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Sex steroid hormones regulate developmental programming in many tissues, including programming gene expression during prenatal development. While estradiol is known to regulate placentation, little is known about the role of testosterone and androgen signaling in placental development despite the fact that testosterone rises in maternal circulation during pregnancy and in placenta-induced pregnancy disorders. We investigated the role of testosterone in placental gene expression, and focused on androgen receptor (AR). Prenatal androgenization decreased global DNA methylation in gestational day 90 placentomes, and increased placental expression of AR as well as genes involved in epigenetic regulation, angiogenesis, and growth. As AR complexes with histone lysine demethylases (KDMs) to regulate AR target genes in human cancers, we also investigated if the same mechanism is present in the ovine placenta. AR co-immunoprecipitated with KDM1A and KDM4D in sheep placentomes, and AR-KDM1A complexes were recruited to a half-site for androgen response element (ARE) in the promoter region of VEGFA. Androgenized ewes also had increased cotyledonary VEGFA. Finally, in human first trimester placental samples KDM1A and KDM4D immunolocalized to the syncytiotrophoblast, with nuclear KDM1A and KDM4D immunostaining also present in the villous stroma. In conclusion, placental androgen signaling, possibly through AR-KDM complex recruitment to AREs, regulates placental VEGFA expression. AR and KDMs are also present in first trimester human placenta. Androgens appear to be an important regulator of trophoblast differentiation and placental development, and aberrant androgen signaling may contribute to the development of placental disorders.

The objective of this study was to evaluate various equine follicle-stimulating hormone (eFSH) treatment protocols and the effect of “follicle coasting” on ovulation and embryo recovery rates in mares. Cycling mares (n = 40) were randomly assigned to one of four groups 7 days after ovulation: (1) 12.5 mg eFSH twice daily until follicles were 35 mm or larger; (2) 12.5 mg eFSH twice daily until follicles were 32 mm or larger; (3) 12.5 mg eFSH twice daily for 3.5 days followed by 12.5 mg eFSH enriched with luteinizing hormone (LH) twice daily until follicles were 35 mm or larger; and (4) 25 mg eFSH once daily until follicles were 32 mm or larger. Mares in groups 1 and 3 were injected with human chorionic gonadotropin (hCG) (2500 IU intravenously) at the end of eFSH treatment, whereas mares in groups 2 and 4 were given hCG approximately 42 and 54 hours, respectively, after the last eFSH treatment (“follicle coasting”). Nonsurgical embryo collection was performed 6.5 to 7.5 days after ovulation. Each mare experienced a nontreated estrous cycle before being reassigned to a second treatment. Ovulation rates for mares in treatment groups 1 to 4 were 3.3 ± 0.4, 4.1 ± 0.4, 3.5 ± 0.4, and 2.8 ± 0.4 (mean ± SEM; P P > .05). The overall embryo recovery rate was 2.1 ± 1.5 embryos per flush. In summary, ovulation rate was higher for mares treated with eFSH (3.4 ± 0.4) compared with non-treated controls (1.1 ± 0.2). Ovulation rate in mares in which hCG was delayed (follicle coasting) was higher ( P

Summary Cryosurvival of cells is reduced if the cooling rate used is suboptimal. If cells cool too rapidly, intracellular water will freeze, causing intracellular ice crystals. However, if spermatozoa are cooled too slowly, excessive cellular dehydration occurs, causing irreversible damage to cellular compartments. In addition, cryoprotectants are added to the freezing diluent to protect cells from damage during cryopreservation. This study was conducted to determine the optimal cooling rate for stallion spermatozoa frozen in the presence of three different cryoprotectants. Spermatozoa were frozen in a skim milk, egg yolk diluent containing 4% glycerol, and ethylene glycol or dimethyl formamide at 10 different cooling rates ranging from 5°C/min to 50°C/min. The percentage of viable spermatozoa was higher for spermatozoa cooled at 10°C/min than at 50°C/min ( P P

The objective of Experiment 1 was to determine a dose and frequency of gonadotropin-releasing hormone (GnRH) antagonist administration to effectively suppress serum luteinizing hormone (LH) concentration and to delay ovulation when administered to mares. The objectives of Experiment 2 were 1) to determine the effects of subcutaneous or intravenous administration of a GnRH antagonist or oral altrenogest on serum LH concentration in the estrual mare; and 2) to determine the effectiveness of human chorionic gonadotropin (hCG) in inducing ovulation in mares with suppressed LH concentrations. In Experiment 1, mares ( N = 20) were randomly assigned and treated with either 5% mannitol (control, single subcutaneous injection, 1 mL, at time 0; n = 5); low-dose GnRH antagonist (single subcutaneous injection, 0.01 mg/kg, at time 0; n = 5); frequent low-dose GnRH antagonist (subcutaneous injections, 0.01 mg/kg, at 0, 6, 18, and 24 hours; n = 5); or high-dose GnRH antagonist (single subcutaneous injection, 0.04 mg/kg, at time 0; n = 5). Both the frequent low-dose and high-dose GnRH antagonist treatments resulted in significantly lower LH concentrations compared with controls at 90, 102, and 114 hours after treatment ( P P P

Practical application of sex-selected spermatozoa in the horse industry would be greatly improved by the ability to develop simplified methods for shipping, storing, and inseminating sex-selected spermatozoa. Acceptable pregnancy rates have been achieved using fresh sex-sorted stallion sperm, however many stallion owners are reluctant to send their stallions to the sorter location for collection during the breeding season. Furthermore, the technology would be more applicable if the hysteroscopic insemination technique was not necessary for adequate pregnancy rates. Hysteroscopic insemination requires expensive equipment and specially trained personnel. In the present study, stallion sperm were sex-sorted after being stored at either 5 degrees C or 15 degrees C for 18 h. Twenty million sex-sorted sperm were then inseminated using one of two insemination techniques: the hysteroscopic method or the rectally guided, deep-uterine technique. Results were determined based on 16-day pregnancy status. A first-cycle pregnancy rate of 72% (18/25) was achieved when sperm were shipped at 15 degrees C, sex-sorted, and then inseminated using the hysteroscopic method. With these results, it can be concluded that stallions are not necessary at the sorter location to achieve acceptable fertility with sex-sorted sperm. There was a tendency for more mares to become pregnant when sperm were shipped at 15 degrees C prior to sorting, when compared to shipment at 5 degrees C. Similarly, there was a tendency for more mares to become pregnant when hysteroscopic insemination was utilized, when compared to the rectally guided, deep-uterine technique. These trends suggest that if larger group numbers were available, significant differences between the treatments may be revealed.

