Developmental Stage, Solid Food Introduction, and Suckling Cessation Differentially Influence the Comaturation of the Gut Microbiota and Intestinal Epithelium in Rabbits.

BACKGROUND: In mammals, the establishment around weaning of a symbiotic relationship between the gut microbiota and its host determines long-term health. OBJECTIVES: The aim of this study was to identify the factors driving the comaturation of the gut microbiota and intestinal epithelium at the suckling-to-weaning transition. We hypothesized that the developmental stage, solid food ingestion, and suckling cessation contribute to this process. METHODS: From birth to day 18, Hyplus rabbits were exclusively suckling. From day 18 to day 25, rabbits were 1) exclusively suckling; 2) suckling and ingesting solid food; or 3) exclusively ingesting solid food. The microbiota (16S amplicon sequencing), metabolome (nuclear magnetic resonance), and epithelial gene expression (high-throughput qPCR) were analyzed in the cecum at days 18 and 25. RESULTS: The microbiota structure and metabolic activity were modified with age when rabbits remained exclusively suckling. The epithelial gene expression of nutrient transporters, proliferation markers, and innate immune factors were also regulated with age (e.g., 1.5-fold decrease of TLR5). Solid food ingestion by suckling rabbits had a major effect on the gut microbiota by increasing its α diversity, remodeling its structure (e.g., 6.3-fold increase of Ruminococcaceae), and metabolic activity (e.g., 4.6-fold increase of butyrate). Solid food introduction also regulated the gene expression of nutrient transporters, differentiation markers, and innate immune factors in the epithelium (e.g., 3-fold increase of nitric oxide synthase). Suckling cessation had no effect on the microbiota, while it regulated the expression of genes involved in epithelial differentiation and immunoglobulin transport (e.g., 2.5-increase of the polymeric immunoglobulin receptor). CONCLUSIONS: In rabbits, the maturation of the microbiota at the suckling-to-weaning transition is driven by the introduction of solid food and, to a lesser extent, by the developmental stage. In contrast, the maturation of the intestinal epithelium at the suckling-to-weaning transition is under the influence of the developmental stage, solid food introduction, and suckling cessation.

A multi-omics dataset of the response to early plant polysaccharide ingestion in rabbits.

The transition from a milk-based diet to exclusive solid feeding deeply modifies microbiota-host crosstalk. Specifically, early ingestion of plant polysaccharides would be one of the main nutritional components to drive host-microbiota-interaction. To capture the effects of polysaccharides early-life nutrition (starch vs rapidly fermentable fiber) on the holobiont development, we investigated on the one hand the gut bacteriome and metabolome and on the other hand the transcriptome of two host gut tissues. Rabbit model was used to study post-natal co-development of the gut microbiota and its host around weaning transition. The assessment of the microbial composition of the gut appendix together with the caecum was provided for the first time. Gene expression signatures were analyzed along the gut (ileum and caecum) through high-throughput qPCR. The data collected were completed by the analysis of animal growth changes and time-series assessment of blood biomarkers. Those accessible and reusable data could help highlight the gut development dynamics as well as biological adaptation processes at the onset of solid feeding.

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.

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.

Standardized Natural Citrus Extract dietary supplementation influences sows' microbiota, welfare, and preweaning piglets' performances in commercial rearing conditions.

We investigated the effect of the Standardized Natural Citrus Extract (SNCE; Nor-Spice AB, Nor-Feed SAS, France) on the microbiota of the sows and on the weight gain of their piglets. Fifty sows were randomly divided into two groups: a control group (23 sows) with a standard diet and a SNCE group (27 sows) with a standard diet supplemented with 2,500 ppm of SNCE. Supplementation occurred 10 d before and 5 d after farrowing. Fecal samples from 16 sows (8 randomly selected sows of each dietary treatment) were collected for the fecal microbiota analysis 5 d after farrowing. The supplementation of SNCE increases the amount of cultivable Lactobacillus threefold in vitro. Microbial DNA was extracted from the fecal samples for sequencing of the 16S rRNA gene. The SNCE, which affected the microbiota as a discriminant analysis, was able to separate the microbial communities of the eight sows that received SNCE from the three control sows with 21 Operational Taxonomic Units (area under the ROC curve = 96%). SNCE also reduced the interval between farrowing and the first dejection of the sow and increased their feed intake (P-value < 0.05). Furthermore, feeding the sows with SNCE improved the weight gain of the piglets in the first week of life. These results show that SNCE supplementation allows to enhance zootechnical performances of peripartum' sows, possibly due to the modulation of the microbiota transmitted to the piglets.

