Host-Parasite list updating of Ciliates and fermentation in the digestive tract of wild miscellaneous herbivores in South Africa (RSA).

This article is dedicated to the Author, Burk Dehority, who became very ill with cancer. Fortunately he confirmed or corrected all identifications on ciliates in this article. This paper was 80% finished when he became ill. He unfortunately passed away in February 2016.

The effect of acid drinking water on rumen protozoa in the blesbok (Damaliscus dorcas phillipsi).

Rumen contents were collected from ten adult female blesbok, five from a mine area with only acid drinking water available and five from a control group consuming normal, non-polluted drinking water. The mean concentration of total protozoa in the normal water group was almost double that in the acid drinking water group, 24.9 x 10(3) versus 14.7 x 10(3). Percent of Entodinium was higher and Diplodinium lower in those animals drinking the acid water. The number of different protozoa species present in animals from both locations was fairly similar. Diplodinium bubalidis, Ostracodinium gracile and Diplodinium consors were present in the highest percentage in the normal water group, 18.8, 18.4 and 17.7 %, respectively. The same three species, plus Entodinium dubardi, were also highest in the acid water group, O. gracile, 21.3 %; D. consors, 12.6 %; E. dubardi, 11.4 % and D. bubalidis, 10.3 %. Seventeen species of protozoa found in this study were a new host record for the blesbok, bringing the total number of species reported from the blesbok to 29.

Magnesium requirement of some of the principal rumen cellulolytic bacteria.

Information available on the role of Mg for growth and cellulose degradation by rumen bacteria is both limited and inconsistent. In this study, the Mg requirements for two strains each of the cellulolytic rumen species Fibrobacter succinogenes (A3c and S85), Ruminococcus albus (7 and 8) and Ruminococcus flavefaciens (B34b and C94) were investigated. Maximum growth, rate of growth and lag time were all measured using a complete factorial design, 2(3)×6; factors were: strains (2), within species (3) and Mg concentrations (6). R. flavefaciens was the only species that did not grow when Mg was singly deleted from the media, and both strains exhibited a linear growth response to increasing Mg concentrations (P<0.001). The requirement for R. flavefaciens B34b was estimated as 0.54 mM; whereas the requirement for R. flavefaciens C94 was >0.82 as there was no plateau in growth. Although not an absolute requirement for growth, strains of the two other species of cellulolytic bacteria all responded to increasing Mg concentrations. For F. succinogenes S85, R. albus 7 and R. albus 8, their requirement estimated from maximum growth was 0.56, 0.52 and 0.51, respectively. A requirement for F. succinogenes A3c could not be calculated because there was no solution for contrasts. Whether R. flavefaciens had a Mg requirement for cellulose degradation was determined in NH3-free cellulose media, using a 2×4 factorial design, 2 strains and 4 treatments. Both strains of R. flavefaciens were found to have an absolute Mg requirement for cellulose degradation. Based on reported concentrations of Mg in the rumen, 1.0 to 10.1 mM, it seems unlikely that an in vivo deficiency of this element would occur.

Chemotaxis toward carbohydrates and peptides by mixed ruminal protozoa when fed, fasted, or incubated with polyunsaturated fatty acids.

In contrast to the well-characterized chemotaxis and migratory behavior between the dorsal and ventral locations of the rumen by isotrichids, we hypothesized that chemotaxis toward soluble nutrients maintains entodiniomorphid protozoa in the particulate fraction. The objectives of these experiments were to compare the dose-responsive chemotaxis (1) toward different glucose concentrations when ruminal samples were harvested from fed versus fasted cows; (2) toward increasing concentrations of glucose compared with xylose when protozoa were harvested from a fed cow; (3) toward peptides of bacterial, protozoal, and soy origin; and (4) toward glucose when mixed ruminal protozoa were previously incubated for 0, 3, or 6h in the presence of emulsified polyunsaturated fatty acids (PUFA; Liposyn II, Hospira, Lake Forest, IL). In experiment 1, isotrichid protozoa decreased chemotaxis toward increasing glucose concentration when cows were fasted. Entodiniomorphids exhibited chemotaxis to similar concentrations of glucose as did isotrichids, but to a lesser magnitude of response. In experiment 2, xylose was chemotactic to both groups. Xylose might draw fibrolytic entodiniomorphid protozoa toward newly ingested feed. In contrast, even though isotrichids should not use xylose as an energy source, they were highly chemoattracted to xylose. In experiment 3, entodiniomorphids were not selectively chemoattracted toward bacterial or protozoal peptides compared with soy peptides. In experiment 4, despite isotrichid populations decreasing in abundance with increasing time of incubation in PUFA, chemotaxis to glucose remained unchanged. In contrast, entodiniomorphids recovered chemotaxis to glucose with increased time of PUFA incubation. Current results support isotrichid chemotaxis to sugars but also our hypothesis that a more moderate chemotaxis toward glucose and peptides explains how they swim in the fluid but pass from the rumen with the potentially digestible fraction of particulates.

