Feeding live cultures of Enterococcus faecium and Saccharomyces cerevisiae induces an inflammatory response in feedlot steers.

Two experiments were conducted to investigate the effects of oral supplementation of the lactic-acid-producing bacterium Enterococcus faecium EF212 alone or in combination with Saccharomyces cerevisiae (yeast) on mediators of the acute phase response in feedlot steers. Eight fistulated steers were used to study the effects of E. faecium alone or with yeast in a crossover design with 2 Latin squares, 4 steers within each square, and 2 periods. The length of each period was 3 wk, with a 10-d adaptation and an 11-d measurement period. The experimental diet contained 87% steam-rolled barley, 8% whole-crop barley silage, and 5% supplement (DM basis). In Exp. 1, treatments were control vs. the lactic-acid-producing bacterium E. faecium (6 x 10(10) cfu/d). In Exp. 2, treatments were control vs. E. faecium (6 x 10(10) cfu/d) and S. cerevisiae (6 x 10(10) cfu/d). The bacteria and yeast supplements were blended with calcium carbonate to supply 6 x 10(10) cfu/d when top-dressed into the diet once daily at the time of feeding (10 g/d). Steers fed the control diet received only carrier (10 g/d). Blood samples were collected from the jugular vein on d 17 and 21 of each period, and serum amyloid A (SAA), lipopolysaccharide binding protein (LBP), haptoglobin, and alpha1-acid glycoprotein (alpha1-AGP) were measured. Supplementation of feed with E. faecium had no effect on concentrations of SAA, LBP, haptoglobin, or alpha1-AGP in plasma compared with those of controls. However, feeding E. faecium and yeast increased (P = 0.02) plasma concentrations of SAA, LBP, and haptoglobin but had no effect on plasma alpha1-AGP. In conclusion, oral supplementation of E. faecium alone had no effect on the mediators of the acute phase response that were measured, whereas feeding of E. faecium and yeast induced an inflammatory response in feedlot steers fed high-grain diets. Further research is warranted to determine the mechanism(s) by which E. faecium and yeast stimulated production of acute phase proteins in feedlot steers.

Effects of bacterial direct-fed microbials and yeast on site and extent of digestion, blood chemistry, and subclinical ruminal acidosis in feedlot cattle.

Two studies were conducted to determine whether a bacterial direct-fed microbial (DFM) alone or with yeast could minimize the risk of acidosis and improve feed utilization in feedlot cattle receiving high-concentrate diets. Eight ruminally cannulated steers, previously adapted to a high-concentrate diet, were used in crossover designs to study the effects of DFM on feed intake, ruminal pH, ruminal fermentation, blood characteristics, site and extent of digestion, and microbial protein synthesis. Steers were provided ad libitum access to a diet containing steam-rolled barley, barley silage, and a protein-mineral supplement (87, 8, and 5% on a DM basis, respectively). In Exp. 1, treatments were control vs. the lactic-acid producing bacterium Enterococcus faecium EF212 (EF; 6 x 10(9) cfu/d). In Exp. 2, treatments were control vs EF (6 x 10(9) cfu/d) and yeast (Saccharomyces cerevisiae; 6 x 10(9) cfu/d). Supplementing feedlot cattle diets with EF in Exp. 1 increased (P < 0.05) propionate and (P < 0.05) decreased butyrate concentrations, decreased the nadir of ruminal pH (P < 0.05), enhanced the flow of feed N (P < 0.10) to the duodenum but reduced that of microbial N (P < 0.10), reduced (P < 0.10) intestinal digestion of NDF, and increased (P < 0.10) fecal coliform numbers. Other than the increase in propionate concentrations that signify an increase in energy precursors for growth, the other metabolic changes were generally considered to be undesirable. In Exp. 2, providing EF together with yeast abolished most of these undesirable effects. Combining EF with yeast increased the DM digestion of corn grain incubated in sacco, but there were no effects on altering the site or extent of nutrient digestion. The diets used in this study were highly fermentable, and the incidence of subclinical ruminal acidosis, defined as steers with ruminal pH below 5.5 for prolonged periods of time, was high. Supplementing the diet with EF, with or without yeast, had limited effects on reducing ruminal acidosis. It seems that cattle adapted to high-grain diets are able to maintain relatively high feed intake and high fiber digestion despite low ruminal pH. The Enterococcus faecium bacterium and yeast used in this study were of limited value for feedlot cattle already adapted to high-grain diets.

