Relationship between true digestibility of dietary phosphorus and gastrointestinal bacteria of goats.

The present research was conducted to evaluate the connection between the true digestibility of Phosphorus (TDP) in diet and bacterial community structure in the gastrointestinal tract (GIT) of goats. Twenty-eight Nubian goats were chosen and metabolic experiment was conducted to analyze TDP of research animals. Eight goats were grouped into the high digestibility of phosphorus (HP) phenotype, and another 8 were grouped into the low digestibility of phosphorus (LP) phenotype. And from the rumen, abomasum, jejunim, cecum and colon content of the goats, bacterial 16S rRNA gene amplicons were sequenced. In the rumen 239 genera belonging to 23 phyla, in abomasum 319 genera belonging to 30 phyla, in jejunum 248 genera belonging to 36 phyla, in colon 248 genera belonging to 25 phyla and in cecum 246 genera belonging to 23 phyla were noticed. In addition, there was a significant correlation between the TDP and the abundance of Ruminococcaceae_UCG-010, Ruminococcus_2, Ruminococcaceae_UCG-014, Selenomonas_1 and Prevotella in the rumen, Lachnospiraceae_ND3007_group, Saccharofermentans, Ruminococcus_1, Ruminococcaceae_UCG-014, Lachnospiraceae_XPB1014_group and Desulfovibrio in the abomasum, Prevotella, Clostridium_sensu_stricto_1, Fibrobacter, Desulfovibrio and Ruminococcus_2 in the jejunum, Ruminococcaceae_UCG-014 in the colon, and Desulfovibrio in the cecum. Present research trial recommended that the community of gastrointestinal microbiota is a factor affecting TDP in goats.

A metagenomics investigation of carbohydrate-active enzymes along the goat and camel intestinal tract

Studies of the digestive microbiota of ruminant animals most often focus on the bacterial diversity in the rumen or the feces of the animals, but little is known about the diversity and functions of their distal intestine. Here, the bacterial microbiota of the distal intestinal tract of two goats and two camels was investigated by metagenomics techniques. The bacterial taxonomic diversity and carbohydrate-active enzyme profile were estimated for samples taken from the small intestine, the large intestine, and the rectum of each animal. The bacterial diversity and abundance in the small intestine were lower than in the rectal and large intestinal samples. Analysis of the carbohydrate-active enzyme profiles at each site revealed a comparatively low abundance of enzymes targeting xylan and cellulose in all animals examined, similar to what has been reported earlier for sheep and therefore suggesting that plant cell wall digestion probably takes place elsewhere, such as in the rumen

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Microbial diversity in the rumen, reticulum, omasum, and abomasum of yak on a rapid fattening regime in an agro-pastoral transition zone.

The ruminant digestive system harbors a complex gut microbiome, which is poorly understood in the case of the four stomach compartments of yak. High-throughput sequencing and quantitative PCR were used to analyse microbial communities in the rumen, reticulum, omasum, and abomasum of six domesticated yak. The diversity of prokaryotes was higher in reticulum and omasum than in rumen and abomasum. Bacteroidetes predominated in the four stomach compartments, with abundance gradually decreasing in the trend rumen > reticulum > omasum > abomasum. Microorganism composition was different among the four compartments, all of which contained high levels of bacteria, methanogens, protozoa and anaerobic fungi. Some prokaryotic genera were associated with volatile fatty acids and pH. This study provides the first insights into the microorganism composition of four stomach compartments in yak, and may provide a foundation for future studies in this area.

Structural and Functional Characterization of a Ruminal ß-Glycosidase Defines a Novel Subfamily of Glycoside Hydrolase Family 3 with Permuted Domain Topology.