Effects of wortmannin, sodium nitroprusside, insulin, genistein, and guanosine triphosphate on chemotaxis and cell growth of Entodinium caudatum, Epidinium caudatum, and mixed ruminal protozoa.

The mechanisms by which ruminal protozoa sense and migrate toward nutrients are not fully understood. Chemotaxis by many diverse eukaryotic cells is mediated by phosphatidylinositol-3-kinase, which is highly conserved in receptor tyrosine kinase (RTK) signaling pathways and consistently inhibited by wortmannin. In experiment 1a, increasing the concentration of wortmannin inhibited cell growth nonlinearly at 24h of a culture of the rumen protozoan Entodinium caudatum, but high variability prevented growth inhibition of Epidinium caudatum from reaching significance. In experiment 1b, increasing the insulin concentration recovered 24-h cell counts for both cultures, depending on wortmannin concentration. In experiment 2, addition of sodium nitroprusside (Snp; activator of protein kinase G for cilial beat reversal in nonrumen ciliate models) at 500µM or wortmannin at 200µM in beakers containing rumen fluid decreased random swimming by mixed entodiniomorphids into capillary tubes (inserted into beakers) containing saline. Both Snp and wortmannin increased chemotaxis into tubes containing glucose compared with the beaker control. For isotrichids, beaker treatments had no response. Glucose increased chemotaxis, but peptides decreased chemotaxis even when combined with glucose. In experiment 3, we assessed preincubation of genistein (a purported RTK blocker in nonrumen ciliate models) at 40 or 400µM in beakers and guanosine triphosphate (GTP; a universal chemorepellent in nonrumen ciliate models, perhaps mediated through an RTK) at 10 or 100µM combined with glucose in capillary tubes. Neither genistein nor GTP affected chemotaxis toward glucose for entodiniomorphids. However, GTP at 100µM reduced chemotaxis toward glucose for isotrichids. After the animal is fed, isotrichids that are depleted in glycogen migrate to the dorsal area of the rumen, and the rapid uptake of sugars is enhanced through strong chemotaxis but can be reversed by peptides or GTP. In contrast, entodiniomorphids are less intensely chemoattracted to glucose than isotrichids but are chemoattracted to peptides. Entodiniomorphids' chemoattraction appears to be integrated with slower but prolonged availability of energy from digesting starch and fiber.

Ionized calcium requirement of rumen cellulolytic bacteria.

Ionized calcium (Ca(+2)) appears to be required by the 3 predominant species of rumen cellulolytic bacteria, Fibrobacter succinogenes, Ruminococcus flavefaciens, and Ruminococcus albus. The present study evaluated the role of ionized calcium in growth and cellulose digestion. Maximum growth or rate and extent of digestion and lag time were the criteria used to evaluate Ca(+2) requirements. All cultures except F. succinogenes A3c grew when repeatedly transferred in a medium without added Ca(+2). As Ca(+2) concentration increased in cellobiose medium, the rate of growth increased and lag time decreased for F. succinogenes A3c, whereas F. succinogenes S85 exhibited increases in both maximum growth and rate of growth. No responses in any of the criteria were observed for the ruminococci in cellobiose medium. Both strains of F. succinogenes had an absolute requirement for Ca(+2) with cellulose as the only substrate. For strain A3c the requirement was 0.36 to 0.42 mM and for S85, >0.64 mM. Increases in extent of cellulose degradation occurred with all strains of ruminococci as Ca(+2) concentration increased; however, degradation in Ca(+2)-free medium was similar to that of F. succinogenes with Ca(+2). Although the ruminococci presumably have cellulosomes that require Ca(+2) in their structure, such was not evident in our studies. The function of Ca(+2) in cellulose degradation by F. succinogenes is unknown, but may be related to the secretion or activation of their cellulolytic enzymes. Based on reported concentrations of Ca(+2) in the rumen, it seems unlikely that an in vivo deficiency would occur for these bacteria.

