Search

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.

Reproductive efficiency is critically dependent on embryo survival, establishment of a successful pregnancy and placental development. Recent advances in gene editing technology have enabled investigators to use gene knockdown and knockout approaches to better understand the role of hormone signaling in placental function and fetal growth and development. In this review, an overview of ruminant placentation will be provided, including recent data highlighting the role of histone lysine demethylase 1A and androgen signaling in ruminant placenta and pregnancy. Studies in ruminant placenta establish a role for histone lysine demethylase 1A in controlling genetic networks necessary for important cellular events such as cell proliferation and angiogenesis, as well as androgen receptor signaling during early placentation.

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.

Mammalian gamete maturation requires extensive signaling between germ cells and their surrounding somatic cells. In the ovary, theca cells, mural granulosa cells, cumulus cells and the oocyte all secrete factors throughout follicle growth and maturation that are critical for ovulation of a high-quality oocyte with the competence to develop into an embryo. Similarly, maturation of sperm occurs as it transits the epididymis during which epididymal epithelium and sperm exchange secretory factors that are required for sperm to gain motility and fertility. Recent studies in a variety of species have uncovered the presence of cell-secreted vesicles in follicular fluid (microvesicles and exosomes) and epididymal fluid (epididymosomes). Moreover, these cell-secreted vesicles contain small non-coding regulatory RNAs called microRNAs, which can be shuttled between maturing gametes and surrounding somatic cells. Although little is known about the exact mechanism of how microRNAs are loaded into these cell-secreted vesicles or are transferred and modulate gene expression and function in gametes, recent studies clearly suggest that cell-secreted vesicle microRNAs play a role in oocyte and sperm maturation. Moreover, a role for cell-secreted vesicular microRNAs in gamete maturation provides for novel opportunities to modulate and discover new diagnostic markers associated with male or female fertility. This manuscript provides an overview of cell-secreted vesicles in ovarian follicular fluid and epididymal fluid and microRNAs and discusses recent discoveries on the potential function of cell-secreted vesicles as carriers of microRNAs in oocyte and sperm maturation.

This study examined the effects of fish meal supplementation on spatial distribution of lipid microdomains and lateral mobility of prostaglandin F2α (FP) receptors on cell plasma membranes of the bovine corpus luteum (CL). Beef cows were stratified by BW and randomly assigned to receive a corn gluten meal supplement (n = 4) or fish meal supplement (n = 4) for 60 d to allow incorporation of fish meal-derived omega-3 fatty acids into luteal tissue. Ovaries bearing the CL were surgically removed between days 10 to 12 after estrus corresponding to approximately day 60 of supplementation. A 200-mg sample of luteal tissue was analyzed for fatty acid content using gas-liquid chromatography (GLC). The remaining tissue was enzymatically digested with collagenase to dissociate individual cells from the tissue. Cells were cultured to determine the effects of dietary supplementation on lipid microdomains and lateral mobility of FP receptors. Luteal tissue collected from fish meal-supplemented cows had increased omega-3 fatty acids content (P < 0.05). Lipid microdomain total fluorescent intensity was decreased in dissociated luteal cells from fish meal-supplemented cows (P < 0.05). Micro and macro diffusion coefficients of FP receptors were greater for cells obtained from fish meal-supplemented cows (P < 0.05). In addition, compartment diameter of domains was larger, whereas resident time was shorter for receptors from cells obtained from fish meal-supplemented cows (P < 0.05). Data indicate that dietary supplementation with fish meal increases omega-3 fatty acid content in luteal tissue causing disruption of lipid microdomains. This disruption leads to increased lateral mobility of the FP receptor, increased compartment sizes, and decreased resident time, which may influence prostaglandin signaling in the bovine CL.

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.

