Protozoal Protein Research Articles

In vitro Study of Urtica cannabina and Leymus chinensis on Rumen Microbial Fermentation and Gas Production

Background: Urtica cannabina, an unconventional forage, is widely distributed in northern China. It has high nutritional values that make it suitable for the ruminant’s feeding requirments as compared to Leymus chinensis. The current study was designed to evaluate varying ratios of Urtica cannabina and Leymus chinensis in the feeding diet and to see the effects on rumen fermentation and gas production in vitro. Methods: The study was designed into five treatments based on the different ratios of U. cannabina and L. chinensis: 0:100, 30:70, 50:50, 70:30 and 100:0 categorized into five groups from A-E. To detect the rumen fermentation parameters, the culture medium was collected at 1, 3, 6, 12 and 24 h. Result: Gas production of groups A and C was increased than other groups at 24h (P less than 0.05), whereas the rate of gas production (c) was also increased in group A (P less than 0.05). The pH values at 1, 3, 6 and 24 h were increased in groups A and C with higher values in group C at 24h (P less than 0.05). The ammonia concentration was increased in groups D and E at 3, 6, 12 and 24 h, with the lower values in group C at 24h (P less than 0.05). The concentration of bacterial and protozoal proteins was also observed higher in groups A and C at 1 and 24 h, with highest value in group C at 24 h (P less than 0.05). In summary, as for Urtica cannabina to Leymus chinensis ratios are concerned, 50:50 is an optimal ratio for rumen fermentation in vitro, which increases the gas production and microbial protein synthesis.

Macromolecular Targets of Antiparasitic Germacranolide Sesquiterpenoids: An In Silico Investigation.

The parasitic protozoal infections leishmaniasis, human African trypanosomiasis, and Chagas disease are neglected tropical diseases that pose serious health risks for much of the world's population. Current treatment options suffer from limitations, but plantderived natural products may provide economically advantageous therapeutic alternatives. Several germacranolide sesquiterpenoids have shown promising antiparasitic activities, but the mechanisms of activity have not been clearly established. The objective is to use in silico screening of known antiparasitic germacranolides against recognized protozoal protein targets in order to provide insight into the molecular mechanisms of activity of these natural products. Conformational analyses of the germacranolides were carried out using density functional theory, followed by molecular docking. A total of 88 Leishmania protein structures, 86 T. brucei protein structures, and 50 T. cruzi protein structures were screened against 27 antiparasitic germacranolides. The in-silico screening has revealed which of the protein targets of Leishmania spp., Trypanosoma brucei, and Trypanosoma cruzi are preferred by the sesquiterpenoid ligands.

Effects of Piper sarmentosum extract supplementation on growth performances and rumen fermentation and microflora characteristics in goats.

This study was carried out to investigate the effect of diet Piper sarmentosum extract (PSE) on the growth performance, antioxidant properties, rumen fermentation and microflora in goats. Forty Hainan black goats with similar body weight were divided into four groups with supplementation of PSE in the concentrate at 0, 300, 600 and 1,200mg/kg, respectively, and fed for 56days. Results showed that average daily gain (ADG) was higher and feed intake/body gain (F/G) was lower in goats fed with PSE at 300mg/kg (p<.05). The activities of glutathione peroxidase (GSH-Px) and total antioxidant capacity (T-AOC) in the serum of goats differed among treatments and were greatest linearly when PSE was added at 1,200mg/kg (p<.05). The level ofmalondialdehyde (MDA) in the serum of goats differed among treatments and was lowest linearly when PSE was added at 1,200mg/kg (p<.05). The level of protozoal protein in the rumen of goats differed among treatments and was lowest linearly when PSE was added at 1,200mg/kg (p<.05). The concentrations of ruminal acetic acid and valeric acid and the ratio of acetate to propionate were reduced with PSE supplementation (p<.05). Protozoa, fungi, Ruminococcus flavefaciens and Fibrobacter succinogenes contents differed among treatments and were lowest linearly when PSE was added at 1,200mg/kg (p<.05). Thus, supplementation of PSE at 300-1,200mg/kg to goat concentrate is recommended for improving antioxidative ability and rumen efficiency and reducing protozoal content of goat.

