Behavior and heart rate variability after intranasal administration of oxytocin in Holstein steers.

Oxytocin (OXT) is a neuropeptide that regulates memory, emotion, stress response, and behavior in the brain. In our previous study with cattle, we demonstrated the anti-stress effect of intracerebroventricularly administered OXT on the central nervous system. However, it is important to investigate the effects of this peptide after intranasal administration, as it offers convenience and non-invasiveness for practical use. Therefore, this study investigated the effects of intranasal OXT on the behavior and autonomic nervous system of Holstein steers. The experiment followed a within-subjects design, including a total of six steers. Each steer received intranasal administration of either 1 mL of saline (SAL), 100 µg OXT (OXT100), or 200 µg OXT (OXT200). However, due to some issues, the sample size for the OXT200 group was reduced to five. After these treatments, we conducted electrocardiography recordings to analyze heart rate variability (HRV) and also made behavioral observations for 90 min. OXT200 tended to increase the time spent ruminating while lying down (Steel's multiple comparison test; P=0.053). In contrast, OXT treatment did not affect HRV indices. In conclusion, the current OXT dosage did not significantly affects behavior or the autonomic nervous system. However, the observed tendency to increase rumination may suggest a central effect of OXT.

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.

Intracerebroventricularly administered oxytocin attenuated cortisol secretion, but not behavioral responses, during isolation in Holstein steers.

In rodents, intracerebroventricular oxytocin administration attenuated hypothalamo-pituitary-adrenal (HPA) responses and anxiety behavior during stress. We examined the effects of intracerebroventricular injection of oxytocin on isolation-induced stress responses in cattle. In a methodological test, we determined the dosage of oxytocin applied in a main test which did not induce an increase in plasma cortisol concentration or stereotyped behaviors. In a main test, 5 steers aged from 199 to 250 days were assigned to the following three treatments randomly: T1, no isolation after injection of 200 microl of artificial cerebrospinal fluid (aCSF); T2, isolation after aCSF injection; and T3, isolation after 0.5 microg of oxytocin in 200 microl aCSF injection. The isolation was conducted by leaving the experimental steer alone in its stall for one hour while its peers were taken outside. In T2, the isolation induced a rapid increase in plasma cortisol concentration. The maximum %-changes from the pre-isolation value were significantly attenuated by oxytocin injection (T2 vs. T3, p<0.05). The isolation also induced an increase in the frequency (number of occurrences/1 hr isolation) of vocalizations and body orientation changes, and a decrease in the percentage of time spent lying and ruminating. The effect of oxytocin on these behavioral responses to isolation was not apparent. These results indicate that intracerebroventricularly injected oxytocin at low dose attenuated the cortisol response to isolation in steers while the effect on behavior was very small in this experimental condition.

Effects of intravenous tryptophan infusion on thermoregulation in steers exposed to acute heat stress.

This study was conducted to investigate the effect of tryptophan (TRP) supply on the thermoregulatory responses via brain serotonin (5-HT) in cattle. In period 1, 12 Holstein steers were kept under a constant room temperature (22°C) and were administered the intravenous (i.v.) infusion of saline or TRP (38.5 mg/kg/2 h). Changes in rectal temperature (RT), 5-HT concentration in the cerebrospinal fluid (CSF), and other factors involved in thermoregulation were measured. In period 2, the steers received the same treatments as in period 1; however, the room temperature was elevated from 22°C to 33°C during i.v. infusion and maintained at 33°C for 3 h. 5-HT concentration in CSF increased following TRP infusion in both periods, and RT significantly decreased following TRP infusion only in period 2. The effect of TRP on respiration rate and plasma prolactin and total triiodothyronine concentrations was not significant. These results suggest that increase in TRP supply can attenuate increase in RT in response to acute heat stress through the increase in brain 5-HT, followed by presumable increase in evaporative heat loss from the skin surface in cattle. It is possible that the increase in peripheral blood TRP metabolites could also participate in the hypothermic effect of TRP.

The effects of l-DOPA and sulpiride on growth hormone secretion at different injection times in Holstein steers.

