Disruption of the primocolonizing microbiota alters epithelial homeostasis and imprints stem cells in the colon of neonatal piglets.

The gut microbiota plays a key role in the postnatal development of the intestinal epithelium. However, the bacterial members of the primocolonizing microbiota driving these effects are not fully identified and the mechanisms underlying their long-term influence on epithelial homeostasis remain poorly described. Here, we used a model of newborn piglets treated during the first week of life with the antibiotic colistin in order to deplete specific gram-negative bacteria that are transiently dominant in the neonatal gut microbiota. Colistin depleted Proteobacteria and Fusobacteriota from the neonatal colon microbiota, reduced the bacterial predicted capacity to synthetize lipopolysaccharide (LPS), and increased the concentration of succinate in the colon. The colistin-induced disruption of the primocolonizing microbiota was associated with altered gene expression in the colon epithelium including a reduction of toll-like receptor 4 (TLR4) and lysozyme (LYZ). Our data obtained in porcine colonic organoid cell monolayers suggested that these effects were not driven by the variation of succinate or LPS levels nor by a direct effect of colistin on epithelial cells. The disruption of the primocolonizing microbiota imprinted colon epithelial stem cells since the expression of TLR4 and LYZ remained lower in organoids derived from colistin-treated piglet colonic crypts after several passages when compared to control piglets. Finally, the stable imprinting of LYZ in colon organoids was independent of the H3K4me3 level in its transcription start site. Altogether, our results show that disruption of the primocolonizing gut microbiota alters epithelial innate immunity in the colon and imprints stem cells, which could have long-term consequences for gut health.

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.

Saccharomyces cerevisiae boulardii CNCM I-1079 supplementation in finishing male pigs helps to cope with heat stress through feeding behaviour and gut microbiota modulation.

Pigs subjected to heat stress (HS) decrease their feed intake and growth. The objectives of the experiment were to determine the effects of live yeast (LY) supplementation (Saccharomyces cerevisiae var boulardii CNCM I-1079) on feeding behaviour, energy metabolism and faecal microbiota composition of finishing boars (n 10) housed in a respiration chamber at thermoneutrality (7 d at 22°C) or during HS (seven plus six days at 28°C). Dietary LY supplementation increased DM intake (P = 0·01) whatever the ambient temperature, whereas HS decreased feed intake whatever the dietary supplementation (P = 0·01). Dietary LY supplementation increased the number of meals (P = 0·02). Energy retention was higher with dietary LY supplementation (P < 0·01) but decreased during HS (P < 0·01). The skin temperature of the supplemented pigs was lower at thermoneutrality and increased during HS to a lesser extent than that of non-supplemented pigs (P < 0·01). Faecal microbiota composition was determined using 16S rRNA gene sequencing. Treponema, Christensenellaceae R-7, Ruminococcaceae UCG-002, Rikenellaceae RC9, Clostridium sensu stricto 1 and Romboutsia genera and some bacteria belonging to Alloprevotella, Oxalobacter and Anaeroplasma genera were more abundant under HS. LY supplementation attenuated HS effects on Romboutsia abundance, while decreasing the abundance of some bacteria from Ruminoccocus, Coprococcus, Peptococcus and Oxalobacter genera and increasing the abundance of beneficial bacteria from Lactococcus and Subdoligranulum genera. Our results suggest that higher level of the keystone species Ruminococcus bromii at thermoneutrality may be one of the causes for higher energy retention observed under subsequent HS.

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.

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.

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.

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.

Quantitative feed restriction rather than caloric restriction modulates the immune response of growing rabbits.

