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To more clearly understand the equine gonadotrope response to kisspeptin and gonadotropin releasing hormone (GnRH), peripheral LH and FSH were quantified in diestrous mares after treatment with either equine kisspeptide (eKp-10, 0.5 mg iv), GnRH (25 μg iv), or a combination thereof every 4 h for 3 days. The following observations were made: 1) a diminished LH and FSH response to eKp-10 and GnRH was observed by Day 3, but was not different by treatment, 2) a decrease in basal LH concentration was observed from Day 1 to Day 3 for the eKp-10, but not the GnRH treated mares, 3) there was no change in basal FSH with either treatment. Additionally, pre-treatment with GnRH antagonist (antide 1.0 mg iv) eliminated any measurable change in LH after eKp-10 (1.0 mg iv) treatment. Both GnRH and kisspeptin are Gαq/11 coupled receptors, therefore quantifying the rise in intracellular calcium following treatment with cognate ligand allows simultaneous assessment of receptor activation. Direct stimulation of equine primary pituitary cells with GnRH and/or eKp-10 demonstrates three distinct populations of pituitary cells: one population responded to both eKp-10 and GnRH, a second, independent population, responded to only eKp-10, and a third population responded only to GnRH. These populations were confirmed using co-immunofluorescence of hemipituitaries from mares in diestrus. Although the rise in peripheral LH concentration elicited by eKp-10 is dependent on GnRH, this work suggests that kisspeptin also has a specific and direct effect on the equine gonadotrope, independent of GnRH.

MicroRNAs (miRNAs) are a class of small endogenous single-stranded noncoding RNA molecules that have important roles in several biological processes. Research in human and laboratory animals has shown that miRNAs can regulate genes associated with type 2 diabetes mellitus 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 (IR) horses would have a different circulating miRNA profile than those that are healthy. Fifteen nonpregnant mares housed at the Virginia Tech Middelburg Agricultural Research and Extension Center were evaluated for insulin sensitivity, with the frequent sampling intravenous glucose tolerance test. Selected mares, representing the most insulin-sensitive (IS, n = 3) and IR (n = 3) states, and paired for age, weight, and body condition, underwent miRNA profiling. Serum samples were collected, miRNA extracted, and microarray analysis performed to investigate the presence and relative amount of 340 equine miRNAs. Confirmation by quantitative real-time polymerase chain reaction revealed that miRNA was present in the serum of all animals. Results demonstrated different miRNA profiles between groups: Six miRNAs were expressed only in IS mares, five miRNAs were found to have lower quantity in IR mares relative to the IS ones, and three miRNAs were higher quantity in IR mares relative to the IS ones. The novel results of this preliminary study suggest potential new tools that could be developed for the diagnosis and treatment of metabolic syndrome in horses.

The equine placenta is critical in maintaining pregnancy. Not only does it provide essential nutrients and gasses, but it also functions as an endocrine, paracrine, and autocrine organ. For example, it is one of the main sources of estrogen and progesterone in the latter stages of pregnancy. In addition to supplying hormones required for development and pregnancy maintenance, hormones supplied by both the mare and fetus act on the placenta to influence its development and function. The equine placenta possesses both estrogen and progesterone receptors, and a great deal is known about these hormones' effects on placental development. However, very little is known about how, or if, androgens interact with the placenta to affect placental development and/or maintenance. Androgen receptor (AR) has been identified in the placenta of humans, cows, sheep, and rats; however, little is known about its exact functions related to the placenta. The focus of this study was to confirm the presence of AR in the equine placenta. Three term equine placentas from normal pregnancies were utilized in this study. Reverse transcription polymerase chain reaction was used to identify mRNA encoding AR, and Western blotting confirmed the presence of AR protein within the equine placenta. The results of this study provide new insight into the presence of AR, similar to other species including the human, bovine, ovine, and murine placenta.

