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BACKGROUND Dietary calcium and phosphorus are required for bone and muscle development. Deficiencies of these macrominerals reduce bone mineral and muscle accretion potentially via alterations of mesenchymal stem cell (MSC) and satellite cell (SC) activities. OBJECTIVES With increasing interest in the role of early-life events on lifetime health outcomes, we aimed to elucidate the impact of dietary calcium and phosphorus, from deficiency through excess, on MSC and SC characteristics during neonatal development. METHODS Neonatal pigs [30 females, 1-d-old, 1.46 ± 0.04 kg body weight (BW)] were fed milk replacers for 16 d that were isonitrogenous and isocaloric with a consistent ratio of calcium to phosphorus, but either 25% deficient (calcium: 0.78%; phosphorus: 0.60%; CaPD), adequate (calcium: 1.08%; phosphorus: 0.84%; CaPA), or 25% in excess (calcium: 1.38%; phosphorus: 1.08%; CaPE) of calcium and phosphorus requirements based on sow-milk composition and extrapolation from NRC requirements for older pigs. BW and feed intake were recorded daily. Blood was collected for serum phosphorus, parathyroid hormone (PTH), and fibroblast growth factor 23 (FGF23) determination. Humeri were collected for MSC isolation and radii/ulnae bone were collected for analysis. Longissimus dorsi muscle was collected for SC isolation and analysis. RESULTS There was 4.6% increase in bone ash percentage in CaPE- versus CaPD-fed pigs (P

Dietary protein intake during the neonatal period is critical for lifetime muscle development. Appropriate nutrition during the neonatal period may have lifelong impacts on animal growth and muscle development. We hypothesized that minor changes (25% ± adequacy) in dietary protein during the neonatal phase of life will impact future lean growth potential. The goal of this project was to determine the effects of a diet with a 25% deficiency (D), adequacy (A), and 25% excess in protein (E) on neonatal muscle hypertrophy. Dietary crude protein level in the adequate group (27%) was based upon sow’s milk composition and an extrapolation of NRC recommendations. Thirty, individually housed, 1-day-old piglets were divided into 3 groups and fed milk replacers with a 25% deficiency (D), adequacy (A), or 25% excess in protein (E) over a 17-day period. Body weight and feed intake were recorded daily, and a section of the Longissimus dorsi were collected for molecular and histological evaluation at the end of the experiment. Piglets fed E had significantly greater final body weight, (P

Pork quality is evaluated by commercial processors along the ventral surface of whole boneless loins. However, in retail settings boneless chops are evaluated individually for quality. The objective was to determine the variability in marbling attributable to chop location, sex, and sire line. The population of pigs evaluated were sired by commercial duroc boars selected for meat quality (MQ; n=96) or lean growth (LG; n=96) and equally split between sexes. Upon slaughter and fabrication, bone-in chops were removed from four locations along the loin (A=6th rib, B=10th rib, C=last rib, D=4th lumbar). Pairs of chops from each location were used for visual color, visual marbling, and subjective firmness, Warner-Bratzler shear force, and determination of moisture and extractable lipid (IMF). Variability across location, sex, and sire line was measured using a Levene’s test and the VARCOMP procedure of SAS was used to evaluate the proportion of variability each contributed to total variance. Barrows produced chops with greater IMF (3.64%) and variability (P< 0.001) than gilts (3.20%; P< 0.001). Chops from MQ pigs exhibited greater IMF (4.02%) and variability (P< 0.001) than LG (2.82%; P< 0.001). Chops from location A (3.80%) and D (3.77%) had greater IMF than B (3.34%; P< 0.001) with all three locations greater than C (2.77%; P< 0.001). Variance (s2) also differed (A=1.44% B=1.59% C=1.05% D=2.18%; P< 0.05) across chop locations. Of the variability in IMF, 33.0% was attributed to sire line, 10.16% to chop location and 4.01% to sex, with 52.83% unaccounted for. Location A chops were the most tender (2.57kg; P< 0.001) and C chops the least tender (2.93kg; P< 0.01). No differences in variability (s2; P=0.40) of tenderness were observed across chop location (A=0.31kg B=0.24kg C=0.24kg D=0.23kg). In conclusion, chop location, sex, and sire line all contribute to the amount and variability of pork loin marbling. Supported by National Pork Board Grant #18-118.

