R. D. Randel

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.

Interrelationships among growth, endocrine, immune, and temperament variables in neonatal Brahman calves.

Interrelationships among growth, endocrine, immune, and temperament variables were assessed in neonatal Brahman calves. The velocity upon exiting a working chute (exit velocity) of an animal was measured and used as an objective indicator of temperament to classify calves as calm, intermediate, or temperamental. Calves (n = 116) were weighed weekly between d 0 and 21 to 24, and blood samples were collected for plasma and serum on d 0, 1, 2, 7, 14, and 21 to 24 after birth to measure concentrations of immunoglobulins, cortisol, and epinephrine (EPI). Body weight increased from d 0 through d 21 to 24 (P < 0.001) with bulls (n = 60) having greater BW than heifers (n = 56; P = 0.02). Serum concentrations of cortisol were greatest on d 0 before declining (P < 0.001) over the ensuing 21 to 24 d and were not related to temperament (P = 0.89) or sex (P = 0.97). Concentrations of EPI were affected by time, with an increase in EPI concentrations in temperamental bulls between 2 and 14 d of age (P < 0.008). Concentrations of EPI were not affected by temperament (P = 0.44) or sex (P = 0.68). Serum immunoglobulin concentrations peaked on d 1 before declining (P < 0.01) but were not related to temperament (P = 0.40 to 0.68). Of the stress hormones measured (cortisol and EPI), only cortisol was associated with the early performance of the calf. Calf BW at d 21 to 24 and BW gain were positively associated with serum immunoglobulin concentrations, yet negatively associated with concentrations of cortisol. Serum immunoglobulin concentrations were negatively correlated with cortisol concentrations (r = -0.28; P = 0.003), yet positively associated with EPI concentrations (r = 0.51; P = 0.003). During the neonatal period in this study, there was no relationship of temperament with passive immunity or stress hormone concentrations; however, growth was positively associated with passive immunity and negatively associated with stress hormones. Measuring exit velocity as early in life as d 21 to 24 fails to accurately predict temperament at weaning in over 40% of Brahman calves. Our conclusion is that measurement of exit velocity should be done nearer to the time of weaning than to birth. These data can be beneficial in developing best management practices for young calves.

In vivo intra-luteal implants of prostaglandin (PG) E1 or E2 (PGE1, PGE2) 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-17β 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.Lysophosphatidic acid (LPA), a pleiotropic signalling lipid is assuming growing significance in osteoblast biology. Although committed osteoblasts from several mammalian species are receptive to LPA far less is known about the potential for LPA to influence osteoblast formation from their mesenchymal progenitors. An essential factor for both bone development and post-natal bone growth and homeostasis is the active metabolite of vitamin D3, calcitriol (D3). Previously we reported how a combination of LPA and D3 synergistically co-operated to enhance the differentiation of immature human osteoblasts. Herein we provide evidence for the formation of human osteoblasts from multiple, primary human bone marrow derived stromal (stem) cells (hBMSCs). Importantly osteoblast development from hBMSCs only occurred when LPA was administered as a complex with albumin, its natural carrier. Collectively our findings support a co-operative role of LPA and D3 in osteoblastogenesis, findings which may aid the development of novel treatment strategies for bone repair.

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-17β 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.

Effects of endocannabinoid 1 and 2 (CB1; CB2) receptor agonists on luteal weight, circulating progesterone, luteal mRNA for luteinizing hormone (LH) receptors, and luteal unoccupied and occupied receptors for LH in vivo in ewes

