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Twenty-four multiparous and fifteen first lactation Holstein cows averaging 263 days in milk and weighing 614 kg were fed diets adequate or deficient in ruminal nitrogen (N), based on predictions of the Cornell Net Carbohydrate and Protein System (CNCPS). After adjustment to a low crude protein (CP) total mixed rations (TMR; 12.6% CP), the cows were allocated to 13 blocks based on lactation number, milk production, body condition score, and body weight. Within each block, cows were randomly assigned to one of the 3 treatment (TRT) diets (9.4, 11.1 and 14.1% CP for TRT 1, 2, and 3, respectively). All diets contained the same proportion of high moisture corn, chopped grass hay, and minerals, with urea substituted for corn silage as needed to reach the three CP levels. The TRT diets were then fed to the cows for 4 wk. Milk production was significantly affected by TRT: 15.5, 18.8, and 21.7 kg/d for TRT diets 1, 2, and 3, respectively. DMI was increased significantly as the percentage of CP increased from 9.4 to 14.1% CP: 17.6, 20.0, and 21.2 kg/d for TRT diets 1,2, and 3, respectively. CNCPS predictions for production (with and without the N adjustment for ruminal N deficiency) of metabolizable protein (MP) allowable milk were compared with observed milk production. Using the average individual weekly cow data from all 3 TRT, we found that the CNCPS accounted for 72 and 68% of the variation in MP allowable milk without and with the N deficiency adjustment, respectively. The overall mean bias without the N adjustment was 3.3 kg of milk (over prediction model bias of 14.6%, P < 0.001), and the N adjustment reduced the model over-prediction bias to 0.01 kg of milk (P = 0.96).

We conducted a lactation trial with a fresh forage diet in order to evaluate 1) the effects of monensin on nitrogen metabolism, and 2) the Cornell Net Carbohydrate and Protein System (CNCPS). Thirty Holstein cows in midlactation (eight fitted with ruminal fistulas) were gradually introduced to a fresh forage diet. A concentrate mix based on corn meal was fed before the a.m. and p.m. milking times 0730 and 1730 h, then the fresh forage was fed at 0830 and 1830 h. Fifteen cows each were allocated to a control (no monensin) and a treatment group receiving 350 mg/cow per day of monensin in the p.m. concentrate feeding. A 7-d fecal and urine collection period and a 3-d rumen sampling period were conducted with the fistulated cows. After the lactation study was concluded, the fistulated cows were fed forage regrowth and a 3-d rumen sampling period was repeated. Monensin increased milk production by 1.85 kg. Milk fat and protein concentrations decreased and milk fat and protein yields increased, but the effects were nonsignificant. Monensin did not significantly affect DMI. Ruminal ammonia and the acetate-to-propionate ratio decreased with the addition of monensin in both fed forages. Monensin decreased fecal N output, and increased apparent N digestibility by 5.4%. Because of the decrease in ruminal ammonia and increase in apparent N digestibility, we concluded monensin was sparing amino acids from wasteful rumen degradation with a fresh forage diet. The precision of the CNCPS in predicting performance was high (r2 = 0.76), and the bias was low (overprediction of 3.6%). These results indicate that the CNCPS can be used for dairy cows consuming fresh forage and gives realistic predictions of performance.

Escherichia coli O157:H7 is a pathogenic bacterium that causes acute illness in humans, but mature cattle are not affected. E. coli O157:H7 can enter the human food supply from cattle via fecal contamination of beef carcasses at slaughter. Previous attempts to correlate the incidence of E. coli O157:H7 with specific diets or feeding management practices gave few statistically significant or consistent findings. However, recent work indicates that cattle diets may be changed to decrease fermentation acid accumulation in the colon. When fermentation acids accumulate in the colon and pH decreases, the numbers of acid-resistant E. coli increase; acid-resistant E. coli are more likely to survive the gastric stomach of humans. When cattle were fed hay for a brief period (<7 d), acid-resistant E. coli numbers declined dramatically. Other workers have shown that brief periods of hay feeding can also decrease the number of cattle shedding E. coli O157:H7, and a similar trend was observed if cattle were taken off feed and exposed to simulated transport. These observations indicate that cattle feeding management practices may be manipulated to decrease the risk of foodborne illness from E. coli, but further work will be needed to confirm these effects.