Rumen ciliated protozoa decrease generation time and adjust 18S ribosomal DNA copies to adapt to decreased transfer interval, starvation, and monensin.

Defaunation studies have documented decreased ammonia concentrations associated with reduced microbial protein recycling and wastage of dietary protein, whereas many methods to suppress protozoa can reduce feed intake or depress ruminal organic matter or fiber digestibility. Therefore, more research is needed to optimize dietary conditions that improve protozoal growth and ruminal outflow relative to autolysis and recycling. Response in growth rate to ruminal outflow was simulated by abrupt changes in transfer interval of batch cultures, and substrate availability was evaluated by feeding without or with abrupt addition of monensin, which was postulated to inhibit digestive vacuole function. In experiment 1, Entodinium caudatum, a mix of Entodinium species, Epidinium caudatum, or Ophryoscolex caudatus cultures rapidly adjusted their generation times to approach respective changes in transfer interval from 3 to 2 or 1 d (cultures were always fed at 24-h intervals). Monensin (0.25 microM) consistently delayed this response. To evaluate a metabolic upshift associated with feeding or a downshift associated with substrate depletion, experiment 2 used real-time PCR to quantify protozoal 18S rRNA gene (rDNA) copies that were expressed relative to cell numbers or to the cellular constituents N and nucleic acids after feeding without or with monensin (0.5 microM). The 18S rDNA copies per milligram of nucleic acids were least for Ophryoscolex compared with the other cultures. When averaged over cultures (no culture x treatment interaction), 18S rDNA copies per unit of nucleic acids decreased at 16 h when cultures were starved but increased with feeding unless monensin uncoupled availability of consumed substrate. Rumen protozoal growth increased in response to decreased transfer interval in experiment 1. Substrate availability appeared to initiate metabolic responses preparing for cell growth, explaining how cultures could rapidly adjust to decreasing transfer interval in experiment 2. Because feeding was not coupled with transfer in experiment 2, however, a metabolic control probably arrested cell division to prevent overgrowth relative to substrate availability.

Improved in vitro procedure for maintaining stock cultures of three genera of rumen protozoa.

To maintain stock cultures of rumen protozoa, studies were initiated to explore possible methods for keeping the protozoa viable without feeding every day. Cultures of Entodinium caudatum, Epidinium caudatum, Enoploplastron triloricatum, and Entodinium exiguum were used to study the effect of not feeding for 1 or 2 d. The study lasted 88 d, and although bacterial concentrations decreased when cultures were not fed for 2 d (over the weekend), they recovered quickly with subsequent daily feedings. The exception was Enoploplastron triloricatum, which showed a gradual decline over the entire study. Addition of streptomycin to the media had little effect on maintaining bacterial concentrations in all cultures except Entodinium caudatum, in which the overall mean concentration was greater (P < 0.01). No differences in pH or bacterial concentrations were found between cultures fed daily and those held without feed for 2 d, with or without streptomycin. For maintaining protozoal cultures (10-mL volumes) over a long period without feeding on weekends, the following schedule is proposed: transfer and feed 0.12 mL of 1.5% ground wheat-1% orchardgrass on Monday; feed 0.12 mL of 1.5% ground wheat-1% orchardgrass on Tuesday, Wednesday, and Thursday; transfer and feed 0.5 mL of 1.5% ground wheat-1% orchardgrass on Friday; do not feed on Saturday and Sunday.

