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.

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.

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.

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.

Temporal changes in histomorphology and gene expression in goat testes during postnatal development.

Testicular cell proliferation and differentiation is critical for development of normal testicular function and male reproductive maturity. The objective of the current study was to evaluate histoarchitecture and expression of genes marking specific cells and important functions as well as testosterone production of the developing goat testes. Testes were harvested from Alpine bucks at 0, 2, 4, 6, and 8 mo of age (n = 5/age group). Paired testes weight increased from 2 to 4 (P < 0.001) and 4 to 6 mo (P < 0.01). The greatest increases in seminiferous tubule and lumen diameters and height of the seminiferous epithelium occurred between 2 and 4 mo (P < 0.001). Genes expressed in haploid germ cells (Protamine1 [PRM1], Outer Dense Fiber protein 2 [ODF2], and Stimulated by Retinoic Acid gene 8 [STRA8]) increased dramatically at the same time (P < 0.001). Expression of other genes decreased (P < 0.05) during testicular maturation. These genes included P450 side chain cleavage (CYP11A1), Sex determining region Y-box 9 (SOX9), Insulin-like Growth Factor 1 Receptor (IGF1R), and Heat Shock Protein A8 (HSPA8). The Glutathione S-Transferase A3 (GSTA3) gene, whose product was recently recognized as a primary enzyme involved in isomerization of androstenedione in man and livestock species including goats, sheep, cattle, pigs, and horses, uniquely peaked in expression at 2 mo (P < 0.05). Follicle-Stimulating Hormone Receptor (FSHR) mRNA abundance tended to steadily decrease with age (P = 0.1), while Luteinizing Hormone Receptor (LHCGR) mRNA abundance in testes was not significantly different across the ages. Testosterone content per gram of testicular tissue varied among individuals. However, testosterone content per testis tended to increase at 6 mo (P = 0.06). In conclusion, major changes in cellular structure and gene expression in goat testes were observed at 4 mo of age, when spermatogenesis was initiated. Male goats mature rapidly and represent a good model species for the study of agents that enhance or impair development of testicular functions.

Reproduction Symposium: hypothalamic neuropeptides and the nutritional programming of puberty in heifers.

Nutrition during the juvenile period has a major impact on timing reproductive maturity in heifers. Restricted growth delays puberty, whereas elevated BW gain advances the onset of puberty. The initiation of high-frequency episodic release of GnRH and, consequently, LH during the peripubertal period is crucial for maturation of the reproductive axis and establishment of normal estrous cycles. Nutritional signals are perceived by metabolic-sensing cells in the hypothalamus, which interact with estradiol-receptive neurons to regulate the secretory activity of GnRH neurons. The orexigenic peptide, neuropeptide Y (NPY), and the anorexigenic peptide derived from the proopiomelanocortin (POMC) gene, melanocyte-stimulating hormone α (αMSH), are believed to be major afferent pathways that transmit inhibitory (NPY) and excitatory (αMSH) inputs to GnRH neurons. The neuropeptide kisspeptin is considered a major stimulator of GnRH secretion and has been shown to mediate estradiol's effect on GnRH neuronal activity. Kisspeptin may also integrate the neuronal pathways mediating the metabolic and gonadal steroid hormone control of gonadotropin secretion. Recent studies in our laboratories indicate that functional and structural changes in the pathways involving NPY, POMC, and kisspeptin neurons occur in response to high rates of BW gain during the juvenile period in heifers. Changes include regulation of expression in NPY, POMC, and KISS1 and plasticity in the neuronal projections to GnRH neurons and within the neuronal network comprising these cells. Moreover, an intricate pattern of differential gene expression in the arcuate nucleus of the hypothalamus occurs in response to feeding high concentrate diets that promote elevated BW gain. Genes involved include those controlling feeding intake and cell metabolism, neuronal growth and remodeling, and synaptic transmission. Characterizing the cellular pathways and molecular networks involved in the mechanisms that control the timing of pubertal onset will assist in improving existing strategies and facilitate the development of novel approaches to program puberty in heifers. These include the use of diets that elevate BW gain during strategic periods of prepubertal development.

Use of a stair-step compensatory gain nutritional regimen to program the onset of puberty in beef heifers.