Grain feeding often causes a decrease in ruminal pH, and experiments were conducted to define the role of pH in regulating the acetate to propionate ratio and production of CH4. Cows that were fed 90% concentrate had lower ruminal pH values (6.22 vs. 6.86), higher VFA concentrations (85 vs. 68 mM), and lower acetate to propionate ratios (2.24 vs. 4.12) than did cows that were fed forage only. When mixed ruminal bacteria from cows that were fed 90% concentrate or 100% forage were incubated (48 h) with hay (10 g/L) or cracked corn (5 g/L) in a medium containing bicarbonate (38 mM) and tricarballylate (50 mM), the final pH values were less than 0.3 units lower than the initial pH. At final pH values less than 5.7, hay fermentation was inhibited, the acetate to propionate ratio and CH4 production declined more than twofold, and the inoculum source was without effect. Small amounts of H2 were detected at pH values less than 5.5. Total VFA production from cracked corn decreased when pH declined, but only if the inoculum was obtained from cows that were fed 90% concentrate. The acetate to propionate ratio of cracked corn incubations declined from 1.2 to 0.6 when final pH was decreased from 6.5 to 5.3, and CH4, as a percentage of total VFA production, also decreased. At pH values less than 5.3, the acetate to propionate ratio of cracked corn increased more than fourfold, and large amounts of H2 could be detected. Over the final pH range of 6.5 to 5.3, CH4 production was highly correlated with acetate to propionate ratio, which was dependent on pH and substrate (CH4 = 0.02 + 0.05 pH; r2 = 0.80). Calculations based on the differences between pH 6.5 and 5.8 indicated that as much as 25% of the decrease in acetate to propionate ratio could be explained by the effect of pH alone.

The gastric stomach of humans is a barrier to food-borne pathogens, but Escherichia coli can survive at pH 2.0 if it is grown under mildly acidic conditions. Cattle are a natural reservoir for pathogenic E. coli, and cattle fed mostly grain had lower colonic pH and more acid-resistant E. coli than cattle fed only hay. On the basis of numbers and survival after acid shock, cattle that were fed grain had 10(6)-fold more acid-resistant E. coli than cattle fed hay, but a brief period of hay feeding decreased the acid-resistant count substantially.

When steers (n = 4) were fed increasing amounts of concentrate (0, 45, or 90% of DM) and decreasing amounts of forage, the VFA concentration increased (P < .001) and ruminal pH, acetate:propionate ratio, and dissociated ammonia declined (P < .001). Acetate:propionate ratio and dissociated ammonia were highly correlated (r2 = .82 and .65, respectively) with ruminal pH. In vivo acetate:propionate ratio was highly correlated (r2 = .78) with the capacity of the bacteria to produce methane from H2 and CO2 in vitro, and in vivo pH-dissociated ammonia was correlated (r2 = .59) with the capacity of the bacteria to produce ammonia from protein hydrolysate. The role of pH in regulating methane and ammonia production was supported by the effect of pH in vitro. When bacteria from cattle fed concentrate or forage were incubated at pH values from 6.5 to 5.7, methane production decreased (P < .001) from 48 to 7 nmol x mg protein(-1) x min(-1) and from 14 to 2 nmol x mg protein(-1) x min(-1), respectively. The reduction in in vitro pH (6.5 to 5.7) also decreased (P < .001) the rates of ammonia production, but only if the bacteria were obtained from cattle fed forage (28 to 15 nmol x mg protein(-1) x min(-1)). Bacteria from cattle fed 90% concentrate had similar (P > .05) rates of ammonia production at pH 6.5 to 5.7 (approximately 12 nmol x mg protein(-1) x min(-1)). These results indicated that ruminal pH affected ruminal methane production, acetate:propionate ratio, deamination, and ammonia concentration.

