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 pile of pipelines: An overview of the bioinformatics software for metabarcoding data analyses.

Environmental DNA (eDNA) metabarcoding has gained growing attention as a strategy for monitoring biodiversity in ecology. However, taxa identifications produced through metabarcoding require sophisticated processing of high-throughput sequencing data from taxonomically informative DNA barcodes. Various sets of universal and taxon-specific primers have been developed, extending the usability of metabarcoding across archaea, bacteria and eukaryotes. Accordingly, a multitude of metabarcoding data analysis tools and pipelines have also been developed. Often, several developed workflows are designed to process the same amplicon sequencing data, making it somewhat puzzling to choose one among the plethora of existing pipelines. However, each pipeline has its own specific philosophy, strengths and limitations, which should be considered depending on the aims of any specific study, as well as the bioinformatics expertise of the user. In this review, we outline the input data requirements, supported operating systems and particular attributes of thirty-two amplicon processing pipelines with the goal of helping users to select a pipeline for their metabarcoding projects.

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.

Early Introduction of Plant Polysaccharides Drives the Establishment of Rabbit Gut Bacterial Ecosystems and the Acquisition of Microbial Functions.

In mammals, the introduction of solid food is pivotal for the establishment of the gut microbiota. However, the effects of the first food consumed on long-term microbiota trajectory and host response are still largely unknown. This study aimed to investigate the influences of (i) the timing of first solid food ingestion and (ii) the consumption of plant polysaccharides on bacterial community dynamics and host physiology using a rabbit model. To modulate the first exposure to solid nutrients, solid food was provided to suckling rabbits from two different time points (3 or 15 days of age). In parallel, food type was modulated with the provision of diets differing in carbohydrate content throughout life: the food either was formulated with a high proportion of rapidly fermentable fibers (RFF) or was starch-enriched. We found that access to solid food as of 3 days of age accelerated the gut microbiota maturation. Our data revealed differential effects according to the digestive segment: precocious solid food ingestion influenced to a greater extent the development of bacterial communities of the appendix vermiformis, whereas life course polysaccharides ingestion had marked effects on the cecal microbiota. Greater ingestion of RFF was assumed to promote pectin degradation as revealed by metabolomics analysis. However, transcriptomic and phenotypic host responses remained moderately affected by experimental treatments, suggesting little outcomes of the observed microbiome modulations on healthy subjects. In conclusion, our work highlighted the timing of solid food introduction and plant polysaccharides ingestion as two different tools to modulate microbiota implantation and functionality. IMPORTANCE Our study was designed to gain a better understanding of how different feeding patterns affect the dynamics of gut microbiomes and microbe-host interactions. This research showed that the timing of solid food introduction is a key component of the gut microbiota shaping in early developmental stages, though with lower impact on settled gut microbiota profiles in older individuals. This study also provided in-depth analysis of dietary polysaccharide effects on intestinal microbiota. The type of plant polysaccharides reaching the gut through the lifetime was described as an important modulator of the cecal microbiome and its activity. These findings will contribute to better define the interventions that can be employed for modulating the ecological succession of young mammal gut microbiota.

A carvacrol-based product reduces Campylobacter jejuni load and alters microbiota composition in the caeca of chickens.

