Leptin prevents fasting-mediated reductions in pulsatile secretion of luteinizing hormone and enhances its gonadotropin-releasing hormone-mediated release in heifers

We tested the hypothesis that leptin could prevent fasting-mediated reductions in pulsatile secretion and modify GnRH-mediated release of LH in heifers approaching puberty. Thirteen crossbred, prepubertal heifers (13.5-16 mo; 280-350 kg) exhibiting frequencies of pulses of LH between 0.67 and 1 pulse/h, were assigned randomly to two groups: 1). control (n = 6), fasted for 72 h with s.c. injections of saline at 12-h intervals, and 2). leptin (n = 7), fasted for 72 h with s.c. injections of oleptin (19.2 microg/kg) at 12-h intervals. Blood samples were collected intensively for 6 h on Days 0 and 3. This was followed on Day 3 with sequential administration of physiological (0.0011 microg/kg, i.v.) and pharmacological (0.22 microg/kg, i.v.) doses of GnRH and additional blood sampling. Leptin treatment increased (P = 0.0003) plasma concentrations of leptin 5-6-fold compared to controls. Fasting caused a marked decline (P = 0.01) between Days 0 and 3 in the frequency of LH pulses in controls; however, this effect was prevented in the leptin group, with pulse frequency increasing (P < 0.008) from Day 0 to 3. Leptin treatment increased GnRH-induced release of LH at both low (P = 0.04) and high (P = 0.02) doses. Plasma insulin and insulin-like growth factor-1 were reduced by fasting and unaffected by leptin. Leptin increased mean concentrations of growth hormone. Results indicate, for the first time, that exogenous leptin can prevent fasting-mediated reductions in the frequency of LH pulses and modify GnRH-mediated release of LH in intact, prepubertal heifers.     

Divergent effects of leptin on luteinizing hormone and insulin secretion are dose dependent

We have shown recently that fasting permits leptin to modulate both luteinizing hormone (LH) and insulin secretion in cows. In rodents, leptin causes divergent effects on LH and insulin release that are dose dependent. To test the hypothesis that leptin effects on LH and insulin secretion in fasted cows are dose related, we examined the effects of various doses of recombinant ovine leptin (oleptin) in mature cows. Twenty ovariectomized beef cows, each bearing an estradiol implant to maintain basal estradiol concentrations, were used. All cows were fasted for 60 hr with free access to water and were assigned randomly to one of four groups (n = 5/group): 1) saline control; 2) leptin, 0.2 microg/kg; 3) leptin, 2.0 microg/kg; and 4) leptin, 20 microg/kg body wt. Blood samples were collected at 10-min intervals for 6 hr on Days 0 and 2, with saline or oleptin injected intravenously immediately after the first intensive sample on Day 2 (54 hr). Leptin caused a dose-related increase (P < 0.001) in mean concentrations of circulating LH. Stimulation of LH release by leptin was significant at the lowest (141% of control) and middle (122% of control) doses used, but no increase was observed for the highest dose. Increased mean concentrations of LH appeared to result from an augmentation of basal secretion, as pulse characteristics were not affected. After 54 hr of fasting, plasma insulin concentrations were lowered (P < 0.01) in all treatment groups compared to Day 0. After leptin injections, plasma insulin concentrations increased (P < 0.01) and reached highest concentrations during the first hour of sampling. However, this increase was sustained for several hours only in the intermediate (2.0 microg/kg) dose group. Collectively, our results show that leptin has potent positive effects on both LH and insulin secretion in fasted cows, but the anterior pituitary and endocrine pancreas appear to become downregulated in the presence of excess ligand.     

Leptin gene expression, circulating leptin, and luteinizing hormone pulsatility are acutely responsive to short-term fasting in prepubertal heifers: relationships to circulating insulin and insulin-like growth factor I(1)

In the present study, we tested the hypothesis that short-term fasting would reduce leptin gene expression, circulating leptin, and LH pulsatility in prepubertal heifers in association with a decrease in circulating concentrations of insulin and insulin-like growth factor I (IGF-I). Twelve prepubertal crossbred heifers (mean +/- SD = 315 +/- 5 kg body weight) were assigned randomly to one of two treatments in two replicates: 1) control; normal feed consumption (n = 6) and 2) fasted; 48 h of total feed restriction (n = 6). Blood samples were collected at 15-min intervals for 8 h on Days 0 and 2 of the experiment and twice on Day 1. Subcutaneous fat samples were collected before treatment onset (Day -1) and at the end of the intensive blood sampling on Day 2. Acute feed restriction markedly reduced leptin mRNA in adipose tissue (P < 0.01) and circulating concentrations of leptin (P < 0.05), IGF-I (P < 0.01), and insulin (P = 0.05) as compared with controls on Day 2. Moreover, the treatment x day interaction (P < 0.076) and within-day contrasts (expressed as a percentage of Day 0 values) revealed that the mean frequency of LH pulses in the fasted group was lower (P < 0.06) than in controls on Day 2. Neither mean concentrations of growth hormone (GH) nor GH secretory dynamics were affected by acute feed restriction. Fasting-mediated decreases in leptin gene expression and circulating leptin, in association with reductions in secretion of IGF-I, insulin, and LH, provide a basis for investigating leptin as a hormone signaling energy status to the central reproductive axis in cattle.     