Substituting oat hay or maize silage for portion of alfalfa hay affects growth performance, ruminal fermentation, and nutrient digestibility of weaned calves.

ObjectiveThe impact of forage feeding strategy on growth performance, ruminal fermentation and nutrient digestibility in post-weaning calves was investigated.MethodsForty-five female Holstein calves (body weight [BW] = 79.79±0.38 kg) were enrolled in the 35-d study at one week after weaning and randomly assigned to one of three dietary treatments. All diets were fed as total mixed ration containing 60% (dry matter [DM] basis) of basal starter feed and 40% (DM basis) of forage, but varied in composition of forage source including i) alfalfa (40% DM, AH); ii) alfalfa hay (26.7% DM)+oat hay (13.3% DM; OH); iii) alfalfa hay (26.7% DM)+corn silage (13.3% DM; WS).ResultsDry matter intake was not different among treatment groups (p>0.05). However, BW (p<0.05) and average daily gain (p<0.05) of calves fed AH and OH were greater than WS-fed calves, whereas heart girth was greater in OH-fed calves than those fed AH and WS (p<0.05). Ruminal fermentation parameters including proportion of butyric acid, acetated-to-propionate ratio, concentration of total volatile fatty acid, protozoal protein, bacterial protein, and microbial protein in rumen were the highest in OH (p<0.05) and the lowest in WS. Compared with the AH and WS, feeding oat hay to postweaning calves increased crude protein digestibility (p<0.05), and decreased duration of diarrhea (p<0.05) and fecal index (p<0.05).ConclusionOur results suggested that partially replacing alfalfa hay with oat hay improved ruminal fermentation, nitrogen utilization, and reduced incidence of diarrhea in post-weaning dairy calves.

Further assessment of the protozoal contribution to the nutrition of the ruminant animal

The flow of protozoa from the reticulo-rumen is lower than expected, due to ability of protozoa to prevent washout through sequestration on feed particles and the rumen epithelium. In order to estimate the distribution of protozoa within the reticulo-rumen and passage to the omasum, Czerkawski (1987) developed a model containing pools for the rumen liquid phase, rumen solid phase, and the omasum. This model was used to estimate loss of protozoa in the omasum as well as the amount of protozoal protein available to the animal in the lower gut. A number of assumptions were incorporated into the model, some of which appear unsupported by current research. This paper represents an update, revision, and re-evaluation of Czerkawski's model, where the assumptions that all protozoa in the ‘attached’ phase are in solid digesta, and that protozoa only leave the rumen in the liquid, have been relaxed. Therefore, the revised model allows for sequestration of protozoa on the rumen epithelium and protozoal passage with particulate outflow. Using experimental observations with inputs within biological limits, the revised model and Czerkawski's original model were verified. The effect of diet on the model was then assessed using inputs from a 100% forage diet and a 35–65% concentrate diet. The extent of sequestration was also varied from complete sequestration, to partial sequestration, and no sequestration. A sensitivity analysis was conducted through a linear regression of perturbed mean inputs versus outputs. The results from the revised model indicate that within the reticulo-rumen, the concentrate diet has a greater fractional flow rate of protozoa from the liquid to solid phase, but a lesser fractional flow rate back to the liquid phase, compared to the forage diet. As well, the concentrate diet has a slower net growth rate of protozoa in the attached phase, compared to the forage diet. In the omasum, the forage diet has a less negative net growth rate, compared to the concentrate diet. The forage diet was also associated with smaller loss of protozoa from the omasum. There are limited data from the omasum to be incorporated into the revised model, especially for quantity of protozoa in the omasum. Further research on quantification of protozoa in the omasum could strengthen the predictions made by the model. Despite this, the revised model found a loss of protozoa in the omasum similar to that suggested by Czerkawski's original model 65–73% versus 66%. The revised model results indicate that efforts to increase protozoal flow to the duodenum should focus on reduced sequestration and increased outflow rate from the rumen, although more research is needed to quantify protozoa in the omasum, and to investigate the role of sequestration onto the wall of the reticulo-rumen.

