In vitro prebiotic potential of agricultural by-products on intestinal fermentation, gut barrier and inflammatory status of piglets.

The inclusion of fibre-rich ingredients in diets is one possible strategy to enhance intestinal fermentation and positively impact gut ecology, barrier and immunity. Nowadays, inulin-type fructans are used as prebiotics in the feed of piglets to manipulate gut ecology for health purposes. Likewise, some by-products could be considered as sustainable and inexpensive ingredients to reduce gut disorders at weaning. In the present study, chicory root and pulp, citrus pulp, rye bran and soya hulls were tested in a three-step in vitro model of the piglet's gastro-intestinal tract combining a pepsin-pancreatin hydrolysis (digestion), a dialysis step using cellulose membranes (absorption) and a colonic batch fermentation (fermentation). The fermentation kinetics, SCFA and microbiota profiles in the fermentation broth were assessed as indicators of prebiotic activity and compared with the ones of inulin. The immunomodulatory effects of fermentation supernatant (FS) were investigated in cultured intestinal porcine epithelial cells (IPEC-J2) by high-throughput quantitative PCR. Chicory root displayed a rapid and extensive fermentation and induced the second highest butyrate ratio after inulin. Citrus pulp demonstrated high acetate ratios and induced elevated Clostridium clusters IV and XIVa levels. Chicory root and pulp FS promoted the intestinal barrier integrity with up-regulated tight and adherens junction gene expressions in comparison with inulin FS. Chicory pulp FS exerted anti-inflammatory effects in cultured IPEC-J2. The novel approach combining an in vitro fermentation model with IPEC-J2 cells highlighted that both chicory root and pulp appear to be promising ingredients and should be considered to promote intestinal health at weaning.

Araceae root and citrus fibers tend to decrease Escherichia coli adhesion and myeloperoxidase levels in weaned piglets.

Introduction: Weaning is a stressful experience in the piglet's life, and it often coincides with impaired gut health. Post-weaning diarrhea in piglets is frequently caused by enterotoxigenic Escherichia coli (E. coli). The first step of an E. coli infection is the adhesion to host-specific receptors present on enterocytes, leading to pro-inflammatory immune responses. The aim of this study was to examine if specific fiber fractions in the piglet diet can prevent E. coli adhesion and subsequent immune responses. Methods: The trial included 200 piglets (Danbred × Piétrain): 10 piglets/pen × 10 pens/dietary treatment × 2 dietary treatments. From weaning until 14 days (d14) post-weaning, piglets were fed a control diet or test diet with 2 kg/ton of a mixture of specific fiber fractions derived from Araceae root and citrus. Afterwards, 1 piglet per pen was euthanized, a section was taken at 75% of small intestinal length and E. coli colonization on the mucosal epithelium was quantified by scraping and conventional plating. From the same small intestinal section, histo-morphological indices were assessed, and mucosal scrapings were analyzed for gene expression of pro- and anti-inflammatory cytokines, and NF-kB. Analyses of specific intestinal bacteria and SCFA were performed on samples of intestinal content (small intestine, caecum, colon). Fecal samples were taken to measure myeloperoxidase (MPO), calprotectin and PAP/RAG3A as biomarkers for intestinal inflammation. Results and discussion: Piglets fed the fiber mixture tended to have decreased E. coli colonization to the mucosal epithelium (5.65 vs. 4.84 log10 CFU/g; P = 0.07), less E. coli in the caecum (8.91 vs. 7.72 log10 CFU/g; P = 0.03) and more Lachnospiraceae in the colon (11.3 vs. 11.6 log10 CFU/g; P = 0.03). Additionally, the fiber mixture tended to increase cecal butyric acid (10.4 vs. 19.1 mmol/kg; P = 0.07). No significant effect on histo-morphological indices and on gene expression of pro- and anti-inflammatory cytokines and NF-kB was observed. The fecal MPO concentration tended to decrease (20.2 vs. 10.4 ng/g; P = 0.07), indicating less intestinal inflammation. In conclusion, this study showed that specific fiber fractions from Araceae root and citrus in piglet weaner diets may decrease the risk of pathogen overgrowth by reducing E. coli adhesion and intestinal inflammation.

Impact of Citrus Pulp or Inulin on Intestinal Microbiota and Metabolites, Barrier, and Immune Function of Weaned Piglets.