It was hypothesized that metabolic programming of processes underlying puberty can be shifted temporally through the use of a stair-step compensatory growth model such that puberty is optimally timed to occur at 11 to 12 mo of age. Forty crossbred beef heifers were weaned at approximately 3.5 mo of age and, after a 2-wk acclimation period, were assigned randomly to 1 of 4 nutritional groups: 1) low control (LC), restricted feed intake of a forage-based diet to promote BW gain of 0.5 kg/d until 14 mo of age, 2) high control (HC), controlled feed intake of a high-concentrate diet to promote BW gain of 1 kg/d until 14 mo of age, 3) stair-step 1 (SS-1), ad libitum feed intake of a high-concentrate diet until 6.5 mo of age followed by restricted access to a high-forage diet to promote BW gain of 0.35 kg/d until 9 mo of age, ad libitum feed intake of a high-concentrate diet until 11.5 mo of age, and restricted intake of a high-forage diet to promote BW gain of 0.35 kg/d until 14 mo of age, and 4) stair-step 2 (SS-2), reverse sequence of SS-1, beginning with restricted access to a high-forage diet. Body weight (every 2 wk) and circulating concentrations of leptin (monthly) were determined throughout the experiment. Concentrations of progesterone in blood samples collected twice weekly beginning at 8 mo of age were used to determine pubertal status. Body weight gain followed a pattern similar to that proposed in our experimental design. Circulating concentrations of leptin increased following distinct elevations in BW but decreased abruptly after feed intake restriction. Survival analysis indicated that the percentage of pubertal heifers in the LC group was lower (P < 0.05) than all other groups throughout the experiment. Although heifers in SS-1 were nutritionally restricted between 6.5 and 9 mo of age, the proportion pubertal by 12 mo of age did not differ (P = 0.36) from that of the HC group, with 80% and 70% pubertal in SS-1 and HC, respectively. In contrast, the proportion of heifers pubertal by 12 mo of age in the SS-2 group (40%) was lower (P < 0.05) than both HC and SS-1. However, by 14 mo of age, 90% of heifers in the SS-2 group had also attained puberty compared to only 40% of the LC group. In summary, these data provide evidence that changes in the nutritional and metabolic status during the early juvenile period can program the onset of puberty that occurs months later, allowing optimal timing of sexual maturation in replacement beef heifers.

Respiratory dysfunction in patients with chronic neck pain - influence of thoracic spine and chest mobility.

Patients with chronic neck pain exhibit various musculoskeletal deficits and respiratory dysfunction. As there is a link between thoracic and cervical spine motion, the aim of this study was to investigate the relationship between thoracic spine and chest mobility with respiratory function and neck disability. Nineteen patients with chronic neck pain (7 male, 46.6 ± 10.5 years) and 19 healthy subjects (7 male, 46.5 ± 9.9 years) participated. Spirometry was conducted to determine maximal voluntary ventilation (MVV), maximal inspiratory (Pimax) and maximal expiratory pressure (Pemax). Thoracic spine mobility was measured using the Spinal Mouse(®). Chest expansion was assessed by subtracting chest circumference during maximal inspiration and expiration. Neck function was investigated by examining range of motion, forward head posture, neck flexor muscle synergy endurance and self-assessment (Neck disability index (NDI)). Correlation analyses and multiple linear regression analyses were conducted using MVV, Pimax and Pemax as independent variables. Thoracic spine mobility during flexion and chest expansion correlated significantly to MVV (r = 0.45 and 0.42), all neck motions (r between 0.39 and 0.59) and neck muscle endurance (rS = 0.36). Pemax and Pimax were related to NDI (r = -0.58 and -0.46). In the regression models, chest expansion was the only significant predictor for MVV, and Pemax was determined by neck muscle endurance. These results suggest that chronic neck pain patients should improve the endurance of the neck flexor muscles and thoracic spine and chest mobility. Additionally, these patients might benefit from respiratory muscle endurance training, possibly by increasing chest mobility and Pemax.

Pharmacologic application of native GnRH in the winter anovulatory mare, II: accelerating the timing of pregnancy.

