Sodium salicylate treatment in early lactation increases whole-lactation milk and milk fat yield in mature dairy cows.

Multiple lines of inquiry have suggested that a high degree of inflammation in early lactation cows is associated with low productivity and increased disease incidence. In addition, some small studies have suggested that milk production increases in response to antiinflammatory treatment in the first week of lactation. Our objective was to determine if administration of sodium salicylate (SS), a nonsteroidal antiinflammatory drug (NSAID), in the first week of lactation changes whole-lactation productivity and retention in the herd. At calving, 78 cows [n=39 primiparous (1P); n=24 second parity (2P); n=15 third parity or greater (3P)] were alternately assigned to either control (CON) or SS treatments for 7 d postpartum. Sodium salicylate treatment was administered via individual water bowls at a concentration of 1.95 g/L, delivering a mean of 123.3±5.5 g of salicylate/d during the 7-d treatment. For the first 21 d of lactation, dry matter intake, water intake, milk yield, and health were monitored daily, and milk samples were collected twice weekly for milk component analysis. Monthly milk yield and component testing through the rest of the lactation provided data to assess long-term responses, and the effects of treatment on the risk of leaving the herd and on 305-d milk, fat, and protein yields were assessed. During the first 21 d of lactation, we observed no differences in morbidity, except for increased risk of metritis in 3P SS cows. Treatment interacted with parity to influence both 305-d milk and milk fat yields, and a tendency for an interaction was detected for 305-d milk protein yield. Milk yield was 2,469±646 kg greater over the lactation in 3P SS cows compared with 3P CON cows (21% increase) and tended to decrease by 8% in 1P cows treated with SS; no effects were detected in 2P cows. Furthermore, 3P SS cows produced 130±23 kg more milk fat over the lactation (30% increase), with no effects detected for 1P or 2P. Treatment with SS tended to increase 305-d milk protein yield in 3P cows by 14%, with no effects in 1P or 2P cows. A tendency for a treatment × parity interaction was also observed for the risk of leaving the herd. First-parity cows treated with SS tended to have greater risk of leaving the herd than controls (30 vs. 6% risk); however, treatment did not alter herd retention in 2P or 3P groups, and SS had no effect on the risk of leaving the herd overall. Results indicate that SS has long-term effects on lactation of mature dairy cows, particularly on fat yield, but may have negative effects for primiparous cows.

Effects of feeding L-carnitine to gilts through day 70 of gestation on litter traits and the expression of insulin-like growth factor system components and L-carnitine concentration in foetal tissues.

We investigated the influence of supplemental L-carnitine on foetal blood metabolites, litter characteristics, L-carnitine concentration in skeletal muscle and insulin-like growth factor (IGF) axis components in foetal hepatic and skeletal muscle tissues at day 40, 55 and 70 of gestating gilts. A total of 59 gilts (body weight = 137.7 kg) received a constant feed allowance of 1.75 kg/day and a top-dress containing either 0 or 50 ppm of L-carnitine starting on the first day of breeding through the allotted gestation length. Foetuses from the gilts fed diets with L-carnitine tended to be heavier (p = 0.06) and the circulating IGF-II tended to be lower (p = 0.09) at day 70, compared with the foetuses from the control gilts. Insulin-like growth factor-I messenger RNA (mRNA) was lower (p = 0.05) in hepatic tissue in the foetuses collected from gilts fed L-carnitine. Free and total carnitine concentration increased (p < 0.05) in the skeletal muscle from the foetuses collected from gilts fed supplemental L-carnitine. This study showed that L-carnitine had beneficial effects on the average foetal weight at day 70 of gestation, associated with changes in the foetal IGF system.

Effects of TNF receptor blockade on in vitro cell survival and response to negative energy balance in dairy cattle.

BACKGROUND: Associative data and some controlled studies suggest that the inflammatory cytokine tumor necrosis factor (TNF) α can induce fatty liver in dairy cattle. However, research demonstrating that TNFα is a necessary component in the etiology of bovine fatty liver is lacking. The aim of this work was to evaluate whether blocking TNFα signaling with a synthetic cyclic peptide (TNF receptor loop peptide; TRLP) would improve liver metabolic function and reduce triglyceride accumulation during feed restriction. RESULTS: Capability of TRLP to inhibit TNFα signaling was confirmed on primary bovine hepatocytes treated with recombinant bovine TNFα and 4 doses of TRLP (0, 1, 10, 50 µmol/L) over 24 h. Next, 4 lactating Holstein cows (parity 1.4 ± 0.5, 433 ± 131 d in milk) in an incomplete Latin rectangle design (3 × 2) were subcutaneously administered with different TRLP doses (0, 1.5, 3.0 mg/kg BW) every 4 h for 24 h, followed by an intravenous injection of TNFα (5 µg/kg BW). Before and for 2 h after TNFα injection, TRLP decreased plasma non-esterified fatty acid (NEFA) concentration (P ≤ 0.05), suggesting an altered metabolic response to inflammation. Finally, 10 non-pregnant, non-lactating Holstein cows (3.9 ± 1.1 yr of age) were randomly assigned to treatments: control (carrier: 57% DMSO in PBS) or TRLP (1.75 mg TRLP /kg BW per day). Treatments were administrated every 4 h for 7 d by subcutaneous injection to feed-restricted cows fed 30% of maintenance energy requirements. Daily blood samples were analyzed for glucose, insulin, ß-hydroxybutyrate, NEFA, and haptoglobin concentrations, with no treatment effects detected. On d 7, cows completed a glucose tolerance test (GTT) by i.v. administration of a dextrose bolus (300 mg glucose/kg BW). Glucose, insulin, and NEFA responses failed to demonstrate any significant effect of treatment during the GTT. However, plasma and liver analyses were not indicative of dramatic lipolysis or hepatic lipidosis, suggesting that the feed restriction protocol failed to induce the metabolic state of interest. Injection site inflammation, assessed by a scorer blinded to treatment, was enhanced by TRLP compared to control. CONCLUSIONS: Although the TRLP inhibited bovine TNFα signaling and altered responses to i.v. administration of TNFα, repeated use over 7 d caused apparent local allergic responses and it failed to alter metabolism during a feed restriction-induced negative energy balance. Although responses to feed restriction seemed atypical in this study, side effects of TRLP argue against its future use as a tool for investigating the role of inflammation in metabolic impacts of negative energy balance.

