Design and biological effects of a vaginally administered gel containing the GnRH agonist, triptorelin, for synchronizing ovulation in swine.

A novel gel formulation was selected for intravaginal delivery of the GnRH agonist (triptorelin) for synchronizing ovulation in pigs. Studies with gilt models were used to assess LH response profiles. The lowest dose of triptorelin that induced the most gilts to show an LH surge was 100 µg in 1.2% methylcellulose gel. This formulation had a similar effect in weaned sows while also advancing ovulation. The timing of administration was evaluated in sows after weaning. Administration at 96 h induced more sows to ovulate (58%) by 48 h compared to treatment at estrus (45%) or for controls (34%), but the desired level of ovulation synchrony was not achieved. As a result, greater doses of triptorelin were tested and 200 µg given at 96 h after weaning, induced 81% of sows to ovulate within 48 h after treatment. The best synchrony of ovulation occurred when given at 96 h after weaning compared to earlier or later intervals. The optimum time to give a single fixed time AI (SFT-AI) after administration of 200 µg of triptorelin in 1.2% gel (OvuGel®) at 96 h after weaning was tested. A SFT-AI at 22 ± 2 h after OvuGel achieved the highest fertility and was practical for staff during the normal work day. In field trials, a SFT-AI 22 ± 2 h after all weaned sows were treated with OvuGel improved (P = 0.04) farrowing rate to 82.5% compared to control sows weaned (80.1%), with no effect on numbers of pigs born alive (12.1). Research continues for identifying the advantages for use of OvuGel in different production systems, and potential application for use in gilts.

Hypothalamic neurons innervating fat tissue in the pig express leptin receptor immunoreactivity.

While leptin receptors have been found in both the autonomic ganglion neurons and the hypothalamic nuclei, studies dealing with the projections from the central nervous system to the adipose tissue have been conducted mainly in laboratory animals. Therefore, the purpose of our study was to establish whether hypothalamic neurons are transsynaptically connected to adipose tissue depots in the pig, and if these neurons express leptin receptor immunoreactivity. Pseudorabies virus (PRV; Bartha's K strain) was introduced in perirenal or subcutaneous adipose tissue depots in domestic pigs. On day 9, animals were euthanized and hypothalami were collected and processed immunohistochemically with primary antisera against PRV and leptin receptor (OBR). PRV-labeled neurons were localized in paraventricular nucleus, supraoptic nucleus and arcuate nucleus following injections in both the perirenal and the subcutaneous adipose tissue depots. Ventromedial nucleus, dorsomedial nucleus and preoptic area-labeled neurons were observed after injection of the PRV into the perirenal adipose tissue, while in the lateral hypothalamic area-labeled neurons projected only to the subcutaneous adipose tissue. The majority of the PRV-labeled neurons simultaneously expressed OBR-immunoreactivity. Our results provide the morphological data on multisynaptic projections from hypothalamus to the fat tissue in the pig and demonstrate that these neurons, located in areas involved in reproductive processes and feeding behavior, may regulate fat tissue metabolism.

Effects of estrus synchronization using Matrix® followed by treatment with the GnRH agonist triptorelin to control ovulation in mature gilts.

Estrus and ovulation responses in Matrix-treated gilts may affect ovulation synchrony in response to triptorelin. In experiment 1, estrus and ovulation measures at 12h intervals after last Matrix feeding (LMF) were analyzed. For the 398 gilts that displayed estrus, 87.4% were detected on Days 6-8 after LMF. Duration of estrus was 24-60h for 85.6% of gilts and negatively correlated with interval from LMF to estrus (r=-0.53, P<0.0001). The estrus to ovulation interval was positively correlated with duration of estrus (r=0.61, P<0.0001). In experiment 2, gilts (n=96) received intravaginal treatment with 2mL of gel containing placebo (Control) at 96h, 200µg of triptorelin at 96h (TRP96), 120h (TRP120) or 144h (TRP144) after LMF. Estrus measures did not differ (P>0.10) among treatments. The proportion of gilts ovulating 32-56h after treatment was greater for TRP120 and TRP144 (P<0.01) compared to other treatments. The treatment to ovulation intervals for all triptorelin treatments were shorter (P<0.001) than Control. In experiment 3, gilts (n=86) received placebo (Control), 100µg (TRP100), 200µg (TRP200), or 400µg (TRP400) of triptorelin at 120h after LMF. There was no effect of treatment (P>0.10) on estrus or on interval from LMF to estrus. The proportion of gilts ovulating by 40, 48 and 56h after treatment increased (P<0.05) with triptorelin compared to Control. Our results indicate that gilts receiving 100-400µg of triptorelin at 120h after LMF had the greatest ovulation synchrony 24-48h following treatment. These studies provide important information for developing a procedure for a single insemination in synchronized gilts.