The effects of the unsaturated degree of long-chain fatty acids on the rumen microbial protein content and the activities of transaminases and dehydrogenase in vitro

AbstractThis study investigated the effects of the degree of unsaturation (unsaturity) of long-chain fatty acids on microbial protein content and the activities of transaminases and dehydrogenase in vitro using goat rumen fluid as the cultural medium. Six types of fatty acids, stearic acid (C18:0, group A, control group), oleic acid (C18:1, n-9, group B), linoleic acid (C18:2, n-6, group C), α-linolenic acid (C18:3, n-3, group D), arachidonic acid (C20:4, n-6, group E), and eicosapentaenoic acid (C20:5, n-3, group F), were tested, and the inclusion ratio of each fatty acid was 3% (w/w) in total of culture substrate. Samples were taken at 0, 3, 6, 9, 12, 18 and 24 h, respectively, during culture for analyses. Compared with stearic acid, linoleic acid, α-linolenic acid, and arachidonic acid increased the bacterial protein content, while oleic acid and eicosapentaenoic acid had no significant effects; the protozoal protein content was reduced for all the unsaturated fatty acids, and the magnitude of the reduction appeared to be associated with the degree of unsaturity of fatty acids. The total microbial protein content was dominantly accounted by the protozoal protein content (about 4–9 folds of the bacterial protein), and only increased by linoleic acid, but reduced by oleic acid, arachidonic acid and eicosapentaenoic acid. There were no significant effects in the activities of both glutamic oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) for all the other fatty acids, except for arachidonic acid which enhanced GOT activity and oleic acid which enhanced GPT activity. The total dehydrogenase activity was positively correlated with the degree of unsaturation of fatty acids. In conclusion, the inclusion of 3% of long-chain unsaturated fatty acids increased bacterial protein content, whereas reduced protozoal protein content and enhanced dehydrogenase activity. The fatty acids with more than three double bonds had detrimental effects on the microbial protein content. This work demonstrates for the first time the effect rule of the unsaturation degree of long-chain fatty acids on the rumen microbial protein in vitro.

Functional conservation of a natural cysteine peptidase inhibitor in protozoan and bacterial pathogens

Cysteine peptidase inhibitor genes ( ICP) of the chagasin family have been identified in protozoan ( Leishmania mexicana and Trypanosoma brucei) and bacterial ( Pseudomonas aeruginosa) pathogens. The encoded proteins have low sequence identities with each other and no significant identity with cystatins or other known cysteine peptidase inhibitors. Recombinant forms of each ICP inhibit protozoan and mammalian clan CA, family C1 cysteine peptidases but do not inhibit the clan CD cysteine peptidase caspase 3, the serine peptidase trypsin or the aspartic peptidases pepsin and thrombin. The functional homology between ICPs implies a common evolutionary origin for these bacterial and protozoal proteins.

Quantitative studies of the in vitro production of pipecolic acid by rumen protozoa and its degradation by rumen bacteria

An in vitro study was conducted to quantitatively investigate the metabolism of pipecolic acid (Pip), a neuromodulator, by mixed rumen bacteria (B), mixed rumen protozoa (P), a combination of B and P (BP), species-enriched rumen protozoal suspension (Polyplastron sp., Diploplastron sp., entodinia and Entodinium caudatum) and pure cultures of several isolates of rumen bacteria (Prevetolla bryantii, Prevetolla albensis, Streptococcus bovis, Veillonella parvula, Megasphaera elsdenii and Ruminococcus albus). Only P produced Pip from L-lysine (1.0 mmol/L L-Lys) at a rate of 83.5 ± 1.6 µmol/L/h and even in BP, Pip was produced from L-Lys by P and increased at a rate of 31.2 ± 3.8 µmol/L/h. Pip production by P was highest when the substrate (L-Lys) concentration was 6 mmol/L and then the rate was 580 ± 36 µmol/L/h. Pipecolic acid production by P suspension enriched with different species of protozoa showed that Polyplastron sp. had the highest Pip production rate of 0.907 ± 0.092 µmol/L/mg protozoal protein per h, and Diploplastron sp. had the lowest rate of 0.55 ± 0.13 µmol/L/mg protozoal protein per h. The addition of D-Lys (1.0 mmol/L) as a substrate to the P suspension revealed that P were also able to produce Pip from D-Lys, though at a lower rate (1/3) compared with L-Lys (1.0 mmol/L), suggesting the presence of epimerases in P. It was confirmed that B were unable to produce Pip from L- or D-Lys. Only B degraded Pip (1.0 mmol/L) after a lag phase at a rate of 56.0 ± 1.5 µmol/L/h. The B suspension was able to degrade D-Lys, though the products were not identified. Pip degradation by pure culture of some species of rumen bacteria showed that P. bryantii and R. albus had the highest rate followed by P. albensis, S. bovis and M. elsdenii with a low rate of Pip degradation. Veillonella parvula showed no ability to degrade Pip. The results suggest that a fairly large proportion of rumen-produced Pip is likely to be absorbed by the host animal before degradation by rumen bacteria.