Practical estrus synchronization schemes are needed for mares. The Ovsynch synchronization protocol for cattle involves the administration of gonadotropin-releasing hormone (GnRH) to induce ovulation or luteinization of dominant follicles during the luteal phase and prostaglandin 7 days later to cause regression of any luteal tissue and development of a preovulatory follicle. An Ovsynch-type synchronization program potentially could be developed for horses if luteinization or ovulation of diestrous follicles occurred in response to GnRH treatment. The objective of this study was to determine if administration of the GnRH agonist, deslorelin acetate, on Day 8 or 12 postovulation would induce luteinization or ovulation of diestrous follicles in the mare. The model used was cycling mares maintained in an artificial luteal phase by administration of a synthetic progestin following prostaglandin-induced luteal regression. On the day of ovulation, 21 light horse mares were randomly assigned to one of three groups: (1) no GnRH, altrenogest from Days 5 to 15 postovulation with prostaglandin on Day 15; (2) GnRH on Day 8, altrenogest from Days 5 to 15 with prostaglandin given on Day 6 to induce luteolysis of the primary corpus luteum, an implant containing 2.1mg of deslorelin acetate inserted on Day 8 and removed on Day 10, with a second prostaglandin treatment on Day 15; (3) GnRH on Day 12, altrenogest from Days 9 to 19, prostaglandin on Day 10, a deslorelin acetate implant injected on Day 12 (subsequently removed on Day 14), and a second dose of prostaglandin administered on Day 19. Follicular development was monitored every other day from Day 5 until a 30-mm sized follicle was observed, and then daily to detection of ovulation. Serum progesterone concentrations were determined daily for 12 consecutive days. Progesterone concentrations in Group 1 remained elevated until approximately Day 12 postovulation. Prostaglandin administration on Day 15 resulted in complete luteolysis in all seven mares. In Group 2, progesterone concentrations in six of seven mares declined to baseline after prostaglandin treatment. No increase in serum progesterone was noted in any of the six mares that were given GnRH on Day 8, including three mares that had diestrous follicles > or =30mm in diameter at the time of treatment. Similarly, progesterone concentrations in six of seven mares in Group 3 declined to baseline after prostaglandin and there was no increase in progesterone after administration of GnRH on Day 12. No ultrasound evidence of luteinization or ovulation of diestrous follicles were noted after GnRH administration in any mares of Group 2 or 3. In conclusion, administration of the GnRH agonist deslorelin acetate to mares failed to induce luteinization or ovulation of diestrous follicles. Consequently, the Ovsynch program (as used in cattle) has little efficacy for synchronization of estrus in mares.

The ability to ship cooled stallion sperm for subsequent freezing at a facility specializing in cryopreservation would be beneficial to the equine industry. Stallion sperm has been centrifuged, cooled to 5 degrees C for 12 h, and frozen without a detrimental effect on motility in a previous study; however, no fertility data were available. Experiment 1 compared the post-thaw motility of sperm cooled for 18 h at 15 or 5 degrees C at either 400 or 200 x 10(6) sperm/mL and then frozen. Storage temperature, sperm concentration, or the interaction of temperature and concentration had no effect on total (TM) and progressive motility (PM) after cooling. Post-thaw TM and PM were higher for control than (P < 0.05) for treated samples. There was no difference in post-thaw TM and PM due to temperature or concentration. Experiment 2 further evaluated procedures for cooling before freezing. Ejaculates were either cooled to 5 degrees C for 18 h and centrifuged, centrifuged at room temperature and then cooled to 5 degrees C for 18 h before freezing, or centrifuged and frozen immediately (control). There was no difference among treatments on post-thaw TM or PM. In Exp. 3, mares were inseminated with semen that had been extended in skim milk-egg yolk without glycerol, centrifuged, resuspended at 200 x 10(6) sperm/mL, cooled to 5 degrees C for 18 h, and then frozen or not cooled for 18 h before freezing (control). Pregnancy rates did not differ for mares receiving semen cooled and then frozen (21 of 30, 70%) or semen frozen directly without prior cooling (16 of 30, 53%). In summary, a procedure was developed for cooling stallion sperm for 18 h before freezing without a resultant decrease in fertility.

The effects of a single or double regimen of exogenous progesterone and estradiol-17beta (P/E, total dose 300 mg P/20 mg E) were investigated in 50 postparturient Quarter Horse mares. In Trial 1, at 1 and 24 h after foaling, mares were injected with progesterone (150 mg) and estradiol-17beta (10 mg) (n = 7) or 0.9% NaCl (control, n = 13). In Trial 2, within 12 h after foaling, mares were injected with progesterone (300 mg) and estradiol-17beta (20 mg) (n = 13) or 0.9% NaCl (control, n = 17). Mares were examined daily by palpation per rectum and transrectal ultrasonography to determine the day of ovulation. The largest cross sectional diameters of each uterine horn and uterine body were measured ultrasonographically on Day 15 postpartum. Mean uterine diameters did not differ between treatment groups (P > 0.05) in Trial 1, Trial 2 or for combined data for both Trials 1 and 2. For mares bred on the first postpartum estrus pregnancy rates did not differ (P > 0.05) between treatment groups (16/18, 89%) and controls (22/30, 81%) nor was there a difference in mean day to first postpartum ovulation (P > 0.05) between treated and control groups in Trial 1, Trial 2 or Trials 1 and 2 combined. However, fewer (P < 0.05) total P/E treated mares (0/20) ovulated prior to Day 10 postpartum than did control mares (6/30). Variance in days to ovulation was lower (P < 0.05) for P/E treated mares (var = 3.73 days) than for control mares (var = 7.64 days) for data combined from Trials 1 and 2.