After spermatogenesis, spermatozoa are not mature and unable to fertilize an oocyte. During transition through the epididymis, spermatozoa mature, and gain fertility and motility. Although epididymal functions are relatively well known, the mechanisms behind maturation are not yet fully understood. Studies indicate proteins are delivered to the spermatozoa during epididymal transit through microvesicles. These microvesicles, called epididymosomes, carry microRNAs (miRNAs). These miRNAs are ∼22 nucleotides in length and regulate mRNA translation. The purpose of this study was to investigate which miRNAs are located within the various regions of stallion epididymal tissue. Three mature stallion epididymides were divided into four sections for miRNA isolation: the caput, proximal corpus, distal corpus and cauda. The contents of the lumen were removed and epididymal tissue was homogenized. Quantitative real-time polymerase chain reaction analysis was used to determine miRNA content. A total of 328 miRNAs were noted in epididymal tissue. A total of 186 of the 346 known equine miRNA transcripts used were found in each section, all four sections also contained miRNAs distinct to specific regions. The cauda contained the greatest number of exclusive miRNA (19) and expressed the greatest number of miRNA (282). This study shows there is indeed a difference in the miRNA profile between regions of the epididymis, and their targeted pathways may effect spermatozoal maturation.

Maternal recognition of pregnancy (MRP) in the mare is not well defined. In a non-pregnant mare, prostaglandin F2&alpha, (PGF) is released on day 14 post-ovulation (PO) to cause luteal regression, resulting in loss of progesterone production. Equine MRP occurs prior to day 14 to halt PGF production. Studies have failed to identify a gene candidate for MRP, so attention has turned to small, non-coding RNAs. The objective of this study was to evaluate small RNA (&lt, 200 nucleotides) content in endometrium during MRP. Mares were used in a cross-over design with each having a pregnant and non-mated cycle. Each mare was randomly assigned to collection day 11 or 13 PO (n = 3/day) and endometrial biopsies were obtained. Total RNA was isolated and sequencing libraries were prepared using a small RNA library preparation kit and sequenced on a HiSeq 2000. EquCab3 was used as the reference genome and DESeq2 was used for statistical analysis. On day 11, 419 ncRNAs, representing miRNA, snRNA, snoRNA, scaRNA, and vaultRNA, were different between pregnancy statuses, but none on day 13. Equine endometrial ncRNAs with unknown structure and function were also identified. This study is the first to describe ncRNA transcriptome in equine endometrium. Identifying targets of these ncRNAs could lead to determining MRP.

Equine maternal recognition of pregnancy (MRP) is a process whose signal remains unknown. During MRP the conceptus and endometrium communicate to attenuate prostaglandin F2&alpha, (PGF) secretion, sparing the corpus luteum and maintaining progesterone production. Recognition of a mobile conceptus by the endometrium is critical by days 14&ndash, 16 post-ovulation (PO), when endometrium produces PGF, initiating luteolysis. The objective of this study was to evaluate endometrial gene expression changes based upon pregnancy status via RNA sequencing. This experiment utilized a cross-over design with each mare serving as both a pregnant and non-mated control on days nine, 11, and 13 PO (n = 3/status/day). Mares were randomly assigned to collection day and pregnancy confirmed by terminal uterine lavage at the time of endometrial biopsy. Total RNA was isolated and libraries prepared using Illumina TruSeq RNA sample preparation kit. Reads were mapped and annotated using HISAT2 and Stringtie. Expression values were evaluated with DESEQ2 (P &le, 0.05 indicated significance). On day nine, 11, and 13 there were 1435, 1435 and 916 significant transcripts, respectively. Multiple genes with splice variants had different expression patterns within the same day. These are the first data to evaluate the endometrial transcriptome during MRP on days nine, 11, and 13.

All epididymal regions are lined with multiple epithelial cell types, each with different functions to provide the luminal environment for spermatozoal maturation. Epithelial cells also create apical blebs, which are released from the apical surface via apocrine secretion and disintegrate in the lumen, thereby releasing epididymosomes. Epididymosomes transport proteins to spermatozoa and contain microRNAs. We hypothesized that epididymosomes also transfer miRNA from epididymal epithelium to spermatozoa. Quantitative real-time polymerase chain reaction was used to determine miRNA profiles of epididymal tissue from caput and cauda, epididymal spermatozoa from caput and cauda, and epididymosomes and from caput, proximal corpus, distal corpus, and cauda. Pathway analysis was performed using DIANA tools on the miRNA unique to caudal spermatozoa. We found 66 newly acquired miRNAs in spermatozoa located in the caudal epididymis. Predicted pathways targeted by these miRNAs suggest a role in cell motility and viability and factors in oocyte and embryo maturation and development. These findings suggest that miRNAs are transported to spermatozoa from epididymal epithelium via epididymosomes.

