Post-natal co-development of the microbiota and gut barrier function follows different paths in the small and large intestine in piglets.

Gut microbiota and intestinal barrier co-develop after birth, establishing a homeostatic state whereby mucosal cells cohabit with commensal bacteria. We hypothesized that this post-natal co-development follows different timings depending on the intestinal site considered. Jejunal, ileal, and colonic luminal contents and mucosa were sampled in suckling piglets at post-natal day (PND) 0, 2, 7, 14, and 28. Jejunal, ileal, and colonic luminal microbiota (evaluated by 16S DNA sequencing followed by beta-diversity analysis) clustered at PND2 but colonic microbiota diverge afterwards (P < .05). Mucosal permeability, evaluated in Ussing chambers, increased with age in the jejunum and ileum (P < .05) but not the colon. Expression of pattern recognition receptor (PRR) exhibited different patterns (gradual or sharp increase, decrease, or no change with age, P < .05) depending on PRR and intestinal site considered. Principal component analysis of mucosa data revealed clear clustering of colonic samples, irrespective of the age and clustering of jejunal and ileal samples, with gradual changes with age. Correlation analysis highlighted three families correlating with mucosal parameters: Enterobacteriaceae in the jejunum, Peptostreptococcaceae in the ileum, and Micrococcaceae in the colon. In conclusion, small and large intestine display close microbiota composition early in life but distinct mucosal phenotype and follow very different post-natal development.

Oral sodium butyrate impacts brain metabolism and hippocampal neurogenesis, with limited effects on gut anatomy and function in pigs.

Butyrate can improve gut functions, whereas histone deacetylase inhibitors might alleviate neurocognitive alterations. Our aim was to assess whether oral butyrate could modulate brain metabolism and plasticity and if this would relate to gut function. Sixteen pigs were subjected to sodium butyrate (SB) supplementation via beverage water or water only [control (C)]. All pigs had blood sampled after 2 and 3 wk of treatment, and were subjected to a brain positron emission tomography after 3 wk. Animals were euthanized after 4 wk to sample pancreas, intestine, and brain for gut physiology and anatomy measurements, as well as hippocampal histology, Ki67, and doublecortin (DCX) immunohistochemistry. SB compared with C treatment triggered basal brain glucose metabolism changes in the nucleus accumbens and hippocampus ( P = 0.003), increased hippocampal granular cell layer volume ( P = 0.006), and neurogenesis (Ki67: P = 0.026; DCX: P = 0.029). After 2 wk of treatment, plasma levels of glucose, insulin, lactate, glucagon-like peptide 1, and peptide tyrosine tyrosine remained unchanged. After 3 wk, plasma levels of lactate were lower in SB compared with C animals ( P = 0.028), with no difference for glucose and insulin. Butyrate intake impacted very little gut anatomy and function. These results demonstrate that oral SB impacted brain functions with little effects on the gut.-Val-Laillet, D., Guérin, S., Coquery, N., Nogret, I., Formal, M., Romé, V., Le Normand, L., Meurice, P., Randuineau, G., Guilloteau, P., Malbert, C.-H., Parnet, P., Lallès, J.-P., Segain, J.-P. Oral sodium butyrate impacts brain metabolism and hippocampal neurogenesis, with limited effects on gut anatomy and function in pigs.

Comparing the intestinal transcriptome of Meishan and Large White piglets during late fetal development reveals genes involved in glucose and lipid metabolism and immunity as valuable clues of intestinal maturity.

