Effects of mixed tocopherols added to milk replacer and calf starter on intake, growth, and indices of stress.

Vitamin E comprises 8 fat-soluble isoforms: α-, ß-, γ-, and δ-tocopherol and α-, ß-, γ-, and δ-tocotrienol. Yet the body preferentially uses α-tocopherol, and only α-tocopherol supplementation can reverse vitamin E deficiency symptoms. However, other isoforms influence many biological functions in the body, including inflammation and stress. Therefore, the study objective was to determine metabolic and performance responses in young calves fed diets containing a constant amount of α-tocopherol and increasing amounts of soybean oil-derived mixed γ- and δ-tocopherols. Holstein calves [n = 48; 2-3 d of age; 40.2 kg of initial body weight (BW), standard error = 0.54] were assigned to receive approximately 0, 5, 10, or 15 mg/kg of BW daily (treatments T0, T1, T2, and T3, respectively) of mixed tocopherols (TMIX) provided in milk replacer (MR) and calf starter. The TMIX liquid contained 86% γδ-tocopherols and 9% α-tocopherol. Milk replacers were formulated to contain approximately 0, 400, 800, or 1,200 mg of TMIX/kg for treatments T0, T1, T2, and T3, respectively. Calf starters were formulated to contain approximately 0, 250, 500, or 750 mg of TMIX/kg for treatments T0, T1, T2, and T3, respectively. Mean consumption of γδ-tocopherols was 0.0, 6.5, 14.3, and 20.5 mg/kg of BW, respectively. Milk replacer contained 24% crude protein (CP) and 20% fat on a dry matter (DM) basis. Calf starters were pelleted and offered for ad libitum consumption from 0 to 56 d. Starters contained 18 to 20% CP and 9 to 12% starch in the DM. On d 28, 4 calves per treatment were randomly selected for slaughter, and necropsy was performed. Samples of liver, duodenum, ileum, and trapezius muscle were collected and stored before analysis for α-, ß-, γ-, and δ-tocopherols and δ-tocotrienol. Data were analyzed using a completely randomized design using mixed model ANOVA with orthogonal polynomials to determine linear and quadratic effects of TMIX. Repeated-measures analyses were performed for data collected over time. Increasing dietary TMIX increased or tended to increase change in hip width at 28 and 56 d, respectively, and improved average daily BW gain and gain-to-feed ratio at 56 d. Increasing TMIX reduced plasma xanthine oxidase at 0 h and tended to reduce concentrations at 24 h following vaccination with 2 commercial vaccines on d 28; however, we detected no effect of TMIX following vaccination on d 56. Concentration of α-tocopherol in skeletal muscle declined quadratically with increasing TMIX, whereas ileal and liver γ-tocopherol increased linearly with increasing TMIX. The number of mucin-2 cells in the ileum increased more than 2-fold in calves fed T3. Addition of mixed tocopherols to diets of young dairy calves improved animal growth and altered indices of antioxidant metabolism.

The effect of delayed feeding post-hatch on caeca development in broiler chickens.

1. Broiler chicks are frequently deprived of food up to 72 h due to uneven hatching rates, management procedures and transportation to farms. Little is known about the effect of delayed feeding due to extended hatching times on the early neonatal development of the caeca. Therefore, the objective of this study was to investigate the developmental changes and effects of a 48-h delay in feed access immediately post-hatch (PH) on the caeca.2. After hatch, birds (Ross 708) were randomly divided into two treatment groups (n = 6 battery pen/treatment). One group (early fed; EF) received feed and water immediately after hatch, while the second group (late fed; LF) had access to water but had delayed access to feed for 48 h. Contents averaging across all regions of the caeca were collected for mRNA expression as well as for histological analysis at -48, 0, 4 h PH and then at 1, 2, 3, 4, 6, 8, 10, 12 and 14 days PH.3. Expression of MCT-1 (a nutrient transporter), Cox7A2 (related to mitochondrial function) IgA, pIgR, and ChIL-8 (immune function) genes was affected by delayed access to feed that was dependent by the time PH. Expression of immune and gut barrier function-related genes (LEAP2 and MUC2, respectively) was increased in LF group. There was no effect of feed delay on expression of genes related to mitochondrial functions in the caeca, although developmental changes were observed (ATP5F1B, Cox4|1). Caecal mucus and muscle thickness were affected by delayed access to feed during caeca development.4. The data suggested a limited effect of delayed feed access PH on the developmental changes in caecal functions. However, the caeca seemed to be relatively resistant to delayed access to feed early PH, with only a few genes affected.

Effect of Holstein genotype on innate immune and metabolic responses of heifers to lipopolysaccharide (LPS) administration.

