Short communication: Insertion of an intravaginal progesterone device at the time of gonadotropin-releasing hormone (GnRH) injection affects neither GnRH-induced release of luteinizing hormone nor development of dominant follicle in early diestrus of lactating dairy cows.

Our objectives were to evaluate the acute effects of a controlled internal drug release (CIDR) insert containing 1.38 g of progesterone (P4) on the release of LH, follicular growth, and circulating concentrations of P4 in cows treated with GnRH at the time of CIDR insertion. Nonpregnant, lactating dairy cows (n=27) were blocked by parity, predicted 305-d mature-equivalent milk production, and body condition score and randomly assigned to 1 of 3 treatments: (1) CIDR insertion concurrent with an injection of 200 µg of GnRH (n=10; 2GP4); (2) CIDR insertion concurrent with an injection of 100 µg of GnRH (n=10; 1GP4); and (3) injection of 100 µg of GnRH (n=7; CON). Prior to onset of treatments, cows were submitted to a presynchronization protocol that consisted of a CIDR insert containing 1.38 g of P4 from d -7 to -2, 25mg of PGF2α on d -2 and -1, and 100 µg of GnRH on d 0. Experimental treatments were applied on d 6, the early luteal phase of the estrous cycle. Concentrations of P4 in plasma were determined on d -2 and 0 and at 0, 15, 30, 60, 120, 240, 345, 600, and 1,200 min relative to treatment on d 6. Concentrations of LH were determined in plasma samples obtained at 0, 15, 30, 60, 120, 240, and 345 min relative to treatment on d 6. Ultrasonography examinations of ovarian structures were performed on d -2, 0, 2, and at 0, 600, and 1,200 min relative to treatment on d 6. Mean concentrations of P4 in the CON group (1.91±0.28 ng/mL) were lower than in 2GP4 (3.40±0.26 ng/mL) and 1GP4 (3.31±0.24 ng/mL) groups, but concentrations in 2GP4 and 1GP4 were similar. Mean concentration of LH in response to the GnRH injection on d 6 was greatest in 2GP4 cows (3.08±0.21 ng/mL) and did not differ between 1GP4 (2.23±0.21 ng/mL) and CON (2.14±0.25 ng/mL) cows. The diameter of the dominant follicle on d 6 was similar among treatments (2GP4=15.34±0.50; 1GP4=15.31±0.50; CON=14.77±0.62 mm). In conclusion, CIDR insertion concurrent with a 100- or 200-µg dose of GnRH neither altered GnRH-induced LH release nor had an acute effect on dominant follicle growth.

Hormonal manipulations in the 5-day timed artificial insemination protocol to optimize estrous cycle synchrony and fertility in dairy heifers.

Objectives were to determine the effects of GnRH at the initiation of the 5-d timed artificial insemination (AI) program combined with 2 injections of PGF2α on ovarian responses and pregnancy per AI (P/AI) in dairy heifers, and the role of progesterone concentrations on LH release and ovulation in response to GnRH. In study 1, heifers received a controlled internal drug release (CIDR) insert containing 1.38 g of progesterone on d 0, an injection of 25 mg of PGF2α and CIDR removal on d 5, and an injection of 100 µg GnRH concurrently with AI on d 8. Heifers were assigned to receive no additional treatment (control; n=559) or an injection of GnRH on d 0 and a second injection of PGF2α on d 6 (G2P; n=547). In study 2, all heifers were treated as described for the control in study 1, and were allocated to receive no additional treatment (control; n=723), an injection of PGF2α on d 6 (NG2P; n=703), or an injection of GnRH on d 0 and an injection of PGF2α on d 6 (G2P; n=718). In study 3, heifers received a CIDR on d 7 after ovulation and were assigned randomly to a low-progesterone (LP; n=6) treatment in which 2 injections of 25 mg of PGF2α each were administered 12h apart, on d 7 and 7.5 after ovulation, or to a high-progesterone (HP; n=12) treatment in which no PGF2α was administered. On d 8, heifers received 100 µg of GnRH and blood was sampled at every 15 min from -30 to 180 min relative to the GnRH for assessment of LH concentrations. Additionally, 94 heifers were assigned to LP or HP and ovulation in response to GnRH was evaluated. In study 1, P/AI was greater for G2P than for the control on d 32 (59.4 vs. 53.5%) and 60 after AI (56.6 vs. 51.3%). In study 2, administration of GnRH on d 0 increased the proportion of heifers with a new corpus luteum on d 5 (control=21.9 vs. NG2P=20.1 vs. G2P=34.4%). Administration of a second PGF2α increased the proportion of heifers with progesterone <0.5 ng/mL at AI (control=83.1 vs. NG2P=93.0 and G2P=87.2%). Pregnancy per AI was greater for G2P than for control and NG2P on d 32 (control=52.9 vs. NG2P=55.0 vs. G2P=61.7%) and 60 (control=49.0 vs. NG2P=51.6 vs. G2P=59.1%). In study 3, HP attenuated LH release and reduced ovulation (19.0 vs. 48.4%) in response to GnRH compared with LP. Combining GnRH and 2 doses of PGF2α in the 5-d timed AI protocol improved follicle turnover, luteolysis, and P/AI in heifers. Elevated concentrations of progesterone suppressed LH release and are linked with the low ovulatory response to the initial GnRH treatment of the protocol.

