Influence of prenatal transportation stress-induced differential DNA methylation on the physiological control of behavior and stress response in suckling Brahman bull calves.

The objective of this experiment was to examine potential differential methylation of DNA as a mechanism for altered behavioral and stress responses in prenatally stressed (PNS) compared with nonprenatally stressed (Control) young bull calves. Mature Brahman cows (n = 48) were transported for 2-h periods at 60 ± 5, 80 ± 5, 100 ± 5, 120 ± 5, and 140 ± 5 d of gestation (Transported group) or maintained as nontransported Controls (n = 48). From the offspring born to Transported and Control cows, a subset of 28-d-old intact bulls (n = 7 PNS; n = 7 Control) were evaluated for methylation of DNA of behavior and stress response-associated genes. Methylation of DNA from white blood cells was assessed via reduced representation bisulfite sequencing methods. Because increased methylation of DNA within gene promoter regions has been associated with decreased transcriptional activity of the corresponding gene, differentially methylated (P ≤ 0.05) CG sites (cytosine followed by a guanine nucleotide) located within promoter regions (n = 1,205) were used to predict (using Ingenuity Pathway Analysis software) alterations to canonical pathways in PNS compared with Control bull calves. Among differentially methylated genes (P ≤ 0.05) related to behavior and the stress response were OPRK1, OPRM1, PENK, POMC, NR3C2, TH, DRD1, DRD5, COMT, HTR6, HTR5A, GABRA4, GABRQ, and GAD2. Among altered (P < 0.05) signaling pathways related to behavior and the stress response were Opioid Signaling, Corticotropin-Releasing Hormone Signaling, Dopamine Receptor Signaling, Dopamine-DARPP32 Feedback in cAMP Signaling, Serotonin Receptor Signaling, and GABA Receptor Signaling. Alterations to behavior and stress response-related genes and canonical pathways supported previously observed elevations in temperament score and serum cortisol through weaning in the larger population of PNS calves from which bulls in this study were derived. Differential methylation of DNA and predicted alterations to behavior and stress response-related pathways in PNS compared with Control bull calves suggest epigenetic programming of behavior and the stress response in utero.

Influence of prenatal transportation stress on innate immune response to an endotoxin challenge in weaned Brahman bull calves†,‡.

The objective of this study was to assess the influence of prenatal stress (PNS) on innate immune responses to an endotoxin challenge in weaned bull calves. Altered innate immune response to lipopolysaccharide (LPS), as characterized by changes in a range of variables was hypothesized in PNS bull calves. Brahman cows (n = 96; 48 stressed by transportation at five stages of gestation and 48 Controls) produced 85 calves, from which 16 uncastrated male (bull) calves from each PNS and Control treatment were selected for an LPS challenge period. Rectal temperature (RT), sickness behavior score (SBS), serum concentrations of cortisol, interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and complete blood count (CBC) variables were assessed in response to intravenous LPS (0.25 µg/kg body weight) administration. Each reported variable increased or decreased following LPS administration. Prior to LPS, PNS bull calves exhibited increased TNF-α, IL-6, and monocyte counts, but decreased IFN-γ, eosinophils, and basophils (p < .05). Compared with Control, in response to LPS, PNS bull calves exhibited greater circulating concentrations of cortisol. PNS bull calves exhibited lower (p < .05) eosinophil and basophil counts at time 0 (time of LPS administration) but similar counts to Control bull calves 2 h after LPS. PNS bull calves exhibited a greater change from baseline for IFN-γ and monocytes in response to LPS administration. No other variables were influenced by prenatal treatment (p > .05). These findings suggest that PNS did not adversely affect basal or induced components of the innate immune response to an immunological challenge. Lay summary Our laboratory studied the influence of prenatal stress (i.e., transportation of pregnant cows) on immune function of bull calves at 8 months of age. This was accomplished by studying aspects of their innate immune response to an immunological challenge. Prenatal stress did not adversely affect basal or induced components of the innate immune response to an immunological challenge.

Prenatal transportation stress alters genome-wide DNA methylation in suckling Brahman bull calves.