Heifers (n = 4/genotype) from unselected (stable genotype since 1964, UH) and contemporary (CH) Holsteins that differed in milk yield (6,200 and 11,100 kg milk/305 d) were used to assess the impact of selection on innate immune and acute-phase response to an endotoxin (lipopolysaccharide; LPS). Jugular catheters were implanted 24 h before LPS administration. Blood samples were collected at -1, -0.5, 0, 1, 2, 3, 4, 6, 8, and 24 h relative to iv administration of 0.5 µg LPS/kg BW. Rectal body temperature (BT) was determined at these sampling times and at 5 and 7 h. Dermal biopsies were collected after the 24 h blood sample and processed to isolate fibroblasts. Plasma was analyzed for tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), serum amyloid A (SAA), xanthine oxidase (XO), and nitrate + nitrite (NOx), cortisol, glucose, and IGF-1 content. Isolated fibroblasts were exposed to IL-1ß or LPS and IL-6 and IL-8 content of culture media determined. Exposure to LPS increased BTs and plasma concentrations of TNF-α, IL-6 SAA, XO, cortisol, and glucose (P < 0.05) in both genotypes. Plasma concentrations of TNF-α, XO, NOx, and glucose did not differ (P > 0.25) between the genotypes, but IL-6 and SAA concentrations were reduced (P < 0.05) in CH relative to UH heifers while cortisol and IGF-1 concentrations tended (P < 0.08) to be reduced in CH heifers. After 36 h exposure to LPS, concentrations of IL-6 were greater (P < 0.05) in culture media from incubations of CH than UH fibroblasts but concentrations of IL-8 did not differ between genotypes. There was a trend (P = 0.08) for IL-8 concentrations to be reduced in media from CH fibroblasts exposed to IL-1ß for 24 h but IL-6 concentrations did not differ between genotypes. Results indicate 50 yr of selection has reduced the robustness of the innate immune and acute-phase response to LPS in the contemporary Holstein heifer.

α-1 acid glycoprotein inhibits insulin responses by glucose oxidation, protein synthesis and protein breakdown in mouse C2C12 myotubes.

Increased plasma α-1 acid glycoprotein (AGP) is correlated with reduced growth rates in neonatal swine. The specific physiological mechanisms contributing to this relationship are unknown. This study was performed to determine if AGP can modify muscle metabolism by examining glucose oxidation and protein synthesis in the C2C12 muscle cell line. Cells were used for experiments 4 days post-fusion as myotubes. Myotubes were exposed to AGP for 24 h, with the last 4 h used to monitor 14C-glucose oxidation or to measure protein synthesis by incorporation of 3H-tyrosine. Treatment of C2C12 myotubes with mouse AGP (100 µg/ml) reduced glucose oxidation (P0.05, n=6 trials), whereas incubation with both AGP and insulin reduced 3H-tyrosine release by 15% (P<0.01, n=6 trials). First, these data indicate that the acute phase protein AGP can interact with the skeletal muscle to reduce glucose oxidation, but this is not the result of an effect on glucose transport. Second, AGP can specifically reduce protein synthesis. Lastly, AGP can inhibit insulin-stimulated glucose oxidation, protein synthesis and breakdown.

Variation in fibroblast expression of toll-like receptor 4 and lipopolysaccharide-induced cytokine production between animals predicts control of bacterial growth but not severity of Escherichia coli mastitis.

Mastitis caused by environmental pathogens such as Escherichia coli is highly problematic to the dairy industry because it incurs substantial cost and tends to be difficult to manage. An effective innate immune response by the host is key to controlling infection, but it should also limit collateral damage to the mammary gland. Between-animal differences in mastitis severity have been attributed to variability in the innate response. In the current study, we used primary dermal fibroblast as a model to rank animals based on composite expression of the toll-like receptor 4 gene (TLR4) and lipopolysaccharide (LPS)-induced IL-8 and IL-6 protein production. Animals ranked as high and low responders (HR and LR, respectively) were then infected with the P4 strain of E. coli to determine how difference in rank would affect response to mastitis. All animals developed an acute response to the infection with varying degrees in severity; however, HR animals had an elevated somatic cell count and fever response at 12 h post-infection and greater production of milk IL-8 at 24 h post-infection. The HR animals were also significantly more capable of limiting bacterial growth. No differences in post-infection milk production or concentrations of milk BSA were measured. The current study indicates that HR animals have an early upregulation in their innate response that is beneficial for bacterial clearance; however, they are equally susceptible to tissue damage caused by an exuberant response to the infection. The dermal fibroblast may be used in conjunction with other cell types to determine how the innate response is regulated to mitigate unnecessary injury to the mammary gland while still effectively clearing the pathogen.

Short communication: Recombinant bacteriophage endolysin PlyC is nontoxic and does not alter blood neutrophil oxidative response in lactating dairy cows.