Kisspeptin activates the hypothalamic-adenohypophyseal-gonadal axis in prepubertal ewe lambs.

The onset of puberty in mammals involves an increase in the pulsatile release of GNRH and LH. The KISS1 gene is essential for pubertal development, and its product, kisspeptin, stimulates the release of LH. The objective of this study was to determine the effects of kisspeptin in the hypothalamic-adenohypophyseal-gonadal axis of prepubertal ewe lambs. Ewe lambs (28 weeks of age) were treated intravenously with saline (control, n=6) or kisspeptin (20 µg kisspeptin; n=6) every hour for 24 h. Kisspeptin stimulated pulse-like release of LH within 15 min following injections, and increased the frequency and amplitude of LH pulses, and mean circulating concentrations of LH and estradiol. A surge-like release of LH was observed in four kisspeptin-treated lambs beginning 17 h after the onset of treatment, and all four lambs had elevated circulating concentrations of progesterone within 5 days post-treatment. However, circulating concentrations of progesterone decreased within 2 days after the initial rise in three of the four ewe lambs, indicating that induced luteal activity was of short duration. The proportion of lambs that were pubertal (defined by circulating concentrations of progesterone above 1 ng/ml for at least 7 days) by 35 weeks of age (8/11) and the mean age at puberty (32 ± 1 weeks) for those reaching puberty within the experimental period did not differ between treatments. Results support a role for kisspeptin in the activation of the hypothalamic-adenohypophyseal axis leading to the onset of puberty in ewe lambs.

Efficacy of embryo transfer in lactating dairy cows during summer using fresh or vitrified embryos produced in vitro with sex-sorted semen.

The objective was to determine whether transfer of fresh or vitrified embryos produced in vitro with sex-sorted semen improves pregnancy and calving rates during summer in lactating dairy cows compared with artificial insemination (AI). Lactating dairy cows (n=722) were enrolled during summer months at 2 commercial dairies in Central Texas and randomly assigned to 1 of 3 treatments: AI with conventional semen (n=227), embryo transfer-vitrified (ET-V; n=279) or embryo transfer-fresh (ET-F; n=216). Embryos were produced in vitro using sex-sorted semen and with Block-Bonilla-Hansen-7 culture medium. For vitrification, grade 1 expanded blastocysts were harvested on d 7 after fertilization and vitrified using the open-pulled straw method. Fresh embryos were grade 1 blastocysts and expanded blastocysts harvested on d 7 after fertilization. Cows were submitted to the Ovsynch56 protocol: d -10 GnRH, d -3 PGF(2α), d -1 GnRH and d 0 timed AI; or Select Synch protocol: d -9 GnRH, d -2 PGF(2α), and AI following detected estrus (day of AI=d 0). On d 7, all cows were examined for presence of a corpus luteum (CL). A vitrified or fresh embryo was transferred to cows with CL in ET-V and ET-F groups. Cows were considered synchronized if progesterone was <1ng/mL on d 0 and a CL was present on d 7. At d 40±7 of gestation, the percentage of cows pregnant was greater for the ET-F compared with the ET-V and AI groups among all cows (42.1 vs. 29.3 and 18.3%, respectively) and synchronized cows (45.5 vs. 31.6 and 24.8%, respectively). Also, the percentage of cows pregnant was greater for the ET-V than the AI group among all cows and tended to be greater among synchronized cows. At d 97±7 of gestation, the percentage of cows pregnant among all cows was greater for ET-F and ET-V groups than for the AI group (36.4 and 25.7 vs. 17.0%, respectively) and the percentage for the ET-F group was greater than for the ET-V group. Among synchronized cows, the percentage of cows pregnant was significantly increased for the ET-F group than for ET-V and AI groups (39.4 vs. 27.8 and 23.1%, respectively) and no difference was found between ET-V and AI groups. No effect of treatment on embryo loss was observed. The percentage of cows with live births was significantly increased for the ET-F than for ET-V and AI groups among all cows (27.5 vs. 17.1 and 14.6%, respectively) and synchronized cows (29.9 vs. 18.5 and 20.0%, respectively). The percentage of cows giving birth to a live heifer was significantly increased for the ET-F and ET-V groups compared with the AI group among all cows (79.1 and 72.5 vs. 50.0%, respectively) and synchronized cows (79.1 and 72.5 vs. 50.0%, respectively). No difference existed between ET-F and ET-V groups for percent live heifer births but both were greater than for the AI group. The transfer of fresh embryos produced in vitro using sex-sorted semen to lactating dairy cows during summer can effectively increase the percentage of cows that establish pregnancy and also the percentage of cows that give birth to a live heifer compared with percentages from AI with conventional semen.

