Ureolytic bacteria in sheep rumen.

Estimates were made of the numbers of viable bacteria in the rumens of sheep receiving different rations. Representative colonies were isolated and tested for urease production. Some urease-positive isolates were characterized and identified. The ureolytic activities of the urease-producing isolates were determined and compared with the activity of rumen fluid. The rations fed to the sheep did not exert a significant influence on the relative numbers of the urease-producting organisms in the rumen. No obligately anaerobic ureolytic bacteria were found. All urease-positive isolates were facultatively anaerobic, Gram-positive, catalase-positive cocci. Out of ten isolates, nine were identified as Staphylococcus saprophyticus and one as Micrococcus varians. The total urease activity of the different isolates based on the lowest numbers in which they were present in the rumen, compared favourably with the urease activity of rumen fluid. The facultatively anaerobic Gram-positive cocci were probably responsible for a large proportion of the urease activity of the rumen fluid. Conditions prevailing in the rumen were found to be conducive to urease production by the isolates tested.     

Adrenergic reactions of sheep rumen in vitro

The alpha and beta-adrenergic responses of the isolated muscle of sheep rumen were analysed by pharmacodynamic methods after administration of alpha and beta-adrenergic agonists and alpha and beta-adrenergic antagonists. It was found that phenylephrine, and in a lower degree propranolol, stimulated contractions of isolated muscle of sheep rumen while adrenaline, noradrenaline, isoprenaline, phenoxybenzamine and regitine inhibited these contractions. Propranolol abolished the dilating (atonic) effect of catecholamines on the isolated muscles of sheep rumen and previous blockade of beta-adrenergic receptors with propranolol reversed the dilating effects of catecholamines. It is concluded that noradrenaline has an ambiceptor effect (similar to that of adrenaline) on the isolated muscle of the rumen.     

Urease activity of adherent bacteria in the sheep rumen

In experiments on six sheep fed on a low protein diet (6.2 g N/day), it was found that the urease activity of the rumen fluid did not change significantly in the first 6 hours after feeding and that it ranged from 45 to 75 nkat.ml-1. The major portion was bound to the bacterial fraction and formed about 70% of total rumen fluid activity. Urease activity determined in food particles with adherent bacteria removed from the rumen before and 3 and 6 hours after feeding ranged from 20 to 26 nkat.g-1 food (wet weight), and on rumen wall samples with adherent bacteria from 30 to 800 nkat per 2.5 cm2 tissue. Again, no significant changes correlated to the time after feeding were found. The results show that urease activity in the sheep rumen is localized on food particles and on rumen wall epithelium with adherent bacteria, as well as in the rumen fluid.     

Isolation and attempted introduction of sugar alcohol-utilizing bacteria in the sheep rumen

R.J. WALLACE AND N.D. WALKER. 1993. Bacteria that use sorbitol, xylitol, maltitol and dulcitol (galactitol) were isolated from the sheep rumen following enrichments in which bacteria were grown in rumen fluid medium where the sugar alcohol was the only added energy source. Only isolates obtained with sorbitol and maltitol grew sufficiently rapidly to be considered for enrichment by the sugar alcohol in vivo. Isolate SS2, a strain of Selenomonas ruminantium var. lactilytica which grew on sorbitol at 0.87 h^-1 was selected for further study and a rifampicin-resistant mutant, SS2/R5, was isolated to facilitate tracking in the mixed population. Despite an initial transient increase in numbers, a significant population of S. ruminantium SS2/R5 failed to establish in sheep which were dosed twice daily with 10 g of sorbitol. Continuous infusion of sorbitol increased numbers only slightly compared with twice-daily dosing. In vitro experiments indicated that strain SS2/R5 grew less well in the presence of other rumen organisms, particularly ciliate protozoa, than in pure culture. Furthermore, the concentration of sorbitol in vivo was lower than predicted from in vitro experiments, indicating that sorbitol was absorbed rapidly from the rumen. Similar observations were made with xylitol, dulcitol and maltitol. Proposed enrichment strategies that use sugar alcohols or other materials to support the growth of introduced bacteria will thus have to take account of the combined problems of microbe-microbe interactions and the loss of the compounds by absorption from the rumen.     

