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Kisspeptin, neurokinin B, and dynorphin are imperative for GnRH/LH pulsatility and reproductive cyclicity. Neurons co-expressing these neuropeptides, KNDy neurons, within the arcuate nucleus of the hypothalamus (ARC) are positioned to integrate energy balance signals from neuronal and glial cells. Energy balance mediates neurokinin B expression in the ARC and LH pulse amplitude. Dynorphin mediates progesterone negative feedback on GnRH neurons. The hypothesis that the number of KNDy-expressing neurons in the ARC of ewes during the luteal phase of the estrous cycle is influenced by energy balance was tested using ovary-intact, mature ewes fed to lose, maintain, or gain body weight. Fluorescent multiplex immunohistochemistry was employed to identify and quantify neurons expressing a single neuropeptide and co-expressing kisspeptin, neurokinin B, and dynorphin in the ARC. Kaminski et al. (1) reported previously that concentrations of insulin and leptin differed between ewes fed to achieve different body weights and that ewes fed to gain body weight had increased concentrations of progesterone in the luteal phase of the estrous cycle. Moreover, tanycyte density and cellular penetration into the ARC are increased in ewes fed to gain body weight (2). Number of neurons in the ARC expressing kisspeptin (14.9 ± 2.7 neurons, 20.9 ± 3.6 neurons, and 51.5 ± 3.3 neurons in ewes fed to lose, maintain, and gain body weight, respectively), neurokinin B (21.5 ± 3.2 neurons, 31.3 ± 4.3 neurons, and 56.0 ± 3.9 neurons in ewes fed to lose, maintain, and gain body weight, respectively), and dynorphin (10.1 ± 2.4 neurons, 14.9 ± 3.2 neurons, and 33.1 ± 2.9 neurons in ewes fed to lose, maintain, and gain body weight, respectively) protein was increased (P < 0.0001) in ewes fed to gain body weight. Number of KNDy neurons in the ARC expressing kisspeptin, neurokinin B, and dynorphin protein was decreased in ewes fed to lose body weight (1.0 ± 0.5 neurons; P = 0.01) and increased in ewes fed to gain body weight (6.7 ± 0.6 neurons; P = 0.0005) when compared to ewes fed to maintain body weight (3.3 ± 0.7 neurons). These findings suggest that expression of kisspeptin, neurokinin B, and dynorphin protein in the ARC during the luteal phase of the estrous cycle may be influenced by nutritionally-induced alterations in circulating concentrations of progesterone that drive changes in morphology and density of tanycytes. Moreover, these results demonstrate that changes in KNDy neurons within the ARC occur as an adaptation to energy balance, potentially regulated divergently by metabolic milieu. References: (1) Kaminski et al., Theriogenology. 2015 83:808-16. (2) Prezotto et al., Domestic Animal Endocrinology. 2020 Accepted.

The pubertal transition of gonadotropin secretion in pigs is metabolically gated. Kisspeptin (KISS1) and neurokinin B (NKB) are coexpressed in neurons within the arcuate nucleus of the hypothalamus (ARC) and are thought to play an important role in the integration of nutrition and metabolic state with the reproductive neuroendocrine axis. The hypothesis that circulating concentrations of luteinizing hormone (LH) and expression of KISS1 and tachykinin 3(TAC3, encodes NKB) in the ARC of female pigs are reduced with negative energy balance was tested using ovariectomized, prepubertal gilts fed to either gain or lose body weight. Restricted feeding of ovariectomized gilts caused a rapid and sustained metabolic response characterized by reduced concentrations of plasma urea nitrogen, insulin, leptin, and insulin-like growth factor-1 and elevated concentrations of free fatty acids. The secretory pattern of LH shifted from one of low amplitude to one of high amplitude, which caused overall circulating concentrations of LH to be greater in restricted gilts. Nutrient-restricted gilts had greater expression of follicle-stimulating hormone and gonadotropin-releasing hormone receptor, but not LH in the anterior pituitary gland. Expression of KISS1 in the ARC was not affected by dietary treatment, but expression of TAC3 was greater in restricted gilts. These data are consistent with the idea that hypothalamic expression of KISS1 is correlated with the number of LH pulse in pig, and further indicate that amplitude of LH pulses may be regulated by NKB in the gilt.