Butyric acid (BA) affects the differentiation of mesenchymal stem cells (MSC) through the activation of different transcriptional pathways. The aim of this study was to determine the effects of BA on proliferation and spontaneous differentiation of porcine bone marrow–derived MSC. Second passage MSC (n = 6) were cultured in either a basal medium (BM, DMEM + 10% FBS), or BM + 2.5 mmol/L BA (BA-2.5) or BM + 5 mmol/L BA (BA-5). Cell proliferation was significantly decreased by both BA-2.5 and BA-5 after 48 h and 72 h (− 55% and − 63%, respectively). To assess the impact of BA on spontaneous differentiation, MSC were cultured for 27 d, with complete media changes every 3 d. At day 27, cells were stained for osteocytic, chondrocytic, and adipocytic differentiation. No terminal differentiation was detected in control MSC, while accumulated small drops of lipids were stained by Oil-Red-O in BA-treated cells. The phenotypic changes were associated with changes in gene expression, determined by qPCR. Treatment with BA modulated the expression of adipocytic differentiation markers: peroxisome proliferator-activated receptor γ and CCAAT/enhancer binding protein α were significantly increased by both BA-2.5 and BA-5 throughout the study, while lipoprotein lipase and fatty acid-binding protein 4 were increased by BA-5 at day 3, and decreased by both BA-5 and BA-2.5 later throughout the study. Osteocalcin and aggrecan mRNA was reduced throughout the experiment by both doses of BA (P

Muscle growth and repair rely on two main mechanisms – myonuclear accretion and subsequent protein accumulation. Altering the ability of muscle resident stem cells (satellite cells) to progress through their myogenic lineage can have a profound effect on lifetime muscle growth and repair. The use of the histone deacetylase (HDAC) inhibitor, butyrate, has had positive outcomes on the invitro promotion of satellite cell myogenesis. In animal models, the use of butyrate has had promising results in treating myopathic conditions as well as improving growth efficiency, but the impact of dietary butyrate on satellite cells and muscle growth has not been elucidated. We investigated the impact of tributyrin, a butyrate prodrug, on satellite cell activity and muscle growth in a piglet model. Satellite cells from tributyrin-treated piglets had altered myogenic potential, and piglets receiving tributyrin had a ~40% increase in DNA:protein ratio after 21days, indicating the potential for enhanced muscle growth. To assess muscle growth potential, piglets were supplemented tributyrin (0.5%) during either the neonatal phase (d1–d21) and/or the nursery phase (d21–d58) in a 2×2 factorial design. Piglets who received tributyrin during the neonatal phase had improved growth performance at the end of the study and had a ~10% larger loin eye area and muscle fiber cross-sectional area. Tributyrin treatment in the nursery phase alone did not have a significant effect on muscle growth or feed efficiency. These findings suggest that tributyrin is a potent promoter of muscle growth via altered satellite cell myogenesis.

The neonatal pig ranks among the most prominent research models for the study of pediatric nutrition and metabolism. Its precocial development at birth affords ready adaptation to artificial rearing systems, and research using this model spans a wide array of nutrients. Sophisticated in vitro and in vivo methodologies supporting both invasive, reduction-science research as well as whole-animal preclinical investigations have been developed. Potential applications may dually benefit both agricultural and medical sciences (e.g., “agrimedical research”). The broad scope of this review is to outline the fundamental elements of the piglet model and to highlight key aspects of relevance to various macronutrients, including lipids, carbohydrates, proteins/amino acids, and calcium/phosphorus. The review examines similarities between piglets and infants and also piglet idiosyncrasies, concluding that, overall, the piglet represents an adaptable and robust model for pediatric nutrition and metabolism research.

In vivo responses to bacterially derived superantigen-like toxins have been difficult to define due to the inherent limitations with rodent models and the relevance that the results obtained from such models may, or may not, have for human pathophysiology. Further the use of challenge doses of superantigen toxins that are lethal or supra-lethal complicates analogies to human exposures which are rarely fatal. Here, we utilize the superantigen, staphylococcal enterotoxin B, at doses that are sublethal in a swine model of toxin-induced incapacitation. Relevant dosing using an animal species for which this toxin is a true superantigen distinguishes this model.

In vivo responses to bacterially derived superantigen-like toxins have been difficult to define due to the inherent limitations with rodent models and the relevance that the results obtained from such models may, or may not, have for human pathophysiology. Further the use of challenge doses of superantigen toxins that are lethal or supra-lethal complicates analogies to human exposures which are rarely fatal. Here, we utilize the superantigen, staphylococcal enterotoxin B, at doses that are sublethal in a swine model of toxin-induced incapacitation. Relevant dosing using an animal species for which this toxin is a true superantigen distinguishes this model.

Salmonella is a foodborne pathogenic bacterium that causes human illnesses and morbidity and mortality in swine. Bacteriophages are viruses that prey on bacteria and are naturally found in many microbial environments, including the gut of food animals, and have been suggested as a potential intervention strategy to reduce Salmonella levels in the live animal. The present study was designed to determine if anti-Salmonella phages isolated from the feces of commercial finishing swine could reduce gastrointestinal populations of the foodborne pathogen Salmonella Typhimurium in artificially inoculated swine. Weaned pigs (n = 48) were randomly assigned to two treatment groups (control or phage-treated). Each pig was inoculated with Salmonella Typhimurium (2 × 10(10) colony forming units/pig) via oral gavage at 0 h and fecal samples were collected every 24 h. Swine were inoculated with a phage cocktail via oral gavage (3 × 10(9) plaque forming units) at 24 and 48 h. Pigs were humanely killed at 96 h, and cecal and rectal intestinal contents were collected for quantitative and qualitative analysis. Fecal Salmonella populations in phage-treated pigs were lower (p 0.09) than controls after 48 h. Phage treatment reduced intestinal populations of inoculated Salmonella Typhimurium in pigs compared to controls at necropsy. Cecal populations were reduced (p = 0.07) by phage treatment1.4 log(10) colony forming units/g digesta, and rectal populations were numerically reduced. The number of pigs that contained inoculated Salmonella Typhimurium was reduced by phage treatment, but a significant (p 0.05) reduction was only observed in the rectum. We conclude that phages can be a viable tool to reduce Salmonella in swine. Further research needs to be performed to determine the most efficacious dosing regimens and the most effective combinations of phages targeting the diverse Salmonella population found in swine before they can enter the food supply.