Evaluation of the effects of different diets on microbiome diversity and fatty acid composition of rumen liquor in dairy goat

Fat supplementation plays an important role in defining milk fatty acids (FA) composition of ruminant products. The use of sources rich in linoleic and α-linolenic acid favors the accumulation of conjugated linoleic acids isomers, increasing the healthy properties of milk. Ruminal microbiota plays a pivotal role in defining milk FA composition, and its profile is affected by diet composition. The aim of this study was to investigate the responses of rumen FA production and microbial structure to hemp or linseed supplementation in diets of dairy goats. Ruminal microbiota composition was determined by 16S amplicon sequencing, whereas FA composition was obtained by gas-chromatography technique. In all, 18 pluriparous Alpine goats fed the same pre-treatment diet for 40±7 days were, then, arranged to three dietary treatments consisting of control, linseed and hemp seeds supplemented diets. Independently from sampling time and diets, bacterial community of ruminal fluid was dominated by Bacteroidetes (about 61.2%) and Firmicutes (24.2%) with a high abundance of Prevotellaceae (41.0%) and Veillonellaceae (9.4%) and a low presence of Ruminococcaceae (5.0%) and Lachnospiraceae (4.3%). Linseed supplementation affected ruminal bacteria population, with a significant reduction of biodiversity; in particular, relative abundance of Prevotella was reduced (−12.0%), whereas that of Succinivibrio and Fibrobacter was increased (+50.0% and +75.0%, respectively). No statistically significant differences were found among the average relative abundance of archaeal genera between each dietary group. Moreover, the addition of linseed and hemp seed induced significant changes in FA concentration in the rumen, as a consequence of shift from C18 : 2n-6 to C18 : 3n-3 biohydrogenation pathway. Furthermore, dimethylacetal composition was affected by fat supplementation, as consequence of ruminal bacteria population modification. Finally, the association study between the rumen FA profile and the bacterial microbiome revealed that Fibrobacteriaceae is the bacterial family showing the highest and significant correlation with FA involved in the biohydrogenation pathway of C18 : 3n-3.     

一株瘤胃源乳酸利用菌的分离鉴定及其体外代谢特性

【目的】从饲喂高精料的本地山羊瘤胃内分离到一株利用乳酸并能产生大量丙酸的菌株L9,并进一步研究了该菌在调控瘤胃微生物发酵中的作用。【方法】采用厌氧培养技术,结合形态、生理生化特性和16SrRNA基因序列分析结果。【结果】该菌株被鉴定为反刍兽新月形单胞菌（Selenomonas ruminantium）。该菌株体外代谢特性研究表明,L9可利用乳酸作为唯一碳源,该菌在24h内可对90mmol／L的乳酸完全降解。体外摸拟瘤胃急性酸中毒的发酵试验结果表明,以淀粉为底物时,与对照组相比,添加菌株L9可显著降低瘤胃微生物体外培养体系中乳酸浓度,提高pH值,提高总挥发性脂肪酸和丙酸浓度,并显著降低乙酸与丙酸的浓度比（P〈0．05）。【结论】结果显示,菌株L9是一株可代谢乳酸,促进丙酸生成,提高总挥发性脂肪酸浓度的有益瘤胃细菌。     

Influence of natural magnesium sources on the in vitro fermentation and protozoan population in the rumen fluid collected from sheep