The objective of this study was to determine the effect of corn supplementation on muscle microRNA (miRNA) profiles. Twelve mares were blocked by weight and BCS and assigned to one of two treatments (6 hd/treatment): 1) control, (basal diet: 9 Kg/hd/d of chopped mixed grass hay and ad libitum mixed grass hay), 2) basal diet supplemented with 454 g/d steam flaked corn individually using feed bags. All mares were placed on the basal diet and in the same pen 14 days prior to the beginning of corn supplementation. Mares were weighed and BCS on d -7 and 28. Muscle biopsies of the gluteus medius were taken from all horses on d0 and d26. Muscle samples were analyzed using real time RT-qPCR for 277 endogenous miRNAs. Raw CT values were normalized with the geometric mean of three consistently appearing endogenous miRNAs. Sixteen miRNAs that consistently appeared in at least 8 horses including 3 relating to metabolic disorders appearing at d0, d26, or both were analyzed. Differential abundances of miRNAs using log transformed fold change and differences between BW and BCS of treatment groups were determined by ANOVA and LS means analysis. There was no difference between BW (P < .05) and BCS (P < .05) between the two treatment groups. Feeding corn lead to higher quantities (P < .05) of mir 133a, 1515p, 6155p, 770, and 99b within muscle compared to control group. Lower quantities of mir 382 and 433 were found within muscle of horses fed corn compared to the control group (P < .05). Based upon the results of this study corn supplementation appears to influence endogenous miRNA expression profiles within muscle of horses.

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.

Histone lysine demethylase 1A is a master regulator of genes necessary for trophoblast cell proliferation. A proper functioning placenta is critical for pregnancy, fetal growth and development and postnatal health. Trophoblast cell proliferation and differentiation is critical for placental development and function. Recently we demonstrated that the histone lysine demethylase KDM1A binds to androgen receptor (AR) in human and sheep trophoblast cells, and targets the same promoter region of vascular endothelial growth factor A (VEGFA), suggesting a role for KDM1A and AR in early placental angiogenesis. The goal of this study was to determine the function of KDM1A during early placental development. We hypothesized that KDM1A regulates genes that are necessary for trophoblast cell proliferation, and early placental development. To this end, both in vitro and in vivo approaches were used in this study. ACH-3P cells (human first trimester trophoblast cells (CT and EVT) fused with the choriocarcinoma cell line AC1-1) were used, and a KDM1A knock out (KO) cell line was generated using CRISPR-Cas 9 based genome editing. KDM1A KO in ACH-3P cells led to significant (P This project was supported by Agriculture and Food Research Initiative Competitive Grant no. 2019-67015-29000 from the USDA National Institute of Food and Agriculture.

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.

Establishing cell lines is a good model for experimental applications to study molecular mechanisms and cell-specific gene expression. Equids have a diffuse epitheliochorial placenta, where the invasive trophoblast is represented by the chorionic girdle (CG) and the noninvasive trophoblast by the allantochorion (AC). Embryonic CG cells are unique to horses and have a crucial role in equine chorionic gonadotropin (eCG) production and maintenance of pregnancy during the first trimester. This study had three objectives: (1) establishing a stable cell line from Day 30 CG cells and AC using lentivirus encoding hTERT; (2) characterisation of Day 30 CG cells and AC cell morphology and expression of eCG α (eCGA) and β (eCGB) subunits, major histocompatibility complex class II (MHCII), and Kisspeptin receptor (KISS1R) in CG and AC cells; (3) investigating eCG protein production invitro from Day 30 CG and AC cells. Three mares (n=3) were used to collect Day 30 conceptuses by non-surgical uterine lavage on Day 30 of pregnancy. All 3 conceptuses were dissected for CG and AC cells then cultured invitro to confluency in cell culture plates. Second-generation lentiviral particles were generated using a three-vector system including transfer vector pLV-hTERT-IRES-hygro, and human telomerase reverse transcriptase (hTERT) lentivirus was utilised to establish stable hygromycin-resistant equine embryonic cell lines. Reverse-transcription PCR (RT-PCR) was used to study gene expression in cells and radioimmunoassay was used to investigate protein presence in the media. We established a hygromycin-resistant Day 30 CG and AC cell lines that express eCGA, eCGB, and hTERT and confirmed using RT-PCR yielding the predicted bands. The cell lines were maintained for 16 passages (7±2 days/passage), 10 of which were cultured after the lentiviral infection steps. Also, we characterised CG cells as fast-growing, large, binucleated, and epithelioid, and AC cells as rapid-growing showing smaller, squamous, mononucleate, epithelioid, and elongated fibroblastic cells. The RT-PCR results showed eCGA and eCGB subunits are expressed by both Day 30 CG and AC cells, but MHCII and KISS1R genes were not expressed in either of cells. Moreover, radioimmunoassay results showed that Day 30 CG cells did produce eCG protein (35.42ngmL−1) invitro earlier than what previous literature has shown. However, Day 30 AC cells did not produce eCG protein (0.042ngmL−1) invitro, and both CG and AC cell lines stopped secreting eCG in the media after the lentiviral infection. To conclude, establishing stable and hygromycin-resistant cell lines from Day 30 equine CG and AC cells using lentivirus encoding pLV-hTERT-IRES-hygro is attainable. Also, equine chorionic gonadotropin eCG protein is produced invitro as early as Day 30 from CG cells.