Technical note: Specific PCR amplification of protozoal 18S rDNA sequences from DNA extracted from ruminal samples of cows.

A protozoa-specific primer (P-SSU-342f) was designed and paired with a eukarya-specific primer to amplify a 1,360-bp fragment of DNA encoding protozoal small subunit (SSU) ribosomal RNA from ruminal fluid of cows fed a mixed forage:grain diet or alfalfa hay. Sequencing of clones showed that P-SSU-342f is specific to ruminal protozoa and, with slight modifications, the primer will be useful for ecological studies of ruminal protozoa.

In vitro growth and starch digestion by Entodinium exiguum as influenced by the presence or absence of live bacteria.

In a preliminary study, the addition of antibiotics was shown to reduce bacterial concentrations in Entodinium exiguum cultures by more than 99% in 4 h, whereas the protozoal population was apparently unaffected. Using this procedure, the growth and amylolytic capability of Entodinium exiguum, in the presence or absence of live bacteria, was studied in vitro. Treatments for Trial 1 were protozoa plus antibiotics (PA), PA plus autoclaved bacteria (PAB), protozoa plus living bacteria (PLB), and only bacteria (BAC). Autoclaved or non-autoclaved cornstarch was used as an energy source. Treatment main effects were as follows: higher concentration of E. exiguum in PLB than in PA or PAB at 24 and 48 h (P < 0.01); PA and PAB were not different (P > 0.05); concentrations of E. exiguum higher in autoclaved cornstarch at 12 h (P < 0.05) but lower than in non-autoclaved cornstarch at 24 and 48 h (P < 0.01); and starch digestion in PLB was higher than in PA and PAB at all time periods, but only greater than BAC up to 24 h (P < 0.01). In Trial 2, only treatments PA, PLB, and BAC were tested. Rice starch and cornstarch were used as substrates. With rice starch, growth was higher in PLB than in PA at 24 and 48 h (P < 0.05). Starch digestion started earlier in PLB with rice starch (P < 0.05) but was complete for both substrates after 24 h. Up to 12 h (autoclaved cornstarch and rice starch) and 24 h (non-autoclaved cornstarch and cornstarch), the sum of digestion by bacteria and protozoa did not equal the extent of digestion in PLB, suggesting some kind of synergism. Total extent of digestion with protozoa was similar between the two sources; however, bacteria digested rice starch faster and to a greater extent than cornstarch. Approximate lag times with rice starch, autoclaved cornstarch, and non-autoclaved cornstarch were 6, 3, and 12 h for bacteria and < 6, 3, and 9 h for protozoa, respectively. Rate of digestion for non-autoclaved cornstarch was similar for bacteria and protozoa, whereas the rate of bacterial digestion was much faster with the other two substrates (autoclaved cornstarch and rice starch).

The rumen ciliate fauna of domestic sheep (Ovis ammon aires) from the Turkish Republic of Northern Cyprus.

Concentration and composition of ciliate protozoa in the families Ophryoscolecidae and Isotrichidae were determined in rumen contents of domestic sheep (Ovis ammon aries) from Cyprus. A total of five genera of Ophryoscolecidae were identified, Metadinium, Enoploplastron, Polyplastron, Epidinium, and Ophrvoscolex, which included six species: Metadinium affine, Enoploplastron triloricatum, Polyplastron multivesiculatum, Epidinium ecaudatum, Epidinium graini, and Ophryoscolex purkynjei. Eight separate forms of Epidinium were identified (E. ecaudatum f. ecaudatum, E. e. f. caudatum, E. e. f. bicaudatum, E. e. f. tricaudatum, E. e. f. quadricaudatum, E. graini f. graini, E. g. f. caudatricoronatum, and E. g. f. caudaquadricoroniatum), along with five forms of Ophryoscolex purkynjei (O. p. f. purkynjei, O. p. f. bifidobicinctus, O. p. f. bifidoquadricinctus, O. p. f. bicoronatus, O. p. f. tricoronatus, and O. p. f. quadricoronatus). Three species of Isotrichidae were observed, Isotricha intestinalis, I. prostoma, and Dasytricha ruminantium. This study reports new host records for three forms of Epidinium graini and Ophryoscolex purkynjei f. bifidobicinctus. The rumen fauna in the family Ophryoscolecidae from Cypriote domestic sheep appear to have limited diversity compared to those from Turkish and Far Eastern (Chinese/Japanese) sheep, while they are more diverse than those found in Western European (Scottish) and North American (Canadian/Alaskan) sheep.