The objective of this experiment was to determine the effect of two types of caustic calcinated magnesite (caustic magnesite (CM) and Agromag (AG)) upon the end products of in vitro fermentation (total gas, methane, total and individual fatty acids, and VFA) and protozoan population in the rumen fluid collected from sheep. Both magnesium additives (CM and AG) as natural products in the dose of 0.01 g were added to the fermentation bottles containing rumen inoculum from sheep and different substrates. Meadow hay (MH), wheat straw (WS), amorphous cellulose (AC) and barley grain (BG) were used as substrates and incubated with the buffered rumen fluid using an in vitro gas measuring technique during 72 h of incubation. The rumen protozoa, Entodinium spp., Trichostomatids and large Entodiniomorphids and the total protozoan concentration were counted after 24 h of incubation. The methane production was significantly decreased with CM or AG, respectively, by 58 or 62% (MH), by 65% (WS), by 52% (AC) and by 58% (BG). The total VFA concentration was significantly lower compared to control for CM plus MH, WS, AC, BG and AG plus WS. The total VFA concentration was significantly higher compared to control for AG plus AC. The effect of the both additives on ciliate population was not uniform and depended on the substrates used and protozoan type. Ciliate population was significantly increased in Entodinium spp. (AG plus BG) and Diploplastron affinae (CM or AG plus BG) compared to control. Tested additives significantly decreased population of Entodinium spp. (AG plus MH or AC), Dasytricha ruminantium (AG plus AC), Ophryoscolex c. tricoronatus, Eremoplastron dilobum and Polyplastron multivesiculatum (CM or AG plus BG). It can be concluded that both natural magnesium sources influenced rumen fermentation patterns and protozoan population in vitro depending on the type of the substrate used; therefore, the relative efficacy of individual tested additive cannot be determined from these experiments. In vivo experiments are required in future.     

Characterization of several bovine rumen bacteria isolated with a xylan medium

Dehority, B. A. (Ohio Agricultural Research and Development Center, Wooster). Characterization of several bovine rumen bacteria isolated with a xylan medium. J. Bacteriol. 91:1724-1729. 1966.-Studies were conducted to characterize eight strains of bacteria isolated from bovine rumen contents, by use of a medium containing xylan as the only added carbohydrate source. Based on morphology, biochemical reactions, nutritional requirements, and fermentation products, five of the eight strains were identified as Butyrivibrio fibrisolvens. Many properties of the remaining three strains resembled Bacteroides ruminicola; however, propionic acid was consistently found as a fermentation product. When the type strains for B. ruminicola subsp. ruminicola and B. ruminicola subsp. brevis were compared with the present isolates, it was found that propionic acid was a normal fermentation product for the type strain B. ruminicola subsp. ruminicola when grown in a 40% rumen fluid-0.5% glucose broth. Production of propionic acid was markedly reduced for all strains when grown in a 20% rumen fluid-1% glucose broth. The three remaining strains were thus placed in the species B. ruminicola, and further classified into the subspecies ruminicola (one strain) and brevis (two strains) on the basis of their requirement for hemin. Although the type strain of B. ruminicola subsp. brevis did not produce propionic acid, both of the present isolates classified as this subspecies produced substantial amounts. One strain of B. ruminicola subsp. brevis had an absolute requirement for volatile fatty acids. Either isobutyric or dl-2-methylbutyric acid would satisfy this requirement, whereas isovaleric acid was ineffective. It is of interest that xylan-fermenting bacteria isolated from 10(-7) and 10(-8) dilutions of rumen contents by use of a xylan medium are similar to the xylan fermenters isolated at the same dilutions with a nonselective medium.     

Effect of whole-plant corn silage treated with lignocellulose-degrading bacteria on growth performance,rumen fermentation,and rumen microflora in sheep

