Ruminal Protozoa Research Articles

Duodenal flow of nitrogen following protozoal inoculation of fauna-free sheep fed a diet supplemented with casein or soybean meal

Fauna-free sheep, equipped with a rumen cannula and a duodenal re-entrant cannula, were fed a corn silage diet supplemented with casein (CA diet) or soybean meal (SBM diet). Eighteen days later the sheep were inoculated via the rumen cannula with a mixed population of ciliate protozoa. Rumen fluid and duodenal digesta were sampled for 4 d before the inoculation and for 13 d following the inoculation. A stable protozoal population was established within 8 d. Protozoa increased (P < 0.05) the digestion of organic matter in the stomach of sheep fed both the CA and the SBM diet, but the effects of supplemental protein and protein × protozoa interaction were not significant (P > 0.05). The stomach digestion of acid detergent fibre was not affected (P > 0.05) by protozoa or by the source of supplementary protein, but the protein × protozoa interaction was significant (P < 0.05). The disappearance of nitrogen from the stomach was lower (P < 0.01) for the SBM diet than for the CA diet and was increased (P < 0.05) for both diets in the presence of protozoa, but the effect of protein × protozoa interaction was not significant (P > 0.05). The ruminal presence of protozoa decreased the flow from the stomach of individual amino acids (from P < 0.05 to P < 0.001) and of nonammonia nitrogen (P < 0.05). However, the decreases were greater (from P < 0.05 to P < 0.001) for the CA diet than for the SBM diet, but the effects of protein × protozoa interaction were not significant (P > 0.05). The ruminal presence of protozoa increased (P < 0.05) the flow from the stomach of ammonia-N for SBM diet but not for CA diet. The effects of protein and of protein × protozoa interaction were not significant (P > 0.05). It was concluded that although ruminal protozoa do not metabolize casein, their effect of decreasing amino acid flow from the stomach can be greater for diets containing casein (soluble protein) as protein supplement than for those containing soybean meal (insoluble protein). Key words: Duodenal flow, nitrogen, protozoa, casein, soybean meal, sheep

In vivo metabolism of 2,2'-diaminopimelic acid from gram-positive and gram-negative bacterial cells by ruminal microorganisms and ruminants and its use as a marker of bacterial biomass

Cells of Bacillus megaterium GW1 and Escherichia coli W7-M5 were specifically radiolabeled with 2,2'-diamino[G-3H]pimelic acid ([3H]DAP) as models of gram-positive and gram-negative bacteria, respectively. Two experiments were conducted to study the in vivo metabolism of 2,2'-diaminopimelic acid (DAP) in sheep. In experiment 1, cells of [3H]DAP-labeled B. megaterium GW1 were infused into the rumen of one sheep and the radiolabel was traced within microbial samples, digesta, and the whole animal. Bacterially bound [3H]DAP was extensively metabolized, primarily (up to 70% after 8 h) via decarboxylation to [3H]lysine by both ruminal protozoa and ruminal bacteria. Recovery of infused radiolabel in urine and feces was low (42% after 96 h) and perhaps indicative of further metabolism by the host animal. In experiment 2, [3H]DAP-labeled B. megaterium GW1 was infused into the rumens of three sheep and [3H]DAP-labeled E. coli W7-M5 was infused into the rumen of another sheep. The radioactivity contents of these mutant bacteria were insufficient to use as tracers, but the metabolism of DAP was monitored in the total, free, and peptidyl forms. Free DAP, as a proportion of total DAP in duodenal digesta, varied from 0 to 9.5%, whereas peptidyl DAP accounted for 8.3 to 99.2%. These data reflect the extensive metabolism of bacterially bound DAP within the gastrointestinal tracts of ruminant animals and serve as a serious caution to the uncritical use of DAP as a marker of bacterial biomass in the digesta of these animals.

Effects of zinc sulfate concentration and feeding frequency on ruminal protozoal numbers, fermentation patterns and amino acid passage in steers.