Thirty to forty percent of ruminant pregnancies are lost during the first third of gestation due to inadequate progesterone secretion. During the estrous cycle, luteinizing hormone (LH) regulates progesterone secretion by small luteal cells (SLC). Loss of luteal progesterone secretion during the estrous cycle is increased via uterine secretion of prostaglandin F(2α) (PGF(2α)) starting on days 12-13 post-estrus in ewes with up to 4-6 pulses per day. Prostaglandin F(2α) is synthesized from arachidonic acid, which is released from phospholipids by phospholipase A2. Endocannabinoids are also derived from phospholipids and are associated with infertility. Endocannabinoid-induced infertility has been postulated to occur primarily via negative effects on implantation. Cannabinoid (CB) type 1 (CB1) or type 2 (CB2) receptor agonists and an inhibitor of the enzyme fatty acid amide hydrolase, which catabolizes endocannabinoids, decreased luteal progesterone, prostaglandin E (PGE), and prostaglandin F(2α) (PGF(2α)) secretion by the bovine corpus luteum in vitro by 30 percent. The objective of the experiment described herein was to determine whether CB1 or CB2 receptor agonists given in vivo affect circulating progesterone, luteal weights, luteal mRNA for LH receptors, and luteal occupied and unoccupied LH receptors during the estrous cycle of ewes. Treatments were: Vehicle, Methanandamide (CB1 agonist; METH), or 1-(4-chlorobenzoyl)-5-methoxy-1H-indole-3-acetic acid morpholineamide (CB2 agonist; IMMA). Ewes received randomized treatments on day 10 post-estrus. A single treatment (500 μg; N=5/treatment group) in a volume of 1 ml was given into the interstitial tissue of the ovarian vascular pedicle adjacent to the luteal-containing ovary. Jugular venous blood was collected at 0 h and every 6-48 h for the analysis of progesterone by radioimmunoassay (RIA). Corpora lutea were collected at 48 h, weighed, bisected, and frozen in liquid nitrogen until analysis of unoccupied and occupied LH receptors and mRNA for LH receptors. Profiles of jugular venous progesterone, luteal weights, luteal mRNA for LH receptors, and luteal occupied and unoccupied LH receptors were decreased (P≤0.05) by CB1 or CB2 receptor agonists when compared to Vehicle controls. Progesterone in 80 percent of CB1 or CB2 receptor agonist-treated ewes was decreased (P≤0.05) below 1 ng/ml by 48 h post-treatment. It is concluded that the stimulation of either CB1 or CB2 receptors in vivo affected negatively luteal progesterone secretion by decreasing luteal mRNA for LH receptors and also decreasing occupied and unoccupied receptors for LH on luteal membranes. The corpus luteum may be an important site for endocannabinoids to decrease fertility as well as negatively affect implantation, since progesterone is required for implantation.

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.

Physiological and metabolic responses of gestating Brahman cows to repeated transportation

This study characterized physiological responses to repeated transportation (TRANS) of gestating cows of differing temperaments. Cows were classified as Calm (C; = 10), Intermediate (I; = 28), or Temperamental (T; = 10). Based on artificial insemination date and pregnancy confirmation, cows were TRANS for 2 h on d 60 (TRANS1), 80 (TRANS2), 100 (TRANS3), 120 (TRANS4), and 140 (TRANS5) ± 5 d of gestation. Indwelling vaginal temperature (VT) monitoring devices were inserted 24 h before each TRANS with VT recorded from 2 h before TRANS and averaged into 5-min intervals through 30 min after TRANS. Serum samples were collected before loading and on unloading from the trailer to determine concentrations of cortisol, glucose, and nonesterified fatty acids (NEFA). Data were analyzed by repeated measures analysis in SAS. Serum cortisol concentrations were affected by temperament ( < 0.001), with T cows having the greater concentrations of cortisol before each TRANS event. All cows (100%) regardless of temperament exhibited elevations in cortisol following each TRANS event. Peak VT was greater ( < 0.001) at TRANS1 relative to all other TRANS events regardless of cow temperament. During TRANS, the T cows tended ( < 0.09) to have greater peak VT (39.86 ± 0.15°C) compared to C (39.41 ± 0.16°C) and I cows (39.55 ± 0.08°C). Area under the VT curve decreased ( = 0.002) from TRANS1 through TRANS5. Pre-TRANS serum glucose concentration at TRANS1 was greater ( < 0.03) for T (68.13 ± 4.31mg/dL) compared to I (53.42 ± 2.78 mg/dL) and C cows (52.76 ± 4.60 mg/dL). The C and I cows had greater changes in NEFA concentration between pre- and post-transport, and T cows showed the least change ( < 0.001). Cow VT and serum glucose concentration decreased in all temperaments ( < 0.01) with repeated TRANS; however, serum NEFA concentration post-TRANS did not vary ( > 0.10) with repeated TRANS events. Serum glucose concentrations were affected ( < 0.02) by a TRANS event by temperament interaction with T cows taking more TRANS events to decrease their change in glucose concentration compared to C and I cows. These results demonstrate that temperament influences physiological responses to stress in gestating Brahman cows. Although repeated transport in our study is confounded with day of gestation, seasonal changes, and learning from repeated handling and transport, repeated transport is a useful model of repeated stress in studying the effects of temperament.