We conducted two growth trials to evaluate the effects of monensin on amino acid sparing. When Holstein steers were fed a 90% concentrate diet supplemented with soybean meal (13.5% CP), the DMI, ADG, and efficiencies of feed and nitrogen utilization were greater than with urea (P < .10). Monensin improved ADG with both nitrogen supplements (P < .01), but the positive effects of monensin on efficiencies of feed (P = .12) and nitrogen (P = .26) utilization were greater for soybean meal than for urea. Increasing amounts of monensin (0, 11, or 22 mg/kg of DM) caused a linear increase in DMI with urea. Diets with soybean had greater intakes than diets with urea (P < .01); the greatest intake was of a soybean diet with monensin at 11 mg/kg of DM. Holstein steers fed soybean meal at 13.5% CP had lower DMI and greater efficiencies of feed and nitrogen utilization than steers fed 16.7% CP (P < .10). Crude protein level had no effect on ADG (P > .10). Monensin always increased the efficiencies of feed and nitrogen utilization (P < .05), but these trends were greater for diets with 16.7 than for those with 13.5% CP. Overall, monensin decreased DMI (P < .01), but this effect was greater for 16.7% than for 13.5% CP. Because the positive effects of monensin on diet NEg (P = .16) and efficiency of nitrogen utilization (P = .26) were greater for soybean meal than for urea, it seemed that monensin was sparing amino acids.

When mixed ruminal bacteria were starved in vitro for 24 h, cellular ATP decreased, but there was little change in cell protein. Starved ruminal bacteria derived most of their ATP from cellular polysaccharide. Because polysaccharide declined at a first-order rate of 23%/h, it was possible to estimate the endogenous polysaccharide utilization rate at various stages of starvation by multiplying the amount of utilizable polysaccharide remaining at each time point by 0.23. The bacteria initially had a rate of soluble carbohydrate fermentation that was > 717 micrograms of hexose equivalent/mg of protein per h. Starvation had little impact on the rate of soluble carbohydrate fermentation until 8 to 12 h, and the endogenous polysaccharide utilization rate was < 10 micrograms of hexose/mg of protein per h. The bacteria digested ball-milled cellulose at a rate of 24 micrograms of hexose/mg of protein per h for 8 to 12 h. Even bacteria that had been starved for 24 h fermented cellulose at a rate of 16 micrograms of hexose/mg of protein per h. The rate of methane production was initially 70 nmol of methane/mg of protein per min. Short periods of starvation (< 12 h) had little impact on methane production, but longer times caused an almost complete inhibition of methanogenesis. The rate of amino acid deamination was initially 31 nmol of ammonia/mg of protein per min, and the critical phase of starvation was again 8 to 12 h. Ruminal bacteria that were harvested at 24 h after feeding had 10-fold less polysaccharide than did bacteria that were harvested at 2 h after feeding, but this polysaccharide supported high rates of soluble carbohydrate and cellulose fermentation, deamination, and methane production.

A ruminal fermentation study was used to investigate the relationship between forage quality and monensin-dependent amino acid-sparing. Two fistulated cows were fed three concentrations of chopped (2.5 to 5.0 cm in length) timothy and alfalfa hays (100:0, 50:50 and 0:100) and two levels of monensin (0 and 350 mg.cow-1.d-1). The diets were offered 12 times per day (9 kg of DM/d), and the rumen reached a steady-state, reducing animal and day variation. Alfalfa hay had 1.4 times more CP and 1.4 times less NDF than timothy hay, and the substitution of timothy with alfalfa increased (P < .05) ruminal ammonia. Monensin had no effect on total ruminal ammonia when timothy hay was present in the diet, and it increased total ruminal ammonia with the 100% alfalfa diet. Effects on ruminal ammonia were, however, confounded by monensin-dependent decreases in ruminal pH (P < .05). Dissociated ammonia, the species most likely to be adsorbed from the rumen, declined when monensin was added to the 100% timothy diet (P < .05). Monensin had no effect on dissociated ammonia if alfalfa was 50% or 100% of the forage, but it counteracted alfalfa-dependent decreases in bacterial protein (P < .05). The idea that monensin could spare amino acids was supported by the observation that monensin decreased (P < .001) the specific activity of deanimation and increased bacterial protein at all combinations of alfalfa and timothy. Increases in bacterial protein could be explained by monensin-dependent increases in total VFA.