Metagenomics has opened up a vast pool of genes for putative, yet uncharacterized, enzymes. It widens our knowledge on the enzyme diversity world and discloses new families for which a clear classification is still needed, as is exemplified by glycoside hydrolase family-3 (GH3) proteins. Herein, we describe a GH3 enzyme (GlyA1) from resident microbial communities in strained ruminal fluid. The enzyme is a ß-glucosidase/ß-xylosidase that also shows ß-galactosidase, ß-fucosidase, α-arabinofuranosidase, and α-arabinopyranosidase activities. Short cello- and xylo-oligosaccharides, sophorose and gentibiose, are among the preferred substrates, with the large polysaccharide lichenan also being hydrolyzed by GlyA1 The determination of the crystal structure of the enzyme in combination with deletion and site-directed mutagenesis allowed identification of its unusual domain composition and the active site architecture. Complexes of GlyA1 with glucose, galactose, and xylose allowed picturing the catalytic pocket and illustrated the molecular basis of the substrate specificity. A hydrophobic platform defined by residues Trp-711 and Trp-106, located in a highly mobile loop, appears able to allocate differently ß-linked bioses. GlyA1 includes an additional C-terminal domain previously unobserved in GH3 members, but crystallization of the full-length enzyme was unsuccessful. Therefore, small angle x-ray experiments have been performed to investigate the molecular flexibility and overall putative shape. This study provided evidence that GlyA1 defines a new subfamily of GH3 proteins with a novel permuted domain topology. Phylogenetic analysis indicates that this topology is associated with microbes inhabiting the digestive tracts of ruminants and other animals, feeding on chemically diverse plant polymeric materials.

Pathophysiological evaluation of subacute ruminal acidosis (SARA) by continuous ruminal pH monitoring.

Evaluation of the radio-transmission pH-measurement system for monitoring the ruminal pH and subacute ruminal acidosis (SARA) in cattle is described. This is done in order to reveal the possible application of this system for detection and pathophysiological research of SARA by continuous ruminal pH measurement. The possibility of using this system for assessment of the ruminal pH in SARA cattle, and the presence of negative correlation between the ruminal pH and ruminal temperature in heathy and SARA cattle were determined. In addition, the 16S rRNA gene pyrosequencing analysis showed that the ruminal microbial community was simpler in SARA cattle, and the bacterial numbers in SARA cattle were lower than those in healthy hay-fed cattle. Concentrate feeding might have reduced the diversity of the ruminal microbial community. Changes in the ruminal microbial community of SARA cattle might be related to the changes in ruminal pH followed by the decrease in the number of some bacteria. Continuous monitoring of the ruminal pH using the radio-transmission pH-measurement system would be applied for detection and prevention of SARA in the field and pathophysiological research of SARA, including ruminal zymology and bacteriology, which have been determined previously by sampling of the ruminal fluid and measuring of ruminal pH.

Effects of dietary linseed oil and propionate precursors on ruminal microbial community, composition, and diversity in Yanbian yellow cattle.

The rumen microbial ecosystem is a complex system where rumen fermentation processes involve interactions among microorganisms. There are important relationships between diet and the ruminal bacterial composition. Thus, we investigated the ruminal fermentation characteristics and compared ruminal bacterial communities using tag amplicon pyrosequencing analysis in Yanbian yellow steers, which were fed linseed oil (LO) and propionate precursors. We used eight ruminally cannulated Yanbian yellow steers (510 ± 5.8 kg) in a replicated 4 × 4 Latin square design with four dietary treatments. Steers were fed a basal diet that comprised 80% concentrate and 20% rice straw (DM basis, CON). The CON diet was supplemented with LO at 4%. The LO diet was also supplemented with 2% dl-malate or 2% fumarate as ruminal precursors of propionate. Dietary supplementation with LO and propionate precursors increased ruminal pH, total volatile fatty acid concentrations, and the molar proportion of propionate. The most abundant bacterial operational taxonomic units in the rumen were related to dietary treatments. Bacteroidetes dominated the ruminal bacterial community and the genus Prevotella was highly represented when steers were fed LO plus propionate precursors. However, with the CON and LO diet plus malate or fumarate, Firmicutes was the most abundant phylum and the genus Ruminococcus was predominant. In summary, supplementing the diets of ruminants with a moderate level of LO plus propionate precursors modified the ruminal fermentation pattern. The most positive responses to LO and propionate precursors supplementation were in the phyla Bacteriodetes and Firmicutes, and in the genus Ruminococcus and Prevotella. Thus, diets containing LO plus malate or fumarate have significant effects on the composition of the rumen microbial community.

