Effect of copulation on estrus duration, LH response, and ovulation in Boer goats.

The objectives of this investigation were to determine the effect of copulation on estrus duration, LH response and ovulation in Boer goats. A controlled randomized study, with two replicates, in which does were divided at each replicate in treatment (COP; n = 12) and control (CON; n = 12) groups was performed. All the does were pluriparous and estrus synchronized with CIDR (progesterone 300 mg) maintained in the vagina for seven days, and received 50 µg of GnRH at device insertion and 5 mg of natural prostaglandin F-2α im at CIDR removal. The COP group received two copulas within the first 4 h of estrus onset, and the CON group was only permitted to be mounted. Estrus was detected twice a day during the first 24 h after pessary removal and then every 4 h by using bucks with canvas apron as teasers, led by leash for 96 h. Blood was collected during all the estrus period after each estrus detection and analyzed for LH by radioimmunoassay (RIA). In addition, at the second replicate ovulation time and number of ovulations were also monitored by transrectal ultrasonography using a linear 7.5 MHz probe beginning 24 h after estrus onset and repeated every 4 h until all the preovulatory follicles disappeared. Estrus onset was 36.7 ±â€¯10.5 h and 35.5 ±â€¯13.6 h for CON and COP groups, respectively (P = 0.82). Estrus duration for the same groups was 40.3 ±â€¯9.9 h and 28.3 ±â€¯4.7 h, respectively (P = 0.001). The LH peak time for the CON group was 17.7 ±â€¯6.3 h, and for the COP group, it was 10.9 ±â€¯2.6 h (P = 0.004). The LH peak magnitude for the same groups was 31.5 ±â€¯16.2 ng/mL and 34.9 ±â€¯20.7 ng/mL, respectively (P = 0.34). The LH peak duration was not different between groups (CON: 7.3 ±â€¯1.6 h versus COP: 7.2 ±â€¯2.4 h; P = 0.94). The first ovulation time for CON and COP groups was 33.7 ±â€¯3.9 h and 29.1 ±â€¯3.2 h (P = 0.05), and the last ovulation time for the same groups was 37.7 ±â€¯3.9 h and 32.6 ±â€¯2.5 h, respectively (P = 0.02). The overall time from LH peak to ovulation was 18.6 ±â€¯4.8 h without differences between groups (CON: 16.3 ±â€¯5.6 h versus COP: 20.6 ±â€¯3.3 h; P = 0.15). The number of ovulations for the CON group was 2.2 ±â€¯0.4, and for COP group, it was 2.1 ±â€¯0.4 (P = 0.96). It was concluded that copulation reduced estrus duration and hastened LH peak and ovulation in Boer goats.

Pubertal Escape From Estradiol Negative Feedback in Ewe Lambs Is Not Accounted for by Decreased ESR1 mRNA or Protein in Kisspeptin Neurons.

In this study, we investigated whether decreased sensitivity to estradiol negative feedback is associated with reduced estrogen receptor α (ESR1) expression in kisspeptin neurons as ewe lambs approach puberty. Lambs were ovariectomized and received no implant (OVX) or an implant containing estradiol (OVX+E). In the middle arcuate nucleus (mARC), ESR1 messenger RNA (mRNA) was greater in OVX than OVX+E lambs but did not differ elsewhere. Post hoc analysis of luteinizing hormone (LH) secretion from OVX+E lambs revealed three patterns of LH pulsatility: low [1 to 2 pulses per 12 hours; low frequency (LF), n = 3], moderate [6 to 7 pulses per 12 hours; moderate frequency (MF), n = 6], and high [>10 pulses per 12 hours; high frequency (HF), n = 5]. The percentage of kisspeptin neurons containing ESR1 mRNA in the preoptic area did not differ among HF, MF, or LF groups. However, the percentage of kisspeptin neurons containing ESR1 mRNA in the mARC was greater in HF (57%) than in MF (36%) or LF (27%) lambs and did not differ from OVX (50%) lambs. A higher percentage of kisspeptin neurons contained ESR1 protein in all regions of the arcuate nucleus (ARC) in OVX compared with OVX+E lambs. There were no differences in ESR1 protein among the HF, MF, or LF groups in the preoptic area or ARC. Contrary to our hypothesis, increases in LH pulsatility were associated with enhanced ESR1 mRNA abundance in kisspeptin neurons in the ARC, and absence of estradiol increased the percentage of kisspeptin neurons containing ESR1 protein in the ARC. Therefore, changes in the expression of ESR1, particularly in kisspeptin neurons in the ARC, do not explain the pubertal escape from estradiol negative feedback in ewe lambs.

