Understanding host-microbiota interactions in the commercial piglet around weaning.

Weaning is a critical period in the life of pigs with repercussions on their health and welfare and on the economy of the swine industry. This study aimed to assess the effect of the commercial early weaning on gut microbiota, intestinal gene expression and serum metabolomic response via an integrated-omic approach combining 16S rRNA gene sequencing, the OpenArray gene expression technology and 1H-NMR spectroscopy. Fourteen piglets from different litters were sampled for blood, jejunum tissue and caecal content two days before (- 2d), and three days after (+ 3d) weaning. A clearly differential ordination of caecal microbiota was observed. Higher abundances of Roseburia, Ruminococcus, Coprococcus, Dorea and Lachnospira genera in weaned piglets compared to prior to weaning showed the quick microbial changes of the piglets' gut microbiota. Downregulation of OCLN, CLDN4, MUC2, MUC13, SLC15A1 and SLC13A1 genes, also evidenced the negative impact of weaning on gut barrier and digestive functions. Metabolomic approach pinpointed significant decreases in choline, LDL, triglycerides, fatty acids, alanine and isoleucine and increases in 3-hydroxybutyrate after weaning. Moreover, the correlation between microbiota and metabolome datasets revealed the existence of metabolic clusters interrelated to different bacterial clusters. Our results demonstrate the impact of weaning stress on the piglet and give insights regarding the associations between gut microbiota and the animal gene activity and metabolic response.

Early socialization and environmental enrichment of lactating piglets affects the caecal microbiota and metabolomic response after weaning.

The aim of this study was to determine the possible impact of early socialization and an enriched neonatal environment to improve adaptation of piglets to weaning. We hypothesized that changes in the microbiota colonization process and in their metabolic response and intestinal functionality could help the animals face weaning stress. A total of 48 sows and their litters were allotted into a control (CTR) or an enriched treatment (ENR), in which piglets from two adjacent pens were combined and enriched with toys. The pattern of caecal microbial colonization, the jejunal gene expression, the serum metabolome and the intestinal physiology of the piglets were assessed before (-2 d) and after weaning (+ 3d). A differential ordination of caecal microbiota was observed after weaning. Serum metabolome suggested a reduced energetic metabolism in ENR animals, as evidenced by shifts in triglycerides and fatty acids, VLDL/LDL and creatine regions. The TLR2 gene showed to be downregulated in the jejunum of ENR pigs after weaning. The integration of gene expression, metabolome and microbiota datasets confirmed that differences between barren and enriched neonatal environments were evident only after weaning. Our results suggest that improvements in adaptation to weaning could be mediated by a better response to the post-weaning stress.

Review: Are we using probiotics correctly in post-weaning piglets?

Intensive farming may involve the use of diets, environments or management practices that impose physiological and psychological stressors on the animals. In particular, early weaning is nowadays a common practice to increase the productive yield of pig farms. Still, it is considered one of the most critical periods in swine production, where piglet performance can be seriously affected and where they are predisposed to the overgrowth of opportunistic pathogens. Pig producers nowadays face the challenge to overcome this situation in a context of increasing restrictions on the use of antibiotics in animal production. Great efforts are being made to find strategies to help piglets overcome the challenges of early weaning. Among them, a nutritional strategy that has received increasing attention in the last few years is the use of probiotics. It has been extensively documented that probiotics can reduce digestive disorders and improve productive parameters. Still, research in probiotics so far has also been characterized as being inconsistent and with low reproducibility from farm to farm. Scientific literature related to probiotic effects against gastrointestinal pathogens will be critically examined in this review. Moreover, the actual practical approach when using probiotics in these animals, and potential strategies to increase consistency in probiotic effects, will be discussed. Thus, considering the boost in probiotic research observed in recent years, this paper aims to provide a much-needed, in-depth review of the scientific data published to-date. Furthermore, it aims to be useful to swine nutritionists, researchers and the additive industry to critically consider their approach when developing or using probiotic strategies in weaning piglets.

Response to a Salmonella Typhimurium challenge in piglets supplemented with protected sodium butyrate or Bacillus licheniformis: effects on performance, intestinal health and behavior,2.