The effects of l-DOPA, a precursor of dopamine (DA), and sulpiride, a D2 -type DA receptor blocker, on growth hormone (GH) and prolactin (PRL) secretion were investigated in steers. Eight Holstein steers (212.8 ± 7.8 kg body weight) were used. Lighting conditions were 12:12 L:D (lights on: 06.00-18.00 hours). Blood samplings were performed during the daytime (11.00-15.00 hours) and nighttime (23.00-03.00 hours). Intravenous injections of drugs or saline were performed at 12.00 hour for the daytime and 00.00 hour for the nighttime, respectively. Plasma GH and PRL concentrations were determined by radioimmunoassay. l-DOPA did not alter the GH secretion when it was injected at 12.00 hour (spontaneous GH level at its peak). On the other hand, l-DOPA increased GH secretion at 00.00 hour (GH level at its trough). Injection of sulpiride suppressed GH secretion at 12.00 hour but did not affect GH levels at 00.00 hour. l-DOPA inhibited and sulpiride stimulated PRL release during both periods. These results suggest that dopaminergic neurons have stimulatory action on GH secretion and inhibitory action on PRL secretion in cattle. In addition, injection time should be considered to evaluate the exact effects on GH secretion due to its ultradian rhythm of GH secretion in cattle.

Expression of mRNAs for interleukin-4, interleukin-6 and their receptors in porcine corpus luteum during the estrous cycle.

There is increasing evidence that inflammatory cytokines regulate corpus luteum (CL) function in many species. The purpose of the present study was to determine whether interleukin (IL)-4 and IL-6 are expressed in the porcine CL, and whether these cytokines influence porcine luteal steroidogenesis. The gene expressions of IL-4, IL-6 and their specific receptors were determined in the CL of Chinese Meishan pigs during the estrous cycle. Moreover, the effects of these cytokines on progesterone (P(4)), estradiol-17beta (E(2)) and prostaglandin (PG) F2alpha secretion by cultured luteal cells were investigated. IL-4 and IL-6 mRNAs were detected in the CL at all luteal stages. Furthermore, mRNAs of the receptors for IL-4 and IL-6 were clearly expressed in the CL throughout the estrous cycle. Real-time PCR analysis revealed that IL-6 receptor (IL-6R) mRNA expression was higher in the regressed CL (days 19-21 after ovulation) than in the CL at other stages (P<0.01). Exposure of cultured luteal cells obtained from mid-stage CL (days 8-11) to IL-6 (1-100 ng/ml), it inhibited P(4) and E(2) secretion by the cells (P<0.05). Although IL-4 (1-100 ng/ml) did not significantly alter P(4) secretion, it inhibited E(2) secretion by the cells (P<0.05). Neither IL-4 nor IL-6 had any effect on PGF2alpha secretion by the cells. These results suggest that IL-4 and IL-6 are locally produced in the porcine CL, and that they inhibit steroid production from luteal cells via their specific receptors. Collectively, both IL-4 and IL-6 may play roles in regulating porcine CL function throughout the estrous cycle.

Effect of melatonin injected into the third ventricle on growth hormone secretion in Holstein steers.

The effects of melatonin (MEL) injection into the third ventricle (3V) on growth hormone (GH) secretion were investigated in conscious Holstein steers. A stainless steel cannula was stereotaxically implanted in the 3V based on the ventriculogram. In Exp. 1, three doses of MEL (100, 300 or 600 microg) were injected into the 3V through the cannula and the GH concentration after the injection was determined. In Exp. 2, intracerebroventricular (icv) and intravenous (iv) injections of MEL (100 microg) and GH-releasing hormone (GHRH; 0.25 microg/kg body weight), respectively, were performed simultaneously to examine the effect of MEL on GHRH-induced GH release. The icv injection of MEL significantly stimulated GH release at 100 microg. The increase in GH concentrations by 100 microg of MEL was persistent. Intravenous injection of GHRH dramatically increased GH release. The injection of MEL did not alter GHRH-induced GH release. These results suggest that MEL stimulates GH secretion possibly through the hypothalamus in cattle.

Secretory pattern and regulatory mechanism of growth hormone in cattle.

The ultradian rhythm of growth hormone (GH) secretion has been known in several animal species for years and has recently been observed in cattle. Although the physiological significance of the rhythm is not yet fully understood, it appears essential for normal growth. In this review, previous studies concerning the GH secretory pattern in cattle, including its ultradian rhythm, are introduced and the regulatory mechanism is discussed on the basis of recent findings.