Intravenous lipopolysaccharide challenge alters ruminal bacterial microbiota and disrupts ruminal metabolism in dairy cattle.

In the present study, three primiparous lactating Holstein cows (260-285 d in lactation) were used in a 3 × 3 Latin square design to assess the effects of three doses (0.0, 0.4 and 0.8 µg/kg body weight) of lipopolysaccharide (LPS, Escherichia coli 0111:B4) on changes in ruminal microbiota and ruminal fermentation. Ruminal pH was linearly decreased (P< 0.001) by LPS challenge, and the concentrations of acetate, propionate, butyrate, total volatile fatty acids and amino N increased linearly (P< 0.001) according to the LPS dose. LPS infusion linearly decreased (P< 0.001) the organic matter degradability of alfalfa hay and soyabean meal in the rumen, but did not affect (P>0.10) the gene expression of Na⁺/K⁺-ATPase and monocarboxylic acid transporter-1, -2 and -4. A plot of principal coordinate analysis based on unweighted UniFrac values and analysis of molecular variance revealed that the structure of ruminal bacterial communities in the control was distinct from that of the ruminal microbiota in the cattle exposed to LPS. At the phylum level, when compared with the control group, LPS infusion in the tested cows linearly increased (P< 0.05) the abundance of Firmicutes, and linearly decreased (P< 0.05) the percentage of Bacteroidetes, Tenericutes, Spirochaetes, Chlorobi and Lentisphaerae. To our knowledge, this is the first study to report that intravenously LPS challenge altered the ruminal bacterial microbiota and fermentation profiles. The present data suggest that systemic LPS could alter ruminal environment and ruminal microbiota composition, leading to a general decrease in fermentative activity.

Ruminal Prevotella spp. may play an important role in the conversion of plant lignans into human health beneficial antioxidants.

Secoisolariciresinol diglucoside (SDG), the most abundant lignan in flaxseed, is metabolized by the ruminal microbiota into enterolignans, which are strong antioxidants. Enterolactone (EL), the main mammalian enterolignan produced in the rumen, is transferred into physiological fluids, with potentially human health benefits with respect to menopausal symptoms, hormone-dependent cancers, cardiovascular diseases, osteoporosis and diabetes. However, no information exists to our knowledge on bacterial taxa that play a role in converting plant lignans into EL in ruminants. In order to investigate this, eight rumen cannulated cows were used in a double 4 × 4 Latin square design and fed with four treatments: control with no flax meal (FM), or 5%, 10% and 15% FM (on a dry matter basis). Concentration of EL in the rumen increased linearly with increasing FM inclusion. Total rumen bacterial 16S rRNA concentration obtained using Q-PCR did not differ among treatments. PCR-T-RFLP based dendrograms revealed no global clustering based on diet indicating between animal variation. PCR-DGGE showed a clustering by diet effect within four cows that had similar basal ruminal microbiota. DNA extracted from bands present following feeding 15% FM and absent with no FM supplementation were sequenced and it showed that many genera, in particular Prevotella spp., contributed to the metabolism of lignans. A subsequent in vitro study using selected pure cultures of ruminal bacteria incubated with SDG indicated that 11 ruminal bacteria were able to convert SDG into secoisolariciresinol (SECO), with Prevotella spp. being the main converters. These data suggest that Prevotella spp. is one genus playing an important role in the conversion of plant lignans to human health beneficial antioxidants in the rumen.

Net transfer of nutrients to the duodenum and disappearance of n-alkanes in the reticulo-rumen and the hindgut of sheep fed grass/legume combinations.

