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Two new species of the genus Scaptolenus LeConte (Coleoptera: Elateridae: Elaterinae: Cebrionini) from Mexico are described and illustrated: Scaptolenus pacofierroi sp. nov., found in the municipality of Aldama, in the state of Tamaulipas and Scaptolenus zuritai sp. nov., collected from the Chamela-UNAM Biological Station, in the state of Jalisco. Their segregation is based on the morphological configuration of their antennae, aedeagus, protibiae and metacoxal lamina. These new species fit well within the vagans-group and a key is presented to distinguish these new species within the vagans group. Additionally, distribution data of these new species are provided and biological data of the genus Scaptolenus are presented and analyzed.

Seedling emergence, survival, morphological and physiological traits, and oxidative stress resistance of southwestern white pine (Pinus strobiformis Engelm.) were studied in response to warming treatments applied during embryogenesis, germination, and early seedling growth. Daytime air temperature surrounding cones in tree canopies was warmed by +2.1°C during embryo development. Resulting seeds and seedlings were assigned to three thermal regimes in growth chambers, with each regime separated by 4°C to encompass the wide range of temperatures observed over space and time across the species’ range, plus the effect of heat waves coupled with a high carbon emissions scenario of climate warming. The embryo warming treatment reduced percent seedling emergence in all germination and growth environments and reduced mortality of seedlings grown in the warmest environment. Warm thermal regimes during early seedling growth increased subsequent seedling resistance to oxidative stress and transpirational water use. Experimental warming during seed development, germination, and seedling growth affected seedling emergence and survival. Oxidative stress resistance, morphology, and water relations were affected only by warming imposed during germination and seedling growth. This work explores potential outcomes of climate warming on multiple dimensions of seedling performance and uniquely illustrates that plant responses to heat vary with plant developmental stage in addition to the magnitude of temperature change.

Impaction fracture subluxation of the pisotriquetral joint producing arthrosis and ulnar triquetral osteochondral nonunion is a cause for ulnar wrist pain in batting athletes. Two cases of adolescent female softball players managed successfully with pisiform and triquetral fragment excision are reported.

We subjected southwestern white pine (Pinus strobiformis Engelm.) seeds to controlled warming treatments to study responses of seed and seedling demographics, and morphological and physiological traits following warming during embryogenesis, germination, and early seedling growth. Daytime air temperature surrounding cones in tree canopies was warmed by +2.1 ◦C during embryo development. Resulting seeds and seedlings were assigned to three thermal regimes in growth chambers, with each regime separated by 4 ◦C. The embryo-warming treatment reduced percent seedling emergence in all germination and growth environments and reduced mortality of seedlings grown in the warmest environment. Warm thermal regimes during early seedling growth increased seedling resistance to oxidative stress and seedling transpirational water use even after applying cooler temperatures and experimental drought. Experimental warming imposed during seed development affected seedling demographic processes, and warming imposed during germination and seedling growth affected stress resistance and water relations. This work illustrates that the study of numerous ecophysiological and functional trait responses to various types of stress across multiple plant developmental stages is essential for understanding the effects of climate warming on forest regeneration processes.

This study was undertaken to further develop our understanding of the links between breed, diet and the rumen microbial community and determine their effect on production characteristics and methane (CH4) emissions from beef cattle. The experiment was of a 2×2 factorial design, comprising two breeds (crossbred Charolais (CHX); purebred Luing (LU)) and two diets (concentrate-straw or silage-based). In total, 80 steers were used and balanced for sire within each breed, farm of origin and BW across diets. The diets (fed as total mixed rations) consisted of (g/kg dry matter (DM)) forage to concentrate ratios of either 500 : 500 (Mixed) or 79 : 921 (Concentrate). Steers were adapted to the diets over a 4-week period and performance and feed efficiency were then measured over a 56-day test period. Directly after the 56-day test, CH4 and carbon dioxide (CO2) emissions were measured (six steers/week) over a 13-week period. Compared with LU steers, CHX steers had greater average daily gain (ADG; P

Sub-acute ruminal acidosis (SARA) can reduce the production efficiency and impair the welfare of cattle, potentially in all production systems. The aim of this study was to characterise measurable postmortem observations from divergently managed intensive beef finishing farms with high rates of concentrate feeding. At the time of slaughter, we obtained samples from 19 to 20 animals on each of 6 beef finishing units (119 animals in total) with diverse feeding practices, which had been subjectively classified as being high risk (three farms) or low risk (three farms) for SARA on the basis of the proportions of barley, silage and straw in the ration. We measured the concentrations of histamine, lipopolysaccharide (LPS), lactate and other short-chain fatty acids (SCFAs) in ruminal fluid, LPS and SCFA in caecal fluid. We also took samples of the ventral blind sac of the rumen for histopathology, immunohistopathology and gene expression. Subjective assessments were made of the presence of lesions on the ruminal wall, the colour of the lining of the ruminal wall and the shape of the ruminal papillae. Almost all variables differed significantly and substantially among farms. Very few pathological changes were detected in any of the rumens examined. The animals on the high-risk diets had lower concentrations of SCFA and higher concentrations of lactate and LPS in the ruminal fluid. Higher LPS concentrations were found in the caecum than the rumen but were not related to the risk status of the farm. The diameters of the stratum granulosum, stratum corneum and of the vasculature of the papillae, and the expression of the gene TLR4 in the ruminal epithelium were all increased on the high-risk farms. The expression of IFN-γ and IL-1β and the counts of cluster of differentiation 3 positive and major histocompatibility complex class two positive cells were lower on the high-risk farms. High among-farm variation and the unbalanced design inherent in this type of study in the field prevented confident assignment of variation in the dependent variables to individual dietary components; however, the CP percentage of the total mixed ration DM was the factor that was most consistently associated with the variables of interest. Despite the strong effect of farm on the measured variables, there was wide inter-animal variation.

The proliferation, differentiation and survival of mononuclear phagocytes depend on signals from the receptor for macrophage colony-stimulating factor, CSF1R. The mammalian Csf1r locus contains a highly conserved super-enhancer, the fms-intronic regulatory element (FIRE). Here we show that genomic deletion of FIRE in mice selectively impacts CSF1R expression and tissue macrophage development in specific tissues. Deletion of FIRE ablates macrophage development from murine embryonic stem cells. Csf1rΔFIRE/ΔFIRE mice lack macrophages in the embryo, brain microglia and resident macrophages in the skin, kidney, heart and peritoneum. The homeostasis of other macrophage populations and monocytes is unaffected, but monocytes and their progenitors in bone marrow lack surface CSF1R. Finally, Csf1rΔFIRE/ΔFIRE mice are healthy and fertile without the growth, neurological or developmental abnormalities reported in Csf1r−/− rodents. Csf1rΔFIRE/ΔFIRE mice thus provide a model to explore the homeostatic, physiological and immunological functions of tissue-specific macrophage populations in adult animals., The lineage-specific receptor CSF1R controls macrophage development and homeostasis. Here the authors show that deletion of a conserved Csf1r enhancer (FIRE) selectively depletes brain microglia and resident macrophages in the epidermis, kidney, heart and peritoneum of otherwise healthy mice.

SUMMARYBone marrow adipose tissue (BMAT) represents >10% of total adipose mass, yet unlike white or brown adipose tissues (WAT or BAT), its role in systemic metabolism remains unclear. Using transcriptomics, we reveal that BMAT is molecularly distinct to WAT but is not enriched for brown or beige adipocyte markers. Instead, pathway analysis indicated altered glucose metabolism and decreased insulin responsiveness in BMAT. We therefore tested these functions in mice and humans using positron emission tomography–computed tomography (PET/CT) with18F-fluorodeoxyglucose, including establishing a new method for BMAT identification from clinical CT scans. This revealed that BMAT resists insulin- and cold-stimulated glucose uptake and is thus functionally distinct to WAT and BAT. However, BMAT displayed greater basal glucose uptake than axial bones or subcutaneous WAT, underscoring its potential to influence systemic glucose homeostasis. These PET/CT studies are the first to characterise BMAT functionin vivoand identify BMAT as a distinct, major subtype of adipose tissue.HIGHLIGHTSBone marrow adipose tissue (BMAT) is molecularly distinct to other adipose subtypes.BMAT is less insulin responsive than WAT and, unlike BAT, is not cold-responsive.Human BMAT has greater basal glucose uptake than axial bone or subcutaneous WAT.We establish a PET/CT method for BMAT localisation and functional analysisin vivo.