Onset of the winter anovulatory period in mares is associated with a marked diminution in adenohypophyseal synthesis and release of LH. Native GnRH, unlike its synthetic agonists, stimulates the synthesis and secretion of LH in mares without pituitary refractoriness. Herein we tested the hypotheses that (1) the average Julian day of pregnancy can be accelerated by up to 2 months in winter anovulatory mares treated continuously with native GnRH beginning on February 1 and (2) mares will sustain luteal function and pregnancy after treatment withdrawal. Forty-two winter anovulatory mares were stratified by age, body condition score, and size of the largest follicle across two locations in a randomized design and assigned to one of three groups (n = 14 per group): (1) CONTROL: untreated, (2) GnRH-14: GnRH delivered subcutaneously in saline at a rate of 100 µg/h for 8 weeks (February 1-March 29) using four consecutive 14-day pumps (Alzet 2ML2), or (3) GnRH-28: GnRH delivered as in (2), but using two 28-day pumps (Alzet 2ML4). On development of a 35-mm follicle and expression of estrus, mares were bred the following day and treated with hCG. Pregnancies were confirmed using transrectal ultrasonography on Days 14, 24, 33, and 45, with blood samples collected to assess luteal function. Mares treated with GnRH (GnRH-14 and GnRH-28) did not differ reproductively in their responses and data were pooled for statistical comparisons. Mares treated with GnRH exhibited marked increases (P ≤ 0.04) in the frequency of development of a 35-mm follicle, submission rate for live cover and/or artificial insemination, ovulation, and pregnancy compared with control mares on treatment Day 56 (March 29). Interval to the first 35-mm follicle was 51.8 ± 4.9 and 19.3 ± 3.5 days (least square mean ± standard error of the mean) for control and GnRH-treated mares, respectively. Interval to pregnancy was 65.3 ± 6.7 and 28.6 ± 4.8 days (least square mean ± standard error of the mean) for control and GnRH-treated mares, respectively, excluding one GnRH-14 mare that failed to become pregnant over four cycles. By the end of the treatment period (March 29), only 21% of control mares were pregnant compared with 79% of GnRH-treated mares. Furthermore, mean serum concentrations of progesterone were similar to (GnRH-28; P = 0.26) or greater than (GnRH-14; P = 0.01) that of control mares from Day 0 to 46 postbreeding. Data illustrate that continuous administration of native GnRH is a highly efficient option for managing seasonal anovulation in mares and could be effectively used in the breeding industry if a user-friendly delivery option were available.

Pharmacologic application of native GnRH in the winter anovulatory mare, I: frequency of reversion to the anovulatory state following ovulation induction and cessation of treatment.

The continuous, subcutaneous infusion of native GnRH into seasonally anovulatory mares stimulates the synthesis and secretion of LH without pituitary refractoriness, offering opportunities to markedly accelerate the timing of ovulation within the operational breeding season. Herein, we tested the hypothesis that ovarian cycles induced in winter anovulatory mares using continuous administration of native GnRH for 28 days, beginning in either early February or early March (North America) would not revert to an anovulatory state after treatment withdrawal. Anovulatory mares received sham pumps (control) or native GnRH (100 µg/h) for 28 days beginning from February 2 or 3 (GnRH-Feb) or March 2 or 3 (GnRH-Mar). Mean concentrations of LH were five- to seven-fold greater during February in the GnRH-Feb group compared with control and GnRH-Mar groups through February and ending on March 2 or 3. However, concentrations of LH returned to the winter baseline within 3 to 11 days after pump removal and all GnRH-Feb mares failed to remain cyclic after treatment withdrawal. Correspondingly, during March, concentrations of LH in the GnRH-Mar group were greater (P < 0.001) than in the control and GnRH-Feb groups during the 28-day treatment period. Follicular growth and frequency of ovulation (6/10 GnRH-Feb; 9/10 GnRH-Mar, 1/11 controls, respectively) were greater (P < 0.01) in GnRH-treated mares. Ovulatory cycles continued in five of nine GnRH-Mar mares that ovulated, with interovulatory intervals of 15 to 24 days; whereas, three of nine mares had extended (33-42 days) interovulatory intervals and one of nine mares had a persistent CL after cessation of treatment. In summary, continuous administration of native GnRH for 28 days, beginning in early February or March, elevated circulating LH adequately to stimulate follicular growth and ovulation up to 60 days earlier than in untreated controls. However, if continuous, subcutaneous infusion of GnRH is selected as the only pharmacologic or managerial intervention, and mares are not pregnant, treatment must be continued at least until the end of March. This will improve the likelihood of a normal interovulatory interval after treatment withdrawal.

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.

Reproductive seasonality in the mare: neuroendocrine basis and pharmacologic control.