The objective of this experiment was to identify genome-wide differential methylation of DNA in young prenatally stressed (PNS) bull calves. Mature Brahman cows (n = 48) were transported for 2-h periods at 60 ± 5, 80 ± 5, 100 ± 5, 120 ± 5, and 140 ± 5 d of gestation or maintained as nontransported Controls (n = 48). Methylation of DNA from white blood cells from a subset of 28-d-old intact male offspring (n = 7 PNS; n = 7 Control) was assessed via reduced representation bisulfite sequencing. Samples from PNS bulls contained 16,128 CG, 226 CHG, and 391 CHH (C = cytosine; G = guanine; H = either adenine, thymine, or cytosine) sites that were differentially methylated compared to samples from Controls. Of the CG sites, 7,407 were hypermethylated (at least 10% more methylated than Controls; P ≤ 0.05) and 8,721 were hypomethylated (at least 10% less methylated than Controls; P ≤ 0.05). Increased DNA methylation in gene promoter regions typically results in decreased transcriptional activity of the region. Therefore, differentially methylated CG sites located within promoter regions (n = 1,205) were used to predict (using Ingenuity Pathway Analysis software) alterations to canonical pathways in PNS compared with Control bull calves. In PNS bull calves, 113 pathways were altered (P ≤ 0.05) compared to Controls. Among these were pathways related to behavior, stress response, metabolism, immune function, and cell signaling. Genome-wide differential DNA methylation and predicted alterations to pathways in PNS compared with Control bull calves suggest epigenetic programming of biological systems in utero.

Use of random regression to estimate genetic parameters of temperament across an age continuum in a crossbred cattle population.

The objective was to estimate genetic parameters of temperament in beef cattle across an age continuum. The population consisted predominantly of Brahman-British crossbred cattle. Temperament was quantified by 1) pen score (PS), the reaction of a calf to a single experienced evaluator on a scale of 1 to 5 (1 = calm, 5 = excitable); 2) exit velocity (EV), the rate (m/s) at which a calf traveled 1.83 m upon exiting a squeeze chute; and 3) temperament score (TS), the numerical average of PS and EV. Covariates included days of age and proportion of Bos indicus in the calf and dam. Random regression models included the fixed effects determined from the repeated measures models, except for calf age. Likelihood ratio tests were used to determine the most appropriate random structures. In repeated measures models, the proportion of B. indicus in the calf was positively related with each calf temperament trait (0.41 ± 0.20, 0.85 ± 0.21, and 0.57 ± 0.18 for PS, EV, and TS, respectively; P < 0.01). There was an effect of contemporary group (combinations of season, year of birth, and management group) and dam age (P < 0.001) in all models. From repeated records analyses, estimates of heritability (h2) were 0.34 ± 0.04, 0.31 ± 0.04, and 0.39 ± 0.04, while estimates of permanent environmental variance as a proportion of the phenotypic variance (c2) were 0.30 ± 0.04, 0.31 ± 0.03, and 0.34 ± 0.04 for PS, EV, and TS, respectively. Quadratic additive genetic random regressions on Legendre polynomials of age were significant for all traits. Quadratic permanent environmental random regressions were significant for PS and TS, but linear permanent environmental random regressions were significant for EV. Random regression results suggested that these components change across the age dimension of these data. There appeared to be an increasing influence of permanent environmental effects and decreasing influence of additive genetic effects corresponding to increasing calf age for EV, and to a lesser extent for TS. Inherited temperament may be overcome by accumulating environmental stimuli with increases in age, especially after weaning.

Effect of oral administration of meloxicam prior to transport on inflammatory mediators and leukoctye function of cattle at feedlot arrival.