Kisspeptides (KiSS) are a recently discovered family of neuropeptides with a central role in regulating the onset of reproductive function in all animals studied to date. We have established biological and physiological evidence for KiSS signaling in the mare. The objective of the current study was to evaluate the physiological and behavioral responses of mares repeatedly given the equine-specific kisspeptpin decapeptide (eKp-10, YRWNSFGLRY-NH(2)) in an effort to shorten the interovulatory period. Administration of eKp-10 (0.5 mg iv every 4 h) to mares beginning on Day 16 postovulation (Group 2) or in estrus (Group 3) did not shorten the mean ± SEM interovulatory interval compared with untreated (Group 1) controls (21.9 ± 1.2, 22 ± 1.2, and 21.5 ± 1.5 days in Groups 1 to 3, respectively; N = 6 per group), nor was there a significant difference in follicle diameter before ovulation among groups, nor number of days treated with eKp-10 for Groups 2 and 3. Mean daily concentrations of FSH, the preovulatory LH surge (timing, mean, and peak concentrations), and mean progesterone concentrations from the newly formed CL were not significantly different among groups. The initiation of treatment was negatively correlated with sexual receptivity (scored 0 to 5: no interest to strong interest) and serum estradiol concentrations, indicating that eKp-10 can significantly disrupt normal sexual receptivity in the estrous mare. This effect on sexual receptivity was short-lived (< 72 h) and the overall change in sexual receptivity score was not significantly different between Groups 2 and 3 (-1.2 ± 0.5 and -1.4 ± 0.4, respectively). However, the day of the cycle that treatment was initiated significant affected the decline in sexual receptivity score, such that the later in the cycle that treatment was initiated, the greater the estimated decrease in sexual receptivity. In conclusion, the linear hypothalamic-pituitary mechanism for KiSS described in other species was not appropriate for the horse and administration of eKp-10 in the seasonally estrous mare may have been outside of the hormone's normal physiological context.

Processing stallion semen for assisted reproductive procedures, such as intracytoplasmic sperm injection (ICSI), requires special considerations regarding cooling, concentrating, and handling of sperm. The aim of experiment 1 was to determine whether cooled semen could be frozen without removal of seminal plasma and at a low sperm concentration while maintaining motile sperm for ICSI selection procedures. In experiment 2, five media for holding stallion sperm were compared to evaluate sperm motility for an interval of time sufficient for ICSI sperm selection procedures. In experiment 1, semen samples from eight stallions were cooled for 24 hours in two extenders, CST (E-Z Mixin-CST “Cool-Store/Transport” Animal Reproduction Systems) and INRA96 (Institut National de la Recherche Agronomique, IMV International Corporation), before being frozen in four freezing diluents, and were evaluated at 0, 45, and 75 minutes after thawing. The cooling extender did not significantly affect sperm motility, but modified French and glycerol egg yolk diluents provided the best sperm motility for frozen–thawed groups. In experiment 2, semen samples from seven stallions were used to test five media for holding sperm. Samples were analyzed for total and progressive motility at hourly intervals. Mean total and progressive motility were not different (P > .05) among groups from 1 through 4 hours. At 5 hours, groups differed (P = .004), with sperm held in Tyrode’s with albumin, lactate, and pyruvate having higher (P < .05) total and progressive motility than all other samples. In conclusion, motile stallion sperm can be obtained after the sperm are cooled for 24 hours, frozen, and thawed; various media are available to maintain sperm motility during equine ICSI selection procedures.