BACKGROUND: Maturity of intestinal functions is critical for neonatal health and survival, but comprehensive description of mechanisms underlying intestinal maturation that occur during late gestation still remain poorly characterized. The aim of this study was to investigate biological processes specifically involved in intestinal maturation by comparing fetal jejunal transcriptomes of two representative porcine breeds (Large White, LW; Meishan, MS) with contrasting neonatal vitality and maturity, at two key time points during late gestation (gestational days 90 and 110). MS and LW sows inseminated with mixed semen (from breed LW and MS) gave birth to both purebred and crossbred fetuses. We hypothesized that part of the differences in neonatal maturity between the two breeds results from distinct developmental profiles of the fetal intestine during late gestation. Reciprocal crossed fetuses were used to analyze the effect of parental genome. Transcriptomic data and 23 phenotypic variables known to be associated with maturity trait were integrated using multivariate analysis with expectation of identifying relevant genes-phenotypic variable relationships involved in intestinal maturation. RESULTS: A moderate maternal genotype effect, but no paternal genotype effect, was observed on offspring intestinal maturation. Four hundred and four differentially expressed probes, corresponding to 274 differentially expressed genes (DEGs), more specifically involved in the maturation process were further studied. In day 110-MS fetuses, Ingenuity® functional enrichment analysis revealed that 46% of DEGs were involved in glucose and lipid metabolism, cell proliferation, vasculogenesis and hormone synthesis compared to day 90-MS fetuses. Expression of genes involved in immune pathways including phagocytosis, inflammation and defense processes was changed in day 110-LW compared to day 90-LW fetuses (corresponding to 13% of DEGs). The transcriptional regulator PPARGC1A was predicted to be an important regulator of differentially expressed genes in MS. Fetal blood fructose level, intestinal lactase activity and villous height were the best predicted phenotypic variables with probes mostly involved in lipid metabolism, carbohydrate metabolism and cellular movement biological pathways. CONCLUSIONS: Collectively, our findings indicate that the neonatal maturity of pig intestine may rely on functional development of glucose and lipid metabolisms, immune phagocyte differentiation and inflammatory pathways. This process may partially be governed by PPARGC1A.

A high-protein formula increases colonic peptide transporter 1 activity during neonatal life in low-birth-weight piglets and disturbs barrier function later in life.

Dietary peptides are absorbed along the intestine through peptide transporter 1 (PepT-1) which is highly responsive to dietary protein level. PepT-1 is also involved in gut homeostasis, both initiating and resolving inflammation. Low-birth-weight (LBW) neonates are routinely fed a high-protein (HP) formula to enhance growth. However, the influence of this nutritional practice on PepT-1 activity is unknown. Intestinal PepT-1 activity was compared in normal-birth-weight (NBW) and LBW piglets. The effect of HP v. normal-protein (NP) formula feeding on PepT-1 activity and gut homeostasis in LBW piglets was evaluated, during the neonatal period and in adulthood. Flux of cephalexin (CFX) across the tissue mounted in Ussing chambers was used as an indicator of PepT-1 activity. CFX flux was greater in the ileum, but not jejunum or colon, of LBW than NBW piglets during the neonatal period. When LBW piglets were formula-fed, the HP formula increased colonic CFX during the 1st week of life. Later in life, intestinal CFX fluxes and barrier function were similar whether LBW pigs had been fed NP or HP formula. However, colonic permeability of HP- but not NP-fed pigs increased when luminal pH was brought to 6·0. The formyl peptide N-formyl methionyl-leucyl-phenylalanine conferred colonic barrier protection in HP-fed piglets. Heat shock protein 27 levels in the colonic mucosa of HP-fed LBW pigs correlated with the magnitude of response to the acidic challenge. In conclusion, feeding a HP formula enhanced colonic PepT-1 activity in LBW pig neonates and increased sensitivity of the colon to luminal stress in adulthood.

The Yucatan minipig model: A new preclinical model of malnutrition in obese patients with acute or chronic diseases.