OBJECTIVE To investigate the effects of meloxicam administration before long-distance transport on inflammatory mediators and leukocyte function of cattle at feedlot arrival. ANIMALS 60 healthy yearling beef steers. PROCEDURES Single-source steers were assigned to a transported (n = 40) or nontransported (20) group. Then, half of the steers within each group were assigned to receive meloxicam (1 mg/kg, PO) or a lactose placebo (1 bolus/steer, PO). All steers were transported approximately 1,300 km overnight to a feedlot; however, the nontransported group was moved before treatment (meloxicam or placebo) administration and allowed a 17-day acclimation period, whereas the transported group was moved immediately after treatment administration on day -1. Blood samples for measurement of inflammatory mediators and leukocyte function were collected from all steers on days -1, 0, and 3. RESULTS For steers that received meloxicam, mean plasma meloxicam concentration for the transported group was significantly greater than that for the nontransported group on day 0. For steers that received the placebo, mean haptoglobin-matrix metalloproteinase-9 complex for the transported group was significantly greater than that for the nontransported group on day 0. Mean haptoglobin concentration, neutrophil L-selectin intensity, and polymorphonuclear leukocyte count for the transported group were significantly greater than those for the nontransported group. Mean substance P concentration for nontransported steers that received meloxicam was significantly lower than that for the other 3 treatment groups. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated meloxicam administration to healthy steers immediately before long-distance transport did not significantly mitigate the effects of transport-induced stress on leukocyte function or inflammatory markers.

Sexually dimorphic innate immunological responses of pre-pubertal Brahman cattle following an intravenous lipopolysaccharide challenge.

This study was designed to characterize potential sexually dimorphic immunological responses following a lipopolysaccharide (LPS) challenge in beef cattle. Six female (heifers) and five male (bulls) Brahman calves (average age=253 ± 19.9 and 288 ± 47.9 days; average body weight=194 ± 11 kg and 247 ± 19 kg for heifers and bulls, respectively) were challenged with LPS (0.25 µg LPS/kg body weight). Following administration of LPS, all cattle displayed increased sickness behavior beginning at 0.5h, with heifers on average displaying less sickness behavior than bulls. A febrile response was observed in all animals following LPS administration, with a maximum response observed from 4 to 5.5h. The average rectal temperature response was greater in heifers than bulls. In all cattle there were elevated serum concentrations of cortisol from 0.5 to 8h, TNF-α from 1 to 2.5h, IL-6 from 2 to 8h, and IFN-γ from 2.5 to 7h after LPS challenge. Additionally, serum concentrations of TNF-α were greater in heifers than bulls from 1.5 to 2h after the LPS challenge. Concentrations of IFN-γ were also greater on average in bulls than heifers. Leukopenia occurred from 1 to 8h, with a decreased neutrophil to lymphocyte ratio for the first 5h among all calves. These data demonstrate the existence of a sexually dimorphic acute-phase response in pre-pubertal Brahman calves. Specifically, heifers may have a more robust acute response to LPS challenge, even though bulls display more signs of sickness.

Effects of intraluteal implants of prostaglandin E1 or E2 on angiogenic growth factors in luteal tissue of Angus and Brahman cows.

Previously, it was reported that intraluteal implants containing prostaglandin E1 or E2 (PGE1 and PGE2) in Angus or Brahman cows prevented luteolysis by preventing loss of mRNA expression for luteal LH receptors and luteal unoccupied and occupied LH receptors. In addition, intraluteal implants containing PGE1 or PGE2 upregulated mRNA expression for FP prostanoid receptors and downregulated mRNA expression for EP2 and EP4 prostanoid receptors. Luteal weight during the estrous cycle of Brahman cows was reported to be lesser than that of Angus cows but not during pregnancy. The objective of this experiment was to determine whether intraluteal implants containing PGE1 or PGE2 alter vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), angiopoietin-1 (ANG-1), and angiopoietin-2 (ANG-2) protein in Brahman or Angus cows. On Day 13 of the estrous cycle, Angus cows received no intraluteal implant and corpora lutea were retrieved, or Angus and Brahman cows received intraluteal silastic implants containing vehicle, PGE1, or PGE2 on Day 13 and corpora lutea were retrieved on Day 19. Corpora lutea slices were analyzed for VEGF, FGF-2, ANG-1, and ANG-2 angiogenic proteins via Western blot. Day-13 Angus cow luteal tissue served as preluteolytic controls. Data for VEGF were not affected (P > 0.05) by day, breed, or treatment. PGE1 or PGE2 increased (P < 0.05) FGF-2 in luteal tissue of Angus cows compared with Day-13 and Day-19 Angus controls but decreased (P < 0.05) FGF-2 in luteal tissue of Brahman cows when compared w Day-13 or Day-19 Angus controls. There was no effect (P > 0.05) of PGE1 or PGE2 on ANG-1 in Angus luteal tissue when compared with Day-13 or Day-19 controls, but ANG-1 was decreased (P < 0.05) by PGE1 or PGE2 in Brahman cows when compared with Day-19 Brahman controls. ANG-2 was increased (P < 0.05) on Day 19 in Angus Vehicle controls when compared with Day-13 Angus controls, which was prevented (P < 0.05) by PGE1 but not by PGE2 in Angus cows. There was no effect (P > 0.05) of PGE1 or PGE2 on ANG-2 in Brahman cows. PGE1 or PGE2 may alter cow luteal FGF-2, ANG-1, or ANG-2 but not VEGF to prevent luteolysis; however, species or breed differences may exist.

