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 β-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.     

Leptin infusion during the early luteal phase in ewes does not affect progesterone production

Infusion of leptin during the ovine follicular phase has been shown to increase progesterone secretion during the subsequent luteal phase. In this study, we have assessed the effects of infusing leptin during the early luteal phase. Infusion of leptin (2.5 microg/h) into the ovarian artery of ewes with ovarian autotransplants (n=5) on day 3 of the luteal phase for 12h did not affect progesterone estradiol or LH concentrations compared to control ewes (n=5). These results suggest no direct effect of leptin on ovarian function at this stage of the estrous cycle.     

Seasonal changes in the interactions among leptin, ghrelin, and orexin in sheep

The adaptation of the physiology of an animal to changing conditions of light and food availability is evident at the behavioral and hormonal levels. Melatonin, leptin, ghrelin, and orexin, which exhibit rhythmic secretion profiles under ad libitum feeding conditions, are sensitive to changes in daylength, forming a tight web of interrelationships in the regulation of energy balance. The aim of this study was to determine the effects of central injections of leptin, ghrelin, and orexin on the reciprocal interactions among these hormones and the influence of photoperiod on these responses. Twenty-four ovariectomized and estradiol-implanted ewes were used in a replicated switchback design. The ewes were assigned randomly to 1 of 6 treatment groups, and the treatments were infused into their third ventricles 3 times at 0, 1, and 2 h, with 0 h being at dusk. The treatments were as follows: 1) control, Ringer-Locke buffer; 2) leptin, 0.5 μg/kg BW; 3) ghrelin, 2.5 μg/kg BW; 4) orexin B, 0.3 μg/kg BW; 5) leptin antagonist, 50 μg/kg BW, then ghrelin, 2.5 μg/kg BW; and 6) leptin antagonist, 50 μg/kg BW, then orexin B, 0.3 μg/kg BW. Blood samples (5 mL) were collected at 15-min intervals for 6 h. The administration of leptin increased (P < 0.05) plasma concentrations of melatonin during short-day (ShD) photoperiods and decreased (P < 0.05) them during long-day (LD) photoperiods, whereas ghrelin decreased (P < 0.05) melatonin concentrations during ShD photoperiod, and orexin had no effect (P > 0.1). Leptin attenuated (P < 0.05) ghrelin concentrations relative to the concentration in controls during ShD. The plasma concentrations of orexin were reduced (P < 0.05) after leptin infusions during LD and ShD photoperiods; however, ghrelin had the opposite effect (P < 0.05) on orexin concentration. Orexin increased (P < 0.05) ghrelin concentrations during LD. Ghrelin and orexin concentrations were increased (P < 0.05) after leptin antagonist infusions. Our data provide evidence that the secretion of leptin, ghrelin, and orexin are seasonally dependent, with relationships that are subject to photoperiodic regulation, and that leptin is an important factor that regulates ghrelin and orexin releases in sheep.     

Effects of human chorionic gonadotropin and cyclic adenosine monophosphate on progesterone secretion by the ovine placenta

Progesterone production by the ovine placenta was investigated between d 80 and 115 of gestation. Serum progesterone concentrations in ewes ovariectomized (ovx) on d 75 of gestation were measured throughout the remainder of gestation, and after the ewes were injected with human chorionic gonadotropin (hCG) or saline on d 80 or 115. In addition, cotyledonary tissue was collected from intact ewes sacrificed on d 80 or 115 and progesterone accumulation was determined during 2 h incubation with or without pregnenolone supplementation and in the presence or absence of hCG or dibutyryl cyclic adenosine monophosphate (dbcAMP). Serum concentrations of progesterone in ovx ewes increased from 3.5 +/- .4 ng/ml on d 80 to 16.4 +/- 2.1 ng/ml on d 115 (P less than .05). That increase was coincident with a 1.5-to 4.5-fold increase in progesterone output by placental tissue in vitro. Addition of pregnenolone enhanced progesterone accumulation in all tissue incubations. Addition of dbcAMP increased progesterone accumulation in the incubation medium only when supplemented with pregnenolone. Human chorionic gonadotropin did not increase placental progesterone secretion in vivo or in vitro. The results confirm the enhanced secretion of progesterone by the ovine placenta between d 80 and 115 of gestation and indicate that the increase results primarily from increased secretory capability per unit of placenta. The tropic mechanism controlling the placental secretion of progesterone remains unclear, but the mechanism may involve elevation of intracellular cAMP.     