Maternal recognition of pregnancy (MRP) in the mare is an unknown process. In a non-pregnant mare on day 14 post-ovulation (PO), prostaglandin F2α (PGF) is secreted by the endometrium causing regression of the corpus luteum. Prior to day 14, MRP must occur in order to attenuate secretion of PGF. The embryo is mobile throughout the uterus due to uterine contractions from day of entry to day 14. It is unknown what signaling is occurring. Literature stated that infusing oil or placing a glass marble into the equine uterus prolongs luteal lifespan and that in non-pregnant mares, serum exosomes contain miRNA that are targeting the focal adhesion (FA) pathway. The hypothesis of this study is embryo contact with endometrium causes a change in abundance of focal adhesion molecules (FA) in the endometrium leading to decrease in PGF secretion. Mares (n = 3/day) were utilized in a cross-over design with each mare serving as a pregnant and non-pregnant (non-mated) control on days 9 and 11 PO. Mares were randomly assigned to collection day and endometrial samples and embryos were collected on the specified day. Biopsy samples were divided into five pieces, four for culture for 24 hours and one immediately snap frozen. Endometrial biopsies for culture were placed in an incubator with one of four treatments: [1] an embryo in contact on the luminal side of the endometrium, [2] beads in contact on the luminal side of the endometrium, [3] peanut oil in contact on the luminal side of the endometrium or [4] the endometrium by itself. Biopsies and culture medium were frozen for further analysis. RNA and protein were isolated from biopsies for PCR and Western blot analysis for FA. PGF assays were performed on culture medium to determine concentration of PGF. Statistics were performed using SAS (P ≤ 0.05 indicated significance). The presence of beads on day 9 impacted samples from pregnant mares more than non-pregnant mares and had very little impact on day 11. Presence of oil decreased FA in samples from pregnant mares on day 9. On day 11, oil decreased FA abundance in samples from non-pregnant mares. Embryo contact caused multiple changes in RNA and protein abundance in endometrium from both pregnant and non-pregnant mares. The PGF secretion after 24 hours with each treatment was also determined. On day 9, there was no change in PGF secretion compared to any treatments. On day 11, presence of peanut oil increased PGF secretion in samples from non-pregnant mares. In samples from non-pregnant mares, presence of an embryo decreased PGF secretion compared to control samples from non-pregnant mares. Results revealed that while beads and peanut oil may impact abundance of FA RNA and protein in endometrial samples, it does not appear to impact PGF secretion. Conversely, embryo contact for 24 hours with endometrium from a non-pregnant mare causes a decrease in PGF secretion. These results suggest that it is not just contact of any substance/object causing attenuation of PGF secretion, but the embryo itself is necessary to decrease PGF secretion.

Wildlife and humans are increasingly competing for resources worldwide, and a diverse, innovative, and effective set of management tools is needed. Controlling abundance of wildlife species that are simultaneously protected, abundant, competitive for resources, and in conflict with some stakeholders but beloved by others, is a daunting challenge. Free-ranging horses (Equus caballus) present such a conundrum and managers struggle for effective tools for regulating their abundance. Controlling reproduction of female horses presents a potential alternative. During 2009-2017, we determined the long-term effectiveness of GnRH vaccine (GonaCon-Equine) both as a single immunization and subsequent reimmunization on reproduction and side effects in free-ranging horses. At a scheduled management roundup in 2009, we randomly assigned 57 adult mares to either a GonaCon-Equine treatment group (n = 29) or a saline control group (n = 28). In a second roundup in 2013, we administered a booster vaccination to these same mares. We used annual ground observations to estimate foaling proportions, social behaviors, body condition, and injection site reactions. We found this vaccine to be safe for pregnant females and neonates, with no overt deleterious behavioral side effects during the breeding season. The proportion of treated mares that foaled following a single vaccination was lower than that for control mares for the second (P = 0.03) and third (P = 0.08) post-treatment foaling seasons but was similar (P = 0.67) to untreated mares for the fourth season, demonstrating reversibility of the primary vaccine treatment. After two vaccinations, however, the proportion of females giving birth was lower (P

