Experimental facility had a greater effect on growth performance, gut microbiome, and metabolome in weaned pigs than feeding diets containing subtherapeutic levels of antibiotics: A case study.

The objective of this study was to define changes in the intestinal metabolome and microbiome associated with growth performance of weaned pigs fed subtherapeutic concentrations of antibiotics. Three experiments with the same antibiotic treatments were conducted on the same research farm but in two different facilities (nursery and wean-finish) using pigs weaned at 20-days of age from the same source herd and genotype, and fed the same diets formulated without antibiotics (NC) or with 0.01% chlortetracycline and 0.01% sulfamethazine (AB). Pigs were weighed and feed disappearance was determined on days (d) 10, 21, and 42 post-weaning to calculate average daily gain (ADG), average daily feed intake (ADFI), and gain:feed (G:F). On d 42, one pig/pen was selected for blood and ileal and cecal content collection. Targeted and untargeted metabolomic profiles were determined in serum and cecal contents using liquid chromatography-mass spectrometry, and composition of bacterial communities in intestinal content samples was determined by sequencing the V4 region of the 16s rRNA gene. Metabolomics and microbiome data were analyzed using diverse multivariate and machine learning methods. Pigs fed AB had significantly greater (P < 0.05) overall ADG and ADFI compared with those fed NC, and pig body weight, ADG, and G:F were also significantly different (P < 0.05) between experiments. Differences (P < 0.05) in serum metabolome along with ileal and cecal microbiome beta diversity were observed between experiments, but there were no differences in microbiome alpha diversity between experiments or treatments. Bacteria from the families Clostridiaceae, Streptomycetaceae, Peptostreptomycetaceae, and Leuconostocaceae were significant biomarkers for the AB treatment. In addition, pigs fed AB had increased serum arginine, histidine, lysine, and phenylalanine concentrations compared with NC. Percentage error from a random forest analysis indicated that most of the variation (8% error) in the microbiome was explained by the facility where the experiments were conducted. These results indicate that facility had a greater effect on growth performance, metabolome, and microbiome responses than feeding diets containing subtherapeutic levels of antibiotics.

Growth Performance, Metabolomics, and Microbiome Responses of Weaned Pigs Fed Diets Containing Growth-Promoting Antibiotics and Various Feed Additives.

The objective of this study was to determine the potential biological mechanisms of improved growth performance associated with potential changes in the metabolic profiles and intestinal microbiome composition of weaned pigs fed various feed additives. Three separate 42 day experiments were conducted to evaluate the following dietary treatments: chlortetracycline and sulfamethazine (PC), herbal blends, turmeric, garlic, bitter orange extract, sweet orange extract, volatile and semi-volatile milk-derived substances, yeast nucleotide, and cell wall products, compared with feeding a non-supplemented diet (NC). In all three experiments, only pigs fed PC had improved (p < 0.05) ADG and ADFI compared with pigs fed NC. No differences in metabolome and microbiome responses were observed between feed additive treatments and NC. None of the feed additives affected alpha or beta microbiome diversity in the ileum and cecum, but the abundance of specific bacterial taxa was affected by some dietary treatments. Except for feeding antibiotics, none of the other feed additives were effective in improving growth performance or significantly altering the metabolomic profiles, but some additives (e.g., herbal blends and garlic) increased (p < 0.05) the relative abundance of potentially protective bacterial genera that may be beneficial during disease challenge in weaned pigs.

Identification of Sinapine-Derived Choline from a Rapeseed Diet as a Source of Serum Trimethylamine N-Oxide in Pigs.

Choline and its metabolites have diverse and important functions in many physiological processes, especially for anabolic metabolism in growth and reproduction. Besides endogenous biosynthesis and direct choline supplement, choline esters in the diet are another source of choline in the body. Phenolic choline esters are a group of unique dietary choline esters rich in the seeds of Brassicaceae plants, among which sinapine is a choline ester of sinapic acid abundant in rapeseed. In this study, 40 nursery pigs were fed with rapeseed-derived feed ingredients (RSF) or soybean meal for 3 weeks (20 pigs/diet). The metabolic fate of sinapine-derived choline in RSF was examined by comparing the distribution of choline and its metabolites in digesta, liver, and serum samples by liquid chromatography-mass spectrometry analysis. The results showed that choline was released from extensive hydrolysis of sinapine in the small intestine. However, sinapine-derived choline did not increase the levels of choline and its major metabolites, including betaine, phosphocholine, and glycerophosphocholine, in the liver and serum. Instead, RSF feeding increased trimethylamine (TMA), the microbial metabolite of choline, in the large intestine and further increased trimethylamine N-oxide (TMAO), the oxidation metabolite of TMA, in the liver and serum. Overall, these results suggested that sinapine-derived choline from rapeseed feeding had limited influences on the post-absorption choline pool as a result of its low bioavailability but may serve as a major source of TMAO through microbial metabolism in nursery pigs. Improving the bioavailability of sinapine-derived choline might have the potential to modify the nutritional values and functionalities of rapeseed meal in swine feeding.