Gonadotropin-releasing hormone is intermittently released from the hypothalamus in consistent patterns from before birth to final maturation of the hypothalamic-pituitary-gonadal axis at puberty. Disruption of this signaling via GnRH vaccination during the neonatal period can alter reproduction at maturity. The objective of this study was to investigate the long-term effects of GnRH-antibody exposure on reproductive maturation and function in elk calves passively exposed to high concentrations of GnRH antibodies immediately after birth. Fifteen elk calves (eight males and seven females) born to females treated with GnRH vaccine or sham vaccine during midgestation were divided into two groups based on the concentration of serum GnRH antibodies measured during the neonatal period. Those with robust (15 pmol 125 I-GnRH bound per mL of serum) titers (N 10; four females and six males) were designated as the exposed group, whereas those with undetectable titers (N 5; three females and two males) were the unexposed group. Onset of puberty, reproductive development, and endocrine function in antibody-exposed and unexposed male and female elk calves were compared. Neonatal exposure to high concentrations of GnRH antibodies had no effect on body weight (P 0.968), endocrine profiles (P 0.05), or gametogenesis in either sex. Likewise, there were no differences between groups in gross or histologic structure of the hypothalamus, pituitary, testes, or ovaries. Pituitary stimulation with a GnRH analog before the second potential reproductive season induced substantial LH secretion in all experimental elk. All females became pregnant during their second reproductive season and all males exhibited similar mature secondary sexual characteristics. There were no differences between exposure groups in hypothalamic GnRH content (P 0.979), pituitary gonadotropin content (P 0.05) or gonadal structure. We concluded that suppressing GnRH signaling through immunoneutralization during the neonatal period likely does not alter long-term reproductive function in this species. Published by Elsevier Inc.

Recombinant equine luteinizing hormone (reLH) was evaluated for its ability to stimulate production of progesterone in cell lines from three species including murine Leydig tumor (MA-10), equine granulosal, and ovine small luteal cells (SLC). The response to reLH was compared with that obtained with human chorionic gonadotropin (hCG), equine chorionic gonadotropin, ovine luteinizing hormone, and equine luteinizing hormone (eLH). Media were collected hourly for 6 hours and assayed for progesterone content through radioimmunoassay. In MA-10 cells, production of progesterone was stimulated above baseline by reLH and hCG (P < .05). Ovine SLC responded to treatment with eLH, reLH, ovine luteinizing hormone, and hCG by increasing production of progesterone above that stimulated by vehicle control (P < .05). Production of progesterone in equine granulosal cells was maximally stimulated by treatment with hCG (P < .05), followed by reLH and eLH (P < .05). In conclusion, reLH elicited a progesterone response in MA-10, ovine SLC, and equine granulosal cells. Thus, reLH stimulates the production of progesterone in cell lines from three species.

This study was undertaken to re-evaluate safety aspects of the commercial modified live virus vaccine against equine viral arteritis (Arvac) in stallions. Ten seronegative stallions were administered a single dose of vaccine, whereas three others were used as controls. Presence of vaccine virus in blood, semen, and nasopharyngeal and conjunctival secretions was determined by virus isolation and reverse transcription-polymerase chain reaction assay. No adverse effects were observed after vaccination. Vaccine virus was detected in buffy-coat cultures of all stallions for an average of 4.3 days, and in nasopharyngeal swabs from 40% of the stallions for an average of 3.2 days. No virus was detected in conjunctival swabs. Small quantities of virus were detected in the semen of one stallion on days 4 and 6 postvaccination. Vaccinated stallions showed a marked increase in neutralizing antibody titers within 5 to 8 days after vaccination. The stallions in the control group remained uninfected. Finding very low levels of virus in the semen of one stallion provides justification for withholding semen from a first-time vaccinated stallion for a minimum of 14 days to avoid the potential risk of virus transmission or having vaccine virus detected in semen frozen from a recently vaccinated stallion. There was no evidence of persistence of vaccine virus in the reproductive tract of the vaccinated stallions. Virus isolation proved more sensitive than reverse transcription-polymerase chain reaction for detection of virus in buffy-coat and nasopharyngeal specimens. Peak antibody responses were reached at a little more than a week after vaccination and these remained undiminished for the remainder of the study.