Absence of an estradiol-induced surge of luteinizing hormone in pigs receiving unvarying pulsatile gonadotropin-releasing hormone stimulation.

The goal of this study was to pharmacologically block central nervous system (CNS) input to gonadotropes in mature ovariectomized gilts to determine the direct actions of estradiol (E2) on pituitary LH release when given at a dose sufficient to elicit a gonadotropin surge. Feeding AIMAX [N-methyl-N'-(1-methyl-2-propenyl)1,2-hydrazinedicarbothioamide; 125 mg/day] for 7 days reduced serum LH concentrations from 1.25 +/- 0.13 (mean +/- SE) to less than 0.18 ng/ml, abolished LH pulses, but did not compromise LH release in response to exogenous GnRH. Serum FSH concentrations were reduced by 27%, whereas serum concentrations of PRL, GH, thyroid hormones and cortisol were not affected after 7 days of AIMAX treatment. Behavior was not altered, aside from a slightly reduced appetite. The LH surge that peaked 48-80 h after injecting E2 benzoate (E2B) into control gilts was blocked in five of eight gilts given AIMAX. Giving GnRH pulses (1 microgram every 45 min) to AIMAX-treated gilts restored mean serum LH concentrations as well as the frequency and amplitude of LH pulses to those of untreated ovariectomized gilts. E2B suppressed the LH response to these GnRH pulses by 88% at 12 h, whereas from 24-96 h after E2B treatment, the LH response to GnRH and mean serum concentrations of LH were again similar to those of controls not given estradiol. These data indicate that induction of the gonadotropin surge by E2 in the gilt requires CNS input. The action of E2 on the pituitary in the presence of unvarying GnRH pulsation may, however, be limited to an early transient inhibition of responsiveness to GnRH, with no subsequent direct stimulation during the period of the surge.

The effect of adrenocorticotropin or hydrocortisone on serum luteinizing hormone concentrations after adrenalectomy and/or ovariectomy in the prepuberal gilt.

The possible role of ACTH and the adrenal gland in modulating LH secretion in prepuberal gilts was studied. Fifty-one gilts, 170-175 days of age, were randomly assigned to 12 treatments in a 2 X 2 X 3 factorial design. The main treatments were adrenalectomy (ADX) or sham-ADX, ovariectomy (OVX) or sham-OVX, and ACTH, hydrocortisone acetate (HCA), or vehicle (V) administration. ACTH, HCA, or V was administered from days 3-10 after surgery. Beginning at 0800 h on day 10, blood samples were collected every 15 min for 16 h via jugular cannulae. At 1600 and 2000 h, all gilts were injected iv with a pharmacological dosage of 400 micrograms GnRH. ADX and OVX did not influence subsequent serum cortisol (CS) concentrations, detected by RIA. HCA elevated serum CS concentrations in all four surgical groups. ACTH treatment elevated serum CS and progesterone concentrations only in the sham-ADX groups. Every 3 days after surgery, all ADX gilts received 10 mg deoxycorticosterone acetate, im, as an electrolyte maintenance treatment, which may have been detected in the peripheral blood by the CS assay since the cross-reaction of deoxycorticosterone with the first antibody of the CS assay was 17.1%. Serum LH concentration, peak frequency, and peak magnitude were greater in V-treated OVX gilts than in V-treated sham-OVX gilts in both ADX and sham-ADX groups. Chronic ACTH treatment blocked the increase in basal serum LH concentration, peak frequency, and peak magnitude after OVX only when the adrenal glands were present. In contrast, HCA blocked the postcastration increase in the basal serum LH concentration and peak magnitude in the presence or absence of the adrenal glands. However, HCA had no effect on the increased frequency of LH peaks that occurred after OVX in V-treated gilts. The serum LH responses after both GnRH challenges were similar for all gilts, and the LH response to the second GnRH challenge was less than that observed after the first challenge. These data indicate that ACTH and HCA suppressed the postcastration increase in LH secretion. However, the effect of ACTH was mediated through the adrenal gland, and the inhibiting influence of ACTH and HCA on LH secretion was apparently not mediated at the pituitary level in the prepuberal gilt.

