Involvement of salsolinol in the suckling-induced oxytocin surge in sheep.

During lactation, the main surge of oxytocin is induced by a suckling stimulus. Previous studies have shown that salsolinol (1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline), a dopamine-derived compound, stimulates both the synthesis and the release of oxytocin in lactating sheep. The objective of the present study was to verify the hypothesis that salsolinol is involved in the mechanism that generates the oxytocin surge that occurs during suckling. Thus, a structural analogue of salsolinol, 1-methyl-3,4-dihydroisoquinoline (1MeDIQ), known to antagonize some of its actions, was infused into the third ventricle of the brain of lactating sheep nursing their offspring. Serial 30-min infusion of 1MeDIQ (4 × 60 µg/60 µL) or vehicle were administered at 30-min interval from 10 AM to 2 PM. The experimental period in every ewe consisted of a nonsuckling period (10 AM-12 PM) and a suckling period (12 PM-2 PM). Blood samples were collected every 10 min, to measure plasma oxytocin concentration by RIA. In control sheep, oxytocin surges of high amplitude were observed during the suckling period. The oxytocin surges induced by suckling were significantly (P < 0.01) diminished in sheep receiving 1MeDIQ infusions as compared to those that received control infusions. However, no significant effect of 1MeDIQ was observed on basal oxytocin release, before suckling. Furthermore, oxytocin release, as measured by the area under the hormone response curve (AUC), was significantly decreased by the administration of 1MeDIQ during the suckling period. This study shows that elimination of the effect of salsolinol within the central nervous system of lactating sheep attenuates the oxytocin surge induced by suckling. Therefore, salsolinol may be an important factor in the oxytocin-stimulating pathway in lactating mammals.

Influence of preterm delivery on ghrelin and obestatin concentrations in maternal plasm, milk and their expression in mammary epithelial cells.

Ghrelin and obestatin are gastrointestinal peptides with a potential role in the programming of metabolism in newborns. The present study aimed to investigate the influence of preterm delivery on ghrelin and obestatin concentrations in the maternal blood plasma and breast milk as well as their gene expressions in the mammary epithelial cells (MECs). On the 3rd day after delivery, milk and plasma samples were collected from mothers that carried to term or gave birth prematurely (< 36 weeks of gestation) and analyzed for ghrelin and obestatin concentrations. MECs isolated from the milk were analyzed for the relative expression of GHRL splice variants. In both groups ghrelin concentrations were significantly lower in milk than in blood plasma. In the preterm group obestatin concentrations were significantly higher in milk than in blood plasma but significantly lower in comparison to that of the control mothers. The expression of GHRL mRNAs was higher (P < 0.05) in MECs isolated from the preterm group as compared to those isolated from control mothers. The concentration of obestatin (but not ghrelin) in the breast milk is dependent on the term of pregnancy. Moreover, the lactating mammary gland is one of the sources of ghrelin and obestatin.

Central Interleukin-1ß Suppresses the Nocturnal Secretion of Melatonin.

In vertebrates, numerous processes occur in a rhythmic manner. The hormonal signal reliably reflecting the environmental light conditions is melatonin. Nocturnal melatonin secretion patterns could be disturbed in pathophysiological states, including inflammation, Alzheimer's disease, and depression. All of these states share common elements in their aetiology, including the overexpression of interleukin- (IL-) 1ß in the central nervous system. Therefore, the present study was designed to determine the effect of the central injection of exogenous IL-1ß on melatonin release and on the expression of the enzymes of the melatonin biosynthetic pathway in the pineal gland of ewe. It was found that intracerebroventricular injections of IL-1ß (50 µg/animal) suppressed (P < 0.05) nocturnal melatonin secretion in sheep regardless of the photoperiod. This may have resulted from decreased (P < 0.05) synthesis of the melatonin intermediate serotonin, which may have resulted, at least partially, from a reduced expression of tryptophan hydroxylase. IL-1ß also inhibited (P < 0.05) the expression of the melatonin rhythm enzyme arylalkylamine-N-acetyltransferase and hydroxyindole-O-methyltransferase. However, the ability of IL-1ß to affect the expression of these enzymes was dependent upon the photoperiod. Our study may shed new light on the role of central IL-1ß in the aetiology of disruptions in melatonin secretion.

Sex-dependent differences in the effect of early weaning on the chosen hormones secretion in sheep during the postnatal transition to puberty--preliminary results.