Concentration of progesterone during the development of the ovulatory follicle: II. Ovarian and uterine responses.

Two experiments evaluated the influence of altering the concentrations of progesterone during the development of the ovulatory follicle on the composition of the follicular fluid, circulating LH and PGF(2α) metabolite (PGFM), and expression of endometrial progesterone receptor and estrogen receptor-α. In both experiments, the estrous cycles were presynchronized (GnRH and progesterone insert followed by insert removal and PGF(2α) 7 d later, and GnRH after 48 h) and cows were then enrolled in 1 of 2 treatments 7 d later (study d -16): high progesterone (HP) or low progesterone (LP). In experiment 1 (n=19), cows had their estrous cycle synchronized starting on study d -9 (GnRH and progesterone insert on d -9, and insert removal and PGF(2α) on d -2). In experiment 2 (n=25), cows were submitted to the same synchronization protocol as in experiment 1, but had ovulation induced with GnRH on study d 0. In experiment 1, plasma was sampled on d -4 and analyzed for concentrations of LH; the dominant follicle was aspirated on d 0 and the fluid analyzed for concentrations of progesterone, estradiol, and free and total IGF-1. In experiment 2, follicular development and concentrations of progesterone and estradiol in plasma were evaluated until study d 16. Uterine biopsies were collected on d 12 and 16 for progesterone receptor and estrogen receptor-α protein abundance. An estradiol/oxytocin challenge for PGFM measurements in plasma was performed on d 16. In experiments 1 and 2, LP cows had lower plasma concentrations of progesterone and greater concentrations of estradiol, and had larger ovulatory follicle diameter (20.4 vs. 17.2mm) at the end of the synchronization protocol than HP cows. Concentration of LH tended to be greater for LP than HP cows (0.98 vs. 0.84 ng/mL). The dominant follicle of LP cows had greater concentration of estradiol (387.5 vs. 330.9 ng/mL) and a lower concentration of total IGF-1 (40.9 vs. 51.7 ng/mL) than that of HP cows. In experiment 2, estradiol and progesterone concentrations did not differ between treatments from d 0 to 16; however, the proportion of cows with a short luteal phase tended to increase in LP than HP (25 vs. 0%). Concentrations of PGFM were greater for LP than HP. Uterine biopsies had a greater abundance of progesterone receptor, and tended to have less estrogen receptor-α abundance on d 12 compared with d 16. An interaction between treatment and day of collection was detected for estrogen receptor-α because of an earlier increase in protein abundance on d 12. Reduced concentrations of progesterone during the development of the ovulatory follicle altered follicular dynamics and follicular fluid composition, increased basal LH concentrations, and prematurely increased estrogen receptor-α abundance and exacerbated PGF(2α) release in the subsequent estrous cycle.

Time of copulation during estrus period on estrus duration and LH response in Boer goats.

The objectives of this investigation were to determine the effect of the time of copulation during the estrus period on estrus duration and luteinizing hormone (LH) response in goats. A controlled randomized study with two replicates (first = n = 12; second = n = 24), in which Boer does were divided at each replicate into three groups, was performed. Copulations at the beginning of estrus (two copulas within the first 4 h after estrus; COP-1; n = 12), copulations around the middle of estrus (two copulas around 16 h after estrus; COP-2; n = 12), and noncopulations (only mounts) throughout the estrus period (control group; CON; n = 12) were performed. Estrus duration for CON group was 41.3 ± 8.2 h; for COP-1, it was 34.0 ± 5.3 h, and for COP-2, it was 39.7 ± 6.9 h (P = 0.04). Differences were detected between COP-1 and CON groups (P = 0.01) and between COP-1 and COP-2 groups (P = 0.05) but not between CON and COP-2 groups (P = 0.56). The LH peak time for the CON group was 20.0 ± 8.0 h; for the COP-1 group, it was 13.0 ± 3.6 h, and for the COP-2 group, it was 20.5 ± 5.8 h (P = 0.04). The COP-1 group was different than the COP-2 (P = 0.02) and CON groups (P = 0.03), and no differences were detected between these last two groups (P = 0.87). It was concluded that copulation reduced estrus duration and hastened the LH peak time only when performed during the beginning of estrus.

Continuous administration of low-dose GnRH in mares I. Control of persistent anovulation during the ovulatory season.