Three experiments were conducted to investigate the effects of the kisspeptin receptor agonist, 1-adamantanecarbonyl-RF-NH2 (RF9) on the secretion of gonadotropins, the role of estradiol-17β in modulating this response, and the metabolic half-life of RF9 in the mare. During the luteal phase of the estrous cycle (experiment 1), light horse mares were treated with sequential I.V. injections of RF9 (0.2 and 0.4 mg/kg) within a 1-hour interval to determine effects on coincident secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH). Dose-dependent increases (P

Genetic variants associated with traits such as age at puberty and litter size could provide insight into the underlying genetic sources of variation impacting sow reproductive longevity and productivity. Genomewide characterization and gene expression profiling were used using gilts from the University of Nebraska-Lincoln swine resource population ( = 1,644) to identify genetic variants associated with age at puberty and litter size traits. From all reproductive traits studied, the largest fraction of phenotypic variation explained by the Porcine SNP60 BeadArray was for age at puberty (27.3%). In an evaluation data set, the predictive ability of all SNP from high-ranked 1-Mb windows (1 to 50%), based on genetic variance explained in training, was greater (12.3 to 36.8%) compared with the most informative SNP from these windows (6.5 to 23.7%). In the integrated data set ( = 1,644), the top 1% of the 1-Mb windows explained 6.7% of the genetic variation of age at puberty. One of the high-ranked windows detected (SSC2, 12-12.9 Mb) showed pleiotropic features, affecting both age at puberty and litter size traits. The RNA sequencing of the hypothalami arcuate nucleus uncovered 17 differentially expressed genes (adjusted 180 d of age). Twelve of the differentially expressed genes are upregulated in the late pubertal gilts. One of these genes is involved in energy homeostasis (), a function in which the arcuate nucleus plays an important contribution, linking nutrition with reproductive development. Energy restriction during the gilt development period delayed age at puberty by 7 d but increased the probability of a sow to produce up to 3 parities ( < 0.05). Identification of pleotropic functional polymorphisms may improve accuracy of genomic prediction while facilitating a reduction in sow replacement rates and addressing welfare concerns.

Widespread use of artificial insemination in swine requires millions of doses of boar semen each year. Subfertility of boars remains a major constraint, which can impact the reproductive efficiency of thousands of sows, so a better understanding of testicular function is needed in order to develop methods to improve semen production. With this in mind, the effects of RFamide-related peptide 3 (RFRP3) and Gonadotropin-releasing hormone-II (GnRH-II) on gonadotropin secretion and testicular function of pigs are reviewed here. Receptors for RFRP3 are present in the pig hypothalamus, adenohypophysis, and testis. Evidence from in vitro studies indicates that RFRP3 could be a hypophysiotropic hormone in the pig by suppressing secretion of GnRH-I from the hypothalamus and luteinizing hormone (LH) from the pituitary gland; however, effects of RFRP3 on in vivo secretion of LH in pigs are minimal. Within the pig testis, RFRP3 suppresses testosterone secretion by inhibiting steroidogenic enzymes. GnRH-II and its receptor (GnRHR-II) are abundant in pig testes. Interstitial cells express GnRHR-II, and exogenous GnRH-II robustly stimulates secretion of testosterone in boars, despite minimal secretion of LH. Data illustrate that GnRH-II directly stimulates secretion of testosterone from the testes of mature boars. Thus, the primary function of RFRP3 and GnRH-II in the boar appears to be autocrine-paracrine inhibition and stimulation, respectively, of testosterone secretion within the testis. A better understanding of changes in the RFRP3 and GnRH-II systems within the testis during development will provide important clues about how to improve the testicular function of boars.

The blood–brain barrier regulates the transport of molecules that convey global energetic status to the feeding circuitry within the hypothalamus. Capillaries within the median eminence (ME) and tight junctions between tanycytes lining the third ventricle (3V) are critical components of this barrier. Herein, we tested the hypothesis that altering the plane of nutrition results in the structural reorganization of tanycytes, tight junctions, and capillary structure within the medial basal hypothalamus. Proopiomelanocortin (POMC) neuronal content within the arcuate nucleus of the hypothalamus (ARC) was also assessed to test whether reduced nutritional status improved access of nutrients to the ARC, while decreasing the access of nutrients of overfed animals. Multiparous, nongestating ewes were stratified by weight and randomly assigned to dietary treatments offered for 75 d: 200% of dietary recommendations (overfed), 100% of dietary recommendations (control), or 60% of dietary recommendations (underfed). The number of POMC-expressing neurons within the ARC was increased (P ≤ 0.002) in underfed ewes. Overfeeding increased (P ≤ 0.01) tanycyte cellular process penetration and density compared with control and underfeeding as assessed using vimentin immunostaining. Immunostaining of tight junctions along the wall of the 3V did not differ (P = 0.32) between treatments. No differences were observed in capillary density (P = 0.21) or classification (P ≥ 0.47) within the ME. These results implicate that changes within the satiety center and morphology of tanycytes within the ARC occur as an adaptation to nutrient availability.