Central infusion of recombinant ovine leptin normalizes plasma insulin and stimulates a novel hypersecretion of luteinizing hormone after short-term fasting in mature beef cows

The present studies tested the hypotheses that short-term fasting would reduce leptin gene expression and circulating concentrations of leptin and insulin in mature, ovariectomized, estradiol-implanted cows and that intracerebroventricular infusions of recombinant ovine leptin (oleptin) would attenuate reductions in insulin concentration and stimulate LH secretion. Ovariectomized cows were assigned to either control (normal fed; n = 6) or fasted (60 h of fasting; n = 7) groups and infused with 200 microg recombinant oleptin three times at hourly intervals on Day 2 (n = 6 per group). Fasting decreased plasma concentrations of insulin (P < 0.01) and leptin (P < 0.04) but, as expected, did not reduce plasma concentrations of glucose or any LH secretion variable. Central infusion of leptin on Day 2 increased (P < 0.01) plasma concentrations of leptin in both control and fasted groups. Concomitantly, leptin treatment increased plasma insulin (P < 0.01) and LH (P < 0.03) concentrations in fasted but not in control cows. Increases in overall mean and baseline concentrations of LH after leptin treatment were the result of an augmentation of the size of LH pulses. The effects of fasting on leptin gene expression and the potential diurnal effects on circulating leptin were examined in a group of cows (n = 12) not treated with leptin. Fasting for 60 h reduced (P < 0.001) leptin gene expression by 30%, and no diurnal effects on circulating leptin were observed. These results indicate that although short-term fasting does not reduce the frequency or amplitude of LH pulses or the concentration of LH in mature cows, this nutritional perturbation clearly sensitizes both the hypothalamic-pituitary axis and endocrine pancreas to exogenous leptin, which in these experiments resulted in heightened secretion of both LH and insulin.     

Leptin acts at the bovine adenohypophysis to enhance basal and gonadotropin-releasing hormone-mediated release of luteinizing hormone: differential effects are dependent upon nutritional history

Recombinant ovine leptin (oleptin) stimulates an acute increase in the secretion of LH in fasted, but not in normal-fed, cows through an augmentation of the magnitude of individual pulses of LH. Herein, we tested the hypothesis that this effect could be accounted for by functional changes at the adenohypophyseal (AP) level. Eleven ovariectomized, estradiol-implanted cows were assigned to one of two dietary groups: normal-fed (n = 6) and fasted (fasted for 72 h; n = 5). After the animals were killed, the adenohypophyses were collected and AP explants were perifused with Krebs-Ringer bicarbonate buffer (KRB) for a total of 6.5 h, including a 2-h treatment at 2.5 h with KRB or increasing doses of oleptin and a challenge at 4.5 h with 50 ng of GnRH. To test for effects of leptin at the hypothalamic level, explants encompassing the medial basal hypothalamus-infundibular complex (HYP) were incubated in KRB alone (control) or in KRB containing 1000 ng of oleptin. Basal release of LH from AP explants treated with leptin was greater (P < 0.02) than that from control-treated explants in fasted, but not in normal-fed, cows. To the contrary, leptin-treated explants from normal-fed, but not from fasted, cows released more (P < 0.001) LH in response to GnRH than control-treated tissues. Neither fasting nor leptin affected (P > 0.1) the secretion of GnRH from HYP explants. These observations support the hypothesis that leptin modulates the secretion of LH in mature cows, to a large extent, by its direct actions at the AP. Differential manifestations of these effects are dependent upon nutritional history.     