Immunocontraception with porcine ZP (pZP) can be an effective means of fertility control in feral horses. Previous studies suggest that antibodies produced after pZP vaccination may both inhibit fertilization and cause follicular dysgenesis. Zonastat-H, PZP-22, and SpayVac are three pZP vaccines proposed for use in horses. Although all these vaccines contain the pZP antigen, variations in antigen preparation and vaccine formulation lead to differences in antigenic properties among them. Likewise, despite numerous efficacy and safety studies of Zonastat-H and PZP-22, the contraceptive mechanisms of SpayVac remain unclear. The preparation of pZP for SpayVac is thought to include more nonzona proteins, making it less pure than the other two vaccines. This may result in increased antigenicity of the vaccine. We therefore investigated the immunoreactivity of serum antibodies from SpayVac-vaccinated mares to equine zona protein. Western blot analyses revealed an immunoreactivity of these antibodies to protein isolated from mature equine oocytes, ZP, follicular tissues, and ovarian tissues. Immunohistochemical analyses were used to locate the binding of serum antibodies to the ZP of immature oocytes in ovarian stromal tissue. We also found serum antibodies from SpayVac-treated mares to be predominantly specific for zona protein 3. Collectively, our results suggest a model where serum antibodies produced in response to SpayVac vaccination are immunoreactive to equine zona protein in vitro. Our study lends insight into the contraceptive mechanisms underlying the infertility observed after SpayVac vaccination.

Cryopreservation of equine embryos with conventional slow-cooling procedures has proven challenging. An alternative approach is vitrification, which can minimize chilling injuries by increasing the rates of cooling and warming. The open pulled straw (OPS) and cryoloop have been used for very rapid cooling and warming rates. The objective of this experiment was to compare efficacy of vitrification of embryos in OPS and the cryoloop to conventional slow cool procedures using 0.25 mL straws. Grade 1 or 2 morulae and early blastocysts (or = 300 microm in diameter) were recovered from mares on Day 6 or 7 post ovulation. Twenty-seven embryos were assigned to three cryopreservation treatments: (1) conventional slow cooling (0.5 degrees C/min) with 1.8 M ethylene glycol (EG) and 0.1 M sucrose, (4) vitrification in OPS in 16.5% EG, 16.5% DMSO and 0.5 M sucrose, or (3) vitrification with a cryoloop in 17.5% EG, 17.5% DMSO, 1 M sucrose and 0.25 microM ficoll. Embryos were evaluated for size and morphological quality (Grade 1 to 4) before freezing, after thawing, and after culture for 20 h. In addition, propidium iodide (PI) and Hoechst 33342 staining were used to assess percent live cells after culture. There were no differences (P0.1) in morphological grade or percent live cells among methods. Mean grades for embryos after culture were 2.9 +/- 0.2, 3.1 +/- 0.1, and 3.3 +/- 0.2 for conventional slow cooling, OPS and cryoloop methods, respectively. Embryo grade and percent live cells were correlated, r = 0.66 (P0.004). Thus OPS and the cryoloop were similarly effective to conventional slow-cooling procedures for cryopreserving small equine embryos.

A recent report suggested administration of altrenogest during the follicular phase could postpone ovulation. Based on these results, two questions were generated. We first hypothesized that by initiating a altrenogest treatment earlier in the estrous cycle, a greater and/or more consistent delay in ovulation would result. Second, we hypothesized that exposure to elevated progestin concentrations might alter viability of the ovulatory follicle and oocyte. The focus of the first experiment was to determine if initiation of altrenogest treatment at different stages of the estrous cycle would yield a more predictable time to ovulation, whereas the second experiment was designed to determine whether mares receiving altrenogest during estrus had compromised fertility. In the first experiment thirty mares of mixed light breed, ranging in age from 5-15 years, were randomly assigned to one of three groups. The two treated groups received altrenogest (0.088 mg/kg of body weight) for two days once a follicle of 30 or 35 mm in diameter was detected. Control mares were not treated. Mares treated with altrenogest whether initiated at the detection of a 30 or 35 mm follicle demonstrated similar (P>.05) day to ovulation interval when adjusted to 35 mm (5.4 and 5.6 days, respectively). Both treated groups demonstrated a delayed interval (P

MicroRNAs (miRNAs) are small, non-coding RNAs that are differentially expressed throughout the body, including the endometrium and chorion during gestation. Although they are produced by individual tissues, many of these miRNAs are excreted into circulation, either within exosomes or attached to protein carrier molecules. Circulating miRNAs are surprisingly stable, with an average half-life of 5 days. These characteristics suggest that circulating miRNAs have the potential to be powerful biomarkers following the characterisation of the normal population of miRNAs during pregnancy. Additionally, evaluation techniques must be considered, as some miRNAs are extremely sensitive to varying collection and analysis techniques. To evaluate the population of miRNAs in both pregnant and nonpregnant mares, whole blood was collected from mares at 10 months of gestation (n = 3), as well as at 9 days of diestrus (n = 3 for microarray; n = 4 for qPCR). Blood was collected using PAXgene blood miRNA kits (Qiagen, Valencia, CA, USA), and then stored at –20°C until use. RNA extraction was performed following the manufacturer’s instructions. Two different analysis platforms were compared. Quantitative RT-PCR (Lightcycler 480; Roche, Indianapolis, IN, USA) was used to evaluate 346 distinct miRNAs, whereas microarray (LC Sciences, Houston, TX, USA) was used to evaluate 275 miRNAs. In total, 222 of these miRNAs were analysed on both platforms for comparison purposes. Because of the large number of miRNAs evaluated, the robust response screening analysis method (JMP ver. 11.1; SAS Institute Inc., Cary, NC, USA) was used, and multiple comparisons were based upon a false discovery rate of P