Mechanisms governing the timing of puberty in pigs are poorly understood. A genome-wide association study for age at first estrus in pigs identified candidate genes including neuropeptide FF receptor 2 (NPFFR2), which is a putative receptor for RFamide-related peptides (RFRP). RFRP has been shown to negatively regulate secretion of reproductive hormones from hypothalamic and pituitary tissue of pigs in culture. Here, the porcine NPFFR2 gene was further screened and four potentially functional variants were identified to be associated with age at first estrus in pigs (1,288 gilts). The RFRP neurons in the porcine hypothalamus were localized in the paraventricular and dorsomedial nuclei with RFRP fibers in the lateral hypothalamic area. There were marked changes in expression of NPFF receptors in the anterior pituitary gland and hypothalamus of gilts beginning with the peripubertal period. The hypothesis that NPFF receptor function is related to secretion of luteinizing hormone (LH) in gilts was tested with various NPFF receptor ligands. The NPFF receptor antagonist RF9 stimulated a pulse-like release of LH in prepubertal gilts. The putative NPFF receptor agonist RFRP3 modestly suppressed LH pulses in ovariectomized (OVX) prepubertal gilts. A porcine-specific RFRP2 failed to have an effect on LH secretion in OVX prepubertal gilts despite its high degree of homology to avian gonadotropin-inhibitory hormone. Results indicate that an RFRP system is present in the pig and that NPFFR2 is important for pubertal onset in gilts. It is not clear if this regulation involves major control of LH secretion or another unknown mechanism.

RFamide-related peptide 3 (RFRP3) has been implicated in regulating reproduction and growth. This regulation appears to be dependent upon sex, species, physiological status, and developmental stage. The objective of the present study was to evaluate the effects of RFRP3 on circulating concentrations of luteinizing hormone (LH) and growth hormone (GH) in mature boars. The hypothesis was RFRP3 would reduce circulating concentrations of LH and increase concentrations of GH. Meishan boars (716.6±2.8 days of age; 125.0±12.4kg BW) were randomly assigned to treatment: saline (n=4) or RFRP3 (8.5mg; n=5). Plasma was collected at 15-min intervals during 3 periods: pre-treatment, treatment, and post-treatment. During the treatment period, saline or RFRP3 were administered at 15-min intervals. Treatment was administered as a loading dose of 5mg RFRP3, followed by seven repeated injections of 0.5mg RFRP3. Pulsatile secretion of LH and GH were not affected by saline treatment. Mean concentrations of LH in RFRP3-treated boars were greater (P

Maternal nutrient restriction during gestation can exert long-term negative effects on offspring health and performance. Arginine supplementation may rescue some of the negative effects elicited by maternal nutrient restriction. We tested the hypothesis that maternal arginine supplementation during gestation would rescue deleterious effects of nutrient restriction on in vitro O2 consumption in the liver and jejunum and hypothalamic protein expression of proopiomelanocortin (POMC), neuropeptide Y (NPY), agouti-related peptide (AgRP), and neuronal nitric oxide synthase (nNOS), and the colocalization of nNOS and active phosphor-signal transducer and activator of transcription 3 (pSTAT3) in female offspring. Multiparous ewes were assigned to dietary treatment at 54 d of gestation: 100% of requirements (Con), 60% of control (Res), or Res plus rumen-protected arginine (Res-Arg; 180 mg/kg). At parturition, offspring were immediately removed from their dam and placed on a common diet. At 54 ± 4 d of age, female lambs (n = 6 per treatment) were weighed, the liver and jejunum were weighed, and samples were collected for in vitro measurement of O2 consumption. The hypothalamus was collected to determine protein expression of POMC, NPY, AgRP, and nNOS, and the colocalization of nNOS and pSTAT3 (n = 3, 4, and 4 for Con, Res, and Res-Arg, respectively). Hepatic consumption of O2 in vitro (mol/min/liver) was decreased (P = 0.04) in the Res and Res-Arg group compared with Con. Intensity of staining for NPY-containing fibers tended to decrease (P = 0.10) in Res and Res-Arg compared with Con. Number of POMC neuronal cells in the arcuate nucleus (ARC) of the hypothalamus decreased (P ≤ 0.03) in the Res group compared with Res-Arg. These observations demonstrate that maternal nutrient restriction decreases energy utilization in the liver and number of POMC cells in the ARC of offspring. Supplementation of arginine to the gestating ewe failed to influence hepatic use of energy in lambs from Res ewes. Numbers of POMC-containing cells were increased in the ARC in lambs from ewes restricted to 60% of nutritional requirements and supplemented with rumen-protected arginine, potentially influencing feeding behavior and hepatic energy metabolism.