Reproductive seasonality in the mare is characterized by a marked decline in adenohypophyseal synthesis and secretion of LH beginning near the autumnal equinox. Thus, ovarian cycles have ceased in most mares by the time of the winter solstice. Endogenous reproductive rhythms in seasonal species are entrained or synchronized as a result of periodic environmental cues. In the horse, this cue is primarily day length. Hence, supplemental lighting schemes have been used managerially for decades to modify the annual timing of reproduction in the mare. Although a full characterization of the cellular and molecular bases of seasonal rhythms has not been realized in any species, many of their synaptic and humoral signaling pathways have been defined. In the mare, neuroendocrine-related studies have focused primarily on the roles of GnRH and interneuronal signaling pathways that subserve the GnRH system in the regulatory cascade. Recent studies have considered the role of a newly discovered neuropeptide, RF-related peptide 3 that could function to inhibit GnRH secretion or gonadotrope responsiveness. Although results that used native peptide sequences have been negative in the mare and mixed in all mammalian females, new studies that used an RFRP3 antagonist (RF9) in sheep are encouraging. Importantly, despite continuing deficits in some fundamental areas, the knowledge required to control seasonal anovulation pharmacologically has been available for >20 yr. Specifically, the continuous infusion of native GnRH is both reliable and efficient for accelerating reproductive transition and is uniquely applicable to the horse. However, its practical exploitation continues to await the development of a commercially acceptable delivery vehicle.

Secretion of luteinizing hormone into pituitary venous effluent of the follicular and luteal phase mare: novel acceleration of episodic release during constant infusion of gonadotropin-releasing hormone.

We tested the hypothesis that continuous infusion of native GnRH into mares during the estrous cycle, at a dose of 100 µg/h, would elevate circulating concentrations of LH without disrupting the endogenous, episodic pattern of LH release. Ten cyclic mares were assigned to one of two groups (n = 5/group): (1) Control (saline) and (2) GnRH in saline (100 µg/h). On experimental day 0 (3 to 6 d after ovulation), osmotic pumps containing saline or GnRH were placed subcutaneously and connected to a jugular infusion catheter. Blood samples were collected from jugular catheters daily and at 5-min intervals from catheters placed in the intercavernous sinus (ICS) for 8 h on experimental day 4 (luteal phase; 7 to 10 d after ovulation), followed by an additional 6-h intensive sampling period 36 h after PGF(2α)-induced luteal regression (experimental day 6; follicular phase). Treatment with GnRH increased (P < 0.001) concentrations of LH by 3- to 4-fold in the peripheral circulation and 4- to 5-fold in the ICS. Continuous GnRH treatment accelerated (P < 0.01) the frequency of LH release and decreased the interepisodic interval during both luteal and follicular phases. Treatment with GnRH during the luteal phase eliminated the low-frequency, long-duration pattern of episodic LH release and converted it to a high-frequency, short-duration pattern reminiscent of the follicular phase. These observations appear to be unique to the horse. Further studies that exploit this experimental model are likely to reveal novel mechanisms regulating the control of gonadotrope function in this species.

Gene expression in the arcuate nucleus of heifers is affected by controlled intake of high- and low-concentrate diets.

It was hypothesized that a high-concentrate diet fed during early calfhood alters the expression of genes within the arcuate nucleus that subserve reproductive competence. Beef heifers (n = 12) were weaned at approximately 3 mo of age, and after acclimation, were allocated randomly to 1 of 2 nutritional groups: 1) High Concentrate/High Gain (HC/HG), a high concentrate diet fed to promote a gain of 0.91 kg/d; or 2) High Forage/Low Gain (HF/LG), a forage-based diet fed to promote a gain of 0.45 kg/d. Experimental diets were fed under controlled intake for 91 d. At the end of 91 d, heifers were slaughtered by humane procedures, blood samples were collected, brains were removed, liver weights were determined, and rumen fluid was collected for VFA analyses. Tissue blocks containing the hypothalamus were dissected from the brains, frozen, and cut using a cryostat, and frozen sections were mounted on slides. Tissue from the arcuate nucleus (ARC) was dissected from sections for mRNA extraction. Microarray analysis was used to assess genome-wide transcription in the ARC using a 60-mer oligonucleotide 44K bovine expression array. The ADG was greater (P < 0.001) in heifers fed the HC/HG diet than in heifers fed the HF/LG diet. At slaughter, mean propionate to acetate ratios in the ruminal fluid and liver weight as a percentage of BW were increased (P < 0.005) in HC/HG compared with HF/LG heifers. Mean serum concentrations of insulin (P < 0.05) and IGF-1 (P < 0.005) were greater, and leptin tended to be greater (P = 0.1) in HC/HG heifers compared with HF/LG heifers. Approximately 345 genes were observed to be differentially expressed in the HC/HG group with approximately two-thirds of the genes exhibiting increased expression in the HC/HG group. Genes exhibiting decreased expression in the HC/HG group included agouti-related protein and neuropeptide Y, products of which are known to regulate feed intake and energy expenditure. Functional annotation of enriched Gene Ontology terms indicates that a number of biological processes within the hypothalamus are affected by consumption of high-concentrate diets, including those related to control of feed intake, regulation of cellular metabolic processes, receptor and intracellular signaling, and neuronal communication. In summary, dietary treatments shown previously to accelerate the timing of pubertal onset in heifers increased ruminal propionate, promoted enhanced metabolic hormone secretion, and altered gene expression in the ARC.