The influence of early weaning on the cortisol, follicle-stimulating hormone (FSH), luteinizing hormone (LH) and growth hormone (GH) secretion in lambs of both sexes and testosterone (T4) level in male lambs during the postnatal transition to puberty was investigated by radioimmunoassay. It was hypothesized that this influence is long-term and sexually dimorphic. Hence, the effect of weaning at 5 weeks of age in comparison with the weaning at 9 weeks of age on hormone concentra- tions in peripheral blood plasma of 5-, 9-, 12-, and 16-week-old lambs of both sexes was investigated. The cortisol concentrations were greater (P < 0.05) in control and early weaned female lambs than in male lambs at investigated stages. Weaning at 5 weeks of age resulted in the lover (P < 0.05) cortisol secretion in male lambs in contrast to the greater (P < 0.05) cortisol secretion in female lambs at 16 weeks of age. Weaning at 5 weeks of age stimulated (P < 0.001) the FSH secretion, but reduced (P < 0.001) the LH, GH and T4 secretion in 16-week-old male lambs. In female lambs early weaning inhibited (P < 0.05) the FSH secretion at 9 weeks of age, LH secretion after 9 weeks of age and GH secretion after 12 weeks of age. Thus, early weaning results in the sexually dimorphic stress reaction that is more potent and long-lasting in female in contrast to male lambs. This maternal deprivation stress contributes to the inhibition of LH and GH secretion in lambs of both sexes and T4 secretion in male lambs during the postnatal transition to puberty.

Different types of opioid receptors involved in the suppression of LH secretion in lactating sheep.

To learn the involvement of endogenous opioid peptides (EOP) in the regulation of reproductive activity in ruminants, the effects of different opioid antagonists on luteinizing hormone (LH) secretion were determined in sheep during the early stage of lactation. The opioid receptor antagonists: naloxone (all types of receptors, n=5), naloxonazine (µ receptor, n=5), GNTI- (κ receptor, n=5), naltrindole (δ receptor, n=5) or the vehicle (control, n=5) were infused intracerebroventricularly in a series of five 30-min infusions (60µg/60µl) at 30-min intervals. The period of the experiment included the non-suckling (10:00-12.30) and suckling (12.30-15.00) periods. Blood samples were collected from 10.00 to 15.00 at 10-min intervals, and plasma LH concentration was assayed by the radioimmunoassay method. The obtained results showed that blocking of the EOP action within the central nervous system in lactating sheep caused a significant (p<0.001) increase in LH concentration in all treated groups, in comparison to the control. In the naloxone-treated group, a significant (p<0.05) increase in LH secretion also occurred during suckling. The amplitude of LH pulses increased significantly in the naloxonazine- (p<0.01) and naltrindole- (p<0.05) treated ewes compared to the control; there were no significant differences in the frequency of LH pulses among the groups. In conclusion, our study indicates that EOP play a crucial role in the mechanism inhibiting GnRH/LH axis activity in lactating sheep and that the ligands for µ opioid receptor may have the highest inhibitory effect.

The effect of rivastigmine on the LPS-induced suppression of GnRH/LH secretion during the follicular phase of the estrous cycle in ewes.

This study was designed to determine the effect of a potent subcutaneously injected acetylcholinesterase inhibitor, rivastigmine (6mg/animal), on the gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) release during inflammation induced by an intravenous lipopolysaccharide (LPS) (400ng/kg) injection in ewes during the follicular phase of the estrous cycle. The results are expressed as the mean values from -2 to -0.5h before and +1 to +3h after treatment. Rivastigmine decreased the acetylcholinesterase concentration in the blood plasma from 176.9±9.5 to 99.3±15.1µmol/min/ml. Endotoxin suppressed LH (5.4±0.6ng/ml) and GnRH (4.6±0.4pg/ml) release; however, the rivastigmine injection restored the LH concentration (7.8±0.8ng/ml) to the control value (7.8±0.7ng/ml) and stimulated GnRH release (7.6±0.8pg/ml) compared to the control (5.9±0.4pg/ml). Immune stress decreased expression of the GnRH gene and its receptor (GnRH-R) in the median eminence as well as LHß and GnRH-R in the pituitary. In the case of the GnRH and LHß genes, the suppressive effect of inflammation was negated by rivastigmine. LPS stimulated cortisol and prolactin release (71.1±14.7 and 217.1±8.0ng/ml) compared to the control group (9.0±5.4 and 21.3±3.5ng/ml). Rivastigmine also showed a moderating effect on cortisol and prolactin secretion (43.1±13.1 and 169.7±29.5ng/ml). The present study shows that LPS-induced decreases in GnRH and LH can be reduced by the AChE inhibitor. This action of the AChE inhibitor could result from the suppression of pro-inflammatory cytokine release and the attenuation of the stress response. However, a direct stimulatory effect of ACh on GnRH/LH secretion should also be considered.