Despite the potential for its use as an agent of biowarfare or bioterrorism, no approved vaccine against staphylococcal enterotoxin B (SEB) exists. Nontoxic, mutant forms of SEB have been developed; however, it has been difficult to determine the efficacy of such subunit vaccine candidates due to the lack of superantigen activity of native SEB in rodents and due to the limitations of primate models. Since pigs respond to SEB in a manner similar to that of human subjects, we utilized this relevant animal model to investigate the safety and immunogenicity of a triple mutant of SEB carrying the amino acid changes L45R, Y89A, and Y94A. This recombinant mutant SEB (rmSEB) did not possess superantigen activity in pig lymphocyte cultures. Furthermore, rmSEB was unable to compete with native SEB for binding to pig leukocytes. These in vitro studies suggested that rmSEB could be a safe subunit vaccine. To test this possibility, piglets immunized orally with rmSEB formulations experienced no significant decrease in food consumption and no weight loss during the vaccination regimen. Oral vaccination with 1-mg doses of rmSEB on days 0, 7, 14, and 24 resulted in serum IgG and fecal IgA levels by day 36 that cross-reacted with native SEB. Surprisingly, the inclusion of cholera toxin adjuvant in vaccine formulations containing rmSEB did not result in increased antibody responses compared to formulations using the immunogen alone. Taken together, these studies provide additional evidence for the potential use of nontoxic forms of SEB as vaccines.

Dihydroxy-cholecalciferol [1,25(OH)2D3] has been shown to have pleiotropic effects on the differentiation of mesenchymal stem cells (MSC) based on species and culture conditions. We have examined the effects of 1,25(OH)2D3 on the differentiation of porcine MSC under culture conditions designed to promote proliferation in order to attempt to mimic the conditions in young, rapidly growing animals. The MSC were isolated from bone marrow of a young pig and grown in basal media (BM) containing DMEM+10% fetal bovine serum and antibiotics. Cells received either BM, BM+10(-8) M 1,25(OH)2D3 or BM+10(-7) M 1,25(OH)2D3 with complete media changes every 3 days for a total of 12 days of culture. On days 3, 6, 9 and 12, viable cell numbers were determined, and samples were collected for gene expression analysis and cytochemical staining. There was a treatment-based reduction in cell numbers on 6, 9 and 12 days (P

Concern over the environmental effect of P excretion from pig production has led to reduced dietary P supplementation. To examine how genetics influence P utilization, 94 gilts sired by 2 genetic lines (PIC337 and PIC280) were housed individually and fed either a P-adequate diet (PA) or a 20% P-deficient diet (PD) for 14 wk. Initially and monthly, blood samples were collected and BW recorded after an overnight fast. Growth performance and plasma indicators of P status were determined monthly. At the end of the trial, carcass traits, meat quality, bone strength, and ash percentage were determined. Pigs fed the PD diet had decreased (P < 0.05) plasma P concentrations and poorer G:F (P < 0.05) over the length of the trial. After 4 wk on trial, pigs fed the PD diet had increased (P < 0.05) plasma 1,25(OH)2D3 and decreased (P < 0.05) plasma parathyroid hormone compared with those fed the PA diet. At the end of the trial, pigs fed the PD diet had decreased (P < 0.05) BW, HCW, and percentage fat-free lean and tended to have decreased LM area (P = 0.06) and marbling (P = 0.09) and greater (P = 0.12) 10th-rib backfat than pigs fed the PA diet. Additional- ly, animals fed the PD diet had weaker bones and also decreased (P < 0.05) ash percentage and increased (P < 0.05) concentrations of 1α-hydroxylase and parathy- roid hormone receptor mRNA in kidney tissue. Regard- less of dietary treatment, PIC337-sired pigs consumed more feed and gained more BW than their PIC280-sired counterparts (P < 0.05) during the study. The PIC337- sired pigs also had greater (P < 0.05) HCW, larger (P < 0.01) LM area, and tended to have (P = 0.07) greater dressing percentage. Meat from the PIC337-sired pigs also tended to have greater (P = 0.12) concentrations of lactate but decreased (P = 0.07) concentrations of to- tal glucose units 24 h postslaughter. Although plasma 1,25(OH)2D3 concentrations were elevated (P < 0.05) in all the animals fed the PD diet, this elevation due to P deficiency tended (P = 0.09) to be greater in the PIC337-sired pigs after 12 wk on the treatment. The PIC337-sired pigs had stronger (P < 0.01) bones with greater ash percentage than the PIC280-sired pigs. The difference in the strength of the radii between the PIC337-sired pigs fed the PA and PD diets was greater than their PIC280-sired counterparts, which resulted in sire line × treatment interactions (P < 0.05). These data indicate differing mechanisms of P utilization between these genetic lines. Elucidating these mecha- nisms may lead to strategies to increase efficiency of growth in a more environmentally friendly manner.