Direct-fed microbial supplementation on the performance of dairy cattle during the transition period.

The influence of a direct-fed microbial (DFM) on the prepartum period and the effects on production performance during the postpartum period was investigated using 64 multiparous Holstein cows. Two close-up dry cow diets were fed to two groups of 32 cows each starting 21 d precalving as follows: 1) no DFM and 2) DFM. Post-calving cows were fed a lactation ration with or without DFM supplementation.The direct-fed microbial (DFM) supplement contained 2 × 109 viable yeast cells and 5 × 109 cfu of bacteria (Enterococcus faecium) per cow per day, top dressed in a 90-g supplement [corrected].The DMI during the prepartum period was not affected by DFM supplementation. During the postpartum period, DMI, milk yield, and milk protein content were higher for cows receiving DFM supplementation compared with no DFM. Blood glucose and insulin levels were higher and NEFA levels were lower for cows receiving DFM during the postpartum period. These data suggest that targeted DFM supplementation increased DMI and milk production postpartum. Blood metabolite information would suggest this response was associated with more glucose being made available and less fatty acids being mobilized from lipid stores.

Effects of bacterial direct-fed microbials on ruminal fermentation, blood variables, and the microbial populations of feedlot cattle.

A study was conducted to determine whether bacterial direct-fed microbials (DFM) could be used to minimize the risk of acidosis in feedlot cattle receiving high concentrate diets. Six ruminally cannulated steers, previously adapted to a high concentrate diet, were used in a double 3 x 3 Latin square to study the effects of DFM on feed intake, ruminal pH, and ruminal and blood characteristics. Steers were provided ad libitum access to a diet containing steam-rolled barley, barley silage, and a protein-mineral supplement at 87, 9, and 4% (DM basis), respectively. Treatments were as follows: control, Propionibacterium P15 (P15), and Propionibacterium P15 and Enterococcus faecium EF212 (PE). The bacterial treatments (10(9) cfu/g) plus whey powder carrier, or whey powder alone for control, were top-dressed once daily at the time of feeding (10 g/[steer/d]). Periods consisted of 2 wk of adaptation and 1 wk of measurements. Ruminal pH was continuously measured for 6 d using indwelling electrodes. Dry matter intake and ruminal pH (mean, minimum, hours, and area pH < 5.8 or < 5.5) were not affected by treatment (P > 0.05). However, supplementation with P15 increased protozoal numbers (P < 0.05) with a concomitant increase in ruminal NH3 concentration (P < 0.01) and a decrease in the number of amylolytic bacteria (P < 0.05) compared with the control. Streptococcus bovis, enumerated using a selective medium, was numerically reduced with supplementation of PE. Although blood pH and blood glucose were not affected by DFM supplementation, steers fed PE had numerically lower concentrations of blood CO2 than control steers, which is consistent with a reduced risk of metabolic acidosis. Although the bacterial DFM used in this study did not induce changes in DMI or ruminal and blood pH, some rumen and blood variables indicated that the bacterial DFM used in this study may decrease the risk of acidosis in feedlot cattle.

Ruminal supplementation of direct-fed microbials on diurnal pH variation and in situ digestion in dairy cattle.

To evaluate the effect of direct-fed microbial (DFM) concentration on diurnal rumen pH profiles and in situ digestibilities, nine ruminally cannulated cows in early lactation were fed treatments consisting of DFM (Enterococcus faecium, Lactobacillus plantarum, and Saccharomyces cerevisiae) at a level of a) 1 x 10(5) cfu/ml of rumen fluid (10(5)), b) 1 x 10(6) cfu/ml of rumen fluid (10(6)), and c) 1 x 10(7) cfu/ml rumen fluid (10(7)). Treatments were directly administered via rumen cannula once daily. Cows were fitted with pH probes in their cannula and connected to dataloggers, which monitor pH hourly. The experimental period was 21 d: 7-d adjustment, 14-d for pH, and in situ measurements. Cows fed 10(5) were able to sustain a higher nadir pH than were cows fed 10(6) or 10(7). Cows fed 10(5) had a higher digestion rate of high moisture ear corn (HMEC) dry matter. Corn silage digestion was higher for cows fed 10(5) and 10(6) compared with those receiving 10(7). There were no carryover effects of treatment associated with rumen pH when switching from one treatment regimen to the next. Results from this study demonstrate that incorporation of a specific level of DFM aids in reducing diurnal ruminal acidity.