Effects of zinc sulfate (0 vs 1,142 ppm supplemental zinc from zinc sulfate) and feeding frequency (1 x vs 12x daily) on ruminal protozoa numbers, fermentation patterns and amino acid passage were investigated using four ruminally and abomasally cannulated mature Jersey steers in a 4 x 4 Latin square experiment. Steers (530 kg) were fed a 50:50 roughage:concentrate diet at 1.5 times their NEm requirement. Experimental periods were 14 d in duration; ruminal, abomasal and fecal samples were collected at 6-h intervals during the last 3 d of each period. Protozoa numbers tended to be lowest (1.82 x 10(6)/ml) in steers fed zinc 1 x and tended to be highest (3.83 x 10(6)/ml) in steers fed zinc 12 x daily (P less than .10). Frequent feeding decreased ruminal pH .24 units and increased total VFA 20.7%, ammonia 22.7% and ruminal digestion of dietary amino acids (AA) 61.6% (P less than .05). Zinc supplementation decreased ruminal digestion of dietary AA 35.8% (P less than .05) and the abomasal passage of bacterial OM and AA 21.2% (P less than .05) and increased ruminal output of amino acids as a percentage of intake 15.1% (P less than .05). Although it increased escape of dietary AA, zinc sulfate decreased postruminal passage of bacterial AA and resulted in a net negative effect on total postruminal AA passage as a percentage of intake. The effects of zinc on ruminal AA digestion may be more closely related to an interaction of zinc with dietary CP rather than to an effect of Zn on ruminal microbial populations.

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.

In vitro metabolism of 2,2'-diaminopimelic acid from gram-positive and gram-negative bacterial cells by ruminal protozoa and bacteria

Bacillus megaterium GW1 and Escherichia coli W7-M5 were specifically radiolabeled with 2,2'-diamino[G-3H]pimelic acid [( 3H]DAP) as models of gram-positive and gram-negative bacteria, respectively. These radiolabeled bacterial mutants were incubated alone (control) and with mixed ruminal bacteria or protozoa, and the metabolic processes, rates, and patterns of radiolabeled products released from them were studied. Control incubations revealed an inherent difference between the two substrates; gram-positive supernatants consistently contained 5% radioactivity, whereas even at 0 h, those from the gram-negative mutant released 22%. Incubations with ruminal microorganisms showed that the two mutants were metabolized differently and that protozoa were the major effectors of their metabolism. Protozoa exhibited differential rates of engulfment (150 B. megaterium GW1 and 4,290 E. coli W7-M5 organisms per protozoan per h), and they extensively degraded [3H]DAP-labeled B. megaterium GW1 at rates up to nine times greater than those of ruminal bacteria. By contrast, [3H]DAP-labeled E. coli W7-M5 degradation by either ruminal bacteria or ruminal protozoa was more limited. These fundamental differences in the metabolism of the two mutants, especially by ruminal protozoa, were reflected in the patterns and rates of radiolabeled metabolites produced; many were rapidly released from [3H]DAP-labeled B. megaterium GW1, whereas few were slowly released from [3H]DAP-labeled E. coli W7-M5. Most radiolabeled products derived from [3H]DAP-labeled B. megaterium GW1 were peptides of bacterial peptidoglycan origin. The ruminal metabolism of DAP-containing gram-positive and gram-negative bacteria, even with the same peptidoglycan chemotype, is thus likely to be profoundly different.(ABSTRACT TRUNCATED AT 250 WORDS)

Ruminal changes in monensin- and lasalocid-fed cattle grazing bloat-provocative alfalfa pasture.

Microbial and fermentation changes in the rumen in monensin- and lasalocid-fed cattle grazing bloat-provocative alfalfa pasture were studied using genetically bloat-susceptible, ruminally-cannulated adult cattle. Monensin at .66 and .99 mg/kg body weight daily reduced the severity of legume bloat by 41 and 73%, respectively. The same doses of lasalocid reduced bloat by 25 and 12%. Comparison of ruminal contents from animals before treatment with ruminal contents from antibiotic-treated animals showed no differences in pH, ammonia, soluble N, soluble carbohydrate, ethanol-precipitable slime and anaerobic bacterial counts. Monensin treatment decreased protozoal numbers and microbial activity, as evidenced by lower gas production from in vitro fermentation of ground alfalfa hay when compared to pretreatment. Lasalocid had no effect on protozoal counts and in vitro gas production. Addition of monensin or lasalocid (12 micrograms/ml) to in vitro fermentation of chopped, fresh alfalfa reduced microbial activity as evidenced by higher soluble N, lower ammonia concentration and decreased gas production. Monensin reduced the amount of ethanol-precipitable slime and protozoal numbers. Reduction in the severity of bloat when monensin was fed appears to be due to decreased protozoal numbers, which resulted in decreased gas production. Lasalocid did not reduce legume bloat because of its minimal effect on the ruminal protozoa.

Effect of increased levels of urea in the diet on ruminal protozoal counts in four ruminant species.

