Ken-ichi Yayou

Relationships of stress responses with plasma oxytocin and prolactin in heifer calves.

Oxytocin and prolactin are potential candidates for the regulation of behavioral and physiological stress responses in the brain. To investigate the neurobiological basis of individual differences in stress responses in cattle, we examined the association of behavioral and hypothalamo-pituitary-adrenal axis reactivity to acute stressors and basal and stimulated levels of oxytocin and prolactin. Twenty Holstein heifer calves aged 2 weeks were subjected to a 10 min open-field test (OFT) followed by presentation of a feeding bucket for 15 min in the OF. If the calf contacted the bucket, a blast of air was applied to its muzzle (surprise test). Jugular blood samples collected before and after both tests were analyzed for oxytocin, prolactin, and cortisol. Relationships of basal and percent change in oxytocin or prolactin with behavioral responses in each test and percent change in cortisol were analyzed using principal components analysis and Spearman rank correlations. Plasma cortisol and prolactin concentrations were significantly elevated by the tests (p<0.005), but plasma oxytocin concentration did not significantly change (p>0.1). Four principal components explained 56.1% of the total variation: curiosity, general activity, fearfulness, and dependence on humans. Curiosity was inversely correlated with basal oxytocin level (rS=-0.683, p<0.05). General activity was positively correlated with prolactin reactivity (rS=0.448, p<0.05) and inversely with oxytocin reactivity to the novel environment (rS=-0.717, p<0.05). Fearfulness tended to correlate positively with basal oxytocin level (rS=0.583, p<0.1). Dependence on humans correlated with none of the hormonal parameters. The relationships of basal oxytocin level with curiosity and fearfulness for novel environments are of particular interest for future study.

Comparison between the central effects of CRH and AVP in steers

In cattle, the in vivo effects of centrally administered corticotropin-releasing hormone (CRH) or arginine vasopressin (AVP) from the perspective of stress regulation have not been fully elucidated. We compared behavioral, adrenocorticotropic, and autonomic nervous responses to intracerebroventricularly infused bCRH or AVP in steers. Intracerebroventricular infusions of both bCRH and AVP (0.2, 2, 10 and 20 nmol/500 mul/30 min) evoked a dose-related increase in plasma concentrations of ACTH and cortisol. At 10 nmol, the AUC response of cortisol concentration to bCRH tended to be higher than that in response to AVP (p=0.075). There were significant differences among treatments in the total number of head shaking (Friedmans test, chi2=22.79, p=0.004), upright posture (chi2=16.80, p=0.032), head rubbing (chi2=23.93, p=0.002), tongue playing (chi2=27.18, p=6.58E(-4)), and bleating (chi2=26.84, p=7.54E(-4)). AVP 10 and 20 nmol treatments induced more head shaking and tongue playing than vehicle treatment (Nemenyi multiple comparisons: p<0.1). Ten nmol (32.8+/-40.2 times) and 20 nmol (34.8+/-19.9 times) of bCRH induced bleating, but no dosage of AVP induced bleating. These findings indicate that both bCRH and AVP could activate HPA axis in steers when infused intracerebroventricularly and that bCRH was more potent to stimulate HPA axis than AVP. As for the effects on behavioral function, high dosages of both peptides induced stereotyped behaviors and the types of stereotyped behaviors induced were different between bCRH and AVP.

Effects of intracerebroventricular infusions of arginine vasopressin in sheep.

In sheep, neither the in vivo effect of vasopressin administered by a method other than systemic infusion nor the central effects on behavior from the perspective of stress regulation has been fully elucidated in an intact animal. We examined changes in behavioral, adrenocorticotropic, and autonomic nervous functions after intracerebroventricular infusions of arginine vasopressin (AVP) to elucidate its central role. Intracerebroventricular infusions of AVP (0, 0.12, 1.2 and 12 microg/500 microl/30 min) evoked a dose-related increase in plasma cortisol concentration. There were significant treatment-related effects on the total duration of sham-chewing (Friedmans test, X2=12.75, p=.0052), on the total duration of bar-biting (Friedmans test, X2=15.0, p=.0018), and on the total duration of rubbing (Friedmans test, X2=12.0, p=.0074). AVP 12 microg treatment induced a greater degree of sham-chewing and bar-biting than the other three treatments did (Nemenyi multiple comparisons: p<0.1). These findings indicate, together with our previous findings, that AVP has the same corticotropic potential as corticotropin-releasing hormone infused intracerebroventricularly in equal molar concentrations. Although the degree to which central stress signaling pathways are involved in these responses remains speculative, the relationships between stereotypies and central AVP are of particular interest.