Dependence of the lactation duration and efficiency on the season of lambing in relation to the prolactin and melatonin secretion in ewes

Changes in day length and the related secretion of melatonin and prolactin are of particular significance in sheep, as they determine reproductive processes, the last stage of which is lactation. The aim of the present study was to examine the dependence of the lactation duration and efficiency on the season of lambing (January—Group I/June—Group II) in relation to the hormonal status of milking ewes (Polish Longwool). The possibility of artificial extension of the milking period in late-lambing ewes by application of prolonged day length, 16 h of light–8 h of darkness (16L:8D), was introduced additionally (Group III). Measurements of plasma levels of prolactin and melatonin were used as parameters of season-dependent hormonal regulation of milk production in this seasonally breeding species. Although during the first 28 days of lactation sheep of all groups produced similar amounts of milk (based on the weight gains of the lambs), our results clearly showed that during the period of machine milking (from weaning at 56 day to the dry period) the level of milk production in Group I was almost twice that noted in Group II. Thus, the highest milk yield was obtained from sheep during the period when the days became longer, i.e. from March to May, which was accompanied by an increase in prolactin secretion. In both groups, melatonin secretion was found to increase while milk performance parameters and prolactin secretion decreased during the shortening photoperiod. The sustained decreases in milk production and prolactin secretion were also observed during the autumn months in sheep from Group III, despite the low level of melatonin. The present study showed that time of lambing and related photoperiod length could significantly affect the milk production of sheep that have a clear season of sexual activity. Maintenance of June-lambing sheep under the 16L:8D regime failed to extend the high prolactin level and lactation considerably into the autumn months. The spontaneous decrease in prolactin secretion observed under the long artificial photoperiod indicated for a development of refractoriness, which could also limit the lactation period.     

Possible role of histamine in the regulation of secretion of luteinizing hormone in the ewe

Concentrations of histamine were quantified by an enzymatic isotopic assay in different regions of the brain and pituitary gland of gonadal-intact and chronically ovariectomized ewes during the anestrous season. Sera concentrations of LH were confirmed to be elevated in ovariectomized compared with intact animals immediately before tissues were obtained. Areas of the brain that were examined included cerebral cortex, thalamus, pineal gland, hypothalamus (rostral, medial basal, median eminence), midbrain, cerebellum and brain stem. Concentrations of histamine were greatest within the thalamus, pineal gland, medial basal hypothalamus and median eminence. Histamine within the medial basal hypothalamus was greater (P less than .05) in ovariectomized than in ovarian-intact animals. Further experiments were designed to determine the effect of antihistaminic drugs on secretion of LH. Ovariectomized ewes were treated every 6 h (i.m.) for 24 h with diphenhydramine (an antagonist of the H1-receptor for histamine), cimetidine (an H2-receptor antagonist), a combination of the drugs, or vehicle. Twelve hours after initiation of treatments, animals were injected with estradiol. Diphenhydramine depressed (P less than .01) basal serum concentrations of LH and the positive feedback effect of estradiol on serum concentrations of LH. Cimetidine did not influence the pattern of secretion of LH. Diphenhydramine did not alter LHRH-induced release of LH in ovariectomized ewes or basal serum concentrations of LH in ovarian-intact anestrous ewes. We suggest that histamine acts at the level of the central nervous system through an H1-receptor mechanism to control secretion of LH in female sheep.     