BACKGROUND & AIMS: Malnutrition can develop in patients with obesity suffering from acute or chronic illness or after obesity surgery, promoting sarcopenic obesity. A better understanding of this pathophysiology and the development of new therapeutics for chronic diseases, that are often complicated with malnutrition and obesity, justify the development of new animal experimental models close to the human physiology. This study aims to characterize the effects of obesity and underfeeding on Yucatan obese minipigs, assessing its validity as a preclinical model for obesity-related malnutrition. METHODS: Sixteen 30-month-old Yucatan minipigs were divided into two groups for 8 weeks: a standard diet group (ST, n = 5) and an obesogenic diet group (OB, n = 11). After 8 weeks, the OB group was further divided into two sub-groups: a standard diet group (OB-ST, n = 5) and a low-calorie/low-protein diet group (OB-LC/LP, n = 6) for 8 weeks. Body composition by CT-Scan and blood parameters were monitored, and trapezius muscle biopsies were collected to analyse signaling pathways involved in protein turnover and energy metabolism. RESULTS: At W8, OB-ST animals exhibited significantly higher body weight (+37.7%, p = 0.03), muscle mass (+24.9%, p = 0.02), and visceral fat (+192.0%, p = 0.03) compared to ST. Trapezius cross sectional area (CSA) normalized to body weight was lower in OB-ST animals (-15.02%, p = 0.017). At W16, no significant changes were observed in protein turnover markers, although REDD1 increased in OB-ST (96.4%, p = 0.02). After 8 weeks of low-caloric/low protein diet, OB-LC/LP showed decreased body weight (-9.8%, p = 0.03), muscle mass (-6.5%, p = 0.03), and visceral fat (-41.5%, p = 0.03) compared to OB-ST animals. Trapezius fiber CSA significantly decreased in OB-LC/LP (-36.1%, p < 0.0001) and normalized to body weight (-25.4%, p < 0.0001), combined to higher ubiquitinated protein content (+38.3%, p = 0.02). CONCLUSION: Our data support that the Yucatan minipig model mimics nutritional and skeletal muscle phenotypes observed in obese patients, with or without protein-energy malnutrition. It also reproduces muscle atrophy observed in chronic diseases or post-obesity surgery, making it a promising preclinical model for obesity-related malnutrition.

Exploration of fMRI brain responses to oral sucrose after Roux-en-Y gastric bypass in obese yucatan minipigs in relationship with microbiota and metabolomics profiles.

BACKGROUND & AIMS: In most cases, Roux-en-Y gastric bypass (RYGBP) is an efficient intervention to lose weight, change eating behavior and improve metabolic outcomes in obese patients. We hypothesized that weight loss induced by RYGBP in obese Yucatan minipigs would induce specific modifications of the gut-brain axis and neurocognitive responses to oral sucrose stimulation in relationship with food intake control. METHODS: An integrative study was performed after SHAM (n = 8) or RYGBP (n = 8) surgery to disentangle the physiological, metabolic and neurocognitive mechanisms of RYGBP. BOLD fMRI responses to sucrose stimulations at different concentrations, brain mRNA expression, cecal microbiota, and plasma metabolomics were explored 4 months after surgery and integrated with WGCNA analysis. RESULTS: We showed that weight loss induced by RYGBP or SHAM modulated differently the frontostriatal responses to oral sucrose stimulation, suggesting a different hedonic treatment and inhibitory control related to palatable food after RYGBP. The expression of brain genes involved in the serotoninergic and cannabinoid systems were impacted by RYGBP. Cecal microbiota was deeply modified and many metabolite features were differentially increased in RYGBP. Data integration with WGCNA identified interactions between key drivers of OTUs and metabolites features linked to RYGBP. CONCLUSION: This longitudinal study in the obese minipig model illustrates with a systemic and integrative analysis the mid-term consequences of RYGBP on brain mRNA expression, cecal microbiota and plasma metabolites. We confirmed the impact of RYGBP on functional brain responses related to food reward, hedonic evaluation and inhibitory control, which are key factors for the success of anti-obesity therapy and weight loss maintenance.

Is there adaptation of the exocrine pancreas in wild animal? The case of the Roe deer.

BACKGROUND: Physiology of the exocrine pancreas has been well studied in domestic and in laboratory animals as well as in humans. However, it remains quite unknown in wildlife mammals. Roe deer and cattle (including calf) belong to different families but have a common ancestor. This work aimed to evaluate in the Roe deer, the adaptation to diet of the exocrine pancreatic functions and regulations related to animal evolution and domestication. RESULTS: Forty bovine were distributed into 2 groups of animals either fed exclusively with a milk formula (monogastric) or fed a dry feed which allowed for rumen function to develop, they were slaughtered at 150 days of age. The 35 Roe deer were wild animals living in the temperate broadleaf and mixed forests, shot during the hunting season and classified in two groups adult and young. Immediately after death, the pancreas was removed for tissue sample collection and then analyzed. When expressed in relation to body weight, pancreas, pancreatic protein weights and enzyme activities measured were higher in Roe deer than in calf. The 1st original feature is that in Roe deer, the very high content in pancreatic enzymes seems to be related to specific digestive products observed (proline-rich proteins largely secreted in saliva) which bind tannins, reducing their deleterious effects on protein digestion. The high chymotrypsin and elastase II quantities could allow recycling of proline-rich proteins. In contrast, domestication and rearing cattle resulted in simplified diet with well digestible components. The 2nd feature is that in wild animal, both receptor subtypes of the CCK/gastrin family peptides were present in the pancreas as in calf, although CCK-2 receptor subtype was previously identified in higher mammals. CONCLUSIONS: Bovine species could have lost some digestive capabilities (no ingestion of great amounts of tannin-rich plants, capabilities to secrete high amounts of proline-rich proteins) compared with Roe deer species. CCK and gastrin could play an important role in the regulation of pancreatic secretion in Roe deer as in calf. This work, to the best of our knowledge is the first study which compared the Roe deer adaptation to diet with a domesticated animal largely studied.