Alterations in adipogenic and mitogenic activity of porcine serum in response to hypophysectomy.

The importance of the pituitary in postnatal regulation of peripheral preadipocyte proliferation and differentiation was examined by hormone supplementation of hypophysectomized pig serum in primary cultures of preadipocyte and stromal-vascular cells derived from rat inguinal adipose tissue. Hypophysectomized pig serum promoted at least 25% less preadipocyte proliferation, less differentiation of sn-glycerol-3-phosphate dehydrogenase activity, and less histochemical differentiation than serum from intact pigs. Porcine GH supplementation of hypophysectomized serum-stimulated [3H]thymidine incorporation by preadipocytes and stromal cells and also histochemical differentiation of preadipocytes, but not enzymatic differentiation. Insulin-like growth factor I (IGF-I) stimulated [3H]thymidine incorporation by preadipocytes and stromal cells. Enzyme differentiation by developing cells was stimulated by IGF-I. Hydrocortisone supplementation of hypophysectomized serum inhibited [3H]thymidine incorporation and stimulated enzymatic differentiation. Thyroid hormones (T3 and T4) stimulated [3H]thymidine incorporation by preadipocytes in a dose-responsive manner when supplemented to hypophysectomized serum. Thyroid hormones stimulated differentiation of enzyme activity at the lowest concentrations examined. The mitogenic effects of GH, IGF-I, and T4 were not specific to the preadipocyte population, since the stromal-vascular cells responded in a similar manner. However, hypophysectomy resulted in a specific reduction in preadipocyte proliferation while stimulating multiplication of stromal-vascular cells. These results suggest that these hormones are nonspecific mitogens in adipose tissue, while unidentified factors of pituitary origin may be important for the specific regulation of proliferation of preadipocytes. Additionally, hypophysectomy appears to remove mitogenic inhibitors that are specific for the stromal-vascular cells.

Immunocytochemical distribution of catecholamine-synthesizing neurons in the hypothalamus and pituitary gland of pigs: tyrosine hydroxylase and dopamine-beta-hydroxylase.

This study describes the distribution of catecholaminergic neurons in the hypothalamus and the pituitary gland of the domestic pig, Sus scrofa, an animal that is widely used as an experimental model of human physiology in addition to its worldwide agricultural importance. Hypothalamic catecholamine neurons were identified by immunocytochemical staining for the presence of the catecholamine synthesizing enzymes, tyrosine hydroxylase and dopamine-beta-hydroxylase. Tyrosine hydroxylase-immunoreactive perikarya were observed in the periventricular region throughout the extent of the third ventricle, the anterior and retrochiasmatic divisions of the supraoptic nucleus, the suprachiasmatic nucleus, the ventral and dorsolateral regions of the paraventricular nucleus and adjacent dorsal hypothalamus, the ventrolateral arcuate nucleus, and the posterior hypothalamus. Perikarya ranged from parvicellular (10-15 microns) to magnocellular (25-50 microns) and were of multiple shapes (rounded, fusiform, triangular, or multipolar) and generally had two to five processes with branched arborization. No dopamine-beta-hydroxylase immunoreactive perikarya were observed within the hypothalamus or in the adjacent basal forebrain structures. Both tyrosine hydroxylase- and dopamine-beta-hydroxylase-immunoreactive fibers and punctate varicosities were observed throughout areas containing tyrosine hydroxylase perikarya, but dopamine-beta-hydroxylase immunoreactivity was very sparse within the median eminence. Within the pituitary gland, only tyrosine hydroxylase fibers, and not dopamine-beta-hydroxylase immunoreactive fibers, were located throughout the neurohypophyseal tract and within the posterior pituitary in both pars intermedia and pars nervosa regions. Generally, the location and patterns of both catecholamine-synthesizing enzymes were similar to those reported for other mammalian species except for the absence of the A15 dorsal group and the very sparse dopamine-beta-hydroxylase immunoreactive fibers and varicosities in the median eminence in the pig. These findings provide an initial framework for elucidating behavioral and neuroendocrine species differences with regard to catecholamine neurotransmitters.

