Relationship between feed efficiency and gut microbiota in laying chickens under contrasting feeding conditions.

The gut microbiota is known to play an important role in energy harvest and is likely to affect feed efficiency. In this study, we used 16S metabarcoding sequencing to analyse the caecal microbiota of laying hens from feed-efficient and non-efficient lines obtained by divergent selection for residual feed intake. The two lines were fed either a commercial wheat-soybean based diet (CTR) or a low-energy, high-fibre corn-sunflower diet (LE). The analysis revealed a significant line x diet interaction, highlighting distinct differences in microbial community composition between the two lines when hens were fed the CTR diet, and more muted differences when hens were fed the LE diet. Our results are consistent with the hypothesis that a richer and more diverse microbiota may play a role in enhancing feed efficiency, albeit in a diet-dependent manner. The taxonomic differences observed in the microbial composition seem to correlate with alterations in starch and fibre digestion as well as in the production of short-chain fatty acids. As a result, we hypothesise that efficient hens are able to optimise nutrient absorption through the activity of fibrolytic bacteria such as Alistipes or Anaerosporobacter, which, via their production of propionate, influence various aspects of host metabolism.

A large database linking the rumen bacterial composition and milk traits in Lacaune sheep.

Ruminants are able to produce food for human consumption from plants, thanks to rumen bacteria. Bacteria are able to transform feed to microbial proteins and to biohydrogenate unsaturated fatty acids, contributing directly to fine milk composition. The database consists of daily records of milk yield, somatic cell score and 17 milk components such as fatty acids and proteins from 795 Lacaune dairy ewes. Ruminal samples were extracted from ewes using a gastric tube and sequenced to determine the bacterial composition by metabarcoding 16S rRNA gene on a next-generation sequencing platform. From bioinformatics analysis, 9,536,442 sequences were retained and re-grouped into 2,059 affiliated OTUs, represented by 751 to 168,617 sequences. Overall, 2,059 OTUs from 795 samples were attributed to 11 phyla. The most representative phyla were Bacteroidota (50.6%) and Firmicutes (43.6%), and the most abundant families were Prevotellaceae (37.9%), Lachnospiraceae (18.1%), Ruminococcaceae (8.97%). Both shared datasets will be useful for researchers to study the link between rumen bacteria and milk traits and to propose solutions to improve animal production and health.

Host genetic control on rumen microbiota and its impact on dairy traits in sheep.

BACKGROUND: Milk yield and fine composition in sheep depend on the volatile and long-chain fatty acids, microbial proteins, vitamins produced through feedstuff digestion by the rumen microbiota. In cattle, the host genome has been shown to have a low to moderate genetic control on rumen microbiota abundance but a high control on dairy traits with heritabilities higher than 0.30. There is little information on the genetic correlations and quantitative trait loci (QTL) that simultaneously affect rumen microbiota abundance and dairy traits in ruminants, especially in sheep. Thus, our aim was to quantify the effect of the host genetics on rumen bacterial abundance and the genetic correlations between rumen bacterial abundance and several dairy traits, and to identify QTL that are associated with both rumen bacterial abundance and milk traits. RESULTS: Our results in Lacaune sheep show that the heritability of rumen bacterial abundance ranges from 0 to 0.29 and that the heritability of 306 operational taxonomic units (OTU) is significantly different from 0. Of these 306 OTU, 96 that belong mainly to the Prevotellaceae, Lachnospiraceae and Ruminococcaceae bacterial families show strong genetic correlations with milk fatty acids and proteins (absolute values ranging from 0.33 to 0.99). Genome-wide association studies revealed a QTL for alpha-lactalbumin concentration in milk on Ovis aries chromosome (OAR) 11, and six QTL for rumen bacterial abundances i.e., for two OTU belonging to the genera Prevotella (OAR3 and 5), Rikeneleaceae_RC9_gut_group (OAR5), Ruminococcus (OAR5), an unknown genus of order Clostridia UCG-014 (OAR10), and CAG-352 (OAR11). None of these detected regions are simultaneously associated with rumen bacterial abundance and dairy traits, but the bacterial families Prevotellaceae, Lachnospiraceae and F082 show colocalized signals on OAR3, 5, 15 and 26. CONCLUSIONS: In Lacaune dairy sheep, rumen microbiota abundance is partially controlled by the host genetics and is poorly genetically linked with milk protein and fatty acid compositions, and three main bacterial families, Prevotellaceae, Lachnospiraceae and F082, show specific associations with OAR3, 5, 15 and 26.