BACKGROUND: Short-term feed restriction strategies are used in rabbits to reduce postweaning digestive disorders, but little is known about the involvement of the immune system in these beneficial effects. OBJECTIVE: In the present study, the consequences of feed and energy restriction on immune response were investigated. METHODS: At weaning, 320 male and female rabbits were assigned to 4 groups differing in dietary digestible energy (DE) concentrations and intake levels: a low-energy ad libitum-feed (LE100) group, a low-energy restricted-feed (LE75) group, a high-energy ad libitum-feed (HE100) group, and a high-energy restricted-feed (HE75) group. The high-energy groups consumed 10.13 MJ DE/kg of feed, whereas the low-energy groups consumed 9.08 MJ DE/kg (formulated values). Intake amounts for the restricted groups were 75% those of the ad libitum groups. Rabbits consumed these diets until age 63 d, after which they consumed feed ad libitum for 9 d. Ten rabbits per group and per age were killed at ages 42, 50, 63, and 72 d. Spleens and appendixes were weighed; Peyer's patch surface area was determined by image analysis; plasma total immunoglobulin (Ig) G and anti-ovalbumin IgG; and fecal and plasma IgA concentrations were determined by ELISA; and ileal expressions of cytokines were measured by quantitative reverse transcriptase-polymerase chain reaction at ages 50 and 63 d. RESULTS: The relative weight and size of the lymphoid organs were not affected by treatments. Concentrations of plasma total IgA (-41% at 63 d and -29% at 72 d), IgG (-22% at 72 d), and anti-ovalbumin IgG (-41% at 63 d) were lower with feed restriction. Fecal IgA concentrations were lower with quantitative restriction (-40%, -52%, and -65% at age 42, 50, and 63 d, respectively) and energy restriction (-56%, -46%, and -73% at ages 50, 63, and 72 d, respectively). Feed-restricted rabbits tended to have greater expressions of interleukin (IL) 1ß and IL-2 and lower expressions of tumor necrosis factor α (P < 0.1). CONCLUSION: These results demonstrated that, in rabbits, restriction and, to a lesser extent, dietary energy concentration modulate gut immunity.

MilkOligoThesaurus, a dataset of mammalian milk oligosaccharide synonyms.

There is a growing interest in milk oligosaccharides (MOs) because of their numerous benefits for newborns' and long-term health. A large number of MO structures have been identified in mammalian milk. Mostly described in human milk, the oligosaccharide richness, although less broad, has also been reported for a wide range of mammalian species. The structure of MOs is particularly difficult to report as it results from the combination of 5 monosaccharides linked by various glycosidic bonds forming structurally diverse and complex matrices of linear and branched oligosaccharides. Exploring the literature and extracting relevant information on MO diversity within or across species appears promising to elucidate structure-function role of MOs. Currently, given the complexity of these molecules, the main issues in exploring literature to extract relevant information on MO diversity within or across species relate to the heterogeneity in the way authors refer to these molecules. Herein, we provide a thesaurus (MilkOligoThesaurus) including the names and synonyms of MOs collected from key selected articles on mammalian milk analyses. MilkOligoThesaurus gathers the names of the MOs with a complete description of their monosaccharide composition and structures. When available, each unique MO molecule is linked to its ID from the NCBI PubChem and ChEBI databases. MilkOligoThesaurus is provided in a tabular format. It gathers 245 unique oligosaccharide structures described by 22 features (columns) including the name of the molecule, its abbreviation, the chemical database IDs if available, the monosaccharide composition, chemical information (molecular formula, monoisotopic mass), synonyms, its formula in condensed form, and in abbreviated condensed form, the abbreviated systematic name, the systematic name, the isomer group, and scientific article sources. MilkOligoThesaurus is also provided in the SKOS (Simple Knowledge Organization System) format. This thesaurus is a valuable resource gathering MO naming variations that are not found elsewhere for (i) Text and Data Mining to enable automatic annotation and rapid extraction of milk oligosaccharide data from scientific papers; (ii) biology researchers aiming to search for or decipher the structure of milk oligosaccharides based on any of their names, abbreviations or monosaccharide compositions and linkages.

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.

Culture of Piglet Intestinal 3D Organoids from Cryopreserved Epithelial Crypts and Establishment of Cell Monolayers.