Dietary fiber sources and non-starch polysaccharide-degrading enzymes modify mucin expression and the immune profile of the swine ileum.

Due to their complex chemical and physical properties, the effects and mechanisms of action of natural sources of dietary fiber on the intestine are unclear. Pigs are commonly fed high-fiber diets to reduce production costs and non-starch polysaccharide (NSP)-degrading enzymes have been used to increase fiber digestibility. We evaluated the expression of mucin 2 (MUC2), presence of goblet cells, and ileal immune profile of pigs housed individually for 28 days and fed either a low fiber diet based on corn-soybean meal (CSB, n = 9), or two high fiber diets formulated adding 40% corn distillers' dried grains with solubles (DDGS, n = 9) or 30% wheat middlings (WM, n = 9) to CSB-based diet. Pigs were also fed those diets supplemented with a NSP enzymes mix (E) of xylanase, ß-glucanase, mannanase, and galactosidase (n = 8, 10, and 9 for CSB+E, DDGS+E and WM+E, respectively). Feeding DDGS and WM diets increased ileal MUC2 expression compared with CSB diet, and this effect was reversed by the addition of enzymes. There were no differences in abundance of goblet cells among treatments. In general, enzyme supplementation increased gene expression and concentrations of IL-1ß, and reduced the concentrations of IL-4, IL-17A and IL-11. The effects of diet-induced cytokines on modulating intestinal MUC2 were assessed in vitro by treating mouse and swine enteroids with 1 ng/ml of IL-4 and IL-1ß. In accordance with previous studies, treatment with Il-4 induced Muc2 and expansion of goblet cells in mouse enteroids. However, swine enteroids did not change MUC2 expression or number of goblet cells when treated with IL-4 or IL-1ß. Our results suggest that mucin and immune profile are regulated by diet in the swine intestine, but by mechanisms different to mouse, emphasizing the need for using appropriate models to study responses to dietary fiber in swine.

Fecal Hyodeoxycholic Acid Is Correlated With Tylosin-Induced Microbiome Changes in Growing Pigs.

The changes in the gut microbiome play an important role in the promoting effects of antibiotics, such as tylosin, to the health, and productivity of farm animals. Microbial metabolites are expected to be key mediators between antibiotics-induced microbiome changes and growth-promoting effects. The objective of this study was to extend the identification of tylosin-responsive microbes to the identification of tylosin-responsive metabolites in growing pigs. The feeding trial was conducted on a commercial farm using two pens of pigs fed diets with and without tylosin (40 mg/kg of diet). Fecal samples were collected from 10 pigs per pen at weeks 10, 13, 16, 19, and 22 of age, and subsequently analyzed using liquid chromatography-mass spectrometry (LC-MS) analysis. The multivariate model of LC-MS data showed that time-dependent changes occurred in the fecal metabolome of both control and tylosin-treated pigs. More importantly, the metabolomic profiles were similar between the tylosin treatment and control groups in weeks 10 and 22, but diverged during weeks 13-19. Subsequent analyses of the fecal metabolites contributing to the separation of two groups of pigs showed that hyodeoxycholic acid (HDCA), together with tylosin and its metabolites in feces, was greatly increased during weeks 13-19 (P < 0.05) in the group of pigs fed tylosin. The integration of current metabolomics data and the microbiome data from a previous study revealed the consistency between HDCA and a specific genus of microbes in the Clostridia family. Further studies are required to determine the causative relations between tylosin-elicited changes in HDCA and the microbiome as well as the role of HDCA in the growth promoting effects of tylosin.

Survival of porcine epidemic diarrhea virus (PEDV) in thermally treated feed ingredients and on surfaces.