Three experiments were conducted during the operational breeding season to confirm that continuous, subcutaneous infusion of low-dose GnRH would not disrupt established estrous cycles (Experiment 1), and test the hypotheses that a similar treatment would stimulate secretion of LH and induce development of ovulatory follicles in persistently anovulatory mares (Experiments 2 and 3). Treatment with GnRH (5 microg/h) increased (P<0.001) serum P4 during the luteal phase (7.7+/-0.5 versus 6.4+/-0.5 ng/mL), tended to increase serum LH (2.6+/-0.27 versus 1.9+/-0.25 ng/mL), and did not modify interovulatory intervals. In Experiment 2, GnRH treatment (2.5-5 microg/h) of persistently anovulatory mares increased (P<0.001) serum LH compared to controls (0.5+/-0.08 versus 0.1+/-0.03 ng/mL), with all GnRH-treated and no Control mares ovulating. Mares exhibiting Delayed Recrudescence (n=29) or Lactational Anovulation (n=18), were assigned randomly in Experiment 3 to receive either (1) GnRH/GnRH (n=23); 2.5 microg GnRH/h for 14 d (Period I) and 5 microg/h during the subsequent 28 d (Periods II and III); or (2) Control/GnRH (n=24); no treatment during Period I (control period) and GnRH treatments as in 1 during Periods II and III. Percentage of mares ovulating and pregnant during Period I was greater (P<0.05) for GnRH-treated than Control mares. Thereafter, cumulative ovulation frequency (85%), pregnancy (72%) and cycles/conception (1.3+/-0.2) were similar between groups; however, interval to conception was reduced (P<0.01) by 10.3 d in GnRH/GnRH compared to Control/GnRH.

Interaction of dietary energy source and body weight gain during the juvenile period on metabolic endocrine status and age at puberty in beef heifers.

Using a previously established model for nutritional acceleration of puberty, beef heifers ( = 48; 1/2 Angus × 1/4 Hereford × 1/4 Brahman) were used in a replicated 2 × 2 factorial design to examine the effects of diet type (high forage [HF] vs. high concentrate [HC]) and rate of BW gain (low gain [LG], 0.45 kg/d, vs. high gain [HG], 0.91 kg/d) on key metabolic hormones and age at puberty. After weaning at 14 ± 1 wk of age, heifers were assigned randomly to be fed HC-HG, HC-LG, HF-HG, or HF-LG ( = 12/group) beginning at 4 mo of age for 14 wk. Heifers were then switched to a common growth diet until puberty. Average daily gain was greater ( < 0.04) during the dietary treatment phase in HG heifers (0.81 ± 0.06 kg/d) than in LG heifers (0.43 ± 0.06 kg/d), and there was no diet type × rate of gain interaction. Puberty was achieved at a younger age (54.5 ± 1.8 wk) in both HG groups than in LG groups (60.2 ± 1.9 wk; < 0.04), but dietary energy source (HC vs. HF) did not influence this variable. Moreover, mean BW at puberty did not differ by diet type or rate of gain during the dietary treatment phase. Nonetheless, heifers fed HC-HG exhibited a striking increase ( < 0.0001) in serum leptin beginning at 26 ± 1 wk of age and remained elevated ( < 0.01) throughout the remainder of the experimental feeding phase compared to all other treatments. However, serum leptin in HC-HG dropped precipitously when heifers were switched to the common growth diet and did not differ from that of other groups thereafter. Overall mean concentrations of serum glucose were greater ( < 0.006) in HG heifers than in LG during the dietary treatment phase, with serum insulin also greater ( < 0.04) in HG than in LG only during weeks 20, 22, and 30. Mean serum IGF-1 was not affected by dietary type or rate of BW gain. We speculate that failure of the marked increase in serum leptin observed in HC-HG heifers during the dietary treatment phase to further accelerate puberty compared to HF-HG occurred because of its abrupt decline at the onset of the common growth phase, thus attenuating the temporal cue for activation of the reproductive neuroendocrine system.

Evaluation of the influence of prenatal transportation stress on GnRH-stimulated luteinizing hormone and testosterone secretion in sexually mature Brahman bulls.