Recent studies describe an association between poor iron status and obesity in humans, although the mechanism explaining this relationship is unclear. The present study aimed to determine the effect of moderate iron deficiency and physical activity (PA) on body composition in an animal model. Male Sprague-Dawley rats consumed iron-adequate (IA; 40 mg/kg) or moderately iron-deficient (ID; 9 mg/kg) diets ad libitum for 12 wk. Rats were assigned to 4 treatment groups (n = 10 per group): IA, sedentary (IAS); IA, PA (IAPA); ID, sedentary (IDS); or ID, PA (IDPA). Activity involved running on motorized running wheels at 4 m/min for 1 h/d for 5 d/wk. After 12 wk, ID rats were not anemic, but body iron stores were reduced as indicated by diminished (P < 0.05) femur iron compared with IA rats. Treatment group did not affect body weight or feed consumption. However, fat mass was greater (P < 0.05) in IDS rats (38.6 +/- 6.7%) than IAS (31.8 +/- 2.9%), IAPA (31.8 +/- 2.0%), and IDPA (32.8 +/- 4.5%) rats. Furthermore, lean body mass was diminished in IDS rats (58.7 +/- 6.8%) compared with IAS (65.6 +/- 3.0%), IAPA (65.6 +/- 2.1%), and IDPA (64.7 +/- 4.5%) rats. Thus, moderate iron deficiency may cause increased body fat accretion in rats and PA attenuates that effect.

A study was conducted to determine the influence on broiler chicken growth and laying hen performance of chemical and physical traits of corn kernels from different hybrids. A total of 720 male 1-d-old Ross-308 broiler chicks were allotted to floor pens in 2 replicated experiments with a randomized complete block design. A total of 240 fifty-two-week-old Hy-Line W-36 laying hens were allotted to cages in a randomized complete block design. Corn-soybean meal diets were formulated for 3 broiler growth phases and one 14-wk-long laying hen phase to be marginally deficient in Lys and TSAA to allow for the detection of differences or correlations attributable to corn kernel chemical or physical traits. The broiler chicken diets were also marginally deficient in Ca and nonphytate P. Within a phase, corn- and soybean-based diets containing equal amounts of 1 of 6 different corn hybrids were formulated. The corn hybrids were selected to vary widely in chemical and physical traits. Feed consumption and BW were recorded for broiler chickens every 2 wk from 0 to 6 wk of age. Egg production was recorded daily, and feed consumption and egg weights were recorded weekly for laying hens between 53 and 67 wk of age. Physical and chemical composition of kernels was correlated with performance measures by multivariate ANOVA. Chemical and physical kernel traits were weakly correlated with performance in broiler chickens from 0 to 2 wk of age (P

Corn kernel composition may affect its nutritive value and, thus, pig growth performance and carcass characteristics. The objective of this study was to determine the influence of the chemical and physical traits of corn kernels from different hybrids on the growth performance and carcass characteristics of pigs. A total of 288 crossbred pigs were grown in a 3-phase program from 21 kg of BW until slaughter at 113 kg of BW with 12 pens (4 pigs/pen) per dietary treatment. Target BW for each phase were 20 to 40 kg (grower 1), 40 to 80 kg (grower 2), and 80 to 120 kg (finisher). In each phase, diets were formulated to be marginally deficient in Lys, TSAA, Ca, Na, and nonphytate P to improve the likelihood of detecting differences in perfor- mance due to corn hybrid. Each of 6 corn hybrids repre- sented a wide range of kernel chemical and physical traits and was substituted for corn in a common diet formulation on an equal weight basis to make the 6 dietary treatments. Physical and chemical composition of the kernels were analyzed and correlated with perfor- mance measures by multivariate ANOVA. Kernel den- sity was correlated with i.m. fat (IMF) content in LM (r = −0.35, P < 0.05). Stenvert grinding time was corre- lated (P < 0.05) with ADG during the grower 1 phase (r = 0.26), ADFI during the grower 2 phase (r = 0.27), final BW (r = 0.27), and IMF (r = −0.36). The amylose content of the cornstarch was correlated (P < 0.05) with ADG during the grower 2 phase (r = −0.28) and with BW at the end of the grower 2 phase (r = −0.27). The NDF content of the kernels was correlated (P < 0.05) with ADG during the finisher phase (r = −0.30), final BW (r = −0.33), and number of days to market (r = 0.31). The ADF content of the kernels was correlated (P < 0.05) with ADG during the grower 1 phase (r = −0.26), final BW (r = −0.26), and IMF (r = 0.31). The correlations of performance measure variation with individual ker- nel hybrid physical and chemical traits were statisti- cally significant yet not large enough to base corn hybrid selection for feeding pigs on any single kernel chemical or physical trait.