Maternal recognition of pregnancy is a physiological process that primarily describes endometrial responses to a conceptus. Recognition of a conceptus prevents the release of prostaglandin F(2α) , thereby ensuring survival of the corpus luteum and continued progesterone production. Exactly how this occurs in the mare is poorly understood. Because prostaglandin F(2α) is a pro-inflammatory hormone, we hypothesized that differential gene expression in the endometrium at the time of maternal recognition reflects an anti-inflammatory event leading to decreased prostaglandin F(2α) secretion. Mares were inseminated, and endometrial biopsies were recovered from pregnant mares on Day 18 post-ovulation. In subsequent estrous cycles, mares were not inseminated and Day 18 post-ovulation endometrial biopsies were collected (non-pregnant control, matched per individual). Endometrial gene expression profiles were examined by screening an Affymetrix equine GeneChip containing probes specific for genes related to inflammatory processes. Microarray analysis revealed 118 genes that were up-regulated and 93 genes that were down-regulated (P 0.001) at least 1.5-fold in the endometrium of pregnant versus non-pregnant mares. Quantitative, real-time RT-PCR confirmed the microarray results for three up-regulated genes homologous to TSC22D3, PPAPDC2, and KLF6, and three down-regulated genes homologous to ESR1, MARCKSL1, and EPSTI1 (P 0.05). It is concluded that the presence of the equine embryo induces differential gene expression in the endometrium of Day 18 pregnant mares, and that these genes are associated with inflammatory processes and pathways involving cellular growth and proliferation. The results from this study provide important new insights into endometrial gene expression in response to early equine pregnancy.

Interferon tau (IFNT) from the ovine conceptus has paracrine actions on the endometrium that alter release of prostaglandin F(2alpha) (PGF) and protect the corpus luteum (CL). Antiviral activity in uterine vein blood and expression of interferon-stimulated genes (ISGs) in CL is greater in pregnant than in nonpregnant ewes. We hypothesized that IFNT contributes to antiviral activity in uterine vein blood and has endocrine actions on the CL. Preadsorption of IFNT with antiserum against recombinant ovine (ro) IFNT revealed that antiviral activity in uterine vein blood from pregnant ewes was mediated by IFNT. Endocrine actions of IFNT were examined after infusing either roIFNT or bovine serum albumin (BSA; 200 microg/24 h; mini-osmotic pump) into the uterine vein of nonpregnant ewes from Day 10 to Day 11 postestrus. The abundance of ISG15 mRNA and protein was greater in CL (P0.05) from ewes receiving 24-h roIFNT infusion compared to that from ewes receiving 24-h BSA infusion. Injection of PGF at 12 h following insertion of mini-osmotic pumps resulted in a decline in serum progesterone concentrations 6 through 12 h later in BSA-infused ewes; however, in roIFNT-infused ewes, a similar decline in progesterone concentrations at 6 h was followed by recovery to control values at 12 h. Ewes then received infusions (200 microg/day) of either roIFNT or BSA for 7 days beginning on Day 10 of the estrous cycle. All BSA-infused ewes returned to estrus by Day 19, whereas 80% of roIFNT-infused ewes maintained luteal-phase concentrations of progesterone through Day 32. In conclusion, IFNT is released from the uterus into the uterine vein and acts through an endocrine mechanism to induce ISGs in the CL and delay luteolysis.

There is increasing evidence that the corpus luteum has an important role in regulating its own demise. A series of experiments was performed to study the effects of luteal concentrations of progesterone on the functions of steroidogenic luteal cells. In the first experiment, steroidogenic small luteal cells (SLCs) were separated from endothelial cells, and it was determined that it was the SLCs that contained receptors for oxytocin. Treatment with progesterone (95 muM) for as little as 1 h decreased (P0.05) the percentage of SLCs responding to oxytocin (10 muM) with an increase in intracellular concentrations of calcium, and this effect continued for the duration of the experiment. In a second experiment, the response to oxytocin was increased (P0.05) by 3 h (but not 1 h) following progesterone removal, with a further increase by 16 h. The ability of 1 muM prostaglandin F(2 alpha) (PGF(2 alpha)) to increase intracellular concentrations of calcium was also decreased (P0.05) by progesterone treatment. By 3 h following removal of progesterone, the percentage of steroidogenic large luteal cells (LLCs) responding to PGF(2 alpha) was increased and not different from that observed in cells 16 h after progesterone removal. Finally, cyclodextrins (methyl-beta cyclodextrin [M beta CD]) were used to remove cholesterol from the plasma membrane of luteal cells, and M beta CD loaded with cholesterol was used to put cholesterol back into the plasma membrane of progesterone-treated cells. Treatment with M beta CD reduced (P0.05) the responsiveness of SLCs to oxytocin and LLCs to PGF(2 alpha). Use of cholesterol-loaded M beta CD returned the responsiveness of both SLCs and LLCs treated with progesterone to that observed in vehicle (no progesterone)-treated controls. In summary, intraluteal concentrations of progesterone inhibit the ability of oxytocin to increase intracellular concentrations of calcium in SLCs and the ability of PGF(2 alpha) to increase intracellular concentrations of calcium in LLCs. The highest concentration of progesterone appears to act by influencing cholesterol content of the luteal cell membranes.

Zonadhesin is a rapidly evolving protein in the sperm acrosome that confers species specificity to sperm-zona pellucida adhesion. Though structural variation in zonadhesin likely contributes to its species-specific function, the protein has not previously been characterized in organisms capable of interbreeding. Here we compared properties of zonadhesin in several animals, including the horse (Equus caballus), donkey (E. asinus), and Grevy's zebra (E. grevyi) to determine if variation in zonadhesin correlates with ability of gametes to cross-fertilize. Zonadhesin localized to the apical acrosomes of spermatozoa from all three Equus species, similar to its localization in other animals. Likewise, in horse and donkey testis, zonadhesin was detected only in germ cells, first in the acrosomal granule of round spermatids and then in the developing acrosomes of elongating spermatids. Among non-Equus species, D3-domain polypeptides of mature, processed zonadhesin varied markedly in size and detergent solubility. However, zonadhesin D3-domain polypeptides in horse, donkey, and zebra spermatozoa exhibited identical electrophoretic mobility and detergent solubility. Equus zonadhesin D3-polypeptides (p110/p80 doublet) were most similar in size to porcine and bovine zonadhesin D3-polypeptides (p105). Sequence comparisons revealed that the horse zonadhesin precursor's domain content and arrangement are similar to those of zonadhesin from other large animals. Partial sequences of horse and donkey zonadhesin were much more similar to each other (>99% identity) than they were to orthologous sequences of human, pig, rabbit, and mouse zonadhesin (52%–72% identity). We conclude that conservation of zonadhesin D3-polypeptide properties correlates with ability of Equus species to interbreed.