Mastitis is the leading cause of antimicrobial use on dairy farms. The potential for antimicrobial resistance has led to the examination of alternative strategies for controlling mastitis. One such alternative is PlyC, a potent peptidoglycan hydrolase derived from the streptococcal C1 bacteriophage that causes targeted lysis of the cell wall of Streptococcus uberis. At a concentration of 1.0 µg/mL, recombinant PlyC can induce lytic activity, suggesting that a low dose may successfully eliminate infection. We evaluated the dose effect of PlyC (1-50 µg/mL) on cytotoxicity and oxidative response on bovine blood polymorphonuclear leukocytes (PMN) obtained from 12 healthy, mid-lactation primiparous dairy cows. Following incubation at 0.5 and 2 h, cytotoxicity was characterized by measuring lactate dehydrogenase release from isolated cells. Oxidative burst response was characterized as the intensity of chemiluminescence produced in the interaction of reactive oxygen species generated in response to 0 or 1.6 µg/mL of phorbol 12-myristate-13-acetate (PMA) with a luminescent substrate with and without addition to PlyC to the incubation matrix. Data were analyzed as a complete randomized block design using mixed model procedures. Cytotoxicity of PlyC was not affected by concentrations up to 50 µg/mL. As expected, PlyC cytotoxicity on PMN varied across incubation time with greater cell toxicity measured at 2 h of incubation as compared with 0.5 h and is primarily attributed to the short life of PMN ex vivo. Concentrations of PlyC up to 50 µg/mL did not affect oxidative response; however, oxidative response was affected by incubation time and PMA concentration. In summary, varying doses of PlyC are nontoxic as estimated by lactate dehydrogenase release from cells and do not appear to alter PMA-stimulated reactive oxygen species production in bovine PMN. These early observations support continued work on the potential for application of this novel agent in combating mastitis.

The effects of feeding mixed tocopherol oil on whole-blood respiratory burst and neutrophil immunometabolic-related gene expression in lactating dairy cows.

The 4 major tocopherol isoforms differ in their biochemical reactivity and cellular effects due to basic chemical structural differences. Alpha-tocopherol has been well studied regarding effects on bovine polymorphonuclear leukocyte (PMN) function and its involvement in respiratory burst. However, no studies to date have identified the effects of supplementing a mixed tocopherol oil (Tmix) particularly enriched in non-α tocopherol isoforms (i.e., γ- and δ-isoforms) on fundamental immunometabolic changes in dairy cows. Therefore, the objectives of this study were to determine whether short-term feeding of vegetable oil-derived Tmix alters specific biomarkers of metabolism, whole-blood leukocyte populations, respiratory burst, immunometabolic-related gene expression of PMN, or gene expression of isolated PMN when challenged with lipopolysaccharides (LPS). Clinically healthy multiparous lactating Holstein cows (n = 12; 179 ± 17 d in milk, 40.65 ± 3.68 kg of milk yield) were fed Tmix (620 g/d) for 7 consecutive days. Jugular blood (EDTA anticoagulant) was collected from all cows on d 0 before treatment initiation and again on d 7 after Tmix feeding. Total stimulated respiratory burst activity (RBA) and leukocyte populations were assessed in whole blood, and tocopherol isoform concentrations, metabolites, and hormones were measured in plasma. For gene expression analysis, isolated PMN from cows before and after Tmix feeding were incubated with LPS at a final concentration of either 0.0 or 1.5 µg/mL. Feeding of Tmix for 7 d increased the concentrations of α- and γ-tocopherol. The Tmix did not alter plasma insulin but decreased cholesterol. The Tmix did not alter whole-blood RBA or the leukocyte populations. The LPS challenge increased the expression of proinflammatory genes TNFA and IL6. However, Tmix treatment did not alter the patterns of LPS-affected expression of genes (e.g., TNFA, ITGB2, PPARA, and RXRA) associated with the immune or metabolic response. In conclusion, short-term feeding of Tmix may have no negative effect on animal health as Tmix increased α- and γ-tocopherol concentrations in blood and did not impair whole-blood RBA or alter leukocyte populations. The data provide further support that the α- and γ-tocopherol isoforms do not interfere with normal immune or metabolic function.

Reducing gut effects from Cryptosporidium parvum infection in dairy calves through prophylactic glucagon-like peptide 2 therapy or feeding of an artificial sweetener.

Glucagon-like peptide 2 (GLP-2) therapy was shown previously to reduce inflammation-related gut damage from coccidiosis in dairy calves, and feeding of artificial sweetener stimulates GLP-2 secretion from intestinal L cells. The purpose of this study was to determine whether GLP-2 treatment or artificial sweetener feeding beginning 1 wk before an experimental inoculation with the coccidian parasite Cryptosporidium parvum can reduce infection-related intestinal damage in Holstein bull calves. Newborn calves were assigned to 1 of 4 treatment groups of 6 calves each, including noninfected control calves injected s.c. every 12 h with control buffer (CON), infected control calves injected s.c. every 12 h with control buffer (INF), infected calves injected s.c. every 12 h with 50 µg/kg of body weight of GLP-2 (GLP2), and infected calves injected s.c. every 12 h with control buffer and supplemented in the diet with Sucram (Pancosma, Geneva, Switzerland) at 400 mg/kg of dry matter of milk replacer (SUC). Treatments were initiated on d 1, and calves in INF, GLP2, and SUC were orally dosed on d 8 with 12,500 C. parvum oocysts. Fecal scores were recorded daily, plasma was collected on d 1, 8, 12, 15, and 18 to evaluate markers of inflammation, and fecal samples were collected on d 1, 8, and every other day thereafter to determine the presence of oocysts. Calves were euthanized on d 18 for collection of intestinal tissues and histological and gene expression analyses. Relative to CON, calves in INF exhibited an increase in diarrhea severity, increased plasma serum amyloid A concentration on d 15 and 18, reduced intestinal villus height, increased villus apoptosis and crypt cell proliferation, and increased intestinal mRNA expression of MARVELD2 and GPX2. However, calves in SUC and GLP2 had reduced diarrhea severity and fecal C. parvum oocyst shedding, reduced plasma serum amyloid A concentration on d 15 and 18, and, depending on the intestinal segment, increased villus height, reduced crypt cell proliferation, and reduced mRNA expression of MARVELD2, GPX2, and other tight junction proteins relative to INF. Lastly, GLP2 and SUC exhibited increased intestinal mass-to-length ratio and decreased length-to-empty body weight ratio relative to INF. Our findings suggest that GLP-2 and Sucram treatments administered before a low-level C. parvum exposure may contribute to fewer effects on intestinal integrity, morphology, and inflammation in response to infection, and shorter, denser intestines.