Lignification of cellulose limits the effective utilisation of fibre in plant cell wall. Lignocellulose-degrading bacteria secrete enzymes that decompose lignin and have the potential to improve fibre digestibility. Therefore, this study aimed to investigate the effect of whole-plant corn silage inoculated with lignocellulose-degrading bacteria on the growth performance, rumen fermentation, and rumen microbiome in sheep. Twelve 2-month-old male hybrid sheep (Dorper ♂ × small-tailed Han ♀) were randomly assigned into two dietary groups (n = 6): (1) untreated whole-plant corn silage (WPCS) and (2) WPCS inoculated with bacterial inoculant (WPCSB). Whole-plant corn silage inoculated with bacterial inoculant had higher in situ NDF digestibility than WPCS. Sheep in the WPCSB group had significantly higher average daily gain, DM intake, and feed conversion rate than those in the WPCS group (P < 0.05). Furthermore, higher volatile fatty acid concentrations were detected in WPCSB rumen samples, leading to lower ruminal pH (P < 0.05). The WPCSB group showed higher abundance of Bacteroidetes and lower abundance of Firmicutes in the rumen microbiome than the WPCS group (P < 0.05). Multiple differential genera were identified, with Prevotella being the most dominant genus and more abundant in WPCSB samples. Moreover, the enriched functional attributes, including those associated with glycolysis/gluconeogenesis and citrate cycle, were more actively expressed in the WPCSB samples than in the WPCS samples. Additionally, certain glucoside hydrolases that hydrolyse the side chains of hemicelluloses and pectins were also actively expressed in the WPCSB microbiome. These findings suggested that WPCSB increased NDF digestibility in three ways: (1) by increasing the relative abundance of the most abundant genera, (2) by recruiting more functional features involved in glycolysis/gluconeogenesis and citrate cycle pathways, and (3) by increasing the relative abundance and/or expression activity of the glucoside hydrolases involved in hemicellulose and pectin metabolism. Our findings provide novel insights into the microbial mechanisms underlying improvement in the growth performance of sheep/ruminants. However, the biological mechanisms cannot be fully elucidated using only metagenomics tools; therefore, a combined multi-omics approach will be used in subsequent studies.     

Influence of intoxication with organophosphates on rumen bacteria and rumen protozoa and protective effect of clinoptilolite-rich zeolite on bacterial and protozoan concentration in rumen

The effect ofO-ethyl-S-(2-diisopropylaminoethyl) methylthiophosphonate on rumen bacteria and rumen protozoa was investigated in sheep (after premedication with clinoptilolite-rich zeolite and without that premedication). In control animals a decrease in the total concentration of rumen protozoa was observed 3–7 d after intoxication (particularly in small and large ones). In clinoptilolite-rich-zeolite-treated animals only a slight decrease in protozoan numbers occurred during the first hours after the intoxication. Similarly, in every category of rumen bacteria marked differences between the groups were recorded, particularly in concentration of lipolytic bacteria. The results suggest some protective effect of clinoptilolite-rich zeolite for rumen microbiota against the organophosphate poison.     

Influence of tannic acid application on alfalfa hay: in vitro rumen fermentation, serum metabolites and nitrogen balance in sheep

Alfalfa protein is poorly utilised by ruminants due to its rapid degradation in rumen. The objective of the study was to assess the influence of spraying tannic acid (TA) on chopped alfalfa hay on in vitro rumen fermentation and nitrogen (N) retention by sheep. Alfalfa hay with and without TA was fed to sheep to determine nutrient digestibility and N balance. TA was sprayed on chopped alfalfa at three concentrations to determine its effect on in vitro fermentation of dry matter (DM) and N balance in sheep. Final TA concentrations were 0, 30, 60 and 90 g TA per kg DM. The control was sprayed with the same amount of water but without TA. In vitro DM degradation and the production of gas, ammonium-N (NH4-N) and short-chain fatty acid (SCFA) were measured. TA-sprayed alfalfa and the control were fed to sheep to determine nutrient digestibility and N retention. Addition of TA had no influence on the extent and rate of gas production but significantly decreased NH4-N concentration at 30 (P < 0.05), 60 and 90 (P < 0.0001) g/kg DM. Addition of polyethylene glycol (PEG) to TA-sprayed alfalfa increased NH4-N to a level comparable to non-TA-sprayed alfalfa. Spraying of alfalfa with TA significantly decreased (P < 0.05) isovalerate but did not affect the total and individual SCFA acid production. Tannic acid significantly (P < 0.05) reduced in vitro true degradability of DM (IVTD) after 24 h incubation at levels of 60 and 90 g TA per kg DM. Neutral-detergent fibre digestibility (dNDF) after 24 h (P < 0.01), 60 and 90 (P < 0.0001) g TA per kg DM. The effect of TA on either IVTD or dNDF was not significant (P > 0.05) after 48 h of incubation. There was a strong linear relationship between percentage increase in gas production due to PEG and protein precipitation capacity (R2 = 0.94). N digestibility was significantly reduced with all three levels of TA additions. However, the proportion of urine-N to total N output was reduced by adding 60 g (P < 0.05) and 90 g (P < 0.01) TA per kg DM. Serum metabolites and liver enzymes were not affected by TA (P > 0.05). Higher faecal N as the TA level increased indicates incomplete dissociation of tannin-protein complexes post ruminally. Factors affecting dissociation of tannin-protein complexes need further study.     