Intravenous tryptophan administration attenuates cortisol secretion induced by intracerebroventricular injection of noradrenaline.

This study was conducted to investigate the possibility of suppression of stress-induced cortisol (CORT) secretion by tryptophan (TRP) administration and to better understand its regulatory mechanisms by using a noradrenaline (NA) injection into the third ventricle (3V) as a stress model in cattle. A total of 25 Holstein steers with a cannula in the 3V were used. First, the increase in CORT secretion was observed following a NA injection into the 3V in a dose-dependent manner, verifying the appropriateness of this treatment as a stress model of CORT secretion (Experiment 1). The effect of prior-administration of TRP into peripheral blood with a dose that has been demonstrated to increase brain 5-hydroxytryptamine levels on the elevation of plasma CORT induced by NA or corticotropin-releasing hormone (CRH) was then examined (Experiment 2). The prior administration of TRP suppressed NA-induced, but not CRH-induced, CORT elevation. These results suggest that an increase in TRP absorption into peripheral blood could suppress the stress-induced CORT secretion in cattle via the attenuation of the stimulatory effect of NA on the hypothalamic CRH release.

Effects of ghrelin on growth hormone secretion in vivo in ruminants.

Ghrelin, a novel endogenous growth hormone (GH) secretagogue, has been shown to exert very potent and specific GH-releasing activity in rats and humans. However, little is known about its GH-releasing activity and endocrine effects in domestic animals. To clarify the effect of ghrelin on GH secretion in vivo in ruminants, plasma GH responses to intra-arterial and intra-hypothalamic injections of rat ghrelin (rGhrelin) were examined in goats and cattle. The intra-arterial injection of 1 microg/kg BW of rGhrelin in ovariectomized goats failed to stimulate GH release, however, a dosage of 3 microg/kg BW significantly increased plasma GH concentrations (P<0.05). GH levels peaked at 15 min after the injection, then decreased to basal concentrations within 1 h after the injection. However, the secretory response to 3 microg/kg BW of rGhrelin was weaker than that of growth hormone-releasing hormone (GHRH) (0.25 microg/kg BW) (P<0.05). An infusion of 10 nmol of ghrelin into the medial basal hypothalamus (arcuate nucleus) significantly stimulated the release of GH in male calves (P<0.05). GH levels began to rise just after the infusions and peaked at 10 min, then decreased to the basal concentrations within 1 h after the injection. The present results show that ghrelin stimulates GH release in ruminants.

A novel stereotaxic approach to the hypothalamus for the use of push-pull perfusion cannula in Holstein calves.

To determine secretory patterns of growth hormone-releasing hormone (GHRH) and somatostatin (SS) and their roles in the regulation of growth hormone (GH) secretion, a method for collecting hypothalamic perfusates, a push-pull perfusion method was developed in calves. With the use of the stereotaxic apparatus for cattle, a cannula was implanted into the hypothalamus of four male calves based upon cerebral ventriculography. Push-pull perfusates were collected at 10 min intervals for 6h and GHRH and SS concentrations in perfusates and plasma GH concentration were determined by EIAs and RIA, respectively. A cannula was implanted into the hypothalamus based on the image of the third ventricle and maintained for 1 month. GHRH and SS showed pulsatile secretion and the pulses for GHRH and SS were irregular in conscious animals. Neither GHRH nor SS secretion had a clear relationship with GH secretion. In the present study, we thus (1) established a stereotaxic technique for approaching the hypothalamus using cerebral ventriculography for calves, and (2) demonstrated that GHRH and SS secretion were pulsatile but not closely related to GH profile in conscious calves. The technique is useful for the study of the functions of the hypothalamus in the control of pituitary hormones in cattle.

Radiotelemetry recording of electroencephalogram in piglets during rest.