Effects of Salmonella enterica serovar Typhimurium, or serovar Choleraesuis, Lactobacillus reuteri and Bacillus licheniformis on chemokine and cytokine expression in the swine jejunal epithelial cell line, IPEC-J2.

Direct-fed microbials, including Lactobacillus and Bacillus spp., are potential replacements for low dose in-feed antibiotics for swine and other livestock. To understand the function of these microbes in the gut, the current study used pig jejunal epithelial cells (IPEC-J2) to evaluate how Lactobacillus reuteri (LR) and Bacillus licheniformis (BL) differed from Salmonella enterica serovars Typhimurium (ST) or Choleraesuis (SC) in their ability to regulate, stimulate, or modify the proinflammatory mediators, interleukin 8 (IL8), CC chemokine 20 (CCL20), and tumor necrosis factor-alpha (TNFalpha). To optimize the positive control to drive IL8 secretion by IPEC-J2 cells, cells were treated apically with various concentrations of ST (versus control (CTL)) for 1h, followed by a wash. Media containing gentamicin was added and collected at 6h post-treatment. Compared to CTL, 10(8) ST produced maximal IL8 secretion in both the apical and basolateral directions, with significant basolateral polarization (P<0.0001). We next evaluated the time course of IL8 secretion, and IL8, CCL20, and TNFalpha mRNA expression by IPEC-J2 cells treated apically with 10(8) ST, SC, LR, and BL versus CTL. Media and RNA were collected at 1.5, 3.0, and 6.0 h post treatment. Only ST stimulated an increase in IL8 secretion at any time point, with increases in IL8 mRNA at both 3 and 6h (P<0.05). However, BL increased IL8 mRNA at 1.5h (P<0.0001). Neither LR nor SC affected IL8 mRNA expression. CCL20 mRNA was strongly upregulated by ST (P<0.05) and BL (1.5 and 3.0 h; P<0.05), but not LR or SC. Only ST increased TNFalpha mRNA relative to CTL (P<0.05). Two experiments were conducted to determine if pre-exposure of IPEC-J2 cells to LR or BL modified ST induced IL8 secretion. Confluent cells were treated apically overnight with various levels of LR or BL (in separate experiments) followed by ST challenge. Media were collected at 4 (LR experiment) or 5h (BL experiment) post ST. In the LR study, IL8 secretion was increased by ST as compared to CTL (P<0.0001), reduced by LR (P<0.05), and LR+ST co-treatments failed to alter ST stimulated secretion. In the BL experiment, secretion of IL8 was increased by ST (P<0.0001), but blunted basolaterally in BL+ST co-treated wells. The data demonstrate that IPEC-J2 cells increase IL8 secretion in response to ST, and IL8 mRNA in response to ST and BL, but not LR. Furthermore, ST stimulated secretion of IL8 is inhibited basolaterally in the presence of BL.

Expression of Toll-like receptors, interleukin 8, macrophage migration inhibitory factor, and osteopontin in tissues from pigs challenged with Salmonella enterica serovar Typhimurium or serovar Choleraesuis.