Salmonella spp. is one of the worldwide leading causes of food-borne illnesses for which the inclusion of probiotics or organic acids in animal feeds can be useful control methods. Experimental models are utilized to test the efficacy of strategies against pathogens, but they exhibit limitations which may preclude finding sensible evaluation parameters. The objective of this work is to evaluate the efficacy of 2 different feed additives; a Bacillus licheniformis based probiotic and a protected sodium butyrate (SB) salt, using an experimental model of salmonellosis and, second, to explore if behavior analysis can be used as a sensible evaluation tool for additives evaluation. A total of 78 piglets weaned at 24 d, 8.3 kg BW, were used. Seventy-two were placed in 3 rooms of 8 pens (3 animals/pen) with evenly distributed treatments (n = 8): CON, control group with plain diet; PRO, plain diet with 1 kg/t of Proporc (109 cfu of B. licheniformis/kg of feed), and BUT, plain diet with 3 kg/t of Gustor BP70 (2.1 g of partially protected SB salt/kg of feed). Remaining piglets (n = 6) were separated and used as a challenge negative control. The experiment lasted 16 d. After 1 wk of adaptation, animals were challenged with 5 × 108 cfu of Salmonella Typhimurium. One pig per pen was euthanized and sampled at d 4 and 8 post-inoculation (PI). There were no significant differences among treatments for ADFI, ADG, G:F, rectal temperature, fecal consistency, pH, ammonia, short-chain fatty acids and lactic acid concentrations, cytokine TNF-α, Pig-MAP acute-phase proteins and histological parameters. However, both products were equally able to reduce colonization and shedding of Salmonella (P = 0.016 for PRO and BUT vs. CON). In addition, PRO treatment had a positive effect on behavioral displays, particularly exploring (P < 0.05 vs. CON), feeding (P < 0.05 vs. CON and BUT) and other active behaviors (P < 0.05 vs. CON and BUT) in the morning period (0830 to 1030 h). In the afternoon (1400 to 1600 h), the challenge effect was most significant. Pigs were less active after the challenge (P < 0.001), with a decrease in positive contacts (P = 0.004), exploration (P < 0.001) and feeding behaviors (P < 0.001) on d 3 PI, in comparison with before the challenge. Accordingly, many lying conducts increased at d 3 PI (P < 0.05). In conclusion, both treatments had positive effects against Salmonella, and behavior analysis appears to be a sensible tool to be considered.

Lactulose and Lactobacillus plantarum, a potential complementary synbiotic to control postweaning colibacillosis in piglets.

The potential of a prebiotic oligosaccharide lactulose, a probiotic strain of Lactobacillus plantarum, or their synbiotic combination to control postweaning colibacillosis in pigs was evaluated using an enterotoxigenic Escherichia coli (ETEC) K88 oral challenge. Seventy-two weanlings were fed four diets: a control diet (CTR), that diet supplemented with L. plantarum (2 × 10(10) CFU · day(-1)) (LPN), that diet supplemented with 10 g · kg(-1) lactulose (LAC), or a combination of the two treatments (SYN). After 7 days, the pigs were orally challenged. Six pigs per treatment were euthanized on days 6 and 10 postchallenge (PC). Inclusion of lactulose improved the average daily gain (ADG) (P < 0.05) and increased lactobacilli (P < 0.05) and the percentage of butyric acid (P < 0.02) in the colon. An increase in the ileum villous height (P < 0.05) and a reduction of the pig major acute-phase protein (Pig-MAP) in serum (P < 0.01) were observed also. The inclusion of the probiotic increased numbers of L. plantarum bacteria in the ileum and colon (P < 0.05) and in the total lactobacilli in the colon and showed a trend to reduce diarrhea (P = 0.09). The concentrations of ammonia in ileal and colonic digesta were decreased (P < 0.05), and the villous height (P < 0.01) and number of ileal goblet cells (P < 0.05) increased, at day 10 PC. A decrease in plasmatic tumor necrosis factor alpha (TNF-α) (P < 0.01) was also seen. The positive effects of the two additives were combined in the SYN treatment, resulting in a complementary synbiotic with potential to be used to control postweaning colibacillosis.