Effects of Melatonin Implants During Non‐Breeding Season on Sperm Motility and Reproductive Parameters in Rasa Aragonesa Rams

The effect of melatonin implants administered during non‐breeding season in Rasa Aragonesa rams on sperm motility parameters and other reproductive traits was assessed. In a first experiment, two Rasa Aragonesa rams were implanted (with melatonin group M), remaining other two males as control group (C). Semen of each group was collected from 1 May to 23 June, twice or three times a week, and motility parameters were assessed using a computer‐assisted sperm analysis system. Melatonin increased the percentage of progressive motile spermatozoa, particularly during 46–75 days after melatonin implantation (p < 0.01). In experiment 2, M and C in vitro fertilization ability had been determined by zona‐pellucida binding assays, using spermatozoa from experiment 1, obtained 60–70 days after melatonin was implanted. A significantly higher number of spermatozoa attached per oocyte was observed in frozen‐thawed immature ovine oocytes incubated with sperm from M animals than in those incubated with sperm from the C group (p < 0.01). Finally, a field assay (experiment 3) was performed. In this case, five Rasa Aragonesa rams were implanted with melatonin and three remained as control group. Sperm doses from those animals were used for artificial insemination of 2608 Rasa Aragonesa ewes from 39 different farms at non‐breeding season. Fertility, litter size and fecundity were studied. Semen from melatonin implanted rams seemed to increase both fertility and fecundity in ewes inseminated with spermatozoa obtained 46–60 days after implantation (p < 0.1). Thus, melatonin treatment in rams during non‐breeding season modifies sperm motility parameters and seems to improve the fertilization parameters obtained.     

Chronic administration of recombinant ovine leptin in growing beef heifers: effects on secretion of LH, metabolic hormones, and timing of puberty

Serum concentrations of leptin increase linearly from approximately 16 wk before until the week of pubertal ovulation in beef heifers. To test the hypothesis that exogenous leptin can hasten the onset of puberty in heifers, we examined the effects of chronic administration of recombinant ovine leptin (oleptin) on timing of puberty, pulsatile and GnRH-mediated release of LH, and plasma concentrations of GH, IGF-I, and insulin. Fourteen fall-born, prepubertal heifers (Brahman x Hereford, 12 to 13 mo; 304.7+/-4.12 kg) were used. Heifers were stratified by age and BW and assigned randomly to one of two groups (seven animals per group): 1) Control; heifers received s.c. injections of saline twice daily (0700 and 1900) for 40 d; and 2) Leptin; heifers received s.c. injections of oleptin (19.2 microg/kg) twice daily at 0700 and 1900 for 40 d. Blood samples were collected at 10-min intervals for 5 h on. d 0, 5, 10, 20, 30, and 40, and twice daily, just before each treatment injection, throughout the study. On d 41, heifers received i.v. injections of GnRH at 0 (0.0011 microg/kg) and 90 min (0.22 microg/kg), with additional sampling for 5.5 h to examine releasable pools of LH. Diets promoted a gain of 0.32+/-0.09 kg/d, which did not differ between groups. Plasma concentrations of leptin increased markedly in leptin-treated heifers and were greater (P < 0.001) than controls throughout (27.8+/-0.8 vs. 4.9+/-0.12 ng/mL). None of the heifers reached puberty during the experiment, but did so within 45 d of its termination. Mean concentrations of plasma LH, GH, IGF-I, and insulin were not affected by treatment, nor was there an overall effect on the frequency of LH pulses. However, a treatment x day interaction (P = 0.02) revealed that the frequency of LH pulses (pulses/ 5 h) was greater (P = 0.03) in controls (3.6+/-0.36) than in leptin-treated heifers (1.7+/- 0.28) on d 10. Characteristics of GnRH-induced release of LH were not affected by treatment. In summary, chronically administered leptin failed to induce puberty or alter endocrine characteristics in beef heifers nearing the time of expected puberty.     