AIM: This study was conducted to test the ability of a carvacrol-based formulation (Phodé, France) to decrease the C. jejuni caecal load in inoculated broiler chickens and to study the impact of the C. jejuni inoculation alone or combined with the product, on the caecal microbiota. METHODS AND RESULTS: On day 1, chickens were either fed a control feed or the same diet supplemented with a carvacrol-based product. On day 21, the carvacrol-supplemented chickens and half of the non-supplemented chickens were inoculated with C. jejuni (108  CFU). Quantitative PCR was used to quantify C. jejuni in chicken caecal samples and 16S rRNA gene sequencing was carried out at 25, 31 and 35 days of age. A significant decrease of 1.4 log of the C. jejuni caecal load was observed in 35-day-old chickens supplemented with the product, compared to the inoculated and unsupplemented group (p < 0.05). The inoculation with C. jejuni significantly increased the population richness, Shannon and Simpson diversity and altered beta-diversity. Compared to the control group, the C. jejuni inoculation causes significant changes in the microbiota. The carvacrol-based product associated with C. jejuni inoculation increased the diversity and strongly modified the structure of the microbial community. Functional analysis by 16S rRNA gene-based predictions further revealed that the product up-regulated the pathways involved in the antimicrobial synthesis, which could explain its shaping effect on the caecal microbiota. CONCLUSIONS: Our study confirmed the impairment of the caecal bacterial community after inoculation and demonstrated the ability of the product to reduce the C. jejuni load in chickens. Further investigations are needed to better understand the mode of action of this product to promote the installation of a beneficial microbiota to its host. SIGNIFICANCE AND IMPACT OF THE STUDY: Results suggested that this product could be promising to control C. jejuni contamination of broilers.

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.

FROGS: a powerful tool to analyse the diversity of fungi with special management of internal transcribed spacers.

Fungi are present in all environments. They fulfil important ecological functions and play a crucial role in the food industry. Their accurate characterization is thus indispensable, particularly through metabarcoding. The most frequently used markers to monitor fungi are ITSs. These markers are the best documented in public databases but have one main weakness: polymerase chain reaction amplification may produce non-overlapping reads in a significant fraction of the fungi. When these reads are filtered out, traditional metabarcoding pipelines lose part of the information and consequently produce biased pictures of the composition and structure of the environment under study. We developed a solution that enables processing of the entire set of reads including both overlapping and non-overlapping, thus providing a more accurate picture of fungal communities. Our comparative tests using simulated and real data demonstrated the effectiveness of our solution, which can be used by both experts and non-specialists on a command line or through the Galaxy-based web interface.

The intestinal microbial composition in Greylag geese differs with steatosis induction mode: spontaneous or induced by overfeeding.

BACKGROUND: Relationships between microbial composition and steatosis are being extensively studied in mammals, and causal relations have been evidenced. In migratory birds the liver can transiently store lipids during pre-migratory and migratory phases, but little is known about the implications of the digestive microbiota in those mechanisms. The Landaise greylag goose (Anser anser) is a good model to study steatosis in migratory birds as it is domesticated, but is still, from a genetic point of view, close to its wild migratory ancestor. It also has a great ingestion capacity and a good predisposition for hepatic steatosis, whether spontaneous or induced by conventional overfeeding. The conventional (overfeeding) and alternative (spontaneous steatosis induction) systems differ considerably in duration and feed intake level and previous studies have shown that aptitudes to spontaneous steatosis are very variable. The present study thus aimed to address two issues: (i) evaluate whether microbial composition differs with steatosis-inducing mode; (ii) elucidate whether a digestive microbial signature could be associated with variable aptitudes to spontaneous liver steatosis. RESULTS: Performances, biochemical composition of the livers and microbiota differed considerably in response to steatosis stimulation. We namely identified the genus Romboutsia to be overrepresented in birds developing a spontaneous steatosis in comparison to those submitted to conventional overfeeding while the genera Ralstonia, Variovorax and Sphingomonas were underrepresented only in birds that did not develop a spontaneous steatosis compared to conventionally overfed ones, birds developing a spontaneous steatosis having intermediate values. Secondly, no overall differences in microbial composition were evidenced in association with variable aptitudes to spontaneous steatosis, although one OTU, belonging to the Lactobacillus genus, was overrepresented in birds having developed a spontaneous steatosis compared to those that had not. CONCLUSIONS: Our study is the first to evaluate the intestinal microbial composition in association with steatosis, whether spontaneous or induced by overfeeding, in geese. Steatosis induction modes were associated with distinct digestive microbial compositions. However, unlike what can be observed in mammals, no clear microbial signature associated with spontaneous steatosis level was identified.

Gut Microbiota-Derived Metabolite Signature in Suckling and Weaned Piglets.