Two serovars of Salmonella enterica, namely serovar Typhimurium (ST) and serovar Choleraesuis (SC) account for the vast majority of clinical cases of swine salmonellosis worldwide. These serovars are thought to be transmitted among pigs in production settings mainly through fecal-oral routes. Yet, few studies have evaluated effects of these serovars on expression of innate immune targets when presented to pigs via repeated oral dosing in an attempt to model transmission in production settings. Thus, a primary objective of the current experiments was to evaluate expression of Toll-like receptors (TLR) and selected chemoattractive mediators (interleukin 8, IL8; macrophage migration inhibitory factor, MIF; osteopontin, OPN) in tissues from pigs exposed to ST or SC that had been transformed with kanamycin resistance and green (STG) or red (SCR) fluorescent protein to facilitate isolation from pen fecal samples. In vitro studies confirmed that STG and SCR largely (though not completely) retained their ability to upregulate IL8 and CC chemokine ligand 20 (CCL20) in cultured swine jejunal epithelial cells. Transformed bacteria were then fed to pigs in an in vivo study to determine tissue specific effects on mRNA relative expression. Pigs were fed cookie dough inoculated with bacteria on days 0, 3, 7, and 10 with 10(8)CFU STG (n=8) or SCR (n=8), while control (CTL) pigs (n=8) received dough without bacteria. Animals were sacrificed 14 days from the initial bacterial challenge and samples of tonsil, jejunum, ileum, colon, mesenteric lymph node (MLN), spleen, and liver were removed for subsequent RNA isolation. Expression of mRNA in tissues was determined using real-time quantitative PCR and expressed relative to 18S rRNA. Within CTL pigs, when expressed relative to the content in liver, mRNA for all targets demonstrated substantial tissue effects (P<0.001 for all TLR; MIF, and OPN; P<0.05 for IL8). Feeding STG and SCR resulted in significant (P

Effects of Salmonella enterica serovars Typhimurium (ST) and Choleraesuis (SC) on chemokine and cytokine expression in swine ileum and jejunal epithelial cells.

The gastrointestinal epithelium represents a barrier to potentially invasive enteric pathogens, maintains a role in innate immune surveillance, and is a source of both chemokine and cytokine chemotactic mediators in response to bacterial invasion. In the current study, we evaluated cytokine and chemokine mediators known to regulate movement of macrophages (macrophage migration inhibitory factor; MIF), neutrophils (IL8), dendritic cells (CCL20), and epithelial remodeling (osteopontin; OPN) in response to invasive swine enteropathogens Salmonella enterica serovar Typhimurium (ST) or Choleraesuis (SC). For the in vivo experiment, weaned pigs served as uninfected controls (0 h) or were given 3 x 10(9) CFU ST orally. Pigs were sacrificed at 8, 24, 48, and 144 h after inoculation and total RNA was extracted from defined segments of proximal (PI) and distal (DI) ileum. Relative expression of MIF and OPN were not affected by ST. IL8 expression was increased numerically (P = 0.17 for the interaction term) at 24 and 144 h in the PI and these increases accounted for greater expression in the PI relative to the DI (P < 0.05). Relative expression of CCL20 was increased at 24 h after ST (P < 0.05). Next, we evaluated the time course of MIF, IL8, CCL20, and OPN mRNA expression induced by application of lipopolysaccharide (LPS), ST or SC in vitro using pig jejunal epithelial cells (IPEC-J2). Cells were grown to confluency on permeable membranes, and treated apically with LPS (10 ng/mL), ST or SC (10(8)/well). After 1 h, cells were washed to remove LPS or extracellular bacteria, and media containing gentamicin was added to kill remaining extracellular bacteria. Media and RNA were collected at 1.5, 3, and 6 h after treatment. MIF mRNA was not affected by LPS or bacterial treatment. Similarly, IL8 expression was not affected by LPS, but was increased by ST and SC relative to controls at 1.5 and 3 h post exposure (P < 0.05 for all comparisons). Treatment with SC increased CCL20 mRNA relative to controls at 3 h (P < 0.05), while ST increased CCL20 at 1.5, 3, and 6h with maximal expression at 6 h (P < 0.05 for all comparisons). ST and SC increased polarized IL8 secretion. Our data demonstrate that invasive bacterial pathogens in the pig gastrointestinal tract trigger upregulation of selected cytokine and chemokine mediators, but serovars of Salmonella elicited differing patterns of activation in vitro.

Does cortisol bias cytokine production in cultured porcine splenocytes to a Th2 phenotype?

Glucocorticoids are reported to bias the production of cytokines from a type 1 to a type 2 phenotype. However, this dogma has been advanced largely from studies utilizing potent glucocorticoid analogs, particularly dexamethasone (DEX). Although studies utilizing DEX certainly have clinical and pharmacological relevance, DEX is probably not the best glucocorticoid for studies designed to evaluate the interaction and regulation of endogenous corticosteroids with immune cells in vivo in the domestic pig. Functional measures of immune suppression suggest that the pig is relatively resistant to DEX. Furthermore, type II corticosteroid receptors exclusively bind DEX with high affinity, whereas type I receptors, the so-called mineralocorticoid receptors, have a higher affinity for cortisol. In addition, DEX is not bound by serum binding proteins as are endogenous corticosteroids. These issues prompted us to revisit glucocorticoid regulation of type 1 and type 2 cytokines in cultured pig splenocytes and to test the broad hypothesis that cortisol biases cytokine production in favor of a Th2 response. We evaluated interferon gamma (IFNgamma) (also interleukin 2 (IL-2) in one experiment) and interleukin 10 (IL-10) as representative Th1 and Th2 cytokines, respectively. Furthermore, we evaluated macrophage migration inhibitory factor (MIF) because it is reported to be an essential factor in T cell activation; it is also upregulated by glucocorticoids and reported to be a product of Th2 lymphocytes. In general, both IFNgamma and IL-10 were sensitive to cortisol inhibition early in culture. However, IFNgamma ultimately escaped cortisol inhibition, whereas IL-10 continued to be substantially suppressed by high physiological concentrations of cortisol. Similarly, MIF mRNA could be suppressed by cortisol, but only when cortisol was added to cultures after ConA (concanavalin A) stimulation of splenocytes. So, taken together, our studies do not support the hypothesis that cortisol favors a Th2 cytokine profile in cultured pig splenocytes.