The predominant ruminal bacteria that were obtained from a 10(8) dilution of ruminal fluid could be maintained as a mixed population for long periods as long as the bacteria were provided with a complex mixture of carbohydrates. Growth of predominant ruminal bacteria in carbohydrate-limited, ammonia-excess, continuous cultures (0.07/h) had a low requirement for maintenance energy, but the nongrowth energy dissipation of ammonia-limited, carbohydrate-excess, predominant ruminal bacteria was approximately 10-fold higher (0.96 vs. 0.09 mg of hexose equivalent/mg of protein per h, respectively). Mathematical derivations indicated that this additional nongrowth energy dissipation could be accommodated by an energy spilling function that was independent of the growth rate. Peptides and amino acids had little impact on the yield of carbohydrate-limited, ammonia-excess, continuous cultures (0.07/ h), but amino N greatly increased the growth rate and yield of excess-energy batch cultures. The change in growth rate and yield that was dependent on amino N indicated that the energy-excess batch cultures had the same capacity to spill energy as did the ammonia-limited, carbohydrate-excess, predominant ruminal bacteria (0.80 vs. 0.86 mg of hexose equivalent/mg of protein per h, respectively). When the energy-excess batch cultures were provided with amino N, the growth rate increased, the difference in anabolic and catabolic rates was smaller, and less energy was spilled.

Gram-negative, ionophore-resistant ruminal bacteria and Gram-positive, ionophore-sensitive species bound similar amounts of [14C]lasalocid, but neither group bound large amounts of [14C]monensin. Membrane vesicles also bound more lasalocid than monensin (P < .05). The binding was first-order at low cell or vesicle concentrations and saturable at high cell or vesicle densities. Streptococcus bovis was inhibited by both monensin and lasalocid (5 microM), but cells that were re-incubated in medium lacking ionophore grew rapidly. Lasalocid-treated cells grew very slowly when they were resuspended in fresh medium. Based on these results, it seemed that lasalocid had a higher affinity for bacterial membranes than monensin. Mixed bacteria, however, bound nearly equal amounts of [14C]monensin and [14C]lasalocid (P > .05). Monensin binding was greatly reduced when the mixed ruminal bacteria were pretreated with Tris+EDTA (P < .05), but Tris+EDTA did not affect the binding of lasalocid. Mixed ruminal protozoa always took up more lasalocid than monensin (P < .05), but feed particles bound equal amounts of [14C]lasalocid and [14C]monensin (P > .05). Based on the binding capacity of mixed ruminal bacteria, ruminal protozoa, and feed particles, there would be little free ionophore in ruminal fluid.

When nonlactating Holstein cows (685 +/- 59 kg) were fed chopped timothy hay (9% CP, 7.0 kg/d) 12 times daily, the steady-state ruminal ammonia concentration was 2.6 mM, and the specific activity of ammonia production by mixed ruminal bacteria was 27.4 nmol/mg of protein-1.min-1. The addition of soybean meal (53% CP, 1 or 2 kg/d) to the basal diet caused a linear increase in ruminal ammonia (7.0 and 12.4 mM, respectively; P < .001), but there was only a small increase in the specific activity of ammonia production (30.7 and 33.8 nmol/mg of protein-1.min-1, respectively; P < .05). The addition of monensin (350 mg/d) to the diets caused more than a 30% decrease (P < .01) in ruminal ammonia at all levels of soybean supplementation, and there was a similar decrease (P < .001) in the specific activity of ammonia production. Before monensin addition, the most probable number of bacteria that could utilize peptides and amino acids, but not carbohydrates, as an energy source for growth was 5.8 to 7.0 x 10(6)/mL. When monensin was added to the diets, these bacteria decreased (P < .001) nearly 10-fold. Based on these results, it seemed that monensin inhibited highly active amino acid-fermenting ruminal bacteria, and this inhibition, in turn, decreased ruminal amino acid deamination and ammonia production. Because monensin did not increase soluble protein, peptides, or amino acids in ruminal fluid, it did not seem that the decrease in ammonia increased flow of dietary amino N to the lower gut.(ABSTRACT TRUNCATED AT 250 WORDS)