Opioid modulation of FSH, growth hormone and prolactin secretion in the prepuberal gilt.

The role of endogenous opioid peptides (EOP) in modulating GH, prolactin (PRL) and FSH secretion was evaluated in prepuberal (P) gilts. In experiment I, P gilts received 1 (n = 2), 3 (n = 3) or 6 (n = 3) mg naloxone (NAL)/kg body weight i.v. Blood was collected every 15 min for 2 h prior to and 2 h after NAL and an additional 1 h after 100 micrograms gonadotrophin-releasing hormone (GnRH) i.v. In experiment II, P and mature (M) gilts were ovariectomized. Three weeks after ovariectomy, P and M gilts were injected twice a day for 10 days with either 0.85 mg progesterone (P4)/kg body weight or oil vehicle (V), resulting in the following groups: PP4 (n = 11), PV (n = 10), MP4 (n = 11) and MV (n = 10). All gilts received 1 mg NAL/kg body weight on the last day of treatment. Blood samples were collected every 15 min for 4 h before and 2 h after NAL and an additional 1 h after 100 micrograms GnRH i.v. In experiment III, six P and five M gilts were ovariectomized and surgically implanted with intracerebroventricular (i.c.v.) cannulae. Blood was collected every 15 min for 3 h before and 3 h after i.c.v. injection of 500 micrograms morphine in artificial cerebrospinal fluid (CSF) or 250 microliters CSF. In experiment I, all doses of NAL failed to alter PRL secretion, while NAL increased (P less than 0.05) GH secretion in three out of eight gilts. However, NAL suppressed (P less than 0.05) FSH concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of melanocortin-3 and -4 receptor in regulating appetite, energy homeostasis and neuroendocrine function in the pig.

A recently discovered class of receptors, melanocortin-3 and -4 receptor (MC3/4-R), are located within the brain and modulate feed intake in rodents. Stimulation of the receptor (agonist) inhibits feed intake whereas blockade (antagonist) of the receptor increases intake. Our knowledge of factors regulating voluntary feed intake in humans and domestic animals is very limited. i.c.v. administration of an MC3/4-R agonist, NDP-MSH, suppressed (P<0.05) feed intake compared with controls at 12, 24, 48 and 72 h after treatment in growing pigs. Fed pigs were more responsive to the MC3/4-R agonist then fasted animals. However, i.c.v. treatment with MC3/4-R antagonist, SHU9119, failed to stimulate intake. The failure of MC3/4-R antagonist to stimulate feed intake suggests involvement of other brain hormone(s) which antagonize the action of SHU9119 at the MC3/4-R, blocking its stimulatory effect on intake. Treatment with NDP-MSH or SHU9119, across a wide dose range, failed to affect LH and GH secretion, except for the 10 micro g dose of NDP-MSH, which exhibited both a stimulatory and an inhibitory effect on GH secretion in fasted animals. Treatment with agouti-related peptide, a natural brain hormone that blocks the MC3/4R, failed to stimulate feed intake. These results do not support the idea that endogenous melanocortin pays a critical role in regulating feed intake and pituitary hormone secretion in the pig. SHU9119 blocked the NDP-MSH-induced increase in cAMP in HEK293 cells expressing the porcine MC4-R sequence without the missense mutation. The EC(50) and IC(50) values were similar to the human MC4-R, confirming that SHU9119 is a pig MC4-R antagonist. However, pigs were heterozygous for an MC4-R gene missense mutation. It is possible that the MC4-R mutation alters function and this may explain the failure to demonstrate MC3/4-R involvement in modulating feeding behavior and LH and GH secretion in the pig.