Assessment of reductive acetogenesis with indigenous ruminal bacterium populations and Acetitomaculum ruminis.

The objective of this study was to evaluate the role of reductive acetogenesis as an alternative H2 disposal mechanism in the rumen. H2/CO2-supported acetogenic ruminal bacteria were enumerated by using a selective inhibitor of methanogenesis, 2-bromoethanesulfonic acid (BES). Acetogenic bacteria ranged in density from 2.5 x 10(5) cells/ml in beef cows fed a high-forage diet to 75 cells/ml in finishing steers fed a high-grain diet. Negligible endogenous acetogenic activity was demonstrated in incubations containing ruminal contents, NaH13CO3, and 100% H2 gas phase since [U-13C]acetate, as measured by mass spectroscopy, did not accumulate. Enhancement of acetogenesis was observed in these incubations when methanogenesis was inhibited by BES and/or by the addition of an axenic culture of the rumen acetogen Acetitomaculum ruminis 190A4 (10(7) CFU/ml). To assess the relative importance of population density and/or H2 concentration for reductive acetogenesis in ruminal contents, incubations as described above were performed under a 100% N2 gas phase. Both selective inhibition of methanogenesis and A. ruminis 190A4 fortification (>10(5) CFU/ml) were necessary for the detection of reductive acetogenesis under H2-limiting conditions. Under these conditions, H2 accumulated to 4, 800 ppm. In contrast, H2 accumulated to 400 ppm in incubations with active methanogenesis (without BES). These H2 concentrations correlated well with the pure culture H2 threshold concentrations determined for A. ruminis 190A4 (3,830 ppm) and the ruminal methanogen 10-16B (126 ppm). The data demonstrate that ruminal methanogenic bacteria limited reductive acetogenesis by lowering the H2 partial pressure below the level necessary for H2 utilization by A. ruminis 190A4.

Effects of a live yeast culture and enzymes on in vitro ruminal fermentation and milk production of dairy cows.

Live yeast culture (Saccharomyces cerevisiae) grew best on malt extract agar and required incubation under aerobic conditions to maximize the number of viable cells. In sterile, anaerobic ruminal fluid that had been supplemented with malt extract, yeast cells remained viable and metabolically active for up to 48 h, as indicated by the production of ethanol. A supplement containing live yeast and enzymes was fed twice daily with a diet of 50:50 (wt/wt) forage to concentrate (dry matter basis) to continuous fermentors inoculated with mixed ruminal microorganisms. The supplement had no effect on major fermentation acids or pH. After the last supplement with yeast was fed, numbers of yeast immediately decreased in the fermentors and were not detectable after 24 h. In the first of two lactation experiments, Holstein cows in midlactation were offered a diet with corn silage as the primary forage source. Half of the cows received a top-dressing based on corn that contained 10 g/d of the yeast and enzyme supplement. The supplement had no effect on milk production, milk composition, or dry matter intake. In a second lactation experiment, high producing cows in early lactation were fed 0, 10, and 20 g/d of the supplement. Cows fed the control diet produced 36.4 kg of milk/d, and milk production was 39.3 and 38.0 kg/d from cows fed 10 and 20 g of yeast/d, respectively.

Evaluation of health and ruminal variables during adaptation to grain-based diets in beef cattle.