Intestinal organoids are increasingly being used to study the gut epithelium for digestive disease modeling, or to investigate interactions with drugs, nutrients, metabolites, pathogens, and the microbiota. Methods to culture intestinal organoids are now available for multiple species, including pigs, which is a species of major interest both as a farm animal and as a translational model for humans, for example, to study zoonotic diseases. Here, we give an in-depth description of a procedure used to culture pig intestinal 3D organoids from frozen epithelial crypts. The protocol describes how to cryopreserve epithelial crypts from the pig intestine and the subsequent procedures to culture 3D intestinal organoids. The main advantages of this method are (i) the temporal dissociation of the isolation of crypts from the culture of 3D organoids, (ii) the preparation of large stocks of cryopreserved crypts derived from multiple intestinal segments and from several animals at once, and thus (iii) the reduction in the need to sample fresh tissues from living animals. We also detail a protocol to establish cell monolayers derived from 3D organoids to allow access to the apical side of epithelial cells, which is the site of interactions with nutrients, microbes, or drugs. Overall, the protocols described here is a useful resource for studying the pig intestinal epithelium in veterinary and biomedical research.

The Early Life Microbiota Is Not a Major Factor Underlying the Susceptibility to Postweaning Diarrhea in Piglets.

Postweaning diarrhea (PWD) in piglets impair welfare, induce economic losses and lead to overuse of antibiotics. The early life gut microbiota was proposed to contribute to the susceptibility to PWD. The objective of our study was to evaluate in a large cohort of 116 piglets raised in 2 separate farms whether the gut microbiota composition and functions during the suckling period were associated with the later development of PWD. The fecal microbiota and metabolome were analyzed by 16S rRNA gene amplicon sequencing and nuclear magnetic based resonance at postnatal day 13 in male and female piglets. The later development of PWD was recorded for the same animals from weaning (day 21) to day 54. The gut microbiota structure and α-diversity during the suckling period were not associated with the later development of PWD. There was no significant difference in the relative abundances of bacterial taxa in suckling piglets that later developed PWD. The predicted functionality of the gut microbiota and the fecal metabolome signature during the suckling period were not linked to the later development of PWD. Trimethylamine was the bacterial metabolite which fecal concentration during the suckling period was the most strongly associated with the later development of PWD. However, experiments in piglet colon organoids showed that trimethylamine did not disrupt epithelial homeostasis and is thus not likely to predispose to PWD through this mechanism. In conclusion, our data suggest that the early life microbiota is not a major factor underlying the susceptibility to PWD in piglets. IMPORTANCE This study shows that the fecal microbiota composition and metabolic activity are similar in suckling piglets (13 days after birth) that either later develop post-weaning diarrhea (PWD) or not, which is a major threat for animal welfare that also causes important economic losses and antibiotic treatments in pig production. The aim of this work was to study a large cohort of piglets raised in separates environments, which is a major factor influencing the early life microbiota. One of the main findings is that, although the fecal concentration of trimethylamine in suckling piglets was associated with the later development of PWD, this gut microbiota-derived metabolite did not disrupt the epithelial homeostasis in organoids derived from the pig colon. Overall, this study suggests that the gut microbiota during the suckling period is not a major factor underlying the susceptibility of piglets to PWD.

Effect of live yeast supplementation in sow diet during gestation and lactation on sow and piglet fecal microbiota, health, and performance.