Exogenous estradiol reduces inhibition of luteinizing hormone by estradiol in prepubertal heifers

The hypothesis that high levels of exogenous estradiol administered to heifers during the prepubertal period would decrease subsequent negative feedback of estradiol on luteinizing hormone (LH) secretion was tested. Fourteen prepubertal heifers were ovariectomized on Day 0. Ovariectomized heifers received either no further treatment (OVX, n = 4), a single estradiol implant on Day 0 (OVXE, n = 5), or the single implant on Day 0 and two additional implants between Days 16 and 30 (OVXE+ E, n = 5). Ten ovary-intact heifers received either no treatment (INT, n = 5) or were administered the two estradiol implants between Days 16 and 30 (INT+ 5, n = 5). Comparison of LH secretion in OVXE to OVXE+E, and in INT to INT+E resulted in significant time-by-treatment interactions (p less than 0.05 for both). As pubertal age approached, mean concentration of LH (p less than 0.05) and pulse frequency (p less than 0.05) increased more rapidly in OVXE+E and INT+E than in OVXE and INT, respectively. Amplitude of LH pulses was unaffected by treatment. When data were standardized to day of puberty in INT and INT+E heifers, mean LH concentration and LH pulse frequency increased as puberty approached in both groups. These data confirm earlier reports indicating that secretion of LH increases gradually as puberty approaches in heifers. It was concluded that administration of estradiol during the prepubertal period hastened the decline in the subsequent negative feedback of estradiol. Precocious puberty was not induced in ovary-intact females.     

Stimulation of a pulse of LH and reduction in PRL concentration by a physiologic dose of GnRH before, during, and after luteolysis in heifers

A single physiologic dose (5.0 μg) of GnRH was given to 9 heifers each day (Hour 0) beginning on Day 15 postovulation until regression of the corpus luteum. Blood samples were taken each day for Hours -3, -2, -1, 0, 0.25, 0.5, 0.75, 1, 1.25, 1.50, 1.75, 2, 3, 4, and 5. Based on daily progesterone concentrations, data were grouped into phases of before (n=4), during (n=8), and after (n=7) luteolysis. The number of LH pulses with a peak at pretreatment Hours -2 or -1 (0.35 ± 0.12 pulses/sampling session) was less (P<0.0001) than for a pulse peak at posttreatment Hours 1 or 2 (1.0 ± 0.0 pulses/session). The characteristics and effects of LH pulses on progesterone and estradiol were similar between natural (pretreatment) and primarily induced (posttreatment) LH pulses. The same dose of GnRH stimulated an LH pulse with greater (P<0.05) amplitude after luteolysis than during luteolysis. Concentrations of PRL and number and prominence of PRL pulses decreased (P<0.05) between Hours 0 and 2 within each of the phases of before, during, and after luteolysis. The hypothesis that a physiologic dose of GnRH increases the concentration of PRL was not supported; instead, GnRH reduced the concentration of PRL. Results supported the hypotheses that an appropriate dose of GnRH stimulates an LH pulse during luteolysis that is similar to a natural pulse in characteristics and in the effects on progesterone and estradiol.     

Serotonergic activation rescues reproductive function in fasted mice: does serotonin mediate the metabolic effects of leptin on reproduction?

Negative energy balance inhibits reproduction by restraining GnRH secretion. Leptin is a permissive metabolic signal for reproduction, but GnRH neurons do not appear to express leptin receptors, suggesting that interneurons transmit leptin signals to these cells. Serotonin (5HT) has satiety effects similar to those of leptin and alters LH release, and serotonergic neurons, which have been shown to express leptin receptors, terminate on GnRH neurons. We hypothesized that serotonergic neurons convey leptin signals to the reproductive neuroendocrine axis. To test this, mice were fasted for 48 h beginning on Diestrous Day 1. While fasting, mice received saline or leptin every 12 h or the 5HT-selective reuptake-inhibitor fluoxetine once at the start of the fast. Estrous cycles of fasted mice were longer (mean +/- SEM, 10.2 +/- 0.5 days; P < 0.0001) than those of fed mice (4.5 +/- 0.2 days). As previously reported, leptin prevented fasting-induced cycle lengthening (4.6 +/- 0.7 days). Fluoxetine also rescued estrous cycles in fasted mice (4.7 +/- 0.6 days), suggesting that 5HT and leptin have similar positive effects on reproduction. Coadministration of the 5HT 1/2/7 receptor-antagonist metergoline blocked rescue of cycle length by fluoxetine and by leptin. Treating leptin-deficient ob/ob and leptin receptor-deficient db/db mice with fluoxetine did not normalize body weight or rescue fertility, perhaps due to altered serotonergic tone in these animals. Together, these data demonstrate a permissive role for serotonergic systems in the metabolic control of reproduction and are consistent with the hypothesis that serotonergic neurons convey leptin signals to GnRH neurons.     