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.

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.

Developmental changes in hypothalamic Kiss1 expression during activation of the pulsatile release of luteinising hormone in maturing ewe lambs.

Onset of puberty is characterised by a marked increase in the frequency of release of gonadotrophin-releasing hormone (GnRH) and luteinising hormone (LH). The Kiss1 gene plays a critical role in pubertal development, and its product, kisspeptin, stimulates GnRH and LH release. In the present study, we tested the hypothesis that Kiss1 gene expression in the preoptic area (POA) and hypothalamus increases during maturation of the reproductive neuroendocrine axis in association with increased LH pulsatility. Ovariectomised, oestradiol-replaced lambs were euthanised at 25, 30 and 35 weeks of age. Blood samples were collected before euthanasia to characterise the pattern of LH release. Kiss1 mRNA was detected in coronal sections of the POA and hypothalamus and Kiss1-expressing cells were identified on the basis of silver grain density. The mean number of Kiss1-expressing cells in the POA/periventricular (PeV) areas increased from 25 to 30 weeks of age. No further increase at 35 weeks of age was observed, and the changes in Kiss1 expression in the POA/PeV were independent of changes in LH pulse frequency. The mean number of Kiss1-expressing cells in the arcuate (ARC) nucleus did not differ among age groups, although it was greater in the middle ARC of lambs exhibiting increased frequency of LH release. The density of silver grains per cell did not differ among groups in any of the areas studied. The results obtained indicate that the Kiss1 gene is activated in the POA/PeV and ARC of ewe lambs during juvenile development, and that kisspeptin neurones in the middle ARC, in particular, are involved in the acceleration of pulsatile LH release during maturation of the reproductive neuroendocrine axis in ewe lambs.

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.

Neurokinin 3 receptor immunoreactivity in the septal region, preoptic area and hypothalamus of the female sheep: colocalisation in neurokinin B cells of the arcuate nucleus but not in gonadotrophin-releasing hormone neurones.

Recent evidence has implicated neurokinin B (NKB) in the complex neuronal network mediating the effects of gonadal steroids on the regulation of gonadotrophin-releasing hormone (GnRH) secretion. Because the neurokinin 3 receptor (NK3R) is considered to mediate the effects of NKB at the cellular level, we determined the distribution of immunoreactive NK3R in the septal region, preoptic area (POA) and hypothalamus of the ewe. NK3R cells and/or fibres were found in areas including the bed nucleus of the stria terminalis, POA, anterior hypothalamic and perifornical areas, dopaminergic A15 region, dorsomedial and lateral hypothalamus, arcuate nucleus (ARC) and the ventral premammillary nucleus. We also used dual-label immunocytochemistry to determine whether a neuroanatomical basis for direct modulation of GnRH neurones by NKB was evident. No GnRH neurones at any rostral-caudal level were observed to contain NK3R immunoreactivity, although GnRH neurones and fibres were in proximity to NK3R-containing fibres. Because NKB fibres formed close contacts with NKB neurones in the ARC, we determined whether these NKB neurones also contained immunoreactive NK3R. In luteal-phase ewes, 64% +/- 11 of NKB neurones colocalised NK3R. In summary, NK3R is distributed in areas of the sheep POA and hypothalamus known to be involved in the control of reproductive neuroendocrine function. Colocalisation of NK3R in NKB neurones of the ARC suggests a potential mechanism for the autoregulation of this subpopulation; however, the lack of NK3R in GnRH neurones suggests that the actions of NKB on GnRH neurosecretory activity in the ewe are mediated indirectly via other neurones and/or neuropeptides.