Protozoal Sequences may Reveal Additional Isoforms of the 14-3-3 Protein Family

The phylogenetic position of eleven 14-3-3 proteins from five protozoal species was tested relative to other eukaryotic 14-3-3 versions representing many of the previously described isoforms. The protozoal proteins, four from Entodinium caudatum, three from Entameoba histolytica and four from apicomplexan parasites formed clusters closer to the plant and animal epsilon isoforms than to the animal beta, gamma/eta, sigma/theta, and zeta isoforms. This extends the preliminary findings of Wang and Shakes (1996) but data from a wider range of genera are still required to strengthen our hypothesis that the protozoan isoforms may constitute novel isoforms of the 14-3-3 family.

Cross-reactivity of antibodies in human infections by the kinetoplastid protozoa Trypanosoma cruzi, Leishmania chagasi and Leishmania (viannia) braziliensis.

We have detected antibodies, in the sera of Chagas disease, Kala-azar and Mucocutaneous leishmaniasis patients, that bind multiple antigens shared between the three causative agents. The Chagas disease sera showed 98 to 100% positive results by ELISA when the Leishmania braziliensis and Leishmania chagasi antigens were used, respectively. The Kala-azar sera showed 100% positive results with Trypanosoma cruzi or L. braziliensis antigens by immunofluorescence assays. The antibodies in the sera of Mucocutaneous leishmaniasis patients showed 100% positive results by ELISA assays with T. cruzi or L. chagasi antigens. Furthermore, the direct agglutination of L. chagasi promastigotes showed that 95% of Kala-azar and 35% of Mucocutaneous leishmaniasis sera agglutinated the parasite in dilutions above 1:512. In contrast, 15% of Chagas sera agglutinated the parasite in dilutions 1:16 and below. Western blot analysis showed that the Chagas sera that formed at least 24 bands with the T. cruzi also formed 13 bands with the L. chagasi and 17 bands with the L. braziliensis. The Kala-azar sera that recognized at least 29 bands with the homologous antigen also formed 14 bands with the T. cruzi and 10 bands with the L. braziliensis antigens. Finally, the Mucocutaneous leishmaniasis sera that formed at least 17 bands with the homologous antigen also formed 10 bands with the T. cruzi and four bands with the L. chagasi antigens. These results indicate the presence of common antigenic determinants in several protozoal proteins and, therefore, explain the serologic cross-reactions reported here.

Sequestration, migration and lysis of protozoa in the rumen

Summary: A container with nylon and stainless steel mesh screens (10 μm and 20 μm pores, respectively), which allowed the passage of bacteria and soluble substrate but restricted the passage of protozoa, was used to investigate the phenomena of sequestration, migration and lysis of protozoa in the rumen of steers. After feeding, the concentration of Isotrichidae in the rumen increased 8·7-fold within 40 min and then decreased 88% by 4h; however, the concentration of Isotrichidae inside the containers remained almost constant. Fluctuations in concentrations of Isotrichidae were shown to be due to migration and sequestration within the rumen. Ophryoscolecidae did not exhibit the phenomena of sequestration and migration. When steers were fed once a day, about 50% of the decrease in the concentration of Ophryoscolecidae immediately after feeding could be attributed to the dilution effects of feed and water and/or passage out of the rumen. The remaining 50% of the decrease appeared to be from cell lysis, resulting in a wasteful recycling of protozoal protein. In an experiment to determine the effects of feed restriction on concentrations of Ophryoscolecidae, it was shown that decreased concentrations associated with substrate restriction are due to cell lysis rather than to passage out of the rumen.

