A. G. Van Kessel

Effects of Commensal Bacteria on Intestinal Morphology and Expression of Proinflammatory Cytokines in the Gnotobiotic Pig

A germ-free neonatal pig model was established to determine the effects of bacterial colonization by different species on small intestinal morphology and proinflammatory cytokine gene expression. Two experimental groups of 16 pigs were aseptically delivered by cesarian section and allocated into 4 gnotobiotic isolators. Pigs were either maintained germ-free (GF), or were orally inoculated with either a single strain of nonpathogenic Escherichia coli (EC) or Lactobacillus fermentum (LF) or conventionalized with adult porcine feces (CV). After 13 days tissue samples were collected at 5 regions corresponding to 5%, 25%, 50%, 75%, and 95% of the small intestine (SI) length. In Experiment 2, the GF isolator became contaminated with Staphylococcus epidermidis (SE). In general, intestinal responses to bacterial colonization were similar among GF, LF, and SE pigs, and intestinal responses in EC pigs were more similar to CV pigs. Responses to bacterial colonization were most pronounced in the distal SI regions (50%–95%), suggesting that nonmicrobiai factors may be more important in the proximal SI. Relative to CV pigs, the distal intestines of GF, LF, and SE pigs were characterized by long villi, shallow crypts, Increased relative intestinal mass, and decreased lamina propria cellularity, whereas SI morphology was intermediate in EC pigs. Relative expression of proinflammatory cytokines interleukin-1β (IL-1β) and IL-6 generally increased distally in the SI and was highest in EC and CV pigs. We observed regional variation in SI morphology and proinflammatory cytokine expression, which differed with bacterial species. This study demonstrates that bacterial species differentially affect intestinal morphology and expression of proinflammatory cytokines and suggests that neonatal bacterial colonization patterns may have long-term effects on intestinal health and development

Diets supplemented with chickpea or its main oligosaccharide component raffinose modify faecal microbial composition in healthy adults

The effects of diets supplemented with either chickpea or its main oligosaccharide raffinose on the composition of the faecal microbial community were examined in 12 healthy adults (18-65 years) in a randomised crossover intervention study. Subjects consumed their usual diet supplemented with soups and desserts that were unfortified, or fortified with either 200 g/d of canned chickpeas or 5 g/d of raffinose for 3 week periods. Changes in faecal bacterial populations of subjects were examined using 16S rRNA-based terminal restriction fragment length polymorphisms (T-RFLP) and clone libraries generated from the diet pools. Classification of the clone libraries and T-RFLP analysis revealed that Faecalibacterium prausnitzii, reported to be an efficient butyrate producer and a highly metabolically active bacterium in the human intestinal microbiota, was more abundant in the raffinose diet and the chickpea diet compared to the control diet. However, no significant difference was observed in the faecal total short chain fatty acid concentration or in the levels of the components (butyrate, acetate and propionate) with the chickpea diet or the raffinose diet compared to the control diet. Bifidobacterium species were detected by T-RFLP in all three diet groups and quantitative real-time PCR (qPCR) analysis showed a marginal increase in 16S rRNA gene copies of Bifidobacterium with the raffinose diet compared to control (P>0.05). The number of individuals showing TRFs for the Clostridium histolyticum - Clostridum lituseburense groups, which include pathogenic bacteria species and putrefactive bacteria, were lower in the chickpea diet compared to the other two treatments. Diet appeared to affect colonisation by a high ammonia-producing bacterial isolate which was detected in 83%, 92% and 42% of individuals in the control, raffinose and chickpea groups, respectively. Our results indicate that chickpea and raffinose have the potential to modulate the intestinal microbial composition to promote intestinal health in humans.

Nutritional and physiological role of medium-chain triglycerides and medium-chain fatty acids in piglets

Abstract Medium-chain fatty acids (MCFAs) are found at higher levels in milk lipids of many animal species and in the oil fraction of several plants, including coconuts, palm kernels and certain Cuphea species. Medium-chain triglycerides (MCTs) and fatty acids are efficiently absorbed and metabolized and are therefore used for piglet nutrition. They may provide instant energy and also have physiological benefits beyond their energetic value contributing to several findings of improved performance in piglet-feeding trials. MCTs are effectively hydrolyzed by gastric and pancreatic lipases in the newborn and suckling young, allowing rapid provision of energy for both enterocytes and intermediary hepatic metabolism. MCFAs affect the composition of the intestinal microbiota and have inhibitory effects on bacterial concentrations in the digesta, mainly on Salmonella and coliforms. However, most studies have been performed in vitro up to now and in vivo data in pigs are still scarce. Effects on the gut-associated and general immune function have been described in several animal species, but they have been less studied in pigs. The addition of up to 8% of a non-esterified MCFA mixture in feed has been described, but due to the sensory properties this can have a negative impact on feed intake. This may be overcome by using MCTs, allowing dietary inclusion rates up to 15%. Feeding sows with diets containing 15% MCTs resulted in a lower mortality of newborns and better development, particularly of underweight piglets. In conclusion, MCFAs and MCTs offer advantages for the improvement of energy supply and performance of piglets and may stabilize the intestinal microbiota, expanding the spectrum of feed additives supporting piglet health in the post-weaning period.