Glucagon-like peptide 2 and its beneficial effects on gut function and health in production animals.

Numerous endocrine cell subtypes exist within the intestinal mucosa and produce peptides contributing to the regulation of critical physiological processes including appetite, energy metabolism, gut function, and gut health. The mechanisms of action and the extent of the physiological effects of these enteric peptides are only beginning to be uncovered. One peptide in particular, glucagon-like peptide 2 (GLP-2) produced by enteroendocrine L cells, has been fairly well characterized in rodent and swine models in terms of its ability to improve nutrient absorption and healing of the gut after injury. In fact, a long-acting form of GLP-2 recently has been approved for the management and treatment of human conditions like inflammatory bowel disease and short bowel syndrome. However, novel functions of GLP-2 within the gut continue to be demonstrated, including its beneficial effects on intestinal barrier function and reducing intestinal inflammation. As knowledge continues to grow about GLP-2's effects on the gut and its mechanisms of release, the potential to use GLP-2 to improve gut function and health of food animals becomes increasingly more apparent. Thus, the purpose of this review is to summarize: (1) the current understanding of GLP-2's functions and mechanisms of action within the gut; (2) novel applications of GLP-2 (or stimulators of its release) to improve general health and production performance of food animals; and (3) recent findings, using dairy calves as a model, that suggest the therapeutic potential of GLP-2 to reduce the pathogenesis of intestinal protozoan infections.

Neonatal lipopolysaccharide exposure does not diminish the innate immune response to a subsequent lipopolysaccharide challenge in Holstein bull calves.

The innate immune response following experimental mastitis is quite variable between individual dairy cattle. An inflammatory response that minimizes collateral damage to the mammary gland while still effectively resolving the infection following pathogen exposure is beneficial to dairy producers. The ability of a lipopolysaccharide (LPS) exposure in early life to generate a low-responding phenotype and thus reduce the inflammatory response to a later-life LPS challenge was investigated in neonatal bull calves. Ten Holstein bull calves were randomly assigned to either an early life LPS (ELL) group (n=5) or an early life saline (ELS) group (n=5). At 7d of age, calves received either LPS or saline, and at 32d of age, all calves were challenged with an intravenous dose of LPS to determine the effect of the early life treatment (LPS or saline) on the immune response generated toward a subsequent LPS challenge. Dermal fibroblast and monocyte-derived macrophage cultures from each calf were established at age 20 and 27d, respectively, to model sustained effects from the early life LPS exposure on gene expression and protein production of components within the LPS response pathway. The ELL calves had greater levels of plasma IL-6 and tumor necrosis factor-α than the ELS calves following the early life LPS or saline treatments. However, levels of these 2 immune markers were similar between ELL and ELS calves when both groups were subsequently challenged with LPS. A comparison of the in vitro LPS responses of the ELL and ELS calves revealed similar patterns of protein production and gene expression following an LPS challenge of both dermal fibroblast and monocyte-derived macrophage cultures established from the treatment groups. Whereas an early life exposure to LPS did not result in a dampened inflammatory response toward a later LPS challenge in these neonatal bull calves, the potential that exposure to inflammation or stress in early life or in utero can create an offspring with a low-responding phenotype as an adult is intriguing and has been documented in rodents. Further work is needed to determine if an inflammatory exposure in utero in a dairy animal would result in a low-responding innate immune phenotype.

Short communication: Amino acid supplementation and stage of lactation alter apparent utilization of nutrients by blood neutrophils from lactating dairy cows in vitro.