Variation in Rumen Bacteria of Lacaune Dairy Ewes From One Week to the Next.

Bacteria are the most abundant microorganisms in the rumen microbiota and play essential roles, mainly fermenting plant compounds that yield fatty acids. In this study, we aimed at assessing stability of both bacterial composition and of its associations with rumen and milk fatty acids phenotypes over a 1-week period. The study was performed using 118 Lacaune dairy ewes from the INRAE Experimental Unit of La Fage. Rumen and milk samples were obtained from the ewes twice, 1 week apart, and microbiota composition, volatile and long-chain fatty acid concentrations were analyzed. Bacterial composition was assessed using 16S rRNA gene sequencing, and microbiota and fatty acids were analyzed as compositional data. As we worked with relative abundances expressed in a constrained space, the centered log-ratio transformation enabled to transform data to work with multivariate analyses in the Euclidian space. Bacterial composition differed between the 2 weeks of sampling, characterized by different proportions of the two main phyla, Bacteroidetes and Firmicutes. The repeatability of the operational taxonomic units (OTUs) was low, although it varied significantly. However, 66 of them presented a repeatability of over 0.50 and were particularly associated with fatty acid phenotypes. Even though the OTUs from the same bacterial families presented similar correlations to fatty acids in both weeks, only a few OTUs were conserved over the 2 weeks. We proved with the help of sequencing data that there is significant change in microbial composition over a week in terms of abundance of different families of bacteria. Further studies are required to determine the impact of bacterial composition alterations over 1 week, and the specificities of the highly repeatable OTUs.

Evaluation of the Links between Lamb Feed Efficiency and Rumen and Plasma Metabolomic Data.

Feed efficiency is one of the keystones that could help make animal production less costly and more environmentally friendly. Residual feed intake (RFI) is a widely used criterion to measure feed efficiency by regressing intake on the main energy sinks. We investigated rumen and plasma metabolomic data on Romane male lambs that had been genetically selected for either feed efficiency (line rfi-) or inefficiency (line rfi+). These investigations were conducted both during the growth phase under a 100% concentrate diet and later on under a mixed diet to identify differential metabolite expression and to link it to biological phenomena that could explain differences in feed efficiency. Nuclear magnetic resonance (NMR) data were analyzed using partial least squares discriminant analysis (PLS-DA), and correlations between metabolites' relative concentrations were estimated to identify relationships between them. High levels of plasma citrate and malate were associated with genetically efficient animals, while high levels of amino acids such as L-threonine, L-serine, and L-leucine as well as beta-hydroxyisovalerate were associated with genetically inefficient animals under both diets. The two divergent lines could not be discriminated using rumen metabolites. Based on phenotypic residual feed intake (RFI), efficient and inefficient animals were discriminated using plasma metabolites determined under a 100% concentrate diet, but no discrimination was observed with plasma metabolites under a mixed diet or with rumen metabolites regardless of diet. Plasma amino acids, citrate, and malate were the most discriminant metabolites, suggesting that protein turnover and the mitochondrial production of energy could be the main phenomena that differ between efficient and inefficient Romane lambs.

Integrating data from spontaneous and induced trans-10 shift of ruminal biohydrogenation reveals discriminant bacterial community changes at the OTU level.

Introduction: Microbial digestion is of key importance for ruminants, and disturbances can affect efficiency and quality of products for human consumers. Ruminal biohydrogenation of dietary unsaturated fatty acids leads to a wide variety of specific fatty acids. Some dietary conditions can affect the pathways of this transformation, leading to trans-10 fatty acids rather than the more usual trans-11 fatty acids, this change resulting in milk fat depression in dairy cows. Materials and methods: We combined data from an induced and spontaneous trans-10 shift of ruminal biohydrogenation, providing new insight on bacterial changes at different taxonomic levels. A trans-10 shift was induced using dietary addition of concentrate and/or unsaturated fat, and the spontaneous milk fat depression was observed in a commercial dairy herd. Results and discussion: Most changes of microbial community related to bacteria that are not known to be involved in the biohydrogenation process, suggesting that the trans-10 shift may represent the biochemical marker of a wide change of bacterial community. At OTU level, sparse discriminant analysis revealed strong associations between this change of biohydrogenation pathway and some taxa, especially three taxa belonging to [Eubacterium] coprostanoligenes group, Muribaculaceae and Lachnospiraceae NK3A20 group, that could both be microbial markers of this disturbance and candidates for studies relative to their ability to produce trans-10 fatty acids.