This study examined the relationship of prenatal transportation stress (PNS) with exogenous GnRH-induced LH and testosterone secretion in sexually mature Brahman bulls. Brahman cows (n = 96; 48 were stressed by transportation at 5 stages of gestation and 48 were controls) produced a calf crop of 85 calves. All bulls (n = 46) from this calf crop were electroejaculated every 2 wk beginning at a scrotal circumference of 24 cm until sexual maturity (SM; i.e., 500 million sperm/ejaculate). The initial 11 control and 12 PNS bulls to reach SM were selected for the experiment. Within 7-21 d after reaching SM, bulls were fitted with jugular cannulas, from which blood samples were collected at 15-min intervals for 6 h prior to exogenous GnRH administration (10 ng/kg BW; i.v.) and for 6 h after GnRH. Serum concentrations of LH, testosterone, and cortisol were determined by RIA. Age and body weight did not differ ( > 0.1) between PNS and control bulls at the time of the experiment. All bulls responded similarly to exogenous GnRH, indicating no influence of PNS on LH or testosterone response to GnRH. More ( < 0.01) PNS (9 of 11) than control (3 of 12) bulls exhibited an endogenous pre-GnRH LH pulse, and more ( = 0.02) PNS (9 of 11) than control bulls (4 of 12) exhibited a pre-GnRH testosterone response to LH. The average concentration of testosterone during the 60 min (time -60, -45, -30, -15, and 0 min relative to GnRH) immediately preceding GnRH, tended to be greater ( = 0.07) in PNS (1.46 ± 0.30 ng/mL) than control (0.68 ± 0.28 ng/mL) bulls. During that time span serum cortisol was lower ( < 0.01) in PNS (4.00 ± 0.91 ng/mL) than control (7.8 ± 0.87 ng/mL) bulls. A treatment by time interaction ( = 0.03) affected testosterone concentrations from time -240 to 360 min relative to GnRH. Results from this study indicate that PNS did not affect pituitary responsiveness to GnRH or testicular responsiveness to GnRH-induced LH secretion.

Colocalisation of dynorphin a and neurokinin B immunoreactivity in the arcuate nucleus and median eminence of the sheep.

Dynorphin A (DYN)-containing cells play a key role in conveying the negative feedback influence of progesterone upon pulsatile gonadotrophin-releasing hormone (GnRH) secretion in the ewe. A very high percentage of DYN cells in the arcuate nucleus express the progesterone receptor; another population of arcuate nucleus cells that also express steroid receptors in the sheep are those that express the tachykinin peptide, neurokinin B (NKB). Both DYN and NKB fibres have been shown to form close contacts with ovine GnRH cells. Therefore, the present study tested the hypothesis that neurones expressing NKB and DYN represent the same neuronal population in the arcuate nucleus. Confocal microscopic analysis of brain sections processed for dual immunofluorescence revealed that a large majority of DYN neurones in the arcuate nucleus were also immunoreactive for NKB. Likewise, a similar majority of NKB neurones in the arcuate nucleus were immunoreactive for DYN. By contrast, DYN cells in the preoptic area and anterior hypothalamus did not colocalise with NKB, nor did DYN cells in the paraventricular or supraoptic nuclei. Fibres that stained positively for both DYN and NKB were seen in the arcuate nucleus, where they formed close appositions with DYN/NKB-positive neurones, and in the external zone of the median eminence. Taken together with previous findings, these data suggest that a subpopulation of arcuate nucleus neurones coexpressing DYN and NKB mediate the negative feedback influence of progesterone on pulsatile GnRH secretion in the ewe and may also be involved in other feedback actions of gonadal steroids.

Regulatory roles of leptin in reproduction and metabolism: a comparative review.

Leptin plays an important role in signaling nutritional status to the central reproductive axis of mammals and appears to be at least a permissive factor in the initiation of puberty. The expression and secretion of leptin are correlated with body fat mass and are acutely affected by changes in feed intake. Moreover, circulating leptin increases during pubertal development in rodents, human females and heifers. Effects of leptin are mediated mainly via receptor activation of the JAK-STAT pathway; however, activation of alternative pathways, such as MAP kinase, has also been reported. Although the leptin receptor (LR) has not been found on GnRH neurons, leptin stimulates the release of GnRH from rat and porcine hypothalamic explants. Moreover, leptin increases the release of LH in rats and from adenohypophyseal explants and/or cells from full-fed rats and pigs. In contrast, stimulation of the hypothalamic-gonadotropic axis by leptin in cattle and sheep is observed predominantly in animals and tissues pre-exposed to profound negative energy balance. For example, leptin prevents fasting-mediated reductions in the frequency of LH pulses in peripubertal heifers, augments the magnitude of LH and GnRH pulses in fasted cows, and enhances basal secretion of LH in vivo and from adenohypophyseal explants of fasted cows. However, leptin is incapable of accelerating the frequency of LH pulses in prepubertal heifers, regardless of nutrient status, and has no effect on the secretion of GnRH and LH in full-fed cattle or hypothalamic/hypophyseal explants derived thereof. Similar to results obtained with LH, basal secretion of GH from anterior pituitary explants of fasted, but not normal-fed cows, was potentiated acutely by low, but not high, doses of leptin. Mechanisms through which undernutrition hypersensitize the hypothalamic-gonadotropic axis to leptin may involve up-regulation of the LR. However, an increase in LR mRNA expression is not a requisite feature of heightened adenohypophyseal responses in fasted cattle. To date, leptin has not been successful for inducing puberty in ruminants. Future therapeutic uses for recombinant leptin that exploit states of nutritional hypersensitization, and identification of genetic markers for genotypic variation in leptin resistance, are currently under investigation.

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.