Substituted long-chain fatty acids may be useful dietary supplements to suppress ruminal biohydrogenation of unsaturated fatty acids (UFA) and thereby increase the flow of UFA to meat and milk. The aim of this study was to determine if Vernonia galamensis (ironweed, a member of the sunflower family) and vernolic acid (cis-12,13-epoxy-cis-9-octadecenoic acid), the main constituent of the seed oil, affected the biohydrogenation of linoleic acid (LA; cis-9,cis-12-18:2) to rumenic acid (CLA; cis-9, trans-11-18:2), vaccenic acid (VA; trans-11-18:1) and stearic acid (SA; 18:0) by ruminal microorganisms. Ruminal digesta from four sheep receiving a mixed hay-concentrate diet were incubated in vitro with LA (0.2 g/L) ± 0.2 g/L vernolic acid or 5 g/L of dried flowers or leaves of V. galamensis, either alone or combined. Vernolic acid had a substantial effect on LA metabolism, causing decreases in cis-9, trans-11 CLA and VA accumulation as well as SA production (P

Studies on microbial biohydrogenation of fatty acids in the rumen are of importance as this process lowers the availability of nutritionally beneficial unsaturated fatty acids for incorporation into meat and milk but also might result in the accumulation of biologically active intermediates. The impact was studied of adsorption of 22:6n-3 (DHA) to particulate material on its disappearance during 24 h in vitro batch incubations with rumen inoculum. Four adsorbants were used in two doses (1 and 5 mg/ml of mucin, gum arabic, bentonite or silicic acid). In addition, the distribution of 22:6n-3 in the pellet and supernatant of diluted rumen fluid was measured. Bentonite and silicic acid did not alter the distribution of 22:6n-3 between pellet and supernatant nor increased the disappearance of 22:6n-3 during the incubation. Both mucin and gum arabic increased the recovery of 22:6n-3 in the supernatant, indicating that these compounds lowered the adsorption of the fatty acid to ruminal particles. This was associated with an increased disappearance of 22:6n-3, when initial 22:6n-3 was 0.06 or 0.10 mg/ml, and an increased formation of 22:0, when initial 22:6n-3 was 0.02 mg/ml, during the 24 h batch culture experiment. Addition of gum arabic to pure cultures of Butyrivibrio fibrisolvens or Butyrivibrio proteoclasticus did not negate the inhibitory effect of 22:6n-3 on growth. As both mucin and gum arabic provide fermentable substrate for ruminal bacteria, an additional experiment was performed in which mucin and gum arabic were replaced by equal amounts of starch, cellulose or xylan. No differences in disappearance of 22:6n-3 were observed, suggesting that the stimulatory effect of mucin and gum arabic on disappearance of 22:6n-3 most probably is not due to provision of an alternative site of adsorption but related to stimulation of bacterial growth. A relatively high proportion of 22:6n-3 can be reduced to 22:0 provided the initial concentration is low.

Macrophage colony-stimulating factor (CSF1) is an essential growth factor to control the proliferation, differentiation and survival of cells of the macrophage lineage in vertebrates. We have previously produced a recombinant chicken CSF1-Fc fusion protein and administrated it to birds which produced a substantial expansion of tissue macrophage populations. To further study the biology of CSF1 in the chicken, here we generated anti-chicken CSF1 antibodies (ROS-AV181 and 183) using CSF1-Fc as an immunogen. The specific binding of each monoclonal antibody was confirmed by ELISA, Western blotting and immunohistochemistry on tissue sections. Using the anti-CSF1 antibodies, we show that chicken bone marrow derived macrophages (BMDM) express CSF1 on their surface, and that the level appears to be regulated further by exogenous CSF1. By capture ELISA circulating CSF1 levels increased transiently in both layer and broiler embryos around the day of hatch. The levels of CSF1 in broilers was higher than in layers during the first week after hatch. Antibody ROS-AV183 was able to block CSF1 biological activity in vitro and treatment of hatchlings using this neutralising antibody in vivo impacted on some tissue macrophage populations, but not blood monocytes. After anti-CSF1 treatment, CSF1R-transgene reporter expressing cells were reduced in the bursa of Fabricius and cecal tonsil and TIM4+ Kupffer cells in the liver were almost completely ablated. Anti-CSF1 treatment also produced a reduction in overall bone density, trabecular volume and TRAP+ osteoclasts. Our novel neutralising antibody provides a new tool to study the roles of CSF1 in birds., Highlights • Two mouse monoclonal antibodies to chicken CSF1 (ROS-AV181 and 183) were produced. • A capture ELISA assays was developed to quantify native circulating CSF1 levels. • Antibody ROS-AV183 was shown to neutralise the bioactivities of CSF1 in vitro. • Treatment of hatchlings using ROS-AV183 in vivo impacted on chicken macrophage subsets and B-cells. • Anti-CSF1 treatment produced a reduction in overall bone density, trabecular volume and TRAP+ osteoclasts.

In the analysis of gene expression in the brain, the sample labels for the pituitary gland and olfactory bulb were reversed. As a consequence, the clusters in Fig. 10B were mislabeled. A corrected version of Fig. 10 is shown below, along with the corrected figure legend. These corrections have been made to the online version of the article, which now differs from the print version as originally published. A corrected version of Supplemental Table I has also been published online. The current online supplemental material therefore differs from what was originally published online. The relevant text in the Results section under the heading “Network analysis of gene expression in the brain of Csf1r-deficient rats” now reads as follows: Fig. 10B shows the network graph for the combined analysis of the four brain regions; gene lists are provided in Supplemental Table I. The largest cluster was cluster 1 (4283 genes), containing pituitary gland-associated genes. Other region-specific clusters were cluster 3 (olfactory bulb; 878 genes), cluster 5 (hippocampus; 513 genes), and cluster 7 (striatum; 384 genes). The second largest cluster (cluster 2; 1253 genes) contained genes that were more highly expressed in both hippocampus and striatum. Two other main clusters also shared highly expressed genes between tissues: cluster 4 (olfactory bulb, striatum, and hippocampus; 757 genes) and cluster 6 (pituitary gland and olfactory bulb; 471 genes). None of these clusters showed any evidence of genotype association. (Figure Presented).

International audience; Methane emissions from ruminal fermentation contribute significantly to total anthropological greenhouse gas (GHG) emissions. New meta-omics technologies are beginning to revolutionise our understanding of the rumen microbial community structure, metabolic potential and metabolic activity. Here we explore these developments in relation to GHG emissions. Microbial rumen community analyses based on small subunit ribosomal RNA sequence analysis are not yet predictive of methane emissions from individual animals or treatments. Few metagenomics studies have been directly related to GHG emissions. In these studies, the main genes that differed in abundance between high and low methane emitters included archaeal genes involved in methanogenesis, with others that were not apparently related to methane metabolism. Unlike the taxonomic analysis up to now, the gene sets from metagenomes may have predictive value. Furthermore, metagenomic analysis predicts metabolic function better than only a taxonomic description, because different taxa share genes with the same function. Metatranscriptomics, the study of mRNA transcript abundance, should help to understand the dynamic of microbial activity rather than the gene abundance; to date, only one study has related the expression levels of methanogenic genes to methane emissions, where gene abundance failed to do so. Metaproteomics describes the proteins present in the ecosystem, and is therefore arguably a better indication of microbial metabolism. Both two-dimensional polyacrylamide gel electrophoresis and shotgun peptide sequencing methods have been used for ruminal analysis. In our unpublished studies, both methods showed an abundance of archaeal methanogenic enzymes, but neither was able to discriminate high and low emitters. Metabolomics can take several forms that appear to have predictive value for methane emissions; ruminal metabolites, milk fatty acid profiles, faecal long-chain alcohols and urinary metabolites have all shown promising results. Rumen microbial amino acid metabolism lies at the root of excessive nitrogen emissions from ruminants, yet only indirect inferences for nitrogen emissions can be drawn from meta-omics studies published so far. Annotation of meta-omics data depends on databases that are generally weak in rumen microbial entries. The Hungate 1000 project and Global Rumen Census initiatives are therefore essential to improve the interpretation of sequence/metabolic information.