We investigated the use of citrus pulp (CP) as a novel prebiotic capable of exerting microbiota and immunomodulating capacities to alleviate weaning stress. Inulin (IN), a well-known prebiotic, was used for comparison. Hundred and 28 male weaned piglets of 21 days old were assigned to 32 pens of 4 piglets each. Piglets were assigned to one of the four treatments, i.e., control, IN supplemented at 0.2% (IN0.2%), and CP supplemented either at 0.2% (CP0.2%) or at 2% (CP2%). On d10-11 and d31-32 post-weaning, one pig per pen was euthanized for intestinal sampling to evaluate the growth performance, chyme characteristics, small intestinal morphology, colonic inflammatory response and barrier integrity, metabolite profiles [gas chromatography-mass spectrometry (GC-MS), and liquid chromatography-mass spectrometry (LC-MS)], and microbial populations. The IN treatment and the two CP treatments induced higher small intestinal villus height to crypt depth ratios in comparison with the control diet at both sampling times. All treatments decreased acidic goblet cell absolute counts in the crypts in comparison to the control diet of the duodenum on d10-11 and d31-32. The gene expression of ß-defensin 2 was downregulated in colonic tissues following the IN and CP2% inclusion on d31-32. On d31-32, piglets fed with IN and CP0.2% showed lower mRNA levels of occludin and claudin-3, respectively. Not surprisingly, flavonoids were observed in the colon in the CP treatments. Increased colonic acetate proportions on d10-11, at the expense of branched-chain fatty acid (BCFA) levels, were observed following the CP2% supplementation compared to the control diet, inferring a reduction of proteolytic fermentation in the hindgut. The beneficial microbial community Faecalibacterium spp. was promoted in the colon of piglets fed with CP2% on d10-11 (p = 0.04; false discovery rate (FDR) non-significant) and on d31-32 (p = 0.03; FDR non-significant) in comparison with the control diet. Additionally, on d31-32, CP2% increased the relative abundance of Megasphaera spp. compared to control values (p = 0.03; FDR non-significant). In conclusion, CP2% promoted the growth of beneficial bacterial communities in both post-weaning time points, modulating colonic fermentation patterns in the colon. The effects of CP supplementation were similar to those of IN and showed the potential as a beneficial feed supplement to alleviate weaning stress.

Cumulus contributions during bovine fertilization in vitro.

A mandatory step in performing micromanipulation techniques, studying sperm-oocyte interactions and evaluating morphological aspects of oocyte quality is the removal of cumulus cells from oocytes or zygotes at various stages. In cattle, cumulus removal shortly before fertilization in vitro strongly decreases sperm penetration rates. This study was conducted to evaluate the function of the cumulus oophorus during bovine fertilization in vitro. The importance of cumulus secretions during IVF was investigated by inseminating cumulus-denuded oocytes (CDOs) in fertilization medium supplemented with individual cumulus secretions, such as progesterone or hyaluronic acid. None of these substances increased the fertilization rate of CDOs. However, fertilizing CDOs in cumulus-conditioned medium or on a cumulus monolayer partially restored the reduction in fertilization rate (P<0.05). The fertilization rate of CDOs inseminated on a cumulus monolayer further increased when physical contact between the gametes and the monolayer was prevented by fertilizing them inside a culture plate insert placed on the monolayer (P<0.05). Finally, the importance of reactive oxygen species (ROS) generation and O(2) concentration during IVF was studied. Luminol-dependent chemiluminescence revealed a higher ROS load in conditioned medium of cumulus-enclosed oocytes (CEOs) than in that of CDOs after sperm-oocyte co-incubation (P<0.05). Furthermore, lowering the external O(2) concentration from 20 to 5% decreased the fertilization rate of both CEOs and CDOs, but had a higher impact on CEOs (P<0.05). In conclusion, this study provides evidence that the cumulus oophorus benefits the fertilizing ability of penetrating spermatozoa by creating a complex microenvironment of both cumulus secretions and metabolic products around the oocyte. Gap junctional communication between the oocyte and corona cells as well as sperm trapping by the cumulus oophorus seem to be essential factors in supporting fertilization.

Effect of fatty acid composition of the sow diet on the innate and adaptive immunity of the piglets after weaning.

This study investigated whether the immunocompetence of piglets at weaning was modulated by including different sources of n-3 polyunsaturated fatty acids (PUFA) in the maternal diet. From day 73 of gestation until weaning at 4 weeks, 32 pregnant sows were fed a palm oil-based diet (control group) or a diet including 1% linseed oil (C18:3n-3), 1% echium oil (C18:3n-3, C18:4n-3, C18:3n-6) or 1% fish oil (C20:5n-3, C22:6n-3). It was hypothesized that each diet would differently affect immune function through effects such as specific eicosanoid production. Piglets were fed a conventional diet without added n-3 PUFA from weaning until day 35 post-weaning. At weaning and 21 days post-weaning, four piglets per litter were immunized with bovine thyroglobulin. Blood samples were taken from weaning until day 35 post-weaning to determine thyroglobulin-specific antibodies, serum amyloid A (SAA) concentration and fatty acid composition. The fatty acid composition of the maternal diets was reflected in the plasma and red blood cells of the weaned piglets. The onset of the thyroglobulin-specific IgM response differed between dietary groups, with a delay in response for piglets from sows fed the fish oil diet. No significant dietary effects were observed on the thyroglobulin-specific IgG and IgA titres or on SAA concentrations in the piglet serum. Including n-3 PUFA in the maternal diet at the concentrations used in the present study had no major effects on the adaptive and innate immunity of the piglets after weaning.