The ghrelin system follows a precise post-natal development in mini-pigs that is not impacted by dietary medium chain fatty-acids.

The ghrelin-ghrelin receptor (GHSR1) system is one of the most important mechanisms regulating food intake and energy balance. To be fully active, ghrelin is acylated with medium-chain fatty acids (MCFA) through the ghrelin-O-acetyl transferase (GOAT). Several studies reported an impact of dietary MCFA on ghrelin acylation in adults. Our study aimed at describing early post-natal development of the ghrelin system in mini-pigs as a model of human neonates and evaluating the impact of dietary MCFA. Suckled mini-pigs were sacrificed at post-natal day (PND) 0, 2, 5, and 10 or at adult stage. In parallel, other mini-pigs were fed from birth to PND10 a standard or a dairy lipid-enriched formula with increased MCFA concentration (DL-IF). Plasma ghrelin transiently peaked at PND2, with no variation of the acylated fraction except in adults where it was greater than during the neonatal period. Levels of mRNA coding pre-proghrelin (GHRL) and GOAT in the antrum did not vary during the post-natal period but dropped in adults. Levels of antral pcsk1/3 (cleaving GHRL into ghrelin) mRNA decreased significantly with age and was negatively correlated with plasma acylated, but not total, ghrelin. Hypothalamic ghsr1 mRNA did not vary in neonates but increased in adults. The DL-IF formula enriched antral tissue with MCFA but did not impact the ghrelin system. In conclusion, the ghrelin maturation enzyme PCSK1/3 gene expression exhibited post-natal modifications parallel to transient variations in circulating plasma ghrelin level in suckling piglets but dietary MCFA did not impact this post-natal development.

Infant nutrition affects the microbiota-gut-brain axis: Comparison of human milk vs. infant formula feeding in the piglet model.

Early nutrition plays a dominant role in infant development and health. It is now understood that the infant diet impacts the gut microbiota and its relationship with gut function and brain development. However, its impact on the microbiota-gut-brain axis has not been studied in an integrative way. The objective here was to evaluate the effects of human milk (HM) or cow's milk based infant formula (IF) on the relationships between gut microbiota and the collective host intestinal-brain axis. Eighteen 10-day-old Yucatan mini-piglets were fed with HM or IF. Intestinal and fecal microbiota composition, intestinal phenotypic parameters, and the expression of genes involved in several gut and brain functions were determined. Unidimensional analyses were performed, followed by multifactorial analyses to evaluate the relationships among all the variables across the microbiota-gut-brain axis. Compared to IF, HM decreased the α-diversity of colonic and fecal microbiota and modified their composition. Piglets fed HM had a significantly higher ileal and colonic paracellular permeability assessed by ex vivo analysis, a lower expression of genes encoding tight junction proteins, and a higher expression of genes encoding pro-inflammatory and anti-inflammatory immune activity. In addition, the expression of genes involved in endocrine function, tryptophan metabolism and nutrient transport was modified mostly in the colon. These diet-induced intestinal modifications were associated with changes in the brain tissue expression of genes encoding the blood-brain barrier, endocrine function and short chain fatty acid receptors, mostly in hypothalamic and striatal areas. The integrative approach underlined specific groups of bacteria (Veillonellaceae, Enterobacteriaceae, Lachnospiraceae, Rikenellaceae, and Prevotellaceae) associated with changes in the gut-brain axis. There is a clear influence of the infant diet, even over a short dietary intervention period, on establishment of the microbiota-gut-brain axis.