Glucocorticoid regulation of type 1 and type 2 cytokines in cultured porcine splenocytes.

Glucocorticoids are reported to bias cytokines to a Th2 phenotype. However, this dogma has been advanced largely from studies utilizing potent glucocorticoid analogs. The current study was conducted to revisit the issue of glucocorticoid modulation of Th1/Th2 cytokine production and evaluate migration inhibitory factor (MIF) mRNA expression in cultured pig splenocytes treated with physiologically relevant concentrations of cortisol (CORT). Dexamethasone (DEX) was included for comparison. In Experiment 1, DEX, at 150 and 300 nM, suppressed concanavalin (ConA)-stimulated IFNgamma at both 12 and 24 h in culture, and IL-10 at 24h (P<0.05). Both 150 and 300 nM CORT suppressed IL-10 at 24 h (P<0.05), but neither concentration affected IFNgamma at 24 h. In Experiment 2, cells were cultured with a broader range of CORT for 48 h following ConA. Parallel cultures with identical treatments also were conducted in separate plates for evaluation of glucocorticoid regulation of MIF mRNA. IFNgamma was reduced by 300 nM DEX at 12, 24, and 48 h (P<0.05), whereas 150 and 300 nM CORT blunted IFNgamma at 24 h (P<0.05), but not 48 h. ConA increased IL-2 (P<0.01), but none of the steroid treatments affected IL-2. At both 12 and 24 h, IL-10 was reduced by 300 nM DEX and by 150 and 300 nM CORT (P<0.05). ConA increased relative abundance of MIF mRNA (P<0.001), but no steroid treatment affected MIF mRNA. In Experiment 3, steroid additions were delayed by 24 h after ConA, and cytokine concentrations evaluated 48 h later. Again, separate cultures were used for determination of effect of treatments on MIF mRNA. None of the steroid treatments affected IFNgamma, but 300 nM DEX reduced IL-10 (P<0.05). All of the CORT treatments (75-300 nM) reduced MIF mRNA (P<0.05), whereas DEX did not affect MIF mRNA in this experiment. The current experiments suggest that both DEX and high physiological concentrations of CORT can suppress both type 1 and type 2-like cytokines in cultured pig splenocytes. But, IL-10 was generally more sensitive to CORT suppression with increased time in culture than was IFNgamma. In addition, MIF mRNA could be suppressed by delayed addition of CORT to porcine splenocytes. Taken together, the data do not support the hypothesis that CORT directs the cytokine milieu toward a type 2 bias in cultured pig splenocytes.

The influence of duration of photoperiod and hemicastration on growth and testicular and endocrine functions of boars.

Yorkshire boars were used to evaluate the influence of duration of photoperiod and hemicastration on growth and testicular and endocrine functions. At 10 wk of age, 5 hemicastrate (HC) and 5 intact (I) boars were assigned to either 8 or 16 hr of light daily until 6 mo of age. Body weights were recorded biweekly throughout the experiment. Venous cannulae were placed in all boars at 6 mo of age, and serum was collected at 30 min intervals from 0800 to 2000 hr. Gonadotropin releasing hormone (GnRH) was infused at 2000 hr (50 micrograms) and at 2030 hr (250 micrograms), and samples of serum were collected until 2400 hr. The following day, all boars were castrated, and the weights and sperm content of the testes and epididymides were determined. At castration, all pigs were given implants containing testosterone. Two weeks later, pigs were again canulated, and serum was obtained at 15 min intervals for 2 hr. Growth of boars was not significantly affected by duration of photoperiod or number of testes. Duration of photoperiod did not affect weight or sperm content of testes or epididymides. Hemi-castrated boars had greater testicular (P less than .01) and capita-corpora (C-C) epididymal weights (P less than .05) and more testicular and C-C sperm (P less than .01) per testis. Neither average concentrations of luteinizing hormone (LH) nor number and amplitude of pulses of LH were affected by photoperiod treatment. However, HC boars had greater average concentrations of LH (P less than .05) than I boars (.71 +/- .05 vs .52 +/- .05 ng/ml). Hemicastrated boars in 16 hr light daily had greater concentrations of FSH in serum (P less than .05) than 8I, 8HC, and 16I boars. Intact and HC boars had similar concentrations of prolactin (PRL) and testosterone. Similarly, concentrations of PRL and testosterone were not affected by duration of photoperiod. Secretion of LH and testosterone after treatment with GnRH was not significantly affected by duration of photoperiod. In general, HC boars released more LH in response to GnRH treatment than I boars. Concentrations of LH were greater (P less than .05) in HC than I boars at .5, 1, 2, and 3 hr after GnRH and tended (P less than .10) to be elevated at 1.5, 2.5, 3.5 and 4 hr after GnRH. The FSH response to GnRH was greater (P less than .05) for 16HC than 8I, 8HC, or 16I boars.(ABSTRACT TRUNCATED AT 400 WORDS)

Free-running rhythms of adrenocorticotropic hormone (ACTH), cortisol and melatonin in pigs.