A possible role of central serotonin in L‐tryptophan‐induced GH secretion in cattle

To clarify the role of serotonin (5-HT) in the regulatory mechanism of L-tryptophan (TRP)--induced growth hormone (GH) secretion in cattle, changes in 5-HT concentrations in the cerebrospinal fluid (CSF) in the third ventricle (3V) and GH in plasma before and after the peripheral infusion of TRP were determined simultaneously. The direct effect of TRP on GH release from the dispersed anterior pituitary cells was also assessed. A chronic cannula was placed in 3V by stereotaxic surgery, then CSF and blood were withdrawn under physiological conditions. TRP (38.5 mg/kg BW) was infused through an intravenous catheter from 12.00 to 14.00 hours and CSF and blood sampling were performed from 11.00 to 18.00 hours at 1-h intervals. The concentration of 5-HT in CSF was determined by high-performance liquid chromatography with electrochemical detection. GH, melatonin (MEL), and cortisol (CORT) concentrations were measured by radio-immunoassay and enzyme-immunoassay. Concentrations of 5-HT were increased by TRP infusion. The TRP infusion significantly increased GH release. On the other hand, TRP did not stimulate GH release from the bovine pituitary cells. MEL and CORT concentrations were not altered by TRP infusion. These results suggest that TRP induced GH release via the activation of serotonergic neurons in cattle.

L‐DOPA attenuates prolactin secretion in response to isolation stress in Holstein steers

To clarify endocrine responses to psychological stressors in cattle, the effects of isolation from familiar peers on plasma prolactin (PRL) and cortisol (CORT) concentrations, and the effect of 3,4-dihydroxy-L-phenylalanine (L-DOPA), a precursor of dopamine (DA), on stress-induced PRL secretion were determined in Holstein steers. First, the potency of peripheral L-DOPA administration on attenuation of central DA levels was confirmed. Cerebrospinal fluid (CSF) collected from a chronic cannula in the third ventricle and plasma were sampled 1 h before and 3 h after intravenous injection of L-DOPA (100 mg/head). DA concentrations in CSF increased just after L-DOPA injection with subsequent decrease in PRL secretion. Injection of L-DOPA increased CORT secretion. Second, one experimental steer was isolated in its stall by removing its peers for 2 h with or without- pre-injection of L-DOPA. The concentration of PRL was elevated by isolation treatment, whereas the effect of isolation on CORT concentration could not be detected. The increase in PRL concentration after isolation was abolished by pre-injection of L-DOPA. These results suggest that PRL responds to isolation and that DA neurons in the central nervous system may regulate stress-induced PRL secretion in steers.

Effect of intracerebroventricular injections of prolactin‐releasing peptide on prolactin release and stress‐related responses in steers

Some evidence suggests that there might be a species difference in the effect of intracerebroventricularly administered (ICV) prolactin-releasing peptide (PrRP) between rodents and sheep. We compared the levels of cortisol (CORT) and prolactin (PRL), rectal temperature (RT) and behavioral responses to ICV bovine PrRP (bPrRP) in steers. ICV bPrRP (0.2, 2 and 20 nmol/200 µL) tended to evoke a dose-related increase in CORT concentrations and 0.2 nmol of bPrRP induced transient increase in PRL concentrations. A significant time-treatment interaction was observed for the percent change of CORT (P<0.05) and PRL (P<0.05) from pre-injection value. The time-treatment interaction for changes in RT was not significant (P=0.50). There tended to be a difference among the four treatments in terms of maximum change in RT from the pre-injection value between 0 and 90 min (P<0.1). Stress-related behavioral signs were not observed in the present experiment. These findings indicate that ICV bPrRP increased CORT and PRL levels, suggesting that central PrRP might participate in controlling the hypothalamo-pituitary-adrenal axis and PRL release in cattle, unlike sheep. In contrast, central PrRP is unlikely to be involved in controlling the behavior of this species because ICV bPrRP did not induce marked changes in their behavior.

Relationships between postnatal plasma oxytocin concentrations and social behaviors in cattle

We examined the associations between natural individual variations in basal oxytocin (OXT) in postnatal cattle and social behavioral traits. At 1, 2 and 6 weeks of age, the basal OXT exhibited individual variability in 20 Holstein heifer calves. Cluster analysis of mean OXT for these time periods obtained two subgroups: high OXT (HOXT; n = 9) and low OXT (LOXT; n = 11). Social behaviors were observed for 2 days at week 6 after introduction into a four-peer group, and at 10-14 months of age (10 months) immediately and 1 week, 1 month and 5 months after introduction into 11-15 heifers. At week 6, the main effect of the OXT groups was not significant for all social behaviors. At 10 months, there tended to be interactions between the OXT groups and time periods with respect to the frequency of escape behaviors. LOXT heifers exhibited more escape behaviors than HOXT heifers on the first day of the second sociality tests. At 10 months, HOXT heifers exhibited both attacking and affiliative behavior for peers more than LOXT heifers during 5 months after the second social introduction. This suggests that postnatal OXT concentrations may have long-lasting effects on individual differences among social behavioral traits in cattle.