Immunocytochemical localization of the catecholamine-synthesizing enzymes, tyrosine hydroxylase and dopamine-beta-hydroxylase, in the hypothalamus of cattle.

Immunocytochemical staining for the presence of catecholamine synthesizing enzymes, tyrosine hydroxylase and dopamine beta-hydroxylase, was used to characterize the regional distribution of catecholaminergic neurons in the hypothalamus and adjacent areas of domestic cattle, Bos taurus. In steers, heifers and cows, tyrosine hydroxylase-immunoreactive perikarya was located throughout periventricular regions of the third cerebral ventricle, in both anterior and retrochiasmatic divisions of the supraoptic nucleus, suprachiasmatic nucleus, and ventral and dorsolateral regions of the paraventricular nucleus, dorsal hypothalamus, ventrolateral aspects of the arcuate nucleus, along the ventral hypothalamic surface between the median eminence and optic tract, and in the posterior hypothalamus. Immunostained perikarya ranged from small (10-20 microns, parvicellular) to large (30-50 microns, magnocellular) and were of multiple shapes: round, triangular, fusiform or multipolar, often with 2-5 processes of branched arborization. There were no dopamine-beta-hydroxylase immunoreactive perikarya observed within the hypothalamus and adjacent structures. However, both tyrosine hydroxylase and dopamine-beta-hydroxylase immunoreactive fibers and punctate varicosities were observed throughout regions of tyrosine hydroxylase immunoreactivity perikarya. Generally, the location and pattern of hypothalamic tyrosine hydroxylase immunoreactivity and dopamine-beta-hydroxylase immunoreactive were similar to those reported for most other large brain mammalian species, however, there were several differences with commonly used small laboratory animals. These included intense tyrosine hydroxylase immunoreactivity of perikarya within the retrochiasmatic division of the supraoptic nucleus (ventral A15 region), the absence of tyrosine hydroxylase immunoreactive perikarya below the anterior commissure or within the bed nucleus of stria terminalis (absence of the dorsal A15 region), an abundance of tyrosine hydroxylase immunoreactive perikarya within the ependymal layer of the median eminence, heavy innervation of the arcuate nucleus with dopamine-beta-hydroxylase immunoreactive fibers and varicosities, and the paucity of dopamine-beta-hydroxylase immunoreactive throughout the median eminence.

Catecholaminergic region A15 in the bovine and porcine hypothalamus.

Magnocellular perikarya within the retrochiasmatic division of the supraoptic nucleus of bovine and porcine hypothalami were immunoreactive (ir) with antiserum against tyrosine hydroxylase (TH), but not dopamine-beta-hydroxylase (DBH). Few cells in this region were also immunoreactive for vasopressin (VP) or oxytocin (OT). In contrast, the main division of the supraoptic nucleus contained numerous perikarya immunoreactive for VP and OT, but not TH nor DBH. Both the retrochiasmatic and principal divisions of the supraoptic nuclei contained TH- and DBH-ir fibers and varicosities. This region in bovine and porcine hypothalami corresponds to the ventral A15 catecholaminergic (dopamine-producing) cell group.

Prolactin secretion after bromocriptine in the hypophysial stalk-transfected gilt.