A wireless recording system was developed to study the electroencephalogram (EEG) in unrestrained, male Landrace piglets. Under general anesthesia, ball-tipped silver/silver chloride electrodes for EEG recording were implanted onto the dura matter of the parietal and frontal cortex of the piglets. A pair of miniature preamplifiers and transmitters was then mounted on the surface of the skull. To examine whether other bioelectrical activities interfere with the EEG measurements, an electrocardiogram (ECG) or electromyogram (EMG) of the neck was simultaneously recorded with the EEG. Next, wire electrodes for recording movement of the eyelid were implanted with EEG electrodes, and EEG and eyelid movements were simultaneously measured. Power spectral analysis using a Fast Fourier Transformation (FFT) algorithm indicates that EEG was successfully recorded in unrestrained piglets, at rest, during the daytime in the absence of interference from ECG, EMG or eyelid movements. These data indicate the feasibility of using our radiotelemetry system for measurement of EEG under these conditions.

A technique for long-term implantation of a microcatheter into the third ventricle of post-pubertal Chinese Meishan pigs based on ventriculography.

The objectives of this study were to implant a microcatheter into the third ventricle of post-pubertal Chinese Meishan pigs, and to maintain the microcatheter for a long time without causing stress. Fourteen pigs (45-60 kg BW) were used. Each pig was anesthetized and the head was orientated on the stereotaxic apparatus. A radiopaque dye was placed into the ventricle via a guide cannula inserted 3.5 mm forward of the bregma. A microcatheter was inserted into the third ventricle using ventriculography, and fixed with dental cement to a metal-mesh protector and screw anchors embedded into the skull. The opposite end of the microcatheter was externalized from the dorsal neck so that corticotropin-releasing hormone (CRH) could be injected easily. Simultaneously, a catheter was fitted in the jugular vein, and the free end of the catheter was externalized from the dorsal neck. Microcatheter-implanted pigs showed a normal progesterone concentration profile, and a constant cortisol level during at least two estrous cycles. Furthermore, intracerebroventricular injections of CRH (25 microg/500 microl) resulted in an increased plasma cortisol concentration (P < 0.05). Thus, the technique developed in this study enables us to approach the third ventricle in post-pubertal freely-moving pigs effectively over a long time, without causing stress.

Development of a stereotaxic instrument for study of the bovine central nervous system.

We have developed a bovine stereotaxic instrument in this study to be used for Holstein cattle from weanling to adult age. The bovine stereotaxic instrument is derived from the Horsley-Clarke principle. A pair of straight ear-bars and orbital ridge pieces with their carriers, head clamps, AP bars are symmetrically arranged across the head pace. For holding the heavy heads, a hard palate and a mandibular support are designed to be placed on the surface of the base frame. A new manipulator with long working distance has also been made. For ventriculography, a holder inserting an X-ray film cassette is placed just next to the AP bar. Finally, the instrument was combined together with an oil-operated lift, so that it can be adjusted to a proper height. Details of this instrument are described in this paper.

Plasma cortisol and prolactin secretion rhythms in cattle under varying external environments and management techniques.

The secretion rhythms of plasma cortisol (CORT) and prolactin (PRL), hormones related to stress responsiveness and biological rhythm and controlled by light and temperature, were investigated under varying external environments and different management techniques. Serial blood samples were collected from female cattle reared in free-stall and freely fed (FF) conditions (n = 4) or in tie-stall and restricted feeding (RF) conditions (hay and concentrate twice daily, n = 4). Plasma CORT and PRL concentrations, eating behavior, and environmental parameters were analyzed. Cyclic patterns for each parameter were examined using spectral analysis, and correlations between CORT, PRL and other parameters were investigated using cross-spectral analysis. Under FF conditions, CORT secretion was not related to the lighting intensity and eating behavior. However, under RF conditions, the CORT secretion rhythm showed a distinct correlation with lighting intensity and eating behavior. Under FF conditions, the PRL secretion rhythm was similar in all seasons. However, under RF conditions, the PRL rhythm oscillated with high frequency in summer and low frequency in winter, indicating a seasonal change in rhythm. The present study demonstrates that hormone secretion rhythms change under different environments and management techniques.

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.

Effects of exposure to plant-derived odorants on behavior and the concentration of stress-related hormones in steers isolated under a novel environment.