Glutamine is the preferred AA used by polymorphonuclear leukocytes (PMN) during the inflammatory response. However, the effect of other AA on bovine PMN response during inflammation and how this is altered by stage of lactation has not been fully elucidated. The objective of this study was to determine the effect of additional AA supplementation (pool of AA excluding Gln) on AA profiles, gene expression, and inflammatory function of PMN from dairy cows in early and mid lactation in vitro. We used 18 Holstein cows for this study. Polymorphonuclear leukocytes were isolated. Working solutions of AA (0 or 4 mM) and LPS (0 or 50µg/mL) were added to cell populations suspended in RPMI and incubated for 2h at 37°C. We used a subset of samples for gene and protein expression. Concentrations of AA in medium were determined using gas chromatography-mass spectrometry with norleucine as an internal standard. Apparent AA and glucose utilization were calculated by subtracting the concentration after from that of before incubation. Data were analyzed as a randomized block design. Challenge with LPS increased the expression of proinflammatory genes and AA supplementation decreased both the expression of some proinflammatory genes and the media concentrations of tumor necrosis factor-α. Neither stage of lactation, LPS challenge, nor AA supplementation altered the chemotactic or phagocytic abilities of PMN in vitro. Polymorphonuclear leukocytes supplemented with AA had greater concentrations and apparent utilization of most of the supplemented AA, whereas the unsupplemented group had greater apparent utilization of glucose. Alanine was not provided in the media but was present in spent media, and Ile, Gly, and Pro were greater in spent media than in media before incubation indicating synthesis of these AA. Regarding expression of genes involved in nutrient metabolism, the expression of G6PD, coding for the enzyme glucose 6-phosphate dehydrogenase, was increased and that of PDHA1, coding for the enzyme pyruvate dehydrogenase α 1, tended to increase with AA supplementation. Due to the lower concentration of tumor necrosis factor-α in media coupled with a downregulation of several proinflammatory genes, we concluded that AA, rather than Gln, alter the inflammatory response of bovine blood PMN. Independent from Gln, blood PMN from cows in early lactation may use certain AA as their primary carbon source for energy than cows in later lactation. Evaluating cows during the early postpartum period will provide additional information on the effect of stage of lactation and nutrient supplementation on PMN function.

In utero heat stress increases postnatal core body temperature in pigs.

In utero heat stress (IUHS) negatively impacts postnatal development, but how it alters future body temperature parameters and energetic metabolism is not well understood. Future body temperature indices and bioenergetic markers were characterized in pigs from differing in utero thermal environments during postnatal thermoneutral (TN) and cyclical heat stress (HS) exposure. First-parity pregnant gilts ( = 13) were exposed to 1 of 4 ambient temperature (T) treatments (HS [cyclic 28°C to 34°C] or TN [cyclic 18°C to 22°C]) applied for the entire gestation (HSHS, TNTN), HS for the first half of gestation (HSTN), or HS for the second half of gestation (TNHS). Twenty-four offspring (23.1 ± 1.2 kg BW; = 6 HSHS, = 6 TNTN, = 6 HSTN, = 6 TNHS) were housed in TN (21.7°C ± 0.7°C) conditions and then exposed to 2 separate but similar HS periods (HS1 = 6 d; HS2 = 6 d; cycling 28°C to 36°C). Core body temperature (T) was assessed every 15 min with implanted temperature recorders. Regardless of in utero treatment, T increased during both HS periods ( = 0.01; 0.58°C). During TN, HS1, and HS2, all IUHS pigs combined had increased T ( = 0.01; 0.36°C, 0.20°C, and 0.16°C, respectively) compared to TNTN controls. Although unaffected by in utero environment, the total plasma thyroxine to triiodothyronine ratio was reduced ( = 0.01) during HS1 and HS2 (39% and 29%, respectively) compared with TN. In summary, pigs from IUHS maintained an increased T compared with TNTN controls regardless of external T, and this thermal differential may have practical implications to developmental biology and animal bioenergetics.

Glucose supplementation has minimal effects on blood neutrophil function and gene expression in vitro.

During early lactation, glucose availability is low and the effect of glucose supply on bovine polymorphonuclear leukocyte (PMNL) function is poorly understood. The objective of this study was to determine the effect of glucose supplementation on the function and transcriptomic inflammatory response of PMNL from cows in early and mid-lactation in vitro. Twenty Holstein cows in early (n=10; days in milk=17±3.1) and mid-lactation (n=10; days in milk=168±14.8) were used for this study. Jugular blood was analyzed for serum concentrations of nonesterified fatty acids, ß-hydroxybutyrate, and glucose. Polymorphonuclear leukocytes were isolated and diluted using RPMI (basal glucose concentration was 7.2 mM) to different concentrations of PMNL/mL for phagocytosis, chemotaxis, gene expression, and medium analyses. Working solutions of glucose (0 or 4 mM of d-glucose) and lipopolysaccharide (0 or 50µg/mL) were added and tubes were incubated for 120 min at 37°C. Media were analyzed for concentrations of glucose and tumor necrosis factor-α (TNF-α). Data were analyzed in a randomized block (stage of lactation) design. Challenge with lipopolysaccharide increased the expression of the genes encoding for nuclear factor kappa B (NFKB1), IL-10 (IL10), IL1B, IL6, IL8, TNF-α (TNFA), glucose transporter 3 (SLC2A3), and the concentration of TNF-α in medium (147.3 vs. 72.5 pg/mL for lipopolysaccharide and control, respectively). Main effect of stage of lactation was minimal where the expression of IL10 increased for cows in early compared with cows in mid-lactation. After lipopolysaccharide challenge, cows in early lactation experienced more marked increases in the expression of IL6, TNFA, and IL8 when compared with cows in mid-lactation. Glucose supplementation had minimal effects on gene expression where glucose supplementation increased the expression of lysozyme (LYZ). Glucose supplementation increased PMNL phagocytosis but did not alter chemotaxis, morphology, or concentration of TNF-α in the medium. Under the conditions of the experiment, stage of lactation had minimal effects on PMNL response to glucose supply where only the expression of NFKB1 and the production of TNF-α were greater for cows in mid-lactation when compared with early lactation. Metabolic profiles for cows in early lactation did not parallel those for cows during the early postpartum period and may partly explain results for this study. Future studies investigating the effect of glucose supply on bovine PMNL function in vivo and how this may be altered by stage of lactation are warranted.