The purpose of the present study was to evaluate the effects of kisspeptin (KiSS) on LH and FSH secretion in the seasonally estrous mare and to examine the distribution and connectivity of GnRH and KiSS neurons in the equine preoptic area (POA) and hypothalamus. The diestrous mare has a threshold serum gonadotropin response to iv rodent KiSS decapeptide (rKP-10) administration between 1.0 and 500 μg. Administration of 500 μg and 1.0 mg rKP-10 elicited peak, mean, and area under the curve LH and FSH responses indistinguishable to that of 25 μg GnRH iv, although a single iv injection of 1.0 mg rKP-10 was insufficient to induce ovulation in the estrous mare. GnRH and KiSS-immunoreactive (ir) cells were identified in the POA and hypothalamus of the diestrous mare. In addition, KiSS-ir fibers were identified in close association with 33.7% of GnRH-ir soma, suggesting a direct action of KiSS on GnRH neurons in the mare. In conclusion, we are the first to reveal a physiological role for KiSS in the diestrous mare with direct anatomic evidence by demonstrating a threshold-like gonadotropin response to KiSS administration and characterizing KiSS and GnRH-ir in the POA and hypothalamus of the diestrous horse mare.

Experiments were conducted to further our understanding of the cellular and molecular mechanisms that regulate luteal function in ewes. Inhibition of protein kinase A (PKA) reduced (P < 0.05) secretion of progesterone from both small and large steroidogenic luteal cells. In addition, the relative phosphorylation state of steriodogenic acute regulatory protein (StAR) was more than twice as high (P < 0.05) in large vs small luteal cells. Large steroidogenic luteal cells appear to contain constitutively active PKA and increased concentrations of phosphorylated StAR which play a role in the increased basal rate of secretion of progesterone. To determine if intraluteal secretion of prostaglandin (PG) F2alpha was required for luteolysis, ewes on day 10 of the estrous cycle received intraluteal implants of a biodegradable polymer containing 0, 1 or 10 mg of indomethacin, to prevent intraluteal synthesis of PGF2alpha. On day 18, luteal weights in ewes receiving 1 mg of indomethacin were greater (P < 0.05) than controls and those receiving 10 mg were greater (P < 0.05) than either of the other two groups. Concentrations of progesterone in serum were also increased (P < 0.05) from days 13 to 16 of the estrous cycle in ewes receiving 10 mg of indomethacin. Although not required for decreased production of progesterone at the end of the cycle, intraluteal secretion of PGF2alpha appears to be required for normal luteolysis. To ascertain if oxytocin mediates the indirect effects of PGF2alpha on small luteal cells, the effects of 0, 0.1, 1 or 10 mM oxytocin on intracellular concentrations of calcium were quantified. There was a dose-dependent increase (P < 0.05) in the number of small luteal cells responding to oxytocin. Thus, oxytocin induces increased calcium levels and perhaps apoptotic cell death in small luteal cells. Concentrations of progesterone, similar to those present in corpora lutea (approximately 30 microg/g), prevented the increased intracellular concentrations of calcium (P < 0.05) stimulated by oxytocin in small cells. In large luteal cells the response to progesterone was variable. There was no consistent effect of high quantities of estradiol, testosterone or cortisol in either cell type. It was concluded that normal luteal concentrations of progesterone prevent the oxytocin and perhaps the PGF2alpha-induced increase in the number of small and large luteal cells which respond to these hormones with increased intracellular concentrations of calcium. In summary, large ovine luteal cells produce high basal levels of progesterone, at least in part, due to a constituitively active form of PKA and an enhanced phosphorylation state of StAR. During luteolysis PGF2alpha of uterine origin reduces the secretion of progesterone from the corpus luteum, but intraluteal production of PGF2alpha is required for normal luteolysis. Binding of PGF2alpha to receptors on large luteal cells stimulates the secretion of oxytocin which appears to activate PKC and may also inhibit steroidogenesis in small luteal cells. PGF2alpha also activates COX-2 in large luteal cells which leads to secretion of PGF2alpha. Once intraluteal concentrations of progesterone have decreased, oxytocin binding to its receptors on small luteal cells also results in increased levels of intracellular calcium and presumably apoptosis. Increased secretion of PGF2alpha from large luteal cells activates calcium channels which likely results in apoptotic death of this cell type.

MicroRNAs (miRNAs) are a class of small endogenous single stranded, non-coding RNA molecules that have an important role in several biological processes such as gene regulation, proliferation, apoptosis and metabolism. Emerging research in humans and laboratory animals has shown that miRNAs play important roles in regulating genes associated with type II diabetes and metabolic syndrome, and that the levels of specific miRNAs circulating in the bloodstream can serve as potential biomarkers for the diagnosis and prognosis of these diseases. We hypothesized that insulin resistant horses would have a different circulating miRNA profile than those who are healthy. Fifteen non-pregnant mares housed at the Virginia Tech Middleburg Agricultural Research and Extension Center were evaluated for insulin sensitivity with the frequent sampling intravenous glucose tolerance test to determine insulin sensitivity. Six mares representing the most insulin-sensitive (IS, n 1⁄4 3) and insulinresistant (IR, n 1⁄4 3) states, and paired for age, weight, and body condition, underwent miRNA profiling. Serum samples were collected, miRNA was extracted and microarray analysis was performed to investigate the presence and relative amount of 340 equine miRNAs. Paired t-tests were performed to compare miRNA between groups. Confirmation by qRT-PCR clearly demonstrated that miRNA was present in the serum of all animals, with 5 miRNAs present only in IS mares. Included in the latter are mir-302, a described inhibitor of adipogenesis and hepatic insulin resistance; mir-208a,implicated in cardiac metabolism and regulation of metabolic genes involved in energy expenditure; and mir-381, previously shown to be upregulated in obese sheep and their offspring. Eight of the 82 miRNAs recovered from all samples demonstrated differential expression (P < 0.10) between IS and IR groups; 3 were upregulated and 5 were downregulated in IR relative to IS horses. The novel results of this preliminary study suggest a potential new tool that could be developed for the diagnosis and treatment of metabolic syndrome in horses.