Does sow reproduction and piglet performance benefit from the addition of n-3 polyunsaturated fatty acids to the maternal diet?

Good sow reproductive performance and piglet survival are essential for the profitability of the pig industry. Based on basic research of fatty acid and endocrine metabolism, it has been suggested that dietary supplementation with n-3 polyunsaturated fatty acids (PUFA) could enhance reproductive outcome. However, proper requirements for these nutrients in sow diets have not been established. This review examines the literature on the effect of n-3 PUFA in the maternal diet on sow reproduction and piglet performance. Few reported studies have included biochemical analyses, e.g. eicosanoid concentrations or gene expression data, which could help elucidate any link between dietary n-3 PUFA supplementation and reproduction. Additionally, most studies used relatively low numbers of pigs, limiting the validity of the conclusions which can be drawn. In pregnant pigs, supplementing with n-3 PUFA has not been shown to increase significantly the number of embryos (in gilts) or (in most studies) the total number of piglets born (live and/or stillborn), but may prolong gestation, although the literature is not consistent. Most studies found no effect of n-3 PUFA on piglet birth weight, although positive effects on piglet vitality and pre- and post-weaning growth have been reported. In contrast to the impact during pregnancy, low amounts of n-3 PUFA in the lactation diet may increase litter size in the subsequent gestation.

Inhibition of bovine sperm-oocyte fusion by the p-aminophenyl derivative of D-mannose.

Several steps in the process of mammalian fertilization are mediated by carbohydrates. This study investigated the role of the p-aminophenyl derivative of d-mannose (APMP) during bovine fertilization. Inseminating cumulus-oocyte complexes (COCs) in the presence of increasing APMP concentrations resulted in a significant dose-dependent decrease of the fertilization rate (P < 0.05). No negative effect of 50 mM APMP on total sperm motility and progressive motility was found. Subsequently, the fertilization steps at which this blocking effect could be exerted were investigated, i.e., sperm penetration of the cumulus oophorus, sperm-zona binding, acrosome reaction, sperm-oolemma binding, and/or sperm-oocyte fusion. Inseminating cumulus-enclosed and cumulus-denuded oocytes in the presence of 50 mM APMP significantly decreased the fertilization rate to a comparable minimum level (P < 0.05). There was no significant relationship between the number of spermatozoa bound to the zona pellucida and the APMP concentration, and APMP nor d-mannosylated bovine serum albumin (BSA) suppressed or stimulated sperm acrosomal status. Inseminating zona-free oocytes in the presence of 50 mM APMP did not influence sperm-oolemma binding, but significantly inhibited sperm-oocyte fusion (P < 0.05). Preincubating zona-free oocytes with 200 microg/ml Con A but not with 50 mM APMP inhibited the sperm-oocyte fusion rate to the same extent as when the gametes were simultaneously exposed to 50 mM APMP. These data indicate that the blocking effect of APMP on bovine fertilization is mainly due to an inhibition of sperm-oocyte fusion, probably by specific obstruction of the sperm receptor sites that are responsible for the fusion process.

Assessment of different sperm quality parameters to predict in vitro fertility of bulls.

Frozen-thawed semen from six bulls with high (> 60%) and low (20-35%) in vitro fertility was used for studying the predictive value of simple sperm quality tests with respect to in vitro fertilization (IVF) outcome as assessed by pronucleus (PN) formation ability. Sperm quality parameters, such as sperm concentration, motility, progressive motility, live-dead sperm ratio, morphology, membrane integrity, mitochondrial activity and acrosomal status were analysed using both conventional and automatic techniques at three time points during the IVF process, namely after sperm thawing, Percoll differential gradient centrifugation and IVF. Associations between the sperm quality parameters before and after IVF, and PN formation ability were assessed by using linear regression analyses. The percentages of motility, progressive motility and normal morphology determined after sperm thawing, and the percentage of live spermatozoa assessed after Percoll preparation by using nigrosin-eosin (N-E) staining showed a good correlation with PN formation ability, but the regression parameters were borderline not significant. These parameters formed the most reliable basis for predicting IVF outcome. After IVF, the percentage of live spermatozoa determined by using N-E staining was the only sperm quality parameter showing a significant association with the PN formation ability of a given bull. This sperm quality test can be used as a non-invasive method to estimate the PN formation ability of oocytes which are further cultured to assess embryonic development.