COMPARATIVE GUT PHYSIOLOGY SYMPOSIUM: Comparative physiology of glucagon-like peptide-2: Implications and applications for production and health of ruminants.

Glucagon-like peptide-2 (GLP-2) is a 33-amino acid peptide derived from proteolytic cleavage of proglucagon by prohormone convertase 1/3 in enteroendocrine L cells. Studies conducted in humans, in rodent models, and in vitro indicate that GLP-2 is secreted in response to the presence of molecules in the intestinal lumen, including fatty acids, carbohydrates, amino acids, and bile acids, which are detected by luminal chemosensors. The physiological actions of GLP-2 are mediated by its G protein-coupled receptor expressed primarily in the intestinal tract on enteric neurons, enteroendocrine cells, and myofibroblasts. The biological activity of GLP-2 is further regulated by dipeptidyl peptidase IV, which rapidly cleaves the N-terminus of GLP-2 that is responsible for GLP-2 receptor activation. Within the gut, GLP-2 increases nutrient absorption, crypt cell proliferation, and mesenteric blood flow and decreases gut permeability and motility, epithelial cell apoptosis, and inflammation. Outside the gut, GLP-2 reduces bone resorption, can suppress appetite, and is cytoprotective in the lung. Thus, GLP-2 has been studied intensively as a therapeutic to improve intestinal function of humans during parenteral nutrition and following small bowel resection and, more recently, as a treatment for osteoporosis and obesity-related disorders and to reduce cellular damage associated with inflammation of the gut and lungs. Recent studies demonstrate that many biological actions and properties of GLP-2 in ruminants are similar to those in nonruminants, including the potential to reduce intestinal nitro-oxidative stress in calves caused by parasitic diseases such as coccidiosis. Because of its beneficial impacts on nutrient absorption, gut healing, and normal gut development, GLP-2 therapy offers significant opportunities to improve calf health and production efficiency. However, GLP-2 therapies require an extended time course to achieve desired physiological responses, as well as daily administration because of the hormone's short half-life. Thus, practical means of administration and alternative strategies to enhance basal GLP-2 secretion (e.g., through specific feed additives), which are more likely to achieve consumer acceptance, are needed. Opportunities to address these challenges are discussed.

Environmental heat stress modulates thyroid status and its response to repeated endotoxin challenge in steers.

The objective of this study was to evaluate in cattle, the effects of acute exposure to a heat stress (HS) environment on the status of the pituitary (thyrotropin, TSH)-thyroid (thyroxine, T4)-peripheral tissue T4 deiodination (type 1 5'-deiodinase [D1]; triiodothyronine [T3]; reverse-triiodothyronine [rT3]) axis, and the further response of this pituitary-thyroid-peripheral tissue axis (PTTA) to perturbation caused by the induction of the proinflammatory innate immune state provoked by the administration of gram-negative bacteria endotoxin (lipopolysaccharide [LPS]). Ten steers (318 ± 49 kg body weight) housed in controlled environment chambers were subjected to either a thermoneutral (TN: constant 19°C) or HS temperature conditions (cyclical daily temperatures: 32.2°C-40.0°C) for a total period of 9 d. To minimize the effects of altered plane of nutrition due to HS, steers in TN were pair-fed to animals in HS conditions. Steers received 2 LPS challenges 3 d apart (LPS1 and LPS2; 0.2 µg/kg body weight, intravenously, Escherichia coli 055:B5) with the first challenge administered on day 4 relative to the start of the environmental conditioning. Jugular blood samples were collected at 0, 1, 2, 4, 7, and 24 h relative to the start of each LPS challenge. Plasma TSH, T4, T3, and rT3 were measured by radioimmunoassay. Liver D1 activity was measured in biopsy samples collected before the LPS1 (0 h) and 24 h after LPS2. Before the start of LPS1, HS decreased (P < 0.01 vs TN) plasma TSH (40%), T4 (45.4%), and T3 (25.9%), but did not affect rT3 concentrations. In TN steers, the LPS1 challenge decreased (P < 0.01 vs 0 h) plasma concentrations of TSH between 1 and 7 h and T4 and T3 at 7 and 24 h. In HS steers, plasma TSH concentrations were decreased at 2 h only (P < 0.05), whereas plasma T3 was decreased at 7 and 24 h (P < 0.01). Whereas plasma T4 concentrations were already depressed in HS steers at 0 h, LPS1 did not further affect the levels. Plasma rT3 concentrations were increased in all steers at 4, 7, and 24 h after LPS1 (P < 0.01). The patterns of concentration change of T4, T3, and rT3 during LPS2 mirrored those observed in LPS1; the responses in plasma TSH were of smaller magnitude than those incurred after LPS1. The LPS challenges reduced (P < 0.01) hepatic activity of D1 in all animals but no differences were observed between steers subjected to TN or HS environment. The data are consistent with the concept that acute exposure of cattle to a HS environment results in the depression of the pituitary and thyroid components of the PTTA, whereas a normal capacity to generate T3 from T4 in the liver is preserved. The data also suggest that LPS challenge further suppresses all components of the PTTA including liver T3 generation, and these PTTA perturbations are more pronounced in steers that encounter a HS exposure.