Regulation of growth hormone secretion in nursing ewes: an involvement of µ-receptor subtype.

Suckling by newborns induces a surge of lactogenic hormones, that is prolactin and growth hormone (GH), in mother's body, with endogenous opioid peptide (EOP) participating in generation of this surge. The aim of the current study was to investigate which types of opioid receptors are involved in generation of the GH surge in ewes during suckling. A series of intracerebroventricular infusions of opioid receptors antagonists: naloxone (for all types of receptors), naloxonazine (specific for µ receptor) and 5'-guanidinonaltrindole (GNTI--specific for κ receptor) and the vehicle (control) were performed in nursing sheep during the fifth week of lactation. All infusions were carried out in a serial manner: five 30-min infusions (60 µg/60 µl) from 10:00 to 15:00, at 30-min intervals. The period of the experiment consisted of the non-suckling (10:00-12:30) and suckling (12:30-15:00) periods. Simultaneously, blood samples were collected at 10-min intervals to determine plasma GH concentration by radioimmunoassay. Suckling evoked a rapid increase in GH concentration in control ewes. Naloxone and naloxonazine significantly decreased both the basal GH release in the non-suckling period and the suckling-induced GH surge. Specifically, the suppressive effect concerned either the duration or the amplitude of the GH surge. In contrast, GNTI did not significantly affect the GH release. In conclusion, the EOPs may affect the regulatory process of GH secretion in lactating sheep, especially through µ opioid receptor.

Central injection of exogenous IL-1ß in the control activities of hypothalamic-pituitary-gonadal axis in anestrous ewes.

This study was performed to determine the effect of intracerebroventricular (icv) injection of interleukin (IL)-1ß on the gene expression, translation and release of gonadotropin-releasing hormone (GnRH) and the GnRH receptor (GnRHR) gene expression in the hypothalamus of anestrous ewes. In the anterior pituitary gland (AP), the expression of genes encoding: GnRHR, ß subunits of luteinizing hormone (LH) and folliculotropic hormone (FSH) was determined as well as the effect of IL-1ß on pituitary gonadotropins release. The relative mRNA level was determined by real-time PCR, GnRH concentration in the cerebrospinal fluid (CSF) was assayed by ELISA and the plasma concentration of LH and FSH were determined by radioimmunoassay. Our results showed that icv injection of IL-1ß (10 or 50 µg/animal) decreased the GnRH mRNA level in the pre-optic area (POA) (35% and 40% respectively; p ≤ 0.01) and median eminence (ME) (75% and 70% respectively; p ≤ 0.01) and GnRHR gene expression in ME (55% and 50% respectively; p ≤ 0.01). A significant decrease in GnRHR mRNA level in the AP in the group treated with the 50 µg (60%; p ≤ 0.01) but not with the 10 µg dose was observed. The centrally administrated IL-1ß lowered also GnRH concentration in the CSF (60%; p ≤ 0.01) and reduced the intensity of GnRH translation in the POA (p ≤ 0.01). It was not found any effect of icv IL-1ß injection upon the release of LH and FSH. However, the central injection of IL-1ß strongly decreased the LHß mRNA level (41% and 50%; p ≤ 0.01; respectively) and FSHß mRNA in the case of the 50 µg dose (49%; p ≤ 0.01) in the pituitary of anestrous ewes. These results demonstrate that the central IL-1ß is an important modulator of the GnRH biosynthesis and release during immune/inflammatory challenge.

Effects of orexigenic peptides and leptin on melatonin secretion during different photoperiods in seasonal breeding ewes: an in vitro study.