BACKGROUND: Infection with Porcine Epidemic Diarrhea Virus (PEDV) causes vomiting, diarrhea, and dehydration in young pigs. The virus made its first appearance in the U.S. in 2013, where it caused substantial neonatal mortality and economic losses in the U.S. pork industry. Based on outbreak investigations, it is hypothesized that the virus could be transmitted through contaminated feed or contaminated feed surfaces. This potential risk created a demand for research on the inactivation kinetics of PEDV in different environments. Therefore, the objective of this study was to evaluate the survival of PEDV in 9 different feed ingredients when exposed to 60, 70, 80, and 90 °C, as well as the survival on four different surfaces (galvanized steel, stainless steel, aluminum, and plastic). RESULTS: Overall, there were no differences (P > 0.05) in virus survival among the different feed matrices studied when thermally processed at 60 to 90 °C for 5, 10, 15, or 30 min. However, the time necessary to achieve a one log reduction in virus concentration was less (P < 0.05) when ingredients were exposed to temperatures from 70 °C (3.7 min), 80 °C (2.4 min), and 90 °C (2.3 min) compared with 60 °C (4.4 min). The maximum inactivation level (3.9 log) was achieved when heating all ingredients at 90 °C for 30 min. There were no differences in the amount of time necessary to cause a one log reduction in PEDV concentration among the different surfaces. CONCLUSIONS: The results of this study showed that PEDV survival among the 9 feed ingredients evaluated was not different when exposed to thermal treatments for up to 30 min. However, different combinations of temperature and time resulted in achieving a 3 to 4 log reduction of PEDV in all feed ingredients evaluated. Finally, PEDV survival was similar on galvanized steel, stainless steel, aluminum and plastic.

Environmental persistence of porcine coronaviruses in feed and feed ingredients.

Porcine Epidemic Diarrhea Virus (PEDV), Porcine Delta Corona Virus (PDCoV), and Transmissible Gastroenteritis Virus (TGEV) are major threats to swine health and contaminated feed plays a role in virus transmission. The objective of our study was to characterize inactivation of PEDV, PDCoV, and TGEV in various feed ingredient matrices. Samples of complete feed, spray dried porcine plasma, meat meal, meat and bone meal, blood meal, corn, soybean meal, and corn dried distillers grains with solubles were weighed (5 g/sample) into scintillation vials and inoculated with 1 mL of PEDV, PDCoV, or TGEV. Samples were incubated at room temperature for up to 56 days. Aliquots were removed at various time points followed by preparing serial 10-fold dilutions and inoculating in cell cultures to determine the amount of surviving virus. Inactivation kinetics were determined using the Weibull model, which estimates a delta value indicating the time necessary to reduce virus concentration by 1 log. Delta values of various ingredients were compared and analyzed as to their nutrient composition. Soybean meal had the greatest delta value (7.50 days) for PEDV (P < 0.06) as compared with all other ingredients. High delta values (P < 0.001) were observed in soybean meal for PDCoV (42.04 days) and TGEV (42.00 days). There was a moderate correlation between moisture content and the delta value for PDCoV (r = 0.49, P = 0.01) and TGEV (r = 0.41, P = 0.02). There was also a moderate negative correlation between TGEV survival and ether extract content (r = -0.51, P = 0.01). In conclusion, these results indicate that the first log reduction of PDCoV and TGEV takes the greatest amount of time in soybean meal. In addition to this, moisture and ether content appear to be an important determinant of virus survival in feed ingredients.

Comparison of Thermal and Non-Thermal Processing of Swine Feed and the Use of Selected Feed Additives on Inactivation of Porcine Epidemic Diarrhea Virus (PEDV).

Infection with porcine epidemic diarrhea virus (PEDV) causes diarrhea, vomiting, and high mortality in suckling pigs. Contaminated feed has been suggested as a vehicle of transmission for PEDV. The objective of this study was to compare thermal and electron beam processing, and the inclusion of feed additives on the inactivation of PEDV in feed. Feed samples were spiked with PEDV and then heated to 120-145°C for up to 30 min or irradiated at 0-50 kGy. Another set of feed samples spiked with PEDV and mixed with Ultracid P (Nutriad), Activate DA (Novus International), KEM-GEST (Kemin Agrifood), Acid Booster (Agri-Nutrition), sugar or salt was incubated at room temperature (~25°C) for up to 21 days. At the end of incubation, the virus titers were determined by inoculation of Vero-81 cells and the virus inactivation kinetics were modeled using the Weibull distribution model. The Weibull kinetic parameter delta represented the time or eBeam dose required to reduce virus concentration by 1 log. For thermal processing, delta values ranged from 16.52 min at 120°C to 1.30 min at 145°C. For eBeam processing, a target dose of 50 kGy reduced PEDV concentration by 3 log. All additives tested were effective in reducing the survival of PEDV when compared with the control sample (delta = 17.23 days). Activate DA (0.81) and KEM-GEST (3.28) produced the fastest inactivation. In conclusion, heating swine feed at temperatures over 130°C or eBeam processing of feed with a dose over 50 kGy are effective processing steps to reduce PEDV survival. Additionally, the inclusion of selected additives can decrease PEDV survivability.