The Cornell Net Carbohydrate and Protein System (CNCPS) has equations for predicting nutrient requirements, feed intake, and feed utilization over wide variations in cattle (frame size, body condition, and stage of growth), feed carbohydrate and protein fractions and their digestion and passage rates, and environmental conditions. Independent data were used to validate the ability of the CNCPS to predict responses compared to National Research Council (NRC) systems. With DMI in steers, the CNCPS had a 12% lower standard error of the Y estimate (Sy.x) and three percentage units less bias than the NRC system. For DMI in heifers, both systems had a similar Sy.x but the NRC had four percentage units less bias. With lactating dairy cows' DMI, the CNCPS had a 12% lower Sy.x. Observed NEm requirement averaged 5% under NRC and 6% under CNCPS predicted values at temperatures above 9 degrees C but were 18% over NRC and 9% under CNCPS at temperatures under 9 degrees C. Energy retained was predicted with an R2 of .80 and .95 and a bias of 8 and 4% for the NRC and CNCPS, respectively. Protein retained was predicted with an R2 of .75 and .85 with a bias of 0 and -1% for NRC and CNCPS, respectively. Biases due to frame size, implant, or NEg were small. Body condition scores predicted body fat percentage in dairy cows with an R2 of .93 and a Sy.x of 2.35% body fat. The CNCPS predicted metabolizable protein allowable ADG with a bias of 1.6% with a Sy.x of .07 kg compared to values of -30% and .10 kg, respectively for the NRC system.

The Cornell Net Carbohydrate and Protein System (CNCPS) has a submodel that predicts rates of feedstuff degradation in the rumen, the passage of undegraded feed to the lower gut, and the amount of ME and protein that is available to the animal. In the CNCPS, structural carbohydrate (SC) and nonstructural carbohydrate (NSC) are estimated from sequential NDF analyses of the feed. Data from the literature are used to predict fractional rates of SC and NSC degradation. Crude protein is partitioned into five fractions. Fraction A is NPN, which is trichloroacetic (TCA) acid-soluble N. Unavailable or protein bound to cell wall (Fraction C) is derived from acid detergent insoluble nitrogen (ADIP), and slowly degraded true protein (Fraction B3) is neutral detergent insoluble nitrogen (NDIP) minus Fraction C. Rapidly degraded true protein (Fraction B1) is TCA-precipitable protein from the buffer-soluble protein minus NPN. True protein with an intermediate degradation rate (Fraction B2) is the remaining N. Protein degradation rates are estimated by an in vitro procedure that uses Streptomyces griseus protease, and a curve-peeling technique is used to identify rates for each fraction. The amount of carbohydrate or N that is digested in the rumen is determined by the relative rates of degradation and passage. Ruminal passage rates are a function of DMI, particle size, bulk density, and the type of feed that is consumed (e.g., forage vs cereal grain).

The Cornell Net Carbohydrate and Protein System (CNCPS) has a kinetic submodel that predicts ruminal fermentation. The ruminal microbial population is divided into bacteria that ferment structural carbohydrate (SC) and those that ferment nonstructural carbohydrate (NSC). Protozoa are accommodated by a decrease in the theoretical maximum growth yield (.50 vs .40 g of cells per gram of carbohydrate fermented), and the yields are adjusted for maintenance requirements (.05 vs .150 g of cell dry weight per gram of carbohydrate fermented per hour for SC and NSC bacteria, respectively). Bacterial yield is decreased when forage NDF is < 20% (2.5% for every 1% decrease in NDF). The SC bacteria utilize only ammonia as a N source, but the NSC bacteria can utilize either ammonia or peptides. The yield of NSC bacteria is enhanced by as much as 18.7% when proteins or peptides are available. The NSC bacteria produce less ammonia when the carbohydrate fermentation (growth) rate is rapid, but 34% of the ammonia production is insensitive to the rate of carbohydrate fermentation. Ammonia production rates are moderated by the rate of peptide and amino acid uptake (.07 g of peptide per gram of cells per hour), and peptides and amino acids can pass out of the rumen if the rate of proteolysis is faster than the rate of peptide utilization. The protein-sparing effect of ionophores is accommodated by decreasing the rate of peptide uptake by 34%. Validation with published data of microbial flow from the rumen gave a regression with a slope of .94 and an r2 of .88.