The pineal gland (PG) acts as a neuroendocrine transducer of daily and seasonal time through the nocturnal release of melatonin. Here, we examined the interaction of season, orexin, ghrelin, and leptin on melatonin secretion by pineal explants in short-term culture. Glands were collected after sunset from 12 ewes during long days (LD; April and May) and from an additional 12 ewes during short days (SD; October and November). Glands were transected sagittally into strips, with each equilibrated in 2.5 mL of Dulbecco's modified Eagle's medium for 60 min, followed by a 2-h incubation in control medium or medium containing orexin B (10 and 100 ng/mL), ghrelin (10 and 100 ng/mL), or 50 ng/mL of leptin. After a 3-h incubation, some PG explants treated previously with lower doses of orexin or ghrelin were challenged with 50 ng/mL of leptin and those treated with both doses of orexin were challenged with 300 nM of the ß-agonist isoproterenol. One milliliter of medium was harvested and replaced from each well every 30 min. Treatment with the low dose of orexin during LD increased melatonin secretion about 110% (P<0.01); treatment with a high dose increased melatonin secretion about 47% (P<0.001). During the SD period, leptin stimulated (P < 0.05) melatonin secretion slightly compared with mean melatonin concentration in controls. However, together, orexin and leptin depressed (P<0.01) melatonin secretion. Both doses of ghrelin reduced (P < 0.01) melatonin concentration during the SD season compared with control culture. Addition of ghrelin and leptin to culture medium increased (P<0.01) melatonin concentration compared with ghrelin-treated culture and decreased melatonin concentration (P<0.01) compared with leptin-treated culture during SD. Isoproterenol stimulated (P<0.01) melatonin secretion compared with values observed during the pretreatment period. We conclude that orexigenic peptides (orexin B and ghrelin) and an anorectic peptide (leptin) affect PG directly. The responses of PG to those hormones depend on day length. Moreover, secretion of melatonin from the ovine PG is under an adrenergic regulation.

Opioid-salsolinol relationship in the control of prolactin release during lactation.

Endogenous opioid peptides (EOP) and dopamine (DA)-derived salsolinol are implicated in the suckling-induced prolactin surge. The aim of this study was to investigate the relationship between the opioidergic and salsolinergic activity in the mediobasal hypothalamus of nursing sheep. The sheep were infused intracerebroventricularly with opioid receptors antagonists: naloxone (all types of receptors, n=6); naloxonazine (µ receptor, n=6) or the vehicle (control, n=6) in a series of five 30-min infusions (60 µg/60 µl) from 10:00 to 15:00, at 30-min intervals. The period of the experiment included the non-suckling (10:00-12:30) and suckling (12:30-15:00) periods. Simultaneously, a push-pull perfusion of the infundibular nucleus/median eminence was performed in every sheep to study the dopaminergic system activity. Blood samples were also collected at 10-minute intervals to determine plasma prolactin concentration. Both the mean perfusate salsolinol and plasma prolactin concentrations were higher during the suckling vs. non-suckling (P<0.001) period in the control. The perfusate DA concentration was below the detection limit in this group. Treatment with either naloxone or naloxonazine significantly (P<0.01) diminished plasma prolactin concentration, as compared with the controls and blocked the prolactin surge during suckling. In drug-infused sheep, the perfusate salsolinol concentration was below the detection limit but the increased DA and its metabolite 3,4-dihydroxyphenylacetic acid concentrations were observed. In conclusion, the stimulatory action of EOP on prolactin secretion in nursing females is mediated, at least in part, by salsolinol, and the ligands for µ opioid receptor may be the primary factors of this relationship, especially with respect to the suckling-induced prolactin surge.

Effect of LPS on reproductive system at the level of the pituitary of anestrous ewes.

In our research we focused our attention on the effect of the immune stress induced by bacterial endotoxin-lipopolysaccharide (LPS) on the hypothalamic-pituitary-gonadal axis (HPG) at the pituitary level. We examined the effect of intravenous (i.v.) LPS injection on luteinizing hormone (LH) and follicle-stimulating hormone (FSH) release from the anterior pituitary gland (AP) in anestrous ewes. The effect of endotoxin on prolactin and cortisol circulating levels was also determined. We also researched the effect of immune challenge on the previously mentioned pituitary hormones and their receptors genes expression in the AP. Our results demonstrate that i.v. LPS injection decreased the plasma concentration of LH (23%; p < 0.05) and stimulates cortisol (245%; p < 0.05) and prolactin (60%; p < 0.05) release but has no significant effect on the FSH release assayed during 6 h after LPS treatment in comparison with the control levels. The LPS administration affected the genes expression of gonadotropins' ß-subunits, prolactin and their receptors in the AP. Endotoxin injection significantly decreased the LHß and LH receptor (LHR) gene expression (60%, 64%; p < 0.01 respectively), increased the amount of mRNA encoding FSHß, FSH receptor (FSHR) (124%, 0.05; 166%, p < 0.01; respectively), prolactin and prolactin receptor (PRLR) (50%, 47%, p < 0.01; respectively). The presented, results suggest that immune stress is a powerful modulator of the HPG axis at the pituitary level. The changes in LH secretion could be an effect of the processes occurring in the hypothalamus. However, the direct effect of immune mediators, prolactin, cortisol and other components of the hypothalamic pituitary-adrenal (HPA) axis on the activity of gonadotropes has to be considered as well. Those molecules could affect LH synthesis directly through a modulation at all stages of LHß secretion as well as indirectly influencing the GnRHR expression and leading to reduced pituitary responsiveness to GnRH stimulation.