Fish oil (FO) alters ruminal biohydrogenation causing trans fatty acid (FA) intermediates to accumulate, but the effects of 18-carbon polyunsaturated FA supply on ruminal long-chain FA metabolism and microbial communities in cattle fed FO are not well established. Four cows fitted with rumen cannula were used in a 4 × 4 Latin square with 21-d experimental periods to evaluate the effects of FO alone or in combination with plant oils high in 18:2n-6 or 18:3n-3 on rumen microbial ecology and flow of FA at the omasum. Treatments comprised a basal grass silage-based diet containing no additional oil (control) or supplements of FO (200 g/d) or FO (200 g/d) plus 500 g/d of sunflower oil (SFO) or linseed oil (LFO). Flow of FA was determined using the omasal sampling technique. The relative abundance of key biohydrogenating bacteria was assessed by quantitative PCR on 16S rRNA genes in omasal digesta. Fish oil-supplemented treatments increased the amounts of trans-18:1, trans-18:2, and 20- to 22-carbon polyunsaturated FA escaping the rumen. Relative to the control, oil supplements had no effect on the amount of 18:0 leaving the rumen, but LFO decreased the flow of 18:0 at the omasum compared with SFO. Both SFO and LFO increased trans-18:1 relative to FO, whereas LFO resulted in the highest trans-18:2 and 20- to 22-carbon FA flow. Supplements of FO plus plant oils shifted biohydrogenation toward trans-10 18:1 formation. Compared with FO alone, the ruminal metabolism of 22:6n-3 in the rumen of lactating cows is more extensive on diets containing higher amounts of 18-carbon polyunsaturated FA. However, the biohydrogenation of 22:5n-3 was less extensive in LFO than SFO, but showed no difference between FO and diets containing plant oils. Ruminal outflow of 20:5n-3 was not altered when plant oils were added to FO. Alterations in the amount of intermediates at the omasum or ruminal biohydrogenation pathways were not accompanied by major changes in analyzed bacterial populations. In conclusion, dietary supplements of FO alone or in combination with plant oils increase the amount of biohydrogenation intermediates containing 1 or more trans double bonds escaping the rumen, which may have implications for host metabolism and the nutritional quality of ruminant foods.

The testicular vasculature forms a complex network, providing oxygenation, micronutrients, and waste clearance from the testis. The vasculature is also instrumental to testis function because it is both the route by which gonadotropins are delivered to the testis and by which T is transported away to target organs. Whether Sertoli cells play a role in regulating the testicular vasculature in postnatal life has never been unequivocally demonstrated. In this study we used models of acute Sertoli cell ablation and acute germ cell ablation to address whether Sertoli cells actively influence vascular structure and function in the adult testis. Our findings suggest that Sertoli cells play a key role in supporting the structure of the testicular vasculature. Ablating Sertoli cells (and germ cells) or germ cells alone results in a similar reduction in testis size, yet only the specific loss of Sertoli cells leads to a reduction in total intratesticular vascular volume, the number of vascular branches, and the numbers of small microvessels; loss of germ cells alone has no effect on the testicular vasculature. These perturbations to the testicular vasculature leads to a reduction in fluid exchange between the vasculature and testicular interstitium, which reduces gonadotropin-stimulated circulating T concentrations, indicative of reduced Leydig cell stimulation and/or reduced secretion of T into the vasculature. These findings describe a new paradigm by which the transport of hormones and other factors into and out of the testis may be influenced by Sertoli cells and highlights these cells as potential targets for enhancing this endocrine relationship.The testicular vasculature forms a complex capillary bed, interdigitating between the seminiferous tubules to provide oxygenation, delivery of micronutrients, and clearance of waste from the testis. Impairment of the testicular vasculature, for example, the reduction in venous drainage observed in cases of varicocele, causes intratesticular hypoxia and germ cell apoptosis (1). The vasculature is also instrumental to the endocrine function of the testis because it is the route by which pituitary gonadotropins are delivered to the testis to support T production and spermatogenesis (2). Conversely, alongside the lymphatic system, the vascular system is important for transport of T to other body systems; a reduced testis and vascular volume is associated with a reduction in circulating T concentrations (3).Our understanding of the mechanisms by which the testis controls local vascular function in adulthood is extremely limited. There is some evidence that testicular mast cells can influence vascular blood flow through release of 5-hydroxytryptamine (4), but perhaps the most well-studied factor influencing testicular vascular function is T. T is a well-established regulator of testicular vasomotion (rhythmical contraction and relaxation of blood vessels, independent of heartbeat) (5, 6) via direct T-mediated activation of the androgen receptor in smooth muscle cells of the testicular vasculature (7).Speculation that Sertoli cells may influence the testicular vasculature is supported by some indirect evidence (5) and in vitro studies (8), but confirmation of a direct role for Sertoli cells in the regulation of the testicular vasculature in vivo has never been demonstrated unequivocally. Recently we developed a unique model system that uses diphtheria toxin to specifically and acutely ablate Sertoli cells from the testis (9, 10). This model has revealed several important, yet previously unknown, roles that Sertoli cells play in neonatal and adult life (reviewed in reference 11).In this study we used models of acute Sertoli cell ablation and acute germ cell ablation, to address whether Sertoli cells actively influence vascular function in the adult testis. Our findings suggest that Sertoli cells play a key role in supporting the structure of the testicular vasculature and describe a new paradigm by which the transport of hormones and other factors into and out of the testis can be influenced by Sertoli cells and highlights these cells as potential targets for enhancing this endocrine relationship.

Ruminal methane production is among the main targets for greenhouse gas (GHG) mitigation for the animal agriculture industry. Many compounds have been evaluated for their efficacy to suppress enteric methane production by ruminal microorganisms. Of these, nitrate as an alternative hydrogen sink has been among the most promising, but it suffers from variability in efficacy for reasons that are not understood. The accumulation of nitrite, which is poisonous when absorbed into the animal’s circulation, is also variable and poorly understood. This review identifies large gaps in our knowledge of rumen microbial ecology that handicap the further development and safety of nitrate as a dietary additive. Three main bacterial species have been associated historically with ruminal nitrate reduction, namely Wolinella succinogenes, Veillonella parvula and Selenomonas ruminantium, but others almost certainly exist in the largely uncultivated ruminal microbiota. Indications are strong that ciliate protozoa can reduce nitrate, but the significance of their role relative to bacteria is not known. The metabolic fate of the reduced nitrate has not been studied in detail. It is important to be sure that nitrate metabolism and efforts to enhance rates of nitrite reduction do not lead to the evolution of the much more potent GHG, nitrous oxide. The relative importance of direct inhibition of archaeal methanogenic enzymes by nitrite or the efficiency of capture of hydrogen by nitrate reduction in lowering methane production is also not known, nor are nitrite effects on other members of the microbiota. How effective would combining mitigation methods be, based on our understanding of the effects of nitrate and nitrite on the microbiome? Answering these fundamental microbiological questions is essential in assessing the potential of dietary nitrate to limit methane emissions from ruminant livestock.