Mixed ruminal microorganisms from a cow fed timothy hay and concentrate supplement (50:50) were incubated with various protein sources for 15 h (no carbohydrates or growth), and deamination was studied under enzyme-limiting substrate-excess conditions (n = 3). Addition of amphotericin (10 micrograms/ml) killed protozoa and decreased (P less than .05) ammonia production from killed bacteria but it had no effect (P greater than .05) on casein deamination. Monensin (5 micrograms/ml) also killed protozoa; however, it decreased (P less than .05) casein deamination to a much greater extent than amphotericin. Antibacterial antibiotics (penicillin G, polymixin B, cephalosporin C and streptomycin) greatly reduced (P less than .05) ammonia formation from casein. Isolated bacteria always produced more ammonia than isolated protozoa, but the difference was less with heat-treated, particulate proteins. Heated soybean protein was as soluble as heated casein but it was deaminated (P less than .05) at a faster rate by bacteria. Nonammonia-nonprotein N accumulation was greater (P less than .05) with the protozoa than bacteria. When incubations containing bacteria or protozoa were compared with combinations of protozoa and bacteria, the combinations always caused a synergistic increase in ammonia and decrease (P less than .05) in nonammonia-nonprotein N. These results suggest: soluble proteins were primarily degraded by bacteria; protozoa could contribute to the degradation of insoluble, particulate proteins; protozoa were limited in their ability to assimilate peptides (or amino acids); low molecular weight products could be fermented more readily by bacteria and monensin was toxic to protozoa, but decreases in ammonia were primarily due to action of monensin on bacteria.

Two monensin-sensitive bacteria which utilized carbohydrates poorly and grew rapidly on amino acids were isolated from the bovine rumen. The short rods (strain SR) fermented arginine, serine, lysine, glutamine, and threonine rapidly (greater than 158 nmol/mg of protein per h) and grew faster on casein digest containing short peptides than on free amino acids ().34 versus 0.29 h(-1)). Gelatin hydrolysate, an amino acid source containing an abundance of long peptides, was unable to support growth or ammonia production, but there was a large increase in ammonia production if strain SR was cocultured with peptidase-producing ruminal bacteria (Bacteroides ruminicola or Streptococcus bovis). Cocultures showed no synergism with short peptides. Strain SR washed out of continuous culture ().1 h(-1)) at pH 5.9. The irregularly shaped organisms (strain F) deaminated glutamine, histidine, glutamate, and serine rapidly (greater than 137 nmol/mg of protein per min) and grew faster on free amino acids than on short peptides ().43 versus 0.21 h(-1)). When strain F was provided with casein or gelatin hydrolysate and cocultured with peptidase-producing bacteria, there was a more than additive increase in ammonia production. Strain F grew in continuous culture (0.1 h(-1)) when the pH was as low as 5.3. The irregularly shaped cells and short rods were present at less than 10(9)/ml in vivo, but they ahd very high specific activities of ammonia production (greater than 310 nmol of ammonia/mg of protein per min) and could play an important role in ruminal amino acid fermentation.

A relatively simple and sensitive procedure was developed to measure the concentration of peptides in rumen fluid. Feed particles and microorganisms were removed by centrifugation, and the supernatant was treated with perchloric acid (5% final concentration). Perchloric acid precipitated macromolecules that included protein, RNA, and DNA. Perchlorate was subsequently removed by precipitation with an excess of potassium carbonate. Ammonia was removed by boiling the alkaline sample. Supernatant samples were then analyzed for ninhydrin reactive material before and after HCl hydrolysis. Because ninhydrin reaction was 3 to 7.5 times greater after HCl hydrolysis, peptides rather than amino acids were the primary source of nonprotein, nonammonia nitrogen. Rumen fluid from a cow fed timothy hay and concentrate supplement (16% crude protein) contained more than 1200 mg peptides/L (192 mg N/L), 1 h after feeding, and this value declined the prefeeding value of 400 mg/L (64 mg N/L) by 8 h after feeding. Comparison of ninhydrin reactivity with and without HCl hydrolysis indicated that peptides present before feeding contained more peptide bonds than the peptides soon after feeding. High pressure liquid chromatography revealed a variety of peaks soon after feeding and fewer dominant peaks 8 h later. The data suggest that peptide uptake into rumen microorganisms can be a rate-limiting step in ruminal protein degradation.