Health and ruminal variables were intensively measured during adaptation to grain-based diets in 6 beef cattle with fistulated rumens. The cows had been maintained on prairie grass hay-supplemented diets, and were converted to a grain-based finishing ration by feeding each successive diet (diets 1-4, respectively) for a period of 7 days. Each cow was evaluated and samples were obtained 3 times each day for the first 5 days that each diet was fed. Health variables monitored were rectal temperature, pulse, respiratory and rumen motility rates, fecal consistency, demeanor, blood pH, and blood glucose and L (+) lactate concentrations. Ruminal variables monitored were pH and glucose, DL-lactate, and volatile fatty acid concentrations of rumen contents. Data were analyzed by use of a multivariate ANOVA. We determined that most of the health variables were within reference range limits throughout the adaptation period; however, analysis of pulse and respiratory rates indicated that diets 2 and 4 were stressful. Although blood pH continually decreased during feeding of the 4 diets (7.38 to 7.30), blood L (+) lactate and glucose concentrations had large increases only within diet 4. The pH of ruminal contents decreased progressively from 6.8 to 5.3. Rumen glucose concentration was low (< mumol/ml), except with diet 4 in which values were 8 times higher than for other diets. By the end of the study, the ruminal contents of all animals were acidic (pH < 5.5), and, on the basis of higher than background amounts of ruminal glucose and DL-lactate, it was determined that rumen microbial equilibrium had not yet been achieved.(ABSTRACT TRUNCATED AT 250 WORDS)

Vitamin E is not degraded by ruminal microorganisms: assessment with ruminal contents from a steer fed a high-concentrate diet.

Using an in vitro incubation system containing undiluted ruminal contents from a steer fed a high-concentrate, corn-based diet, we examined microbial degradation of DL-alpha-tocopherol acetate (TA). Gas production, pH, and fermentation acid profiles were done in an initial experiment to ensure conditions for reproducible, viable cultures over 24 h. The pH decreased from 5.7 to 4.9, gas production averaged 3.4 mL/mL of ruminal contents, and > 300 mM fermentation acids were produced. We then monitored the fate of TA added to bottles containing ruminal contents. Three methods of TA extraction were tried, of which two were used in experiments. The two methods used were 1) hot ethanol in a Soxhlet apparatus and 2) chloroform/methanol. Each of these was used to extract added TA from a set of three in vitro experiments. Concentrations of TA were determined at 0 h and after 4, 8, and 24 h at 39 degrees C. In the three hot ethanol extracted experiments, TA recoveries were 85% at 0 h. With time of incubation, TA levels either 1) remained constant, 2) decreased then returned to the initial value, or 3) decreased by approximately 50%. These inconsistent results indicated that this extraction method was unacceptable. In the latter three experiments we used a chloroform/methanol extraction method. Recoveries of added TA averaged 96% overall. Thus, the level of TA remained constant during the 24-h period, suggesting that microbial destruction of TA does not occur. Rather, the previously reported losses of vitamin E may be attributable to incomplete extraction of tocopherol from high-concentrate ruminal contents.

Factors affecting the in vitro production of volatile fatty acids by mixed bacterial populations from the bovine rumen.

Factors affecting in vitro ruminal bacterial VFA production were examined. Treatments consisted of high and low initial pH (6.7, 5.7), osmolality (600, 400 mOsm) and concentrations of acetic (40, 0 mM) and propionic acids (20, 0 mM). Response variables measured included the production of acetic, propionic and total VFA, total gas and methane. Initial pH affected (P less than .05) most variables either independently or in combination with one or more of the other factors. Acetic acid production was reduced 40% (P = .03) when initial acetic acid concentrations were 40 mM compared with 0 mM. Also, acetic acid production was less (P less than .01) at low initial pH (5.7) than at high initial pH (6.7). Propionic acid production was greater (P = .05) at high vs low initial acetic acid concentrations. Propionic acid production was greater in response to low vs high initial osmolality, although the magnitude of this difference depended on initial pH (interaction P = .02). Total production of VFA was greater (P less than .01) at high than at low initial pH; however, at low initial pH, no difference (P greater than .05) was observed due to initial osmolality, whereas at high pH, production was greater (interaction P = .04) for low than for high initial osmolality. The diminished production of total VFA at pH 5.7 occurred primarily due to reduced acetic acid production, although increased production of propionic and butyric acids was noted.(ABSTRACT TRUNCATED AT 250 WORDS)

Enumeration of selected anaerobic bacterial groups in cecal and colonic contents of growing-finishing pigs.