The mechanism for maternal recognition of pregnancy (MRP) in horses is unknown. To maintain a pregnancy, a mobile conceptus must be recognized by the uterus before d 14 postovulation (PO). This recognition prevents endometrial secretion of PGF2α on d14 through 16, which would otherwise initiate luteolysis. The objective of this study was to evaluate gene expression in the endometrium of pregnant and nonpregnant mares during and after MRP to identify possible genes involved during this time. Twelve normally cycling mares were used in a crossover design and randomly assigned to a specific collection day. Endometrial samples were collected from a pregnant and nonpregnant (nonmated) mare on cycle d 12, 14, 16, and 18 (n = 3/d) PO. Microarray analysis comparing the endometrial gene expression in pregnant and nonpregnant mares revealed no differences at d 12. Ten genes were identified to have consistently higher or lower expression levels in the endometrium from pregnant versus nonpregnant mares on d 14, 16, and 18 (P < 0.001). The expression of these 10 genes was further analyzed with real-time PCR. d 14, 16, and 18 gene expression patterns were consistent with the microarray analysis, but on d 12, 4 of the 10 were identified as differentially expressed. Endometrial samples were then collected on d 13 PO (n = 3) and processed for western blot and immunohistochemical analysis of 2 proteins due to their reproductive significance. SPLA2 and DKK1 antibody specificity were confirmed via western blot analysis but were not different in samples from pregnant and nonpregnant mares (P = 0.114 and P = 0.514, respectively) and cellular localization was examined by immunohistochemical analysis. This is the first study to describe gene expression and cellular localization in the endometrium at the time of MRP for these genes and suggests that the uterus does not prepare to support a pregnancy until d 14. The function of these genes may be critical in the process of MRP.

Progesterone is a steroid hormone secreted from the corpus luteum (CL), which is responsible for establishment and maintenance of pregnancy. Early embryonic mortality often occurs due to inadequate regulation of uterine prostaglandin (PG) F2α secretion, leading to a decrease in progesterone and loss of pregnancy. The objective of the current study was to determine the effects of fish meal supplementation on luteal sensitivity to intrauterine infusions of PGF2α. Nonlactating beef cows received corn glutenmeal or fishmeal supplementation for 60 days. Cows were administered four intrauterine infusions of 0.25 mL saline at 6-h intervals (n = 6 corn gluten meal; n = 5 fish meal) or two doses of 0.5 mg PGF2α in 0.25 mL saline at 12-h intervals (n = 11 corn gluten meal; n = 11 fishmeal) commencing on days 10 to 12 of the estrous cycle. At time of each infusion, luteal biopsies were collected to determine the effects of supplementation on expression of immediate early and steroidogenic genes involved in cholesterol transport and progesterone biosynthesis. Transrectal ultrasonography was performed to measure diameter of CL, and blood samples were collected to determine serum progesterone. Intrauterine infusion of PGF2α resulted in upregulation or no change in FOS, NR4A1, and 3BHSD and downregulation in LDLR, STARD1, and CYP11A1. Although CL diameter decreased, infusion of PGF2α resulted in functional regression in 91% of cows supplemented with corn gluten meal, and only 46% for fish meal supplemented animals. Results demonstrate that fish meal supplementation alters luteal sensitivity to PGF2α, which may affect fertility.Summary SentenceFish meal supplementation alters luteal sensitivity to prostaglandin F2α in the bovine.

MicroRNAs (miRNAs) are small, non-coding RNAs which are produced throughout the body. Individual tissues tend to have a specific expression profile and excrete many of these miRNAs into circulation. These circulating miRNAs may be diagnostically valuable biomarkers for assessing the presence of disease while minimizing invasive testing. In women, numerous circulating miRNAs have been identified which change significantly during pregnancy-related complications (e.g. chorioamnionitis, eclampsia, recurrent pregnancy loss); however, no prior work has been done in this area in the horse. To identify pregnancy-specific miRNAs, we collected serial whole blood samples in pregnant mares at 8, 9, 10 m of gestation and post-partum, as well as from non-pregnant (diestrous) mares. In total, we evaluated a panel of 178 miRNAs using qPCR, eventually identifying five miRNAs of interest. One miRNA (miR-374b) was differentially regulated through late gestation and four miRNAs (miR-454, miR-133b, miR-486-5p and miR-204b) were differentially regulated between the pregnant and non-pregnant samples. We were able to identify putative targets for the differentially regulated miRNAs using two separate target prediction programs, miRDB and Ingenuity Pathway Analysis. The targets for the miRNAs differentially regulated during pregnancy were predicted to be involved in signaling pathways such as the STAT3 pathway and PI3/AKT signaling pathway, as well as more endocrine-based pathways, including the GnRH, prolactin and insulin signaling pathways. In summary, this study provides novel information about the changes occurring in circulating miRNAs during normal pregnancy, as well as attempting to predict the biological effects induced by these miRNAs.