Absolute quantitation of microbes using 16S rRNA gene metabarcoding: A rapid normalization of relative abundances by quantitative PCR targeting a 16S rRNA gene spike-in standard.

Metabarcoding of the 16S rRNA gene is commonly used to characterize microbial communities, by estimating the relative abundance of microbes. Here, we present a method to retrieve the concentrations of the 16S rRNA gene per gram of any environmental sample using a synthetic standard in minuscule amounts (100 ppm to 1% of the 16S rRNA sequences) that is added to the sample before DNA extraction and quantified by two quantitative polymerase chain reaction (qPCR) reactions. This allows normalizing by the initial microbial density, taking into account the DNA recovery yield. We quantified the internal standard and the total load of 16S rRNA genes by qPCR. The qPCR for the latter uses the exact same primers as those used for Illumina sequencing of the V3-V4 hypervariable regions of the 16S rRNA gene to increase accuracy. We are able to calculate the absolute concentration of the species per gram of sample, taking into account the DNA recovery yield. This is crucial for an accurate estimate as the yield varied between 40% and 84%. This method avoids sacrificing a high proportion of the sequencing effort to quantify the internal standard. If sacrificing a part of the sequencing effort to the internal standard is acceptable, we however recommend that the internal standard accounts for 30% of the environmental 16S rRNA genes to avoid the PCR bias associated with rare phylotypes. The method proposed here was tested on a feces sample but can be applied more broadly on any environmental sample. This method offers a real improvement of metabarcoding of microbial communities since it makes the method quantitative with limited efforts.

Gut microbiota derived metabolites contribute to intestinal barrier maturation at the suckling-to-weaning transition.

In suckling mammals, the onset of solid food ingestion is coincident with the maturation of the gut barrier. This ontogenic process is driven by the colonization of the intestine by the microbiota. However, the mechanisms underlying the microbial regulation of the intestinal development in early life are not fully understood. Here, we studied the co-maturation of the microbiota (composition and metabolic activity) and of the gut barrier at the suckling-to-weaning transition by using a combination of experiments in vivo (suckling rabbit model), ex vivo (Ussing chambers) and in vitro (epithelial cell lines and organoids). The microbiota composition, its metabolic activity, para-cellular epithelial permeability and the gene expression of key components of the gut barrier shifted sharply at the onset of solid food ingestion in vivo, despite milk was still predominant in the diet at that time. We found that cecal content sterile supernatant (i.e. containing a mixture of metabolites) obtained after the onset of solid food ingestion accelerated the formation of the epithelial barrier in Caco-2 cells in vitro and our results suggested that these effects were driven by the bacterial metabolite butyrate. Moreover, the treatment of organoids with cecal content sterile supernatant partially replicated in vitro the effects of solid food ingestion on the epithelial barrier in vivo. Altogether, our results show that the metabolites produced by the microbiota at the onset of solid food ingestion contribute to the maturation of the gut barrier at the suckling-to-weaning transition. Targeting the gut microbiota metabolic activity during this key developmental window might therefore be a promising strategy to promote intestinal homeostasis.

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.

Relationships between sensory and physicochemical measurements in meat of rabbit from three different breeding systems using canonical correlation analysis.

Meat from rabbits reared either according to a standard (STAND) or a high quality norm (LABEL) or a low growth breeding (RUSSE) system were submitted to a sensory evaluation and to a large set of physicochemical measurements (weight of retail cuts, colour parameters, ultimate pH, femur flexure test, Warner-Bratzler shear test, water holding capacities and cooking losses). STAND rabbit meat exhibited the most juicy meat in back and in leg (p<0.01). Leg tenderness significantly decreased (p<0.001) in the rank order STAND>LABEL>RUSSE. Canonical correlation analysis showed strong correlations between physicochemical and sensory variables (R(2)=0.73 and 0.68 between the two first pairs of canonical variates). Especially, sensory tenderness and WB shear test variables assessed on raw longissimus muscle (LL) were correlated. Fibrous attribute in back was correlated with cooking loss in LL. When analysed separately only RUSSE rabbits exhibited the same relations between variables as those calculated in whole dataset.