Siphuak.  2000b. Feeding of cassava hay for lactating cows. Asian-Aus. J. Anim. Sci. 13:478-482.

Williams, A.G. and G.S. Coleman 1992. The Rumen Protozoa. Springer-Verlag, London. 441 pp.

Weimer, P.J.  1998.  Manipulating ruminal fermentation: A microbial ecological perspective. J. Anim. Sci. 76:3114-3122. 

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