Microb Ecol (1989) 17:311-316

MICROBIAL ECOLOGY ~) Springer-VerlagNew York Inc. 1989

Ruminal Microbial Populations and Fermentation Characteristics in Bison and Cattle Fed High- and Low-Quality Forage Gene Towne, T. G. Nagaraja, and R. C. Cochran Department of Animal Sciences and Industry, Kansas State University, Manhattan, Kansas 66506, USA

Abstract. Ruminal microbial populations and fermentation products were compared between two ruminally cannulated bison (375 kg) and two ruminally cannulated Hereford steers (567 kg) on alfalfa or prairie hay diets. Differential media were used to enumerate carbohydrate-specific bacterial subgroups. Voluntary dry matter intake was higher (P -- 0.006) for cattle than for bison fed alfalfa, but prairie hay intake was not different (P = 0.16) between the two species. Volatile fatty acid concentrations, pH, and ruminal ammonia were similar between bison and cattle on both diets. Total anaerobic bacteria and xylanolytic bacterial counts were higher (P < 0.02) in bison than in cattle fed alfalfa. However, with the prairie hay diet, no differences in bacterial counts on any medium were observed between ruminant species. Both bison and cattle possessed a mixed A-B protozoan population with nearly identical protozoan numbers and distribution of genera. The similarities between bison and cattle consuming either highor low-quality forage suggest that any differences in putative forage digestibility between the species are not due to differences in microbial counts.

Introduction Various studies have suggested that American bison (Bison bison) are capable of digesting poor-quality forages more extensively than cattle [8, 9, 16, 17]. The mechanisms responsible for putative differences in digestive efficiency between bison and cattle are unknown, but may involve different ruminal microbial populations. Apparently, high-arctic Svalbard reindeer (Rangifer tarandus platyrhynchus) can subsist on poor-quality forage by harboring a highly specialized ruminal microflora that is particularly effective in fiber degradation [15]. Because bison consistently select lower quality forage than cattle [16], they also may possess high concentrations of fibrolytic microorganisms. The objectives of this study were to compare major ruminal bacterial groups, protozoan species, and fermentation products between bison and cattle consuming low-quality and high-quality forages.


G. Towne et al.

Materials and Methods

Animals, Sampling, and Diets Two ruminally cannulated bison steers (av wt, 375 kg) and two ruminally cannulated Hereford steers (av wt, 567 kg) were penned separately and offered alfalfa hay (22.0% crude protein, 33.9% neutral detergent fiber) twice daily at 12 hour intervals in amounts sufficient to allow ad libitum consumption. Orts were removed and weighed each morning, immediately before feed was offered. After a 14-d adjustment, ruminal samples in one animal were collected 2 hours postfeeding from the mid-dorsal sac, ventral sac, and reticulum with 125-rnl plastic containers capped in situ. Previous data indicated that 2 hours postfeeding was the optimum sampling time for both bisola and cattle [18]. During the next 3 d, the three remaining animals were similarly sampled, and the procedure then was repeated 10 d later. After two sampling replications on an alfalfa diet, the animals were switched to a prairie hay diet (4.4% crude protein, 69.4% neutral detergent fiber). Following a 14-d adaptation, ruminal samples were collected as previously described, and the procedure was then replicated 1 wk later.

Sample Preparation Immediately after each collection, the three ruminoreticular subsamples were transported to a laboratory and combined under oxygen-free CO2, Approximately 20 ml of ruminal contents were pipetted with a wide-orifice pipette into tared flasks containing 50% (vol/vol) formalin. Flasks were reweighed, and additional formalin was added to obtain a 1:1 (wt/wt) dilution of ruminal contents. This mixture was used for protozoan enumeration. The remaining ruminal sample was blended under CO2 for 1 rnin and strained through four layers of cheesecloth. The strained ruminal fluid was used to record pH, analyze for fermentation products, and inoculate various culture media for bacterial enumeration. Duplicate aliquots from the blended ruminal sample were acidified with 25% (wt/vol) rectaphosphoric acid [6] and frozen for subsequent volatile fatty acid (VFA) analysis. Two aliquots also were preserved with 0.1 N HC1 and frozen for ammonia-N (NH~-N) analysis, After thawing and centrifuging, VFA samples were analyzed in duplicate by gas chromatography and NH3-N concentrations were determined in duplicate with an autoanalyzer, using the phenol-hypochlorite reaction.