With worldwide concern over the use of antibiotics in animal agriculture and their contribution to the spread of antibiotic resistance, alternatives to conventional antibiotics are needed. Previous research in our laboratories has shown that colicin E1 is effective against some Escherichia coli strains responsible for postweaning diarrhea (PWD) in vitro. In this study we examined the efficacy of the dietary inclusion of colicin E1 in preventing experimentally induced PWD caused by F18-positive enterotoxigenic E. coli in young pigs. Twentyfour weaned pigs (23 days of age), identified by genotyping to be susceptible to F18-positive E. coli infections, were individually housed and fed diets containing 0, 11, or 16.5 mg colicin E1/kg diet. Two days after the start of the trial, all animals were orally inoculated with 1 10 9 CFU of each of two F18-positive E. coli strains isolated from pigs with PWD. The dietary inclusion of colicin E1 decreased the incidence and severity of PWD caused by F18-positive enterotoxigenic E. coli and improved the growth performance of the piglets. Additionally, the reduced incidence of PWD due to dietary colicin E1, lowered the levels of expression of the genes for interleukin 1 and tumor necrosis factor beta in ileal tissues from these animals. The dietary inclusion of colicin E1 may be an effective alternative to conventional antibiotics in the diets of weaning pigs for the prevention of PWD caused by F18-positive enterotoxigenic E. coli. Postweaning diarrhea (PWD) is a serious threat to the economic success of the swine industry both due to losses as a result of mortalities and due to the reduced growth performance of surviving pigs. It is estimated that 50% of piglet mortality due to diarrhea is attributable to the causative agent of PWD, enterotoxigenic Escherichia coli (ETEC) (18). The ETEC strains most commonly associated with PWD possess either the F4 or the F18 fimibrial type (11, 39). As a result of the significant impact that F18-positive ETEC and other bacterial infections can have on pig production, prophylactic antibiotics are frequently included in the diets of young pigs in an attempt to prevent ETEC colonization and the resulting PWD. An estimated 78% of large swine farms in the United States include subtherapeutic concentrations of antibiotics in the diets for young pigs (36). Despite the use of antibiotic prophylaxis, 40.7% of these farms reported the occurrence of diarrhea caused by E. coli infections (36). The lack of the effective prevention of PWD with the use of prophylactic antibiotics is not surprising because of the frequency and spectrum of antibiotic resistance seen among ETEC strains (7, 23, 25). It is expected that antibiotic resistance will further increase among these strains, based on the overall increase in resistance to antibiotics by ETEC strains over the last 20 years (25).

Myostatin, a member of the transforming growth factor β superfamily, is expressed in skeletal muscle both pre- and postnatally. Myostatin prevents excessive differentiation of myoblasts during muscular development. Loss of function mutations to Myostatin can produce supernumerary muscle fibers, resulting in muscle hyperplasia. Despite the large effects of Myostatin mutations in different cattle breeds, no mutations with major effects have been described in pigs. In this study, we identified single nucleotide polymorphisms (SNPs) in the promoter region of the Myostatin gene, investigated allele frequencies, analyzed gene expression pattern in the longissimus dorsi muscle and performed statistical associations with performance, carcass composition and meat quality traits in pigs. Pigs from different breeds and commercial lines were included in these analysis. Two SNPs, G-847A and A-835G, in complete linkage disequilibrium were identified. Their allele and genotypic frequencies were analyzed and an association study conducted. Determination of Myostatin gene expression was obtained by semi-quantitative real-time PCR and correlated to phenotypic traits. The SNPs were associated ( P P

Enterohemorrhagic Escherichia coli (EHEC) is one of the leading causes of bacterial enteric infections worldwide, causing ∼100,000 illnesses, 3,000 hospitalizations, and 90 deaths annually in the United States alone. These illnesses have been linked to consumption of contaminated animal products and vegetables. Currently, other than thermal inactivation, there are no effective methods to eliminate pathogenic bacteria in food. Colicins are nonantibiotic antimicrobial proteins, produced by E. coli strains that kill or inhibit the growth of other E. coli strains. Several colicins are highly effective against key EHEC strains. Here we demonstrate very high levels of colicin expression (up to 3 g/kg of fresh biomass) in tobacco and edible plants (spinach and leafy beets) at costs that will allow commercialization. Among the colicins examined, plant-expressed colicin M had the broadest antimicrobial activity against EHEC and complemented the potency of other colicins. A mixture of colicin M and colicin E7 showed very high activity against all major EHEC strains, as defined by the US Department of Agriculture/Food and Drug Administration. Treatments with low (less than 10 mg colicins per L) concentrations reduced the pathogenic bacterial load in broth culture by 2 to over 6 logs depending on the strain. In experiments using meats spiked with E. coli O157:H7, colicins efficiently reduced the population of the pathogen by at least 2 logs. Plant-produced colicins could be effectively used for the broad control of pathogenic E. coli in both plant- and animal-based food products and, in the United States, colicins could be approved using the generally recognized as safe (GRAS) regulatory approval pathway.