The effect of bentonite supplementation on ruminal protozoa density and wool growth in sheep either fed roughage based diets or grazing

In penned sheep offered a mainly roughage diet, supplementation with 30, 50 or 60 g/day bentonite consistently increased the density of ruminal protozoa. The effect of 15 g/day bentonite administered via the drinking water to pregnant grazing ewes that were either normally faunated or had been defaunated for 6 months was studied. The wool growth of defaunated sheep at pasture was 25% greater (P &lt; 0.05) than that of faunated animals. The wool growth of the latter was increased 17% (P &lt; 0.15) by the addition of 15 g/day bentonite to their drinking water, while a similar addition of bentonite to defaunated sheep had no effect on wool growth. In this field study, supplementation with bentonite increased the population densities of protozoa free in the ruminal fluid of faunated sheep by 51% (5.6 X 105 v. 3.7 X 105, P&lt;0.07). It is suggested that this allows a greater flow of protozoal protein from the rumen to the intestines, leading to increased wool growth in response to supplements of bentonite.

HPLC Determination of Phosphatidylcholine and Its Application for Estimating Protozoal Protein at Duodenum

Protozoal contribution to the intestinal protein of the ruminants has been estimated by the marker method using 2-aminoethylphosphonic acid (AEP) and phosphatidylcholine (PC) as a specific marker.Utilization of AEP was, however, criticized on its wide distribution in the ruminant diet and bacteria. While PC was demonstrated its high reliability in the elaborate work of JOHN and ULYATT. But no other data is, at present, available. Their method is based on the column chromatography and subsequent phosphorus determination. In this note, we describe direct measurement of PC, by means of an HPLC, in the polar lipid fraction extracted from protozoal and duodenal samples. A preliminary trial of its application for estimating the protozoal protein entering the duodenum of sheep is also reported.

A cyclic nucleotide-independent protein kinase in Leishmania donovani.

Leishmania donovani promastigotes labelled for 2 h with 32Pi incorporated radioactivity into at least 21 different proteins, as determined by SDS/polyacrylamide-gel electrophoresis. Pulse-chase studies with 32Pi demonstrated that the labelled proteins were in a dynamic state: some radiolabelled proteins rapidly disappeared and others appeared after the chase. The possibility of an ectokinase on the parasite was examined; incubation of intact parasites for 10 min at 25 degrees C in an osmotically buffered medium containing [gamma-32P]ATP, but not [alpha-32P]ATP, resulted in the labelling of 10 different protozoal proteins, presumably localized to the surface of the organism's plasma membrane. Intact promastigotes also catalysed the transfer of 32P from [gamma-32P]ATP to histones. The histone-dependent kinase was solubilized by repeated freezing and thawing, and sonication, and purified 118-fold by chromatographing the high-speed (200,000 g, 1 h) supernatant fraction on QAE-Sephadex, Sephadex G-150 and hydroxyapatite columns. The kinase eluted as a single activity peak from all three columns. The partially purified histone-dependent kinase had the following properties: pH optimum, 7.0; optimum temperature, 37 degrees C; Km for mixed calf thymus histone, 0.15 mM; Km for ATP, 0.8 mM; preferred fractionated histone acceptors, H2b greater than H4 greater than H2a greater than H3 (H1 does not serve as an acceptor); optimum activity required 10-20 mM-Mg2+; inhibited 50-80% by 0.01 mM- and 1 mM-Ca2+; activity was not stimulated by calmodulin, cyclic AMP (1 mM) or cyclic GMP (1 mM) nor inhibited by a cyclic AMP-dependent protein kinase inhibitor (50 micrograms/assay); apparent Mr 75,000, as determined by Sephadex G-150 gel filtration chromatography; phosphorylated exclusively serine residues. Protein kinase activity was low in the early exponential phase of the growth curve and increased 6-fold upon entry into the stationary phase.