Apart From the Diet, the Ruminal Microbiota of Lambs Is Modified in Relation to Their Genetic Potential for Feed Efficiency or Feeding Behavior.

Using two successive types of diets (100% concentrate and 67% forage), this study explores the relationship between the ruminal microbiota of 78 Romane lambs and their feed efficiency (residual feed intake trait) or feeding behavior (feeding rate trait). Analysis was carried out phenotypically by correlating feed efficiency or feeding behavior traits with the relative abundance of bacteria at the phylum, family, and genus levels, and then genetically by comparing the microbiota of lambs selected for extreme breeding values for residual feed intake or feeding rate. Our results confirmed the major effect of diet on the ruminal microbiota composition. The microbiota of lambs consuming a forage-based diet was distinguished by higher microbial diversity and also by higher relative abundance of Firmicutes, whereas Bacteriodetes and Actinobacteria were relatively more abundant in the microbiota of lambs consuming a concentrate-based diet. Moreover, the comparison of lambs divergent for residual feed intake breeding values revealed that regardless of diet, more efficient lambs possessed a ruminal microbiota enriched in Coprococcus, Moryella, [Eubacterium] Brachy group, and [Eubacterium] hallii group, but depleted in Lachnospiraceae FD2005 and Shuttleworthia. The connection between microbiota composition and feeding rate was more tenuous, with no link between the abundance of particular genera and lambs genetically divergent for feeding rate.

Compositional analysis of ruminal bacteria from ewes selected for somatic cell score and milk persistency.

Ruminants are dependent on their rumen microbiota to obtain energy from plants. The composition of the microbiome was well-known to be associated with health status, and production traits, but published results are difficult to reproduce due to large sources of variation. The objectives of this study were to evaluate the associations of ruminal microbiota and its association with genetic lines selected by somatic cell score (SCS) or milk persistency (PERS), as well as milk production, somatic cell score, fat and protein contents, and fatty acids and proteins of milk, using the principles of compositional data. A large sample of 700 Lacaune dairy ewes from INRAE La Fage feeding the same diet and belonging to two divergent genetic lines selected for SCS or PERS was used. The ruminal bacterial metagenome was sequenced using the 16S rRNA gene, resulting in 2,059 operational taxonomic units affiliated with 112 genera. The abundance data were centred log-transformed after the replacement of zeros with the geometric Bayesian method. Discriminant analysis of the SCS showed differences between SCS+ and SCS- ewes, while for PERS no difference was obtained. Milk traits as fat content, protein content, saturated fatty acids and caseins of milk were negatively associated with Prevotella (R = [-0.08;-0.16]), Suttonella (R = [-0.09;-0.16]) and Ruminococcus (R = [-0.08;-0.16]), and positively associated with Lachnospiraceae (R = [0.09;0.16]) and Christensenellaceae (R = [0.09;0.16]). Our findings provide an understanding of the application of compositional data to microbiome analysis, and the potential association of Prevotella, Suttonella, Ruminococcaceae and Lachnospiraceae with milk production traits such as milk fatty acids and proteins in dairy sheep.

Enzymatic Study of Linoleic and Alpha-Linolenic Acids Biohydrogenation by Chloramphenicol-Treated Mixed Rumen Bacterial Species.

In the rumen, dietary polyunsaturated fatty acids (PUFA) are reduced by a multistage reaction called biohydrogenation (BH). BH leads to a high proportion of saturated fat in ruminant products, but also products some potential bioactive intermediates like conjugated linoleic and linolenic acids. BH is composed of two kinds of reactions: first an isomerization of PUFA followed by reductions (two for linoleic acid, C18:2n-6; three for α-linolenic acid, C18:3n-3). There is little knowledge about BH enzymes as BH bacterial species are the subject of a lot of studies. Nevertheless, both aspects must be explored to control BH and enhance the fatty acids profile of ruminant products. In the present study, an alternative approach was developed to study the enzymes produced in vivo by mixed ruminal bacteria, using inactivation of bacteria by chloramphenicol, an inhibitor of protein synthesis in prokaryotes, before in vitro incubation. To study C18:2n-6 and C18:3n-3 BH several experiments were used: (1) with different incubation durations (0 to 3) to estimate average rates and efficiencies of all BH reactions, and intermediates production; and (2) with different initial quantities of PUFA (0.25 to 2 mg) to estimate Michaelis-Menten enzymatic parameters, Km and Vmax. A last experiment explored the effect of pH buffer and donor cow diet on C18:2n-6 isomerization pathways. Concerning C18:2n-6 BH, this study confirmed the high saturability of its isomerization, the inhibition of both trans11 and trans10 pathways by a low pH, and the last reduction to stearic acid as the limiting-step. Concerning C18:3n-3, its BH was faster than C18:2n-6, in particular its isomerization (Vmax = 3.4 vs. 0.6 mM/h, respectively), and the limiting-step was the second reduction to t11-C18:1. Besides, our mixed isomerases had a higher affinity for C18:2n-6 than for C18:3n-3 (Km = 2.0 × 10-3 vs. 4.3 × 10-3 M, respectively), but due to their high saturability by C18:2n-6, they had a lower efficiency to isomerize C18:2n-6 than C18:3n-3. Chloramphenicol-treated ruminal fluid would be a meaningful method to study the BH enzymes activities.