Serum concentrations of melatonin in prepubertal gilts exposed to either constant or stepwise biweekly alteration in scotophase

Melatonin (MEL), a hormone known to mediate photoperiodic cues, is secreted from the pineal gland in a circadian fashion in numerous species. The transduction of photoperiodic information into the secretion of MEL, however, remains controversial in the pig. To determine whether domestic pigs have a nocturnal increase in serum melatonin when exposed to equatorial photoperiods only, 24 prepubertal gilts (38.7 ± 0.7 kg; 104.5 ± 0.8 d) and 12 mature ewes, serving as positive controls, were assigned randomly to one of two environmentally regulated rooms. The light (L):dark (D) schedule in one room remained constant (10 L:14 D), while the other room scotophase (darkness duration) was decreased by 1 hr every 2 wk (Experiment 1). After a 2-wk acclimation to each new schedule, 6 ewes and 6 gilts in each room were bled by venipuncture at 2-hr intervals for 22 hr. Experiment 2 was conducted as described for Experiment 1, except that the LD schedule in one room remained constant (15L : 9D) while length of scotophase in the other room was increased by 1 hr every 2 wk. In gilts that were exposed to constant 10L:14D, scotophase MEL in serum averaged 103 ± 13 pg/ml as compared with 57 ± 13 pg/ml in the photophase. Using each gilt's initial photophase value as a statistical covariate, scotophase MEL in the constant 10L:14D schedule was higher (P < 0.001) than photophase MEL. A similar analysis of MEL in gilts exposed to stepwise biweekly decreases in scotophase revealed a scotophase elevation (P < 0.05) in only certain LD schedules (i.e., 12L: 12D and 13L:11D) but the same trend was present throughout all LD schedules. Subjective examination of individual gilt profiles revealed that 56% of gilts had a nocturnal increase in serum MEL in Experiment 1. However, only 10% of the MEL profiles were closely coupled to the environmental LD periods. Overall, mean serum MEL was slightly elevated during scotophase in gilts, but the nocturnal elevation of MEL in gilts was of lesser magnitude and more variable than in ewes. Data from these two experiments suggest that the domestic pig has an inherently weak nocturnal elevation in serum MEL, and the ability to detect these rises is dampened by considerable pig-to-pig variability.     

The Effect of Melatonin on the Secretion of Progesterone in Sheep and on the Development of Ovine Embryos In Vitro

Two experiments were carried out in order to determine whether melatonin can improve secretion of progesterone in vivo, and its effect on embryonic development in vitro. In the first experiment, blood samples were collected from 5 ewes at 15 min intervals for 2 h at 7 and 10 days after withdrawal of progestagen pessaries. The first hour constituted a control period, which ended with an intravenous administration of 3 g/(kg bw)^0.75 melatonin. All the ewes on day 7 and three of the ewes on day 10 showed a progesterone response to melatonin challenge, defined as an increase in the plasma progesterone concentration in at least two consecutive samples during the post-treatment period above the mean+2SD of the values in the pre-treatment period. A paired t-test revealed a significant effect of melatonin on the overall plasma progesterone concentrations before and after the challenge, both on day 7 (pre, 0.61±0.11; post, 0.73±0.13 ng/ml; p<0.01) and day 10 (pre, 1.16±0.19; post, 1.30±0.20 ng/ml; p<0.05). Ninety-one thawed embryos (46 morulae and 45 blastocysts) were used in the second experiment, being cultured with or without 1 g/ml melatonin. If the embryos were blastocysts when the culture started, melatonin increased the percentage that had hatched after 24 h of culture (p<0.01), and there was a lower percentage of degenerated embryos at the end of the incubation period (p<0.05). It may be concluded that melatonin treatment in sheep can increase both fertility and prolificacy by improving luteal function and embryonic survival.     

Changes in the level of endogenous leptin, FSH, 17beta-oestradiol and metabolites during lupin-induced increase in ovulation rate in ewes