An experiment was carried out to examine the effect of increasing the proportion of Wimmera ryegrass hay in a lucerne hay-based diet on net transfer of nutrients to the intestine, and on the disappearance of n-alkanes in the reticulo-rumen and the hindgut of sheep. Following a latin square design, four adult ewes were fed 1:0, 0·33:0·67, 0·67:0·33 and 0:1 proportions of legume and grass. Increasing the proportion of ryegrass in the diet linearly decreased the intake of DM (P= 0·017), organic matter (P= 0·021) and N (P= 0·001). However, neutral-detergent fibre intake was not affected (P= 0·148), nor was its digestibility coefficient (P>0·10). Diet had no effect on duodenal flows of nutrients (P>0·10), although the proportion of N intake (NI) recovered at the duodenum as non-NH3 N (NAN) increased linearly with Lolium rigidum in the diet (P= 0·002). Full recovery of NI as NAN was achieved at NH3 concentrations in the rumen below 110 g/l. Microbial N contribution to NAN varied in a quadratic manner (P< 0·05) with the proportion of grass in the diet, although efficiency of microbial synthesis was not affected (P>0·10). Duodenal recovery of consumed n-alkanes was not affected by diet and was complete for those present in higher concentrations in the forages. Isolated rumen bacteria contained significant amounts of n-alkanes, contributing to the duodenal flow of these hydrocarbons in variable proportions depending on the diet consumed.

Lovastatin-enriched rice straw enhances biomass quality and suppresses ruminal methanogenesis.

The primary objective of this study was to test the hypothesis that solid state fermentation (SSF) of agro-biomass (using rice straw as model); besides, breaking down its lignocellulose content to improve its nutritive values also produces lovastatin which could be used to suppress methanogenesis in the rumen ecosystem. Fermented rice straw (FRS) containing lovastatin after fermentation with Aspergillus terreus was used as substrate for growth study of rumen microorganisms using in vitro gas production method. In the first experiment, the extract from the FRS (FRSE) which contained lovastatin was evaluated for its efficacy for reduction in methane (CH4) production, microbial population, and activity in the rumen fluid. FRSE reduced total gas and CH4 productions (P < 0.01). It also reduced (P < 0.01) total methanogens population and increased the cellulolytic bacteria including Ruminococcus albus, Fibrobacter succinogenes (P < 0.01), and Ruminococcus flavefaciens (P < 0.05). Similarly, FRS reduced total gas and CH4 productions, methanogens population, but increased in vitro dry mater digestibility compared to the non-fermented rice straw. Lovastatin in the FRSE and the FRS significantly increased the expression of HMG-CoA reductase gene that produces HMG-CoA reductase, a key enzyme for cell membrane production in methanogenic Archaea.

Validation of a novel extraction method for studying hexahydro-1,3,5-trinitro-1,3,5 triazine (RDX) biodegradation by ruminal microbiota.

A simple, fast liquid-liquid extraction method was developed for studying hexahydro-1,3,5-trinitro-1,3,5 triazine (RDX) biodegradation using small sample volumes. The method was tested in vitro with anaerobic incubations of RDX with whole rumen fluid (WRF) and a commercial Sporanaerobacter acetigenes strain in methanogenic media for RDX. Additionally, validation experiments were conducted in deionized water in order to show applicability toward various aqueous matrices. Conditions for extraction were as follows: 300 µL of sample were mixed with an equal volume of a 0.34 M ammonium hydroxide solution to reach a basic pH, extracted with a hexane/ethyl acetate 1:1 (v/v) solution (1 mL) and shaken vigorously for 10 s. The resulting organic phase was transferred, then dried under a constant flow of N2 and reconstituted with acetonitrile (300 µL) for HPLC-UV and LC-MS/MS analysis. Percent recovery values were obtained (83-101%) in all matrices for RDX. In WRF (n=3 animals), RDX degradation was observed with almost 100% elimination of RDX after 4 h. The five nitroso and ring cleavage metabolites were observed by mass spectrometry. Liquid cultures of S. acetigenes did not show significant RDX biodegradation activity. RDX extractions from deionized water samples indicated acceptable recoveries with low variability, suggesting suitability of the method for aqueous matrices. Overall, the new method demonstrated acceptable efficiency and reproducibility across three matrices, providing an advantageous alternative for studies where complex matrices and small volume samples are in use.

Ruminal microbe of biohydrogenation of trans-vaccenic acid to stearic acid in vitro.