Comparative growth rates of various rumen bacteria in clarified rumen fluid from cows and sheep fed different diets.

Pure cultures of strains of different species of rumen bacteria were grown in filter-sterilized rumen fluid supplemented with glucose, bicarbonate, and reducing agent (cysteine and sulfide). Growth rates were determined in a series of experiments. Strains of species most abundant in the rumen grew more rapidly than strains of less abundant bacteria. Ammonia, amino acids, and peptides increased growth rates to some extent, but the greatest stimulatory effect for less abundant bacteria was provided by other factors, present in yeast extract. Factors released from lysates of mixed rumen microbes stimulated growth, but their rate of release was slow. It was concluded that, besides energy and nitrogen sources, growth factors of an as-yet-undetermined nature probably play an important role in determining the predominance of different bacterial species in the rumen.     

Comparative growth rates of various rumen bacteria in clarified rumen fluid from cows and sheep fed different diets.

Pure cultures of strains of different species of rumen bacteria were grown in filter-sterilized rumen fluid supplemented with glucose, bicarbonate, and reducing agent (cysteine and sulfide). Growth rates were determined in a series of experiments. Strains of species most abundant in the rumen grew more rapidly than strains of less abundant bacteria. Ammonia, amino acids, and peptides increased growth rates to some extent, but the greatest stimulatory effect for less abundant bacteria was provided by other factors, present in yeast extract. Factors released from lysates of mixed rumen microbes stimulated growth, but their rate of release was slow. It was concluded that, besides energy and nitrogen sources, growth factors of an as-yet-undetermined nature probably play an important role in determining the predominance of different bacterial species in the rumen.     

Influence of dietary acetylated peptides on fermentation and peptidase activities in the sheep rumen

The predominant mechanism of peptide breakdown by rumen micro-organisms is aminopeptidase. Thus acetylation of the N-terminus of peptides inhibits their degradation by rumen micro-organisms in short-term incubations with rumen fluid in vitro . An experiment was undertaken to determine if adaptation of the rumen microbial population would take place when acetylated peptides were fed for a prolonged period, which would enable the microbial population to break down the protected peptides and thus decrease their nutritive value. Three adult sheep, fitted with permanent rumen cannulae, received a maintenance hay/concentrate diet to which was added, at each meal, 20 g of casein enzymic hydrolysate ('peptides') or 20 g of peptides previously treated with acetic anhydride. The diets were fed for 28 d in a 3 × 3 latin square and samples were taken during the last 7 d. Fermentation products and NH₃ concentrations indicated that acetylated peptides remained less degradable than untreated peptides. There was a trend towards increased proteolytic activity with acetylated peptides, and dipeptidase activity increased by 18% and 28%, respectively, compared with untreated peptides and control treatments. Activity against N-acetyl-Ala₂ also increased when acetylated peptides were fed, but it remained only 13% of the rate of Ala₂ hydrolysis. No increase was found in the rate of ammonia production from acetylated peptides in animals receiving acetylated peptides–this rate was 26% of that found with untreated peptides–and acetylated peptides continued to persist for longer in the rumen than untreated peptides after feeding. Thus it was concluded that the rumen microbial population did not adapt to utilize acetylated peptides.