Culture Media Incubated and centrifuged ruminal fluid [13] was used to prepare basal medium for enumeration of total and selected bacterial groups. Total viable anaerobic bacteria were determined on a complete carbohydrate medium [ 13] with purified agar. Selected bacterial groups were enumerated on basal medium containing 0.3% (wt/vol) carbohydrate as the single energy source. Carbohydrate substrates examined were xylan, pectin, and soluble starch. Xylan and pectin were suspended in solution by homogenization for 90 sec. Culture media were made in bulk and dispensed into roll-tubes under CO_?; the tubes then were sealed and autoclaved. Cellulolytic bacteria were cultured in cellulose broth [13] containing a filter paper strip.

Inoculation of Media Serial 10-fold dilutions were made in anaerobic dilution blanks [1 ]. Four replicate roll-tubes were inoculated with 0.5 ml of the apDropriate dilutions into each culture medium and incubated at 39~ for 7 d before colonies were counted.


Ruminal Microbial Populations in Bison and Cattle Table 1.

Intake and ruminal fermentation characteristics in bison and cattle Alfalfa hay

Prairie hay








Intake, g D M - d -~ kg body wt -~ pH Total VFA, mM Acetate, mol/100 tool Propionate, mol/100 tool Butyrate, tool/100 rnol Acetate : propionate ratio Ammonia-N, mg/dl

23.7" 6.38 134.9 72.7 a 15.9 6.9 4.6 41.8

29.6 b 6.42 147.1 70.7 ~ 17.2 7.5 4.1 45.8

1.0 0.05 5.9 0.4 0.4 0.2 0.2 2.9

17.6 6.47 101.0 75.8 12.2 10.6 6.2 0.85

18.2 6.55 103.9 76.4 12.1 10.0 6.3 0.63

0.5 0.06 2.6 0.3 0.3 0.2 0.2 0.12

-.b Means with different superscripts within each diet differ between bison and cattle (P < 0.05) Quadruplicate tubes of cellulose broth were inoculated with 1 ml o f 10 -4 to 10 -1~ dilutions and incubated for 21 d at 39~ Cellulolytic bacteria were enumerated by the most probable number method [11], based upon disintegration of the filter paper strip [3].

Protozoa An aliquot o f the preserved sample was diluted with staining solution containing methyl green in phosphate buffer with 30% (vol/vol) glycerol. Total numbers and generic composition of ciliate protozoa were counted from 20 microscopic fields in a Sedgwick-Rafter counting chamber. Identification of protozoan genera was according to Hungate [10], with supplemental classification based on descriptions from Ogimoto and Imai [12] and Orpin and Mathiesen [14]. Length and width of 20 random ceils from each protozoan species in both bison and cattle were measured with a calibrated ocular micrometer. Individuals in the genus Entodinium were not identified to species, but average cell dimensions were determined from 40 randomly selected ceils. Relative cell volumes were calculated from a rotational ellipsoid formula, assuming that thickness was in constant proportion to width [7].

Statistical Analysis Differences in microbial populations and fermentation products between species were analyzed by analysis of variance using animal within species as the error term. Because the two diets were confounded with time, no statistical comparisons were made between forages. Means for each diet were separated by least significant difference when a significant (P < 0.05) F-test was detected.

Results and Discussion

After adjusting for body weight, cattle consumed higher (P = 0.006) amounts of alfalfa than bison (Table 1); but voluntary consumption of prairie hay was similar (P = 0.16) between species. Ruminal pH, NH3-N, and total VFA concentrations were not different between bison and cattle on either diet. Acetate proportion was higher (P = 0.04) in bison than in cattle fed alfalfa; however, the difference is probably not biologically significant. When consuming alfalfa hay, xylanolytic and total anaerobic bacterial counts were higher (P < 0.02) in bison than in cattle (Table 2). Pectinolytic and


G. T o w n e et al.

Table 2. cattle

R u m i n a l anaerobic bacterial counts from various media in bison and

Alfalfa hay Media Xylan Pectin Starch Complete carbohydrate Cellulose



Prairie hay SE

............................................................. X 109/g 89.9 ~ 60.7 b 16.9 102.9 71.6 16.3 56.7 29.9 8.7 78.2"

45.0 ~,





............................................................. 20.1 37.7 17.0 27.7 39.0 10.5 27.0 33.6 9.5 27.9



................................................... M P N , x 10Vml ................................................... 232.5 205.0 118.7 25.1 8.9 24.0