BACKGROUND: Optimization of the unsaturated fatty acid composition of ruminant milk and meat is desirable. Alteration of the milk and fatty acid profile was previously attempted by the management of ruminal microbial biohydrogenation. The aim of this study was to identify the group of ruminal trans-vaccenic acid (trans-11 C18:1, t-VA) hydrogenating bacteria by combining enrichment studies in vitro. METHODS: The enrichment culture growing on t-VA was obtained by successive transfers in medium containing t-VA. Fatty acids were detected by gas chromatograph and changes in the microbial composition during enrichment were analyzed by denaturing gradient gel electrophoresis (DGGE). Prominent DGGE bands of the enrichment cultures were identified by 16S rRNA gene sequencing. RESULTS: The growth of ruminal t-VA hydrogenating bacteria was monitored through the process of culture transfer according to the accumulation of stearic acid (C18:0, SA) and ratio of the substrate (t-VA) transformed to the product (SA). A significant part of the retrieved 16S rRNA gene sequences was most similar to those of uncultured bacteria. Bacteria corresponding to predominant DGGE bands in t-VA enrichment cultures clustered with t-VA biohydrogenated bacteria within Group B. CONCLUSIONS: This study provides more insight into the pathway of biohydrogenation. It also may be important to control the production of t-VA, which has metabolic and physiological benefits, through management of ruminal biohydrogenation bacterium.

Dose response to cinnamaldehyde supplementation in growing beef heifers: ruminal and intestinal digestion.

The objective of this study was to determine if cinnamaldehyde (CIN) could be used to improve feed intake, digestion, and immune status in growing beef heifers fed high-concentrate diets. The experiment was designed as a 4 x 4 Latin square using 4 ruminally and duodenally cannulated beef heifers with 4 treatments: control (no CIN added), 400 mg/d of CIN (low), 800 mg/d of CIN (medium), and 1,600 mg/d of CIN (high), and four 21-d periods. Feed intake, rumen pH and fermentation characteristics, site and extent of digestion, microbial N synthesis, blood metabolites, and acute phase protein response were measured. The diets consisted of 15% barley silage, 80% dry-rolled barley grain, and 5% supplement (DM basis). Intakes (kg/d) of DM, OM, NDF, starch, and N were quadratically (P = 0.04) changed with increasing CIN supplementation. The amount of OM fermented in the rumen quadratically (P = 0.02) decreased with increasing CIN. Digestibilities (% of intake) of OM, NDF, and N in the rumen were not affected by supplementing with low and medium CIN, but they were reduced by 8% (P = 0.10), 31% (P = 0.05), and 17% (P = 0.05), respectively, with high CIN. Similarly, digestibilities of OM and NDF in the total tract also tended to be reduced by 7% (P = 0.10) and 20% (P = 0.10), respectively, with high CIN because supplementation of CIN had minimal effects on intestinal digestibility. Flows (g/d) of microbial N and other nutrients to the duodenum were not affected by CIN supplementation, even though the amount of ruminal fermented OM varied with level of CIN supplementation. Rumen pH, total VFA concentration, and molar proportions of individual VFA were not affected by CIN. Although concentrations of NEFA (P = 0.06) and triglyceride (P = 0.01) were quadratically changed with increasing CIN supplementation, blood concentrations of glucose and urea N, white blood cell counts, serum amyloid A, and lipopolysaccharide in plasma were not affected by CIN. Plasma haptoglobin numerically (P = 0.11) decreased with the medium dose of CIN fed compared with control. The results indicate that supplementation of a high-concentrate diet with a low dose of CIN resulted in small increases in nutrient availability in the rumen due to increased feed intake and greater ruminal digestion of OM. However, feed intake and ruminal digestion of feeds were adversely affected when a high dose of CIN was used.

Defense mechanisms against toxic phytochemicals in the diet of domestic animals.