Nutritional Programming of Accelerated Puberty in Heifers: Involvement of Pro-Opiomelanocortin Neurones in the Arcuate Nucleus.

The timing of puberty and subsequent fertility in female mammals are dependent on the integration of metabolic signals by the hypothalamus. Pro-opiomelanocortin (POMC) neurones in the arcuate nucleus (ARC) comprise a critical metabolic-sensing pathway controlling the reproductive neuroendocrine axis. α-Melanocyte-stimulating hormone (αMSH), a product of the POMC gene, has excitatory effects on gonadotrophin-releasing hormone (GnRH) neurones and fibres containing αMSH project to GnRH and kisspeptin neurones. Because kisspeptin is a potent stimulator of GnRH release, αMSH may also stimulate GnRH secretion indirectly via kisspeptin neurones. In the present work, we report studies conducted in young female cattle (heifers) aiming to determine whether increased nutrient intake during the juvenile period (4-8 months of age), a strategy previously shown to advance puberty, alters POMC and KISS1 mRNA expression, as well as αMSH close contacts on GnRH and kisspeptin neurones. In Experiment 1, POMC mRNA expression, detected by in situ hybridisation, was greater (P < 0.05) in the ARC in heifers that gained 1 kg/day of body weight (high-gain, HG; n = 6) compared to heifers that gained 0.5 kg/day (low-gain, LG; n = 5). The number of KISS1-expressing cells in the middle ARC was reduced (P < 0.05) in HG compared to LG heifers. In Experiment 2, double-immunofluorescence showed limited αMSH-positive close contacts on GnRH neurones, and the magnitude of these inputs was not influenced by nutritional status. Conversely, a large number of kisspeptin-immunoreactive cells in the ARC were observed in close proximity to αMSH-containing varicosities. Furthermore, HG heifers (n = 5) exhibited a greater (P < 0.05) percentage of kisspeptin neurones in direct apposition to αMSH fibres and an increased (P < 0.05) number of αMSH close contacts per kisspeptin cell compared to LG heifers (n = 6). These results indicate that the POMC-kisspeptin pathway may be important in mediating the nutritional acceleration of puberty in heifers.

Effects of leptin on basal and GHRH-stimulated GH secretion from the bovine adenohypophysis are dependent upon nutritional status.

We have shown recently that leptin modulates at least two aspects of anterior pituitary LH release in ruminants: basal and GnRH-mediated release. To test the hypothesis that leptin directly affects basal and GHRH-mediated GH secretion from the adenohypophysis, we examined the effects of various doses of recombinant ovine leptin (oleptin) on perifused adenohypophyseal (AP) explants and compared responses of tIssues from control and fasted cows. Ten mature, ovariectomized and estradiol-implanted cows were assigned to one of two dietary groups: (1) normal-fed (n=5) and (2) fasted for 72 h (n=5). At the end of the fasting period, cows were euthanized and pituitaries were collected. Adenohypophyseal explants were perifused for a total of 6.5 h, including a 2-h treatment at 2.5 h with Krebs-Ringer bicarbonate buffer containing 0, 5, 10, 50, or 100 ng/ml oleptin, and a challenge with GHRH at 4.5 h. All doses of oleptin greater than 5 ng/ml decreased (P<0.01) basal GH secretion compared with controls in tIssues collected from normal-fed cows. In contrast, GH release from AP explants from fasted cows treated with the lowest dose of oleptin was 28% (P<0.002) higher than control explants, but larger doses had no effect. Leptin caused an inversely related, dose-dependent increase in GHRH-mediated GH release in tIssues from normal-fed cows. Marked increases (P<0.01-P<0.001) in GH release were observed for the 5 and 10 ng/ml oleptin, with lesser (P<0.08) and no effects observed at the 50 and 100 ng/ml doses respectively. In fasted cows, oleptin had no stimulatory effect on GHRH-induced GH release. Results show that leptin can act directly at the anterior pituitary level to modulate GH release, and this effect is dependent upon nutritional history.

Leptin attenuates the acute effects of centrally administered neuropeptide Y on somatotropin but not gonadotropin secretion in ovariectomized cows.

We tested the hypothesis that recombinant ovine leptin would attenuate the acute effects of neuropeptide Y (NPY) on secretion of GH and gonadotropins (LH and FSH) in cows. Ovariectomized cows (n=6) fitted with third ventricle guide cannulas were assigned randomly to each of three groups in a Latin square arrangement: (1) control; saline treatment only, (2) NPY; saline followed by NPY, and (3) L-NPY; leptin pretreatment followed by NPY. Treatments were: s.c. injection of saline or leptin (30 microg/kg BW) at time 0, i.v. injection of saline or leptin (30 microg/kg BW) at 70 min, and intracerebroventricular (i.c.v.) injection of saline or NPY (500 microg) at 90 min. Plasma leptin was elevated (P<0.01) at least four-fold throughout the experiment in the L-NPY group. Mean plasma concentrations of LH declined within 1 h and were lower (P<0.03) than controls in both the NPY and L-NPY groups beginning 2 h after NPY injection. An acute increase in plasma concentrations of GH was observed within 1 h after NPY in the NPY group and mean values were greater (P<0.01) than controls. However, in the L-NPY group, leptin pretreatment attenuated the NPY effect on GH. Treatments had no effect on FSH secretion. Results confirm suppressive and stimulatory effects of NPY on LH and GH secretion, respectively, and indicate that leptin can attenuate the acute effects of NPY on GH secretion in cattle.