The uptake and utilization of Entodinium caudatum, bacteria, free amino acids and glucose by the rumen ciliate Entodinium bursa.

Washed suspensions of Entodinium bursa were incubated anaerobically with Entodinium caudatum, ten species of bacteria and a yeast. The rate of uptake and digestion of these micro-organisms was investigated. Protozoa grown in vivo did not engulf Proteus mirabilis or Klebsiella aerogenes but rapidly took up Bacillus megaterium. Selenomonas ruminantium, Torulopsis glabrata and Streptococcus bovis, although only the last was digested with release of soluble material into the medium. Protozoa grown in vitro engulfed each of the bacteria tested, taking up Megasphaera elsdenii and Proteus mirabilis most rapidly. Individual bacterial species and mixed rumen bacteria were engulfed more rapidly (up to 20 times) by protozoa grown in vivo than those grown in vitro, although the latter digested over 80% of the B. megaterium, Escherichia coli and P. mirabilis taken up. Labelled Ent. caudatum was extensively digested after engulfment by Ent. bursa. Some of the digestion products were released into the medium but individual amino acids were transferred as such from Ent. caudatum protein to Ent. bursa protein. Engulfed bacteria and polysaccharide granules were transferred intact from one protozoon to the other. Free amino acids were also taken up intact from the medium into protozoal protein but there was little biosynthesis of amino acids from glucose. When available for engulfment Ent. caudatum was quantitatively a much more valuable source of amino acids for protein synthesis by Ent. bursa than free amino acids or bacteria.

Negative correlation between protozoal and bacterial levels in rumen samples and its relation to the determination of dietary effects on the rumen microbial population.

The bacterial protein content and protozoal protein content of unfractionated samples from the liquid-small particle phase of the rumen were determined on the basis of direct microscopic measurement of bacteria numbers and protozoa numbers and cell volumes. Standard values of 8.7 X 10(-11) mg of protein per bacterial cell and 5.9 X 10(-11) mg/micron 3 of protozoa cell volume, obtained from analysis of isolated cells, were used to convert the microscopic measurements to an estimate of the protein content of the rumen sample. When the correlation between bacterial and protozoal protein levels was examined within groups of animals, a highly significant negative correlation between these two parameters was found (P less than 0.001). The variation among animals for total (bacterial plus protozoal) microbial protein was smaller than the variation among animals for bacterial or protozoal protein alone. There was also a highly significant positive correlation (P less than 0.001) between protozoal protein level and total microbial protein level. The variation found among animals in total microbial protein level could be reduced by using a regression equation determined for bacterial versus protozoal protein to correct for the different population dynamics of the two groups.

EFFECT OF FORMALDEHYDE TREATMENT OF SOYBEAN MEAL ON RATES OF PROTEIN DEGRADATION AND MICROBIAL PROTEIN CONCENTRATION IN THE BOVINE RUMEN

Four rumen-fistulated cows were fed concentrate:corn silage (40: 60) diets in a 4 × 4 latin square design experiment. The diets differed in the soybean meal or soybean meal treated with 0.1, 0.3 or 0.6% (wt/wt) formaldehyde. Rumen contents were collected hourly from 0900 h to 1600 h with an additional sample at half an hour after the 1100 h feeding on 3 days in the second week of each 2-wk experimental period. The maximum ammonia concentration in rumen contents decreased with increasing levels of formaldehyde used to treat the soybean meal. From 1130 to 1200 h the rates of ammonia appearance in the rumen contents were different (P &lt; 0.05) for all four diets. Treatment of soybean meal with between 0.1 and 0.3% (wt/wt) formaldehyde reduced its in vitro degradation by rumen protease by about 50% compared with the untreated control. The rates of in vivo ammonia appearance and in vitro proteolysis were highly correlated (r = 0.966, P &lt; 0.01) between 1130 h and 1200 h. These rates of rumen ammonia appearance reflected the in vivo rate of proteolysis. Both bacterial and protozoal protein content decreased significantly at the higher levels of formaldehyde treatment. There was a negative correlation (r = − 0.63, P &lt; 0.01) between protozoal and bacterial protein. Key words: Formaldehyde, soybean meal, bovine, rumen, degradation, protease