Plant secondary metabolites (PSMs) are non-nutritional components that occur in numerous feed materials and are able to exert toxic effects in animals. The current article aims to summarize innate defense strategies developed by different animal species to avoid excessive exposure to PSMs. These mechanisms include pre-systemic degradation of PSMs by rumen microbiota, the intestinal barrier including efflux transporters of monogastric species, as well as pre-hepatic and intra-hepatic biotransformation processes. These physiological barriers determine systemic exposure and ultimately the dose-dependent adverse effects in the target animal species. Considering the large number of potentially toxic PSMs, which makes an evaluation of all individual PSMs virtually impossible, such a mechanism-oriented approach could improve the predictability of adverse effects and support the interpretation of clinical field observations. Moreover, mechanistic data related to tissue disposition and excretion pathways of PSMs for example into milk, could substantially support the assessment of the risks for consumers of foods derived from PSM-exposed animals.

Hepatic and extra-hepatic metabolic pathways involved in flubendazole biotransformation in sheep.

Flubendazole (FLBZ) is a broad-spectrum benzimidazole anthelmintic compound used in pigs, poultry and humans. Its potential for parasite control in ruminant species is under investigation. The objective of the work described here was to identify the main enzymatic pathways involved in the hepatic and extra-hepatic biotransformation of FLBZ in sheep. Microsomal and cytosolic fractions obtained from sheep liver and duodenal mucosa metabolised FLBZ into a reduced FLBZ metabolite (red-FLBZ). The keto-reduction of FLBZ led to the prevalent (approximately 98%) stereospecific formation of one enantiomeric form of red-FLBZ. The amounts of red-FLBZ formed in liver subcellular fractions were 3-4-fold higher (P<0.05) compared to those observed in duodenal subcellular fractions. This observation correlates with the higher (P<0.05) carbonyl reductase (CBR) activities measured in the liver compared to the duodenal mucosa. No metabolic conversion was observed following FLBZ or red-FLBZ incubation with sheep ruminal fluid. Sheep liver microsomes failed to convert red-FLBZ into FLBZ. However, this metabolic reaction occurred in liver microsomes prepared from phenobarbital-induced rats, which may indicate a cytochrome P450-mediated oxidation of red-FLBZ. A NADPH-dependent CBR is proposed as the main enzymatic system involved in the keto-reduction of FLBZ in sheep. CBR substrates such as menadione and mebendazole (a non-fluoride analogue of FLBZ), inhibited this liver microsomal enzymatic reaction, which may confirm the involvement of a CBR enzyme in FLBZ metabolism in sheep. This research is a further contribution to the understanding of the metabolic fate of a promissory alternative compound for antiparasitic control in ruminant species.

[The regulation of fatty acid metabolism and other lipid compounds in ruminant animals].

The review deals with peculiarities of molecular mechanisms of metabolism regulation of fatty acids and other lipid components in preruminant and ruminant animals (i.e. before and after formation of the functioning of the mixed microbial population in the rumen). A characteristic of possible biosynthesis regulation processes, transformation, transport, utilization and catabolism of fatty acids with different molecular masses and peculiarities and of triacylglycerols and other lipid substances in the liver compartments, skeletal and heart muscle cells has been shown. Peculiarities of intracellular changes in metabolism of fatty acids and apolipoprotein B in the liver of neonatal calves under development of alimentary enteropathology have been considered. Main factors in the mechanism of regulation of intracellular metabolism of fatty acids and lipid substances have been defined.

Clostridium proteoclasticum: A ruminal bacterium that forms stearic acid from linoleic acid.

The aim of this study was to identify ruminal bacteria that form stearic acid (18 : 0) from linoleic acid (cis-9,cis-12-18 : 2). One 18 : 0-producing isolate, P-18, isolated from the sheep rumen was similar in morphology and metabolic properties to 'Fusocillus' spp. isolated many years ago. Phylogenetic analysis based on nearly full-length 16S rRNA gene sequence (>1300 bp) analysis indicated that the stearate producer was most closely related to Clostridium proteoclasticum B316(T). Clostridium proteoclasticum B316(T) was also found to form 18 : 0, as were other bacteria isolated elsewhere, which occurred in the same family subclass of the low G+C% Gram-positive bacteria, related to Butyrivibrio fibrisolvens. These bacteria are not clostridia, and the ability to form 18 : 0 was present in all strains in contrast to proteolytic activity, which was variable. Production of 18 : 0 occurred in growing, but not in stationary-phase, bacteria, which made detection of biohydrogenating activity difficult, because of the inhibitory effects of linoleic acid on growth.