Short communication: Glucagon-like peptide-2 and coccidiosis alter tight junction gene expression in the gastrointestinal tract of dairy calves.

Tight junction (TJ) proteins are integral factors involved in gut barrier function, and therapy with glucagon-like peptide-2 (GLP-2) enhances gut integrity. Our aim was to assess effects of GLP-2 treatment on mRNA expression of 8 TJ complex proteins in the intestine of dairy calves not infected or infected with Eimeria bovis at 11±3d of age. Mucosal epithelium from jejunum, ileum, and cecum was collected at slaughter from Holstein bull calves assigned to 4 groups: noninfected, buffer-treated (n=5); noninfected, GLP-2 treated (n=4); E. bovis-infected, buffer-treated (n=5); and E. bovis-infected, GLP-2-treated (n=4). Infected calves were orally dosed with 100,000 to 200,000 sporulated E. bovis oocysts on d 0; GLP-2-treated calves received 50 µg of GLP-2/kg of body weight subcutaneously twice daily for 10d beginning on d 18; and buffer-treated calves received an equal injection volume of 0.01 M Na bicarbonate buffer. All calves were killed on d 28. The mRNA expression of coxsackie and adenovirus receptor (CXADR), claudins 1, 2, and 4 (CLDN1, CLDN2, and CLDN4), F11 receptor (F11R), junction adhesion molecule 2 (JAM2), occludin (OCLN), and tight junction protein ZO-1 (TJP1) was determined by real-time quantitative PCR. In jejunum and ileum, an interaction of E. bovis infection and GLP-2 treatment on gene expression was noted. In jejunum of noninfected calves, GLP-2 increased CXADR, CLDN2, OCLN, and TJP1 mRNA expression but had no effect on mRNA expression in infected calves. Treatment with GLP-2 also increased tight junction protein ZO-1 protein expression in jejunum of noninfected calves as determined by immunohistochemistry. In ileum, E. bovis decreased expression of JAM2, OCLN, and TJP1 in buffer-treated calves, and GLP-2 increased TJP1 expression in infected calves. In cecum, E. bovis infection reduced expression of CXADR, CLDN4, F11R, and OCLN, and GLP-2 therapy increased expression of CLDN4, F11R, OCLN, and TJP1. Results are consistent with studies in nonruminants showing decreased expression of TJ complex proteins in the intestinal tract during pathogen-induced diarrhea and increased TJ protein expression in intestinal tissues in response to GLP-2 treatment. In conclusion, E. bovis reduces gene expression of TJ proteins primarily in cecum of calves 28d postinfection, and GLP-2 increases expression of selected TJ genes in intestinal tissues. Use of GLP-2 to improve gut barrier function in ruminants during pathogen-induced diarrhea warrants additional study.

The effect of citrus-derived oil on bovine blood neutrophil function and gene expression in vitro.

Research on the use of natural products to treat or prevent microbial invasion as alternatives to antibiotic use is growing. Polymorphonuclear leukocytes (PMNL) play a vital role with regard to the innate immune response that affects severity or duration of mastitis. To our knowledge, effect of cold-pressed terpeneless Valencia orange oil (TCO) on bovine PMNL function has not been elucidated. Therefore, the objective of this study was to investigate the effect of TCO on bovine blood PMNL chemotaxis and phagocytosis capabilities and the expression of genes involved in inflammatory response in vitro. Polymorphonuclear leukocytes were isolated from jugular blood of 12 Holstein cows in mid-lactation and were incubated with 0.0 or 0.01% TCO for 120min at 37°C and 5% CO2, and phagocytosis (2×10(6) PMNL) and chemotaxis (6×10(6) PMNL) assays were then performed in vitro. For gene expression, RNA was extracted from incubated PMNL (6×10(6) PMNL), and gene expression was analyzed using quantitative PCR. The supernatant was stored at -80°C for analysis of tumor necrosis factor-α. Data were analyzed using a general linear mixed model with cow and treatment (i.e., control or TCO) in the model statement. In vitro supplementation of 0.01% of TCO increased the chemotactic ability to IL-8 by 47%; however, migration of PMNL to complement 5a was not altered. Treatment did not affect the production of tumor necrosis factor-α by PMNL. Expression of proinflammatory genes (i.e., SELL, TLR4, IRAK1, TRAF6, and LYZ) coding for proteins was not altered by incubation of PMNL with TCO. However, downregulation of TLR2 [fold change (FC=treatment/control)=-2.14], NFKBIA (FC=1.82), IL1B (FC=-2.16), TNFA (FC=-9.43), and SOD2 (FC=-1.57) was observed for PMNL incubated with TCO when compared with controls. Interestingly, expression of IL10, a well-known antiinflammatory cytokine, was also downregulated (FC=-3.78), whereas expression of IL8 (FC=1.93), a gene coding for the cytokine IL-8 known for its chemotactic function, tended to be upregulated in PMNL incubated with TCO. Incubation of PMNL with TCO enhanced PMNL chemotaxis in vitro. The expression of genes involved in the inflammatory response was primarily downregulated. Results showed that 0.01% TCO did not impair the function of PMNL in vitro. Future studies investigating the use of TCO as an alternative therapy for treatment of mastitis, including dose and duration, for cows during lactation are warranted.