Screening of extracts from natural feed ingredients for their ability to reduce enterotoxigenic Escherichia coli (ETEC) K88 adhesion to porcine intestinal epithelial cell-line IPEC-J2.

Enterotoxigenic Escherichia coli (ETEC) K88 is the most prevalent enteropathogen in weaned piglets, with the ability to express fimbria F4 and specifically attach to intestinal receptors in the young piglet. The prevention of ETEC K88 adhesion to the epithelium by interfering in this fimbria-receptor recognition provides an alternative approach to prevent the initial stage of disease. The aim of this study is to screen, among different feed ingredients (FI), their ability to reduce ETEC K88 attachment to the porcine intestinal epithelial cell-line (IPEC-J2). The selected FI consisted of products of a vegetable or dairy origin, and microbial by-products, which could be suitable to be included in piglet's diet. Incubation of a mixture of each FI extract with the bacteria on IPEC-J2 monolayer was allowed. After washing with PBS to remove the non-adhered bacteria, the culture medium was added to grow the adhered bacteria and, simultaneously, to keep the cells alive. Then, the bacterial growth was monitored in a spectrophotometer reader for 12h. Casein glycomacropeptide (CGMP), locust bean (LB), exopolysaccharide (EPS) and wheat bran (WB) reduced the number of attached ETEC K88 to IPEC-J2, but no anti-adhesive effect was found for soybean hulls, sugar-beet pulp, locust gum, fructooligosaccharides, inulin, mushroom, mannanoligosaccharides or the fermented product from Aspergillus oryzae. The lineal analysis of dose responses demonstrated lineal activity (P<0.0001) for CGMP, LB, EPS and WB. These in vitro results suggest CGMP, LB, EPS and WB as good candidates to be included in piglet's diet with supported functional activity against colibacillosis.

Use of mannanoligosaccharides and zinc chelate as growth promoters and diarrhea preventative in weaning pigs: Effects on microbiota and gut function.

The efficacy of a commercial source of mannanoligosacharides (BM), organic zinc (BP), or their combination to enhance performance, gastrointestinal health, and immune response in weaned pigs was evaluated. A total of 128 piglets, weaned at 20 +/- 2 d, were housed in 32 pens. Animals received 1 of 4 dietary treatments: a control diet (CT) to which 0.2% of BM, 80 mg/kg of Zn as BP, or both additives (BMP) were added. The experiment lasted for 5 wk including a prestarter period of 2 wk and a starter period of 3 wk. Body weight was recorded and daily feed intake was calculated. Fecal consistency was monitored for the first 21 d. After 2 wk, 32 animals were killed, digesta samples from the stomach, ileum, and cecum were collected, and pH and the short-chain fatty acid profile were determined. Microbiological counts for enterobacteria and lactobacilli were evaluated using quantitative PCR. Histological parameters in the jejunum and immunoglobulin concentrations in serum and ileal digesta were also measured. Both additives improved G:F during the starter period (0.63, 0.69, 0.67, and 0.68 for CT, BM, BP, and BMP, respectively; P < 0.04). Mean fecal score values for the first 21 d were improved by BM and BP, showing decreased values compared with the CT diet (1.22, 0.89, 0.87, and 1.06 for CT, BM, BP, and BMP, respectively; P = 0.002). The addition of BM decreased enterobacteria counts in the jejunum (9.13, 8.05, 8.87, and 7.89 log 16S rRNA gene copies/g of matter for CT, BM, BP, and BMP, respectively; P = 0.05). Empty ileal weight, defined as the segment including the continuous Peyer's patch, tended (P = 0.08) to increase with BP treatment (8.9, 9.6, 11.9, and 10.3 g/kg of BW for CT, BM, BP, and BMP, respectively). Crypt depths in the jejunum were lower in animals fed the combination of the additives (BPM) compared with those fed the control diet (281 vs. 235; P < 0.03). No significant differences were registered in pH, short-chain fatty acids, or serum and ileal immunoglobulin concentrations. The results suggest that the use of BM or BP can improve the efficiency of gain during the starter period.