Secretory patterns of leptin and luteinizing hormone in food-restricted young female sheep

Leptin, the product of the ob gene, has been proposed as a metabolic signal that regulates the secretion of GnRH/LH. This may be critical during prepubertal development to synchronize information about energy stores and the secretion of GnRH/LH. This study aimed to assess the effect of food restriction on the episodic secretion of leptin and LH in young female sheep. Five 20-week-old prepubertal females were fed a low-level diet for 10 weeks to maintain the body weight. Control females of the same age received food ad libitum. Blood samples were collected at 10-min intervals for six hours at 20, 26, and 30 weeks of age, and plasma leptin, LH, insulin and cortisol concentrations were measured. In the control group, no changes were found in pulsatile LH secretion characteristics. Mean LH concentrations and LH amplitude were lower in the food-restricted group than in the control group at 26 and 30 weeks of age. In the control group, pulsatile leptin secretion did not change. When compared to control lambs of the same age, the food-restricted group showed lower mean plasma leptin concentrations, pulse amplitude and plasma insulin levels, after 6 weeks of restriction (week 26), although by week 30, plasma leptin concentrations and plasma insulin rose to those of the control group. Leptin pulse frequency did not change, nor did mean plasma levels of insulin in the control group at any age studied. Mean plasma concentration of cortisol did not change within or between groups. These data suggest that plasma leptin concentrations may not be associated with the onset of puberty under regular feeding and natural photoperiod in lambs. Prolonged food restriction, however, induces metabolic adaptations that allow an increase of leptin during the final period, probably related to the development of some degree of insulin resistance.     

Effect of intracerebroventricular infusion of leptin on the secretory activity of the GnRH/LH axis in fasted prepubertal lambs

Leptin is believed to link metabolic status to reproductive processes. The aim of the present study was to investigate the effect of exogenous leptin on the secretory activity of GnRH/LH system in acutely undernourished prepubertal, female lambs. Merino lambs were randomly divided into four groups, two standard-fed and two fasted for 72 h. One standard and one fasted groups were infused intracerebroventricularly (i.c.v.) with the vehicle; the remaining standard and fasted groups were infused with leptin (25 μg/120 μl/h). Leptin was administered in series of four 1-h infusions at 30-min intervals for 3 consecutive days from 08:30 to 14:00 h. Blood samples were collected on day 0 (before infusions) and on day 3 every 10 min over a 6-h period. Immediately after the experiment, the sheep were slaughtered and brains fixed in situ. Hypothalamic and pituitary tissues were prepared for further immunohistochemical and hybridization in situ analysis. In fasted sheep, increased GnRH levels in the median eminence (P < 0.001) and LHβ levels in the pituitary cells (P < 0.001) plus decreased LHβ mRNA and LH pulsatility in blood plasma were observed (P < 0.05). In leptin-infused fasted sheep, GnRH levels in the median eminence decreased (P < 0.001), LHβ mRNA hybridization signal increased, LHβ levels decreased in the pituitary cells (P < 0.001) and LH pulsatility increased (P < 0.05) in the blood plasma. These results indicate that, in prepubertal sheep, the GnRH/LH axis is sensitive to the fasting signal, that influence of which can be reversed by leptin. Leptin cancels out the suppressing effect of fasting on LH secretion by augmentation of GnRH.     

Progesterone (CIDR)-based timed AI protocols using GnRH, porcine LH or estradiol cypionate for dairy heifers: ovarian and endocrine responses and pregnancy rates

The overall objective was to compare the efficacy of GnRH, porcine LH (pLH) and estradiol cypionate (ECP), in a modified Ovsynch/fixed-time AI (FTAI) protocol that included a controlled internal drug [progesterone] release (CIDR) device. In Experiment 1, heifers received a CIDR on Day -10, and PGF (25mg) on Day -3. At CIDR insertion, heifers received 100 microg of GnRH (n=6), 0.5mg of ECP (n=6), 5.0mg of pLH (n=6) or 2 mL of saline (n=7); these treatments were repeated on Day -1, except for ECP, that was repeated on Day -2, concurrent with CIDR-removal. The 5.0 mg pLH was the least effective with a longer interval to ovulation than the other groups combined (102 versus 64 h; P<0.05). Overall mean LH concentrations (1.6 ng/mL) and area under the curve (AUC) did not differ among treatments, but mean peak LH concentration was lower in heifers given 5 mg of pLH compared to all other groups (4.5 versus 10.3 ng/mL; P<0.05). In Experiment 2, heifers on CIDR-based Ovsynch protocols were given 12.5mg pLH (n=6; pLH-low), 25.0 mg pLH (n=6, pLH-high), or 100 microg GnRH (n=5; control). Heifers in the pLH-high group had greater (P<0.01) plasma LH concentrations (between 12 and 20 h) than GnRH-treated heifers, but the pLH treatments did not differ (P>0.10). Area under the curve for LH (ng/32 h) was at least 50% greater (P<0.01) in pLH-treated heifers compared to GnRH-treated heifers (mean, 41.3, 56.3 and 20.3 for pLH-low, pLH-high and GnRH, respectively). Ovulation occurred in 15 of 17 heifers. Progesterone concentrations were higher on Days 9 and 14 in heifers given 25mg of pLH, suggesting enhanced CL function. In Experiment 3, 240 heifers were assigned to CIDR-based Ovsynch/FTAI protocols. The first and second hormonal treatments (with an intervening PGF treatment on Day -3) were GnRH/GnRH (100 microg), ECP/ECP (0.5 mg), pLH/pLH (12.5 mg) or GnRH/ECP, respectively; pregnancy rates were 58.7, 66.1, 45.9 and 48.3%, respectively (ECP/ECP>both pLH/pLH and GnRH/ECP; P相似文献   