The Yucatan minipig model: A new preclinical model of malnutrition induced by a low-calorie/low-protein diet.

BACKGROUND & AIMS: Severe malnutrition exposes patients to adverse outcomes and a higher mortality risk. The Yucatan minipig, closer to human physiology than murine models could be a pertinent and innovative experimental model for studying the physiopathology and consequences of severe malnutrition. The present study aimed to determine whether a low calorie/low protein diet (LC/LP) can reproduce marasmus malnutrition in minipigs, and to characterize body composition, gut microbiota, malnutrition-related blood parameters, and histological and molecular skeletal muscle patterns. METHODS: Eleven Yucatan minipigs were subjected to two different diets: a standard control diet (ST) (n = 5) and a LC/LP diet (n = 6). LC/LP animals daily received 50% of an isocaloric low-protein diet (10.37 MJ/kg, 8.6% protein). Body composition was measured by computed tomography (CT-scan) before (T0) and after 8 weeks of diet (T8). Trapezius and biceps femoris muscles were sampled at the end of protocol to perform histological and molecular analyses. Gut microbiota composition were was also analyzed at T0 and T8 in fecal samples. RESULTS: Eight weeks of LC/LP diet significantly reduced body weight (-12.3 ± 9.5%, P = 0.03) and gut microbiota richness (i.e. number of observed species) (-10.4 ± 8.3%, P = 0.014) compared to baseline. After 8 weeks, LC/LP animals exhibited a significant reduction of retroperitoneal fat and skeletal muscle surface areas (P = 0.03 and P = 0.047, respectively), whereas these parameters remained unchanged in ST animals. These reductions were associated with lower muscle fiber cross-sectional area (CSA) in trapezius (P < 0.001) and biceps femoris (P = 0.003) in LC/LP animals compared to ST. LC/LP diet promoted an increase of AMP kinase phosphorylation in trapezius and biceps femoris (P = 0.05), but did not affect cytochrome c and COX IV protein content, markers of mitochondrial content. Gene and proteins involved in ubiquitin-proteasome system and apoptosis remained unchanged after 8 weeks of LC/LP diet both in trapezius and biceps femoris. CONCLUSION: All these findings support that this experimental minipig model of severe malnutrition is valid to mimic pathophysiological changes occurring in human protein-energy marasmus malnutrition and muscle atrophy associated with malnutrition, as observed in patients with secondary sarcopenia.

Phaseolin from Phaseolus vulgaris bean modulates gut mucin flow and gene expression in rats.

Dietary protein might modulate mucin flow and intestinal mucin gene expression. Since unheated phaseolin from Phaseolus vulgaris bean is resistant to digestion and increases gut endogenous protein losses, we hypothesised that unheated phaseolin influences mucin flow and gene expression, and that phaseolin heat treatment reverses these effects. The hypothesis was tested using a control diet containing casein as the sole protein source and three other diets with casein being replaced by 33 and 67 % of unheated and 67 % of heated phaseolin. The rats were fed for 6 d and euthanised. Digesta and faeces were collected for determining digestibility and mucin flow. Gut tissues were collected for mucin (Muc1, Muc2, Muc3 and Muc4) and Trefoil factor 3 (Tff3) gene expressions. Colonic mucin flow decreased linearly with increasing the dietary level of unheated phaseolin (P < 0·05). Unheated phaseolin increased N flow in ileum, colon and faeces (P < 0·05), and reduced apparent N digestibility linearly (P < 0·01). Heat treatment reversed all these changes (P < 0·05 to < 0·001), except mucin flow. The expressions of Muc mRNA in gut tissues were influenced by dietary phaseolin level (ileum and colon: Muc3 and Muc4) and thermal treatment (ileum: Muc2; colon: Muc2, Muc3, Muc4 and Tff3) (P < 0·05 to 0·001). In conclusion, phaseolin modulates mucin flow and Muc gene expression along the intestines differentially.

Maternal Supplementation of Food Ingredient (Prebiotic) or Food Contaminant (Mycotoxin) Influences Mucosal Immune System in Piglets.