Nutritional programming of accelerated puberty in heifers: alterations in DNA methylation in the arcuate nucleus.

High rates of body weight gain during the juvenile period appear to program molecular events within the hypothalamus, leading to advancement of puberty. Methylation of DNA, an epigenetic mechanism that controls gene expression, is associated with metabolic programming events and is proposed to play a role in the pubertal process. In this study, DNA methylation was assessed in genomic DNA obtained from the arcuate nucleus (ARC) of juvenile heifers fed to gain body weight at low (0.5 kg/d; low-gain, LG, n = 4) or high (1 kg/d; high-gain, HG, n = 4) rates from 4.5 to 8.5 mo of age (earliest puberty expected at 9 mo of age in HG heifers). Using a custom-designed oligonucleotide array targeted to imprinted genes and genes associated with nutritional inputs and the control of puberty, a comparative-genomic-hybridization array was used to identify differentially methylated regions between LG and HG heifers. Differential methylation of genomic regions associated with altered mRNA expression was observed for genes whose activity has been reported to be involved in the modulation of growth and metabolism (GHR) and puberty (HMGA2). Hence, increased rates of body weight gain during the juvenile period alter the methylation pattern of genomic DNA obtained from the ARC and these changes may be involved in programming the age at puberty in heifers.

Onset of luteolytic action of exogenous prostaglandinF-2α during estrous cycle in goats.

The objectives of these two experiments were to determine the day of onset of luteolysis after exogenous PGF-2α administration during the estrous cycle and the fertility of this synchronized estrus in goats. In the first experiment, during the breeding season, 48 Nubian does were estrous synchronized, using intravaginal sponges impregnated with a progestin, and estrus was detected by vasectomized bucks. The does were divided at random into three groups of 16 does each to be treated at days 2, 3, and 4 of the estrous cycle (estrus = day 0). Then, at each day of injection, the does were again randomly divided to receive a single dose of natural prostaglandin F-2α im (PGF-2α; 5 mg/doe; treatment [TRE] group) or sterile saline solution (control [CON] group; 1 mL/doe). Finally, the following groups were originated: TRE-2, CON-2, TRE-3, CON-3, TRE-4, and CON-4. The overall estrus response after treatment with PGF-2α (TRE group, 70.8%) was higher than saline (CON group, 12.5%, P ≤ 0.001). Estrus response for TRE-2, CON-2, TRE-3, CON-3, TRE-4, and CON-4 was 25% (2 of 8), 12.5% (1 of 8), 87.5% (7 of 8), 12.5% (1 of 8), 100% (8 of 8), and 0% (0 of 8) for the same groups, respectively. Estrus response was different between day 2 and days 3 and 4 (P ≤ 0.04) and not between day 3 and day 4 (P ≥ 0.05). In the second experiment, 15 multiparous Boer does were estrous synchronized with control internal drug release (CIDR, 300 mg progesterone = P4) and PGF-2α and randomly divided to receive one single dose of PGF-2α im at days 2, 3 or 4, after synchronized estrus (n = 5 at each day). The does were detected twice a day for estrus, and blood was collected daily for P4 determination for 11 days after the synchronized estrus. Each doe in estrus was bred by hand mating to a proven male. All the does with a functional corpus/corpora luteum/lutea (CL; ≥1.0 ng/mL of P4) responded to PGF-2α with a drop in P4 levels that either lasted only 24 h for the does that did not show estrus (0.27 ± 0.10 ng/mL; n = 4) or persisted longer in all the does that showed estrus (0.22 ± 0.18 ng/mL; n = 10; P = 0.47). Estrus response for days 2, 3, and 4 was 20% (1 of 5), 80% (4 of 5), and 100% (5 of 5), respectively (P = 0.05). The conception rate was 100%, 100%, and 80% for the same days of administration, respectively (P = 0.64). It was concluded that luteolytic action of PGF-2α begins at day 3 of the estrous cycle by inducing an ovulatory and fertile estrus in goats.