The aim of the study was to determine the changes in the plasma concentration of leptin during lupin feeding-induced increase in the ovulation rate (OR) in ewes. Additionally, alterations in the plasma level of glycogenic amino acids and glucose (as the factors influencing leptin secretion) and the levels of follicle-stimulating hormone (FSH) and 17beta-oestradiol (E-2) (as the hormones regulated by leptin and engaged in recruitment, selection and development of ovulatory follicles) were analysed. Ninety-six female Polish Lowland Sheep were used. All ewes were cyclic and synchronized with PGF2alpha. The ewes were divided into two groups: control (n = 48), fed only with hay, and experimental (n = 48), received additionally lupin (Lupinus angustifolius) grain as a high-protein and a high-energy supplement. They were given lupin from the second to 13th day of the oestrous cycle at increasing doses (150-750 g/day per ewe). On the 11th day of cycle blood samples for analysis of hormones, amino acids and total glucose concentration, were collected from the jugular vein. OR was determined by laparoscopy of ovaries on the sixth day of the following oestrous cycle. Mean OR of ewes supplemented with lupin grain (1.687 +/- 0.463) was 30.67% higher than that of control (1.291 +/- 0.454). In spite of the unchanged body mass, a significant increase (P < or = 0.05) in mean concentration of plasma leptin in the experimental ewes [2.17 +/- 0.15 ng/ml human equivalent (HE)] was found in comparison with control (1.42 +/- 0.12 ng/ml HE). A significantly (P < or = 0.05) higher plasma FSH level in the ewes fed lupin (105.21 +/- 5.87 ng/ml) compared with those fed hay (67.88 +/- 6.03 ng/ml) was also found. However, plasma level of E-2 decreased after lupin feeding. Moreover, in the ewes fed lupin the plasma concentrations of glucose and nine glycogenic amino acids (Gly, Ala, Val, Met, Leu, Ile, Tyr, Phe and Arg) were increased. It can be concluded that lupin feeding exerts the stimulatory effect on the OR in Polish Lowland Sheep. The increase in OR is connected with significantly higher plasma leptin level and coincident with rise in FSH, glycogenic amino acids and glucose concentration. In contrast, the level of plasma E-2 was significantly decreased in lupin-fed ewes.     

Effect of cerebroventricular infusion of insulin and (or) glucose on hypothalamic expression of leptin receptor and pituitary secretion of LH in diet-restricted ewes

The objective was to determine the effect of central infusion of insulin and (or) glucose on hypothalamic expression of leptin receptor and pituitary secretion of LH in the ewe. Twenty-two ovariectomized ewes (32 wk of age) were fitted with two lateral cerebroventricular (LCV) cannulae and fed 33% of NRC requirements for 8 wk. Ewes (n> or =5/group) were then infused, via LCV cannulae, with artificial cerebrospinal fluid (aCSF) or aCSF containing physiological concentrations of insulin (INS), glucose (GLU), or INS + GLU; the mass of each increasing linearly from Day 0 (mass = 0 units/h) to Day 8 (mass of INS = 80 mIU/hr and GLU = 10 mg/hr). Jugular serum was collected every 12 min for 4 hr on Days 0, 2, and 4. Ewes treated with INS or INS + GLU had greater (P<0.06) mean concentrations of LH than aCSF treated ewes on Day 2 (13.8+/-1.8 and 12.5+/-1.3 > 8.0+/-3.3 ng/ml). Furthermore, on Day 4, concentrations of LH in INS treated ewes exceeded that (P<0.07) of aCSF treated ewes (14.8+/-2.0 > 7.4+/-3.0 ng/ml). Expression of NPY mRNA did not differ between treatments (P = 0.87). Leptin receptor mRNA expression was dramatically reduced (P<0.0002) in INS+GLU versus aCSF treated ewes. These data provide evidence to suggest that insulin may be an important component of hypothalamic mechanisms regulating secretion of LH and expression of leptin receptors in undernourished ruminants.     

Fasting-induced changes in ovulation rate, plasma leptin, gonadotropins, GH, IGF-I and insulin concentrations during oestrus in ewes