Despite reports that circulating levels of maternal serum exosomes increase during pregnancy and that placenta-specific microRNAs (miRNAs) have been identified in humans, little is known about exosomes and miRNAs during pregnancy in agriculture animals. In this study, we characterized the expression of 94 miRNAs in ovine placentomes at gestation day (GD) 90 by real-time PCR, and then investigated the presence of these miRNAs in exosome samples isolated from maternal jugular blood in non-pregnant ewes and at GD30 and GD90 and in umbilical blood collected at GD90. In maternal jugular exosome samples, 13 miRNAs were present in lower and 12 miRNAs were present in higher amounts at GD90 compared to non-pregnant (GD0) or GD30. Additionally, 12 miRNAs were present in higher amounts in umbilical venous exosomes compared to umbilical arterial exosomes; only miR-132 was lower in exosomes isolated from umbilical venous blood than from umbilical arterial blood. In placentome samples, miR-34c and miR135a abundance was higher in cotyledon tissue than in caruncle, while miR-183 and miR-379 amounts were higher in caruncle than cotyledon tissue. Only miR-379 was differentially expressed in all serum exosomes and placentome samples. Pathway analysis predicted that differentially expressed maternal serum exosomal miRNAs target Cellular Growth and Proliferation and Organ Development pathways, while umbilical serum exosomal and placentomes miRNAs were predicted to target cellular development and organismal/embryonic development.

During early pregnancy, the conceptus and mare communicate to establish pregnancy. Cell-secreted vesicles (e.g., exosomes) have been reported in serum. Exosomes contain bioactive materials, such as miRNA, that can mediate cell responses. We hypothesized that a) exosomes are present in mare circulation and quantity varies with pregnancy status, b) exosomes contain miRNAs unique to pregnancy status, and c) miRNAs target pathways in endometrium based upon pregnancy status of the mare. First, serum samples were obtained from mares in a crossover design, with each mare providing samples from a pregnant and nonmated control cycle (n = 3/sample day) on Days 12, 14, 16, and 18 postovulation. Flow cytometry revealed the presence of serum microvesicles in mares in two different-sized populations (greater than or less than 100 nm), validated by transmission electron microscopy. Second, serum was collected on Days 9, 11, and 13 (n = 4/day), and endometrial biopsies were collected on Days 11 and 13 (n = 3/day) from pregnant and nonmated mares. Total RNA from serum exosomes was evaluated with quantitative RT-PCR using equine-specific miRNA sequences. A total of 12 miRNAs were found in different quantities on the specified days. Pathway analysis suggested that miRNAs targeted focal adhesion molecules (FAMs). Transcripts corresponding to FAMs were evaluated in endometrial biopsies. Protein levels and localization for PAK6 and RAF1 were further evaluated. Our data suggest that serum exosomes contain miRNA that differ based upon pregnancy status, and may affect mRNA expression related to focal adhesion pathway in the endometrium, with a potential role in maternal recognition of pregnancy.

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.

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 F2α, thereby ensuring survival of the corpus luteum and continued progesterone production. Exactly how this occurs in the mare is poorly understood. Because prostaglandin F2α 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 F2α 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

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.

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 (P < 0.05) 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.