Barley and oat cultivars with diverse carbohydrate composition alter ileal and total tract nutrient digestibility and fermentation metabolites in weaned piglets.

An experiment was conducted to evaluate the effects of cereal carbohydrate form (isolated v. cereal matrix) and level, especially mixed-linked β-glucan (hereafter referred to as β-glucan) and starch amylase/amylopectin ratio on nutrient digestibility and fermentation parameters in the intestines of weaned pigs. Four hulless barley cultivars containing varying β-glucan levels (41 to 84 g/kg) were compared with hulled barley, supplemented or not with a β-glucan concentrate (BBG; 270 g/kg β-glucan) and two oat cultivars for digestibility and fermentation metabolites. Seventy-two weaned piglets (BW = 12.8 ± 1.9 kg) were assigned to one of nine diets composed of 815 g/kg cereal, 60 g/kg whey, 90 g/kg soy protein isolate and 35 g/kg minerals. After 15 days, the pigs were killed, and digesta collected from ileum and colon were analyzed for proximate nutrients, short-chain fatty acids (SCFAs), lactic acid (LA) and ammonia. Ileal and total tract digestibility of proximate nutrients and non-starch polysaccharides (NSPs) were determined using HCl-insoluble ash as a marker. Organic matter (OM) ileal digestibility was greater (P < 0.05) for diets based on hulless barley (77% ± 1.1% on average), as compared with hulled barley (64% ± 1.4%) and oat (58% ± 1.5%). Similar trends were found for total tract OM digestibility, varying from 90% ± 0.3% for hulless barley to 67% ± 0.4% for oat, on average. NSP digestibility differed (P < 0.05) within and between cereal types, ranging from 20% (hulled barley plus 163 g/kg BBG or 40 g/kg β-glucan) to 51% (SB94893 hulless barley cultivar with high β-glucan and high amylose ratio) at the ileum and from 44% (hulled barley) to 84% (SB94893 cultivar) at the total tract level. No dietary effect (P > 0.05) was found for SCFA concentration in ileal contents, whereas in colonic contents, SCFA was lower in pigs fed oat (P < 0.001). LA concentration was greater (P < 0.001) in the colon of pigs fed hulless barley than in pigs fed hulled barley and oat. Expressed per kg carbohydrate (NSP + starch) fermented, the ammonia concentration at the colon was lowest for hulled barley diets (supplemented with β-glucan) and the highest for oat diets. In conclusion, the interaction of both form and level of β-glucan impacted nutrient digestibility and fermentation. Hulless barleys with high soluble NSP such as β-glucan and resistant starch yielded, in general higher SCFA and LA and lower ammonia. Hulless barleys may, therefore, have potential for use in feeding strategies designed to improve gut health in pigs.

Intestinal microbiota differentially affect brush border enzyme activity and gene expression in the neonatal gnotobiotic pig.

To study microbial influence on intestinal development pertaining to nutrient digestion, two separate gnotobiotic experiments were performed, each with 16 piglets allocated to four treatment groups: germfree (GF), monoassociation with Escherichia coli, monoassociation with Lactobacillus fermentum or conventionalization with faecal bacteria (CV). Enzyme activity and gene expression of lactase phlorizin hydrolase (LPH) and aminopeptidase N (APN) were measured in isolated enterocytes, harvested on day 14, using specific substrates and quantitative PCR respectively. Enterocytes of CV pigs had reduced APN activity, but had increased gene expression relative to GF, making the specific activity:mRNA (A:G) ratio dramatically lower (p < 0.05). Similarly, LPH A:G ratio was significantly reduced (p < 0.05) in enterocytes of CV pigs as compared with GF. The results of co-incubation of L. fermentum, E. coli and faecal bacteria with APN indicate a direct relationship between enzyme inactivation and specific A:G ratio in enterocytes. We conclude that enterocyte up-regulation of APN expression occurs as either a direct response to microbial colonization or as a feedback mechanism in response to reduced enzyme activity through microbial degradation. This mechanism may play a role in ensuring effective competition of the host with the intestinal microbiota for available nutrients.

Dose-dependent effects of dietary zinc oxide on bacterial communities and metabolic profiles in the ileum of weaned pigs.