Organic acids, such as citric and sorbic acid, and pure plant-derived constituents, like monoterpens and aldehydes, have a long history of use in pig feeding as alternatives to antibiotic growth promoters. However, their effects on the intestinal barrier function and inflammation have never been investigated. Therefore, aim of this study was to assess the impact of a microencapsulated mixture of citric acid and sorbic acid (OA) and pure botanicals, namely thymol and vanillin, (PB) on the intestinal integrity and functionality of weaned pigs and in vitro on Caco-2 cells. In the first study 20 piglets were divided in 2 groups and received either a basal diet or the basal diet supplemented with OA + PB (5 g/kg) for 2 weeks post-weaning at the end of which ileum and jejunum samples were collected for Ussing chambers analysis of trans-epithelial electrical resistance (TER), intermittent short-circuit current (I SC), and dextran flux. Scrapings of ileum mucosa were also collected for cytokine analysis (n = 6). In the second study we measured the effect of these compounds directly on TER and permeability of Caco-2 monolayers treated with either 0.2 or 1 g/l of OA + PB. Pigs fed with OA + PB tended to have reduced I SC in the ileum (P = 0.07) and the ileal gene expression of IL-12, TGF-β, and IL-6 was down regulated. In the in vitro study on Caco-2 cells, TER was increased by the supplementation of 0.2 g/l at 4, 6, and 14 days of the experiment, whereas 1 g/l increased TER at 10 and 12 days of treatment (P

Microarray technology is now available for many livestock species, and animal scientists are beginning to utilize the technology to address issues of importance to animal agriculture. This review discusses how microarray technology has been applied to study global gene expression changes in skeletal muscle. For example, microarrays have been used to elucidate gene function in knockout mice, evaluate breed differences, evaluate the effects of hormone administration, and evaluate the effects of diet. Data generated from these global gene expression studies are providing new insights to stimulate future hypothesis-driven research.

To develop an effective fermentation system for producing Escherichia coliphytase AppA2, we expressed the enzyme in three inducible yeast systems: Saccharomyces cerevisiae (pYES2), Schizosaccharomyces pombe (pDS472a), and Pichia pastoris (pPICZ αA), and one constitutive system: P. pastoris (pGAPZαA). All four systems produced an extracellular functional AppA2 phytase with apparent molecular masses ranging from 51.5 to 56 kDa. During 8-day batch fermentation in shaking flasks, the inducible Pichia system produced the highest activity (272 units ml−1 medium), whereas the Schizo. pombesystem produced the lowest activity (2.8 units ml−1). The AppA2 phytase expressed in Schizo. pombehad 60–75% lower K m for sodium phytate and 28% higher heat-stability at 65 °C than that expressed in other three systems. However, all four recombinant AppA2 phytases had pH optimum at 3.5 and temperature optimum at 55 °C and similar efficacy in hydrolyzing phytate–phosphate from soybean meal.

Escherichia coli O157:H7 is a virulent foodborne pathogen that causes severe human illness and inhabits the intestinal tract of food animals. Colicins are antimicrobial proteins produced by E. coli strains that inhibit or kill other E. coli. In the present study, the efficacy of three pore-forming colicins (E1, N, and A) were quantified in vitro against E. coli O157:H7 strains 86-24 and 933. Colicins E1 and N reduced the growth of E. coli O157:H7 strains, but the efficacy of each colicin varied among strains. Colicin E1 was more effective against both strains of E. coli O157:H7 than colicins A and N and reduced (P < 0.05) populations of E. coli O157:H7 at concentrations

The objective of this study was to determine possible synergistic effects of supplementing one of three fungal phytases: Aspergillus fumitagus PhyA (AFP),A. niger PhyA (ANP), or Peniophora lyci phytase (PLP) with an Escherichia coli AppA phytase (EP) in diets for pigs. Three experiments, each lasting for 4 wk, were conducted with a total of 106 weanling pigs (5 wk old). The corn-soybean meal basal diet (BD) contained no supplemental inorganic P. In Exp. 1, 35 pigs (8.6 +/- 1.0 kg BW) were fed (as-fed basis) BD + AFP at 750 U/ kg of feed, BD + inorganic P (0.2% P), or BD + PLP at 500, 750, or 1,000 U/kg feed. Pigs fed BD + AFP or BD + 0.2% P had higher (P < 0.05) plasma inorganic P concentrations than those fed BD + PLP at the end of the trial (wk 4). In Exp. 2, 35 pigs (8.1 +/- 0.9 kg BW) were fed BD + AFP, EP, PLP, a 1:1 mix of AFP:EP, or a 1:1 mix of PLP:EP at 500 U/kg. Pigs fed the AFP:EP mixture had growth performance and plasma measures similar to those fed either enzyme alone. Pigs fed the PLP:EP mixture had lower (P < 0.05) plasma alkaline phosphatase activity than those fed BD + PLP. Pigs fed BD + PLP had lower (P < 0.05) plasma inorganic P concentrations than pigs fed BD + EP, and higher (P < 0.05) plasma alkaline phosphatase activity than all other groups at wk 4. In Exp. 3, 36 pigs (9.1 +/- 1.2 kg BW) were fed BD + ANP, EP, or a 1:1 mix of ANP:EP at 500 U/kg feed. Pigs fed the two enzymes together had lower (P < 0.05) plasma inorganic P concentration than those fed BD + EP and lower (P < 0.05) plasma alkaline phosphatase activity than pigs fed BD + ANP at wk 4. In conclusion, although the four phytases showed different effects on plasma P status of weanling pigs, there was no synergistic effect between any of the three fungal phytases and the bacterial phytase on the plasma measures or growth performance under the conditions of the present study.