Blood samples were collected by jugular vein cannula from 6 hypophysial stalk-transected (HST) and 4 intact (INTACT) crossbred gilts at 0800, 0830 and 0900 h. Immediately after the 0900 h sample, thyrotropin releasing hormone (TRH) was injected i.v. to determine anterior pituitary gland response via release of prolactin (PRL). Blood was collected every 15 min for 1 h and every 30 min for an additional 2 h. All gilts received the dopamine (DA) agonist, bromocriptine (CB-154), s.c. at 1600 h and blood sampling and TRH challenge was repeated beginning at 0800 h the next day. Mean serum PRL concentration at 0800, 0830 and 0900 h was termed basal PRL for each day. Before CB-154, basal PRL for HST gilts was greater (P < .01) than for INTACT gilts, whereas, after CB-154, basal PRL were similar among HST and INTACT gilts. Before CB-154, TRH caused peak secretion of PRL of similar magnitude within 15 minutes in both HST and INTACT gilts; PRL decreased to basal concentration by 120 min after TRH in both groups. However, after CB-154, PRL response to TRH was blunted similarly in all gilts. These results indicate that suppression of DA pathways is an antecedent to the physiological mechanism by which a secretagogue, such as TRH, stimulates PRL secretion in the intact pig.

On the site of action of naloxone-stimulated cortisol secretion in gilts.

The increase in serum cortisol concentrations following naloxone administration to female pigs was abolished by hypophysial stalk-transection, even though CRH and ACTH stimulated cortisol release in these animals. We suggest that the opioid antagonist enhances cortisol secretion primarily by a central action in pigs.

N-methyl-D,L-aspartate modulation of luteinizing hormone and growth hormone secretion from pig pituitary cells in culture.

Administration of n-methyl-d, l-aspartate (NMA) to pigs in vivo increased GH and suppressed LH secretion. Cultures of anterior pituitary cells from pigs in the follicular phase (FOL; n = 3) and luteal phase (LUT; n = 3) of the estrous cycle, and ovariectomized (OVX; n = 10) pigs were treated with NMA (10(-4), 10(-6) or 10(-8) M) or the NMA antagonist, 2-amino-5-phosphonopentanoic acid (AP5; 10(-4), 10(-6) or 10(-8) M), to determine if NMA affects the pituitary directly. Secreted LH and GH were measured at 4 h after treatment. Basal LH and GH secretion (control; C) were 1.1 +/- 0.6, 4.4 +/- 2.1 and 5.6 +/- 1.3 ng/well and 5.2 +/- 1.2, 7.5 +/- 1.2 and 5.2 +/- 1.7 ng/well for FOL, LUT and OVX, respectively. Relative to C, 10(-4) M NMA increased (P < 0.001) LH secretion 2.4-, 2.2- and 5.1-fold in FOL, LUT and OVX cultures, respectively. The effect of 10(-4) M NMA was inhibited by 10(-4) M AP5 (P < 0.05) in FOL cultures, but not in OVX cultures. GnRH increased (P < 0.001) LH levels 3.1-, 2.3- and 3.8-fold in FOL, LUT and OVX cultures, respectively. Relative to C, 10(-4), 10(-6) and 10(-8) M NMA increased (P < 0.03) GH secretion 1.5-, 1.5- and 2.3-fold in LUT and 1.7-, 2.3- 2.0-fold in OVX cultures, respectively. AP5 alone or in combination with NMA failed to alter basal GH secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Luteinizing hormone secretion following intracerebroventricular administration of morphine in the prepuberal gilt.

Antagonism of endogenous opioids with naloxone stimulates luteinizing hormone (LH) release in mature but not prepuberal gilts. The present report demonstrates that the opiate agonist morphine (500 micrograms), administered intracerebroventricularly (ICV), reduced LH secretion in both ovariectomized mature and prepuberal gilts. We suggest that opioid receptors are functionally coupled to the GnRH secretory system in prepuberal gilts even though endogenous opioid peptide modulation of LH secretion was not demonstrable in our previous studies.

Intracerebroventricular porcine corticotropin-releasing hormone and cortisol effects on pig immune measures and behavior.