The aim of this experiment was to study the changes in the hormonal status and ovulation rate (OR) evoked by starvation during the follicular phase of the oestrous cycle in ewes. To achieve this goal, 12 female crossbreed sheep were synchronized and then half of them were fasted from the 12th to the 16th day of the oestrous cycle. On the 16th day, analysis of hormones and insulin-like growth factor-I (IGF-I) were performed in 10-min intervals. Then, on the 6th day of the following oestrous cycle, the OR in all ewes was determined by laparoscopy. Fasting reduced significantly (P < 0.05) the OR in ewes (1.25 +/- 0.50) in comparison with control (1.75 +/- 0.50). The drop in the OR was coincident with a significant (P < 0.001) decrease in the plasma concentration and pulse amplitude of leptin (0.29 +/- 0.08 ng/ml versus control 0.53 +/- 0.14 ng/ml), the plasma level of luteinizing hormone (LH) (0.19 +/- 0.06 IU/l versus 0.25 +/- 0.09 IU/l in control; P < 0.05) and the mean frequency of LH pulses (2.0/h versus 2.5/h in control). Fasting resulted also in a significant (P < 0.05) decrease in the plasma concentration and pulse amplitude of follicle stimulating hormone (FSH) in comparison with the control. Simultaneously, a significant (P < 0.001) drop in the IGF-I concentration in the fasted ewes (4.78 +/- 0.91 ng/ml) was found in comparison with control (7.63 +/- 1.85 ng/ml). Also the level of insulin were significantly (P < 0.001) lower in the fasted (178.99 +/- 39.08 pM/l respectively) than in the control sheep (302.66 +/- 49.01 pM/l respectively). Meanwhile, a double increase in the growth hormone (GH) pulses frequency and an augmentation in its plasma concentrations as a result of starvation was found. The obtained results shows that the acute fasting exerts an inhibitory effect on the ovulation rate in ewes coincident with suppression in leptin, FSH and LH secretion and changes in signalization mediated by GH.     

Effects of exogenous estradiol and progesterone on plasma concentrations of leptin in ewes in non-breeding season

Estradiol and progesterone may play a role in controlling leptin secretion by utilizing their receptors in adipocytes and the genomic mechanisms of the leptin gene. This study was conducted to evaluate the effects of exogenous sex steroids on the blood leptin concentrations in ewes in the non-breeding season. Multiparous ewes were fed to maintenance level for their live weights. Blood samples were collected at 12-h intervals from Days -3 to -1 to determine the basal leptin levels (pre-injection period). From Day 0 to Day 5 (injection period), blood sampling continued at 12-h intervals, and the ewes were injected intramuscularly at 24-h intervals with oil, 50 mg progesterone in oil, 1 mg of estradiol in oil, or both steroids in oil. Leptin was measured using a sensitive and specific radioimmunoassay based on recombinant bovine leptin. Overall, plasma concentrations of leptin were not affected by any of the steroid treatments, and there were no differences in the value of leptin between the pre-injection and injection periods among the 4 groups. Therefore, the exogenous estrogen and progesterone used in this study do not have a strong effect on the blood leptin concentrations of ewes in the non-breeding season.     

Control of ovarian follicles activity in the ewe.

During the ovine estrous cycles, three waves of follicular growth, closely associated with the FSH secretion pattern, were observed. The parameters of these follicular waves and the ability of follicles to produce steroids in vitro were studied in various conditions. In vivo, the follicular events were similar between the breeding season and the anestrus, except for the lack of ovulation; but at the end of the breeding season and in anestrus, the follicles lose a big part of their aromatization ability. In ewes carrying the Booroola fecundity gene or Cambridge fecundity gene, the reduction in follicular atresia seems to be one of the main follicular features implicated in the control of high ovulation rate. In vitro, the most relevant difference is an early acquisition of estrogen production ability of small follicles in Booroola fecundity gene barring ewes. Fluoro-gestone-acetate (FGA) pessaries reduced the number of growing follicles; despite this effect disappearing after the sponge withdrawal, the ovulation rate is significantly reduced. But an equine chorionic gonadotrophin (eCG) treatment restores the ovulation rate (OR) by reducing the atresia rate of pre-ovulatory follicles. In similar conditions, a pretreatment of the ewes with melatonin again reduced the atresia rate of large follicles and resulted in an increased ovulation rate. In vitro, FGA blocked aromatization ability, and melatonin inhibited both androstenedione and estradiol production, but a further treatment with eCG partly restores the steroid secretion. Immunization against androstenedione leads to a higher OR, owning to a reduced atresia of large follicles. Daily growth hormone injections for a hole cycle resulted in an increased follicular population and ovulation rate, while FSH plasma levels decreased and the follicle sensitivity to gonadotrophins was reduced.     