The early life period is crucial for the maturation of the intestinal barrier, its immune system, and a life-long beneficial host-microbiota interaction. The study aims to assess the impact of a beneficial dietary (short-chain fructooligosaccharides, scFOS) supplementation vs. a detrimental dietary environment (such as mycotoxin deoxynivalenol, DON) on offspring intestinal immune system developmental profiles. Sows were given scFOS-supplemented or DON-contaminated diets during the last 4 weeks of gestation, whereas force-feeding piglets with DON was performed during the first week of offspring life. Intestinal antigen-presenting cell (APC) subset frequency was analyzed by flow cytometry in the Peyer's patches and in lamina propria and the responsiveness of intestinal explants to toll-like receptor (TLR) ligands was performed using ELISA and qRT-PCR from post-natal day (PND) 10 until PND90. Perinatal exposure with scFOS did not affect the ontogenesis of APC. While it early induced inflammatory responses in piglets, scFOS further promoted the T regulatory response after TLR activation. Sow and piglet DON contamination decreased CD16+ MHCII+ APC at PND10 in lamina propria associated with IFNγ inflammation and impairment of Treg response. Our study demonstrated that maternal prebiotic supplementation and mycotoxin contamination can modulate the mucosal immune system responsiveness of offspring through different pathways.

Validation of a Psychosocial Chronic Stress Model in the Pig Using a Multidisciplinary Approach at the Gut-Brain and Behavior Levels.

Psychological chronic stress is an important risk factor for major depressive disorder, of which consequences have been widely studied in rodent models. This work aimed at describing a pig model of chronic stress based on social isolation, environmental impoverishment and unpredictability. Three groups of animals of both sexes were constituted. Two were exposed to the psychosocial stressors while receiving (SF, n = 12) or not (SC, n = 22) the antidepressant fluoxetine, and a third group (NSC, n = 22) remained unstressed. Animals were observed in home pens and during dedicated tests to assess resignation and anxiety-like behaviors. Brain structure and function were evaluated via proton MRS and fMRI. Hippocampal molecular biology and immunodetection of cellular proliferation (Ki67+) and neuron maturation (DCX+) in the dentate gyrus were also performed. Salivary cortisol, fecal short-chain fatty acids (SCFAs), and various plasmatic and intestinal biomarkers were analyzed. Compared to NSC, SC animals showed more resignation (p = 0.019) and had a higher level of salivary cortisol (p = 0.020). SC brain responses to stimulation by a novel odor were lower, similarly to their hippocampal neuronal density (p = 0.015), cellular proliferation (p = 0.030), and hippocampal levels of BDNF and 5-HT1AR (p = 0.056 and p = 0.007, respectively). However, the number of DCX+ cells was higher in the ventral dentate gyrus in this group (p = 0.025). In addition, HOMA-IR was also higher (p < 0.001) and microbiota fermentation activity was lower (SCFAs, SC/NSC: p < 0.01) in SC animals. Fluoxetine partially or totally reversed several of these effects. Exposure to psychosocial stressors in the pig model induced effects consistent with the human and rodent literature, including resignation behavior and alterations of the HPA axis and hippocampus. This model opens the way to innovative translational research exploring the mechanisms of chronic stress and testing intervention strategies with good face validity related to human.

Addition of dairy lipids and probiotic Lactobacillus fermentum in infant formula programs gut microbiota and entero-insular axis in adult minipigs.

Clinical and animal studies have demonstrated beneficial effects of early consumption of dairy lipids and a probiotic, Lactobacillus fermentum (Lf), on infant gut physiology. The objective of this study was to investigate their long-term effects on gut microbiota and host entero-insular axis and metabolism. Piglets were suckled with a milk formula containing only plant lipids (PL), a half-half mixture of plant lipids and dairy lipids (DL), or this mixture supplemented with Lf (DL + Lf). They were weaned on a standard diet and challenged with a high-energy diet until postnatal day 140. DL and DL + Lf modulated gut microbiota composition and metabolism, increasing abundance of several Clostridia genera. Moreover, DL + Lf specifically decreased the faecal content of 2-oxoglutarate and lysine compared to PL and 5-aminovalerate compared to PL and DL. It also increased short-chain fatty acid concentrations like propionate compared to DL. Furthermore, DL + Lf had a beneficial effect on the endocrine function, enhancing caecal GLP-1 and GLP-1 meal-stimulated secretion. Correlations highlighted the consistent relationship between microbiota and gut physiology. Together, our results evidence a beneficial programming effect of DL + Lf in infant formula composition on faecal microbiota and entero-insular axis function.

