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.

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.

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.

Effects of diet and hindgut defaunation on diet digestibility and microbial concentrations in the cecum and colon of the horse.

The effects of diet and hindgut defaunation (removal of protozoa from the hindgut) on diet digestibility (Trial 1) and on total and cellulolytic bacterial and fungal concentrations in the cecum and colon (Trial 2) were investigated. A high-forage (HF) diet, 90% alfalfa hay-10% concentrate, or a higher-concentrate (HC) diet, 60% alfalfa hay-40% concentrate, was limit-fed. In Trial 1, defaunation resulted in a slight decrease in DM digestibility (P < .1) and had no effect on cellulose digestibility. Dry matter digestibility was higher (P < .001) with the HC diet; however, no differences were observed in cellulose digestion. For the faunated periods, protozoal concentrations were similar in the cecum and greater in the colon for both diets (P < .05). A diet x location interaction was observed for the genera Buetschlia and Blepharocorys. In Trial 2, defaunation had no effect on either total or cellulolytic bacterial concentrations in the cecum or colon. Total bacterial concentrations were higher (P < .06) in the colon when ponies were fed the HC diet. Defaunation did not affect total fungal concentrations in the cecum; however, fungal concentrations in the colon were slightly higher (P < .1) when the ponies were defaunated. Diet had no effect on total or cellulolytic fungal concentrations. Both total and cellulolytic fungal concentrations were approximately 10-fold higher in the colon than in the cecum (P < .01). Protozoa do not seem to play an essential role in the fermentation of feedstuffs in the equine hindgut.

A comparative study of feeding behavior and digestive function in dairy goats, wool sheep and hair sheep.

An intake and digestibility study was conducted with three groups (six animals per group) of yearling wether dairy goats (four Toggenburg, two Alpine), wool sheep (Targhee X Dorset) and hair sheep (St. Croix). Body weight (BW) ranged from 42 to 52 kg, averaging 47 kg. All animals were penned individually and given ad libitum access to a mixture of alfalfa-smooth bromegrass hay in pelleted, chopped or long form. Each group contained three ruminally cannulated animals. There were no apparent differences in the composition of feed consumed among goats, wool sheep and hair sheep, and no significant animal type X forage form interactions for any of the variables evaluated. Significant differences were observed in dry matter intake (DMI) between wool sheep, hair sheep and goats: 3.17%, 2.66% and 2.23% of BW, respectively (P less than .05). Daily water intake (WI) was greatest for wool sheep (P less than .05), but not different between hair sheep and goats. Total digestibility of dry matter (DM) and all fiber fractions were similar among animal types. For the cannulated animals, ruminal content weight and total ruminal volume were greatest for wool sheep (P less than .05). Ruminal acid detergent lignin (ADL) turnover was greater in wool and hair sheep than goats (P less than .05), but no differences were apparent for dry matter or neutral detergent fiber (NDF) turnover. For all animals, DMI, DMI/BW, digestible DMI and WI were greater for pelleted than chopped and long hay (P less than .05). Total ruminal volume, contents weight (on an absolute or BW basis) and fluid volume were lower in the cannulated animals consuming pelleted hay (P less than .05). Ruminal DM turnover rate was faster on pelleted than long hay, while DM turnover rate on chopped hay was intermediate. Turnover of ADL was faster on pelleted than chopped or long hay (P less than .05), but there were no differences among forage forms in NDF turnover rate. Fluid turnover rate was faster on pelleted and chopped than on long hay (P less than .05). Under the conditions of this study, no apparent differences were observed among animal types in the nutrient composition of feed consumed, ruminal or total tract digestibilities or rate of passage for dry matter. However, feeding behavior or selectivity differences under natural grazing conditions may deviate from what has been observed in confinement.

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.

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).

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.

Interactions between Fibrobacter succinogenes, Prevotella ruminicola, and Ruminococcus flavefaciens in the digestion of cellulose from forages.

The synergistic and inhibitory interactions observed between Fibrobacter succinogenes A3c, Prevotella ruminicola H2b, and Ruminococcus flavefaciens B34b in the digestion of forage cellulose were studied in detail. Orchardgrass and alfalfa hays, both at two maturity stages, were used as substrates. Sequential inoculation procedures were developed whereby a second inoculation was made after the initial fermentation was killed. Total cellulose digestion from sequential addition of the organisms was then compared to values obtained in simultaneous co-culture. When the noncellulolytic P. ruminicola was co-cultured with either of the two cellulolytic species (F. succinogenes or R. flavefaciens) forage cellulose digestion numerically increased over that of the cellulolytic species alone. In contrast, decreases from co-culture values were noted with sequential addition of the organisms. When F. succinogenes and R. flavefaciens were co-cultured, cellulose digestion was reduced compared to F. succinogenes alone. However, no such reduction was observed when the organisms were added sequentially. Further experiments indicated that this inhibitory activity is only produced when the organisms are co-cultured and is stable to autoclaving at 121 degrees C for 20 min. Inhibition of this type could be the result of bacterocin production by one of the organisms; however, most bacterocins are destroyed by autoclaving.