Luteinizing hormone and growth hormone secretion in ewes infused intracerebroventricularly with neuropeptide Y

Neuropeptide Y (NPY) provides an important hypothalamic link between nutritional status and neuroendocrine mechanisms regulating growth and reproduction. The objective of the following series of experiments was to determine the effects of single or continuous administration of NPY on secretion of luteinizing hormone (LH) and (or) growth hormone (GH). In experiment 1, four ovariectomized (OVX) ewes and four OVX + estrogen-treated ewes each received, in a 4 x 4 Latin Square arrangement of treatments, a single injection of 0, 0.5, 5, or 50 microg NPY via an intracerebroventricular (i.c.v.) cannulae to determine the effects on secretion of GH. NPY significantly elevated serum GH at the 50 microg dose regardless of estrogen exposure (P = 0.003). In experiment 2, eight OVX ewes were infused i.c.v. with NPY or saline (n = 4/trmt) continuously for 20 h in a linearly increasing dose, ending at 50 microg/h NPY. Blood samples were collected via jugular cannulae every 10 min during hour -4-0 (interval 1, pre-treatment), hour 6-10 (interval 2) and hour 16-20 (interval 3) relative to the initiation of infusion (0 h). Mean LH and LH pulse frequency were lower in NPY- versus saline-infused ewes during intervals 2 and 3 (P < 0.01), but NPY had no discernable effect on serum GH (P > 0.10). In experiment 3, four OVX ewes were continuously infused with NPY as in experiment 2, except that the maximum 50 microg/h dose was achieved after only 10 h of infusion. Blood samples were collected every 10 min, beginning 4 h before and continuing until 4h after the NPY infusion. Mean serum LH changed significantly over time (P = 0.0001), decreasing below pre-treatment levels by hour 3 of NPY infusion (P < 0.01), and returning to pre-treatment concentrations following the end of infusion (P > 0.15). Serum GH also changed significantly over time (P < 0.001). Mean GH levels tended to be greater than pre-treatment levels by hour 2 of infusion (P < 0.08), but thereafter returned to basal levels. Serum GH also increased following the end of NPY infusion (P < 0.03). From these data we conclude that NPY exerts a persistent inhibitory effect on secretion of LH, and may stimulate the secretion of GH during the initiation and cessation of infusion of NPY. These observations support a role for NPY in mediating the effects of undernutrition on both LH and GH, and also provide evidence for potential mechanisms by which leptin, acting through NPY, may stimulate the secretion of GH.     

Humoral and cellular factors affecting the neutrophil response of the locally immunised mammary gland to staphylococcal infection

Locally immunising the non-lactating ovine mammary gland by infusing killed Staphylococcus aureus enhances neutrophil accumulation in mammary secretion during subsequent staphylococcal infection. Immunological factors influencing this increased neutrophil response were studied in the present experiments. Glands locally immunised with killed Brucella abortus supported a greater neutrophil response to staphylococcal infection than did glands immunised with killed S. aureus. An enhanced neutrophil response to staphylococcal infection was recorded in 3 of 7 ewes locally immunised with zymosan. Passive immunisation by intramammary infusion of secretions from immunised glands conferred an enhanced neutrophil response during staphylococcal infection. Absence of haemolytic complement in secretions of immunised glands suggested complement was not implicated in the response. Secretions of immunised glands contained elevated concentrations of mononuclear cells. When exudates rich in mononuclear cells were established by infusion of endotoxin into non-immunised glands there was no increase in the neutrophil response to subsequent staphylococcal infection. However, when the mononuclear cell concentration was elevated by intramammary infusion of staphylococcal antigens in systemically immunised ewes there was an increase in the neutrophil response to subsequent infection. Thus humoral and cellular characteristics of the locally immunised mammary gland influence the kinetics of the neutrophil influx during staphylococcal infection.     