A phytase gene (appA) from Escherichia coli was cloned into Streptomyces lividans and expressed as an extracellular protein which was then compared with the same enzyme expressed in Pichia pastoris. The phytase expressed in S. lividans was not glycosylated and had a molecular mass of 45 kDa. Compared with the glycosylated phytase expressed in P. pastoris, this non-glycosylated phytase was 25–50% less active (p

Intestinal microbiota of infants differ in response to gestational age, delivery mode and feeding regimen. Dietary supplementation of probiotic bacteria is one method of promoting healthy populations. We examined the impact of a novel probiotic strain of Bifidobacterium longum (AH1206) on the health, growth and development of neonatal pigs as a model for infants. Day-old pigs were fed milk-based formula containing AH1206 at 0, 10⁹, or 10¹¹ CFU/d for 18 d (n=10/treatment). Differences were not detected in growth, organ weights or body temperatures (P>0.1); however pigs fed the high dose showed a small (2%) reduction in feed intake. Bacterial translocation was not affected as indicated by total anaerobic and aerobic counts (CFU) in samples of spleen, liver and mesenteric lymph nodes (P>0.1). Feeding AH1206 had no effects on fecal consistency, but increased the density of B. longum in the cecum. Ileal TNF expression tended to increase (P=0.08) while IL-10 expression increased linearly (P=0.01) with supplementation. Based upon findings in the suckling piglet model, we suggest that dietary supplementation with B. longum (AH1206) may be safe for human infants based on a lack of growth, development or deleterious immune-related effects observed in piglets.

Background and Aims The clinical onset and severity of intestinal disorders in humans and animals can be profoundly impacted by early life stress. Here we investigated the impact of early weaning stress in pigs on intestinal physiology, clinical disease, and immune response to subsequent challenge with enterotoxigenic F18 E. coli (ETEC). Methodology Pigs weaned from their dam at 16 d, 18 d, and 20 d of age were given a direct oral challenge of F18 ETEC at 26 d of age. Pigs were monitored from days 0 to 4 post-infection for clinical signs of disease. On Day 4 post-ETEC challenge, ileal barrier function, histopathologic and inflammatory cytokine analysis were performed on ileal mucosa. Results Early weaned pigs (16 d and 18 d weaning age) exhibited a more rapid onset and severity of diarrhea and reductions in weight gain in response to ETEC challenge compared with late weaned pigs (20 d weaning age). ETEC challenge induced intestinal barrier injury in early weaned pigs, indicated by reductions in ileal transepithelial electrical resistance (TER) and elevated FD4 flux rates, in early weaned pig ileum but not in late weaned pigs. ETEC-induced marked elevations in IL-6 and IL-8, neutrophil recruitment, and mast cell activation in late-weaned pigs; these responses were attenuated in early weaned pigs. TNF levels elevated in ETEC challenged ileal mucosa from early weaned pigs but not in other weaning age groups. Conclusions These data demonstrate the early weaning stress can profoundly alter subsequent immune and physiology responses and clinical outcomes to subsequent infectious pathogen challenge. Given the link between early life stress and gastrointestinal diseases of animals and humans, a more fundamental understanding of the mechanisms by which early life stress impacts subsequent pathophysiologic intestinal responses has implications for the prevention and management of important GI disorders in humans and animals.

The efficacies of colicins E1 and N against Escherichia coli strains responsible for postweaning diarrhea and edema disease, two of the most prevalent disease problems for pigs in the United States, were determined in vitro. These proteins may provide an environmentally sound means for the prevention of these infections in swine.