The response of gastrointestinal microbiota to avilamycin, butyrate, and plant extracts in early-weaned pigs.

An experiment was designed to evaluate the effects of 3 different additives on the gastrointestinal microbiota of early-weaned pigs. Early-weaned (18 to 22 d; n = 32) pigs (6.0 +/- 0.10 kg of BW) from 8 litters were randomly distributed into 8 pens. Each pen was assigned 1 of 4 dietary treatments: a prestarter or control diet, the control diet with 0.04% avilamycin (AB), with 0.3% sodium butyrate, or with 0.03% plant extract mixture (XT; standardized mixture with 5% (wt/wt) carvacrol extracted from Origanum spp., 3% cinnamaldehyde extracted from Cinnamonum spp., and 2% capsicum oleoresin from Capsicum annum). At the end of the experimental period, 8 pigs per treatment were killed, and samples of their intestinal content were taken. The total bacterial load along the gastrointestinal tract (GIT; stomach, jejunum, cecum, and distal colon) and the lactobacilli and enterobacteria in the jejunum and cecum were measured by quantitative PCR. The total microbial counts along the GIT did not differ among the diets, but there was an increase in the lactobacilli:enterobacteria ratio in the cecum of the piglets on the XT diet (P = 0.003). Restriction fragment length polymorphism of the PCR-amplified V3, V4, and V5 regions of the 16S rDNA gene showed changes in the structure of the microbial community in the jejunum. Dendrograms grouped animals by diets; control with 0.3% sodium butyrate was the treatment that promoted the biggest changes in the microbial ecosystem, followed by AB and then XT. Biodiversity increased when using additives compared with the control diet (P = 0.002). Microbial metabolic activity along the hindgut was studied using the concentration of purine bases and carbohydrase activities. Different patterns for purine bases were observed between diets (diet x intestinal section, P = 0.01). The control diet reached a maximum purine base concentration at the end of the colon, whereas that of the AB diet was reached at the cecum. We could not detect any cellulase or xylanase activities in animals of this age. Appreciable amylase and amylopectinase activities were found, but they did not differ between diets. The results suggest that the effects of avilamycin, butyrate, or the plant extract would not be related to a reduction in the number of total bacteria inhabiting different sections of the GIT but rather to changes in the ecological structure and metabolic activity of the microbial community.

Effects of butyrate, avilamycin, and a plant extract combination on the intestinal equilibrium of early-weaned pigs.

We evaluated the effects of 3 additives, sodium butyrate (AC), avilamycin (AB), and a combination of plant extracts (XT), on the productive performance and the intestinal environment of the early-weaned pig. The XT was a standardized mixture with 5% (wt/wt) carvacrol (from Origanum spp.), 3% cinnamaldehyde (from Cinnamonum spp.), and 2% capsicum oleoresin (from Capsicum annum). Pigs (n = 32) weaned at 18 to 22 d of age with an initial BW of 6.0 +/- 0.10 kg were allocated to 8 pens that, in turn, were allocated to 4 treatments. The treatments included a basal diet (CT) or the basal diet supplemented with 0.3% of AC, 0.04% of AB, or 0.03% of XT. Productive performance was determined during the initial 14 d postweaning. On d 19 and 21 of the experiment, the pigs were killed to allow collection of digesta and intestinal tissue to evaluate variables indicative of aspects of the gastrointestinal environment. Treatments AB and AC improved G:F (P = 0.012 and 0.003, respectively) compared with the CT. Butyrate included in the diet was only detected in the stomach but not in cranial jejunum. When compared with CT, AC produced a lower ileal starch digestibility (P = 0.002) and a lower whole-tract OM and starch digestibility (P = 0.001 and 0.003, respectively), related to a lower VFA concentration in the cranial colon (P = 0.082) and a numerically reduced branched VFA percentage in the rectum. The AB treatment diminished propionate production in caudal colon (P = 0.002) and rectum (P = 0.012) compared with CT. The AC group exhibited deeper crypt depth in the jejunum without variations in villus height compared with CT (P = 0.042). The AC and AB groups also increased goblet cell presence in the colon (P = 0.001 and 0.032, respectively). On the other hand, AB and XT diminished intraepithelial lymphocytes in the jejunum (P = 0.003 and 0.034, respectively). The XT increased lymphocyte presence in the colon (P = 0.003). These results show the important influence of AB and AC on productive performance and on pig gut dynamics. The intestinal modifications observed for AB and AC compared with CT suggest distinct modes of action for each additive.