Preliminary study on the requirements of Entodinium exiguum and E. caudatum for live or dead bacteria when cultured in vitro.

Whether live bacteria are required to culture the rumen protozoa Entodinium exiguum and E. caudatum in vitro was studied. Treatments were protozoa plus antibiotics (PA), PA plus autoclaved bacteria (PAB) or protozoa plus live bacteria (PLB). Generation times at 24 h were 22.8 and 31.0 h for E. exiguum and E. caudatum. Protozoal concentrations were unaffected by the absence of bacteria up to 48 h. After 72 h, E. exiguum, concentrations were higher in PLB than PA or PAB. With E. caudatum differences between PLB and PA were only observed at 96 h. Thus, a requirement for live bacteria appears to be manifested in culture periods longer than 48 (E. exiguum) and 72 (E. caudatum) h. Although differences between PLB and PAB indicate a metabolic dependence for bacteria or a long-term antibiotic effect, non-significant differences between PAB and PA suggest that the effect is also related to a nutritive bacterial contribution.

Effect of feeding frequency on bacterial and fungal concentrations, pH, and other parameters in the rumen.

Three sheep were fed a pelleted high-roughage diet either once, 6, or 24 times per day in a 3 x 3 Latin square trial. During each 21-d period, 14 d were allowed for adaptation followed by a 7-d collection period, in which samples for microbial counts were taken on d 1 and 5 and several rumen parameters were measured on d 2 and 6. Bacterial concentrations were not different between feeding frequencies on the first sampling day but were higher (P < 0.05) on the second sampling day when the sheep were fed 24 times a day. Fungal concentrations were not different among feeding frequencies on either sampling day. No effects of feeding frequency were observed for the concentration of cellulolytic bacteria or fungi. On d 2, ruminal volume was larger (P < 0.05) with six feedings than with one feeding and fluid volume turnover was greater (P < 0.05) when sheep were fed 24 times per day. Rumen pH values were higher (P < 0.01) on both d 1 and 5 when the sheep were fed once a day and the percentage of rumen dry matter was highest (P < 0.02) with 24 feedings. These findings would suggest that if the same amount of a given diet is fed daily, the number of feedings does not markedly affect microbial concentrations, rumen volume, or liquid turnover time.

Antibiosis between ruminal bacteria and ruminal fungi.

Cellulose digestion, bacterial numbers, and fungal numbers were monitored over time in vitro by using a purified cellulose medium with and without antibiotics (penicillin and streptomycin). All fermentations were inoculated with a 1:10 dilution of whole rumen contents (WRC). Without antibiotics, cellulose digestion was higher (P < 0.01) at 24, 30, 48, and 72 h; fungi had almost disappeared by 24 h, while bacterial concentrations increased over 100-fold in 24 h and then decreased gradually up to 72 h. In those fermentations with added antibiotics, fungal concentrations increased 4-fold by 30 h and up to 42-fold at 72 h; bacterial concentrations were markedly reduced by 24 h and remained low through 72 h. Similar results were obtained with ground alfalfa as a substrate. In further studies, the in vitro fermentation of purified cellulose without antibiotics was stopped after 18 to 20 h, and the microbial population was killed by autoclaving. Antibiotics were added to half of the tubes, and all tubes were reinoculated with WRC. After 72 h, extensive cellulose digestion had occurred in those tubes without antibiotics, as compared to very low cellulose digestion with added antibiotics. The extent of this inhibition was found to increase in proportion to the length of the initial fermentation period, suggesting the production of a heat-stable inhibitory factor or factors. The inhibitory activity was present in rumen fluid, could be extracted from lyophilized rumen fluid (LRF) with water, and was stable in response to proteolytic enzymes. In addition, the water-extracted residue of LRF was found to contain growth factor activity for rumen fungi in vitro.