Feeding probiotics like live yeast Saccharomyces cerevisiae var. boulardii (SB) in pig diets has been suggested to preserve health and reduce antibiotic use during critical periods like weaning. This study was conducted to determine whether SB added to the diet of sows during the last 2 mo of gestation and the 4 wk of lactation may contribute to support the health and performance of piglets before and after weaning through changes in sow physiology, milk composition, and fecal microbiota. Crossbred sows (n = 45) from parity 1 to 9 were allocated to two dietary treatments: Control (n = 23) and SB (n = 22). Sows in the SB group were fed the same standard gestation and then lactation diet as the Control sows but with the addition of SB at 1 × 109 colony-forming units/kg of feed. Piglets were weaned under challenging conditions consisting of mixing of litters, no pen cleaning, and a 2-h period of nonoptimal temperature exposure. Blood and feces were collected from sows on days 28 and 113 of gestation and days 6 (feces only) and 28 of lactation, and from piglets on days 6 (feces) and 28 of lactation and day 5 after weaning. Colostrum was collected during parturition and milk on day 6 of lactation. Supplementation of sow diets with SB influenced the fecal microbiota of the sows and their piglets. Five days after weaning, the alpha-diversity was lower (P < 0.05) in piglets from SB sows than in piglets from Control sows. Analysis of microbiota with partial least square discriminant analysis discriminated feces from SB sows from that of Control sows at 110 d of gestation (29.4% error rate). Piglet feces could also be discriminated according to the diet of their mother, with a better discrimination early after birth (day 6 of lactation) than after weaning (day 5 postweaning, 3.4% vs. 12.7% error rate). Five days after weaning, piglets had greater white blood cell count, plasma haptoglobin concentration, and oxidative stress than before weaning (P < 0.001). Nevertheless, SB supplementation in sow diets had no effect (P > 0.05) on most of health criteria measured in blood and growth performance of piglets during lactation and the postweaning period. Moreover, dietary supplementation of SB to sows did not elicit any changes (P > 0.05) in their reproductive performance, metabolic and health status, nor in the concentration of immunoglobulins and nutrients in colostrum and milk. In the present experimental conditions, feeding SB to sows influenced sow and piglet microbiota with no consequences on their health and performance.

Feeding live yeast Saccharomyces cerevisiae var. boulardii (SB) in pig diets is recommended to promote a better health and reduce antibiotic use during critical periods like weaning. Our study was conducted to determine if SB added to the diet of sows during the last 2 mo of gestation and the 4 wk of lactation may contribute to support the health and performance of their piglets before and after weaning. We hypothesized that live SB supplementation to the sows may help improve the health and metabolic status of the sows and consequently the quality of milk and microbiota provided to the piglets. Supplementation of sow diet with SB during gestation and lactation induced modifications in the fecal microbiota of sows and their piglets. For piglets, the effects of SB fed to their mother were still observed 5 d after weaning. These modifications were, however, associated with changes neither in piglet ability to cope with the stress of weaning nor in milk nutritional and immune composition.

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.

The phenotype of the gut region is more stably retained than developmental stage in piglet intestinal organoids.

Intestinal organoids are innovative in vitro tools to study the digestive epithelium. The objective of this study was to generate jejunum and colon organoids from suckling and weaned piglets in order to determine the extent to which organoids retain a location-specific and a developmental stage-specific phenotype. Organoids were studied at three time points by gene expression profiling for comparison with the transcriptomic patterns observed in crypts in vivo. In addition, the gut microbiota and the metabolome were analyzed to characterize the luminal environment of epithelial cells at the origin of organoids. The location-specific expression of 60 genes differentially expressed between jejunum and colon crypts from suckling piglets was partially retained (48%) in the derived organoids at all time point. The regional expression of these genes was independent of luminal signals since the major differences in microbiota and metabolome observed in vivo between the jejunum and the colon were not reproduced in vitro. In contrast, the regional expression of other genes was erased in organoids. Moreover, the developmental stage-specific expression of 30 genes differentially expressed between the jejunum crypts of suckling and weaned piglets was not stably retained in the derived organoids. Differentiation of organoids was necessary to observe the regional expression of certain genes while it was not sufficient to reproduce developmental stage-specific expression patterns. In conclusion, piglet intestinal organoids retained a location-specific phenotype while the characteristics of developmental stage were erased in vitro. Reproducing more closely the luminal environment might help to increase the physiological relevance of intestinal organoids.

Impact of feed restriction and fragmented feed distribution on performance, intake behaviour and digestion of the growing rabbit.