Post‐glucose load changes of plasma key metabolite and insulin concentrations during pregnancy and lactation in ewes with different susceptibility to pregnancy toxaemia

Insulin resistance during late gestation may act as a predisposing factor of ovine pregnancy toxaemia (OPT). To evaluate the insulin action on energy metabolism in ewes with different susceptibilities to OPT, intravenous glucose tolerance tests (1 mmol glucose/kg body weight) were performed in 5.6 ± 0.7 year old, slightly underfed German Blackheaded Mutton ewes [high‐risk (HR) ewes] and 2.5 year old, overnourished Finnish Landrace ewes [low‐risk (LR) ewes] during mid and late pregnancy, during early lactation and during the dry period. Plasma samples were analysed for glucose, insulin, non‐esterified fatty acids (NEFA) and β‐hydroxybutyrate (β‐HB). The glucose elimination rate and the glucose‐stimulated first‐phase insulin secretion were significantly (p < 0.05) lower in the HR, in relation to the LR group combining the data of all gestational stages. The basal rate of lipolysis was significantly increased in the HR ewes during late pregnancy, but the NEFA clearance after the glucose load was similar in both groups during all reproductive stages. Plasma β‐HB concentrations decreased only in the LR ewes after the glucose load during late pregnancy. Results indicate an insulin resistance in the HR ewes regarding the glucose utilization and the ketone body formation during late pregnancy. The insulin resistance in the HR ewes may represent one predisposing factor responsible for the susceptibility to OPT. Further scientific work is necessary to elucidate whether this insulin resistance was due to breed, age or nutritional state.     

Nocturnal melatonin and prolactin plasma concentrations in sheep selected for fertility in autumn lambing'.

Ewes selected for fertility in autumn lambing were used to evaluate correlated responses in nocturnal hormone levels. Four jugular blood samples were obtained during nighttime in August from each of 113 selected and 69 control ewes. Melatonin levels were lower for selected ewes (143 +/- 14 pg/mL) than for control ewes (184 +/- 13 pg/mL), and melatonin levels decreased with increases in estimated breeding values (EBV) for fertility (-2.23 +/- 0.79 pg mL(-1) x %(-1)). Prolactin levels were higher for selected ewes (90 +/- 7 ng/mL) than for control ewes (52 +/- 7 ng/mL), but significant line x ewe age interaction was also observed, with smaller differences in prolactin levels in 2-yr-old and older ewes (74 +/- 7 vs. 56 +/- 9 ng/mL for select and control ewes, respectively; P < 0.20 before and P = 0.05 after logarithmic transformation). Prolactin levels increased with both fertility EBV (1.23 +/- 0.53 ng mL(-1) x %(-1)) and maternal birth weight EBV (9.0 +/- 4.0 ng x ml(-1) kg(-1)). Heritability estimates were 0.43 (P < 0.02) for melatonin levels and 0.11 (P > 0.25) for prolactin levels. Thus, we conclude that selection for fertility in autumn lambing has affected patterns of melatonin and prolactin secretion during the dark phase.     

Renal function studies in normal and toxemic pregnant sheep

Renal blood and plasma flow, glomerular filtration rate (GFR) and maximal tubular transport of PAH (TmPAH) were measured in nonpregnant and twin-pregnant sheep. Twin-pregnant animals were studied during normal pregnancy as well as during ovine pregnancy toxemia artificially produced by starvation. All animals were surgically prepared with aortic, post caval and renal vein cannulas at least one week prior to experimentation. Total renal blood and plasma flow was found to be elevated during pregnancy, but if expressed on the basis of body weight no changes were noted. Starvation and the resultant development of hypoglycemia and hyperketonemia caused a 25-30% decline in renal blood and plasma flow. GFR in pregnant fed sheep (193 ml/min or 2.7 ml/kg.min) was significantly higher (P less than .001) than that of nonpregnant ewes (118 or 2.3 ml/kg min). During ovine pregnancy toxemia the GFR was significantly (P less than .001) diminished (142 ml/min or 2.0 ml/kg min). TmPAH also was significantly higher (179 mg/min or 2.5 mg/kg min) in pregnant animals when compared to nonpregnant ewes (98 mg/min or 1.9 mg/kg min.), but starvation had no effect on Tm PAH in pregnant sheep. It thus appears that a functional renal hypertrophy occurs during pregnancy which is similar to that which follows unilateral nephrectomy or renal disease. During ovine pregnancy toxemia the diminution of renal function probably results from the metabolic derangements and is thus not comparable to human preeclampsia.