κ-Opioid Receptor Is Colocalized in GnRH and KNDy Cells in the Female Ovine and Rat Brain.

Kisspeptin-neurokinin B-dynorphin (KNDy) cells of the hypothalamus are a key component in the neuroendocrine regulation of GnRH secretion. Evidence in sheep and other species suggests that dynorphin released by KNDy cells inhibits pulsatile GnRH secretion by acting upon κ-opioid receptors (KOR). However, the precise anatomical location and neurochemical phenotype of KOR-expressing cells in sheep remain unknown. To this end, we determined the distribution of KOR mRNA and protein in the brains of luteal phase ewes, using an ovine specific KOR mRNA probe for in situ hybridization and an antibody whose specificity we confirmed by Western blot analyses and blocking peptide controls. KOR cells were observed in a number of regions, including the preoptic area (POA); anterior hypothalamic area; supraoptic and paraventricular nuclei; ventromedial, dorsomedial, and lateral hypothalamus; and arcuate nucleus. Next, we determined whether KOR is colocalized in KNDy and/or GnRH cells. Dual-label immunofluorescence and confocal analysis of the KNDy population showed a high degree of colocalization, with greater than 90% of these neurons containing KOR. Surprisingly, GnRH cells also showed high levels of colocalization in sheep, ranging from 74.4% to 95.4% for GnRH cells in the POA and medial basal hypothalamus, respectively. Similarly, 97.4% of GnRH neurons in the POA of ovariectomized, steroid-primed female rats also contained immunoreactive KOR protein. These findings suggest that the inhibitory effects of dynorphin on pulsatile GnRH secretion may occur either indirectly by actions upon KOR within the KNDy population and/or directly via the activation of KOR on GnRH cells.

Continuous presence of male on estrus onset, estrus duration, and ovulation in estrus-synchronized Boer goats.

The objectives of the present study were to assess the effect of permanent contact of teasers without copulation on the interval from controlled internal drug release (CIDR) removal to estrus onset, estrus duration, ovulation time, number of ovulations, and interval from CIDR removal to ovulation time on estrus-synchronized Boer goats. During the fall season, a controlled randomized design experiment with two groups, control (CON; n = 18) and treatment (TRE; n = 18), was performed. The TRE group was maintained permanently in a pen with an aproned buck immediately after CIDR removal. The CON group was maintained in a different pen without permanent exposure to the male. All females were estrus synchronized with CIDR maintained in the vagina for 7 days and received 50 µg of GnRH im at device insertion and 5 mg of natural prostaglandin F-2α at device removal. Females were considered to be in estrus when they accepted mounting by the aproned bucks. Estrus was detected four times a day after CIDR removal (at 6 AM, 12 noon, 6 PM, and 12 midnight) using bucks with canvas apron as teasers. The ovulation time and number of ovulations were assessed by transrectal ultrasonography starting 24 hours after estrus onset and repeated every 6 hours until complete ovulation was detected. The estrus onset for the CON group was 44.0 ± 8.3 hours and for the TRE group, it was 37.0 ± 7.7 hours (P = 0.01). Estrus duration from the CON group was 43.7 ± 9.2 hours and for the TRE group, it was 38.3 ± 6.6 hours (P = 0.05). The first, last, and mean ovulation times for the CON group were 32.4 ± 5.3, 38.4 ± 3.4, and 35.4 ± 3.9 hours, and for the TRE group, the times were 31.8 ± 2.8, 36.7 ± 3.0, and 35.8 ± 3.6 hours, respectively (P = 0.85, P = 0.23, and P = 0.82, respectively). The number of ovulations for the CON and TRE groups was 2.6 ± 0.7 and 2.6 ± 0.6 ovulations, respectively (P = 0.96). The interval time for CIDR removal to ovulation for the CON group was 79.2 ± 8.2 hours and for the TRE group, the interval time was 73.2 ± 6.2 hours (P = 0.05). It was concluded that the permanent presence of male without copulation with estrus-synchronized does hastened estrus onset, reduced estrus duration, and decreased the interval time from CIDR removal to ovulation without modification of ovulation time and number of ovulations in Boer goats.