The effects of intracerebroventricular (icv) administration of porcine corticotropin-releasing hormone (pCRH) and cortisol on the immune system and behavior were examined in domestic pigs. In Experiment 1, 50 micrograms of pCRH in 200 microliters of saline or 200 microliters of vehicle was administered i.c.v. at 0600 h. Blood samples were obtained at 0600 (prior to injection), 0700, and 0800 h. Plasma cortisol concentrations were higher at 1 and 2 h after pCRH than after saline. Generally, pCRH failed to effect NK cytotoxicity or lymphocyte proliferation in response to phytohemagluttin (PHA). However, 1 h postinjection, pigs administered pCRH i.c.v. had marginally lower NK activity than control pigs. Pigs injected with pCRH had substantially lower neutrophil chemotaxis (CHTX) than the control pigs at 1 and 2 h postinjection. As blood cortisol concentration increased, neutrophil CHTX decreased. Pigs injected i.c.v. with pCRH had higher neutrophil numbers and neutrophil:lymphocyte ratios than control pigs. Percentage of lymphocytes was higher among control than treated pigs. Central pCRH increased overall activity, particularly walking, standing, licking, rooting, and increased activity-related sequences (e.g., sit, walk and stand, walk), but reduced complex oral/nasal sequences (e.g., root, lick). In Experiment 2, pigs were injected i.c.v. with 10 micrograms of cortisol in 200 microliters of saline or with vehicle at 0600 h. Administration of cortisol failed to effect NK cytotoxicity, lymphocyte proliferation, CHTX, or leukocyte distribution. Pigs given cortisol had no apparent change in behavior. These data indicate leukocyte distribution and specific neutrophil function in pigs were significantly modulated by stress-related hormones of the hypothalamic-pituitary-adrenal axis and complexity of behavioral sequences (pigs repeating certain behavioral sequences) associated with increased activity was reduced. Oral/nasal stereotypies (as seen among confined sows) were not elevated among pigs given i.c.v. pCRH. CRH given by i.c.v. administration may serve as a better model for acute rather than chronic stress.

Central nervous system peptide and amino acid modulation of luteinizing hormone and prolactin secretion in the pig.

We recently demonstrated that pulsatile LH secretion is associated with pulsatile gonadotropin releasing hormone (GnRH) in the pig. Endogenous opioid peptide (EOP) inhibition of pulsatile LH and prolactin (PRL) secretion is dependent on reproductive status and development of this EOP system is a brain maturational process independent of the ovary. Once sexual maturation has occurred, EOP then become part of a progesterone dependent system and EOP inhibit a noradrenergic component of this system. During lactation, EOP also inhibit pulsatile LH, but stimulate PRL secretion. N-methyl-d,l-aspartate (NMA), an agonist of the excitatory amino acids (EAA), aspartate and glutamate, suppressed LH secretion in gilts pretreated with progesterone or vehicle. Both the EOP agonist, morphine (MOR), and the EOP antagonist, naloxone (NAL), delayed emergence and time to maximum serum LH concentration of the estradiol-induced LH surge in prepuberal and mature gilts, respectively. Therefore, EOP may normally have both a permissive as well as an inhibitory role in the LH surge mechanism. Although a norepinephrine synthesis inhibitor failed to alter basal PRL secretion, the PRL increase after NAL was suppressed in progesterone-treated ovariectomized (OVX) gilts. NAL suppressed the PRL response to NMA in OVX gilts pretreated with oil vehicle or progesterone, indicating that NMA stimulation of PRL secretion is mediated through the EOP system.

Growth hormone releasing factor decreases long form leptin receptor expression in porcine anterior pituitary cells.

Pituitary cells from six pigs, 180-200 days of age, were studied in primary culture to determine if growth hormone releasing factor (GRF) affects long form leptin receptor (Ob-Rl) expression. On Day 4 of culture, 10(5) live cells per well were challenged with either 0, 10(-6), 10(-7) or 10(-8)M [Ala15]-hGRF-(1-29)NH(2) (3 wells per treatment per pig). Secretion of growth hormone (GH) into the media and pituitary Ob-Rl mRNA expression were determined at 4 h after treatment. Media were analyzed for GH by radioimmunoassay, and total RNA was isolated from cells for determination of Ob-Rl expression by semi quantitative reverse transcription PCR. Basal GH concentration was 32+/-2 ng per 10(5) cells per well (n=18 wells) for 4 h. Relative to control at 4 h, 10(-6),10(-7) and 10(-8)M GRF increased (P<0.01) GH secretion by 151, 129 and 120%, but decreased (P<0.05) Ob-Rl expression by 32, 50 and 38%, respectively. These results indicate that GRF directly modulates Ob-Rl expression at the level of the pituitary, and thereby playing a role in regulating pituitary sensitivity to leptin.