Synchronization of oestrus in mares remains a challenge to practitioners using Assisted Reproductive Technologies. The research objective was to compare adverse reactions and reproductive parameters in mares treated with different sources of progestins for oestrus synchronization. Mixed breeds of mares with a mean age of 3 years (range 2–20) were used. Groups were: 1) altrenogest 0.044 mg kg–1 BW PO daily for 10 days (D) (Regumate, Merck, White House Station, NJ, USA) (n = 30), 2) Long-acting progesterone (LAP4) (BET Pharmacy, Lexington, KY, USA) (n = 30) 10 cc IM once in the neck, and 3) Controlled intravaginal drug release (CIDR-B 1.9 g P4; Zoetis, Kirkland, QC, Canada) (n = 15) vaginal insert for 10 days. Mares were randomly assigned to treatment and evaluated using transrectal reproductive ultrasonography on Days 1 (treatment initiation), 5, 10, and daily during oestrus until ovulation (Ov). On Day 10, mares were given 250 µg of cloprostenol (PG) (Estrumate, Merck) IM, and when a follicle (F) >35 mm was detected 2500 IU hCG (Chorulon, Intervet, Millsboro, MD, USA) was administered IM. Adverse reactions were scored as follows: Regumate – any reaction at any time; LAP4 – the injection site was inspected and scored as: 0, no reaction; 1, mild slight raised area; 2, moderate reaction 5 cm; 3, severe reaction 6–10 cm; 4, very severe reaction >10 cm on Day 5 post-injection; CIDR categorical scores were: 0, no discharge; 1, mild vaginal discharge on CIDR at withdrawal; 2, moderate discharge on tail or vulva; 3, severe urine scald or visible discharge; 4, very severe thickened inflamed skin from urine scald or discharge. Chi-squared test at P

Recent studies demonstrated that zwitterionic buffers could be used for satisfactory storage of equine embryos at 5 degrees C. The success of freezing embryos is dependent upon size and stage of development. Morulae and blastocysts

The objectives were to compare embryo development rates after transfer into inseminated recipients, vitrified thawed oocytes collected from super-stimulated versus non-stimulated mares. In vivo matured oocytes were collected by transvaginal, ultrasound guided follicular aspiration from super-stimulated and non-stimulated mares 24-26 h after administration of hCG. Oocytes were cultured for 2-4 h prior to vitrification. Cryoprotectants were loaded in three steps before oocytes were placed onto a 0.5-0.7 mm diameter nylon cryoloop and plunged directly into liquid nitrogen. Oocytes were thawed and the cryoprotectant was removed in three steps. After thawing, oocytes were cultured 10-12 h before transfer into inseminated recipients. Non-vitrified oocytes, cultured 14-16 h before transfer, were used as controls. More oocytes were collected from 23 non-stimulated mares (20 of 29 follicles), than 10 super-stimulated mares (18 of 88 follicles; P < 0.001). Of the 20 oocytes collected from non-stimulated mares, 12 were vitrified and 8 were transferred as controls. After thawing, 10 of the 12 oocytes were morphologically intact and transferred into recipients resulting in one embryonic vesicle on Day 16 (1 of 12 = 8%). Fourteen oocytes from super-stimulated mares were vitrified, and 4 were transferred as controls. After thawing, 9 of the 14 oocytes were morphologically intact and transferred into recipients resulting in two embryonic vesicles on Day 16 (2 of 14 = 14%). In control transfers, 7 of 8 oocytes from non-stimulated mares and 3 of 4 oocytes from super-stimulated mares resulted in embryonic vesicles on Day 16. The two pregnancies from vitrified oocytes resulted in healthy foals.

Since the mechanism by which equine maternal recognition of pregnancy occurs remains unknown, and gene expression evaluations have failed to uncover any obvious candidates, we have begun to explore the role of small ncRNAs. Small ncRNAs (< 200 nt) do not code for protein and are involved in regulation of RNA processing and function. We postulate these small ncRNA, i.e., microRNA (miRNA) and small nucleolar RNA (snoRNA), play a role in regulation of equineMRP. For example, miRNAs are known to regulate mRNA through inhibition of translation or RNA degradation. SnoRNAs are responsible for rRNA processing through methylation and / or pseudouridylation, as well as alternative splicing. Our previous data revealed differences in miRNA content in serum of pregnant versus non-pregnant mares during the period of maternal recognition of pregnancy. The objective of this study was to identify all small ncRNA present in equine endometrial tissue during maternal recognition of pregnancy. This experiment utilized a crossover design with each mare serving as a pregnant and non-mated control (n 1⁄4 3/day) at days 11 and 13 post-ovulation. Mares were randomly assigned to a collection day and each provided endometrial samples from a pregnant and non-pregnant cycle. Endometrial biopsies were snap frozen and stored until RNA isolation. Total RNA was isolated using homogenization in Tri Reagent, and precipitated with chloroform