Pharmacological levels of zinc oxide (ZnO) can improve the health of weaning piglets and influence the intestinal microbiota. This experiment aimed at studying the dose-response effect of five dietary concentrations of ZnO on small intestinal bacteria and metabolite profiles. Fifteen piglets, weaned at 25 ± 1 days of age, were allocated into five groups according to body weight and litter. Diets were formulated to contain 50 (basal diet), 150, 250, 1000 and 2500 mg zinc/kg by adding analytical-grade (>98% purity) ZnO to the basal diet and fed ad libitum for 14 days after a 7-day adaptation period on the basal diet. Ileal bacterial community profiles were analysed by denaturing gradient gel electrophoresis and selected bacterial groups quantified by real-time PCR. Concentrations of ileal volatile fatty acids (VFA), D- and L-lactate and ammonia were determined. Species richness, Shannon diversity and evenness were significantly higher at high ZnO levels. Quantitative PCR revealed lowest total bacterial counts in the 50 mg/kg group. Increasing ZnO levels led to an increase (p = 0.017) in enterobacteria from log 4.0 cfu/g digesta (50 mg/kg) to log 6.7 cfu/g digesta (2500 mg/kg). Lactic acid bacteria were not influenced (p = 0.687) and clostridial cluster XIVa declined (p = 0.035) at highest ZnO level. Concentration of total, D- and L-lactate and propionate was not affected (p = 0.736, p = 0.290 and p = 0.630), but concentrations of ileal total VFA, acetate and butyrate increased markedly from 50 to 150 mg/kg and decreased with further increasing zinc levels and reached low levels again at 2500 mg/kg (p = 0.048, p = 0.048 and p = 0.097). Ammonia decreased (p < 0.006) with increasing dietary ZnO level. In conclusion, increasing levels of dietary ZnO had strong and dose-dependent effects on ileal bacterial community composition and activity, suggesting taxonomic variation in metabolic response to ZnO.

Effects of Peripartum Propylene Glycol Supplementation on Nitrogen Metabolism, Body Composition, and Gene Expression for the Major Protein Degradation Pathways in Skeletal Muscle in Dairy Cows

Early-lactating dairy cows mobilize body protein to provide amino acids that are directed toward gluconeogenesis and milk protein synthesis. Propylene glycol (PG) is a precursor of ruminal propionate, and feeding PG has been reported to improve energy supply by increasing blood glucose. Our hypothesis was that feeding PG could spare body protein by providing an alternative source of carbon for gluconeogenesis. The major objectives of this study were 1) to delineate the effects of pre- and postpartum PG supplementation in transition dairy cows on whole-body nitrogen balance, urinary 3-methylhistidine (3-MH) excretion, body composition, and gene expression profiles for the major protein degradation pathways in skeletal muscle; and 2) to characterize the changes in body protein metabolism during the periparturient period. Sixteen pregnant cows (7 primiparous and 9 multiparous) were paired based on expected calving dates and then randomly assigned within each pair to either a basal diet (control) or basal diet plus 600 mL/d of PG. Diets were fed twice daily for ad libitum intake, and PG was fed in equal amounts as a top dress from d -7 to d 45. All measurements were conducted at 3 time intervals starting at d -14 +/- 5, d 15, and d 38 relative to calving. Propylene glycol had no effect on whole-body N balance, urinary 3-MH excretion, or body composition. However, N balance was lower at d 15 and 38, compared with d -14. Urinary excretion of 3-MH was lower at d -14 than at d 15 and 38. Supplemental PG had no effect on body weight (BW) and all components of empty BW. On average, cows fed both diets mobilized 19 kg of body fat and 14 kg of body protein between d -14 and d 38. Supplemental PG had no effect on mRNA abundance in skeletal muscle for m-calpain, and the 14-kDa ubiquitin-carrier protein E2 (14-kDa E2) and proteasome 26S subunit-ATPase components of the ubiquitin-mediated proteolytic pathway; however, PG supplementation downregulated mRNA expression for mu-calpain at d 15, and tended to downregulate mRNA expression for ubiquitin at d 15 and 38. Relative to calving, mRNA abundance for m- and mu-calpain, ubiquitin, and 14-kDa E2 were greater at d 15 compared with d -14 and d 38. In summary, these results indicate that transitional effects on whole-body metabolism and gene expression for the Ca(2+)-dependent and ubiquitin-mediated proteolytic pathways in skeletal muscle were more pronounced than those elicited by PG supplementation.