Sexually dimorphic stress and pro-inflammatory cytokine responses to an intravenous corticotropin-releasing hormone challenge of Brahman cattle following transportation.

This study was designed to characterize potential sexually dimorphic stress and immunological responses following a corticotropin-releasing hormone (CRH) challenge in beef cattle. Six female (heifers) and six male (bulls) Brahman calves (264 ± 12 d of age) were administered CRH intravenously (0.5 µg of CRH/kg body mass) after which serum concentrations of cortisol increased from 0.5 h to 4 h. From 1 h to 4 h after CRH administration, serum cortisol concentrations were greater in heifers than in bulls. In all cattle, increased serum concentrations of TNF-α, IL-6 and IFN-γ were observed from 2.5 h to 3 h after CRH, with greater concentrations of IFN-γ and IL-6 in heifers than bulls. Heifer total leukocyte counts decreased 1 h after CRH administration, while bull leukocyte counts and percent neutrophils decreased 2 h after CRH administration. Heifers had greater rectal temperatures than bulls, yet rectal temperatures did not change following administration of CRH. There was no effect of CRH administration on heart rate. However, bulls tended to have increased heart rate 2 h after CRH administration than before CRH. Heifer heart rate was greater than bulls throughout the study. These data demonstrate that acute CRH administration can elicit a pro-inflammatory response, and cattle exhibit a sexually dimorphic pro-inflammatory cytokine and cortisol response to acute CRH administration.

In vivo intra-luteal implants of prostaglandin (PG) E1 or E2 (PGE1, PGE2) prevent luteolysis in cows. II: mRNA for PGF2α, EP1, EP2, EP3 (A-D), EP3A, EP3B, EP3C, EP3D, and EP4 prostanoid receptors in luteal tissue.

Previously, it was reported that chronic intra-uterine infusion of PGE(1) or PGE(2) every 4h inhibited luteolysis in ewes by altering luteal mRNA for luteinizing hormone (LH) receptors and unoccupied and occupied luteal LH receptors. However, estradiol-17ß or PGE(2) given intra-uterine every 8h did not inhibit luteolysis in cows, but infusion of estradiol+PGE(2) inhibited luteolysis. In contrast, intra-luteal implants containing PGE(1) or PGE(2) in Angus or Brahman cows also inhibited the decline in circulating progesterone, mRNA for LH receptors, and loss of unoccupied and occupied receptors for LH to prevent luteolysis. The objective of this experiment was to determine how intra-luteal implants of PGE(1) or PGE(2) alter mRNA for prostanoid receptors and how this could influence luteolysis in Brahman or Angus cows. On day-13 Angus cows received no intra-luteal implant and corpora lutea were retrieved or Angus and Brahman cows received intra-luteal silastic implants containing Vehicle, PGE(1), or PGE(2) and corpora lutea were retrieved on day-19. Corpora lutea slices were analyzed for mRNA for prostanoid receptors (FP, EP1, EP2, EP3 (A-D), EP3A, EP3B, EP3C, EP3D, and EP4) by RT-PCR. Day-13 Angus cow luteal tissue served as pre-luteolytic controls. mRNA for FP receptors decreased in day-19 Vehicle controls compared to day-13 Vehicle controls regardless of breed. PGE(1) and PGE(2) up-regulated FP gene expression on day-19 compared to day-19 Vehicle controls regardless of breed. EP1 mRNA was not altered by any treatment. PGE(1) and PGE(2) down-regulated EP2 and EP4 mRNA compared to day-19 Vehicle controls regardless of breed. PGE(1) or PGE(2) up-regulated mRNA EP3B receptor subtype compared to day-19 Vehicle control cows regardless of breed. The similarities in relative gene expression profiles induced by PGE(1) and PGE(2) support their agonistic effects. We conclude that both PGE(1) and PGE(2) may prevent luteolysis by altering expression of mRNA for prostanoid receptors, which is correlated with changes in luteal mRNA for LH receptors reported previously in these same cows to prevent luteolysis.