In the domestic pig, a circadian rhythm of plasma cortisol occurs, with greatest concentrations in the morning and lowest concentrations in the afternoon. However, photic entrainment of the rhythms of ACTH and melatonin in pigs have not been defined clearly. This experiment was designed to evaluate free-running rhythms of ACTH, cortisol and melatonin in pigs housed in constant light (LL) and constant darkness (DD). Twelve crossbred barrows, maintained under ambient photoperiod, were catheterized and tethered individually in two environmentally controlled rooms, one with LL and the other with DD. For animals in LL, fluorescent lights provided 202 +/- 15 (mean +/- standard deviation) lux of light at 65 cm above the floors. Incandescent nightlights equipped with 7 watt red bulbs provided 7 +/- 2 lux and were illuminated continuously in both rooms. Pigs were given at least 14 d exposure to LL and DD, then samples of plasma and serum were obtained at hourly intervals for 48 hr. Plasma was assayed for ACTH, and serum for cortisol and melatonin. Periodograms were constructed to analyze the data. For this type of analysis, a statistic, Qp, is calculated, and circadian periodicity is suggested if maximum Qp (Qp max) occurs at or near 24 hr. The period of the free-running rhythms (tau) at Qp max for ACTH, cortisol and melatonin for pigs in LL (23.80 +/- .01, 23.78 +/- .01, and 23.21 +/- .02 hr, respectively) did not differ significantly from those for pigs in DD (23.39 +/- .01, 23.20 +/- .01, and 22.55 +/- .02 hr, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of corticotropin-releasing hormone, lysine vasopressin, oxytocin, and angiotensin II on adrenocorticotropin secretion from porcine anterior pituitary cells.

The aim of this study was to determine the ability of corticotropin-releasing hormone (CRH), lysine vasopressin (LVP), oxytocin (OT), and angiotensin II (AII) to stimulate adrenocorticotropin (ACTH) secretion from porcine anterior pituitary (AP) cells in vitro and to evaluate the role of protein kinase C (PKC) in the interaction between CRH and LVP. In this study, porcine AP cells were enzymatically and mechanically dispersed, cultured (150,000 cells/well) for 4 d, and then challenged with doses of various neuropeptides for 3 hr. CRH (10(-7)-10(-10) M) was the most potent of the peptides tested in stimulating ACTH release from porcine AP cells. In fact, none of the other peptides consistently affected ACTH concentrations relative to basal levels. However, LVP potentiated CRH action, even though by itself, it failed to stimulate ACTH production. Neither OT or AII potentiated CRH-stimulated ACTH release from porcine AP cells. To determine whether the inter-action between CRH and LVP was regulated partially by the protein Kinase C (PKC) pathway, we challenged AP cells in a 30-min incubation with 10(-7) M staurosporine (ST), a treatment predicted to decrease PKC activity. Then, cells were washed and challenged with 10(-9) M LVP, 10(-9) M CRH, and 10(-9) M CRH + LVP. Treatment with ST decreased (P < 0.05) CRH + LVP-stimulated ACTH release. To further demonstrate an interaction between protein kinase A (PKA) and PKC transduction pathways in the observed synergism between CRH and LVP to enhance ACTH secretion, we also challenged AP cells with 10(-7) M phorbol 12, 13-myristate acetate (PMA) and 5 microM forskolin (FOR) for 3 hr. This treatment was predicted to enhance PKA and PKC activities, respectively, and thereby enhance ACTH concentrations. Challenging cells with FOR + PMA enhanced (P < 0.001) ACTH release above basal concentrations, but more important, it increased (P < 0.001) ACTH concentration above that elicited by either drug given alone. Taken together, our in vitro studies support the conclusion that CRH is the principal regulator of ACTH secretion in the pig. In contrast to the results in most other species evaluated, vasopressin alone did not affect ACTH release. However, LVP can enhance the effectiveness of CRH in releasing ACTH, and this enhancement appears to rely, at least in part, on the activation of the PKC signal transduction pathway.

Contribution of the photoperiod to circadian variations in serum cortisol and melatonin in boars.