Effect of day length on cause of mortality, leg health, and ocular health in broilers

An experiment was conducted to study the effect of day length, sex, and genotype (Ross × Ross 308 and 708) on mortality causes, bird mobility, footpad health, and ocular size, with 4 trials within the experiment. Four graded day lengths were chosen to allow the study of relationship between day length and health parameters, including 14L:10D, 17L:7D, 20L:4D, and 23L:1D. The primary statistical tools used to assess the day length relationships were regression analysis (Proc Reg and RSReg of SAS). Data were also analyzed as a 4 (lighting program) × 2 (sex) × 2 (genotype) factorial arrangement. Total mortality, as well as mortality due to metabolic and skeletal disease, decreased linearly with increasing inclusion of darkness (7- to 32-, 7- to 38-, and 7- to 48-d periods). Infectious disorders were quadratically related to day length (7- to 48-d period only), with birds under 20L having the highest level. Day length was linearly or quadratically related to average gait score in a positive fashion, and the incidence of birds falling in painful gait score categories increased linearly with increasing day length. Average footpad lesion scores increased with increasing day length (28 and 35 d). The 23L photoperiod resulted in heavier eye weights than other lighting programs. Males had a higher mortality and morbidity rate and a higher average gait score than females. Average footpad score was lower for males than females (28 and 35 d). Overall mortality was higher for 308 than 708 broilers; hence, levels of specific mortality causes were higher. Average gait scores were lower for 308 than 708 birds in 2 of the 3 time periods measured and footpad lesions were higher. To conclude, many aspects of broiler health improve with decreasing day length.

Effect of immunization against somatostatin in the pregnant ewe on growth and endocrine status of the neonatal lamb.

Absorption of somatostatin (SRIF) specific antibodies from colostrum of ewes actively immunized against SRIF may improve growth rate of the neonatal lamb by neutralizing the inhibitory effects of SRIF on pituitary and thyroid function. Growth and endocrine parameters in the offspring of SRIF immunized (SI) and control (C) crossbred ewes were examined. Lamb weight was recorded at birth and twice each week to 24 days of age. Blood samples were collected prior to first suckle and twice each week. At 21 to 24 days of age, in separate experiments, lambs were infused with glucose (0.29 g/kg), arginine (0.25 g/kg) or thyrotropin-releasing hormone (TRH; 0.33 microgram/kg). A strong correlation (R = 0.88; P less than .01) was observed between anti-SRIF titre in the ewe at parturition and in the lamb at 3 days of age. No effect on lamb birth weight (SI 4.28 +/- 0.27 kg; C 4.35 +/- 0.23 kg) was observed. At 24 days of age cumulative gain in SI lambs (5.4 +/- 0.32 kg) was greater (P less than .05) than in C lambs (4.5 +/- 0.32 kg). The growth hormone secretory responses to glucose or arginine were not affected by treatment. Plasma IGF-I, plasma thyroxine (T4) and the plasma thyrotropin and T4 responses to TRH were not different between treatments. Plasma triiodothyronine (T3) was higher (P less than .05) in SI (2.46 +/- .10 ng/ml) than in C (2.01 +/- .05 ng/ml) lambs, however, the plasma T3 response to TRH was lower in SI lambs. Plasma glucose (mg/dl) was higher (P less than .05) in SI (118.4 +/- 1.7) than in C (106.0 +/- 4.0) lambs. Plasma insulin was not affected by treatment. Increased plasma T3 and glucose concentrations during SRIF immunoneutralization in the neonate lamb may be important factors contributing to the growth response observed.

Differences in intestinal mucin dynamics between germ-free and conventionally reared chickens after mannan-oligosaccharide supplementation

A germ-free (GF) chicken model was used to test 2 hypotheses: 1. microbial colonization of the gastrointestinal tract (GIT) influences mucin gene expression and mucin types; and 2. mannan oligosaccharide (MOS) supplementation affects GIT cells directly, without bacteria mediation, compared with bacterial-mediated effect (i.e., indirectly). Gnotobiotic isolators were used: 1) GF, 2) with a single bacteria population, and 3) conventionalized by exposure to cecal bacterial contents. Each was divided to 2 diet groups: with or without MOS (2 kg/t) for 1 wk. Results show that the absence of bacteria in the GIT caused a reduction in neutral and acidic goblet cell (GC) number and density, an increase in sulfated mucin, absence of sialylated GC, and reduced mucin 2 mRNA expression in the small intestine of GF compared with conventional birds. These results indicate a reduced development of mucin production and secretion in the absence of GIT bacteria implying a less mature small intestine mucosa, supporting our first hypothesis. Results from the single bacteria population group were not conclusive and did not support any of the hypotheses. Supplementation of MOS, regardless of microbial presence, caused a reduction in neutral GC number and density but increased neutral GC area. The MOS caused different effects on acidic mucins in conventional and GF birds, causing a reduction in sialylated GC number (conventional) and a reduction in sulfated GC density (GF), all supporting a direct effect of MOS in GF animals, in addition to an indirect effect via gut microflora.