Mares are generally inseminated with 500 million progressively motile fresh sperm and approximately 1 billion total sperms that have been cooled or frozen. Development of techniques for low dose insemination would allow one to increase the number of mares that could be bred, utilize stallions with poor semen quality, extend the use of frozen semen, breed mares with sexed semen and perhaps reduce the incidence of post-breeding endometritis. Three low dose insemination techniques that have been reported include: surgical oviductal insemination, deep uterine insemination and hysteroscopic insemination. Insemination techniques: McCue et al. [J. Reprod. Fert. 56 (Suppl.) (2000) 499] reported a 21% pregnancy rate for mares inseminated with 50,000 sperms into the fimbria of the oviduct. Two methods have been reported for deep uterine insemination. In the study of Buchanan et al. [Theriogenology 53 (2000) 1333], a flexible catheter was inserted into the uterine horn ipsilateral to the corpus luteum. The position of the catheter was verified by ultrasound. Insemination of 25 million or 5 million spermatozoa resulted in pregnancy rates of 53 and 35%, respectively. Rigby et al. [Proceedings of 3rd International Symposium on Stallion Reproduction (2001) 49] reported a pregnancy rate of 50% with deep uterine insemination. In their experiment, the flexible catheter was guided into position by rectal manipulation.More studies have reported the results of using hysteroscopic insemination. With this technique, a low number of spermatozoa are placed into or on the uterotubal junction. Manning et al. [Proc. Ann. Mtg. Soc. Theriogenol. (1998) 84] reported a 22% pregnancy rate when 1 million spermatozoa were inserted into the oviduct via the uterotubal junction. Vazquez et al. [Proc. Ann. Mtg. Soc. Theriogenol. (1998) 82] reported a 33% pregnancy rate when 3.8 million spermatozoa were placed on the uterotubal junction. Recently, Morris et al. [J. Reprod. Fert. 188 (2000) 95] utilized the hysteroscopic insemination technique to deposit various numbers of spermatozoa on the uterotubal junction. They reported pregnancy rates of 29, 64, 75 and 60% when 0.5, 1, 5 and 10 million spermatozoa, respectively, were placed on the uterotubal junction. Insemination of sex-sorted spermatozoa: One of the major reasons for low dose insemination is insemination of X- or Y-chromosome-bearing sperm. Through the use of flow cytometry, spermatozoa can be accurately separated into X- or Y-bearing chromosomes. Unfortunately, only 15 million sperms can be sorted per hour. At that rate, it would take several days to sort an insemination dose containing 800 million to 1 billion spermatozoa. Thus, low dose insemination is essential for utilization of sexed sperm. Lindsey [Hysteroscopic insemination with low numbers of fresh and cryopreserved flow-sorted stallion spermatozoa, M.S. Thesis, Colorado State University, Fort Collins, CO, USA, 2000] utilized either deep uterine insemination or hysteroscopic insemination to compare pregnancy rates of mares inseminated with sorted, fresh stallion sperm to those inseminated with non-sorted, fresh stallion sperm. Hysteroscopic insemination resulted in more pregnancies than ultrasound-guided deep uterine insemination. Pregnancy rate was similar for mares bred with either non-sorted or sex-sorted spermatozoa. In a subsequent study, Lindsey et al. [Proceedings of 5th International Symposium on Equine Embryo Transfer (2000) 13] determined if insemination of flow-sorted spermatozoa adversely affected pregnancy rates and whether freezing sex-sorted spermatozoa would result in pregnancies. Mares were assigned to one of four groups: group 1 was inseminated with 5 million non-sorted sperms using hysteroscopic insemination; group 2 was inseminated with 5 million sex-sorted sperms using hysteroscopic insemination; group 3 was inseminated with non-sorted, frozen-thawed sperm; and group 4 was inseminated with sex-sorted frozen sperm. Pregnancy rates were similar for mares inseminated with non-sorted fresh sperm, sex-sorted fresh sperm and non-sorted frozen sperm (40, 37.5 and 37.5%, respectively). Pregnancy rates were reduced dramatically for those inseminated with sex-sorted, frozen-thawed sperm (2 out of 15, 13%). These studies demonstrated that hysteroscopic insemination is a practical and useful technique for obtaining pregnancies with low numbers of fresh spermatozoa or low numbers of frozen-thawed spermatozoa. Further studies are needed to determine if this technique can be used to obtain pregnancies from stallions with poor semen quality. In addition, further studies are needed to develop techniques of freezing sex-sorted spermatozoa.

Equine pituitary extract (EPE), has been reported to induce multiple ovulation in mares, however ovulation rates are poor in comparison to those obtained in other species. Attempts to improve the effectiveness of EPE for induction of superovulation in cyclic mares has focused on daily frequency of EPE treatment. Two experiments were performed to compare the ovarian response of cyclic mares given EPE once or twice-daily. Mares were assigned to one of two treatment groups 6 to 8 days after ovulation: prostaglandin was given once and EPE (25 mg) was given once daily (Group 1) or twice daily (Group 2). In Experiment 1, more (P0.05) folliclesor = 35 mm were detected in mares treated with EPE twice daily (6.1 +/- 3.1) than in mares treated once a daily (2.0 +/- 0.6). In a second experiment, the embryo recovery rates of mares given the two EPE protocols used in Experiment 1 were compared. The number of ovulations per mare was higher (P0.05) for mares treated twice-daily (7.1 +/- 5.1, range 3 to 18) than for mares treated once daily (2.4 +/- 1.8, range 1 to 6). The number of embryos produced per mare was higher (P0.05) in mares in Group 2 (3.5) than in Group 1 (1.6). Although it is not clear whether the increased ovulation rate is due specifically to dose or frequency, twice-daily administration of a high dose of EPE significantly improved follicular development, ovulation and embryo recovery over the standard treatment of once-daily injection.