Addition of hyaluronan, a nonsulfated glycosaminoglycan, to fresh and frozen thawed human semen results in substantial retention of motility over time. Hyaluronan also has been reported to preserve postthaw viability and maintain membrane stability of boar spermatozoa. Therefore, experiments were designed to investigate the use of a commercially available hyaluronan (Map-5, Bioniche Animal Health, Inc., Athens, GA) in freezing extender for cryopreservation of equine spermatozoa. In experiment 1, aliquots from ejaculates were supplemented before freezing with one of four levels of hyaluronan: 100 μg/mL, 200 μg/mL, 400 μg/mL, and 1000 μg/mL along with an untreated control. No differences in sperm motility, assessed by computer-assisted sperm motility analysis (CASA), were found for any treatment at times 0, 30, or 60 minutes postthaw. Decreases in motility were noted in the highest hyaluronan group (1,000 μg/mL) after 90 and 120 minutes of incubation. Sperm viability, as assessed using SYBR-14/propidium iodide staining, was decreased ( P P P

The standard treatment for superovulation of mares is to administer equine follicle-stimulating hormone (eFSH) for 4 to 5 days to stimulate multiple follicles and human chorionic gonadotropin (hCG) to induce synchronous ovulations. Objectives of this study were: (1) to determine whether a short-term (3-day) eFSH treatment protocol would result in similar ovulation and embryo recovery rates compared with the standard eFSH protocol; (2) to determine the efficacy of a decreasing dose of eFSH (step-down protocol) on ovulation rate and embryo recovery; (3) to compare the efficacy of hCG and recombinant equine luteinizing hormone (reLH) for inducing ovulation in FSH-treated mares; and (4) to compare embryo recovery rates and embryo size when mares are flushed at 6.5 or 7.0 days after ovulation. Forty light-horse mares were used in 2005 (experiment 1) and 20 different mares were used in 2006 (experiment 2). In experiment 1, mares were randomly assigned to one of three treatment groups: (1) untreated controls, (2) standard eFSH treatment (12.5 mg intramuscularly twice daily), and (3) 3-day eFSH treatment. In experiment 2, mares were randomly assigned to one of four treatments: (1) untreated controls, (2) standard eFSH protocol, (3) 3-day eFSH treatment, and (4) step-down eFSH treatment (12.5 mg twice daily day 1, 8.0 mg twice daily day 2, 4.0 mg twice daily day 3). Within each treatment, mares were given either hCG (2,500 IU) or equine LH (750 mg, EquiPure LH; reLH) to induce synchronized ovulations. Embryo recovery was performed either 6.5 or 7.0 days after ovulation. In experiment 1, numbers of preovulatory follicles and ovulations were less for mares in the 3-day treatment group than the standard group, but were greater than for controls. Embryo recovery per flush was higher in the standard group (2.6) than the 3-day eFSH treatment (0.8) or control groups (0.8). In experiment 2, the number of preovulatory follicles and number of ovulations were greater in the standard and 3-day treatment groups than in control and step-down groups. The percent embryo recovery per ovulation and mean embryo grade were similar for all groups; however, the embryo recovery per flush was higher for mares in the standard treatment than controls (1.3 vs 0.6) but was similar to the 3-day (1.1) and step-down (0.8) treatments. Embryo recovery was similar for flushes performed on days 6.5 and 7.0 post-ovulation. The percentage of control mares ovulating within 48 hours in response to hCG or reLH was similar. In contrast, a higher percentage of eFSH-treated mares ovulated within 48 hours in response to reLH than hCG (92% vs 71%). In both years, the 3-day eFSH treatment protocol resulted in a greater number of preovulatory follicles and a greater number of ovulations than untreated controls. Unfortunately, the increased ovulation rate for mares administered eFSH for 3 days did not result in a greater number of embryos recovered per flush in either year. Use of a step-down eFSH treatment protocol resulted in fewer preovulatory follicles, fewer ovulations, and fewer embryos as compared with the standard eFSH treatment. In conclusion, the standard eFSH treatment resulted in a greater embryo recovery rate per cycle than either the 3-day or step-down treatment protocols. Recombinant equine LH was more effective than hCG in causing ovulation in eFSH-treated mares.

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 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 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 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.

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

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