Mixed ruminal bacteria (n = 4) were incubated in anaerobic media for 24 h in vitro with either hay, corn meal, protein hydrolyzate or hydrogen gas as the substrate. The ionophore monensin and the polypeptide antibiotic bacitracin were added to the incubation flasks at concentrations ranging from 0 to 10 or 40 mg/liter. As was expected, monensin decreased methane production, increased the ratio of propionate to acetate and decreased the deamination of amino acids. Monensin had little effect on methane production, however, if hydrogen gas was the fermentation substrate. Bacitracin, another gram-positive antibiotic with a distinctly different cellular target, was somewhat less potent than monensin, but it produced strikingly similar responses. This similarity of fermentation patterns suggested that monensin action in the rumen is probably due to its activity as a gram-positive antibiotic, and that any gram-positive antibiotic not suppressed by resistance may produce fermentation effects similar to those of monensin. The cellular action of monensin as an ionophore in membranes is probably little more than a means of inhibiting sensitive species. Many gram-positive antibiotics have little affect on ciliate protozoa or coccidia.

When mixed ruminal bacteria were provided with growth rate limiting amounts of mixed carbohydrates, more than 50 mg ammonia/L were required for maximal protein synthesis. Microbial protein synthesis declined when ammonia concentration was less than 50 mg/L and unfermented carbohydrates increased. Ammonia starvation also decreased growth efficiency. Intracellular ammonia increased as a linear function of extracellular ammonia, but the intracellular concentration was always at least 160 mg/L higher than the extracellular concentration. Maximal protein synthesis was not observed until intracellular ammonia was greater than 220 mg/L. The concentration gradient of ammonia across cell membranes ranged from 15-fold to 1.8-fold and indicated that some of the ruminal bacteria may have active transport mechanisms for ammonia. These concentration gradients were, however, far less than those reported for bacteria from other habitats. The ruminal bacteria left more than 12 mg ammonia/L when carbohydrates were still available, and this observation was consistent with the assumption that active ammonium transport was not readily or maximally induced.

Experiments were conducted to ascertain the effect of dietary protein (type and amount) and feeding frequency on peptide concentration and flow from the rumen of lactating Holstein cows. When rations supplemented with soybean meal were fed 12 times/d and CP was 14.5, 17.1, or 20.6%, the estimated flow of N from the rumen was 22, 33, and 34 g/d, respectively. When the soybean meal was autoclaved to decrease protein solubility (14.4, 16.9, and 19.3% CP the flow of peptide N was 19, 26, and 31 g/d, respectively. In both of these experiments rumen volume and dilution rate remained relatively constant throughout the day, and flow was proportional to concentration. If the cows were fed rations containing 17.8% CP once a day, the peptide concentration and dilution rate were highest 2 h after feeding and declined significantly thereafter. Replacement of half the soybean protein with either extruded soybean meal or fish meal decreased the flow of peptide N from the rumen (19 versus 14 and 15 g/d, respectively). Because the total flow of soluble protein and ammonia as well as peptides was less if cows were fed once a day than 12 times per day, increased feeding frequency appeared to increase ruminal proteolysis.

Rumen contents were obtained from a cow fed 2.5 kg concentrates and 2.5 kg timothy hay twice daily, and the mixed rumen bacterial inoculum (pH 6.3) was separated from large feed particles and protozoa by centrifugation. Bacteria were incubated in artificial media at pH 6.7 and 6.0. When starch, sucrose, cellobiose, xylan, pectin, or a mix of carbohydrates were provided to pH 6.7 incubations at 1 mM/h for 10 h, fermentation was carbohydrate-limited, less than 20% of the carbohydrate was converted to lactate, molar ratio of methane to hexose fermented was as great as 49%, and pH remained constant. When initial pH was lowered to 6.0, methane production was nearly eliminated, volatile fatty acid production was reduced, and there was a small decrease (less than .5) in final pH. At pH 6.0, lactate production was increased in all incubations except xylan and pectin, and pH had a greater effect on acid production than the type of carbohydrate provided. Bacterial protein synthesis was reduced 34 to 69% when initial pH was 6.0, but these reductions were greater than decreases in carbohydrate utilization, increases in lactate, and associated decreases in adenosine 5'-triphosphate production. Because less adenosine 5'-triphosphate was used for protein synthesis, it appeared that low pH diverted energy to nongrowth functions.