Relationship between dietary unsaturation and vitamin E in poultry.

Vitamin E requirements are linked to dietary polyunsaturated fatty acids (PUFA) content as a result of the protective effect of vitamin E from lipid peroxidation. On the other hand, it has been suggested that dietary PUFA interfere with vitamin E absorption. A 4 x 4 factorial study was planned to assess the effect of dietary vitamin E inclusion level (0, 100, 200 and 400 mg/kg) and degree of unsaturation (15, 34, 45 and 61 g PUFA/kg) on vitamin E apparent absorption and tissue deposition in poultry. A total of 192 female broiler chickens were used. A digestibility balance was carried out between 19 and 23 days of age to calculate apparent absorption of fat and vitamin E. The livers of 96 animals were obtained at 44 days of age for vitamin E determination. Increasing dietary levels of vitamin E reduced its apparent absorption. The more saturated diet reduced fat and vitamin E apparent absorption while PUFA levels from 34 to 61 g/kg did not modify this parameter but reduced the hepatic vitamin E concentration, suggesting a greater systemic use of this vitamin. These results suggest that PUFA do not limit vitamin E absorption, although they may increase its degradation in the gastrointestinal tract.

Large bowel fermentation of maize or sorghum-acorn diets fed as a different source of carbohydrates to Landrace and Iberian pigs.

Twenty-four finishing pigs (twelve Iberian and twelve Landrace) were used in a growing and slaughtering experiment. Animals were fed two diets differing in their ingredients, maize (diet C) or sorghum-acorn (diet A). At an average weight of 107.0 kg pigs were slaughtered and hindgut digesta sampled to study the effect of breed and diet on large bowel fermentation. Flows of digesta to the hindgut compartment were estimated based on an indigestible flow marker (Cr2O3) and were higher in Iberian than in Landrace pigs (P<0.001), and higher in animals fed diet A than diet C (P=0.07). The higher flows in Iberian pigs were mainly associated with a higher voluntary feed intake (3.50 v. 2.70 kg/d, P<0.01) and lower ileal digestibility of NSP (-12.8 v. 47.8, P<0.01). Differences between diets were mainly associated with a lower ileal digestibility of starch from diet A (89.2 v. 96.9 %, P=0.06), although no differences in the resistant starch content were observed in vitro. Fermentation of different carbohydrates through the large bowel showed that NSP-glucose had lower digestibility in Iberian than in Landrace pigs (62.5 v. 94.2 %, P<0.001), but no differences were observed in starch, or other NSP-fibre fractions (arabinose, xylose and galactose). The type and amount of carbohydrates reaching the large bowel were related to the diet but also to breed, and promoted differences in the fermentative activity associated with different volatile fatty acid patterns and changes in microbial enzymic activity.

Urinary excretion of purine derivatives as an index of microbial-nitrogen intake in growing rabbits.