Effect of prolonged high-concentrate feeding on ruminal protozoa concentrations.

Five ruminally cannulated steers, with ad libitum access to feed, were gradually adapted from an all-forage diet to a 75% concentrate diet over a 6-wk period. Three animals were then randomly assigned to an all-concentrate diet (87% whole corn) and the other two were fed a 90% concentrate plus 10% forage diet. These diets were fed for 17 wk and then reversed between groups for 11 additional weeks. Over the last 22 wk, addition of 10% forage to the all-concentrate diet had no effect on the concentration of total protozoa; however, Isotricha and Epidinium percentages increased (P < .05). Although concentrations varied markedly, protozoa persisted throughout the entire period of high-concentrate feeding (both diets) in three of the animals. In contrast, the other two animals were defaunated most of the time, except for the sporadic appearance of Entodinium species for 1- or 2-wk intervals. Average pH in these latter two animals was lower (P < .05) during the entire 28-wk high-concentrate feeding period. Because these two animals had a lower ruminal pH than the other three, even when fed all forage, it seems that the ruminal environment varies between individual animals. Thus, the maintenance of a protozoal population in animals fed high-concentrate diets may be related to physiological conditions such as rate and extent of salivary production, rate of fluid and particulate matter passage within each animal, and so on.

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.

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.

Occurrence of 2-aminoethylphosphonic acid in feeds, ruminal bacteria and duodenal digesta from defaunated sheep.

A quantitative method of analysis for 2-aminoethylphosphonic acid (AEP) was developed using reverse-phase HPLC. The detection limit for AEP was 15 nM, and the detector response (peak area) was linear from AEP levels up to 100 microM (R = .99). Mean recovery of AEP added to strained ruminal fluid from faunated sheep was 98.2%. When AEP was added to a fermentation mixture at a concentration of 22.6 micrograms/ml, 78% disappeared during a 24-h incubation. 2-Aminoethylphosphonic acid was readily detected in preparations of mixed ruminal ciliate protozoa as well as in mixed and pure strains of ruminal bacteria, feedstuffs, and ruminal fluid and duodenal digesta from defaunated sheep. The occurrence of AEP in feed and bacterial hydrolysates was confirmed by organic phosphorus analyses. The concentration of AEP in mixed ruminal protozoa was three times greater than its concentration in mixed ruminal bacteria (4,304 vs 1,383 micrograms/g DM, respectively). The AEP values for pure ruminal bacterial cultures ranged from 733 micrograms/g DM in Bacteroides succinogenes B21a to 1,166 micrograms/g DM in Butyrivibrio fibrisolvens H17c. Ruminal fluid and duodenal digesta from defaunated sheep contained AEP concentrations of 30 micrograms/ml and 90 micrograms/g DM, respectively. The concentration of AEP in feedstuffs ranged from 25 micrograms/g DM in wheat straw to 263 micrograms/g DM in oats. Because AEP occurrence is not limited to ruminal ciliate protozoa, it is of little value as a marker for protozoal presence in or passage out of the rumen.

Effect of supplemental sodium bicarbonate on nutrient digestibilities and ruminal pH measured continuously.

A technique was used to monitor continuously ruminal pH using a strip-chart recording pH meter. Ruminal pH measurements were made in four ruminal-cannulated crossbred wether lambs (ag initial weight, 42.5 kg). For l.5 h daily, lambs were given ad libitum access to 50% concentrate-50% chopped orchardgrass hay diets supplements with 0, l.5, 3.0 and 4.5% sodium bicarbonate (NaHCO3). A split-plot Latin-square design was used to evaluate NaHCO3 level and day of adaptation on the percentage of time (%T) that ruminal pH was less than 6.6, 6.2, 5.8, 5.4 and 5.0. No effect due to day of adaptation existed for ruminal pH measurements (P more than .10), while the effect of dietary NaHCO3 level was quadratic (P more than .01) for the %T that ruminal pH was less than 5.4. To evaluate the effects of NaHCO3 on nutrient digestion, the same diets were fed to eight wether lambs (avg initial weight, 38 kg) at 85% of their ad libitum intake in a replicated 4 x 4 Latin-square digestion trial. Digestibilities of dry matter (DM), organic matter, N and fiber fractions were not different due to level of NaHCO3 (P greater than .10). Ash digestibility increased with increasing levels of dietary NaHCO3 (P less than .01). Four ruminal-cannulated mature Hereford steers were also given ad libitum access to the diets in a split-plot Latin-square trial to evaluate effects of dietary NaHCO3 level on ruminal pH and in situ digestion of soybean meal N and orchardgrass DM. During incubation of the dacron bags for 3, 6, 9, 12, 24 and 36 h also increased linearly with increasing level of NaHCO3. Ruminal solid and liquid dilution rates were not affected by level of supplemental NaHCO3 (P greater than .10). The results of these trials suggest that increasing level of dietary NaHCO3 greatly increases the proportion of time ruminal pH is above critical levels for ruminal protein and dry matter digestion, but does not affect total tract nutrient digestion when 50% concentrate diets are fed.