Steroid hormone feedback on LH in prepubertal Holstein heifers

A significant dose-response relationship between gonadotropin-releasing hormone (GnRH) and time to luteinizing hormone (LH) peak, peak serum LH and total serum LH was obtained in prepubertal Holstein heifers (28 weeks of age) (Experiment 1). For the second experiment, the effect of steroid feedback on the anterior pituitary was determined. A steady infusion of saline, estradiol-17 beta or progesterone was maintained for 24 h while GnRH, in various schemes, was administered 8 h after the beginning of steroid infusion. Estradiol-17 beta infusion (2.08 micrograms/h), although it did not affect peripheral concentrations of estrogen, caused an LH release 24 to 30 h later in 37.5% of the heifers. This amount of exogenous estrogen did not affect the LH response to a single GnRH (4 micrograms) challenge. When the same GnRH dosage (4 micrograms) was administered 6 times at hourly intervals, the heifers infused with estradiol had a lower response after the first 2 injections of GnRH and a greater response after the last 4 injections than heifers infused with saline. When GnRH was infused (4 micrograms/h) for 6 h, beginning 8 h after steroid infusion, estradiol infusion caused a significantly higher peak LH and total LH release than an infusion of either saline or progesterone (7.3 micrograms/h). The progesterone infusion had no effect on the GnRH-stimulated LH release. We conclude that prepubertal dairy heifers have an anterior pituitary capable of responding to the feedback effect of estrogen in a positive manner.     

Enhancing effect of acute fasting on ethanol suppression of pulsatile luteinizing hormone release via an estrogen-dependent mechanism in Holstein heifers

This study was conducted to determine whether acute fasting in Holstein heifers enhances the suppressive effect of an intravenous injection of ethanol on pulsatile LH release (LH pulse) and, additionally, to establish whether or not the mechanism is estrogen-dependent. After estrus synchronization (Day 0 = estrus), 29 heifers were either fasted (fasting group; n = 14) or fully fed as a control (control group; n = 15) from Days 1 to 4. On Day 4, blood samples were taken at 10-min intervals for 4 h before (pre-injection period) and after (post-injection period) an intravenous injection of 1.5 mL of saline, 1.5 mL of ethanol , or 35 mg of tamoxifen dissolved in 1.5 mL of ethanol . We analyzed the mean LH level, the number of LH peaks per 4 h, and the amplitude of LH peaks. No differences were observed in the LH pulse in the pre-injection period between the control and the fasting group. However, in the post-injection period, compared with the saline injected control heifers, ethanol suppression of the LH pulse was observed in the number of LH peaks of the ethanol injected control heifers and in all pulse parameters of the ethanol injected fasting heifers. Furthermore, tamoxifen inhibited suppression of ethanol on LH pulse was observed in the control and fasting heifers injected with tamoxifen dissolved in ethanol. It was concluded that acute fasting in Holstein heifers has an enhancing effect on ethanol inhibition of the LH pulse and that the mechanism may be estrogen-dependent.     