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.

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.

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.

To investigate the influence of mater- nal Se supply and plane of nutrition on lamb morbidity, mortality, and passive transfer of IgG, pregnant ewe lambs were used in 2 experiments with 2 × 3 factorial treatment arrangements. Supplementation of Se began at breeding and was either adequate Se (ASe, 9.5 μg/ kg of BW) or high Se (HSe, 81.8 μg/kg of BW) in Exp. 1 or ASe (11.5 μ g/kg of BW) or HSe (77.0 μ g/kg of BW) in Exp. 2. On d 50 or 40 of gestation for Exp. 1 or 2, respectively, ewes were assigned randomly to 1 of 3 nutritional planes: 60% (RES), 100% (control, CON), or 140% (HI) of NRC requirements. This resulted in the following treatments: ASe-RES, ASe-CON, ASe- HI, HSe-RES, HSe-CON, and HSe-HI. Upon parturi- tion, lambs were separated from their dams and serum samples obtained. Lambs were fed artificial colostrum for the first 20 h and then placed on milk replacer and grain pellets until completion of the study (Exp. 1, 57 d; Exp. 2, 21 d). Twenty-four hours after parturition, lamb serum samples were collected for IgG analysis. All lambs were reared similarly and morbidity and mortal- ity assessed. Main effects were considered significant when P ≤ 0.05. In Exp. 1, there was a Se × plane of nutrition interaction (P ≤ 0.01) for lamb morbidity from birth to weaning and for 24-h IgG concentration. Lambs from ASe-RES and HSe-HI ewes were treated more frequently (P < 0.01) for respiratory and gastro- intestinal disease, and lambs from HSe-HI ewes had the smallest (P < 0.01) 24-h serum IgG concentration. In Exp. 1, lambs from HI ewes also had the greatest (P < 0.01) mortality rates from birth to weaning compared with lambs from CON and RES ewes. In Exp. 2, there was an effect (P < 0.01) of maternal plane of nutrition with lambs from RES ewes having increased 24-h IgG compared with lambs from CON and HI ewes. There was no effect of maternal Se supplementation on lamb 24-h IgG in Exp. 2; however, there was a Se × plane of nutrition interaction (P < 0.01) for morbidity. From birth to 21 d of age, lambs from ASe-CON ewes had fewer (P < 0.01) treatment days compared with lambs from any of the other treatment groups. There also tended (P = 0.08) to be an effect of maternal Se supple- mentation on lamb mortality with increased mortality observed in lambs from HSe ewes. Results from the studies show a restricted maternal plane of nutrition can increase lamb serum IgG concentration. Selenium results were not consistent between the 2 experiments and may be due to differences in maternal Se.