This study was conducted to investigate physiological and behavioral effects of the exposure to plant-derived odorants in Holstein steers (8 month of age) under the acute stress of social isolation in a novel environment. Each steer was tethered in a new room alone and exposed to one of three odorants: a mixture of equal amounts of trans-2-hexenal and cis-3-hexenol (green odor, GO; n = 5), essential oil of grapefruits (EOG; n = 5) and solvent (SOL; n = 6). Behaviors were recorded and blood samples were taken at intervals of 10-15 min for 6 h. Compared with SOL, exposure to GO or EOG decreased the frequency of vocalization (P < 0.05), shortened the latency period before the onset of rumination (P < 0.05) and increased the duration of rumination (P < 0.05). Plasma cortisol concentrations in steers exposed to plant-derived odorants (GO and EOG) during 90-130 min from commencement of sampling were significantly lower (P < 0.05) compared with SOL. These results suggest that appeasing effects of plant-derived odorants on behavioral and physiological stress responses previously reported in laboratory rodents could be also be expected in steers.

Effects of intracerebroventricularly administered carbetocin on social behavior in Holstein steers.

To shed light on the role of central oxytocin (OXT) in regulating social behavior in cattle, the impact of intracerebroventricularly administered OXT agonist, carbetocin (CBT), on the social behavior of a group of familiar steers was investigated. In the first experiment, we determined the dose response of intracerebroventricularly administered CBT (0.5, 5 or 50 nmol) on plasma cortisol level and behavior using 7 steers aged from 6 to 10 months. Five of the steers were assigned to the second experiment. CBT (50 or 200 nmol/200 µl) in artificial cerebrospinal fluid (aCSF) or aCSF (200 µl) was injected into the third ventricle. Immediately after the injection, the animal and two peers were taken outside to the adjacent paddock. Thirty minutes later, maintenance and social behaviors of the animal were observed for 2 hr. CBT had no effect either on the basal cortisol level or on the maintenance and the abnormal behavior in steers with their movement restricted by a stanchion stall in the first experiment. However, in the same steers with no movement restrictions in the second experiment, CBT facilitated lying, probably because of its sedative effect via OXT receptor activation, which disturbed some aspects of social behavior. These results suggest that central OXT receptor activation might not affect social behavior itself among "familiar members", because the stimulation of the central OXT system by intracerebroventricular administration of CBT did not facilitate social behavior between familiar steers.

Plasma melatonin secretion rhythms in cattle under varying seasonal conditions.

The purpose of the present study was to investigate the secretion cycles of melatonin (MEL) in cattle over the course of four seasons. Four female Japanese Shorthorn cattle under free-stall and ad libitum feeding conditions were used, and plasma MEL concentrations were measured over a 48 h period at 1 h intervals. The time-series data were analyzed by spectral analysis, and the cycle hour was determined. Data indicated that the secretion cycle for MEL was approximately 23.5 h for all four seasons. The area under the curve of MEL from start to end of experiment for 48 h did not differ significantly among the four seasons. However, the duration of high MEL secretion which defined the duration time of the values were more than 10 pg/mL and differed significantly among the four seasons. In conclusion, this study, which was the first to use spectral analysis to evaluate the cyclic rhythm of MEL in cattle, revealed that MEL secretion cycles did not differ among the seasons. These findings are inconsistent with previous study results in that previous reports suggested that the MEL secretion cycle differed under different lighting conditions.

Biological rhythms related to metabolism in Japanese Shorthorn cattle under varying environments and management techniques.

Plasma insulin (INS), thyroxin (T4 ), glucose (GLU), non-esterified fatty acid (NEFA), blood urea nitrogen (BUN), rectal temperature (RT) and eating behavior were evaluated in Japanese Shorthorn cattle under varying external environments and management techniques. Serial blood collection and assessments of RT and eating behavior were performed over 48 h in the spring, summer, autumn and winter in four female cattle reared under either free-stall and ad libitum feeding (FA) conditions or tie-stall and restricted feeding (TR) conditions. Cycle patterns for each parameter were examined using spectral analysis, and correlations between parameters were investigated using cross-spectral analysis. Rhythms for all parameters, except eating behavior and T4 , did not differ significantly among the varied external environments and between management techniques, although seasonal differences in the concentration or value of parameters were observed. An approximate 3- or 4-h rhythm cycle detected in T4 , GLU, NEFA, BUN, and RT might be the common metabolic rhythm. Under both conditions, the metabolite levels showed strong correlations with eating behavior. Moreover, GLU positively correlated with INS at lag time of 0 h, as did eating behavior and RT.