Four pubertal (25 wk) crossbred boars were used to evaluate the role of the photoperiod in the organization of secretion of cortisol and melatonin throughout a 24-hr period. Boars were exposed to a 16-hr photoperiod in an environmentally controlled room and temperature was kept constant at 22 C. For the first 15 d, the onset of the photophase (light phase) and scotophase (dark phase) was 0200 hr and 1800 hr, respectively (ON2). During the final 15 d, the onset of the photophase and scotophase was advanced by 6 hr (0800 hr and 2400 hr, respectively; ON8). Samples of serum were obtained from catheterized boars at hourly intervals for 24 hr between the 14th to 15th d in both ON2 and ON8 photoperiods. Samples of serum were frozen and later assayed for cortisol and melatonin. The data were normalized to the clock time of onset of the photophase, fit to polynomial equations, and the profiles of cortisol and melatonin in ON2 and ON8 were tested for heterogeneity of regression. A circadian rhythm of cortisol was observed in both ON2 and ON8 and the fitted profiles were parallel, suggesting that the rhythm had shifted with the 6-hr shift in lights-on, lights-off. Generally, higher concentrations were observed during the late subjective evening and scotophase, whereas lower concentrations occurred about 6 to 10 hr after onset of the photophase. Although the profiles of melatonin did not differ in ON2 and ON8, there was no evidence of a nocturnal rise in melatonin in either treatment. It is suggested that some characteristic of the photoperiod organizes the circadian rhythm of cortisol in boars, but melatonin is not secreted in a rhythmic fashion in long photoperiods.

Serum hormones during the estrous cycle and estrous behavior in heifers after administration of propylthiouracil and thyroxine.

Two experiments were conducted to assess the characteristics of thyroid-stimulating hormone (TSH) during the estrous cycle and to examine the effects of experimentally altered thyroid status on estrous behavior and hormonal concentrations in heifers. In Exp. 1, normal profiles of luteinizing hormone (LH), follicle-stimulating hormone (FSH), and TSH were examined on days 2, 12, and 19 of the estrous cycle (day 0 = estrus) in six Holstein heifers. Baseline concentrations of LH were 1.5- to 2.1-fold higher on day 2 (P = .07) and day 19 (P < .01) than on day 12. Pulse frequency of LH was higher (P < .05) on days 12 and 19 than on day 2. For FSH, baseline concentrations were similar on all 3 d, but pulse frequency was higher (P < .05) on day 19 than on day 2 or day 12. Overall and baseline concentrations of TSH were similar on all 3 d. Pulse frequency of TSH was higher (P < .05) on day 12 than on day 19, but both were similar to that on day 2. In Exp. 2, secretion of TSH in Holstein heifers was altered by administering (i.v.; one-half of the daily dose given twice daily) either phosphate-buffered saline (SAL; n = 6), propylthiouracil (PTU; 20 mg/100 kg body weight [BW]; n = 6), or T4 (1 mg/100 kg BW; n = 5) from day 4 until the next estrus. Duration of estrous cycles during and after treatments, percentages of heifers exhibiting estrus, BW, and average daily gain were unaffected by treatment. Concentrations of TSH were higher (P < .05) in the afternoon than in the morning and higher (P = .06) in PTU heifers than in T4 heifers. Treatment with T4 during the treatment estrous cycle and for up to 6 d into the next estrous cycle increased (P < .01) concentrations of T4 and T3 compared to SAL and PTU. Treatment with PTU during the treatment cycle increased concentrations of T4 (P < .05) and TSH (P = .06) compared to SAL. We conclude that TSH secretion was pulsatile, demonstrated a diurnal change, and varied inconsistently during the estrous cycle. Altering thyroid hormones and TSH secretion in heifers seemed to have no effect on estrous behavior or estrous cycles.

Serum hormones in response to estradiol and (or) progesterone in ovariectomized cows after thyroidectomy.

To evaluate the effect of gonadal steroid treatment and thyroidectomy on concentrations of gonadotropins and thyroid-stimulating hormone in the bovine, nonlactating Holstein cows were either thyroidectomized and ovariectomized (THYOVEX; n = 6) or ovariectomized only (OVEX; n = 4), and subsequently treated with no gonadal steroids (control), estradiol-17 beta (E2), progesterone (P4), or P4+E2 in a 2 x 4 factorial experiment. Averaged across steroid treatments, baseline concentrations of luteinizing hormone (LH; P < .05) and follicle-stimulating hormone (FSH; P < .10) were higher in THYOVEX cows than in OVEX cows. Pulse frequencies and amplitudes of LH and FSH did not differ between THYOVEX and OVEX cows. Secretion of TSH was pulsatile and all concentrations and pulsatile characteristics of TSH were increased (P < .05) in THYOVEX compared to OVEX cows. Treatment with E2 and P4 decreased (P < .05) baseline concentrations and magnitude of LH pulses, whereas P4+E2 increased (P < .01) pulse frequency of LH and FSH. Amplitude of LH and FSH pulses were not affected by treatment with either steroid. Treatment with P4+E2 decreased (P < .05) baseline concentrations of TSH, whereas pulse frequency, and magnitude and amplitude of TSH pulses were not altered by treatment with steroids. Mean concentrations of LH and FSH were similar during 48 hr after termination of E2 and P4+E2 treatments, but concentrations of TSH were higher (P = .06) after P4+E2 than after E2. Secretion of TSH showed a diurnal variation, with the lowest concentrations in the morning and highest in the afternoon. These results indicate that thyroidectomy influenced secretion of gonadotropins in OVEX cows.

Effect of endotoxin on interleukin-6 secretion and messenger ribonucleic acid in porcine anterior pituitary cells.