Strongyle Infection and Gut Microbiota: Profiling of Resistant and Susceptible Horses Over a Grazing Season.

Gastrointestinal strongyles are a major threat to horses' health and welfare. Given that strongyles inhabit the same niche as the gut microbiota, they may interact with each other. These beneficial or detrimental interactions are unknown in horses and could partly explain contrasted susceptibility to infection between individuals. To address these questions, an experimental pasture trial with 20 worm-free female Welsh ponies (10 susceptible (S) and 10 resistant (R) to parasite infection) was implemented for 5 months. Fecal egg counts (FEC), hematological and biochemical data, body weight and gut microbiological composition were studied in each individual after 0, 24, 43, 92 and 132 grazing days. R and S ponies displayed divergent immunological profiles and slight differences in microbiological composition under worm-free conditions. After exposure to natural infection, the predicted R ponies exhibited lower FEC after 92 and 132 grazing days, and maintained higher levels of circulating monocytes and eosinophils, while lymphocytosis persisted in S ponies. Although the overall gut microbiota diversity and structure remained similar during the parasite infection between the two groups, S ponies exhibited a reduction of bacteria such as Ruminococcus, Clostridium XIVa and members of the Lachnospiraceae family, which may have promoted a disruption of mucosal homeostasis at day 92. In line with this hypothesis, an increase in pathobionts such as Pseudomonas and Campylobacter together with changes in several predicted immunological pathways, including pathogen sensing, lipid metabolism, and activation of signal transduction that are critical for the regulation of immune system and energy homeostasis were observed in S relative to R ponies. Moreover, S ponies displayed an increase in protozoan concentrations at day 92, suggesting that strongyles and protozoa may contribute to each other's success in the equine intestines. It could also be that S individuals favor the increase of these carbohydrate-degrading microorganisms to enhance the supply of nutrients needed to fight strongyle infection. Overall, this study provides a foundation to better understand the mechanisms that underpin the relationship between equines and natural strongyle infection. The profiling of horse immune response and gut microbiota should contribute to the development of novel biomarkers for strongyle infection.

Production of trans and conjugated fatty acids in dairy ruminants and their putative effects on human health: A review.

Consumption of milk and dairy products is important in Western industrialised countries. Fat content is an important constituent contributing to the nutritional quality of milk and dairy products. In order to improve the health of consumers, there is high interest in improving their fatty acid (FA) composition, which depends principally on rumen and mammary metabolism. This paper reviews the lipid metabolism in ruminants, with a particular focus on the production of trans and conjugated linoleic acids (CLA) and conjugated linolenic acids (CLnA) in the rumen. After the lipolysis of dietary lipids, an extensive biohydrogenation of unsaturated FA occurs by rumen bacteria, leading to numerous cis and trans isomers of 18:1, non-conjugated of 18:2, CLA and CLnA. The paper examines the different putative pathways of ruminal biohydrogenation of cis9-18:1, 18:2n-6, 18:3n-3 and long-chain FA and the bacteria implicated. Then mechanisms relative to the de novo mammary synthesis are presented. Ruminant diet is the main factor regulating the content and the composition of milk fat. Effects of nature of forage and lipid supplementation are analysed in cows and small ruminants species. Finally, the paper briefly presents the effects of these FA on animal models and human cell lines. We describe the properties of ruminant trans 18:1, when compared to industrial trans 18:1, CLA and CLnA on human health from meta-analyses of intervention studies and then explore the underlying mechanisms.

Microbial ecology of the rumen evaluated by 454 GS FLX pyrosequencing is affected by starch and oil supplementation of diets.