Effect of copulation on estrus duration and ovulation time in goats.

The objective of the present study was to evaluate the effect of copulation on estrus duration and ovulation in goats. During the fall season, 14 multiparous Boer does were estrous synchronized with controlled internal drug release (300 mg), maintained in the vagina for 7 days, and received 50 µg of intramuscular GnRH device insertion and 5 mg of natural intramuscular PGF2α at device removal. The does were randomly divided into two equal groups: a treatment group (TRE; n = 7) and a control group (CON; n = 7). The TRE group received two copulas by fertile bucks within the first 4 hours of estrus onset, and the CON group received only mounts by the same males equipped with canvas aprons. Estrus detection was performed every 12 hours after controlled internal drug release removal within the first 24 hours and then every 4 hours for 5 days. Estrus was defined when a doe accepted mounting by the bucks equipped with canvas aprons. Each doe in estrus got the first transrectal ultrasonography at 24 hours after estrus onset and then every 4 hours until all the preovulatory follicles ovulated. Estrus onset for the TRE and CON groups was 40.3 ± 17.4 (mean ± standard deviation) and 43.3 ± 12.2 hours (P = 0.72), respectively. Estrus duration for the same groups was 28.6 ± 5.4 and 36.7 ± 5.3 hours (P = 0.02), respectively. The mean ovulation time for the TRE and CON groups was 31.4 ± 2.2 and 35.7 ± 3.7 hours (P = 0.04), respectively. The proportion of ovulations that occurred after the end of estrus in the TRE group was higher than in the CON group (86% vs. 33%, respectively; P = 0.05). The number of ovulations for the TRE group was 2.1 ± 0.7; for the CON group, there were 2.2 ± 0.5 ovulations (P = 0.92). It was concluded that copulation by a buck at the beginning of estrus reduced estrus duration and hastened the ovulation time in Boer goats.

Elevated body weight gain during the juvenile period alters neuropeptide Y-gonadotropin-releasing hormone circuitry in prepubertal heifers.

Increased body weight (BW) gain during the juvenile period leads to early maturation of the reproductive neuroendocrine system. We investigated whether a nutritional regimen that advances the onset of puberty leads to alterations in the hypothalamic neuropeptide Y (NPY) circuitry that are permissive for enhanced gonadotropin-releasing hormone (GnRH) secretion. It was hypothesized that NPY mRNA and NPY projections to GnRH and kisspeptin neurons are reduced in heifers that gain BW at an accelerated rate, compared with a lower one, during the juvenile period. Heifers were weaned at approximately 4 mo of age and fed diets to promote relatively low (0.5 kg/day; low gain [LG]) or high (1.0 kg/day; high gain [HG]) rates of BW gain until 8.5 mo of age. Heifers that gained BW at a higher rate exhibited greater circulating concentrations of leptin and reduced overall NPY expression in the arcuate nucleus. The proportion of GnRH neurons in close apposition to NPY fibers and the magnitude of NPY projections to GnRH neurons located in the mediobasal hypothalamus were reduced in HG heifers. However, no differences in NPY projections to kisspeptin neurons in the arcuate nucleus were detected between HG and LG heifers. Results indicate that a reduction in NPY innervation of GnRH neurons, particularly at the level of the mediobasal hypothalamus, occurs in response to elevated BW gain during the juvenile period. This functional plasticity may facilitate early onset of puberty in heifers.

Reciprocal changes in leptin and NPY during nutritional acceleration of puberty in heifers.

Feeding a high-concentrate diet to heifers during the juvenile period, resulting in increased body weight (BW) gain and adiposity, leads to early-onset puberty. In this study, we tested the hypothesis that the increase in GnRH/LH release during nutritional acceleration of puberty is accompanied by reciprocal changes in circulating leptin and central release of neuropeptide Y (NPY). The heifers were weaned at 3.5 months of age and fed to gain either 0.5 (Low-gain; LG) or 1.0 kg/day (High-gain; HG) for 30 weeks. A subgroup of heifers was fitted surgically with third ventricle guide cannulas and was subjected to intensive cerebrospinal fluid (CSF) and blood sampling at 8 and 9 months of age. Mean BW was greater in HG than in LG heifers at week 6 of the experiment and remained greater thereafter. Starting at 9 months of age, the percentage of pubertal HG heifers was greater than that of LG heifers, although a replicate effect was observed. During the 6-h period in which CSF and blood were collected simultaneously, all LH pulses coincided with or shortly followed a GnRH pulse. At 8 months of age, the frequency of LH pulses was greater in the HG than in the LG group. Beginning at 6 months of age, concentrations of leptin were greater in HG than in LG heifers. At 9 months of age, concentrations of NPY in the CSF were lesser in HG heifers. These observations indicate that increased BW gain during juvenile development accelerates puberty in heifers, coincident with reciprocal changes in circulating concentrations of leptin and hypothalamic NPY release.

