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.

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.

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.

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.

The effectiveness of vasopressin as an ACTH secretagogue in cattle differs with temperament.

By using the temperament selection criterion of exit velocity (EV), cattle typically exhibiting hypercortisolism and a blunted response to exogenous corticotrophin-releasing hormone (CRH) can be identified via individual behavioral responses to handling. To further characterize hypothalamic-pituitary-adrenal (HPA) axis dysfunction associated with bovine temperament, the present study compared pituitary and adrenal activity, following stimulation with exogenous vasopressin (VP), in steers with an excitable or calm temperament. Serial blood samples were collected via indwelling jugular cannula for 6h preceding and 6h following administration of a VP bolus. Plasma concentrations of adrenocorticotropic hormone (ACTH) and cortisol were quantified by RIA to determine pituitary and adrenal responsiveness within temperament groups. Cortisol concentrations in excitable steers during the pre-challenge period revealed an increased initial adrenal reactivity to interactions with humans. Subsequent acclimation to the experimental surroundings yielded greater baseline cortisol concentrations in the cattle with an excitable temperament. Pituitary stimulation with VP resulted in a greater ACTH output from the excitable compared to the calm animals. The data presented herein provide additional evidence that HPA axis function in cattle of an excitable temperament may be akin to a state of chronic stress. The bovine temperament model may be of further use to both decipher mechanisms associated with HPA dysfunction and to elucidate physiological phenotypes or pathologies that have parallels in other species.

Functional characteristics of the bovine hypothalamic-pituitary-adrenal axis vary with temperament.

Hypothalamic-pituitary-adrenal (HPA) axis function, in Brahman heifers of differing temperament, was evaluated using separate challenges with CRH and ACTH. Exit velocity (EV) measurement was used to classify heifer temperament as calm [C; consisted of 6 slowest heifers (EV=1.05+/-0.05 m/s)] or temperamental [T; 6 fastest heifers (EV=3.14+/-0.22 m/s)]. During the 6 h prior to CRH challenge, areas under the ACTH (P=0.025) and cortisol (P<0.001) curves were greater in the temperamental heifers. Baseline cortisol (P<0.001) but not ACTH (P=0.10) differed between temperament groups. Following CRH challenge, areas under the ACTH (P=0.057) and cortisol (P<0.01) response curves were greater in the calm animals. The same animals were subjected to an ACTH challenge 14 d following their utilization in the CRH stimulation experiment. Prior to ACTH challenge, baseline cortisol concentrations were higher (P<0.001) in the temperamental heifers (T=18+/-2.6, C=4.3+/-0.6 ng/mL). Following ACTH administration, area under the cortisol response curve was greater (P=0.07) in the calm heifers. After declining below baseline concentrations during the post-challenge recovery period, cortisol in temperamental animals was again greater (P=0.02) than in the calm heifers. These data demonstrate that cattle with an excitable temperament exhibit increased stress responsiveness to handling, increased baseline adrenal function but not increased basal pituitary function, and a muted responsiveness to pharmacological stimulus. Thus, functional characteristics of the HPA axis vary with animal temperament.

In vivo progestin treatments inhibit nitric oxide and endothelin-1-induced bovine endometrial prostaglandin (PG) E (PGE) secretion in vitro.

Synchronization of estrus with progestins in cows has been reported to inhibit nitric oxide (NO) and endothelin-1 (ET-1)-stimulated bovine luteal PGE secretion without affecting prostaglandin F2alpha (PGF2alpha) secretion in vitro [Weems YS, Randel RD, Tatman S, Lewis A, Neuendorff DA, Weems CW. Does estrous synchronization affect corpus luteum (CL) function? Prostaglandins Other Lipid Mediat 2004;74:45-59]. Two experiments were conducted to determine the effects of NO donors, endothelin-1 (ET-1), and NO synthase (NOS) inhibitors on bovine caruncular endometrial secretion of PGE and PGF2alpha in vitro. In Experiment 1, estrus was synchronized in Brahman cows with Synchromate-B ear implants, which contained the synthetic progestin norgestamet. Days 14-15 caruncular endometrial slices were weighed, diced, and incubated in vitro with treatments. Treatments (100 ng/ml) were: Vehicle (control), l-NAME (NOS inhibitor), l-NMMA (NOS inhibitor), DETA (control), DETA-NONOate (NO donor), sodium nitroprusside (NO donor), or ET-1. In Experiment 2, estrus was synchronized in Brahman cows with either Lutalyse (PGF2alpha) or a controlled intravaginal drug releasing device (CIDR-containing progesterone) or estrus was not synchronized. Days 14-15 caruncular endometrial slices were weighed, diced, and incubated in vitro with treatments. Treatments (100 ng/ml) were: vehicle, l-NAME, l-NMMA, DETA, DETA-NONOate, sodium nitroprusside, SNAP (NO donor) or ET-1. Tissues were incubated in M-199 for 1h without treatments and with treatments for 4 and 8h in both experiments. Media were analyzed for concentrations of PGE and PGF2alpha by radioimmunoassay (RIA). Hormone data in Experiments 1 and 2 were analyzed by 2x7 and 3x2x8 factorial design for ANOVA, respectively. Concentrations of PGE and PGF2alpha in media increased (P< or =0.05) from 4 to 8 h regardless of treatment group in Experiment 1, but did not differ (P> or =0.05) among treatments. In Experiment 2, concentrations of PGE and PGF2alpha increased (P< or =0.05) with time in all treatment groups of all three synchronization regimens. DETA-NONOate, SNAP, and sodium nitroprusside (NO donors) and ET-1 increased caruncular endometrial (P< or =0.05) secretion of PGE2 in unsynchronized and Lutalyse synchronized cows, but not when estrus was synchronized with a CIDR (P> or =0.05). No treatment increased (P> or =0.05) PGF2alpha in any synchronization regimen. It is concluded that norgestamet in Synchromate-B ear implants or progesterone in a CIDR alters NO or ET-1-induced secretion of PGE by bovine caruncular endometrium and could interfere with implantation by altering the PGE:PGF2alpha ratio resulting in increased embryonic losses during early pregnancy.

Influence of season of birth on growth and reproductive development of Brahman bulls.

Seasonal effects on reproduction are more dramatic in Bos indicus than Bos taurus cattle. This experiment evaluated reproductive development of fall- (n=7) versus spring- (n = 10) born Brahman bulls to determine if season of birth affects reproductive development. Measurements of growth and reproductive development began after weaning and continued at bi-weekly intervals until each bull reached sexual maturity. Different stages of sexual development were classified according to characteristics of the ejaculate and included first sperm in the ejaculate, puberty (> 50 x 10(6) sperm/ejaculate), and sexual maturity (two ejaculates with > 500 = 10(6) sperm/ejaculate). Average daily increases in all measured traits were similar in fall- and spring-born bulls and there were no differences in age, body weight, scrotal circumference, or paired testis volume between groups at first sperm or puberty. However, fall-born bulls were older (P < 0.05) than spring-born bulls at sexual maturity (553 days versus 481 days, respectively) as the interval between puberty and sexual maturity was longer (P < 0.05) in fall- than in spring-born bulls (82 days versus 54 days, respectively). The prolonged interval between puberty and sexual maturity in fall-born calves coincided with a short photoperiod (winter) whereas the short interval between puberty and sexual maturity in spring-born calves coincided with a long photoperiod (summer). In conclusion, season of birth affected sexual development; photoperiod might be involved in regulating testicular function immediately after puberty in Brahman bulls.