The possible involvement of salsolinol and hypothalamic prolactin in the central regulatory processes in ewes during lactation.

Salsolinol, a dopamine-related compound and prolactin-producing cells were found in the ovine hypothalamus. This study was designed to test the hypothesis that salsolinol, acting from the CNS level, is able to stimulate pituitary prolactin release as well as prolactin mRNA expression in the anterior pituitary cells (AP) and in the mediobasal hypothalamus (MBH) in lactating ewes. The intracerebroventricular infusions of salsolinol in two doses, total of 50 ng or 5 µg, were performed in a series of five 10-min infusions at 20-min intervals. All infusions were made from 12:30 to 15:00 and the pre-infusion period was from 10:00 to 12.30 h. The prolactin concentration in plasma samples, collected every 10 min, was determined by radioimmunoassay; prolactin mRNA expression in AP and MBH tissues was determined by real-time PCR. The obtained results showed that salsolinol infused at the higher dose significantly (p < 0.001) increased plasma prolactin concentration in lactating ewes, when compared with the concentration noted before the infusion and with that in lactating controls. In lactating ewes, the relative levels of prolactin mRNA expression in the AP and MBH were up to twofold and fivefold higher respectively than in non-lactating ewes (p < 0.05). In our experimental design, salsolinol did not significantly affect the ongoing process of prolactin gene expression in these tissues. We conclude that in ewes, salsolinol may be involved, at least, in the process of stimulation of prolactin release during lactation and that hypothalamic prolactin plays an important role in the central mechanisms of adaptation to lactation.

Seasonal effects of central leptin infusion on secretion of melatonin and prolactin and on SOCS-3 gene expression in ewes.

Recent studies have demonstrated photoperiodic changes in leptin sensitivity of seasonal mammals. Herein, we examined the interaction of season (long days (LD) versus short days (SD)) and recombinant ovine leptin (roleptin) on secretion of melatonin and prolactin (PRL) and on mRNA expression of suppressor of cytokine signaling-3 (SOCS-3) in the medial basal hypothalamus (MBH) in sheep. Twenty-four Polish Longwool ewes, surgically fitted with third ventricle (IIIV) cannulas, were utilized in a replicated switchback design involving 12 ewes per season. Within-season and replicate ewes were assigned randomly to one of three treatments (four ewes/treatment) and infused centrally three times at 0, 1 and 2 h beginning at sunset. Treatments were 1) control, Ringer-Locke buffer; 2) L1, roleptin, 0.5 microg/kg BW; and 3) L2, roleptin, 1.0 microg/kg BW. Jugular blood samples were collected at 15-min intervals beginning immediately before the start of infusions and continued for 6 h. At the end of blood sampling, a washout period of at least 3 days elapsed before ewes were re-randomized and treated with one of the treatments described above (four ewes/treatment). Ewes were then killed and brains were collected for MBH processing. Leptin treatments increased (P<0.001) circulating leptin concentrations compared with controls during both seasons in a dose-dependent manner. Overall, mean plasma concentrations of melatonin were greater (P<0.001) during LD than SD. However, leptin treatments increased melatonin concentrations during SD in a dose-dependent manner and decreased it during LD. Similarly, plasma concentrations of PRL were greater (P<0.001) during LD than SD. However, unlike changes in melatonin, circulating PRL decreased (P<0.001) in response to leptin during LD. Semi-quantitative PCR revealed that leptin increased (P<0.001) SOCS-3 expression in the MBH region during LD in a dose-dependent manner. Data provide evidence that secretion of photoperiodic hormones such as melatonin and PRL are inversely regulated by leptin during SD and LD. However, the increase in expression of SOCS-3 in the MBH during LD compared with SD fails to fully explain these effects.

Nutritional value and physiological effects of soya-free diets fed to rats during growth and reproduction.