To provide a comprehensive examination of the bacterial diversity in the rumen content of cows fed different diets, high-throughput 16S rRNA gene-based pyrosequencing was used. Four rumen fistulated nonlactating Holstein cows received 12 kg of dry matter per day of four diets based on maize silage during four periods: the low-starch diet (22% starch, 3% fat); the high-starch diet, supplemented with wheat plus barley (35% starch, 3% fat); the low-starch plus oil diet, supplemented with 5% of sunflower oil (20% starch, 7.6% fat) and the high-starch plus oil diet (33% starch, 7.3% fat). Samples were taken after 12 days of adaptation, 5 h postfeeding. Whatever the diet, bacterial community of sieved rumen contents was dominated by Firmicutes and Bacteroidetes. Lachnospiraceae, Ruminococcaceae, Prevotellaceae, and Rikenellaceae families were highly present and were clearly affected by cow diet. The highest abundance of Prevotellaceae and the lowest abundance of Ruminococcaceae and Rikenellaceae were found with the high-starch plus oil diet. Dietary starch increased the relative abundance of only three genera: Barnesiella, Oribacterium and Olsenella, but decreased the relative abundances of several genera, with very significant effects for Rikenellaceae_RC9 and Butyrivibrio-Pseudobutyrivibrio. Oil alone had a limited effect, but interestingly, starch plus oil addition differently affected the bacterial populations compared to starch addition without oil.

Effects of fat source and dietary sodium bicarbonate plus straw on the conjugated linoleic acid content of milk of dairy cows.

The effects of fat source (0.7 kg of fatty acids from extruded soybeans or palmitic acid), of sodium bicarbonate (0.3 kg) plus straw (1 kg) and the interaction of these treatments on the content of conjugated linoleic acid (CLA) in the milk of dairy cows were examined. During nine weeks a group of 10 cows received a ration with palmitic acid and bicarbonate plus straw (ration PAB). During three periods of three weeks a second group of 10 cows received successively a ration with extruded soybeans and bicarbonate plus straw (ration ESB), a ration with palmitic acid without bicarbonate or straw (ration PA), and a ration with extruded soybeans without bicarbonate or straw (ration ES). Rations ES and ESB increased the content of polyunsaturated fatty acids in milk, but decreased milk fat content, compared to rations PAB and PA. Ration ESB led to the greatest milk CLA content, by a synergy between the high amount of dietary fat, and the action of bicarbonate plus straw, favouring trans11 isomers of CLA and C18:1, presumably via a ruminal pH near neutrality. Ration ES favoured trans10 isomers, not desaturated in the mammary gland, so that the milk CLA content was lower than with ration ESB, and resulted in the lowest milk fat content. In conclusion, a ration supplemented with both extruded soybeans and bicarbonate plus straw, was an efficient way to increase the CLA content in the milk of dairy cows.

Rates and efficiencies of reactions of ruminal biohydrogenation of linoleic acid according to pH and polyunsaturated fatty acids concentrations.

Data from a previous study about the effects of pH and of linolenic acid (C18:3n-3) and linoleic acid (C18:2n-6) concentrations on C18:2n-6 biohydrogenation in ruminal cultures were used to calculate the rates and efficiencies of the three reactions of C18:2n-6 biohydrogenation (isomerisation of C18:2n-6 to CLA; reduction of CLA to trans-octadecenoic acids; reduction of trans-octadecenoic acids to stearic acid). First, low pH was confirmed to inhibit isomerisation and was shown to inhibit the second reduction, leading to an accumulation of vaccenic acid. This later effect had only been observed in some in vivo studies using high concentrate diets, because in in vitro experiments, the very low pH frequently used depresses isomerisation which consequently generates very low amount of substrates for reductions whose variations become difficult to ascertain. Second, C18:2n-6 at high concentration was confirmed to saturate its own isomerisation and the increase of CLA production due to high initial C18:2n-6 was shown to inhibit the two subsequent reductions. Third, C18:3n-3 at high concentrations was confirmed to inhibit C18:2n-6 isomerisation. Moreover, the second reduction was shown to be saturated, probably by all trans-octadecenoic acids intermediates of C18:2n-6 and C18:3n-3 biohydrogenation, leading to an accumulation of trans-octadecenoic acids, especially vaccenic acid. This fatty acid is partly desaturated into CLA in the mammary gland, which explains the synergy between C18:2n-6 and C18:3n-3 for milk CLA noticed by others in vivo. This approach helped explain the actions of pH and of C18:2n-6 and C18:3n-3 concentrations on C18:2n-6 biohydrogenation and allows some explanations about differences noticed between studies.