Background: Rumen microbes metabolize 22:6n-3. However, pathways of 22:6n-3 biohydrogenation and ruminal microbes involved in this process are not known. In this study, we examine the ability of the well-known rumen biohydrogenating bacteria, Butyrivibrio fibrisolvens D1 and Butyrivibrio proteoclasticus P18, to hydrogenate 22:6n-3. Results: Butyrivibrio fibrisolvens D1 failed to hydrogenate 22:6n-3 (0.5 to 32 mu g/mL) in growth medium containing autoclaved ruminal fluid that either had or had not been centrifuged. Growth of B. fibrisolvens was delayed at the higher 22:6n-3 concentrations; however, total volatile fatty acid production was not affected. Butyrivibrio proteoclasticus P18 hydrogenated 22:6n-3 in growth medium containing autoclaved ruminal fluid that either had or had not been centrifuged. Biohydrogenation only started when volatile fatty acid production or growth of B. proteoclasticus P18 had been initiated, which might suggest that growth or metabolic activity is a prerequisite for the metabolism of 22:6n-3. The amount of 22:6n-3 hydrogenated was quantitatively recovered in several intermediate products eluting on the gas chromatogram between 22:6n-3 and 22:0. Formation of neither 22:0 nor 22:6 conjugated fatty acids was observed during 22:6n-3 metabolism. Extensive metabolism was observed at lower initial concentrations of 22:6n-3 (5, 10 and 20 mu g/mL) whereas increasing concentrations of 22:6n-3 (40 and 80 mu g/mL) inhibited its metabolism. Stearic acid formation (18:0) from 18:2n-6 by B. proteoclasticus P18 was retarded, but not completely inhibited, in the presence of 22:6n-3 and this effect was dependent on 22:6n-3 concentration. Conclusions: For the first time, our study identified ruminal bacteria with the ability to hydrogenate 22:6n-3. The gradual appearance of intermediates indicates that biohydrogenation of 22:6n-3 by B. proteoclasticus P18 occurs by pathways of isomerization and hydrogenation resulting in a variety of unsaturated 22 carbon fatty acids. During the simultaneous presence of 18:2n-6 and 22:6n-3, B. proteoclasticus P18 initiated 22:6n-3 metabolism before converting 18:1 isomers into 18:0.

Concentrations of Na(+), K(+) and Ca(2+) in the growth medium were varied within limits normally found in vivo to determine how cation concentrations affect the sensitivity of ruminal bacteria to the ionophores, monensin (a Na(+)/H(+) and K(+)/H(+) exchanger) and tetronasin (Ca(2+)/H(+)). High [Na(+)] (172 mM cf. 137 mM in control medium) enhanced the efficacy of monensin towards Eubacterium ruminantium 2388, Streptococcus bovis C277, Lactobacillus casei LB17 and Prevotella albensis M384. High [K(+)] (35 mM cf. 19 mM) alone caused a decreased potency of both ionophores, except with L. casei. Added Ca(2+) (7.4 cf. 2.8 mM) increased the potency of tetronasin when [Na(+)] was low. High [Na(+)] alone also potentiated the efficacy of tetronasin. Monensin caused intracellular [Na(+)] and [K(+)] to be decreased in the most sensitive of these organisms, E. ruminantium, whereas only intracellular [Ca(2+)] fell with tetronasin. The changes were small; however, Δp fell by only 20 mV after 2 h when ionophores caused immediate cessation of growth. ATP concentrations fell by 77% and 75% with monensin and tetronasin, respectively. Thus, altering cation concentrations might be used to potentiate the efficacy of ionophores, by increasing the rate of energy expenditure to maintain ionic homoeostasis in sensitive bacteria.

Inclusion of a daisy plant, Chrysanthemum coronarium, in a dairy sheep diet has been reported to result in increased concentrations of health-promoting rumenic acid (RA; cis-9,trans-11 CLA) and vaccenic acid (VA; trans-11-18:1) in milk. The aims of the present study were to determine if the reported change in milk fatty acid composition was the result of the effects of C. coronarium on the biohydrogenation of linoleic acid (LA; cis-9,cis-12-18:2) by ruminal microorganisms, and to investigate which constituents of C. coronarium may be responsible for the observed effects. Ruminal digesta from four sheep receiving a mixed hay-concentrate diet were incubated in vitro with LA in the presence or absence of dried whole-plant C. coronarium var. Primrose Gem. Rates of LA disappearance and stearic acid (SA: 18:0) production decreased as a result of C. coronarium addition, and VA accumulation doubled. Chrysanthemum parthenium and Chrysanthemum vulgare had much smaller effects on biohydrogenation. C. coronarium added to cultures of the only known ruminal SA-forming bacterium, Butyrivibrio proteoclasticus, also inhibited LA metabolism by, but not growth of, this species. Lipid analysis indicated that C coronarium var. Primrose Gem had a high content of a-linolenic acid (LNA; cis-9,cis-12,cis-15-18:3; 8.79 mg/g DM) compared to the other samples (

1. Plants and their biologically active chemical constituents, sometimes called secondary metabolites or bioactives, present numerous opportunities for the improvement of livestock production by inclusion in the diet. 2. Many such plant derived materials have well established therapeutic values in man; however, their potential as feed additives in animal production, particularly of poultry, remains largely unexploited. 3. There is increasing evidence indicating that they can be efficient in controlling diseases, and plant bioactives may also influence production parameters such as feed efficiency and product quality. 4. It has been reported that they may even replicate some of the effects of antibiotic growth promoters, which were banned from use in Europe from 2006. 5. This review assesses the status of plant bioactives in poultry production and their mode of action on avian physiology, particularly in the digestive tract.

Compounds that mimic vertebrate hormone responses are found throughout the environment, and some are implicated in endocrine disruption. Endocrine disruption has been found in humans, wildlife, and even in the partnership of plants and root symbionts. Most endocrine disruption occurs in estrogenic systems. Estrogens, like other steroid hormones, binds a transcription factor known as a nuclear receptor to regulate gene transcription. Recent research has shown that there are other signaling mechanisms for steroid hormones that involve kinase pathways and G protein-coupled receptors. Mounting evidence suggests estrogen mimics can also act by these pathways which work outside the nucleus. Differential expression of these pathways across cell types, and differential affinity for these pathways by diverse compounds may explain some patterns of endocrine disruption and disease.

Despite the fact that the ruminant diet is rich in polyunsaturated fatty acids (PUFA), ruminant products - meat, milk and dairy - contain mainly saturated fatty acids (SFA) because of bacterial lipolysis and subsequent biohydrogenation of ingested PUFA in the rumen. The link between SFA consumption by man and coronary heart disease is well established. In contrast, ruminant products also contain fatty acids that are known to be beneficial to human health, namely conjugated linoleic acids (CLAs). The aims of research in this field have been to understand the microbial ecology of lipolysis and biohydrogenation and to find ways of manipulating ruminal microbes to increase the flow of PUFA and CLA from the rumen into meat and milk. This review describes our present understanding of the microbial ecology of ruminal lipid metabolism, including some apparently anomalous and paradoxical observations, and the status of how the metabolism may be manipulated and the possible consequential effects on other aspects of ruminal digestion. Intuitively, it may appear that inhibiting the ruminal lipase would cause more dietary PUFA to reach the mammary gland. However, lipolysis releases the non-esterified fatty acids that form the substrates for biohydrogenation, but which can, if they accumulate, inhibit the whole process. Thus, increasing lipase activity could be beneficial if the increased release of non-esterified PUFA inhibited the metabolism of CLA. Rumen ciliate protozoa do not carry out biohydrogenation, yet protozoal lipids are much more highly enriched in CLA than bacterial lipids. How could this happen if protozoa do not metabolise PUFA? The answer seems to lie in the ingestion of plant organelles, particularly chloroplasts, and the partial metabolism of the fatty acids by contaminating bacteria. Bacteria related to Butyrivibrio fibrisolvens are by far the most active and numerous biohydrogenating bacteria isolated from the rumen. But do we misunderstand the role of different bacterial species in biohydrogenation because there are uncultivated species that we need to understand and include in the analysis? Manipulation methods include dietary vegetable and fish oils and plant-derived chemicals. Their usefulness, efficacy and possible effects on fatty acid metabolism and on ruminal microorganisms and other areas of their metabolism are described, and areas of opportunity identified.