Response of prepubertal Bos taurus and Bos indicus x Bos taurus heifers to melengestrol acetate with or without gonadotropin-releasing hormone

The effectiveness of treatments to induce estrus in prepubertal beef heifers was evaluated. Angus x Hereford (n = 148) and Brahman x Hereford (n = 148) heifers were sorted after weaning by body weight into light and heavy weight blocks. Heifers were assigned to diets, calculated to reach a target weight of 55% or 65% of their projected mature weight by the start of breeding. Cyclicity was determined after a 160-d observation period and from concentrations of progesterone in serum determined 10 d before and on the day that treatments began to induce puberty. The remaining nonpubertal heifers, with concentrations of progesterone in serum of less than 1 ng/ml (0 or 10 d before treatment), were assigned randomly within breed and nutrition group to either a melengestrol acetate + saline (MGA+S) or MGA + gonadotropin-releasing hormone (MGA+GnRH) treatment. Prepubertal Angus x Hereford heifers (n = 11) and Brahman x Hereford heifers (n = 49) were fed 0.5 mg MGA for 7 d. Forty-eight hours after MGA, heifers were injected with 500 ug s.c. GnRH or 5 ml of saline. Blood samples were collected from all prepubertal heifers every 3 d after GnRH or saline for 30 d. There was no difference between treatments in the proportion of heifers that exhibited estrus by Day 7 after treatment. However, a larger (P<0.05) proportion of MGA+S-treated heifers exhibited estrus within 14 d after treatment than MGA+GnRH-treated heifers (87 vs 63%). Among heifers that exhibited estrus during that time period, the proportion with increased progesterone was higher (P<0.10) for the MGA+GnRH group than for the MGA+S group (71 vs 41%, Day 7; 79 vs 54%, Day 14). There was no difference in conception rate at first service between treatment groups. Thirty-seven and 53%, respectively, of the MGA+S and MGA+GnRH-treated heifers had short estrous cycles after treatment, and 44 and 50%, respectively, of those short cycles were repeated. Pregnancy rates at the end of 45 d were numerically higher for MGA+S heifers than for MGA+GnRH treated counterparts (63 vs 53%).     

Effects of leptin on gonadotropin-releasing hormone release from hypothalamic-infundibular explants and gonadotropin release from adenohypophyseal primary cell cultures: further evidence that fully nourished cattle are resistant to leptin

In rodents and pigs, leptin stimulates the release of gonadotropin-releasing hormone (GnRH) from hypothalamus, gonadotropins from adenohypophyseal (AP) explants and cells, and luteinizing hormone (LH) from full-fed animals. In the current studies, we investigated whether leptin could stimulate the release of GnRH from bovine hypothalamic-infundibular (HYP) explants and gonadotropins from bovine adenohypophyseal cells. In Experiment 1A, HYP explants collected from 17 bulls and seven steers were incubated with Krebs-Ringer bicarbonate buffer (KRB) containing 0, 10, 100, or 1000 ng/ml recombinant ovine leptin (oleptin) for 30 min after a 3-h period of equilibration. None of the doses of leptin affected (P > 0.05) GnRH release into the media. In Experiment 1B, HYP explants collected from six steers were incubated with KRB containing 0 or 1000 ng/ml oleptin for two consecutive 30-min periods and challenged with 60 mM K(+) afterwards. Leptin did not affect (P > 0.05) basal or K(+)-stimulated release of GnRH. In Experiment 2, adenohypophyses from steers were collected at slaughter and cells dispersed and cultured for 4 days. On day 5, cells were treated with media alone (control) or media containing 10(-11), 10(-10), 10(-9), and 10(-8)M oleptin. Three independent replications were performed. None of the doses of leptin stimulated (P > 0.05) the release of LH. Although leptin at 10(-11), 10(-10), and 10(-9)M increased (P < 0.03) slightly the release of FSH compared to control-treated cells in one replicate, this effect was not confirmed in the other two replicates. Results support the hypothesis that leptin has limited effects on the release of GnRH and gonadotropins in full-fed cattle and reiterate important species differences in responsiveness to leptin.     

Differential requirement for pulsatile LH during the follicular phase and exposure to the preovulatory LH surge for oocyte fertilization and embryo development in cattle