Leptin and its role in the central regulation of reproduction in cattle.

Leptin, a 16kDa product of the adipose obese (ob) gene, has been shown to contribute to the regulation of energy metabolism, feeding behavior, and reproduction in several monogastric species, including humans. Recent reports have provided evidence that the leptin gene is functionally relevant in cattle and sheep, and may contribute to an array of important reproductive events, including puberty. Leptin gene expression and circulating leptin increase markedly during sexual maturation in heifers reaching puberty during late spring or early summer. In addition, serum leptin concentrations increased by over 30% from early winter to the summer solstice in mature cows, and also increased with significant changes in adiposity. However, only limited changes in circulating leptin have been observed during the estrous cycle. Short-term fasting of growing peripubertal heifers causes marked reductions in leptin gene expression and circulating leptin, concomitant with declines in LH pulse frequency, and serum concentrations of insulin and IGF-1. Although short-term fasting of mature cows in excellent body condition is without effects on LH pulse frequency, it has remarkably similar metabolic effects to those observed in heifers. Moreover, ICV administration of recombinant oleptin resulted in a marked hypersecretion of LH in fasted cows, and in vitro studies using both hypothalamic and anterior pituitary explants have provided evidence that this effect is at the pituitary level. Paradoxically, ICV administration of oleptin normalized circulating insulin in fasted cows but hleptin was without effect on insulin in estradiol-implanted wethers. Collectively, work in cattle and sheep indicates that leptin can modulate both the hypothalamic-pituitary axis and endocrine pancreas under defined nutritional conditions. Additional work to more fully characterize these roles is clearly warranted and could lead to the development of novel strategies for modifying reproductive potential in food-producing species.

Comparison of three approaches for synchronization of ovulation for timed artificial insemination in Bos indicus-influenced cattle managed on the Texas gulf coast.

Our objectives were to compare the relative efficacies of three protocols designed to synchronize ovulation for timed artificial insemination (AI) of predominantly Brahman-influenced cows and heifers. In Exp. 1, 273 Brahman x Hereford (F1) cows at three locations were stratified by BW, body condition score (BCS), age, and days postpartum and assigned randomly to three treatments: 1) Syncro-Mate-B (SMB), 2) norgestomet-prostaglandin (NP), and 3) Ovsynch. The management goal required that cows have a minimum BCS of 5 and be at least 36 d postpartum (PP) at treatment onset. However, final results included 23 cows (8.4%) whose BCS fell below 5. In Exp. 2, 286 pubertal beef heifers were stratified by BW and BCS and allocated randomly to the three treatments. Heifers were predominantly Brahman crossbred (n = 265; Brahman x Hereford, F1; Santa Cruz) or purebred Brahman-influenced (Santa Gertrudis) with a smaller number (n = 21) of Hereford heifers also included. For both experiments, SMB treatment consisted of a 9-d norgestomet ear implant plus an estradiol valerate/norgestomet injection on d 0. Norgestomet-prostaglandin-treated females were implanted with a SMB implant without the estradiol valerate/norgestomet injection at the time of implant insertion and received 25 mg prostaglandin F2alpha (PGF) i.m. 2 d before implant removal. Ovsynch consisted of 100 microg GnRH i.m. on d 1, 25 mg PGF i.m. on d 8, and a second GnRH injection on d 10. Beginning on d 9, calves were removed for 48 h in Exp. 1. Cattle in SMB and NP groups in both experiments were timed-inseminated 48 to 54 h after implant removal and at 12 to 24 h after the second GnRH injection (Ovsynch). Timed AI conception rates did not differ between the SMB (45.1%) and Ovsynch (42.4%) groups; however, conception rate in the NP group tended (P < 0.12) to be lower overall than in the other groups due to a reduced (P < 0.05) conception rate in cows that were < 60 d PP at treatment onset. Conversely, timed-AI conception was greatest (P < 0.056) in NP (54.7%) compared with SMB (40.4%) and Ovsynch (39.1%) for heifers in Exp. 2. We conclude that in mature, suckled beef cows with Brahman genetic influence, SMB and Ovsynch perform similarly when cow eligibility relies primarily on BCS and minimum days PP. The NP treatment results in lower conception in cows < 60 d PP compared with SMB and Ovsynch. However, in nulliparous Brahman-influenced heifers that are confirmed to be pubertal, NP may be superior to the other two treatments for timed AI.