Characterisation of Early-Life Fecal Microbiota in Susceptible and Healthy Pigs to Post-Weaning Diarrhoea.

Early-life microbial exposure is of particular importance to growth, immune system development and long-lasting health. Hence, early microbiota composition is a promising predictive biomarker for health and disease but still remains poorly characterized in regards to susceptibility to diarrhoea. In the present study, we aimed to assess if gut bacterial community diversity and composition during the suckling period were associated with differences in susceptibility of pigs to post-weaning diarrhoea. Twenty piglets from 5 sows (4 piglets / litter) were weaned in poor housing conditions to challenge their susceptibility to post-weaning diarrhoea. Two weeks after weaning, 13 pigs exhibited liquid faeces during 2 or 3 days and were defined as diarrhoeic (D) pigs. The other 7 pigs did not have diarrhea during the whole post-weaning experimental periodand were defined as healthy (H) pigs. Using a molecular characterisation of fecal microbiota with CE-SSCP fingerprint, Next Generation Sequencing and qPCR, we show that D and H pigs were mainly discriminated as early as postnatal day (PND) 7, i.e. 4 weeks before post-weaning diarrhoea occurence. At PND 7 H pigs displayed a lower evenness and a higher abundance of Prevotellaceae, Lachnospiraceae, Ruminocacaceae and Lactobacillaceae compared to D pigs. The sPLS regression method indicates that these bacterial families were strongly correlated to a higher Bacteroidetes abundance observed in PND 30 H pigs one week before diarrhoea. These results emphasize the potential of early microbiota diversity and composition as being an indicator of susceptibility to post-weaning diarrhoea. Furthermore, they support the health promoting strategies of pig herds through gut microbiota engineering.

Presence and localization of CCK receptor subtypes in calf pancreas.

This study was undertaken to confirm the presence of CCK receptor subtypes in calf pancreas and establish their cellular localization. Using specific antibodies against CCKA and CCKB receptors, somatostatin, glucagon and insulin, we were able to confirm by Western blot the presence of both CCK receptor protein subtypes in the calf pancreas as a 80-85-kDa CCKA receptor and 40-45-kDa CCKB receptor. By immunofluorescence, the CCKB receptor colocalizes with the islets' somatostatin delta cells, confirming what was previously shown in other species, as well as on ductal cells. We could not reproduce in the calf its colocalization with glucagon alpha cells as observed in human and rat. Any specific localization of CCKA receptors with our multiple antibodies failed. Our observation that the CCKB receptor subtype is specifically localized on pancreatic delta cells as well as on ductal cells lets us support the hypothesis that in this species, CCK could be involved in somatostatin metabolism as well as hydrelatic secretion; its effect on enzyme secretion would be indirect.

The role of luminal gastrin in the regulation of pancreatic juice secretion in preruminant calves.

The effect of luminal gastrin on the secretion of pancreatic juice was studied in seven conscious preruminant calves employing luminal infusions of gastrin and cholecystokinin (CCK)-9 and pharmacological CCK1 and CCK2 receptor blocks with antagonists. The study was performed in the preprandial and prandial states. Pharmacological blocking of the CCK2 receptor, like that of the CCK1 receptor, resulted in reduction of pancreatic postprandial secretion and increased the duration of the prandial pattern of duodenal electrical activity. Exogenous luminal gastrin, like luminal CCK-9, enhanced the secretion of pancreatic juice proteins, though the overall effect of gastrin was weaker than that of CCK-9. The effect was inhibited by infusion of CCK2 but also by CCK1 receptor antagonist. In conclusion, duodenal luminal gastrin can stimulate exocrine pancreatic secretion by a mechanism that depends on CCK2 receptors in calves. Involvement of the CCK1 receptor in this mechanism needs further investigation. Prandial pancreatic secretory and duodenal motility cycles can be regulated by endogenous gastrin release.

Bovine pancreatic secretion in the first week of life: potential involvement of intestinal CCK receptors.