Effect of feeding frequency on the diurnal changes in microbial protein, volatile fatty acids and ammonia contents of the rumen of sheep

SUMMARYDiurnal variations in microbial protein nitrogen, total volatile fatty acids and ammonia were examined in two wethers fed once and twice daily with 0·4 kg of concentrateand 0·4 kg hay/day. The total protein nitrogen varied during the day from 76·9 to 130·2 mg/100 ml of rumen fluid. The greatest amount of protein nitrogen was noted immediately before feeding and the smallest 4 h after feeding. The protein nitrogen of rumen protozoa formed about 39·8–53·4% of total protein nitrogen in rumen fluid. Volatile fatty acids varied from 74·0 to 130·5 m-mol/1 of rumen fluid and ammonia from 187·0 to 305·6 mg/1. The higher concentration of total and protozoal protein nitrogen and of VFA was observed when animals were fed twice daily. The changing of feeding frequency from once to twice daily caused an increase of ammonia concentration in the rumen of only one wether. The results obtained are discussed.

Ruminal nitrogen metabolism and the passage of amino acids to the duodenum in sheep receiving diets containing hay and concentrates in various proportions.

AbstractThree sheep fitted with rumen cannulas and three sheep fitted with duodenal re‐entrant cannulas in addition to rumen cannulas were given, in successive periods, 700 g d−1 of five diets consisting of chopped hay and a concentrate mixture of rolled barley and flaked maize (50:50) in the ratios of 1:0, 5:2, 3:4, 1:6, and 0:1. For bacteria isolated from the rumen of sheep fitted with rumen cannulas only, there were no significant differences in either the content of nitrogen in their dry matter or in the amino acid composition, but the content of α‐ϵ‐diaminopimelic acid varied significantly (P &lt; 0.05) between diets, mean values ranging from 39 to 50 kg−1 nitrogen. As the proportion of concentrate in the diet increased, the intake of nitrogen was progressively reduced from 11.9 to 9.8 g d−1 and this was accompanied by an increase in the amount of organic matter apparently digested in the rumen, from 296 to 402 g d−1. But there were no significant differences between diets in either the ruminal concentration of ammonia‐nitrogen (mean values varied from 78 to 96 mg litre−1) or in the duodenal passage of total nitrogen, which ranged from 12.1 to 14.7 g d−1 with a peak value for the 5:2 diet. Rates of synthesis of bacterial protein in the rumen were 186, 228, 124 and 94 g crude protein per kg organic matter apparently digested in the rumen for the 1:0, 5:2, 3:4, 1:6 and 0:1 diets respectively, the rates for the 1:6 and 0:1 diets being significantly (P &lt; 0.05) lower than those for the other diets. The 0:1 diet was associated with a low proportion, 0.5, of bacterial protein in the duodenal crude protein and it is suggested that with this diet, protozoal protein made an appreciable contribution to the crude protein passing from the rumen.

The contribution of protozoa to the protein entering the duodenum of sheep.

1. Four sheep, each fitted with a rumen fistula and a re-entrant cannula at the proximal duodenum were fed a semi-purified diet containing urea as the only nitrogen source. The quantities of total protozoal amino acid-N (TPAN) present in the rumen and entering the duodenum were determined when the mean rumen dilution rate (D) was low (0.034/h) and when D was increased to 0.078/h by the intraruminal infusion of artificial saliva. 2. Increasing the dilution rate had no significant effect upon the proportions of TPAN present in the total microbial amino-acid-N (TMAN) of the rumen fluid and duodenal digesta. With both dilution rates the mean proportion of TPAN in the duodenal TMAN (0.24) was markedly less than the equivalent proportion (0.45) found in the rumen fluid. 3. The daily flow of TPAN, as measured at the duodenal cannula at both dilution rates were equivalent to only 41% of the flow of TPAN as predicted from measurements of rumen outflow, indicating that a substantial proportion of rumen protozoal protein was retained within the rumen.