The effect of lighting conditions on the rhythmicity of growth hormone secretion in Holstein steers

Growth hormone (GH) secretion regularity and the effects of lighting condition and GH-releasing hormone (GHRH) on GH release were determined in steers. First, steers were kept under 12:12 L : D conditions (light: 06.00-18.00 hours). The animals were then subjected to a 1-h advancement in lighting on/off conditions (05.00 and 17.00 hours, respectively). Blood was sampled for 24 h at 1-h interval on the seventh day of each condition. Second, GHRH was injected intravenously (IV) at 12.00 and 00.00 hours under 12:12 L : D and blood was sampled at 15-min interval for 4-h (1 h before and 3 h after the injection). Plasma GH concentrations were measured by a radioimmunoassay. Periodicity of GH secretory profile was calculated by power spectrum analysis using the maximum entropy method. Plasma GH concentrations showed a characteristic pattern consisting of four distinct peaks. Mean periodicity of GH secretory profile was 5.7 h, and it was not altered by any change in lighting conditions. IV injection of GHRH increased GH secretion during the day and night. The increase in GH secretory volume after GHRH injection during the night was equal to that during the day. The present results suggest that GH secreted from the anterior pituitary have regularity in steers.

Effect of oxytocin, prolactin-releasing peptide, or corticotropin-releasing hormone on feeding behavior in steers.

As a preliminary step to elucidate the involvement of central neurotransmitters in the dip in voluntary feed intake during the perinatal period in cows, we investigated the effect of intracerebroventricular (ICV) administration of oxytocin, prolactin-releasing peptide (PrRP), or corticotropin-releasing hormone (CRH), the central functions of all of which undergo drastic changes during the perinatal period, on feed intake in steers. Thirty minutes before the onset of feeding, the treatment solution was injected into the third ventricle through an implanted cannula, and feeding-related behaviors were observed for 1 h after the onset of feeding. Neither ICV oxytocin (5 and 50 μg) nor PrRP (2 and 20 nmol) reduced feed intake (n=6). Twenty nanomoles of bovine CRH noticeably inhibited feeding behavior compared with vehicle treatment (n=5, p<0.05). Fifty micrograms of oxytocin reduced latency to the first water access after feeding onset (p<0.1), which may be because of the stimulation of arginine vasopressin V1b receptor by the high dose of oxytocin. We conclude that CRH inhibits feeding behavior by its central action in this species, although this could also be an indirect effect due to the increased expression of abnormal behaviors caused by CRH. Central administration of neither oxytocin nor PrRP reduced feed intake in steers. Although the effects of sex steroids need to be examined, it appears that increased activity of oxytocin, and possibly PrRP, during the perinatal period does not contribute to the dip in voluntary feed intake in this species. On the other hand, it makes sense that suppressed central CRH activity during the perinatal period should act in the direction of maintaining or even increasing food intake to aid a steady supply of energy to the fetus or offspring. We thus speculate that CRH is not a prime candidate involved in the dip in voluntary feed intake during the perinatal period in cows.

Effects of intracerebroventricular administration of neuromedin U or neuromedin S in steers

Although neuromedin U (NMU) and neuromedin S (NMS) are reported to modulate stress responses mainly through corticotropin-releasing hormone system in rodents, the in vivo effects of centrally administered NMU or NMS on stress regulation have not been fully elucidated in cattle. We examined adrenocorticotropic hormone levels, body temperature, and behavioral responses to intracerebroventricularly (ICV) administered rat NMU or rat NMS in steers. ICV NMU and NMS (0.2, 2, and 20 nmol/200 microl) evoked a dose-related increase in plasma cortisol concentrations (CORT). There was a significant time-treatment interaction for the time course of CORT (p<0.001). ICV NMU evoked a dose-related increase in rectal temperature (RT). There was a significant time-treatment interaction for the change in RT from pre-injection value (p<0.05). There was a significant difference among treatments in the percentage of time spent lying (Friedmans test, chi(2)=15.6, p<0.01) and in the total number of head shaking (Friedmans test, chi(2)=14.49, p<0.01). A high dose of NMS tended to shorten the duration of lying and increase the number of head shaking. These findings indicate that both central NMU and NMS might participate in controlling the hypothalamo-pituitary-adrenal axis, that central NMU might participate in controlling body temperature, and that central NMS is likely to be involved in behavioral activation in cattle.