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 objective of this experiment was to determine the effects of flow cytometric sorting and freezing on stallion sperm fertility. A 2 x 2 factorial design was used to delineate effects of flow sorting and freezing spermatozoa. Oestrus was synchronised (July-August) in 41 mares by administering 10 ml altrenogest (2.2 mg/ml) per os for 10 consecutive days, followed by 250 microg cloprostenol i.m. on Day 11. Ovulation was induced by administering 3,000 iu hCG i.v. either 6 h (fresh spermatozoa) or 30 h (frozen/thawed spermatozoa) prior to insemination. Mares were assigned randomly to one of 4 sperm treatment groups. Semen was collected from 2 stallions with an artificial vagina and processed for each treatment. Treatment 1 (n = 10 mare cycles) consisted of fresh, nonsorted spermatozoa and Treatment 2 (n = 16 mare cycles) of fresh, flow sorted spermatozoa. Spermatozoa to be sorted were stained with Hoechst 33342 and sorted into X- and Y-chromosome-bearing populations based on DNA content using an SX MoFlo sperm sorter. Treatment 3 (n = 16 mare cycles) consisted of frozen/thawed nonsorted spermatozoa (frozen at 33.5 x 106 sperm/ml in 0.25 ml straws) and Treatment 4 (n = 15 mare cycles) of flow sorted frozen/thawed spermatozoa (frozen at 64.4 x 10(6) sperm/ml). Concentrations of sperm in both cryopreserved treatments were adjusted, based on predetermined average post-thaw motilities, so that each insemination contained approximately 5 x 10(6) motile spermatozoa. Hysteroscopic insemination of 5 x 10(6) motile spermatozoa in a volume of 230 microd was used for all treatments. Pregnancy was determined ultrasonographically 16 days postovulation. No differences were found (P>0.1) in the pregnancy rates for mares inseminated with fresh nonsorted (4/10 = 40.0%), fresh flow sorted (6/16 = 37.5%), frozen/thawed nonsorted (6/16 = 37.5%) and flow sorted frozen/thawed spermatozoa (2/15 = 133%). Pregnancy rates tended (P = 0.12) to be lower following insemination of frozen/thawed flow sorted spermatozoa. Further studies are needed with a larger number of mares to determine if fertility of flow sorted frozen/thawed spermatozoa can be improved.

The objectives of this study were 1) to compare pregnancy rates resulting from 2 methods of insemination using low sperm numbers and 2) to compare pregnancy rates resulting from hysteroscopic insemination of 5 x 106 nonsorted and 5 x 106 spermatozoa sorted for X- and Y-chromosome-bearing populations (flow sorted). Semen was collected with an artificial vagina from 2 stallions of known acceptable fertility. Oestrus was synchronised (June to July) in 40 mares, age 3-10 years, by administering 10 ml altrenogest orally for 10 consecutive days, followed by 250 microg cloprostenol i.m. on Day 11. All mares were given 3000 iu hCG i.v. at the time of insemination to induce ovulation. Mares were assigned randomly to 1 of 3 treatment groups: mares in Treatment 1 (n = 10) were inseminated with 5 x 10(6) spermatozoa deposited deep into the uterine horn with the aid of ultrasonography. Mares in Treatment 2 (n = 10) were inseminated with 5 x 10(6) spermatozoa deposited onto the uterotubal junction papilla via hysteroscopic insemination. Mares in Treatment 3 (n = 20) were inseminated using the hysteroscopic technique with 5 x 10(6) flow sorted spermatozoa. Spermatozoa were stained with Hoechst 33342 and sorted into X- and Y-chromosome-bearing populations based on DNA content using an SX MoFlo sperm sorter. Pregnancy was determined ultrasonographically at 16 days postovulation. Hysteroscopic insemination resulted in more pregnancies (5/10 = 50%) than did the ultrasound-guided technique (0/10 = 0%; P 0.05) when hysteroscopic insemination was used for sorted (5/20 = 25%) and nonsorted spermatozoa (5/10 = 50%). Therefore, hysteroscopic insemination of low numbers of flow sorted stallion spermatozoa resulted in reasonable pregnancy rates.

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 antiviral activity of interferon (IFN) increases in uterine vein serum (UVS) during early pregnancy in sheep. This antiviral activity in UVS collected on Day 15 of pregnancy is blocked by anti-IFN-tau (anti-IFNT) antibodies. Conceptus-derived IFNT was hypothesized to induce IFN-stimulated gene (ISG) expression in endometrium and extrauterine tissues during pregnancy. To test this hypothesis, blood was collected from ewes on Days 12-16 of the estrous cycle or pregnancy. Serum progesterone was >1.7 ng/ml in pregnant (P) and nonpregnant (NP) ewes until Day 13, then declined to

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.

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

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.

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.

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.

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

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.

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.

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.