Selected anaerobic bacterial groups in cecal and colonic contents of clinically healthy pigs fed a corn-soybean meal production diet were determined at sacrifice after 4, 8, and 11 weeks on feed, corresponding to intervals within the growing-finishing growth period. By using ruminal fluid-based media, the densities of the culturable anaerobic population; the cellulolytic, pectin-fermenting, pectin-hydrolyzing, xylan-fermenting; and the xylan-hydrolyzing, sulfate-reducing, and methanogenic bacterial populations were estimated. An analysis of variance was performed on these bacterial group variables to examine the effects of phase (weeks on feed), site (cecum or colon), or the interaction of phase with site. The population of total anaerobic bacteria was twice as dense in the colon as it was in the cecum (2 x 10(10) versus 1 x 10(10)/g [wet weight]; P = 0.001). The proportion of cellulolytic bacteria was lower at 4 weeks on feed than at 8 or 11 weeks (23 versus 32%; P = 0.026), while the proportion of pectin-fermenting bacteria depended on the interaction of phase with site (P = 0.021). The numbers of sulfate-reducing bacteria were significantly higher in the colon than in the cecum (6 x 10(7) versus 3 x 10(7); P = 0.014), as were methanogenic bacteria (19 x 10(7) versus 0.6 x 10(7); P = 0.0002). The remaining bacterial groups were stable with respect to phase and site. The results suggest that except for density differences, the microbial communities of the pig cecum and colon are similar in composition throughout the growing-finishing phase.

Enrichment and isolation of Acetitomaculum ruminis, gen. nov., sp. nov.: acetogenic bacteria from the bovine rumen.

Five strains of acetogenic bacteria were isolated by selective enrichment from the rumen of a mature Hereford crossbred steer fed a typical high forage diet. Suspensions of rumen bacteria, prepared from contents collected 7 h postfeeding, blended and strained through cheesecloth, were incubated in a minimal medium containing 10% clarified rumen fluid under either H2:CO2 (80:20) or N2:CO2 (80:20) headspace atmosphere. The selection criterion was an increment of acetate in the enrichments incubated under H2:CO2. Periodically, the enrichment broths were plated onto agar media and presumed acetogenic bacteria subsequently were screened for acetate production. Selected acetogenic bacteria utilized a pressurized atmosphere of H2:CO2 to form acetate in quantities 2 to 8-fold higher than when grown under N2:CO2. All presumptive acetogenic isolates were derived from either the 10(-7) or 10(-8) dilutions of rumen contents. All 5 strains were Gram-positive rods, and all utilized formate, glucose and CO. One strain required, and all were stimulated by, rumen fluid. No spores were observed with phase-contrast microscopy and two strains were motile. No methane was detected in the headspace of pure cultures grown under either gas phase. The isolation of these bacteria indicates that acetogenic bacteria are inhabitants of the rumen of the bovine fed a typical diet and suggests that they may be participants in the utilization of hydrogen in the rumen ecosystem. Strain 139B (= ATCC 43876) is named Acetitomaculum ruminis gen. nov., sp. nov. and is the type strain of this new species.

Postprandial changes in methanogenic and acidogenic bacteria in the rumens of steers fed high- or low-forage diets once daily.

Four ruminally fistulated Hereford steers (400 kg) were fed two isocaloric diets at 1.5 x maintenance once daily in a repeated measurement crossover experiment. Postprandial changes in hydrogen-oxidizing, carbon dioxide-reducing bacterial groups were monitored. The methanogenic bacterial populations were present at densities of 4 x 10(8) to 8 x 10(8)/g of ruminal contents on either the high- or low-forage diet. Numbers remained constant postprandially on the high-forage diet but showed a distinct rise and fall with the once-daily feeding of the low-forage diet. Presumed hydrogen- and carbon dioxide-utilizing, acid-producing (acidogenic) bacteria were present between 2 x 10(8) and 12 x 10(8)/g of ruminal contents, with the density of the low-forage population being twofold higher than that of the high-forage population. Acidogenic bacteria exhibited similar postprandial changes on both diets, with the predominant shift being associated with the feeding event. This is the first study which documents the postfeeding trends in ruminal methanogenic bacteria on specified, production-level diets. It is also the first study to suggest that other hydrogen-oxidizing, carbon dioxide-reducing bacteria which produce acid instead of methane are present at high population densities in the normally fed adult ruminant.

Postprandial trends in estimated ruminal digesta polysaccharides and their relation to changes in bacterial groups and ruminal fluid characteristics.