Hypothalamic distribution, adenohypophyseal receptor expression, and ligand functionality of RFamide-related peptide 3 in the mare during the breeding and nonbreeding seasons.

RFamide-related peptide 3 (RFRP3), the mammalian homologue of avian gonadotropin-inhibitory hormone, has been shown to negatively regulate the secretion of LH and may contribute to reproductive seasonality in some species. Herein, we examined the presence and potential role of the RFRP3-signaling system in regulating LH secretion in the mare during the breeding and nonbreeding seasons. Hypothalamic NPVF mRNA (the precursor mRNA for RFRP3) was detected at the level of the dorsomedial nucleus and paraventricular nucleus, but expression did not change with season. A greater number of RFRP3-expressing cells was observed throughout the rostral-caudal extension of the dorsomedial nucleus. Furthermore, adenohypophyseal expression of the RFRP3 receptor (NPFFR1) during the winter anovulatory season did not differ from that during either the follicular or luteal phases of the estrous cycle. When tested in primary adenohypophyseal cell culture or in vivo during both the breeding and nonbreeding seasons, neither equine nor ovine peptide sequences for RFRP3 suppressed basal or GnRH-mediated release of LH. However, infusion of RF9, an RFRP3 receptor-signaling antagonist, into seasonally anovulatory mares induced a robust increase in secretion of LH both before and following continuous treatment with GnRH. The results indicate that the cellular machinery associated with RFRP3 function is present in the equine hypothalamus and adenohypophysis. However, evidence for functionality of the RFRP3-signaling network was only obvious when an antagonist RF9 was employed. Because GnRH-induced release of LH was not affected by RF9, its actions may occur upstream from the gonadotrope to stimulate or disinhibit secretion of GnRH.

Reduced progesterone concentration during growth of the first follicular wave affects embryo quality but has no effect on embryo survival post transfer in lactating dairy cows.

Fertility of lactating dairy cows is associated with reduced progesterone (P(4)) concentration compared with nonlactating animals. The objective of the current study was to determine whether P(4) during growth of the first follicular wave (FFW) affects embryo quality. Lactating Holstein cows at 33±3 days post partum were allocated to one of three treatments. Cows in the FFW and FFW with P(4) (FFWP) treatments started the superstimulation protocol on day 1 of the estrous cycle and second follicular wave (SFW) cows started the superstimulation protocol on estrous cycle day 7. Cows were superstimulated with 400  mg of NIH-FSH-P1 (FSH) given twice daily for 5 days, two prostaglandin F(2α) (PGF(2α)) injections given with the ninth and tenth injections of FSH, GNRH given 48  h after the first PGF(2α) injection, and timed insemination 12 and 24  h after the GNRH injection. Cows in the FFWP treatment received two intravaginal P(4) inserts during the superstimulation. Embryos were recovered 6.5 days after artificial insemination and excellent/good and fair embryos were frozen and transferred. Blood was sampled daily from estrous cycle day 0 until insemination from donor cows. During the superstimulation protocol, P(4) was (P<0.01) greatest for SFW cows followed by FFWP and FFW cows respectively. The percentage of embryos-oocytes from SFW and FFWP cows classified as excellent/good and fair embryos was (P=0.02) greater than those of FFW cows. Pregnancy per embryo transfer was not (P≥0.73) affected by embryo donor treatment. Reduced embryo quality of cows induced to ovulate the follicles from the first follicular wave is a consequence of reduced P(4) during follicle growth.

Neuroendocrine pathways mediating nutritional acceleration of puberty: insights from ruminant models.