A screening experiment was conducted to evaluate the potential of 450 plant species as antimethanogenic additives in ruminant feeds. Effects of addition of these plants, which were incorporated to the fermentation substrate as a dry powder, on ruminal fermentation, fibre digestion and methane production were studied in vitro in batch cultures of mixed rumen microorganisms. Serum bottles containing 500 mg of substrate (500 g alfalfa hay/kg, 400 g grass hay/kg and 100 g barley grain), 50 mg of the plant additive tested and 50 ml of buffered rumen fluid (10 ml sheep rumen fluid + 40 ml culture medium) were incubated at 39 °C for 24 h. After incubation, gas and methane production, pH and volatile fatty acid (VFA) concentration in the incubation medium and dry matter and neutral detergent fibre disappearance were recorded. Of the 450 samples tested, 35 decreased methane production by more than 15% versus those with corresponding control cultures and, with 6 of these plant additives, the depression in methane production was more than 25%, with no adverse effects on digestibility, total gas and VFA production. With these six samples, incubations were repeated to confirm their effects on methane production in vitro . Some candidates, in particular Rheum nobile and Carduus pycnocephalus , consistently decreased methane production without adversely affecting other parameters of the rumen fermentation.

Conjugated linoleic acids (CLA) are health-promoting fatty acids found in foods derived from ruminant products that are formed in the rumen during bacterial biohydrogenation of linoleic acid (LA). Although selective antimicrobials might increase CLA production by manipulation of ruminal microflora, feeding of antibiotic growth promoters to livestock is declining due to fears of development of antibiotic resistance in human pathogens. This has initiated a search for alternatives, including plants containing bioactive compounds, which can have similar positive effects on ruminal microflora, but without negative impact on animal and human health. In this study we investigated effects of natural compounds in Australian plants on the bacteria and processes involved in ruminal biohydrogenation. Ethanolic extracts and essential oils were obtained from 91 Australian plants collected during 2004/2005 in Western Australia. Minimal Inhibitory Concentrations for the main bacterial species involved in rumen biohydrogenation were established using an agar dilution method, and effects of selected candidates on LA biohydrogenation was examined using an in vitro system with a mixed rumen bacterial population. A wide range of the plant extracts had selective inhibitory effect towards Clostridium proteoclasticum (which forms stearate from LA) without affecting Butyrivibrio fibrisolvens (which forms CLA and vaccenic acid, but not stearate). However, only a few plants, including Acacia iteaphylla and Kennedia eximia, inhibited LA metabolism or stearate formation in the mixed bacterial community in vitro. Further experiments are needed to investigate effects of selected candidates on CLA production in vivo.

The recent EU ban of growth-promoting antibiotics in animal production was based on fears concerning antibiotic resistance being transmitted to human pathogens. This paper explores the adaptation mechanism of a common ruminal bacterium, Prevotella bryantii, to one of the banned compounds, flavomycin (flavophospholipol).Growth in the presence of flavomycin (2 and 20 microg ml(-1)) was characterized by a concentration-dependent increase in the length of the lag phase, which decreased after previous flavomycin exposure. From growth patterns on solid medium, decreased sensitivity appeared to be due to a whole-population adaptation. Proteomic analysis indicated upregulation of three native proteins occurred following flavomycin adaptation. Further analysis of two of these proteins resulted in no database matches, suggesting that they may be species-specific. Flavomycin adaptation also resulted in co-adaptation to bacitracin and vancomycin.Adaptation of P. bryantii to flavomycin, which also resulted in co-adaptation to bacitracin and vancomycin, may involve an increased availability of undecaprenyl pyrophosphate.The use of flavomycin, and similar growth-promoting antibiotics, in animal production may prompt adaptive responses in ruminal bacteria which can significantly change their antibiotic sensitivity.

Recent advances in chromatographic identification of CLA isomers, combined with interest in their possible properties in promoting human health (e.g., cancer prevention, decreased atherosclerosis, improved immune response) and animal performance (e.g., body composition, regulation of milk fat synthesis, milk production), has renewed interest in biohydrogenation and its regulation in the rumen. Conventional pathways of biohydrogenation traditionally ignored minor fatty acid intermediates, which led to the persistence of oversimplified pathways over the decades. Recent work is now being directed toward accounting for all possible trans-18:1 and CLA products formed, including the discovery of novel bioactive intermediates. Modern microbial genetics and molecular phylogenetic techniques for identifying and classifying microorganisms by their small-subunit rRNA gene sequences have advanced knowledge of the role and contribution of specific microbial species in the process of biohydrogenation. With new insights into the pathways of biohydrogenation now available, several attempts have been made at modeling the pathway to predict ruminal flows of unsaturated fatty acids and biohydrogenation intermediates across a range of ruminal conditions. After a brief historical account of major past accomplishments documenting biohydrogenation, this review summarizes recent advances in 4 major areas of biohydrogenation: the microorganisms involved, identification of intermediates, the biochemistry of key enzymes, and the development and testing of mathematical models to predict biohydrogenation outcomes.

Aims: To investigate the mode of action of a blend of essential oil compounds on the colonization of starch-rich substrates by rumen bacteria. Methods and Results: Starch-rich substrates were incubated, in nylon bags, in the rumen of sheep organized in a 4 × 4 latin square design and receiving a 60 : 40 silage : concentrate diet. The concentrate was either high or low in crude protein, and the diet was supplemented or not with a commercial blend of essential oil compounds (110 mg per day). The total genomic DNA was extracted from the residues in the bags. The total eubacterial DNA was quantified by real-time PCR and the proportion of Ruminobacter amylophilus, Streptococcus bovis and Prevotella bryantii was determined. Neither the supplementation with essential oil compounds nor the amount of crude protein affected the colonization of the substrates by the bacteria quantified. However, colonization was significantly affected by the substrate colonized. Conclusions: The effect of essential oils on the colonization of starch-rich substrates is not mediated through the selective inhibition of R. amylophilus. Significance and Impact of the Study: This study enhances our understanding of the colonization of starch-rich substrates, as well as of the mode of action of the essential oils as rumen manipulating agents.

Aims: All members of the ruminal Butyrivibrio group convert linoleic acid (cis-9,cis-12-18 : 2) via conjugated 18 : 2 metabolites (mainly cis-9,trans-11-18 : 2, conjugated linoleic acid) to vaccenic acid (trans-11-18 : 1), but only members of a small branch, which includes Clostridium proteoclasticum, of this heterogeneous group further reduce vaccenic acid to stearic acid (18 : 0, SA). The aims of this study were to develop a real-time polymerase chain reaction (PCR) assay that would detect and quantify these key SA producers and to use this method to detect diet-associated changes in their populations in ruminal digesta of lactating cows. Methods and Results: The use of primers targeting the 16S rRNA gene of Cl. proteoclasticum was not sufficiently specific when only binding dyes were used for detection in real-time PCR. Their sequences were too similar to some nonproducing strains. A molecular beacon probe was designed specifically to detect and quantify the 16S rRNA genes of the Cl. proteoclasticum subgroup. The probe was characterized by its melting curve and validated using five SA-producing and ten nonproducing Butyrivibrio-like strains and 13 other common ruminal bacteria. Analysis of ruminal digesta collected from dairy cows fed different proportions of starch and fibre indicated a Cl. proteoclasticum population of 2–9% of the eubacterial community. The influence of diet on numbers of these bacteria was less than variations between individual cows. Conclusions: A molecular beacon approach in qPCR enables the detection of Cl. proteoclasticum in ruminal digesta. Their numbers are highly variable between individual animals. Significance and Impact of the Study: SA producers are fundamental to the flow of polyunsaturated fatty acid and vaccenic acid from the rumen. The method described here enabled preliminary information to be obtained about the size of this population. Further application of the method to digesta samples from cows fed diets of more variable composition should enable us to understand how to control these bacteria in order to enhance the nutritional characteristics of ruminant-derived foods, including milk and beef.