The pleiotropic cytokine interleukin-6 (IL-6) is produced in and secreted from anterior pituitary (AP) cells of a number of species. Bacterial endotoxin (END) may enhance the transcription of IL-6 and its secretion from the AP. In the studies presented here, we evaluated pig AP cells for the presence of IL-6 mRNA. In addition, because we had observed previously that END stimulated the secretion of prostaglandin E2 from cultured porcine AP cells, the effects of the inhibition of END-stimulated cyclooxygenase products on IL-6 mRNA abundance and the secretion of IL-6 were evaluated. In the first experiment, RNA was extracted from cultured pig AP cells that had been treated with END for 0.5 or 1 hr and subjected to reverse transcription followed by polymerase chain reaction and hybridization after Southern transfer. Bands of expected amplified product size, corresponding to IL-6, were observed only from cells treated with END, although specific hybridization was observed from both control and END-treated wells. In the next experiment, RNA was extracted from cultured AP cells treated with END or END in the presence of the cyclooxygenase inhibitor indomethacin (IND). Amplification of the expected product could be observed from all cultured cells except those treated with IND. However, hybridization data indicated that IND did not eliminate IL-6 mRNA entirely. Next, we measured IL-6 secretion from cultured AP cells exposed to END or END and IND. Treatment with END stimulated IL-6 secretion (P < 0.001) above controls, whereas IND blocked END stimulation of IL-6 secretion (P < 0.001). Finally, using immunostaining, we confirmed the presence of CD14, an END receptor, in cultured pig AP cells. These studies clearly establish the presence of IL-6 mRNA and secretion of the cytokine from cultured porcine AP cells. In addition, END stimulates the secretion of IL-6, perhaps through cells expressing CD14, and END-stimulated IL-6 secretion appears to be mediated by products of the cyclooxygenase pathway.

Changes in circulating insulin-like growth factor-I, insulin-like growth factor binding proteins, and leptin in weaned pigs infected with Salmonella enterica serovar Typhimurium.

One of the hallmarks of the pathophysiology of enteric disease in young pigs is reduced growth performance. This reduction in growth is associated with changes in the endocrine somatotropic growth axis. Our laboratory previously demonstrated that circulating insulin-like growth factor-I (IGF-I) was reduced in pigs infected with Salmonella enterica serovar Typhimurium (S. typhimurium) while circulating growth hormone remained unchanged. The objective of the current study was to determine if infection with S. typhimurium also was associated with changes in circulating IGF binding proteins (IGFBP). In addition, pigs experiencing active enteric disease have reduced feed intake. Because this inappetence may be related to systemic appetite reduction signals, we also evaluated circulating leptin in pigs undergoing active S. typhimurium-induced enteric disease. Crossbred pigs were penned in environmentally controlled rooms with free access to feed and water. Following an acclimation period, pigs were gavaged with 10(10) cfu of S. typhimurium (SAL; n=6) or were given a similar volume of sterile growth media (CON; n=6). Rectal temperatures and feed intakes were measured daily through 168 h to track the time course of the response to S. typhimurium infection. Samples of serum were obtained by jugular venipuncture at 0, 24, 48, 96 and 168 h after infection. Sera were frozen until evaluation for IGF-I by immunoradiometric assay (IRMA). In addition, sera were subjected to western ligand blotting utilizing 125I-IGF-I and 125I-IGF-II. Images were evaluated for total IGFBP and IGFBP-3 by densitometric analyses. Rectal temperature was increased in SAL pigs 24h post-infection (P<0.001) but not at other times. Feed intake was reduced in SAL pigs during the intervals 24-72 h (P<0.001) and 96-144 h (P<0.05) after infection. Serum IGF-I, expressed as a percentage of the 0 h concentration, was reduced in SAL pigs versus CON pigs at 48 h (28.1+/-18.7% versus 102.2+/-17.1%; P<0.01) and 96 h (20.0+/-18.7% versus 128.4+/-17.0%; P<0.0001) post-infection. Both total IGFBP and IGFBP-3, as estimated by ligand blotting, also were reduced in infected pigs at 48 h postchallenge (P<0.05). IGFBPs were similar between the two treatments at other sampling times. Concentrations of IGFBP-3 also were estimated utilizing an IRMA for human IGFBP-3. Serum IGFBP-3 was reduced in S. typhimurium-infected pigs at 24 h (P<0.01), 48 h (P<0.001), 96 h (P<0.001), and 168 h (P<0.05). Serum leptin levels were similar between SAL and CON pigs. The data suggest that swine enteric disease is associated with reduced circulating IGF-I and reductions in total IGFBP and IGFBP-3. However, serum leptin was not affected by enteric disease challenge.

Dietary L-carnitine increases plasma leptin concentrations of gestating sows fed one meal per day.