Ruminal characteristics, microbial populations, and digestive capabilities of newly weaned, stressed calves.

Eight ruminally fistulated steers, 7 to 8 mo old, were used in a completely randomized, 2 x 2 factorial experiment to determine the effects of energy density and protein source in receiving diets on in situ DM, NDF, and N disappearance, concentrations of ruminal bacteria, protozoa, ammonia, and pH. Two energy densities (1.80 and 1.48 Mcal/kg of NEm) and two protein sources (spray-dried blood meal [SDBM] and soybean meal [SBM]) were compared. Fistulated steers were weaned, transported by truck, and held in a sale barn before their arrival at the feedlot. On d 0 (day of arrival at the feedlot), DMI was 62% of DMI on d 7 after arrival. Overall, feeding a high-energy diet resulted in lower (P < .01) in situ DM disappearance (DMD) of orchardgrass than feeding a low-energy diet at both 24 and 48 h. In situ 24-h DMD averaged 46.6% on d-3 and 41.6% on d 0, whereas 48-h in situ DMD on d -3 and 0 averaged 58.2 and 58.6%, respectively, indicating the ruminal microbial population was not inhibited in its ability to digest available substrate. Additionally, there were no differences (P > .10) in 48-h in situ NDF disappearance between d -3 and 0 (58.8 vs 57.8%), respectively. No differences (P > .10) occurred in the concentration of total bacteria, or cellulolytic bacteria, due to feed and water deprivation. Concentration of total protozoa was lower (P < .05) on d 0 than at any other time. Entodinium averaged 72.5% of genera before weaning, and more than 90% of genera found on all treatments by d 21. Diplodinium and Epidinium percentages tended to decline after weaning. Isotricha concentrations were low and Dasytricha were eliminated after d 7. In conclusion, the concentration of ruminal bacteria and the ability to digest available substrate were not decreased immediately after weaning, trucking, and 24 h of feed and water deprivation.

The influence of restricted feeding on site and extent of digestion and flow of nitrogenous compounds to the duodenum in steers.

The effects of restricted feeding on site and extent of digestion, flow of N-containing compounds to the duodenum, and ruminal metabolism were determined. In Trial 1, corn silage-based diets were fed at one of three intakes to three ruminally and duodenally fistulated steers (420 +/- 4.4 kg BW) in a repeated 3 x 3 Latin square experiment. Feeding levels were 100% of ad libitum intake, 90% of ad libitum intake, and 80% of ad libitum intake. Site of digestion was not affected by restricted feeding; however, extent of DM, NDF, and apparent N digestion were increased (P < .03) 2.8, 4.1, and 6.2 percentage units for each 1-kg restriction in DM intake. Restricted feeding increased (P < .06) ruminal pH and ammonia concentrations, decreased (P < .05) ruminal liquid dilution rate, and had no major effects on bacterial concentrations. In Trial 2, all-concentrate diets based on whole shelled corn were fed at 100, 90, or 80% of ad libitum intake to six ruminally and duodenally fistulated steers (440 +/- 4.1 kg BW) in a replicated 3 x 3 Latin square experiment. Restricted feeding increased (P < .06) apparent ruminal DM digestion; however, true ruminal OM digestibility was not affected. Extent of apparent N digestion increased 3.0 percentage units for each 1-kg restriction in DMI. Ruminal pH was not affected by restricted feeding; however, ruminal ammonia concentrations increased (P < .06) and ruminal liquid dilution rate decreased (P < .04) with restricted feeding. In both Trials, total N flow to the duodenum was approximately 100% of N intake for the 80% of ad libitum intake. Flow of N exceeded N intake in a linear (P < .01) manner as DMI increased. Decreases in duodenal N flow when intake was restricted resulted from decreases (P < .01) in bacterial N flowing to the duodenum, and also in decreases (P < .01) in non-ammonia, non-bacterial N flow when whole shelled corn-based diets were fed. We concluded that restrictions in digestible energy intake are less than the DM restriction when corn silage-based diets are fed but are equal to the DM restriction when whole shelled corn-based diets are fed. Also, restricted feeding strategies reduce the flow of N to the duodenum from both bacterial and feed origins.