The objective of this study was to understand the effects of fumarate addition on methane (CH4) and VFA production in the rumen through a meta-analysis of its effects on ruminal batch cultures. Because the reduction of fumarate to succinate can draw electrons away from ruminal methanogenesis, fumarate has been studied as a potential feed additive to decrease CH4 production in ruminants. The average decrease in CH4 in batch cultures was of 0.037 micromol/micromol of added fumarate, which is considerably lower than 0.25 micromol/micromol, the decrease predicted from the stoichiometry of the reactions involved. One reason that fumarate was not effective at decreasing CH4 in batch cultures was that only an average of 48% of added fumarate appeared to be converted to propionate. Secondly, the incorporation of reducing equivalents in the conversion of fumarate to propionate was almost entirely offset by their release from an average of 20% of added fumarate that appeared to be converted to acetate. Thermodynamic calculations indicated that the conversion of added fumarate to both propionate and acetate was feasible. Fumarate appears to be more effective in decreasing CH4 production and increasing propionate in continuous culture than in batch culture. This suggests that microbial adaptation to fumarate metabolism can be important. Variation in populations of fumarate-reducers, methanogens, and protozoa could all be involved. Fumarate supplementation for an extended period may result in the amplification of otherwise small populations of fumarate-reducers. Addition of some of these organisms may be helpful to improve fumarate conversion to propionate. Strategies based on enhancing the rumen's capacity to convert fumarate to propionate by maintaining a low fumarate concentration have been effective. Thermodynamic considerations should be taken into account when designing strategies for CH4 abatement through the addition of external electron acceptors.

Albumin, as an antifouling agent preventing non-specific adsorption, is widely applied in biosensor construction and in the design of nanocarriers for theranostic applications. However, albumin is not completely inert and it interacts with many endogenous and exogenous compounds. The present work investigates the interactions of bovine serum albumin (BSA) with phenol red dye (PR), a common component of cell-growth media. PR was found to bind to BSA, with the affinity constant KA = (2.5 ± 0.8) × 105 L mol−1, and that it quenches the intrinsic BSA fluorescence through the Forster non-radiative resonance energy transfer (FRET) via static modes, with critical FRET distance of R0 = 2.65 nm. Synchronous fluorescence spectroscopy measurements indicated a conformational transition of BSA in the presence of PR to a conformation with tyrosine residues surrounded by a more hydrophobic environment. Thermodynamic parameters, ΔH°,ΔG° and ΔS°, determined using fluorescence and isothermal titration microcalorimetry, indicated that entropic contribution played a major role at ambient temperature. It was also found that common metal ions increased KA but Ca2+ and Fe3+ decreased it.

Dietarycis-9,trans-11-conjugated linoleic acid (CLA) is generally thought to be beneficial for human health. Fish oil added to ruminant diets increases the CLA concentration of milk and meat, an increase thought to arise from alterations in ruminal biohydrogenation of unsaturated fatty acids. To investigate the mechanism for this effect,in vitroincubations were carried out with ruminal digesta and the main biohydrogenating ruminal bacterium,Butyrivibrio fibrisolvens. Linoleic acid (LA) or α-linolenic acid (LNA) was incubated (1·67g/l) with strained ruminal digesta from sheep receiving a 50:50 grass hay–concentrate ration. Adding fish oil (up to 4·17g/l) tended to decrease the initial rate of LA (P=0·025) and LNA (P=0·137) disappearance, decreased (PPtrans-11-18:1. Concentrations of EPA (20:5n-3) or DHA (22:6n-3), the major fatty acids in fish oil, were low (100mg/l or less) after incubation of fish oil with ruminal digesta. Addition of EPA or DHA (50mg/l) to pure cultures inhibited the growth and isomerase activity ofB. fibrisolvens, while fish oil had no effect. In contrast, similar concentrations of EPA and DHA had no effect on biohydrogenation of LA by mixed digesta, while the addition of LA prevented metabolism of EPA and DHA. Neither EPA nor DHA was metabolised byB. fibrisolvensin pure culture. Thus, fish oil inhibits ruminal biohydrogenation by a mechanism which can be interpreted partly, but not entirely, in terms of its effects onB. fibrisolvens.

Effects of a specific preparation of essential oil compounds on rumen fermentation were determined using four mature sheep, each fitted with a permanent rumen cannula and receiving 110 mg per day of dietary essential oils compounds (EO; CRINA RUMINANTS, AKZO Nobel Surface Chemistry Ltd, Hertfordshire, UK). The basal ration comprised 1 kg DM per day of a 40:60 (DM) concentrate:forage mix fed in two equal meals. The forage was grass silage and the concentrate contained crushed barley grain, rapeseed meal, soyabean meal, molasses and a mineral and vitamin supplement (730, 125, 50, 80 and 15 g/kg fresh weight, respectively). Unsupplemented and EO-supplemented diets were fed in a 2×2 design with 6-week periods. EO inhibited ruminal degradation of soyabean meal N from dacron bags incubated in situ but not heat-treated rapeseed meal or hay. Deamination of amino acids measured in vitro in rumen fluid removed from the sheep decreased by 25% (P

Protein synthesis and turnover in ruminal micro-organisms were assessed by stable-isotope methods in order to follow independently the fate of amino acid (AA)-C and -N in different AA. Rumen fluid taken from sheep receiving a grass hay–concentrate diet were strained and incubatedin vitrowith starch–cellobiose–xylose in the presence of NH3and 5 g algal protein hydrolysate (APH)/l, in incubations where the labels were15NH3, [15N]APH or [13C]APH. Total15N incorporation was calculated from separate incubations with15NH3and [15N]APH, and net N synthesis from the increase in AA in protein-bound material. The large difference between total and net AA synthesis indicated that substantial turnover of microbial protein occurred, averaging 3·5 %/h. Soluble AA-N was incorporated on average more extensively than soluble AA-C (70v.50 % respectively,P=0·001); however, incorporation of individual AA varied. Ninety percent of phenylalanine-C was derived from the C-skeleton of soluble AA, whereas the incorporation of phenylalanine-N was 72 %. In contrast, only 15 % aspartate-C + asparagine-C was incorporated, while 45 % aspartate-N+asparagine-N was incorporated. Deconvolution analysis of mass spectra indicated substantial exchange of carboxyl groups in several AA before incorporation and a condensation of unidentified C2and C4intermediates during isoleucine metabolism. The present results demonstrate that differential labelling with stable isotopes is a way in which fluxes of AA synthesis and degradation, their biosynthetic routes, and separate fates of AA-C and -N can be determined in a mixed microbial population.

Elevated low-density lipoprotein cholesterol (LDL-C) is a treatable, heritable risk factor for cardiovascular disease. Genome-wide association studies (GWASs) have identified 157 variants associated with lipid levels but are not well suited to assess the impact of rare and low-frequency variants. To determine whether rare or low-frequency coding variants are associated with LDL-C, we exome sequenced 2,005 individuals, including 554 individuals selected for extreme LDL-C (>98(th) or

Incubations were carried out with a batch culture system to study the effects of different N sources on the fermentation by ruminal micro-organisms from Merino sheep of two fibre substrates derived from feedstuffs that differed in their fermentation rate. The substrates were neutral-detergent fibre (NDF) from barley straw and sugarbeet pulp. N sources were ammonia (NH4Cl) and peptides (Trypticase). Three treatments were made by replacing ammonia-N with peptide-N at levels of 0 (AMMO), 33 (PEPLOW) and 66 % (PEPHIGH) of total N. There were no differences (P>0·05) between treatments in NDF degradation for both the barley straw and the sugarbeet pulp. Peptides increased (PPPPPPP>0·05) by the presence of peptides compared with the AMMO treatment, but values were greater for the PEPHIGH compared with the PEPLOW N source, reaching statistical significance (PPPP>0·05) by N source. These results indicate that N forms other than ammonia are needed for maximal growth of fibre-digesting ruminal micro-organisms.