Summary Fistulated sheep, goats, cattle and buffaloe's were used to study the effect of substituting urea for 0, 36, 55 or 100% of cottonseed cake protein on the total counts, pool size and composition of protozoal population in the rumen. Highest protozoal counts were obtained on the 0 and the 100% urea-N diets and the lowest protozoal counts were observed in 55% urea-N diet. Sheep and goats had higher protozoal counts than buffalo and cattle. Protozoal pool size in the rumen of sheep was greater than of goats and that of buffalo was greater than for cattle. Increasing urea-N in the diet decreased growth rate of protozoa in vitro. The growth rate of protozoa in vitro was affected by postprandial time, but not by animal species. A mixture of Entodinia, Isotricba, Diplodinium and Polyplastron species was found in the rumen contents of all animal species. Entodinia sp. were predominant in all animals on the four diets. Increasing urea-N in the diet increased the percentage of Entodinia and reduced the other species so that negligible numbers of Isotricba, Diplodinium and Polyplastron were observed in 100% urea-N diet. Increased levels of urea increased the generation time (hr) of protozoa in sheep, goats, cattle and buffaloes.

Microbial Species Including Ureolytic Bacteria from the Rumen of Cattle Fed Purified Diets

The rumen microbial populations were studied in cattle fed either natural or purified diets. The purified diets contained either isolated soy protein or 1 of 4 nonprotein nitrogen (NPN) sources. Four-hundred and three strains of bacteria isolated at random from a nonselective medium (264 strains) and a lactobacillus medium (139 strains) were presumptively identified and tested for urease activity. There were 28 and 2 ureolytic strains among the total bacteria isolated from the nonselective and lactobacillus medium, respectively. The ureolytic strains represented a wide variety of bacteria. Among the total population, the most predominant presumptively identified groups of bacteria grown on nonselective medium were anaerobic lactobacilli and Bacteroides amylophilus. The latter group's concentrations in rumen contents were higher in the steers fed either urea or urea phosphate than in steers fed biuret or uric acid. The percentage of facultative anaerobic and H2S-producing ruminal bacteria were significantly greater for steers fed biruet than for steers fed the other NPN sources. Steers fed uric acid had significantly more cellulolytic bacteria than steers fed urea and urea phosphate and steers fed urea had significantly more amylolytic bacteria than steers fed urea phosphate. Cattle fed a 30% roughage-70% concentrate natural diet contained significantly more ruminal protozoa and gramnegative rod-shaped bacteria but contained significantly fewer cocci than cattle fed urea or isolated soy supplemented purified diets.

Feed Intake and Gains of Beef Cattle as Affected by Source and Level of Nitrogen in High-Energy Rations

Feed intake and gains were measured in an experiment using 20 individually fed grade Angus receiving two levels and two sources of nitrogen in ground-pelleted rations containing 25% low quality grass hay as the only roughage. The experiment was patterned after Lucas' extra-period Latin-square change-over design using 28-day evaluation periods following 14-day adjustment periods. Rations St (1) and Su (2) were high-energy low-protein treatments providing a comparison of corn starch and cane sugar as important sources of energy; rations StC (3) and SuC (4) were high-energy high-protein treatments formulated by adding protein to rations St and Su by replacing part of the starch and sugar with cottonseed meal; and ration U (5) was formulated by adding urea to ration St at the level necessary to make the protein equivalent equal to rations StC and SuC. Body weight and metabolic body size had no appreciable effect on feed intake within the range of animal weights in this experiment. Animals when on higher protein or protein equivalent gained faster, more efficiently, and consumed more feed than when on low protein. There was no significant difference between the gains while the animals were receiving the low-protein rations. There was more increase in feed intake when cottonseed meal was added to the sugar ration than when added to the starch ration, although without added protein feed intake of the sugar ration was lowest. Daily gains (lb.) for rations (1) through (5) were: 0.02a; 0.02a; 1.73b; 2.10bc; and 2.26c; and daily feed consumptions were: 11.5b; 9.5a; 17.3d; 17.7d; and 15.1c, (figures with the same a, b or c superscript in each case are not significantly different). Consumption of rations intended to be high-energy and low-protein was very low indicating an effect of protein level on voluntary feed intake. The ration supplemented with urea supported the fastest and most efficient gains. Ruminal protozoal distributions were normal except for animals when fed ration St in which the protozoan Epidinium ecaudatum was found in large numbers; animals when fed StC contained unusually high numbers of species of Diplodinium; and for low populations of the holotrich Isotricha in all rations fed.