Postweaning feed restriction preserves rabbit digestive health after weaning, but the underlying physiological mechanisms are not yet understood. To elucidate whether the feeding intake pattern modification related to feed restriction might be involved, we studied the effects of both feed intake quantity and intake frequency. Animals were allotted at weaning (28 d old) in a 2 × 2 factorial design: feed intake quantity (AL = ad libitum vs R = 75% of AL) and fragmented feed distribution (FFD) (1 vs 13 distributions), thus forming four groups (AL1, AL13, R1 and R13). New Zealand White growing rabbits were used from weaning to slaughter (70 d old), to analyse mortality, morbidity, performance, intake behaviour, digestion and microbial activity. Seven days after starting feed restriction (35 d old, group R1), rabbits consumed 44% of the feed within 2 h, 65% in 4 h and in 7 h over 95%. Over the 28-70 d period, mortality was low (5.3%) while morbidity averaged 18.5% and neither was affected by treatment. However, FFD tended to decrease the morbidity rate during the first 14 days after weaning (P = 0.06). Feed conversion (28-70 d) was improved by restriction (+15%, P < 0.001) and by FFD (+5%, P < 0.001). Nutrient digestibility was improved by restriction (+10% for energy, P < 0.01), but not by FFD. Fragmented feed distribution led to a lower stomachal pH, in the antrum (1.48 vs 2.13, P < 0.001) and in the fundus (1.52 vs 2.63, P < 0.001), while a higher pH was found in the caecum (6.07 vs 5.86, P < 0.001). Butyrate proportion in the caecum was reduced by four units for restricted groups. Fragmented feed distribution reduced the caecal VFA concentration by 23% within restricted rabbit groups only. A similar interaction between intake level and FFD was observed for fibrolytic activity (cellulase and xylanase). The diversity of caecal bacterial community was not modified by either of the two factors studied. Globally, fragmented meals have no major impacts on the caecal microbial activity, diversity, and thus would not be implicated in the better resistance of restricted rabbit to digestive troubles.

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.

Comparison of the archaeal community in the fermentative compartment and faeces of the cow and the rabbit.

The archaeal community in the fermentative compartment and faeces of the cow and the rabbit were compared by analysis capillary electrophoresis single-stranded conformation polymorphism (CE-SSCP) profiles of 16S rRNA genes. Ruminal and faecal contents were sampled in five cows for three weeks. Hard and soft faeces were collected in 14 rabbits for three consecutive weeks and caecal contents were sampled in the third week. The archaeal community differed according to the host species (ANOSIM-R=0.53 and 0.72 respectively for the comparison of the fermentative compartments and faeces; P<0.001) and to the location within the digestive tract of both species (ANOSIM-R=0.37, 0.52 respectively for the cow and the rabbit; P<0.001). In both species, the archaeal community of the digestive tract was stable over weeks and varied very little between individual animals. The structure (NS) and the richness index (9.9+/-2.7, 10.1+/-3.1 respectively, NS) of the archaeal community were similar for the caecal content and the soft faeces which permitted to use the latter as a representative indicator.

Molecular analysis of the bacterial community in digestive tract of rabbit.

This work aimed to study the stability over time of the bacterial community in caecum and faeces of the rabbit (diversity index and structure) without experimental disturbance and to evaluate its relationships with environmental parameters. Soft and hard faeces of 14 rabbits were sampled for 5 weeks while caecal content was sampled on the 3rd week (by surgery) and the 5th week (at slaughter). Bacterial communities were assessed by studying CE-SSCP profiles of 16S rRNA genes fragments. Redox potential, pH, NH3-N concentration and volatile fatty acid concentrations were measured in the caecum. Data showed that bacterial communities of soft and hard faeces barely differed from that of the caecum (ANOSIM-R<0.25; p<0.05). Without disturbance, the bacterial communities of faeces were stable over time (ANOSIM-R<0.25; p<0.001). However, the bacterial communities of caecum and faeces were affected by the surgery (ANOSIM-R=0.22-0.33; p<0.001). The caecal content was an acidic (pH=6.03+/-0.33) and an anaerobic environment (redox potential=-160+/-43 mV). Only the redox potential was correlated with the diversity index of the bacterial community of the caecum (R(2)=0.35; p<0.05) and no environmental parameters were correlated to its structure.

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.