Dynamics of Progesterone, TNF- α , and a Metabolite of PGF2 α in Blood Plasma of Beef Cows following Embryo Transfer.

Lactating beef cows previously synchronized for estrus (d 0) were assigned to four treatments to assess their effectiveness in increasing blood progesterone (P4) and its effects on tumor necrosis factor-α (TNF-α) and prostaglandin F2α (PGF2α) after the transfer of embryos. At the time of transfer (d 7), cows received no treatment (control; n = 16), a controlled internal drug releasing device (CIDR; n = 16), human chorionic gonadotropin (hCG; n = 15), or gonadotropin releasing hormone (GnRH; n = 15). Blood samples were taken on d 7, 14, and 21 for analysis of P4 and tumor necrosis factor-α (TNF-α). Blood was collected (every 15 min for 2 h) in half the animals in each treatment group on d 14 and the remaining half on d 21 for analysis of prostaglandin F2α metabolite (PGFM). Retention rates were 56.2, 62.5, 46.7, and 13.3% for cows in the control, CIDR, hCG, and GnRH groups, respectively. Progesterone was greater (P ≤ 0.05) in cows receiving hCG compared to others on d 14. Progesterone in all treatment groups increased from d 7 to d 14 and declined (P ≤ 0.05) from d 14 to d 21. Contrary to pregnant cows, P4 and TNF-α declined from d 7 to d 21 in nonpregnant cows (P ≤ 0.05). Although PGFM increased by d 21, there was no difference between pregnant and nonpregnant cows.

Blastocystis tropism in the pig intestine.

Blastocystis has been reported in pig feces but the sites of development in the gastrointestinal tract are unknown. The present study was undertaken to determine predilection sites of Blastocystis in 11 naturally infected pigs examined at 20 weeks of age. At necropsy, feces and contents of the duodenum, jejunum, ileum, and cecum were examined by immunofluorescence (IFA) microscopy and PCR and tissues from these sites as well as the proximal and distal colon were processed for histology from pigs 1 to 5. Feces were examined by IFA microscopy, and segments from the jejunum and ileum were processed for histology from pigs 6 to 11. Multiple sections were cut from each tissue segment, and each was stained with the following: hematoxylin and eosin, polyclonal rabbit antibody to Blastocystis, and ParaFlor B monoclonal antibody to Blastocystis. Blastocystis was detected in feces of all 11 pigs by IFA microscopy and determined by PCR and gene sequencing to be subtype 5 for pigs 1-5. Blastocystis was also detected in the lumen contents removed from the cecum of pigs 1-5 examined by IFA microscopy and in the cecum of pigs 4 and 5 by PCR. Blastocystis was also observed in tissue sections from the jejunum of 7 of the 11 pigs, in the proximal and distal colon of pigs 1-5, and in the cecum of 4 of these 5 pigs but was not detected in the duodenum or ileum of any pigs. In tissue sections, Blastocystis was found primarily in the lumen usually associated with digested food debris, sometimes in close proximity or appearing to adhere to the epithelium, but no stages were found to penetrate the epithelium or the lamina propria.

Sensitivity analysis of the relative biological effectiveness predicted by the local effect model.

The relative biological effectiveness (RBE) is a central quantity in particle radiobiology and depends on many physical and biological factors. The local effect model (LEM) allows one to predict the RBE for radiobiologic experiments and particle therapy. In this work the sensitivity of the RBE on its determining factors is elucidated based on monitoring the RBE dependence on the input parameters of the LEM. The relevance and meaning of all parameters are discussed within the formalism of the LEM. While most of the parameters are fixed by experimental constraints, one parameter, the threshold dose Dt, may remain free and is then regarded as a fit parameter to the high LET dose response curve. The influence of each parameter on the RBE is understood in terms of theoretic considerations. The sensitivity analysis has been systematically carried out for fictitious in vitro cell lines or tissues with α/ß = 2 Gy and 10 Gy, either irradiated under track segment conditions with a monoenergetic beam or within a spread out Bragg peak. For both irradiation conditions, a change of each of the parameters typically causes an approximately equal or smaller relative change of the predicted RBE values. These results may be used for the assessment of treatment plans and for general uncertainty estimations of the RBE.