The objectives were to investigate effects of nutritional plane and Se supply during gestation on yield and nutrient composition of colostrum and milk in first parity ewes. Rambouillet ewe lambs (n = 84, age = 240 ± 17 d, BW = 52.1 ± 6.2 kg) were allocated to 6 treatments in a 2 × 3 factorial array. Factors included Se [adequate Se (ASe, 11.5 µg/kg of BW) or high Se (HSe, 77.0 µg/kg of BW)] initiated at breeding, and nutritional plane [60 (RES), 100 (CON), or 140% (HIH) of requirements] initiated at d 40 of gestation. Ewes were fed individually from d 40, and lambs were removed at parturition. Colostrum was milked from all ewes at 3 h postpartum, and one-half of the ewes (n = 42) were transitioned to a common diet meeting lactation requirements and mechanically milked for 20 d. Colostrum yield was greater (P = 0.02) for HSe ewes than ASe, whereas CON had greater (P < 0.05) colostrum yield than RES and HIH. Colostrum Se (%) was greater (P < 0.01) for HSe than ASe. Colostrum from ewes fed HSe had less (P = 0.03) butterfat (%), but greater (P ≤ 0.05) total butterfat, solids-not-fat, lactose, protein, milk urea N, and Se than ASe. Colostrum from HIH ewes had greater (P ≤ 0.02) solids-not-fat (%) than RES, whereas RES had greater (P ≤ 0.04) butterfat (%) than CON and HIH. Colostrum from ewes fed the CON diet had greater (P = 0.01) total butterfat than HIH. Total solids-not-fat, lactose, and protein were greater (P < 0.05) in colostrum from CON than RES and HIH. Ewes fed HSe had greater (P < 0.01) milk yield (g/d and mL/d) than ASe, and CON and HIH had greater (P < 0.01) yield than RES. Milk protein (%) was greater (P ≤ 0.01) in RES compared with CON or HIH. Ewes fed HSe had greater (P < 0.01) milk Se (µg/g and mg/d) than ASe on each sampling day. Milk from CON and HIH ewes had greater (P < 0.01) total solids-not-fat, lactose, protein, and milk urea N than RES. Total Se was greater (P = 0.02) in milk from ewes fed the CON diet compared with RES. Somatic cell count and total somatic cells were greater (P ≤ 0.05) in milk from CON than RES. A cubic effect of day (P ≥ 0.01) was observed for milk yield (g and mL). Butterfat, solids-not-fat, lactose, milk urea N, and Se concentration responded quadratically (P ≤ 0.01) to day. Protein (%), total butterfat, and total Se, and somatic cells (cells/mL and cells/d) decreased linearly (P < 0.01) with day. Results indicate that gestational nutrition affects colostrum and milk yield and nutrient content, even when lactational nutrient requirements are met.

To investigate the maternal plane of nutrition and role of Se yeast on foaling variables and passive transfer of IgG, 28 Quarter Horse mares were used in a study with a randomized complete block design. Mares were blocked by expected foaling date and assigned randomly within block to dietary treatments. Dietary treatments were arranged as a 2 x 2 factorial with 2 planes of nutrition, pasture or pasture + grain mix (fed at 0.75% of BW on an as-fed basis) and 2 concentrations of Se yeast (0 or 0.3 mg/kg of DMI). This resulted in 4 treatments: pasture (PA), pasture + Se (PS), pasture + grain mix (PG), and pasture + grain mix + Se (PGS). Assuming DMI at 2% of BW, the mares fed PA and PS received approximately 100% of the calculated NRC (2007) DE requirements, whereas PG and PGS received 120%. Selenium supplementation began 110 d before the estimated foaling date, and all dietary treatments were terminated at parturition. At parturition, foaling variables were recorded. Additionally, placental weight was recorded and 2 samples from each placenta were collected for analysis of DNA, RNA, and protein. Colostrum was obtained for fat, protein, milk urea N, somatic cell count, and IgG analyses. Foal blood samples were collected at 0, 6, 12, 18, and 24 h after parturition for IgG analysis. There was no effect (P >or= 0.21) of Se or plane of nutrition on foaling variables; however, foal BW as a percentage of mare BW tended (P = 0.10) to be reduced in foals from mares on grain mix (PG and PGS; 7.6%) compared with mares not fed grain mix (PA and PS; 8.0%). There was also no effect (P >or= 0.20) of Se or plane of nutrition on placental cell number (mg of DNA/g), potential cellular activity (RNA:DNA), expulsion time, or weight. However, mares fed supplemental Se (PS and PGS) had decreased (P = 0.02) placental cell size (24.1 mg of protein/mg of DNA) compared with mares not fed supplemental Se (PA and PG; 32.5 mg of protein/mg of DNA). There was also no effect (P >or= 0.18) of Se or plane of nutrition on colostral fat, protein, milk urea N, or somatic cell count. However, mares fed grain mix (PG and PGS) had less (P = 0.03) colostral IgG (76.5 g/L) compared with mares not fed grain mix (PA and PS; 126.6 g/L). Foals from mares fed grain (PG and PGS) tended (P = 0.06) to have less overall serum IgG (13.6 g/L) compared with foals from mares not fed grain (PA and PS; 15.3 g/L). These data indicate that the maternal diet during the last one-third of gestation affects placental efficiency and colostral IgG.