The gut microbiota plays a key role in intestinal development at the suckling-to-weaning transition. The objective of this study was to analyze the production of metabolites by the gut microbiota in suckling and weaned piglets. We studied piglets raised in two separate maternity farms and weaned at postnatal day 21 in the same farm. The fecal metabolome (1H nuclear magnetic resonance) and the microbiota composition (16S rRNA gene amplicon sequencing) and its predicted functions (PICRUSt2) were analyzed in the same piglets during the suckling period (postnatal day 13) and 2 days after weaning (postnatal day 23). The relative concentrations of the bacterial metabolites methylamine, dimethylamine, cadaverine, tyramine, putrescine, 5-aminovalerate, succinate, and 3-(4-hydroxyphenylpropionate) were higher during the suckling period than after weaning. In contrast, the relative concentrations of the short-chain fatty acids acetate and propionate were higher after weaning than during the suckling period. The maternity of origin of piglets also influenced the level of some bacterial metabolites (propionate and isobutyrate). The fecal metabolome signatures observed in suckling and weaned piglets were associated with specific microbiota-predicted functionalities, structure, and diversity. Gut microbiota-derived metabolites, which are differentially abundant between suckling and weaned piglets (e.g., short-chain fatty acids and biogenic amines), are known to regulate gut health. Thus, identification of metabolome signatures in suckling and weaned piglets paves the way for the development of health-promoting nutritional strategies, targeting the production of bacterial metabolites in early life.

Culture of rabbit caecum organoids by reconstituting the intestinal stem cell niche in vitro with pharmacological inhibitors or L-WRN conditioned medium.

Intestinal organoids are self-organized 3-dimensional (3D) structures formed by a single layer of polarized epithelial cells. This innovative in vitro model is highly relevant to study physiology of the intestinal epithelium and its role in nutrition and barrier function. However, this model has never been developed in rabbits, while it would have potential applications for biomedical and veterinary research. Here, we cultured rabbit caecum organoids with either pharmacological inhibitors (2Ki medium) or L-WRN cells conditioned medium (L-WRN CM) to reconstitute the intestinal stem cell niche in vitro. Large spherical organoids were obtained with the 2Ki medium and this morphology was associated with a high level of proliferation and stem cells markers gene expression. In contrast, organoids cultured with L-WRN CM had a smaller diameter; a greater cell height and part of them were not spherical. When the L-WRN CM was used at low concentration (5%) for two days, the gene expression of stem cells and proliferation markers were very low, while absorptive and secretory cells markers and antimicrobial peptides were elevated. Epithelial cells within organoids were polarized in 3D cultures with 2Ki medium or L-WRN CM (apical side towards the lumen). We cultured dissociated organoid cells in 2D monolayers, which allowed accessibility to the apical compartment. Under these conditions, actin stress fibers were observed with the 2Ki medium, while perijonctionnal localization of actin was observed with the L-WRN CM suggesting, in 2D cultures as well, a higher differentiation level in the presence of L-WRN CM. In conclusion, rabbit caecum organoids cultured with the 2Ki medium were more proliferative and less differentiated than organoids cultured with L-WRN CM. We propose that organoids cultured with the 2Ki medium could be used to rapidly generate in vitro a large number of rabbit intestinal epithelial stem cells while organoids cultured with the L-WRN CM used at low concentration represent a suitable model to study differentiated rabbit epithelium.

Dietary composition and yeast/microalgae combination supplementation modulate the microbial ecosystem in the caecum, colon and faeces of horses.