Regulation of adrenocorticotropin secretion in vitro by anterior pituitary corticotrophs from fallow deer (Dama Dama)

The actions of corticotropin-releasing hormone (CRH), vasopressin (VP), the synthetic glucocorticoid dexamethasone (DEX), and mifepristone (RU 486), a glucocorticoid antagonist, on the secretion of adrenocorticotropin (ACTH) by cultured fallow deer corticotrophs were studied in vitro. On Day 5 of primary culture, corticotrophs were challenged for up to 4 hr with medium alone (Control), CRH, VP, DEX, forskolin (FSK), phorbol ester (TPA), cyclic AMP (cAMP), and/or RU 486 at various concentrations and combinations. CRH, VP, FSK and TPA each stimulated (P < 0.01) the secretion of ACTH in dose- and time-related manners. Relative to Control, CRH at 0.001 and 0.1 microM and VP at 0.01 and 1 microM increased (P < 0.01) medium concentration of ACTH by 7.3-, 13.5-, 3.7- and 9.0-fold, respectively. There was a treatment x incubation time interaction (P < 0.01) such that at 30-min posttreatment, CRH-induced ACTH secretion tended (P < 0.10) to be less than that obtained via VP treatment, whereas at 1, 3, and 4 hr posttreatment, medium concentration of ACTH from cells treated with 0.1 microM CRH was greater (P < 0.05) than that in cells treated with 1 microM VP. At equimolar doses of 0.01 and 0.1 microM, CRH was 3.4- and 3.0-fold more potent (treatment x dose, P < 0.05) than VP. Cotreatment with 1 microM DEX reduced (P < 0.001) the stimulatory effects of CRH (0.1 microM), VP (1 microM), FSK (10 microMs), TPA (0.1 microM), and cAMP (0.001 M). However, the coaddition of RU 486 (1 microM) to the CRH plus DEX- and the FSK plus DEX-treated wells partially negated the inhibitory effects of DEX. RU 486 completely negated the inhibitory effects of DEX on the VP-, TPA-, and cAMP-stimulated secretion of ACTH. These data indicate that CRH is a more potent stimulator of ACTH secretion than is VP in primary culture of fallow deer pituitary cells. This study also demonstrates the utility of an in vitro culture system to investigate stress-related hormonal interactions in cervids.

Prenatal transportation stress alters temperament and serum cortisol concentrations in suckling Brahman calves

This experiment examined the relationship between prenatal stress and subsequent calf temperament through weaning. The prenatal stressor used was repeated transportation of pregnant Brahman cows for 2 h at 60 ± 5, 80 ± 5, 100 ± 5, 120 ± 5, and 140 ± 5 d of gestation. Prenatally stressed calves ( = 41) were compared with controls ( = 44; dams did not undergo transportation during pregnancy) from 2 wk of age until weaning (average age at weaning = 174.8 ± 1.3 d). Temperament was defined by pen score (PS; 1 = calm and 5 = excitable), exit velocity (EV; m/sec), and temperament score (TS; (PS + EV)/2) and was recorded for each calf on d -168, -140, -112, -84, -56, -28, and 0 relative to weaning (d 0 = weaning). Cortisol concentrations were determined in serum samples obtained on d -168, -140, -28, and 0 relative to weaning. Birth weight and weaning weight were not different between treatment groups ( > 0.1). Pen score was greater ( = 0.03) in prenatally stressed calves (2.84 ± 0.21) relative to controls (2.31 ± 0.21). Exit velocity was greater ( < 0.01) in prenatally stressed calves (2.1 ± 0.14 m/sec) than in controls (1.61 ± 0.14 m/sec). Exit velocity was affected by a treatment × calf sex interaction ( = 0.04) and was greater in prenatally stressed females. Exit velocity was also affected by day ( < 0.0001). Temperament score was greater ( = 0.01) in prenatally stressed calves (2.45 ± 0.16) than in controls (1.95 ± 0.16). Temperament score was affected by day ( < 0.01). Basal cortisol concentrations were greater ( = 0.04) in prenatally stressed calves (15.87 ± 1.04 ng/mL) than in controls (13.42 ± 1.03 ng/mL). Basal cortisol concentrations were greater ( < 0.01) in females (16.61 ± 1.06 ng/mL) than in males (12.68 ± 1.02 ng/mL). Cortisol concentrations were positively correlated ( < 0.01) with PS ( = 0.55, < 0.01), EV ( = 0.4, < 0.01), and TS ( = 0.55, < 0.01). Overall, suckling Brahman calves that were prenatally stressed were more temperamental and had greater circulating serum concentrations of cortisol than control calves.

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.