To investigate the maternal plane of nutrition and role of Se yeast on muscle Se concen- tration, plasma glutathione peroxidase (Gsh-Px) activ- ity, and colostrum Se concentration in mares and their foals, 28 Quarter Horse mares (465 to 612 kg of BW, and 6 to 19 yr of age) were used in a study with a ran- domized complete block design. Mares were blocked by expected foaling date and randomly assigned to dietary treatments within blocks. Dietary treatments were ar- ranged as a 2 × 2 factorial with 2 planes of nutrition, pasture or pasture + grain mix (fed at 0.75% of BW on an as-fed basis) and 2 concentrations of Se yeast supplementation (0 or 0.3 mg/kg of DMI), resulting in 4 treatments: pasture, pasture + grain mix, pasture + grain mix + Se, or pasture + Se. Mares fed diets of pas- ture and pasture + Se received approximately 100% of the calculated NRC (2007) DE requirements, whereas mares fed diets of pasture + grain mix and pasture + grain mix + Se received 120%. Selenium supplemen- tation began 110 d before the estimated foaling date and treatments were terminated at parturition. Blood and muscle (biopsy) samples were collected on d 0 and then every 14 or 28 d, respectively, thereafter until par- turition. Additionally, BW, BCS, and rump fat (RF) were recorded every 14 d. At parturition, colostrum, foal plasma, and foal muscle samples were collected and sampling continued every 14 d for plasma and every 28 d for muscle until d 56. Mare BW, BCS, and RF were affected by plane of nutrition (P ≤ 0.02), but not by Se supplementation. Mares fed the grain mix had greater (P < 0.05) BW, BCS, and RF measurements throughout the experiment. Mare plasma, muscle, and colostrum Se concentrations were greater (P < 0.01) in mares fed Se. Mares fed the grain mix had greater plasma Se (P = 0.02) than mares on pasture alone. Mare and foal plasma Gsh-Px concentrations were not affected by treatment. Foal plasma and muscle Se con- centrations were greater when dams were fed the sup- plemental grain mix (P = 0.04 and 0.02, respectively) and supplemental Se (P < 0.001). Results indicated that maternal plane of nutrition and Se supplementa- tion affected mare and foal plasma, muscle, and colos- trum Se concentrations, but not Gsh-Px activity.

Three healthy horses were fed the beta-adrenergic agonist feed additive zilpaterol at a dosage of 0.17 mg/kg body weight to study zilpaterol elimination kinetics. Soon after ingestion of zilpaterol, the horses developed skeletal muscle tremors and tachycardia. A 75 to 87.5% reduced dose of zilpaterol was fed to the horses 24 hours after the initial dose; administration was discontinued thereafter. The horses exhibited restlessness, muscle tremors, and profuse sweating 20 to 25 minutes after ingestion of zilpaterol. Tachycardia developed within 40 minutes and took up to 2 weeks to resolve. Muscle tremors lasted up to 1 week. The most pronounced derangements in serum biochemistry were increased activities of lactic dehydrogenase, creatine kinase, and aspartate transferase, indicating muscle damage. The most severely affected horse also had transient azotemia, hematuria, and proteinuria, suggesting renal damage. All three horses recovered without treatment and were clinically normal 2 to 3 weeks after the initial dose of zilpaterol. Because of their anabolic properties, beta-adrenergic feed additives are considered a risk for abuse in performance horses, despite the absence of Food and Drug Administration approval for such use. Oral administration of zilpaterol to horses at the dosage indicated for use in cattle may result in prolonged adverse effects, including tachycardia, muscle tremors, and renal damage.

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.

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.

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 (P0.001) 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 (P0.01) 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.

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.

Three horses were dosed with dietary zilpaterol and the urine concentrations measured from withdrawal day 0 to withdrawal day 21. The analyses were carried out using both enzyme-linked immunosorbent assay (ELISA) and an ultraperformance liquid chromatography with triple-quadrupole-tandem mass spectrometric detection (UPLC-MS/MS). The UPLC-MS/MS method was developed to provide rapid analysis with positive analyte identification by following three product ions and computing the two independent ion ratios. When urinary zilpaterol concentrations were between 0.2 and 2 ng/mL, the ELISA had interday recoveries of 114-120% with coefficients of variation (CV) of