~b Means with different superscripts within each diet differ between bison and cattle (P < 0.05)

amylolytic bacterial counts also tended to be higher in bison than in cattle (P = 0.13 and P = 0.19, respectively). However, no differences in colony counts of any bacterial groups were detected between ruminant species fed prairie hay. It would appear than on poor-quality forages, the putative digestive advantage of bison compared to cattle is not directly associated with higher fibrolytic bacterial numbers. Low ruminal nitrogen concentrations probably inhibited bacterial growth when animals were consuming prairie hay compared to alfalfa. On poor-quality forages, ruminal nitrogen can become limiting, and bacteria that utilize energy sources more readily available than cellulose may assimilate most of the NH3-N [2]. Hawley et al. [8] postulated that bison have a greater capacity than cattle to recycle urea, which would reduce competition for ruminal nitrogen and benefit cellulolysis. Similarities in cellulolytic bacterial counts, however, suggest that any additional urea recycling in bison was not significantly beneficial to the microbial population. Similar fibrolytic bacterial counts, however, do not necessarily imply that the microbial populations in bison and cattle have comparable capacities for fiber degradation. Some cellulolytic bacteria are more efficient at cellulose digestion than others [4]. It is possible that proportions of more active cellulolytic bacteria (e.g., Bacteroides succinogenes) could differ between bison and cattle. Also, the proportions of ruminal cellulolytic bacteria shift from predominantly Butyrivibrio fibrisolvens to more active Ruminococcus spp. as NH 3 concentrations increase [20]. Thus, the potential for microbial fiber degradation could be different between bison and cattle, despite similar numbers of fibrolytic bacteria. Total protozoan numbers, cell volume, and concentrations of protozoan genera were not different between bison and cattle on either diet (Table 3). The homogeneity of protozoan numbers and species distribution indicates an absence of any host specificity between bison and cattle. The presence or absence of some ruminal protozoan species is interrelated with the protozoan population type possessed by the host. Protozoan populations are separated into two basic categories: type-A, identified by the presence of Polyplastron multivesiculatum, Ophryoscolex spp., and Metadinium affine;

Rurninal Microbial P o p u l a t i o n s in Bison a n d Cattle Table 3.


N u m b e r s a n d v o l u m e o f ciliate protozoa in b i s o n a n d cattle Alfalfa hay


Dasytricha lsotricha Microcetus Charonina Entodinium Diplodinium Eudiplodinium Metadiniurn Ostracodinium Epidinium Ophryoscolex Polyplastron Total protozoa Total v o l u m e



Prairie h a y SE

............................................................... • 10Vg 7.0 3.2 0.8 4.8 2.9 0.7 8.6 19.1 14.0 1.9 9.5 3.9 173.5 187.2 8.5 < 0.1 1.6 1.1 2.5 3.2 2.0 2.2 7.3 3.3 < 0.1 5.4 0.5 < 0.1 3.8 0.7 0 5.7 1.0 1.6 2.5 0.4 202.1






............................................................... 36.1 50.6 7.5 3.0 3.2 0.9 8.9 9.5 2.3 83.2 29.0 25.6 83.8 162.0 34.2 4.2 6.0 2.2 9.8 5.7 3.8 19.1 8.6 3.0 1.7 3.8 1.5 4.2 1.9 2.9 0 2.2 0.8 2.5 3.8 1.6 356.5



......................................................... x 108 ~m3/g ......................................................... 87.7 160.1 13.5 186.6 186.7 26.5

and type-B, characterized by Epidinium ecaudaturn, Eudiplodinium bovis, and Eu. rnaggii [5]. Other protozoan species apparently coexist satisfactorily in either population type, but if intermixed, Polyplastron multivesiculatum will selectively consume the type-B species, irrevocably changing a type-B into a type-A population [5]. However, the transformation of population types is not always complete, and mixed A-B populations, consisting of type-A species coexisting with low concentrations of Eudiplodinium maggii and Epidinium ecaudatum, often persist. In this study, both bison and cattle possessed a mixed A-B population with nearly identical protozoan numbers and genera distribution. Previous comparative studies indicated that the bison had initially possessed a type-B protozoan population, whereas the cattle had a mixed A-B type [18]. Although the bison were maintained in a separate pasture, it is possible that accidental contact with nearby cattle cross-inoculated type-A protozoa into the bison; however, Ophryoscolex spp., a type-A protozoan common in cattle, did not become established in the bison. Although we observed no difference in protozoan numbers between ruminant species, significantly higher protozoan concentrations and cell volume have been reported in bison as compared to cattle [ 18]. However, those bison possessed a type-B population, which is typical of wild bison [19]. That implies that type-B populations can support higher protozoan numbers and cell volume than mixed A-B populations. With both high-quality and low-quality diets, no major differences were observed in ruminal fermentation characteristics or microbial popuIations between bison and cattle. Although anaerobic fungi were not determined, homogeneous bacterial and protozoan populations suggest that any putative digestive superiority of bison is not related to microbial counts. However, since the bison in this study possessed a protozoan population type different from that


G. Towne et al.

typically observed in wild bison, the possibility that u n c o n t a m i n a t e d bison could possess separate and distinct m i c r o b i a l populations, which could potentially e n h a n c e r u m i n a l f o r a g e d e g r a d a t i o n , c a n n o t b e d i s c o u n t e d .