Mixed cultures of rumen bacteria were inoculated into anaerobic buffer solutions containing mixed carbohydrates, casein and ammonia, and rates of bacterial growth, protein degradation, ammonia formation or utilization and lactate production were determined. Bacterial growth rate was varied by the provision of excess carbohydrate (one large dose at the onset of the incubation) or limited carbohydrate (small doses every hour or every 2 hr). When carbohydrate was limited, growth rate was slow, the extent of protein degradation was small and lactate did not accumulate in the fermentation vessels. Lactate production and protein degradation were also negligible during the initial phases of the high carbohydrate, fast growth rate incubations, but large increases in each were seen after 3 hours. Microscopic examination of the fast growth incubations revealed large numbers of small ovoid cells similar to Streptococcus bovis, while tha slow growth incubations exhibited a variety of morophological types and very few small ovoid cells. Because the lactic acid and morphological data suggested that proliferation of S. bovis might be responsible for rapid proteolysis, effects of gram-positive antibiotics were examined. When compared against a fast growth control, both thiopeptin (5 ppm) and monensin (5ppm) were found to decrease protein degradation, but the inhibition by thiopeptin (50%) was greater than that by monensin (13%). The ratios of protein degraded to bacteria protein synthesized were .659, .362 and .628 for the control, thiopeptin and monensin treatments, respectively. Actively proteolytic strains of S. bovis were isolated from fast growth incubations, and subsequent experiments showed that the ratio of protein degraded to bacterial protein synthesized was approximately 1.50. Collectively, the data indicate that S. bovis is a very proteolytic rumen bacterium.

Pure cultures of monensin-sensitive ruminal bacteria were involved in both the production and utilization of pyro-glutamate (pyrrolidone carboxylic acid), a novel product of glutamine fermentation, but the effect of monensin on pyro-glutamate accumulation in mixed cultures had not been assessed. Mixed ruminal bacteria (n = 3) fermented glutamine (40 mM) at a rapid rate (63 nmol/[mg protein-min]) and there was a transient increase in pyro-glutamate (3 mM). Monensin (5 ppm) decreased the rate of glutamine fermentation 70% and greatly enhanced the conversion of glutamine to pyro-glutamate (50 vs 7%). Glutamate was fermented at nearly the same rate as glutamine, and monensin eliminated glutamate fermentation. Glutamate was never converted to pyro-glutamate. Trypticase, a pancreatic digest of casein, contained pyro-glutamate, and a significant fraction (30%) of its N was never converted to ammonia. Pyro-glutamate utilization was reduced by monensin and low pH (less than 6.0). Based on these in vitro observations, pyro-glutamate could be a significant end-product of glutamine degradation when animals are fed monensin.

The active vitamin D metabolite 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] causes marked suppression of both pre-proparathyroid hormone messenger RNA (pre-proPTH mRNA) and parathyroid hormone (PTH) secretion. These effects are dose dependent and reversible when tested in an in vitro primary tissue culture cell system using normal bovine parathyroid cells. In the current studies, the precursors of 1,25(OH)2D3 and the related metabolite 24,25-dihydroxyvitamin D3 [24,25(OH)2D3], were used in the same culture system to test for possible regulatory effects. The results were compared with identically prepared cells exposed to 1,25(OH)2D3. In short-term studies (30-120 minutes), none of the vitamin D-related compounds produced any effect on PTH secretion. In long-term studies (24-48 hours, using primary tissue culture in the presence of test agents), neither vitamin D3 nor 25(OH)D3 affected PTH secretion or pre-proPTH mRNA over the concentration range 10(-11)-10(-7) M. On the other hand, 24,25(OH)2D3 produced significant suppression of both pre-proPTH mRNA (77% of control, P less than .01) and PTH secretion (75% of control, P less than .005) at 10(-7) M. By comparison, 10(-11) M 1,25(OH)2D3 produced levels of suppression (25-30%) of both pre-proPTH mRNA and PTH secretion comparable to 10(-7) M 24,25(OH)2D3, while even greater suppression (40-50%) occurred at 10(-9)-10(-7) M 1,25(OH)2D3. From these studies, we conclude that vitamin D3 and 25(OH)D3 do not have significant effects on PTH synthesis and secretion over the range of doses tested.(ABSTRACT TRUNCATED AT 250 WORDS) [ABSTRACT FROM AUTHOR]