Wallace, R. J., Newbold, C. J., Bequette, B. J., MacRae, J. C., Lobley, G. E. (2001). Increasing the flow of protein from ruminal fermentation - Review. Asian-Australian Journal of Animal Science, 14 (6), 885-893.

Samples of foliage from multipurpose leguminous trees (MPT) which had been selected as potential feed supplements for ruminants were examined for their chemical composition and in situ degradation characteristics, and were compared with alfalfa (Medicago sativa) hay and teff (Eragrostis abyssinica) straw. Organic matter (OM), acid detergent fibre (ADF), neutral detergent fibre (NDF), nitrogen, neutral detergent nitrogen, acid detergent lignin (ADL), soluble phenolics, NDF-bound proanthocyanidins and in vitro digestibility were determined in Acacia angustissima, Chamaecytisus palmensis (Tagasaste), Leucaena leucocephala, two cultivars of Sesbania sesban and Vernonia amygdalina (bitter leaf). The MPT all had a nutrient content, particularly in terms of N (up to 39.5 g available N per kg dry matter (DM)) similar to alfalfa hay, which would be suitable for supplementing teff straw, which had a high fibre, but low N (4.0 g available N per kg DM) content. In situ nylon bag digestion and in vitro gas production analyses were carried out to assess microbial degradation characteristics. The MPT were highly degradable in situ, however gas production in vitro decreased as the MPT:teff straw ratio increased in A. angustissima, indicating that antimicrobial components were present in this species. None of the chemical estimations were correlated with antimicrobial properties. It is concluded that some of the MPT tested may prove to be useful dietary supplements for ruminants receiving poor quality forages like teff straw, as has been found in other studies. However, chemical analysis alone will be of limited value in predicting the nutritive value of a new MPT which contains antimicrobial components or material toxic to the animal itself.

International audience; We report for the first time the cloning and characterisation of a protozoal enzyme involved in plant cell wall polysaccharide degradation. A cDNA library was constructed from the ruminal protozoan Polyplastron multivesiculatum and a stable clone expressing xylanase activity was isolated. The encoded enzyme belongs to the glycoside hydrolase family 11, and phylogenetic analysis indicates a closer relationship with catalytic domains from Gram-positive bacteria than the other fibrolytic eukaryotes from the rumen, the anaerobic fungi.

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.

The potential for two complementary fragments of DNA from a clone from the ruminal bacterium Prevotella albensis to encode sequences with homology to at least part of functional proteins is described. One strand contains a sequence with high homology to dnaK, a member of the hsp70 family, and the other strand contains a sequence with some homology to glutamate dehydrogenase genes. Overlapping of these two genes on opposite strands has been reported in eukaryotic species, and is now reported for the first time in a bacterial species. Further investigation of previously described dnaK genes demonstrates that it is more widespread than might be anticipated, with all thirty other dnaK genes investigated also retaining long sequences encoding at least part of a sequence with high homology to a glutamate dehydrogenase gene.

The influence of different N sources on fermentation rate andde novoamino acid synthesis by rumen micro-organisms was investigatedin vitrousing rumen fluid taken from four sheep receiving a mixed diet comprising (g/kg DM): grass hay 500, barley 299·5, molasses 100, fish meal 91, minerals and vitamins 9·5. Pancreatic casein hydrolysate (P; comprising mainly peptides with some free amino acids; 10 g/l), free amino acids (AA; casein acid hydrolysate + added cysteine and tryptophan; 10 g/l), or a mixture of L-proline, glycine, L-valine and L-threonine (M; 0·83 g/l each) were added to diluted (1:3, v/v), strained rumen fluid along with15NH4Cl (A; 1·33 g/l) and 6·7 g/l of a mixture of starch, cellobiose and xylose (1:1:1, by weight). P and AA, but not M, stimulated net gas production after 4 and 8 h incubation (P< 0·05) in comparison with A alone. P increased microbial-protein synthesis (P< 0·05) compared with the other treatments. All of the microbial-N formed after 10 h was synthesizedde novofrom15NH3in treatment A, and the addition of pre-formed amino acids decreased the proportion to 0·37, 0·55, and 0·86 for P, AA, and M respectively.De novosynthesis of amino acids (0·29, 0·42 and 0·69 respectively) was lower than cell-N. Enrichment of alanine, glutamate and aspartate was slightly higher than that of other amino acids, while enrichment in proline was much lower, such that 0·83–0·95 of all proline incorporated into particulate matter was derived from pre-formed proline. Glycine, methionine, lysine, valine and threonine tended to be less enriched than other amino acids. The form in which the amino acids were supplied, as P or AA, had little influence on the pattern ofde novosynthesis. When the concentration of peptides was decreased, the proportion of microbial-N formed from NH3increased, so that at an initial concentration of 1 g peptides/l, similar to the highest reported ruminal peptide concentrations, 0·68 of cell-N was formed from NH3. Decreasing the NH3concentration at 1·0 g peptides/l caused proportionate decreases in the fraction of cell-N derived from NH3, from 0·81 at 0·53 g NH3-N/l to 0·40 at 0·19 g NH3-N/l. It was concluded that different individual amino acids are synthesizedde novoto different extents by mixed rumen micro-organisms when pre-formed amino acids are present, and that the source of N used for synthesis of cell-N and amino acids depends on the respective concentrations of the different N sources available; however, supplementing only with amino acids whose synthesis is lowest when pre-formed amino acids are present does not stimulate fermentation or microbial growth.

Six reductive acetogenic bacteria from a variety of ruminal and non-ruminal environments were investigated for their ability to prevent the accumulation of methane when added to rumen fluid incubated in vitro. Acetitomaculum ruminis, Eubacterium limosum strains ATCC 10825 and ATCC 8486, Ruminococcus productus ATCC 35244, and two acetogenic bacteria, Ser 5 and Ser 8 isolated from 20 h old lambs, were grown in media containing glucose. The bacteria retained the ability to produce acetate from H2 when incubated in the absence of sugar, while no acetate was produced in the absence of H2. When cultures of the acetogens were added to incubations of mixed rumen microorganisms in vitro, providing between 0.05 and 0.13 mg of acetogen protein/ml, methane production decreased by about 5% after 24 h with E. limosum ATCC 8486 and Ser 5, while the other bacteria had no effect on methane production. Increasing the concentration of E. limosum ten-fold did not cause a further decrease in methane production. When E. limosum ATCC 8486 and Ser 5 were added to cultures of mixed ruminal microorganisms in the presence of 2-bromoethanesulfonic acid, which inhibited methane formation and caused H2 to accumulate, both bacteria caused substantial increases in acetate production and decreased H2 formation. It was concluded that although acetogenic bacteria can utilise H2 and CO2 to form acetate in the rumen when methanogenesis is inhibited, even large concentrations of acetogenic bacteria cannot compete for H2 with methanogenic archaea under normal circumstances. # 1999 Elsevier Science B.V. All rights reserved.

Nine different accessions of Sesbania sesban were examined for their toxicity to ciliate protozoa from sheep receiving a mixed grass hay–barley/fishmeal concentrate in Aberdeen, Scotland. Several accessions were much more toxic than those (ILRI 10865, ILRI 15036) identified and tested in a previous study. Two highly toxic accessions and one less toxic accession were compared with ILRI 15036 in a feeding trial in Debre Zeit, Ethiopia with sheep receiving a sululta hay–wheat bran diet. No suppression of protozoal numbers occurred in response to S. sesban supplementation (200 g/day). When rumen fluid removed from sheep on the supplemented diet was tested in vitro, protozoal activity appeared to be inhibited less by S. sesban in the mixed population than in either washed protozoa from the same rumen fluid or than had occurred in Scottish sheep. It was concluded that dietary, microbial or animal factors in the Ethiopian sheep had caused the destruction of the anti-protozoal material present in even the most potent accessions of S. sesban .