Role of leptin in modulating the hypothalamic-pituitary axis and luteinizing hormone secretion in the prepuberal gilt.

Three experiments (EXP) were conducted to test the hypothesis that leptin modulates LH, GnRH, and neuropeptide Y (NPY) secretion. In EXP I, prepuberal gilts received intracerebroventricular (i.c.v.) leptin injections and blood samples were collected. In EXP II, anterior pituitary cells from prepuberal gilts in primary culture were challenged with 10(-14), 10(-13), 10(-12), 10(-11), 10(-10), 10(-9), 10(-8), 10(-7), or 10(-6) M leptin individually or in combinations with 10(-10), 10(-9), and 10(-8) M GnRH. In EXP III, hypothalamic-preoptic area (HYP-POA) explants were placed in perfusion system and exposed to 0 (n=5), 10(-12) M (n=4), 10(-10) M (n=4), 10(-8) M (n=4), or 10(-6) M (n=5) human recombinant leptin (LEP) for 30 min. In EXP I, serum LH concentrations were unaffected by leptin treatment. In EXP II, all doses of leptin increased LH secretion except for 10(-12) and 10(-7) M. Only 10(-7), or 10(-13) M leptin in combination with 10(-8) or 10(-9) M GnRH, respectively, suppressed LH secretion. In EXP III, prior to leptin, media GnRH concentrations were similar across treatments. Media GnRH concentrations increased after 10(-12), 10(-10), and 10(-8) M leptin compared to control. Leptin treatment failed to influence NPY secretion across treatments. These results indicate that components of the neuroendocrine axis that regulate GnRH and LH secretion are functional and leptin sensitive before the onset of puberty. Other neural peptides in addition to NPY may mediate the acute effects of leptin on the GnRH-LH system and lastly, the inability of i.c.v. leptin treatment to increase LH secretion may in part be related to stage of sexual maturation and associated change in negative feedback action of estradiol on LH secretion.

Opioid modulation of gonadotropin releasing hormone release from the hypothalamic preoptic area in the pig.

Two experiments (Exp) were conducted to examine in vitro the release of gonadotropin releasing hormone (GnRH) from the hypothalamus after treatment with naloxone (NAL) or morphine (MOR). In Exp 1, hypothalamic-preoptic area (HYP-POA) collected from 3 market weight gilts at sacrifice and sagittally halved were perifused for 90 min prior to a 10 min pulse of morphine (MOR; 4.5 x 10(-6) M) followed by NAL (3.1 x 10(-5) M) during the last 5 min of MOR (MOR + NAL; n = 3). The other half of the explants (n = 3) were exposed to NAL for 5 min. Fragments were exposed to KCl (60 mM) at 175 min to assess residual GnRH releasability. In Exp 2, nine gilts were ovariectomized and received either oil vehicle im (V; n = 3); 10 micrograms estradiol-17 beta/kg BW in 42 hr before sacrifice (E; n = 3); .85 mg progesterone/kg BW in twice daily for 6 d prior to sacrifice (P4; n = 3). Blood was collected to assess pituitary sensitivity to GnRH (.2 microgram/kg BW) on the day prior to sacrifice. On the day of sacrifice HYP-POA explants were collected and treated as described in Exp 1 except tissue received only NAL. In Exp 1, NAL increased (P < .05) GnRH release. This response to NAL was attenuated (P < .05) by coadministration of MOR. Cumulative GnRH release after NAL was greater (P < .05) than after MOR + NAL. All tissues responded similarly to KCl with an increase (P < .05) in GnRH release.(ABSTRACT TRUNCATED AT 250 WORDS)