The aim of this study was to evaluate pancreatic juice secretion of calves in the first postnatal days, and determine a potential involvement of cholecystokinin (CCK) and intestinal CCK receptor in its regulation. Nine neonatal Friesian calves (five controls and four treated intraduodenally with FK480, a CCK-A receptor antagonist) were surgically fitted with a pancreatic duct catheter and a duodenal cannula before the first colostrum feeding. Collections of pancreatic juice and duodenal luminal pressure recordings were started early after recovery from anaesthesia and continued for 6 days. From day 2 or 3 of life, periodic fluctuations in pancreatic secretions were observed in concert with duodenal myoelectric motor complex (MMC) and variations in plasma pancreatic polypeptide (PP) concentrations. Intraduodenal administration of FK480 reduced pancreatic juice secretion while intravenous infusion of CCK had no effect. Immunocytochemistry indicated an association of mucosal CCK-A and -B receptors with neural components of the small intestine. In conclusion, periodic activity of the exocrine pancreas exists in neonatal calves soon after birth and local neural intestinal CCK-A receptors could be partly responsible for the modulation of neonatal calf pancreatic secretion.

Maternal 18:3n-3 favors piglet intestinal passage of LPS and promotes intestinal anti-inflammatory response to this bacterial ligand.

We recently observed that maternal 18:3n-3 increases piglet jejunal permeability. We hypothesized that this would favor intestinal lipopolysaccharide (LPS) passage and alter gut immune system education toward this bacterial ligand. Sows were fed 18:3n-3 or 18:2n-6 diets throughout gestation and lactation. In each litter, two piglets were given oral Gram-negative spectrum antibiotic from post-natal day (PND) 14 to 28. All piglets were weaned on a regular diet at PND28. 18:3n-3 piglets exhibited greater jejunal permeability to FITC-LPS at PND28. Levels of 18:3n-3 but neither 20:5n-3 nor 20:4n-6 were greater in mesenteric lymph nodes (MLN) of 18:3n-3 piglets. Jejunal explant or MLN cell cytokine responses to LPS were not influenced by the maternal diet. Antibiotic increased jejunal permeability to FITC-LPS and lowered the level of 20:5n-3 in MLN, irrespective of the maternal diet. At PND52, no long-lasting effect of the maternal diet or antibiotic treatment on jejunal permeability was noticed. 18:3n-3 and 20:4n-6 levels were greater and lower, respectively, in MLN of 18:3n-3 compared to 18:2n-6 piglets. IL-10 production by MLN cells in response to LPS was greater in the 18:3n-3 group, irrespective of the neonatal antibiotic treatment. IL-8 secretion by jejunal explants in response to LPS was lower in antibiotic-treated 18:3n-3 compared to 18:2n-6 piglets. Finally, proportion of MHC class II(+) antigen-presenting cells was greater in 18:3n-3 than 18:2n-6 MLN cells. In conclusion, maternal 18:3n-3 directs the intestinal immune response to LPS toward an anti-inflammatory profile beyond the breastfeeding period; microbiota involvement seems dependent of the immune cells considered.

Is caseinomacropeptide from milk proteins, an inhibitor of gastric secretion?

The aim of this work was to study, in vivo, the effect of the ingestion of not glycosylated caseinomacropeptide (CMP) on gastric secretion. In Experiments #1 and #2, 7 calves fitted with a gastric pouch received either a diet without CMP (C diet) or C diet in which CMP was introduced (equal to and 5 folds that of CMP quantity contained in cow milk, diets CMP1 and CMP5, respectively). In Experiment #3, 2 calves (with gastric pouch) were fed C diet followed by an "iv perfusion" of CMP. In Experiment #4, 25 calves fed either C, CMP1 or CMP5 diets were fitted with a blood catheter for sample collections. The quantities of daily gastric secretions seemed few modified by CMP ingestion but the profile of these secretions was changed along the day. The most important result is that CMP can inhibit gastric secretions (mainly hydrochloric acid) stimulated by the meal, but there was no dose-dependent response. No similar observations were obtained after perfusion of CMP in jugular vein. CMP was not detected in blood. Results obtained in our experiments are not in favor of its significant intestinal absorption. Gastrin, somatostatin and VIP could be implicated in the mechanisms of regulation.