Three experiments were carried out to establish a response model between intake and urinary excretion of purine compounds. In Expt 1 the relationship between the intake of purine bases (PB) and the excretion of total purine derivatives (PD) was determined in seven growing rabbits with a mean initial live weight (LW) of 2.03 (SE 0.185) kg, aged 70 d, each fitted with a wooden neck collar to prevent caecotrophagy. They were fed on five experimental diets formulated with different levels of nucleic acids (0.00, 3.75, 7.50, 11.25, 15.00 g yeast-RNA/kg diet). The relationship between intake of purine (x, mumol/kg W0.75) and total urinary PD excretion (y, mumol/kg W0.75), y= 0.56 + 0.67x (r2 O.86; RSD 0.338), indicated that about 70% of duodenal PB were recovered as urinary PD and that the endogenous contribution was constant and independent of dietary PB supply. Endogenous excretion of PD (allantoin and uric acid) was measured in a second experiment using six rabbits fed on a purine-free diet and fitted with neck collars to avoid caecotrophagy. Basal daily urinary excretion values for allantoin and uric acid were 532 (SE 33.9) and 55 (SE 7.3) mumol/kg W0.75 respectively; xanthine and hypoxanthine were not found in urine samples and therefore the sum of allantoin and uric acid should comprise the total excretion of PD (588 (SE 40.1) mumol/kg W0.75). The xanthine oxidase (EC 1.2.3.2) activity in plasma, liver, duodenum, jejunum and kidney was measured in a third experiment. The activities of xanthine oxidase in duodenal and jejunal mucosa, liver and kidney were: 0.61 (SE 0.095), 0.37 (SE 0.045), 0.035 (SE 0.001) and 0 units/g fresh tissue respectively and in plasma 2.96 (SE 0.094) units/1. The results show that urinary excretion of PD may be a useful tool to estimate duodenal PB input and microbial protein intake once the relationship between PB and N has been established in caecal micro-organisms.

Excretion of endogenous and exogenous purine derivatives in sheep: effect of increased concentrate intake.

The present study examined the endogenous urinary excretion of purine derivatives (PD; allantoin, uric acid and xanthine plus hypoxanthine) in fed animals. Four Rasa Aragonesa ewes fitted with simple cannulas in the rumen and proximal duodenum were used. Animals were given a lucerne (Medicago sativa) hay diet, as sole feed (A) or supplemented, respectively, with 220 (B), 400 (C), and 550 (D) g rolled barley grain/d following a 4 x 4 random factorial design. Duodenal flow of purine bases (PB) was determined by the dual-phase marker system. 15N was infused continuously into the rumen to label exogenous or microbial PB. Duodenal PB flow and urinary excretion of PD increased with digestible organic matter intake showing a constant recovery of duodenal PB. The isotope dilution of PD in urine samples confirmed the presence of an endogenous fraction, originating from tissues, that increased from 115.2 (SE 5.84) mumol/kg W0.75 for the basal diet to 304.2 (SE 7.6) mumol/kg W0.75 at the highest level of duodenal PB.

Endogenous purine and pyrimidine derivative excretion in pregnant sows.

The present experiment was carried out to study the endogenous losses of purine and pyrimidine derivatives from pregnant sows. Three pregnant and three non-pregnant Large White x Landrace sows were fed on a purine-free diet composed of starch, glucose, sucrose and vegetable oil, with casein as the protein source. The experiment began, for the six animals, after diagnosis of pregnancy and was divided into six 12 d periods. Urine was collected during the first 3 d of each experimental period by means of a urethral catheter for determination of allantoin, uric acid, xanthine, hypoxanthine and pseudouridine concentrations. In the absence of dietary nucleic acids (NA), allantoin and, as a consequence, excretion of total purine derivatives (PD) decreased significantly to a constant value (128.3 (SE 7.07) mumol/kg metabolic live weight (W0.75) per d), an amount assumed to represent endogenous excretion. Excretion of uric acid (38.7 (SE 2.15) mumol/kg W0.75 per d), hypoxanthine (21.0 (SE 2.58) mumol/kg W0.75 per d) and xanthine (11.2 (SE 0.83) mumol/kg W0.75 per d) were not affected by the experimental treatment, although there was a significant decrease in hypoxanthine excretion in pregnant sows (from 25.5 to 5.2 mumol/kg W0.75 per d) compared with non-pregnant sows (from 26.7 to 44.8 mumol/kg W0.75 per d). Creatinine excretion was not affected by pregnancy and was used as an internal urinary marker. Purine excretion, either expressed as mumol/kg W0.75 per d or as the ratio PD: creatinine, was not affected by experimental treatment, although an apparent increase in pseudouridine excretion, a modified unsalvageable catabolite of RNA-pyrimidine, was found in late pregnancy (3.6 v. 5.2 mol/100 mol creatinine in non-pregnant sows compared with pregnant sows at 102 d collection.