In a diurnal study, feedstuff and digesta polysaccharides, ruminal bacterial carbohydrate-fermenting groups, and selected ruminal fluid characteristics (ruminal pH, ammonia and volatile fatty acids) were measured in ruminal-cannulated Holstein steers fed high- or low-forage diets at maintenance level intake once daily. A procedure for the sequential extraction of soluble sugar, starch, pectin, hemicellulose and cellulose from feedstuffs was developed to measure these carbohydrates in dietary and ruminal digesta samples. Recovery of dry matter (determined chemically) using this scheme was 60 to 70%. Data were obtained within the ranges of those in the literature for similar feedstuffs and(or) by similar methods. Dietary analysis by the sequential method yielded total recovery across all carbohydrate fractions of 87 and 81% for the high- and low-forage diets, respectively, and similar recoveries were obtained for the digesta samples. Analytical variation was small (less than or equal to 15% CV), which permitted comparison of the carbohydrate profiles of the digesta over time. From these values, total ruminal digesta polysaccharide content was calculated and, when plotted over time, indicated that the disappearance per fraction corresponded with theoretical curves for ruminal fermentation of major feedstuff components. The postprandial variation within the bacterial population carbohydrate-fermenting groups was small, but changes were consistent with digesta component fermentation. Xylan- and cellulose-fermenting groups followed a pattern compatible with the disappearance of these polysaccharides from the rumen. In contrast, soluble sugar-fermenting groups predominated at all times despite the rapid rise and fall of these components in the digesta. Ruminal fluid pH, ammonia and total carbohydrate supported the digesta and bacterial trends observed. The data are interpreted to suggest that once daily maintenance feeding of high- or low- forage diets permits detection of digesta sugar and polysaccharide changes, supports a relatively stable microbial population while specific groups increase and decrease with the availability of substrate, and results in few differences in ruminal fluid traits.

Enumeration and presumptive identification of bacteria from the small intestine of sheep.

Microflora of the small intestine of sheep was examined. Samples of tissue and digesta were taken from four sites along the small intestine (3, 8, 13, and 18 m from the pylorus) and maintained under anaerobic conditions. The pH of the digesta taken at the 3rd, 8th, 13th, and 18th m sections were 5.44, 6.37, 7.24, and 6.79. Digesta and tissue samples were diluted serially and plated within the anaerobic glove box on complex medium containing rumen fluid. In addition, each sample was plated on the same medium and incubated aerobically. As determined by growth under anaerobic conditions, viable counts of the digesta ranged from 5 X 10(4) to 7 X 10(6) cells/g, whereas counts for the tissue ranged from 2 X 10(2) to 4 X 10(4) cells/g. Viable counts determined aerobically for the digesta ranged from 1 X 10(4) to 5 X 10(6) cells/g, whereas the counts for the tissue ranged from 5 X 10 to 6 X 10(4) cells/g. Tentative identification of 50 isolates from samples of tissue and digesta in the 13th m section revealed a predominance of streptococcal organisms and rod shaped organism morphologically similar to Propionibacterium. An indigenous population of acid-tolerant bacteria with the ability to ferment simple sugars and starch was in the small intestine.

Changes of bacterial numbers and carbohydrate fermenting groups during in vitro rumen incubations with feedstuff materials.

Short in vitro incubations of mixed ruminal bacteria with feedstuff materials were conducted. In each series of experiments, the rumen bacterial population was divided into equal portions. One portion was washed anaerobically by centrifugation in a mineral buffer prior to initiation of incubation, whereas the other portion was not washed. Changes of carbohydrate group composition of the populations, of bacterial cell composition, and of incubation fluid characteristics were monitored over 24 h. Washing the bacterial inoculum did not affect adversely the ensuing feed fermentation and resulted in lower background carbohydrates and fermentation acids. Unwashed bacterial incubations had a greater tendency to produce more lactate. When the feedstuff substrate contained more soluble nutrients, the microbial population exhibited batch culture type growth, and bacteria fermenting soluble carbohydrate predominated. When alfalfa hay was the sole substrate, a more balanced fermentation resulted with respect to both carbohydrate group composition and fermentation acid production. Implications of these findings in relation to use of in vitro methods to estimate digestibility of feedstuff are addressed.

Diurnal variations in bacterial numbers and fluid parameters in ruminal contents of animals fed low- or high-forage diets.