Wallace, R. J., Atasoglu, C., Newbold, C. J. (1999). Role of peptides in rumen microbial metabolism - Review. Asian-Australian Journal of Animal Science, 12 (1), 139-147.

The influence of sodium fumarate on rumen fermentation was investigatedin vitrousing batch and semi-continuous cultures of mixed rumen micro-organisms taken from three sheep receiving a basal diet of hay, barley, molasses, fish meal and a mineral–vitamin supplement (500, 299·5, 100, 91 and 9·5 g/kg DM respectively). Batch cultures consisted of 10 ml strained rumen fluid in 40 ml anaerobic buffer containing 200 mg of the same feed given to the sheep. Sodium fumarate was added to achieve a final concentration of 0, 5 or 10 mmol/l, as a result of the addition of 0, 250 or 500 μmol, equivalent to 0, 200 and 400 g/kg feed. CH4production at 24 h (360 μmol in the control cultures) fell (P< 0·05) by 18 and 22 μmol respectively (SED 7·5). Total gas production was increased by the addition of fumarate without significant accumulation of H2. Substantial increases in acetate production (92 and 194 μmol; SED 26·7,P< 0·01) were accompanied by increases in propionate formation (212 and 396 μmol; SED 13·0,P< 0·001). Longer-term effects of fumarate supplementation on ruminal fermentation and CH4production were investigated using the rumen simulation technique (Rusitec). Eight vessels were given 20 g basal diet/d, and half of them received a supplement of fumarate (disodium salt) over a period of 19 d. The response to the daily addition of 6·25 mmol sodium fumarate was a decrease in CH4production of 1·2 mmol (SED 0·39,P< 0·05), equivalent to the consumption of 4·8 mmol H2, and an increase in propionate production of 4·9 mmol (from 10·4 to 15·3 (SED 1·05) mmol/d,P< 0·01). The inhibition of CH4production did not decline during the period of time that fumarate was added to the vessels. Thus, the decrease in CH4corresponded well to the fraction of the fumarate that was converted to propionate. Fumarate had no significant (P> 0·05) effect on total bacterial numbers or on the number of methanogenic archaea, but numbers of cellulolytic bacteria were increased (8·8v. 23·9 (SED 2·49) × 105per ml,P< 0·01). Fumarate also increased DM digestibility of the basal diet after 48 h incubation (0·476v. 0·508 (SED 0·0123),P< 0·05). Thus, it was concluded that sodium fumarate may be a useful dietary additive for ruminants, because it diverts some H2from CH4production and because it is able to stimulate proliferation of cellulolytic bacteria and digestion of fibre.

Nsereko, V. L., Rooke, J. A., Newbold, C. J., Wallace, R. J. (1998). Influence of protease inhibitors on nitrogen distribution in ensiled perennial ryegrass and the utilisation of silage nitrogen for growth by rumen bacteria in vitro. Animal Feed Science and Technology, 76 (1-2), 51-63.

The influence of peptides and amino acids on ammonia assimilation and de novo synthesis of amino acids by three predominant noncellulolytic species of ruminal bacteria, Prevotella bryantii B 1 4, Selenomonas ruminantium HD4, and Streptococcus bovis ES1, was determined by growing these bacteria in media containing 15 NH 4 Cl and various additions of pancreatic hydrolysates of casein (peptides) or amino acids. The proportion of cell N and amino acids formed de novo decreased as the concentration of peptides increased. At high concentrations of peptides (10 and 30 g/liter), the incorporation of ammonia accounted for less than 0.16 of bacterial amino acid N and less than 0.30 of total N. At 1 g/liter, which is more similar to peptide concentrations found in the rumen, 0.68, 0.87, and 0.46 of bacterial amino acid N and 0.83, 0.89, and 0.64 of total N were derived from ammonia by P. bryantii , S. ruminantium , and S. bovis , respectively. Concentration-dependent responses were also obtained with amino acids. No individual amino acid was exhausted in any incubation medium. For cultures of P. bryantii , peptides were incorporated and stimulated growth more effectively than amino acids, while cultures of the other species showed no preference for peptides or amino acids. Apparent growth yields increased by between 8 and 57%, depending on the species, when 1 g of peptides or amino acids per liter was added to the medium. Proline synthesis was greatly decreased when peptides or amino acids were added to the medium, while glutamate and aspartate were enriched to a greater extent than other amino acids under all conditions. Thus, the proportion of bacterial protein formed de novo in noncellulolytic ruminal bacteria varies according to species and the form and identity of the amino acid and in a concentration-dependent manner.

Acetylation is a potential method for protecting dietary peptides from degradation by rumen micro-organisms. As a first step in determining the nutritive value of acetylated peptides, their disappearance in the small intestine of sheep and their ability to support growth in a rat bioassay were measured.15N-labelled peptides were prepared from lucerne which had been grown with15N-labelled (NH4)2SO4in the absence ofRhizobium. Peptides were prepared by enzymic hydrolysis of the extracted protein. Two peptide preparations were made using different proteinase mixtures. These mixtures contained peptides with an average molecular weight of 559 and 522 Da. They were treated with acetic anhydride, which resulted in 85 and 88% modification respectively, and their uptake from the small intestine was determined by injecting 1 g of untreated or acetylated peptides in a Cr-EDTA solution into the jejunum of two sheep fitted with jejunal catheters and ileal cannulas. Ileal digesta were collected and analysed for Cr and15N. The uptake of dialanine (Ala2) and N-acetyl-Ala2were compared in a similar way. The disappearance of15N from lucerne peptides was high (88 and 93% respectively) and this was not affected significantly by acetylation (86 and 87%). Corresponding values for Ala2and N-acetyl-Ala2were both 96%, as measured by HPLC. It was therefore concluded that acetylation did not affect the uptake of peptides from the small intestine in sheep. Two feeding trials were carried out with rats. The first trial was carried out with a protein-free diet to which was added 10% lactalbumin or 5% lactalbumin and then a mixture of methionine-free amino acids, either alone or supplemented with Met, Gly-Met or acetylated Gly-Met. The rats grew equally well on all sources of Met, but failed to grow significantly on the mixture of Met-free amino acids. In the second trial the diet contained casein as 5·9% of the basal diet. Additional casein, pancreatic casein hydrolysate (peptides) and acetylated pancreatic casein hydrolysate (acetylated peptides) were compared as sources of amino acids, at inclusion rates of 100 g/kg final diet. Feed intake was similar with casein and peptides treatments, but was depressed by 23% with acetylated peptides. Live weight gain was 15 and 75% lower with the peptides and acetylated peptides diets respectively. Addition of lysine, arginine or histidine did not restore feed intake or weight gain of rats receiving acetylated peptides, but feed intake was restored immediately when peptides replaced acetylated peptides. When intake was restricted to 9 g/d and acetylated casein hydrolysate replaced half of the protein in the diet, rats gained weight less rapidly (1·44 v: 1·09 g/d) and retained less N, such that only 0·36 of the acetylated peptide-N was calculated to remain available to the animal. This N retention compared with 0·70 for unmodified casein. Thus, the rat bioassay indicated that certain specific peptides may well be of high nutritive value following acetylation, but that there may be problems of inappetance and inefficient utilization with acetylated peptide mixtures.

Yeast increased the total viable count of bacteria on a medium containing rumen fluid and soluble sugars by 38% (P < 0.05) and the cellulolytic population increased by 49%, although the increase was not statistically significant (P > 0.05). Microscopic counts indicated a 25% increase in total bacteria (P < 0.05). Bacterial counts with mannitol as sole energy source also increased (P < 0.05), indicating an increased Selenomonas population; that was not in itself sufficient to account for the increase in the total bacterial count. The protozoal population was significantly lower in yeast supplemented vessels (P < 0.001), which may account partly for the increased bacterial numbers. Key words: Yeast culture, probiotics, rumen bacteria, Rusitec