Starchy diets can induce hindgut dysbiosis in horses. The present study evaluated the impact of a yeast (Saccharomyces cerevisiae) and microalgae (Aurantiochytrium limacinum) supplementation on caecal, colonic and faecal microbial ecosystem and on blood inflammatory parameters of horses fed high-fibre or high-starch diets. Six fistulated geldings in a 2 × 2 Latin-square design were alternatively supplemented and received during each period 100 % hay (4 weeks) followed by a 56/44 hay/barley diet (3 weeks). Caecal, colonic and faecal samples were collected 4 h after the morning meal three times per diet, at 5-d intervals, to measure bacterial composition and microbial end products. Blood was simultaneously collected for measuring inflammatory markers. The starchy diet clearly modified the microbial ecosystem in the three digestive segments, with an increase of the amylolytic function and a decrease of the fibrolytic one. However, no effect of the diet was observed on the blood parameters. When horses were supplemented, no significant change was found in lipopolysaccharides, PG-E2, serum amyloid A concentrations and complete blood count neither in cellulose-utilising, starch-utilising and lactate-utilising bacteria concentrations nor in the volatile fatty acids and lactate concentrations and pH. Under supplementation, relative abundance of Family XIII Clostridiales increased in caecum and faeces irrespective of diet and relative abundance of Veillonellaceae was higher during the hay/barley diet in colon and faeces. Most variations of faecal bacterial taxa under supplementation were not observed in the hindgut. However, all variations suggested that supplementation could increase fibrolytic function whatever the diet and limit dysbiosis when the horses' diet changed from high fibre to high starch.

Diversity and Co-occurrence Pattern Analysis of Cecal Microbiota Establishment at the Onset of Solid Feeding in Young Rabbits.

This study aimed to evaluate how the feeding strategy of rabbit kits at the onset of solid feed intake could affect ecological diversity and co-occurrence patterns of the cecal bacterial community. From birth to 18 days of age kits were exclusively milk-fed, and between 18 and 35 days the young rabbits also had access to solid feed. After weaning at (35 days), young rabbits were exclusively fed solid feed. Three experimental feeds were used: a high concentrate diet [H: 10.16 MJ digestible energy (DE)/kg and 15.3% crude protein (CP)], a low concentrate diet (L: 9.33 MJ DE/kg and 14.7% CP) and a reproductive female diet (R: 10.57 MJ DE/kg and 17.3% CP). The rabbit kits (n = 357) were divided into three groups, differing by the diet received during two periods: from 18 to 28 and from 28 to 49 days of age. In the groups LL and HH, rabbit kits were fed L or H diets, respectively, during both periods. Kits in the group RL received feeds R and L from 18 to 28 and 28 to 49 days of age, respectively. Cecal bacterial communities of 10 rabbits per group were carried out at 18, 28, 35, 43 and 49 days of age by MiSeq Illumina sequencing 16S rRNA encoding genes. Between 18 and 28 days of age, solid feed intake was higher in the group RL compared to the other two groups (+24%; P < 0.01). Overall, 13.4% of the OTUs detected were present in the cecal ecosystem from 18 to 49 days old, whereas 17.4% were acquired with the onset of solid feeding and kept from 28 days on. Exclusive milk consumption constrains the bacterial community toward a similar structure but high phylogenetic beta-diversity. Introduction of solid feed induced a sharp change of microbial community structure and decreased phylogenetic diversity. A strong relationship in bacterial community network occurred only from 43 days on. Our feeding strategy at the onset of solid feed ingestion exhibited only a moderate effect on the microbial community structure (P = 0.072), although the LL group seemed to reach faster maturity compared to the two other groups.

The MACADAM database: a MetAboliC pAthways DAtabase for Microbial taxonomic groups for mining potential metabolic capacities of archaeal and bacterial taxonomic groups.