Feasibility of using lignin isolated from forages by solubilization in acetyl bromide as a standard for lignin analyses.

Lignin concentration can be measured in plants by the acetyl bromide-soluble lignin spectrophotometric method; however, as with any spectrophotometric method, a reliable standard is needed. In the present experiments, lignin was extracted from each of the forages under study with the acetyl bromide reagent. The lignin isolated with acetyl bromide (LIAB) was then used as the reference standard in the acetyl bromide-soluble lignin (ABSL) analysis, which was compared with the acid detergent lignin (ADL) and potassium permanganate lignin (PerL) lignin analyses. Two maturity stages of each of the following forages were analyzed: Medicago sativa, Cynodon dactylon var. Coastal, Panicum maximum var. Centenário and var. Colonião, Cynodon plectostachyus, Pennisetum purpureum, Setaria nandi, and Avena sativa. In addition, one wood sample, Eucalyptus sp., was analyzed. In general, ABSL values were highest (P < 0.001), followed by PerL and ADL, which also differed from each other (P < 0.001). Correlations with in vitro dry matter digestibility of samples were highest with the ABSL method. Absorption spectra of LIAB, either from plants of different maturity stages or from different vegetable species, suggested the presence of differences among some of the lignins.

Feasibility of using total purines as a marker for ruminal bacteria.

A procedure for measuring total purine content of mixed ruminal bacteria was adapted for use in the determination of purines in pure cultures of ruminal bacteria. Recovery of adenine and guanine, alone or in mixture, was quite variable. The problem was traced to solubility of the silver salt of adenine in the acid wash solution. When the precipitating solution was used as the wash, recovery of the purines was over 97%. Recovery of a 1:1 mixture of adenine and guanine added to yeast RNA was 100.6+/-3.2%. Purine, protein, and bacterial concentrations were determined for 10 pure cultures of ruminal bacteria: Butyrivibrio fibrisolvens, D16f, H10b, and H17c; Fibrobacter succinogenes B21a; Lachnospira multiparus D25e; Lactobacillus lactis ARD26e; Prevotella ruminicola H15a; Ruminococcus albus 7; Ruminococcus flavefaciens B34b; and Streptococcus bovis ARD5d. The CV for the most-probable-number (MPN) assay (bacterial concentrations), purine analysis, and protein analysis were 55.86, 5.25 and 6.52%, respectively. Considerable variation was found among bacterial species and strains when purine and protein concentrations were compared as the amount per individual cell. More consistent values were obtained when these components were expressed on a dry matter basis. Purine:protein ratios for the 10 pure cultures ranged from .023 to .1299, with a mean value of .0883. For samples of mixed bacteria separated from ruminal fluid, this ratio was found to average .0306, which is approximately one-third of the value for the pure cultures. The value determined for the mixed bacterial sample is similar to previously reported values. Based on the ratio obtained with the pure cultures, the microbial protein flow out of the rumen has probably been overestimated in most previous reports. Limited studies suggest that the samples of mixed ruminal bacteria used as a standard are probably contaminated with feed particles containing protein, which results in lower purine:protein ratios.

Effects of cross-inoculation from elk and feeding pine needles on the protozoan fauna of pregnant cows: occurrence of Parentodinium africanum in domestic U.S. cattle (Bos taurus).

Consumption of pine needles tends to cause abortion in domestic cattle but not in elk. The present study was undertaken to determine whether this difference was associated with the rumen microbial population. After emptying the rumen, pregnant cattle were inoculated with either elk or cattle rumen contents. For those cows fed the pine needle diet, there was no difference in abortion rate between those inoculated with rumen contents from either elk or cattle. Protozoal concentrations and number of genera were observed to decrease markedly in all cows fed the diet containing pine needles. The cycloposthiid ciliate Parentodinium africanum was observed in rumen contents from several of the domestic cattle (Bos taurus). Concentrations ranged from 1.4 to 130.6 x 10(4) per ml of rumen contents, which comprised 4.6 to 80.3% of the total ciliate population. Mean dimensions of this species were: length, 33.4 microns; width, 19.7 microns; length/width ratio, 1.70, which were similar to those previously reported for this species from Bos indicus in Brazil. This is the first observation of P. africanum, originally observed and described in stomach contents of the hippopotamus, either in Bos taurus or in any host in the northern hemisphere.