Age at puberty, total fat and conjugated linoleic acid content of carcass, and circulating metabolic hormones in beef heifers fed a diet high in linoleic acid beginning at four months of age.

In the current study, we hypothesized that diets high in linoleic acid would increase conjugated linoleic acid (CLA) tissue content, reduce adiposity and leptin production, and result in an increase in the age at puberty in heifers. Heifers were weaned and blocked by body weight (heavy, n = 10, and light, n = 10) and allocated randomly within block to receive isocaloric and isonitrogenous diets with either added fat (HF, n = 10) or no added fat (C, n = 10) from 4 mo of age until post-pubertal slaughter. Whole sunflower seed (55% oil; 70% linoleic acid) was used as the fat source in HF diets and provided 5% added fat from the start of the study until heifers weighed 250 +/- 8 kg, at which time added fat was increased to 7% of dry matter until slaughter. Body weights were recorded weekly, and blood samples were collected weekly for total cholesterol and hormone analyses. Puberty was confirmed based on serum concentrations of progesterone and ultrasonographic confirmation of corpora lutea. Heifers were slaughtered at 325 +/- 10 d of age, and longissimus muscle between the 9th and 11th rib was collected and analyzed to estimate carcass composition. Subcutaneous and kidney, pelvic, and heart fat were collected at slaughter for fatty acid analyses. The HF heavy group tended (P < 0.10) to reach puberty later than all other groups, and one HF light heifer did not reach puberty during the study. Linoleic acid and cis-9, trans-11 CLA tissue contents were higher (P < 0.03) in HF heifers than controls, but neither total carcass fat nor percentage of dry matter differed by dietary group, although the percentage of protein tended (P < 0.10) to be lower in HF heifers. Mean serum concentrations of leptin did not differ due to diet; however, leptin increased (P < 0.01) linearly as puberty approached. Circulating concentrations of growth hormone and insulin-like growth factor I increased or remained relatively constant between wk 2 to 10 of feeding, and then declined (P < 0.01) until the onset of puberty. Serum IGF-I was lower (P < 0.01) in heifers receiving the HF diet. Mean serum concentrations of insulin and total cholesterol increased (P < 0.01) with time in both groups, but only total cholesterol was increased by the HF diet (P < 0.05). Results indicate that diets high in linoleic acid fed to growing beef heifers beginning early in life have little or no effect on total carcass fat, circulating leptin, or age at puberty despite measurable increases in CLA accumulation.

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 leptin and its adipose gene expression during pubertal development, the estrous cycle, and different seasons in cattle.

Circulating concentrations of leptin and IGF-I, leptin gene expression, and serum binding of [126I]ovine leptin in cattle during pubertal development, as well as leptin gene expression and circulating concentrations of leptin during the estrous cycle and different calendar seasons, were investigated. Multivariate regression analysis was utilized to evaluate temporal changes in BW, leptin mRNA, and serum concentrations of IGF-I and leptin normalized to the week of puberty (Exp. 1). Body weight accounted for most of the variation associated with the onset of puberty in the full regression model (R2 = 0.99; P < 0.01). However, serum leptin was closely related to changes in BW (r = 0.85; P < 0.02) and in the absence of BW was most predictive of pubertal onset (r2 = 0.73; P < 0.01). Mean concentrations of leptin increased (P < 0.0001) linearly from 16 wk before until the wk of pubertal ovulation in yearling heifers reaching sexual maturation from early spring to midsummer. Leptin mRNA transformed to a percent of the value at puberty increased (P < 0.02) as puberty approached, but serum leptin and leptin mRNA values were not well correlated. We found no evidence of leptin-binding proteins in serum of developing heifers. Combined mean serum concentrations of IGF-I (ng/mL) during periods III and IV (-9 wk to wk of puberty; 216.6 +/- 9) were 21% higher (P < 0.0001) than combined mean concentrations of IGF-I during periods I and II (-19 to wk of puberty; 193 +/- 10). In mature heifers and cows (Exp. 2), serum leptin tended to decrease (P = 0.10) during the late luteal/early follicular phase of the estrous cycle, which corresponded to a reduction (P < 0.03) in adipocyte leptin gene expression. In mature ovariectomized cows, serum concentrations of leptin increased (P < 0.001) by 34% from early winter to the summer solstice and remained unchanged throughout the remainder of the year (Exp. 3). Results from these studies indicate that marked increases in both circulating leptin and leptin gene expression occur in developing heifers during pubertal development and are associated with increases in serum IGF-I and BW. Seasonal effects on circulating leptin observed in mature cows from winter to summer could also plausibly account for a portion of the prepubertal rise in serum leptin observed in heifers.