The objective of the study was to assess the effects of substitution milk and egg for soya products in breeding diets for rats, with concomitant decrease of the dietary protein level and supplementation with amino acids. Soya-containing (S) and two soya-free (NS and NSA) diets were evaluated as protein and energy sources, and their effects on reproductive performance during two cycles, and on the quality of the offspring were assessed. Organ weights were registered in females and blood parameters were determined in males. In the offspring males from S and NS groups, plasma LH, testosterone and prolactin levels were measured on the 22nd and the 60th day of life. The S diet contained more protein of smaller concentration of methionine and cystine and lower biological value than both NS and NSA diets and promoted similar post-weaning growth rate, similar body weight changes of dams during gestation and lactation and slightly lower mating efficiency. Within each reproductive cycle, the number and individual and total body weight of newborn and weanling pups did not differ but in two cycles mean number of neonates per litter and mean litter weight were significantly lower on S than on NSA diet. Plasma concentration of hormones did not differ in 22-day-old offspring males while in the older ones LH and prolactin levels were higher in animals fed on S than on NS diet. It is concluded that replacing soya protein by milk and egg protein with concomitant lowering dietary protein level and amino acid supplementation does not impair the growth rate and tends to improve reproductive performance. Feeding soya-free vs. soya-containing diets differentiates hormonal status of young males.

The intrapituitary endocrine events during maturation and timing of puberty in the female sheep.

The aim of this study was to determine the maturational activity of gonadotroph cells, the site of synthesis, storage and release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in Polish Merino female sheep born after the summer solstice. The actual time of puberty of these lambs was delayed until the following breeding season, when they were 14 months old. Changes were examined in 12 peripubertal (30-, 52-week-old) and pubertal (Days 15 and 17 of the second ovarian cycle) females. Histomorphological and functional changes in the gonadotroph population were assayed with hybridohistochemistry, immunohistochemistry, computer-assisted image analysis and radioimmunoassay. The percentage of the adenohypophyseal area (PAA) occupied by gonadotrophs containing LHbeta-mRNA was higher and the LH plasma concentration and pulse frequency were lower in the 52-week-old sheep in comparison with the 30-week-old sheep (P<0.05). The PAA occupied by immunoreactive (ir)-LHbeta-cells remained stable at the 30th and 52nd weeks of age and then increased at the pubertal follicular phase. The PAA occupied by ir-FSHbeta-cells was higher in the 52-week-old sheep compared with the 30-week-old sheep and then lower at the pubertal follicular phase (P<0.05). The PAA occupied by gonadotrophs containing LHbeta-mRNA or FSHbeta-mRNA was lower at the pubertal follicular phase in comparison with the 52nd week of age (P<0.05). In pubertal sheep, the PAA occupied by gonadotrophs containing LHbeta-mRNA or FSHbeta-mRNA was higher and the PAA occupied by ir-LHbeta or ir-FSHbeta-cells was lower at the preovulatory phase in comparison with the follicular phase of the cycle (P<0.05). In conclusion, the photoperiodic suspension of gonadotroph population's maturational functions has been observed at the level of LH storage and release but not at the level of LH synthesis during the expected time of puberty in female sheep of an aseasonal breed such as Merino. The findings show the heterogeneity in the patterns of LH and FSH post-transcriptional processing during the period of peripubertal/pubertal transition, explained by the different intrapituitary regulation at the level of post-transcriptional synthesis and storage rather, than at the level of release. Altogether, intrapituitary mechanisms of ovine maturation could have the histomorphological feature. Our observations prompt the hypothesis that the female lamb may be able to transduce changes in day length into the appropriate endocrine cues for sexual maturation after attainment by the pituitary gonadotroph population the full peripubertal efficiency, manifested by the sufficient storage of LH.

The role of orexin A in the control of prolactin and growth hormone secretions in sheep--in vitro study.