Progress in genome sequencing and bioinformatics opens up new possibilities, including that of correlating genome annotations with functional information such as metabolic pathways. Thanks to the development of functional annotation databases, scientists are able to link genome annotations with functional annotations. We present MetAboliC pAthways DAtabase for Microbial taxonomic groups (MACADAM) here, a user-friendly database that makes it possible to find presence/absence/completeness statistics for metabolic pathways at a given microbial taxonomic position. For each prokaryotic 'RefSeq complete genome', MACADAM builds a pathway genome database (PGDB) using Pathway Tools software based on MetaCyc data that includes metabolic pathways as well as associated metabolites, reactions and enzymes. To ensure the highest quality of the genome functional annotation data, MACADAM also contains MicroCyc, a manually curated collection of PGDBs; Functional Annotation of Prokaryotic Taxa (FAPROTAX), a manually curated functional annotation database; and the IJSEM phenotypic database. The MACADAM database contains 13 509 PGDBs (13 195 bacterial and 314 archaeal), 1260 unique metabolic pathways, completed with 82 functional annotations from FAPROTAX and 16 from the IJSEM phenotypic database. MACADAM contains a total of 7921 metabolites, 592 enzymatic reactions, 2134 EC numbers and 7440 enzymes. MACADAM can be queried at any rank of the NCBI taxonomy (from phyla to species). It provides the possibility to explore functional information completed with metabolites, enzymes, enzymatic reactions and EC numbers. MACADAM returns a tabulated file containing a list of pathways with two scores (pathway score and pathway frequency score) that are present in the queried taxa. The file also contains the names of the organisms in which the pathways are found and the metabolic hierarchy associated with the pathways. Finally, MACADAM can be downloaded as a single file and queried with SQLite or python command lines or explored through a web interface.

Fumonisin-Exposure Impairs Age-Related Ecological Succession of Bacterial Species in Weaned Pig Gut Microbiota.

Pigs are highly affected by dietary mycotoxin contamination and particularly by fumonisin. The effects of fumonisin on pig intestinal health are well documented, but little is known regarding its impact on gut microbiota. We investigate the effects of the fumonisin (FB1, 12 mg/kg feed) on the fecal microbiota of piglets (n = 6) after 0, 8, 15, 22, and 29 days of exposure. A control group of six piglets received a diet free of FB1. Bacterial community diversity, structure and taxonomic composition were carried out by V3⁻V4 16S rRNA gene sequencing. Exposure to FB1 decreases the diversity index, and shifts and constrains the structure and the composition of the bacterial community. This takes place as early as after 15 days of exposure and is at a maximum after 22 days of exposure. Compared to control, FB1 alters the ecological succession of fecal microbiota species toward higher levels of Lactobacillus and lower levels of the Lachnospiraceae and Veillonellaceae families, and particularly OTUs (Operational Taxonomic Units) of the genera Mitsuokella, Faecalibacterium and Roseburia. In conclusion, FB1 shifts and constrains age-related evolution of microbiota. The direct or indirect contribution of FB1 microbiota alteration in the global host response to FB1 toxicity remains to be investigated.

Probiotics Strains Modulate Gut Microbiota and Lipid Metabolism in Mule Ducks.

BACKGROUND: Livestock production should respond to societal, environmental and economic changes. Since 2006 and the ban on antibiotics as growth factors in European Union, the use of probiotics has become widespread and has demonstrated the effect of intestinal microbiota on the performance of farm animals. OBJECTIVE: The aim of this study was to investigate the effect of supplementation with Lactobacillus salivarius (as a probiotics strain or combined with other strains) on zootechnical performance, metabolic and immune gene expression and intestinal microbiota diversity in mule ducks using high-throughput sequencing and real-time PCR. METHOD: The mule ducks were reared for 79 days and overfed for 12 days with or without probiotics. Samples were collected at 14 (starting period) and 91 days (end of overfeeding period), 3 hours post feeding. RESULTS: Irrespective of digestive content, age, level of feed intake or supplementation with probiotics, Firmicutes, Proteobacteria and Bacteroidetes were the dominant phyla in the bacterial community in mule ducks. At 14 days, both the ileal and cecal samples were dominated by Firmicutes (in particular the Clostridiales order). Overfeeding induced a shift between Clostridiales and Lactobacillales in the ileal samples whereas in the cecal samples, the relative abundance of Firmicutes decreased. Overfeeding also induced hepatic over-expression of Fatty Acid Synthase (FAS) and of the lipid transporter gene Fatty Acid Binding Protein 4 (FABP4). This increase in lipid metabolism genes is associated with a decrease in inflammatory response. CONCLUSION: Finally, probiotic supplementation had only a slight impact on gene expression and microbiota diversity, both at 14 days and after overfeeding.