Differential carbohydrate media and anaerobic replica plating techniques were used to assess the degrees of diurnal variations in the direct and viable cell counts as well as the carbohydrate-specific subgroups within the mixed rumen bacterial populations in cattle fed maintenance (metabolizable energy) levels of either a high-forage or a high-concentrate diet once daily. The rumen was sampled at 1 h before feeding and 2, 4, 8, 12, and 16 h after feeding, and selected microbiological parameters of the isolated bacterial populations were assessed. Corresponding samples of ruminal fluid were assayed for fermentation acids, carbohydrate, ammonia, and pH changes. The data showed that regardless of diet, total bacterial numbers remained fairly constant throughout the day. The number of viable bacteria declined 40 to 60% after feeding and then increased to a maximum at 16 h postfeeding. Changes occurred in the carbohydrate-specific subgroups within the bacterial populations, and some of the changes were consistent with a predicted scheme of ruminal feedstuff carbohydrate fermentation. Regardless of diet, however, soluble-carbohydrate-utilizing bacteria predominated at all times. Xylan-xylose and pectin subgroups respectively comprised about one-half and one-third of the population when the high-forage diet was given. These subgroups, along with the cellulolytics, constituted lesser proportions of the population when the high-concentrate diet was given. The cellulolytic subgroup was the least numerous of all subgroups regardless of diet but followed a diurnal pattern similar to that predicted for cellulose fermentation. There were few diurnal variations or differences in bacterial cell compositions and ruminal fluid parameters between diets. The observed similarities and dissimilarities of the rumen bacterial populations obtained when the two diets were given are discussed. The data are consistent with the versatility and constancy of the rumen as a stable, mature microbial system under the specific low-level feeding regimens used.

1,4-Naphthoquinone and other nutrient requirements of Succinivibrio dextrinosolvens.

Three strains of Succinivibrio dextrinosolvens isolated from the rumen of cattle or sheep under diverse conditions grew well in a minimal medium containing glucose, minerals, cysteine, methionine, leucine, serine, ammonia, 1,4-naphthoquinone, p-aminobenzoic acid, and bicarbonate-carbonic acid buffer, pH 6.7. When menadione or vitamin K5 was substituted for 1,4-naphthoquinone, the growth rate was somewhat depressed. Growth was poor with vitamin K1 and ammonia, further addition of the amino acids aspartic acid, arginine, histidine, and tryptophan was necessary for good growth of type strain 24, but the other two strains grew well only in media containing ammonia. Strains C18 and 22B produced urease and grew well when ammonia replaced urea. When urea replaced ammonia, strain 24 grew poorly and urease activity could not be detected. Strain 24 required no B-vitamins, but the other two strains were stimulated by p-aminobenzoic acid. The methionine requirement was not placed by vitamin B12, betaine, or homocysteine. Cysteine was replaced by sulfide in strain 24 but less well in the other two strains. Very poor growth was obtained when sulfate replaced cysteine. The half-saturation constant for ammonia during growth of S. dextrinosolvens is more than 500 microM, a much higher value than that of many rumen bacteria.

Differential carbohydrate media and anaerobic replica plating techniques in delineating carbohydrate-utilizing subgroups in rumen bacterial populations.

A basal (BC) medium devoid of added carbohydrates, a complete (CC) medium containing nine carbohydrates were developed for enumerating rumen bacteria. The colony counts on the BC medium were 85 to 100% of those obtained on the CC medium. These colonies were pinpoint size (less than or equal to mm in diameter) but increased in size (2 to 5 mm in diameter) when carbohydrates were subsequently added. With the CC medium or other media tested, the colony counts were 20 to 50% higher on plates than on roll tubes and were about 35% of the direct cell counts. The lower colony counts on roll tubes were shown to result primarily from the loss of viability due to heat stress. The DC media were found by plating techniques to be suitable for differentiating mixed rumen bacterial populations into subgroups based upon carbohydrate utilization as shown by differences in subgroup profiles found within solid and liquid fractions of rumen contents, within rumen contents from animals fed high-forage and high-grain diets, and by correct colony formations by pure cultures of rumen bacteria on appropriate DC media. With simple modifications and use of an anaerobic glove box, replica plating methods and the CC and DC media were found to be a suitable means of rapidly determining the range of utilizable carbohydrate energy sources of rumen bacteria.