In vivo intra-luteal implants of prostaglandin (PG) E(1) or E(2) (PGE(1), PGE(2)) prevent luteolysis in cows. I. Luteal weight, circulating progesterone, mRNA for luteal luteinizing hormone (LH) receptor, and occupied and unoccupied luteal receptors for LH.

Previously, it was reported that chronic intra-uterine infusion of PGE(1) or PGE(2) every four hours inhibited luteolysis in ewes. However, estradiol-17ß or PGE(2) given intra-uterine every 8h did not inhibit luteolysis in heifers, but infusion of estradiol+PGE(2) inhibited luteolysis in heifers. The objective of this experiment was to determine whether and how intra-luteal implants containing PGE(1) or PGE(2) prevent luteolysis in Angus or Brahman cows. On day-13 post-estrus, Angus cows received no intra-luteal implant and corpora lutea were retrieved or Angus and Brahman cows received intra-luteal silastic implants containing Vehicle, PGE(1), or PGE(2) and corpora lutea were retrieved on day-19. Coccygeal blood was collected daily for analysis for progesterone. Breed did not influence the effect of PGE(1) or PGE(2) on luteal mRNA for LH receptors or unoccupied or occupied luteal LH receptors did not differ (P>0.05) so the data were pooled. Luteal weights of Vehicle-treated Angus or Brahman cows from days-13-19 were lower (P<0.05) than those treated with intra-luteal implants containing PGE(1) or PGE(2). Day-13 Angus luteal weights were heavier (P<0.05) than Vehicle-treated Angus cows on day-19 and luteal weights of day-13 corpora lutea were similar (P>0.05) to Angus cows on day-19 treated with intra-luteal implants containing PGE(1) or PGE(2). Profiles of circulating progesterone in Angus or Brahman cows treated with intra-luteal implants containing PGE(1) or PGE(2) differed (P<0.05) from controls, but profiles of progesterone did not differ (P>0.05) between breeds or between cows treated with intra-luteal implants containing PGE(1) or PGE(2). Intra-luteal implants containing PGE(1) or PGE(2) prevented (P<0.05) loss of luteal mRNA for LH receptors and unoccupied or occupied receptors for LH compared to controls. It is concluded that PGE(1) or PGE(2) alone delays luteolysis regardless of breed. We also conclude that either PGE(1) or PGE(2) prevented luteolysis in cows by up-regulating expression of mRNA for LH receptors and by preventing loss of unoccupied and occupied LH receptors in luteal tissue.

Temperament influences endotoxin-induced changes in rectal temperature, sickness behavior, and plasma epinephrine concentrations in bulls.

This study was designed to determine the influence of temperament on endotoxin-induced changes in body temperature, sickness behavior, and stress hormone concentrations in cattle. Brahman bulls were selected based on temperament score measured 28 d prior to weaning. In dwelling recording devices were used to monitor rectal temperature, and jugular catheters were used to collect blood samples to determine cortisol and epinephrine concentrations before and after LPS administration (0.5 µg/kg body weight). Temperamental bulls had the lowest peak rectal temperature and sickness behavior scores relative to the Calm and Intermediate bulls. Prior to the administration of LPS, Temperamental bulls had greater cortisol and epinephrine concentrations than Calm or Intermediate bulls. Cortisol concentrations increased following LPS administration but were not affected by temperament. Epinephrine concentrations peaked 1 h after LPS administration in Calm bulls. Temperamental bulls did not exhibit an epinephrine response to LPS challenge. These data demonstrate that the temperament of calves can modulate the physiological, behavioral, and endocrine responses of pre-pubertal Brahman bulls to endotoxin challenge. Specifically, temperament differentially affected the rectal temperature, sickness behavior and epinephrine, but not cortisol, responses to LPS challenge.