The pubertal process is characterized by an activation of physiological events within the hypothalamic-adenohypophyseal-gonadal axis which culminate in reproductive competence. Excessive weight gain and adiposity during the juvenile period is associated with accelerated onset of puberty in females. The mechanisms and pathways by which excess energy balance advances puberty are unclear, but appear to involve an early escape from estradiol negative feedback and early initiation of high-frequency episodic gonadotropin-releasing hormone (GnRH) secretion. Hypothalamic neurons, particularly neuropeptide Y and proopiomelanocortin neurons are likely important components of the pathway sensing and transmitting metabolic information to the control of GnRH secretion. Kisspeptin neurons may also have a role as effector neurons integrating metabolic and gonadal steroid feedback effects on GnRH secretion at the time of puberty. Recent studies indicate that leptin-responsive neurons within the ventral premammillary nucleus play a critical role in pubertal progression and challenge the relevance of kisspeptin neurons in this process. Nevertheless, the nutritional control of puberty is likely to involve an integration of major sensor and effector pathways that interact with modulatory circuitries for a fine control of GnRH neuron function. In this review, observations made in ruminant species are emphasized for a comparative perspective.

Dynorphin immunoreactive fibers contact GnRH neurons in the human hypothalamus.

Dynorphin, an endogenous opioid peptide, mediates progesterone-negative feedback on gonadotropin-releasing hormone (GnRH) neurons in other species. The role of dynorphin in humans is unclear. The objective of this study was to determine if dynorphin fibers have close contacts with GnRH neurons in humans. Dual-label immunocytochemistry was performed on postmortem human hypothalamic tissue. The majority of GnRH neurons, 87.5%, had close contacts with dynorphin fibers and multiple close contacts were common, 62.5%. There were no regional differences between the hypothalamus and preoptic area in the distribution of close contacts. More close contacts were identified on the GnRH dendrites compared to the cell bodies (P < .001), but this difference was not significant when corrected for length. In conclusion, dynorphin fibers form close contacts with GnRH neurons in humans. This neuroanatomical evidence may suggest that dynorphin has effects on GnRH regulation in humans as seen in other species.

Orphanin FQ: evidence for a role in the control of the reproductive neuroendocrine system.

Orphanin FQ (OFQ), also known as nociceptin, is a member of the endogenous opioid peptide family that has been functionally implicated in the control of pain, anxiety, circadian rhythms, and neuroendocrine function. In the reproductive system, endogenous opioid peptides are involved in the steroid feedback control of GnRH pulses and the induction of the GnRH surge. The distribution of OFQ in the preoptic area and hypothalamus overlaps with GnRH, and in vitro evidence suggests that OFQ can inhibit GnRH secretion from hypothalamic fragments. Using the sheep as a model, we examined the potential anatomical colocalization between OFQ and GnRH using dual-label immunocytochemistry. Confocal microscopy revealed that approximately 93% of GnRH neurons, evenly distributed across brain regions, were also immunoreactive for OFQ. In addition, almost all GnRH fibers and terminals in the external zone of the median eminence, the site of neurosecretory release of GnRH, also colocalized OFQ. This high degree of colocalization suggested that OFQ might be functionally important in controlling reproductive endocrine events. We tested this possibility by examining the effects of intracerebroventricular administration of [Arg(14), Lys(15)] OFQ, an agonist to the OFQ receptor, on pulsatile LH secretion. The agonist inhibited LH pulse frequency in both luteal phase and ovariectomized ewes and suppressed pulse amplitude in the latter. The results provide in vivo evidence supporting a role for OFQ in the control of GnRH secretion and raise the possibility that it acts as part of an ultrashort, autocrine feedback loop controlling GnRH pulses.

Expression of leptin receptor and suppressor of cytokine signaling-3 genes in adenohypophysis of normal-fed and fasted cows.

Expression of leptin receptor (LR) and suppressor of cytokine signaling (SOCS)-3 genes was investigated in normal-fed and fasted cows. Fasting did not affect LR mRNA, but increased SOCS-3 mRNA in the adenohypophysis, suggesting that heightened responsiveness of fasted cows to leptin is not dependent upon alterations in LR or SOCS-3 mRNA in the adenohypophysis.

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.