Effect of select nitrocompounds on ruminal fermentation; an initial look at their potential to reduce economic and environmental costs associated with ruminal methanogenesis.

Methane production by ruminal microbes during the digestion of feedstuffs is an inefficient process resulting in losses of 2-12% of the gross energy consumed by ruminants. Presently, we report the effect of three inhibitors on ruminal methane production in vitro. Mixed populations of ruminal microbes collected from cannulated cows maintained on an alfalfa hay:corn diet (50:50) were incubated at 39 degrees C for 24 h under a 100% carbon dioxide gas phase in closed tubes with 72 mM added sodium formate. Cultures were supplemented with 12 mM 2-nitropropanol, nitroethane or nitroethanol (experiment 1) or with 2, 12 or 24 mM nitroethane or a combination of 12 mM nitroethane and 4 mM nitroethanol (experiment 2). Control cultures containing no added nitrocompound were incubated simultaneously with treated incubations. Methane concentrations were reduced (P<0.05) from those measured in control incubations (27.6 +/- 2.1 and 17.7 +/- 0.8 micromol/ml; mean +/- SD for experiments 1 and 2, respectively) by at least 57% and as much as 94% in the nitrocompound supplemented incubations. By comparison, the widely fed methane inhibitor, monensin, typically reduces ruminal methane production by about 33%. Concentrations of volatile fatty acids and ammonia that accumulated in the nitrocompound supplemented incubations were not markedly affected compared to those produced by control cultures despite the reductions in methane produced. Hydrogen accumulated only slightly in cultures supplemented with the nitrocompounds. These results demonstrate that 2-nitropropanol, nitroethane and nitroethanol inhibit ruminal methane production. Further research is warranted to determine the mechanisms responsible for this inhibition and to see if these inhibitors can be used in practical application to reduce economic and environmental costs associated with ruminal methanogenesis.

Degradation of fresh ryegrass by methanogenic co-cultures of ruminal fungi grown in the presence or absence of Fibrobacter succinogenes.

The ability of five ruminal fungi in syntrophic co-culture with the methanogen Methanobrevibacter smithii to degrade perennial ryegrass ( Lolium perenne) stem fragments and leaf blades was studied to determine the susceptibilities of non-autoclaved fresh tissues to fungal degradation. Autoclaving did not significantly increase fungal degradation of stem fragments but strongly increased degradation of leaf blades by a species of Caecomyces. In methanogenic co-cultures, non-autoclaved stem fragments were degraded more extensively by Neocallimastix frontalis and Piromyces isolates than by Caecomyces isolates. The N. frontalis and Piromyces isolates showed the greatest rates of stem degradation. When interactions between Fibrobacter succinogenes and methanogenic co-cultures of fungi growing on ryegrass stem were investigated, N. frontalis inhibited F. succinogenes. This has not been observed previously. In contrast, a Caecomyces species interacted positively with F. succinogenes to increase stem degradation, suggesting that F. succinogenes and Caecomyces spp. may have complementary fibrolytic activities. All five fungi tested failed to grow on fresh non-autoclaved leaf blades. In a repeat experiment with leaves from a separate harvest, leaf blades were degraded by N. frontalis but not by a Caecomyces species. We suggest that ryegrass leaf blades may contain natural anti-fungal compounds. Our results confirm the superiority of fungi in the degradation of intact stem and indicate that in vitro studies with non-autoclaved forage tissues may yield new information on forage factors affecting rumen microbes.

Fusobacteriosis in captive wild-caught pronghorns (Antilocapra americana).

An outbreak of Fusobacterium necrophorum-induced septicemia occurred in a group of 40 captive wild-caught pronghorns (Antilocapra americana). Primary pododermatitis or necrotic stomatitis progressed to produce fatal septicemia with metastatic lesions in the forestomachs, lung, liver, and cecum in 38 of the animals. Two remaining animals were euthanatized because of chronic pododermatitis. Housing the animals in a pasture previously used by bovids and heavy rains with persistence of ground water pools in the pasture were contributing factors in the pathogenesis of this outbreak.