Acknowledgements. Contribution no. 89-154-J from the Kansas Agricultural Experiment Station. Appreciation is extended to the Kansas Department of Wildlife and Parks for donating the bison. Partial funding for this project was provided by C. Owensby.

References 1. Anderson KL, Nagaraja TG, Morrill JL, Avery TB, Galitzer SJ, Boyer JE (1987) Ruminal microbial development in conventionally or early-weaned calves. J Anita Sci 64:1215-1226 2. Bryant MP, Robinson IM (1961) Studies on the nitrogen requirements of some ruminal cellulolytic bacteria. Appl Microbiol 9:96-103 3. Cochran WF (1950) Estimation ofbacterial densities by means ofthe "most probable number." Biometrics 6:105-109 4. Dehority BA, Scott HW (1967) Extent of cellulose and hemicellulose digestion in various forages by pure cultures of rumen bacteria. J Dairy Sci 50:1136-1141 5. Eadie JM (1967) Studies on the ecology of certain rumen ciliate protozoa. J Gen Microbiol 49:175-194 6. Erwin ES, Marco G J, Emery EM (1961) Volatile fatty acid analyses of blood and rumen fluid by gas chromatography. J Dairy Sci 44:1768-1771 7. Harmeyer J, Hill H (1964) Das protozoenvolumen im Panseninhalt bei Ziege und Guanako. Zentralbl. Veterinaermed., Reihe A 11:493-501 8. Hawley AWL, Peden DG, Reynolds HW, Stricklin WR (1981) Bison and cattle digestion of forages from the Slave River Lowlands, Northwest Territories, Canada. J Range Manage 34: 126-130 9. Hawley AWL, Peden DG, Stricklin WR (1981) Bison and Hereford steer digestion of sedge hay. Can J Anim Sci 61:165-174 10. Hungate RE (1978) The tureen protozoa. In: Kxeier JP (ed) Parasitic protozoa, Vol 2. Academic Press, New York, pp 655-695 11. Mann SO (1968) An improved method for determining cellulolytic activity in anaerobic bacteria. J Appl Bacteriol 31:241-246 12. Ogimota K, lmai S (1981) Atlas of rumen microbiology. Japan Sci Soc Press, Tokyo 13. Olumeyan DB, Nagaraja TG, Miller GW, Frey RA, Boyer JE (1986) Rumen microbial changes in cattle fed diets with or without salinomycin. Appl Environ Microbiol 51:340-345 14. Orpin CG, Mathiesen SD (1986) Microcetus lappusgen, nov., sp. nov.: new species ofciliated protozoon from the bovine rumen. Appl Environ Microbiol 52:527-530 15. Orpin CG, Mathiesen SD, Greenwood Y, Blix AS (1985) Seasonal changes in the ruminal microflora of the high-arctic Svalbard reindeer (Rangifer tarandus platyrhynchus). Appl Environ Microbiol 50:144-151 16. Peden DG, Van Dyne GM, Rice RW, Hansen RM (1974) The trophic ecology of Bison bison L. on shortgrass plains. J Appl Ecol 11:489-497 17. Richmond RJ, Hudson RJ, Christopherson RJ (1977) Comparison of forage intake and digestibility by American bison, yak and cattle. Acta Theriol 22:225-230 18. Towne G, Nagaraja TG, Cochran RC, Harmon DL, Owensby CE, Kaufman DW (1988) Comparisons of ruminal fermentation characteristics and microbial populations in bison and cattle. Appl Environ Microbiol 54:2510-2514 19. Towne G, Nagaraja TG, Kemp KK (1988) Ruminal ciliated protozoa in bison. Appl Environ Microbiol 54:2733-2736 20. Van Gylswyk NO (1970) The effect of supplementing a low-protein hay on the cellulolytic bacteria in the rumen of sheep and on the digestibility of cellulose and hemicellulose. J Agric Sci, Camb 74:169-180

Ruminal microbial populations and fermentation characteristics in bison and cattle fed high- and low-quality forage.

Ruminal microbial populations and fermentation products were compared between two ruminally cannulated bison (375 kg) and two ruminally cannulated Her...
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