In an effort to develop a sustainable platform for manufacturing protein-based vaccine candidates, we expressed a triple mutant of staphylococcal enterotoxin B carrying the L45R, Y89A, and Y94A modifications in transgenic soybean seeds (soy-mSEB). Soy-mSEB possessed no detectable superantigen activity in vitro . We found that this soybean-derived, nontoxic mutant of SEB could be stably expressed, stored in seeds for extended periods at room temperature without degradation, and easily purified from contaminating soy proteins. Vaccination of pigs with purified soy-mSEB, or the identical triple mutant expressed in Escherichia coli ( E. coli -mSEB), resulted in high antibody titers against the native toxin in immunized animals. In fact, titers were indistinguishable regardless of the immunogen used, demonstrating the equivalence of soy-mSEB and E. coli -mSEB vaccinations. Antisera from either immunized group were able to block native SEB superantigen activity in an in vitro neutralization assay. Similar results were obtained when immunized animals were challenged with a sublethal dose of native toxin. Significant reductions in toxin-induced serum cytokine levels were observed in soy-mSEB- and E. coli -mSEB-immunized pigs compared to control animals. The reductions in SEB-induced cytokine responses were similar regardless of the immunogen used for vaccination. Surprisingly, however, some clinical symptoms, such as prostration, lethargy, emesis, and/or diarrhea, were still observed in all immunized animals. These studies demonstrate the potential for soybean-derived proteins as a platform technology for sustainable vaccine manufacturing and the usefulness of a sublethal challenge model in pigs for evaluating the efficacy of potential SEB vaccine candidates.

Satellite cell activity is necessary for postnatal skeletal muscle growth. Severe phosphate (PO4) deficiency can alter satellite cell activity, however the role of neonatal PO4 nutrition on satellite cell biology remains obscure. Twenty-one piglets (1 day of age, 1.8 ± 0.2 kg BW) were pair-fed liquid diets that were either PO4 adequate (0.9% total P), supra-adequate (1.2% total P) in PO4 requirement or deficient (0.7% total P) in PO4 content for 12 days. Body weight was recorded daily and blood samples collected every 6 days. At day 12, pigs were orally dosed with BrdU and 12 h later, satellite cells were isolated. Satellite cells were also cultured in vitro for 7 days to determine if PO4 nutrition alters their ability to proceed through their myogenic lineage. Dietary PO4 deficiency resulted in reduced (P < 0.05) sera PO4 and parathyroid hormone (PTH) concentrations, while supra-adequate dietary PO4 improved (P < 0.05) feed conversion efficiency as compared to the PO4 adequate group. In vivo satellite cell proliferation was reduced (P < 0.05) among the PO4 deficient pigs, and these cells had altered in vitro expression of markers of myogenic progression. Further work to better understand early nutritional programming of satellite cells and the potential benefits of emphasizing early PO4 nutrition for future lean growth potential is warranted. Published version

The effects of dietary calcium (Ca) deficiency on skeletal integrity are well characterized in growing and mature mammals; however, less is known about Ca nutrition during the neonatal period. In this study, we examined the effects of neonatal Ca nutrition on bone integrity, endocrine hormones, and mesenchymal stem cell (MSC) activity. Neonatal pigs (24 6 6ho f age) received either a Ca-adequate (1.2 g/100 g) or an ~40% Ca-deficient diet for 18 d. Ca deficiency reduced (P , 0.05) boneflexural strength and bone mineral density without major differences in plasmaindicators of Ca status. There were no meaningful differences in plasma Ca, phosphate (PO4), parathyroid hormone, or 1,25-dihydroxycholecalciferol due to Ca nutrition throughout the study. Calcium deficiency also reduced (P , 0.05) the in vivo proliferation of MSC by ~50%. In vitro studies utilizing homologous sera demonstrated that MSC activity was affected (P , 0.05) by both the Ca status of the pig and the sera as well as by their interaction. The results indicate that neonatal Ca nutrition is crucial for bone integrity and suggest that early-life Ca restriction may have long-term effects on bone integrity via programming of MSC. J. Nutr. 141: 373‐379, 2011.

Although mesenchymal stem cells (MSC) and satellite cells are essential for postnatal muscle and bone development and phosphate (PO(4)) restriction reduces both muscle and skeletal tissue growth, no research to our knowledge has investigated the possible mechanism by which this mineral may affect early cell programming. Twenty piglets obtained at 1 d of age (1.8 +/- 0.3 kg) received either a PO(4)-adequate diet or a 25% less PO(4)-available diet over a 15-d trial. Feed intake and body weight were recorded daily and blood samples collected every 5 d. After 15 d, pigs were given an intraperitoneal injection of bromodeoxyuridine 4 h prior to tissue collection. As expected, PO(4) deficiency resulted in reduced growth (P < 0.05), feed conversion efficiency (P < 0.05), and bone mineral content (P < 0.05), as well as lower plasma concentrations of both PO(4) (P < 0.01) and parathyroid hormone (P < 0.05). In addition to these classical indicators of PO(4) deficiency, there was also reduced proliferation of both MSC (P < 0.01) and satellite cells (P < 0.05) in vivo. The expression of osteocalcin mRNA in bone marrow was also 2-fold greater (P < 0.01) within the PO(4)-adequate treatment group. These data indicate that in addition to reductions in muscle and bone growth, dietary PO(4) affects proliferation of tissue-specific stem cells in vivo. Nutritional programming of tissue-specific stem cells by dietary PO(4) may have profound implications for life-long growth potential.