The requirement for pulsatile LH and the LH surge for the acquisition of oocyte fertilizing potential and embryo developmental competency was examined in Zebu heifers. Follicular growth was superstimulated using the GnRH agonist-LH protocol in which pulsatile LH and the preovulatory LH surge are blocked. In experiment 1, heifers were assigned on Day 7 of the estrous cycle to receive: group 1A (n = 5), 1.5 mg norgestomet (NOR) implant; group 1B (n = 5), GnRH agonist implant. Follicular growth was superstimulated with 2x daily injections of FSH from Day 10 (a.m.) to Day 13 (p.m.), with PGF2alpha injection on Day 12 (a.m.). Heifers were ovariectomized on Day 15 (a.m.) and oocytes were placed immediately into fertilization, without 24 h maturation. Respective cleavage and blastocyst development rates were: group 1A, 0/64 oocytes (0%) and 0/64 (0%); group 1B, 34/70 oocytes (48.6%) and 2/70 (2.9%). In experiment 2, heifers were assigned on Day 7 of the estrous cycle to receive: group 2A (n = 10), 1.5 mg NOR implant; group 2B (n = 10), GnRH agonist implant; group 2C (n = 10), GnRH agonist implant. Follicular growth was superstimulated as in experiment 1 above. Heifers in groups 2A and 2B received an injection of 25 mg LH on Day 14 (p.m.) and all heifers were ovariectomized on Day 15 (a.m.); oocytes were placed immediately into fertilization without 24 h maturation. Cleavage rates were similar for heifers in group 2A (84/175 oocytes, 48.0%), group 2B (61/112 oocytes, 54.5%) and group 2C (69/163, 42.3%). Blastocyst development rates were similar for heifers in group 2A (22/175 oocytes, 12.6%) and group 2B (25/112 oocytes, 22.3%) and lower (P < 0.05) for heifers in group 2C (9/163 oocytes, 5.5%). Oocytes obtained from heifers treated with GnRH agonist, without injection of exogenous LH, underwent cleavage indicating that neither pulsatile LH nor the preovulatory LH surge are obligatory for nuclear maturation in cattle oocytes. Exposure to a surge-like increase in plasma LH increased embryo developmental competency indicating that the preovulatory LH surge promotes cytoplasmic maturation. The findings have important implications for controlling the in vivo maturation of oocytes before in vitro procedures including nuclear transfer.     

Effect of purified llama ovulation-inducing factor (OIF) on ovarian function in cattle

Two experiments were designed to determine the effect of purified ovulation inducing factor (OIF) on ovarian function in cattle. In Experiment 1, prepubertal heifers (n = 11 per group) were treated on Day 5 (Day 0 = day of follicular wave emergence) of the follicular wave with an intramuscular dose of saline (1 mL), GnRH (100 μg), or purified OIF (1 mg/100 kg body weight). Ovulation occurred in 9/11 heifers treated with GnRH, and 1/11 heifers in each of the OIF- and saline-treated groups (P < 0.05). Compared to saline-treated controls, OIF treatment was associated with a smaller dominant follicle diameter (P < 0.01), a rise in plasma FSH concentration (P < 0.1), and earlier emergence of the next follicular wave (P < 0.05). In Experiment 2, sexually mature heifers were given either GnRH or purified OIF on Days 3, 6 or 9 of the first follicular wave (i.e., early growing, early static, or late static phase of the dominant follicle; n = 5 per group per day), or were untreated (n = 10). In heifers treated with OIF on Day 6, the dominant follicle diameter profile tended to be smaller than in controls, and was associated with a rise (P < 0.05) in plasma FSH concentrations. A similar rise in FSH was detected after OIF treatment on Day 9. Compared to untreated controls, treatment with OIF and GnRH was associated with a larger CL diameter (Days 3 and 6 groups; P < 0.05) and a greater concentration of plasma progesterone (Days 6 and 9 groups; P < 0.05). Treatment with purified OIF did not induce ovulation in heifers, but hastened new follicular wave emergence in prepubertal heifers, influenced follicular dynamics in a phase-specific manner in mature heifers, and was luteotrophic.     

Response to GnRH on day 6 of the estrous cycle is diminished as the percentage of Bos indicus breeding increases in Angus, Brangus, and Brahman x Angus heifers

Angus (n=6), Brangus (5/8 Angus x 3/8 Brahman, n=6), and Brahman x Angus (3/8 Angus x 5/8 Brahman, n=6) heifers exhibiting estrous cycles at regular intervals were used to determine if the percentage of Bos indicus breeding influenced the secretory patterns of LH in response to a GnRH treatment on Day 6 of the estrous cycle. Heifers were pre-synchronized with a two-injection PGF(2 alpha) protocol (25 mg i.m. Day -14 and 12.5 mg i.m. Day -3 and -2 of experiment). Heifers received 100 microg GnRH i.m. on Day 6 of the subsequent estrous cycle. Blood samples were collected at -60, -30, and -1 min before GnRH and 15, 30, 60, 90, 120, 150, 180, 240, 300, 360, 420, and 480 min after GnRH to determine concentrations of serum LH. Estradiol concentrations were determined at -60, -30, and -1 min before GnRH. On Day 6 and 8, ovaries were examined by ultrasonography to determine if ovulation occurred. On Day 13, heifers received 25 mg PGF(2 alpha) i.m. and blood samples were collected daily until either the expression of estrus or Day 20 for heifers not exhibiting estrus to determine progesterone concentrations. There was no effect (P>0.10) of breed on ovulation rate to GnRH as well as size of the largest follicle, mean estradiol, and mean corpus luteum volume at GnRH. Mean LH was greater (P<0.05) for Angus (7.0+/-0.8 ng/mL) compared to Brangus (4.6+/-0.8 ng/mL) and Brahman x Angus (2.9+/-0.8 ng/mL), which were similar (P>0.10). Mean LH peak-height was similar (P>0.10) for Brangus (13.9+/-3.4 ng/mL) compared to Angus (21.9+/-3.4 ng/mL) and Brahman x Angus (8.0+/-3.4 ng/mL), but was greater (P<0.05) for Angus compared to Brahman x Angus. Interval from GnRH to LH peak was similar (P>0.10) between breeds. As the percentage of Bos indicus breeding increased the amount of LH released in response to GnRH on Day 6 of the estrous cycle decreased.     