Thirty-four sows (parity=1.8; BW=206 kg) were used to determine the influence of L-carnitine and/or chromium tripicolinate on plasma leptin concentrations of gestating sows fed one meal daily. Treatments were arranged in a 2 x 2 factorial with main effects of carnitine (0 or 50 ppm) and chromium (0 or 200 ppb). Diets were fed for approximately 167 days (through one gestation, the following lactation, the interval from weaning to estrus, and 28 days into the following gestation) prior to blood collection. Leptin concentration was determined in plasma that was collected at feeding, every 15 min for the first 3h after feeding, and at 6, 9, 15, 20, and 24h after feeding. Sows fed diets containing carnitine had greater (P<0.02) overall mean plasma leptin concentrations and greater (P<0.05) leptin concentrations at 2.25, 3, 6, 15, 20, and 24h after feeding compared to sows fed either the control diet or the diet containing chromium. Leptin concentrations of sows fed diets containing carnitine also were greater (P<0.05) than control sows at 2.5 and 2.75 h postprandial and greater than (P<0.05) sows fed diets with both carnitine and chromium at 6h after feeding. Chromium had no effect (P>0.10) on plasma leptin concentration. These results suggest that dietary carnitine, but not chromium, increases circulating leptin in gestating sows fed one meal per day. These results may help to explain the improvements in reproductive function previously observed from feeding sows diets containing carnitine.

Pituitary function in the acute phase response in domestic farm animals: cytokines, prostaglandins, and secretion of ACTH.

Contained in this report is a review of available data on pituitary cytokines in domestic species of agricultural importance. The concept is advanced that the pituitary gland is essential to appropriate generation of host defense mechanisms and thus should be considered among other tissues contributing to innate immunity. The functions of these intrapituitary cytokines, principally IL-6, are discussed in the context of potential regulation of the pituitary-adrenal axis (ACTH secretion) via intrapituitary PGE2 generation during the acute-phase response to infectious/inflammatory stimuli. Data from other species are cited as appropriate for comparative purposes and elaboration of proposed mechanisms. However, the scope of the review is not intended to comprehensively cover the vast literature on proinflammatory cytokines and prostaglandins generated peripherally and centrally during host responses to inflammatory stimuli.

GnRH antagonist inhibition of luteinizing hormone secretion and ovulation in the pig.

A GnRH antagonist, Ac-D-p-Cl-Phe1,2, D-Trp3, D-Arg6, D-Ala10 GnRHb (Organon), was utilized to determine the effective dosage and duration to inhibit LH secretion in the pig. In a preliminary trial, barrows received either 10, 50, or 250 micrograms/kg BW of the GnRH antagonist. Secretion of LH was inhibited within 30 min for a duration of 12 h with the 100 micrograms/kg dose but persisted for greater than 48 h with the 250 micrograms/kg treatment. A second study determined effectiveness of the antagonist for inhibiting ovulation in cyclic gilts. At first detection of standing estrus, cyclic gilts were treated with either saline (control), 100, or 200 micrograms/kg BW of the GnRH antagonist (GnRH1). A second group of GnRH antagonist gilts received 200 micrograms/kg BW of the GnRH antagonist approximately 8 h prior to standing estrus (GnRH2). The GnRH1-treatment failed to inhibit or delay ovulation. Ovulation was inhibited and estrous cycles lengthened in GnRH2-treated gilts. These preliminary results suggest that ovulation in the gilt can be inhibited if the GnRH antagonist is administered prior to the LH surge.

Influence of prepartum exposure of beef heifers to winter weather on concentrations of plasma energy-yielding substrates, serum hormones and birth weight of calves.

The influence of exposure to ambient winter weather conditions (WW) during the final 90 d of gestation on serum hormones, plasma substrates, and birth weight of calves was evaluated in spring-calving, primiparous beef heifers. At of 192+/-14 d of gestation, heifers were assigned by expected calving date, breed, and sire of fetus to one of two treatments. Thirteen heifers were assigned to thermoneutral environment (TN; 12 degrees C) and housed in temperature-controlled rooms. Heifers in WW (n=16) were maintained outdoors in drylots without access to shelter and given additional dietary energy when average weekly windchill fell below -6.7 degrees C. Body weights, hip-heights and samples of serum and plasma were obtained biweekly until heifers were relocated, approximately 7 d prior to expected calving. Polynomial response curves for concentrations of glucose and nonesterified fatty acids (NEFA) in plasma and cortisol in serum were not influenced by treatment. However, average concentrations of NEFA in plasma were increased (P<0.02) and glucose tended to be higher (P=0.13) in WW heifers compared to TN heifers (172.5+/-8.9 vs 136.9+/-7.7 micromol/L and 87.8+/-2.4 vs 83.3+/-2.7 mg/100ml for NEFA and glucose, respectively). Time trends of concentrations of estradiol in serum (P<0.01) and hip-height to weight ratios were different (P<0.05) for WW and TN. Birth weights of calves from TN heifers were greater (P<0.06) than calves from WW heifers (42.3+/-2.0 vs 36.9+/-1.8 kg), but average calving difficulty scores were similar for both heifer groups (3.2+/-0.5 vs 2.7+/-0.4). These data suggest that exposing spring-calving cows to reduced effective ambient temperatures during the final 90 d of pregnancy may elevate energy-yielding metabolites in plasma and alter endocrine function. These changes may contribute to reduced birth weight of calves.