Orexin A may play a special role in animals' sensitivity to the day length changes such as sheep. The localization of mRNA for prepro-orexin in the ovine hypothalamus was found to correspond to the pattern described in rodents. The results of that research also showed that the expression of the orexin gene depends on the length of a day and is higher during short days. Other study revealed that mRNA for orexin receptors (OxR)1 and OxR2 shows strong expression in the anterior, intermediate and posterior pituitary lobes of the rat. In addition, it was also found that in the anterior pituitary, OxR1 is more strongly expressed than OxR2. These observations indicate that the pituitary gland is capable of receiving the orexin signal. The aim of the study was to determine the interaction of season and orexin A on PRL and GH secretion by pituitary explants in short-term culture. Studies were carried out on pituitaries explants collected from lactating Polish Longwool sheep during the long (LD, May, n=5) and short day (SD, December, n=5). Glands were transected saggitally into halves, with each incubated in 2.5 ml of M-199 for 180-min in medium containing either 0 or 1000 ng/ml of orexin A. Treatment with orexin during LD increased significantly the secretion of PRL (P < 0.01) and GH (P < 0.05), compared to controls. In cultures from glands collected during SD, orexin significantly (P < 0.05) decreased the secretion of both hormones, compared to controls. We conclude that the secretion of PRL and GH from the ovine pituitary gland is negatively responsive to orexin A during SD; whereas orexin may stimulate PRL and GH secretion during LD.

Centrally administered PD 140.548 N-methyl-D-glucamine prevents the autonomic responses to duodenal pain in sheep.

Cholecystokinin (CCK) released in the CNS inhibits the analgesic action of exogenous opioids and may antagonize analgesia resulting from the activation of an endogenous pain inhibitory system. The aim of this study was to analyse the central action of PD 140.548 N-methyl-D-glucamine--a peptide antagonist of a specific peripheral type CCK receptor--on animal behaviour, catecholamines (CA) and cortisol concentration, as well as clinical symptoms of visceral pain induced by duodenal distension (DD). A 5 min distension of the duodenum wall, using a 10 cm long balloon filled with 40 and/or 80 ml of water (DD 40 and/or DD 80) at animal body temperature, produced a significant increase in plasma CA and cortisol levels, an increase in the heart rate, hyperventilation and other clinical symptoms (inhibition of rumen motility, bleating, teeth grinding, prostration, urination, defecation) that may be related to pain, proportionally to the degree of intestinal distension. Intracerebroventricular administration of PD 140.548 at the dose of 1 or/and 2 mg in toto 10 min before applying DD 40 completely blocked the increase in blood plasma cortisol, epinephrine (E), norepinephrine (NE) and dopamine (DA) concentration. It is suggested that the central inhibitory action of CCK antagonist on the cortisol and catecholamine release produced by visceral pain is due to the inhibition of peripheral CCK1 type receptors in the central centrifugal descending pain facilitatory system in sheep perhaps via the hypothalamic-pituitary-adrenal axis.

Pancreatic secretion differs according to the genotype of growing pigs.

The objective of this study was to investigate the secretion of pancreatic enzymes and antibacterial activity in weaned pigs of three pure breeds, Pietrain, Duroc and Polish synthetic line 990, to look for eventual differences related to the genotype. Six male pigs of each breed, about 24 kg mean body weight, were equipped with chronic pancreatic duct catheters and duodenal cannulas to assess pure pancreatic juice, and jugular vein catheters for blood withdrawal. Pancreatic juice was collected before and after the morning feeding. Protein output and enzyme activities revealed two distinct profiles: strong manifestation of the prandial phase in Pietrain and line 990 pigs, and weak manifestation in Duroc. The antibacterial activity did not follow the enzyme kinetics, and it was the strongest in pancreatic juice from Pietrain pigs. Postprandial insulinaemia was reduced in the order of: line 990>Pietrain>Duroc. A slight (not significant) tendency towards a reduction of leptin after feeding in synthetic line 990 corresponded with elevated secretion of pancreatic enzymes and plasma insulin. The presented results suggest that the prandial secretion of pancreatic juice differs according to genotype, and the differences may be in part related to release of insulin.

Intracerebroventricular administration of PD 140.548 N-methyl-D-glucamine attenuates the release of cortisol and catecholamines induced by duodenal distension in the sheep.