FROGS: Find, Rapidly, OTUs with Galaxy Solution.

Motivation: Metagenomics leads to major advances in microbial ecology and biologists need user friendly tools to analyze their data on their own. Results: This Galaxy-supported pipeline, called FROGS, is designed to analyze large sets of amplicon sequences and produce abundance tables of Operational Taxonomic Units (OTUs) and their taxonomic affiliation. The clustering uses Swarm. The chimera removal uses VSEARCH, combined with original cross-sample validation. The taxonomic affiliation returns an innovative multi-affiliation output to highlight databases conflicts and uncertainties. Statistical results and numerous graphical illustrations are produced along the way to monitor the pipeline. FROGS was tested for the detection and quantification of OTUs on real and in silico datasets and proved to be rapid, robust and highly sensitive. It compares favorably with the widespread mothur, UPARSE and QIIME. Availability and implementation: Source code and instructions for installation: https://github.com/geraldinepascal/FROGS.git. A companion website: http://frogs.toulouse.inra.fr. Contact: geraldine.pascal@inra.fr. Supplementary information: Supplementary data are available at Bioinformatics online.

Phylogenesis and Biological Characterization of a New Glucose Transporter in the Chicken (Gallus gallus), GLUT12.

In mammals, insulin-sensitive GLUTs, including GLUT4, are recruited to the plasma membrane of adipose and muscle tissues in response to insulin. The GLUT4 gene is absent from the chicken genome, and no functional insulin-sensitive GLUTs have been characterized in chicken tissues to date. A nucleotide sequence is predicted to encode a chicken GLUT12 ortholog and, interestingly, GLUT12 has been described to act as an insulin-sensitive GLUT in mammals. It encodes a 596 amino acid protein exhibiting 71% identity with human GLUT12. First, we present the results of a phylogenetic study showing the stability of this gene during evolution of vertebrates. Second, tissue distribution of chicken SLC2A12 mRNA was characterized by RT-PCR. It was predominantly expressed in skeletal muscle and heart. Protein distribution was analysed by Western blotting using an anti-human GLUT12 antibody directed against a highly conserved region (87% of identity). An immuno-reactive band of the expected size (75kDa) was detected in the same tissues. Third a physiological characterization was performed: SLC2A12 mRNA levels were significantly lowered in fed chickens subjected to insulin immuno-neutralization. Finally, recruitment of immuno-reactive GLUT12 to the muscle plasma membrane was increased following 1h of intraperitoneal insulin administration (compared to a control fasted state). Thus insulin administration elicited membrane GLUT12 recruitment. In conclusion, these results suggest that the facilitative glucose transporter protein GLUT12 could act in chicken muscle as an insulin-sensitive transporter that is qualitatively similar to GLUT4 in mammals.

Evolutionary origin of bone morphogenetic protein 15 and growth and differentiation factor 9 and differential selective pressure between mono- and polyovulating species.

Bone morphogenetic protein 15 (BMP15) and growth and differentiation factor 9 (GDF9) are TGFbeta-like oocyte-derived growth factors involved in ovarian folliculogenesis as critical regulators of many granulosa cell processes and ovulation rate. Ovarian phenotypic effect caused by alterations in BMP15 and GDF9 genes appears to differ between species and may be relevant to their mono- or polyovulating status. Through phylogenetic analysis we recently showed that these two paralogous genes are strongly divergent and in rapid evolution as compared to other members of the TGFbeta superfamily. Here, we evaluate the amino acid substitution rates of a set of proteins implicated in the ovarian function, including BMP15 and GDF9, with special attention to the mono- or polyovulating status of the species. Among a panel of mono- and polyovulating mammals, we demonstrate a better conservation of some areas in BMP15 and GDF9 within mono-ovulating species. Homology modeling of BMP15 and GDF9 homodimer and heterodimer 3-D structures was suggestive that these areas may be involved in dimer formation and stability. A phylogenetic study of BMP15/GDF9-related proteins reveals that these two genes diverged from the same ancestral gene along with BMP3 and GDF10, two other paralogous genes. A substitution rate analysis based on this phylogenetic tree leads to the hypothesis of an acquisition of BMP15/GDF9-specific functions in ovarian folliculogenesis in mammals. We propose that high variations observed in specific areas of BMP15 and GDF9 in polyovulating species change the equilibrium between homodimers and heterodimers, modifying the biological activity and thus allowing polyovulation to occur.