Rumen ciliates of white-tailed deer (Odocoileus virginianus), axis deer (Axis axis), sika deer (Cervus nippon) and fallow deer (Dama dama) from Texas.

Samples of rumen contents from 33 white-tailed deer (Odocoileus virginianus), 31 axis deer (Axis axis), 26 sika deer (Cervus nippon), and 25 fallow deer (Dama dama) were collected from four study areas in central Texas. The geometric mean concentration of total protozoa was 50.2 x 10(4) per ml, with no differences between species (P > 0.36). White-tailed deer had a higher percentage of Entodinium and lower percentage of Diplodiniinae (P < 0.01) than the other deer species, which were not different from each other. Occurrence of Epidinium, Isotricha, and Dasytricha was sporadic and did not differ among deer species. Numerous new host records of protozoan species were observed: white-tailed deer--four; axis deer--five; sika deer--five; fallow deer--four. This brings the total number of protozoan species identified in each deer species to: white-tailed--eight; axis--12; sika--15; fallow--16. For all species combined, protozoan concentration were 7.5 to 11-fold higher (P < 0.01) from Area 4, which differed from the other three areas by having a stream that allowed deer to have free access to water. Criteria used for identification of medium-size Eudiplodinium species were evaluated.

Effect of ruminal cellulolytic bacterial concentrations on in situ digestion of forage cellulose.

To evaluate the effects of ruminal cellulolytic bacterial concentrations on in vivo cellulose digestion, varying percentages of flaked soybean hulls were substituted for orchardgrass hay in high-forage diets fed to sheep. In two experiments, total and cellulolytic ruminal bacterial concentrations were not affected by diet. No differences were found for in situ digestion of forage cellulose in the first experiment; however, in Exp. 2, ruminal pH and in situ cellulose digestion were lower (P<.01) with a 40% soybean hull diet. In Exp. 3 with four sheep, two diets were compared, one containing 19.6% cellulose from alfalfa meal and the other 64.3% purified wood cellulose. Ruminal pH was lower (P<.02), 9 and 24 h after feeding, for the high-cellulose diet. Total bacterial concentrations did not change with diet; however, the concentration of cellulolytic bacteria increased (P<.05) when the higher cellulose diet was fed. In situ cellulose digestion was not different between diets. In Exp. 4, 3% sodium bicarbonate was added to the high-cellulose diet, and it was fed twice a day. No differences were observed in pH between diets (P>.42). However, the concentration of total ruminal bacteria increased (P<.06), the concentration of cellulolytic bacteria increased (P<.03), and the percentage of cellulolytic bacteria increased (P<.04) when the buffered high-cellulose diet was fed. In situ digestion of alfalfa cellulose at 30 h was not different between diets (P>.60). These data indicate that the concentration of cellulolytic bacteria is not the limiting factor in the digestion of cellulose in the rumen.

Generation times of Epidinium caudatum and Entodinium caudatum, determined in vitro by transferring at various time intervals.

Generation times were determined in vitro with a pure culture of Epidinium caudatum and a mixed culture of Epidinium caudatum and Entodinium caudatum. Measurement of logarithmic growth from a small inoculum for Epidinium caudatum alone, or in coculture, resulted in generation times of 30.8 and 19.5 h, respectively. Epidinium concentrations, either alone or in coculture, were maintained when cultures were transferred every 12 h; however, concentrations decreased rapidly with transfers at 4, 6, or 8 h. For Entodinium caudatum, a generation time of 16.3 h was obtained from measurement of logarithmic growth. Based on sequential transfer data at varying time intervals, Epidinium caudatum and Entodinium caudatum seem to be capable of doubling in approximately 12 to 13 h. These values are markedly less than those previously reported and help explain the ability of these protozoa to maintain themselves in the rumen.