Endocrine mechanisms of puberty in heifers: estradiol negative feedback regulation of luteinizing hormone secretion

The hypothesis that luteinizing hormone (LH) secretion in prepubertal females is responsive to estradiol negative feedback and that decreased feedback occurs as puberty approaches was tested in heifers. In the first experiment, seven heifers were maintained prepubertal by dietary energy restriction until 508 days of age (Day 0). All heifers were placed on a high-energy diet on Day 0 at which time they received no additional treatment (CONT), were ovariectomized (OVX) or were ovariectomized and subcutaneously implanted with estradiol-17 beta (OVX-E2). This feeding regimen was used to synchronize reproductive state in all heifers. A second experiment was performed with 16 prepubertal heifers using the same treatments at 266 days (Day 0) of age (CONT, OVX and OVX-E2) but no dietary intake manipulation. In both experiments, LH secretion increased rapidly following ovariectomy in OVX heifers. In the initial experiment, LH secretion was maintained at a low level in OVX-E2 heifers until a synchronous rapid increase was noted coincidental with puberty in the CONT heifer. In the second experiment, LH secretion increased gradually in OVX-E2 heifers and attained castrate levels coincidental with puberty in CONT heifers. A gradual increase in LH secretion occurred as puberty approached in CONT heifers. These results indicate that: a) LH secretion in prepubertal heifers is responsive to estradiol negative feedback; and b) estradiol negative feedback decreases during the prepubertal period in beef heifers.     

Close synchrony of ovulation in superstimulated heifers that have a downregulated anterior pituitary gland and are induced to ovulate with exogenous LH

The synchrony of ovulation was examined in superstimulated heifers that had a downregulated pituitary gland and which were induced to ovulate by injection of exogenous LH. The pituitary was downregulated and desensitized to GnRH by treatment with the GnRH agonist deslorelin. Nulliparous heifers (3.5 yr old) at random stages of the estrous cycle were assigned to 1 of 3 groups, and on Day -7 received the following treatments: Group 1 (control, n = 8), 1 norgestomet ear implant; Group 2 (GnRH agonist, n = 8); Group 3 (GnRH agonist-LH protocol, n = 8), 2 deslorelin ear implants. Ovarian follicle growth in all heifers was superstimulated with twice-daily intramuscular injections of FSH (Folltropin-V): Day O, 40 mg (80 mg total dose); Day 1, 30 mg; Day 2; 20 mg; Day 3, 10 mg. On Day 2, all heifers were given a luteolytic dose of PGF (7 A.M.), Norgestomet implants were removed from heifers in Group 1 (6 P.M.). Heifers in Group 3 were given an injection of 25 mg, i.m. porcine LH (Lutropin) on Day 4 (4 P.M.). Ovarian follicle status was monitored at 8-h intervals from Day 3 (8 A.M.) to Day 6 (4 P.M.) using an Aloka Echo Camera and 7.5 MHz transducer. Heifers in Groups 2 and 3 exhibited estrus earlier (P < 0.05) than heifers in Group 1. Heifers in Group 2 did not have a preovulatory LH surge and they did not ovulate. Individual control heifers in Group 1 ovulated between 12 A.M. on Day 5 and 8 A.M. on Day 6. Heifers with deslorelin implants and injected with LH in Group 3 ovulated between 4 P.M. on Day 5 and 8 A.M. on Day 6. It was confirmed that superstimulated heifers with GnRH agonist implants can be induced to ovulate with LH. It was also demonstrated that ovulation is closely synchronized after injection of LH. Thus, a single, fixed-time insemination schedule could be used in a GnRH agonist-LH superovulation protocol, with significant practical and economic advantages for superovulation and embryo transfer programs.