The aim of this study was to determine the influence of mechanically induced duodenal distension (DD) and PD 140.548 N-methyl-D-glucamine (a specific peptide antagonist of a CCK1 receptor) premedication on mechanographical reticulo-ruminal activity, animal general behaviour, catecholamines (CA) and the blood plasma cortisol levels, as well as the clinical symptoms of visceral pain induced by DD in sheep. After 24 h fasting, 6 animals, Polish merino sheep were praeanaesthetised by i.m. injection of ketamine (20 mg x kg(-1) b.w.) and anaesthetised with i.v. infusion of pentobarbital (20 mg x kg(-1) b.w.) and a permanent stainless steel cannula (gate cannula) was inserted inside the lateral cerebral ventricle (controlled by cerebrospinal fluid efflux) 10 mm above the bregma and 5 mm laterally from the midline suture using stereotaxic method. Under the same general anaesthesia and analgesia a T-shaped silicon cannula, was inserted into the duodenum (12 cm from pylorus) and a second one was inserted into the dorsal sac of the rumen. During 7 consecutive days after surgery each animal was treated i.m. with procaine penicillin (300000 I.U..kg(-1) b.w.), dihydrostreptomycine (DHS, 10 microg x kg(-1) b.w.), prednisolone acetate 1.2 mg x kg(-1) b.w.) together and i.m. injection of ketamine (20 mg x kg(-1) b.w.), separetely. The influence of PD 140.548 N-methyl-D-glucamine on the unfavourable effects of duodenal distension using a 10 cm long balloon filled with 40 and 80 ml (DD40 and DD80) water at animal body temperature was investigated in this study. Five minutes DD40 and DD80 caused an immediate and compete inhibition of the reticulo-ruminal frequency, a significant increase in plasma CA and cortisol levels, an increase in the heart rate, hyperventilation and other symptoms of pain, proportionally to the degree of intestinal distension. Intracerebroventricular (i.c.v.) administration of PD 140.548 alone at a dose of 0.25, 0.5, 1 or 2 mg in toto did not significantly change the reticulo-ruminal motility, CA and cortisol concentrations, but 10 min after the i.c.v. infusion (or 10 min before DD) at a dose 1 and 2 mg in toto , it completely blocked the increase of blood plasma cortisol, epinephrine (E), norepinephrine (NE) and dopamine (DA) concentrations for 20 min. In the some time it prevented the reticulo-ruminal atony provocked by DD. It is concluded that PD 140.548 N-methyl-D-glucamine--an antagonist of the central CCK1 receptor can be an effective analgesic agent in duodenal pain. This action is due to the inhibition of peripheral CCK1 type receptor in the central descending nerve pathway, facilitating pain transmission in sheep perhaps in the hypothalamic-pituitary-adrenal axis.

The effects of prolonged, intracerebroventricular prolactin treatment on luteinizing hormone secretion, catecholaminergic activity and estrous behavior in ewes.

Searching for the role of prolactin (PRL) in controlling gonadotropic axis activity in sheep, we studied the effects of prolonged, intracerebroventricular (i.c.v.) PRL infusion on luteinizing hormone (LH) secretion and catecholaminergic activity in the hypothalamic infundibular nuclei/median eminence (IN/ME) in sexually active ewes during the periovulatory period. Three groups of animals received the following treatments: 1). i.c.v. infusion of PRL at a dose of 200 microg/day (Lower dose, n = 5); 2). i.c.v. infusion of PRL at a dose of 400 microg/day (Higher dose, n = 6), and 3). i.c.v. infusion of the vehicle (control, n = 5). Each dose of PRL was infused in a pulsatile manner, 4 x 50 microg/h and 4 x 100 microg/h, in 30-min intervals, respectively, during four consecutive days before oncoming ovulation. The estrous behavior of ewes following treatments was also monitored as a determinant of the GnRH/LH surge. Two series of blood collections were made in every ewe, the first on the day preceding the infusion (day 0 of the experiment), the second on the day after the infusion (day 5 of the experiment). In addition, on day 5 of the experiment, perfusions of the IN/ME were made by the push-pull method, either in control or lower dose-treated animals. It was shown that a significant (p < 0.01, p < 0.001) increase in tonic LH secretion during the periovulatory period remained in ewes irrespective of the kind of infusion. No statistical differences were found in LH pulse frequency, amplitude, or in the length of the pulse when compared with values from day 0 and 5 of the experiment within each group. A significant (p < 0.001) increase in IN/ME perfusate concentrations of dopamine and noradrenaline metabolites was noted in PRL-treated ewes in comparison with those in the control. The estrous behavior in PRL-treated animals was delayed for a few days, 3.80 +/- 0.80 days at the lower dose (p < 0.01), and 2.83 +/- 0.98 days at the higher dose (p < 0.05) in comparison with the control, 0.20 +/- 0.20 days. These data indicate that maintenance of an increased PRL concentration within the central nervous system (CNS) for a few days before oncoming ovulation has no inhibitory effect on tonic LH secretion. A few-day shift of the preovulatory GnRH/LH surge, as determined by estrous behavior, might, however, be a consequence of the PRL-induced increase in catecholamine turnover in the IN/ME.