Positive selection in bone morphogenetic protein 15 targets a natural mutation associated with primary ovarian insufficiency in human.

Bone Morphogenetic Protein 15 (BMP15) is a TGFß-like oocyte-derived growth factor involved in ovarian folliculogenesis as a critical regulator of many granulosa cell processes. Alterations of the BMP15 gene have been found associated with different ovarian phenotypic effects depending on the species, from sterility to increased prolificacy in sheep, slight subfertility in mouse or associated with primary ovarian insufficiency (POI) in women. To investigate the evolving role of BMP15, a phylogenetic analysis of this particular TGFß family member was performed. A maximum likelihood phylogenetic tree of several TGFß/BMP family members expressed by the ovary showed that BMP15 has a very strong divergence and a rapid evolution compared to others. Moreover, among 24 mammalian species, we detected signals of positive selection in the hominidae clade corresponding to F146, L189 and Y235 residues in human BMP15. The biological importance of these residues was tested functionally after site directed-mutagenesis in a COV434 cells luciferase assay. By replacing the positively selected amino acid either by alanine or the most represented residue in other studied species, only L189A, Y235A and Y235C mutants showed a significant increase of BMP15 signaling when compared to wild type. Additionally, the Y235C mutant was more potent than wild type in inhibiting progesterone secretion of ovine granulosa cells in primary culture. Interestingly, the Y235C mutation was previously identified in association with POI in women. In conclusion, this study evidences that the BMP15 gene has evolved faster than other members of the TGFß family and was submitted to a positive selection pressure in the hominidae clade. Some residues under positive selection are of great importance for the normal function of the protein and thus for female fertility. Y235 represents a critical residue in the determination of BMP15 biological activity, thus indirectly confirming its role in the onset of POI in women.

Deleted in malignant brain tumor 1 is secreted in the oviduct and involved in the mechanism of fertilization in equine and porcine species.

Oviductal environment affects preparation of gametes for fertilization, fertilization itself, and subsequent embryonic development. The aim of this study was to evaluate the effect of oviductal fluid and the possible involvement of deleted in malignant brain tumor 1 (DMBT1) on IVF in porcine and equine species that represent divergent IVF models. We first performed IVF after pre-incubation of oocytes with or without oviductal fluid supplemented or not with antibodies directed against DMBT1. We showed that oviductal fluid induces an increase in the monospermic fertilization rate and that this effect is canceled by the addition of antibodies, in both porcine and equine species. Moreover, pre-incubation of oocytes with recombinant DMBT1 induces an increase in the monospermic fertilization rate in the pig, confirming an involvement of DMBT1 in the fertilization process. The presence of DMBT1 in the oviduct at different stages of the estrus cycle was shown by western blot and confirmed by immunohistochemical analysis of ampulla and isthmus regions. The presence of DMBT1 in cumulus-oocyte complexes was shown by western blot analysis, and the localization of DMBT1 in the zona pellucida and cytoplasm of equine and porcine oocytes was observed using immunofluorescence analysis and confocal microscopy. Moreover, we showed an interaction between DMBT1 and porcine spermatozoa using surface plasmon resonance studies. Finally, a bioinformatic and phylogenetic analysis allowed us to identify the DMBT1 protein as well as a DMBT1-like protein in several mammals. Our results strongly suggest an important role of DMBT1 in the process of fertilization.