Acute IGF-I infusion stimulates protein synthesis in skeletal muscle and other tissues of neonatal pigs.

Studies have shown that protein synthesis in skeletal muscle of neonatal pigs is uniquely sensitive to a physiological rise in both insulin and amino acids. Protein synthesis in cardiac muscle, skin, and spleen is responsive to insulin but not amino acid stimulation, whereas in the liver, protein synthesis responds to amino acids but not insulin. To determine the response of protein synthesis to insulin-like growth factor I (IGF-I) in this model, overnight-fasted 7- and 26-day-old pigs were infused with IGF-I (0, 20, or 50 microg. kg(-1). h(-1)) to achieve levels within the physiological range, while amino acids and glucose were clamped at fasting levels. Because IGF-I infusion lowers circulating insulin levels, an additional group of high-dose IGF-I-infused pigs was also provided replacement insulin (10 ng. kg(-0.66). min(-1)). Tissue protein synthesis was measured using a flooding dose of L-[4-(3)H]phenylalanine. In 7-day-old pigs, low-dose IGF-I increased protein synthesis by 25-60% in various skeletal muscles as well as in cardiac muscle (+38%), skin (+24%), and spleen (+32%). The higher dose of IGF-I elicited no further increase in protein synthesis above that found with the low IGF-I dose. Insulin replacement did not alter the response of protein synthesis to IGF-I in any tissue. The IGF-I-induced increases in tissue protein synthesis decreased with development. IGF-I infusion, with or without insulin replacement, had no effect on protein synthesis in liver, jejunum, pancreas, or kidney. Thus the magnitude, tissue specificity, and developmental change in the response of protein synthesis to acute physiological increases in plasma IGF-I are similar to those previously observed for insulin. This study provides in vivo data indicating that circulating IGF-I and insulin act on the same signaling components to stimulate protein synthesis and that this response is highly sensitive to stimulation in skeletal muscle of the neonate.

Stimulation of protein synthesis by both insulin and amino acids is unique to skeletal muscle in neonatal pigs.

In neonatal pigs, the feeding-induced stimulation of protein synthesis in skeletal muscle, but not liver, can be reproduced by insulin infusion when essential amino acids and glucose are maintained at fasting levels. In the present study, 7- and 26-day-old pigs were studied during 1) fasting, 2) hyperinsulinemic-euglycemic-euaminoacidemic clamps, 3) euinsulinemic-euglycemic-hyperaminoacidemic clamps, and 4) hyperinsulinemic-euglycemic-hyperaminoacidemic clamps. Amino acids were clamped using a new amino acid mixture enriched in nonessential amino acids. Tissue protein synthesis was measured using a flooding dose of L-[4-(3)H]phenylalanine. In 7-day-old pigs, insulin infusion alone increased protein synthesis in various skeletal muscles (from +35 to +64%), with equivalent contribution of myofibrillar and sarcoplasmic proteins, as well as cardiac muscle (+50%), skin (+34%), and spleen (+26%). Amino acid infusion alone increased protein synthesis in skeletal muscles (from +28 to +50%), also with equivalent contribution of myofibrillar and sarcoplasmic proteins, as well as liver (+27%), pancreas (+28%), and kidney (+10%). An elevation of both insulin and amino acids did not have an additive effect. Similar qualitative results were obtained in 26-day-old pigs, but the magnitude of the stimulation of protein synthesis by insulin and/or amino acids was lower. The results suggest that, in the neonate, the stimulation of protein synthesis by feeding is mediated by either amino acids or insulin in most tissues; however, the feeding-induced stimulation of protein synthesis in skeletal muscle is uniquely regulated by both insulin and amino acids.