The ability to ship cooled stallion semen to a facility that specializes in cryopreservation of spermatozoa would permit stallions to remain at home while their semen is cryopreserved at facilities having the equipment and expertise to freeze the semen properly. To accomplish this goal, methods must be developed to freeze cooled shipped semen. Three experiments were conducted to determine the most appropriate spermatozoal extender, package, time of centrifugation, spermatozoal concentration and length of time after collection that spermatozoa can be cooled before cryopreservation. In the first experiment, spermatozoa were centrifuged to remove seminal plasma, resuspended in either a skim milk extender, a skim milk-egg yolk-sugar extender or a skim milk-egg yolk-salt extender, cooled to 5 degreesC and frozen in 0.5- or 2.5-mL straws either 2.5 or 24 h after cooling. Samples frozen 2.5 h after cooling had higher percentages of progressively motile (PM) spermatozoa (27%) than samples frozen 24 h after cooling (10%; P0.05). Samples frozen 2.5 h after cooling in skim milk extenders containing egg yolk had higher percentages of PM spermatozoa (average 32%) than did spermatozoa frozen in extender containing skim milk alone (average 16%; P0.05). The percentages of PM spermatozoa frozen in 0.5- or 2.5-mL straws were similar (21 and 28%, respectively; P0.05). In the second experiment, spermatozoa were centrifuged to remove seminal plasma either before (25 degreesC) or after cooling (5 degreesC), and spermatozoa were frozen after being cooled to 5 degreesC for 2, 6, or 12 h. The percentages of PM spermatozoa were higher (P0.05) for spermatozoa centrifuged before cooling (30%) than for spermatozoa centrifuged after cooling (19%). Spermatozoa centrifuged at 25 degreesC then cooled for 12 h to 5 degreesC had higher (P0.05) post-thaw progressive motility (23%) compared to spermatozoa cooled for 12 h and centrifuged at 5 degreesC (13%). In the third experiment, spermatozoa were centrifuged for seminal plasma removal, resuspended at spermatozoal concentrations of 50,250 or 500 x 10(6)/mL, cooled to 5 degreesC for 12 h and then frozen. Samples with spermatozoa packaged at 50 or 250 x 10(6)/mL had higher (P0.05 percentages of PM spermatozoa (25 and 23%) after freezing than did samples packaged at 500 x 10(6) spermatozoa/mL (17%). We recommend that semen be centrifuged at 25 degreesC to remove seminal plasma, suspended to 250 x 10(6) spermatozoa/ml and held at 5 degreesC for 12 h prior to freezing.

INTRODUCTION Gonadotropic hormones are often utilized in assisted reproductive technology protocols. Human chorionic gonadotropin (hCG) is commonly used to induce ovulation in the equine. However, several studies have shown that horses become less responsive to hCG during the course of a breeding season apparently due to production of antibodies to the human protein. A hormone, that induces ovulation but does not activate the antibody response, would provide a desirable alternative to hCG. A new single chain recombinant equine luteinizing hormone (reLH), with a linker protein between the a and b subunits, was recently developed. Recombinant eLH was evaluated for its ability to stimulate production of progesterone in cell lines from three species including murine Leydig tumor (MA10), equine granulosal, and ovine small luteal cells (SLC). The objective of the current study was to evaluate the ability of reLH to stimulate production of progesterone compared to the response induced by other well characterized glycoprotein hormones including equine chroinic gonadotropin (eCG), equine LH (eLH), ovine LH (oLH), and hCG. We hypothesized that production of progesterone from cells treated with reLH will resemble the secretory profile of progesterone stimulated by hGC and it will be higher that the induced by the other secretagoges evaluated.

s / Journal of Equine Veterinary Science 33 (2013) 321-399 371 reproductive cyclicity in obese broodmares maintained on pasture. Eighteen healthy, light horse mares were matched by age and assigned to obese control (OBC; n1⁄45, mean BCS1⁄47.4 0.3), obese supplemented with 5g/d resveratrol (OBR; n1⁄47, mean BCS1⁄47.4 0.2) or non-obese control (NOC; n1⁄46, mean BCS1⁄45.4 0.1) treatments. Control horses received the resveratrol carrier placebo andNOChorseswere managed to maintain constant BCS throughout the trial. Across three consecutive estrous cycles, morphometric measurements were collected biweekly and follicular dynamics were evaluated via transrectal ultrasonography every other day to determine estrous cycle and follicular phase length, follicle size and number, and time of ovulation. Frequently-sampled intravenous glucose tolerance tests were conducted during luteal phase prior to supplementation and after third ovulation. Insulin and glucose kinetics were analyzed via minimal model. For all parameters, repeated measures (parametric or nonparametric, as warranted) techniques were used to determine treatment effect. Resveratrol supplementation had no discernible effect on reproductive parameters (P>0.05). Regardless of treatment (OBC or OBR), obese mares had more (7 vs. 0) hemorrhagic anovulatory follicles and tended (P1⁄40.07) to have longer inter-ovulatory intervals than NOC. Neither resveratrol treatment nor time on study influenced BCS, or bodyweight, rump fat thickness or neck circumference (raw data or data adjusted for animal size). Insulin sensitivity and disposition index were neither affected by resveratrol supplementation nor initial BCS, although as a whole, horses became more insulin resistant over time (P

Abiotic inputs such as photoperiod and temperature can regulate reproductive cyclicity in many species. When humans perturb this process by intervening in reproductive cycles, the ecological consequences may be profound. Trophic mismatches between birth pulse and resources in wildlife species may cascade toward decreased survival and threaten the viability of small populations. We followed feral horses (Equus caballus) in three populations for a longitudinal study of the transient immunocontraceptive porcine zona pellucida (PZP), and found that repeated vaccinations extended the duration of infertility far beyond the targeted period. After the targeted years of infertility, the probability of parturition from post-treated females was 25.6% compared to 64.1% for untreated females, when the data were constrained only to females that had demonstrated fertility prior to the study. Estimated time to parturition increased 411.3 days per year of consecutive historical treatment. Births from untreated females in these temperate latitude populations were observed to peak in the middle of May, indicating peak conception occurred around the previous summer solstice. When the post-treated females did conceive and give birth, parturition was an estimated 31.5 days later than births from untreated females, resulting in asynchrony with peak forage availability. The latest neonate born to a post-treated female arrived 7.5 months after the peak in births from untreated females, indicating conception occurred within 24-31 days of the winter solstice. These results demonstrate surprising physiological plasticity for temperate latitude horses, and indicate that while photoperiod and temperature are powerful inputs driving the biological rhythms of conception and birth in horses, these inputs may not limit their ability to conceive under perturbed conditions. The protracted infertility observed in PZP-treated horses may be of benefit for managing overabundant wildlife, but also suggests caution for use in small refugia or rare species.