Estrogens Regulate Placental Angiogenesis in Horses.

A sufficient vascular network within the feto-maternal interface is necessary for placental function. Several pregnancy abnormalities have been associated with abnormal vascular formations in the placenta. We hypothesized that growth and expansion of the placental vascular network in the equine (Equus caballus) placenta is regulated by estrogens (estrogen family hormones), a hormone with a high circulating concentration during equine gestation. Administration of letrozole, a potent and specific inhibitor of aromatase, during the first trimester (D30 to D118), decreased circulatory estrone sulfate concentrations, increased circulatory testosterone and androstenedione concentrations, and tended to reduce the weight of the fetus (p < 0.1). Moreover, the gene expression of CYP17A1 was increased, and the expression of androgen receptor was decreased in the D120 chorioallantois (CA) of letrozole-treated mares in comparison to that of the control mares. We also found that at D120, the number of vessels tended to decrease in the CAs with letrozole treatment (p = 0.07). In addition, expression of a subset of angiogenic genes, such as ANGPT1, VEGF, and NOS2, were altered in the CAs of letrozole-treated mares. We further demonstrated that 17ß-estradiol increases the expression of ANGPT1 and VEGF and increases the angiogenic activity of equine endothelial cells in vitro. Our results from the estrogen-suppressed group demonstrated an impaired placental vascular network, suggesting an estrogen-dependent vasculogenesis in the equine CA during the first trimester.

Intrahost Selection Pressure Drives Equine Arteritis Virus Evolution during Persistent Infection in the Stallion Reproductive Tract.

Equine arteritis virus (EAV) is the causative agent of equine viral arteritis (EVA), a reproductive and respiratory disease of horses. Following natural infection, 10 to 70% of infected stallions can become carriers of EAV and continue to shed virus in the semen. In this study, sequential viruses isolated from nasal secretions, buffy coat cells, and semen of seven experimentally infected and two naturally infected EAV carrier stallions were deep sequenced to elucidate the intrahost microevolutionary process after a single transmission event. Analysis of variants from nasal secretions and buffy coat cells lacked extensive positive selection; however, characteristics of the mutant spectra were different in the two sample types. In contrast, the initial semen virus populations during acute infection have undergone a selective bottleneck, as reflected by the reduction in population size and diversifying selection at multiple sites in the viral genome. Furthermore, during persistent infection, extensive genome-wide purifying selection shaped variant diversity in the stallion reproductive tract. Overall, the nonstochastic nature of EAV evolution during persistent infection was driven by active intrahost selection pressure. Among the open reading frames within the viral genome, ORF3, ORF5, and the nsp2-coding region of ORF1a accumulated the majority of nucleotide substitutions during persistence, with ORF3 and ORF5 having the highest intrahost evolutionary rates. The findings presented here provide a novel insight into the evolutionary mechanisms of EAV and identified critical regions of the viral genome likely associated with the establishment and maintenance of persistent infection in the stallion reproductive tract.IMPORTANCE EAV can persist in the reproductive tract of infected stallions, and consequently, long-term carrier stallions constitute its sole natural reservoir. Previous studies demonstrated that the ampullae of the vas deferens are the primary site of viral persistence in the stallion reproductive tract and the persistence is associated with a significant inflammatory response that is unable to clear the infection. This is the first study that describes EAV full-length genomic evolution during acute and long-term persistent infection in the stallion reproductive tract using next-generation sequencing and contemporary sequence analysis techniques. The data provide novel insight into the intrahost evolution of EAV during acute and persistent infection and demonstrate that persistent infection is characterized by extensive genome-wide purifying selection and a nonstochastic evolutionary pattern mediated by intrahost selective pressure, with important nucleotide substitutions occurring in ORF1a (region encoding nsp2), ORF3, and ORF5.

Fetal-fluid proteome analyses in late-term healthy pregnant mares and in mares with experimentally induced ascending placentitis.

Characterisation of fetal fluids in healthy and disease states of pregnant mares can help to unravel the pathophysiology and to identify putative markers of disease. Thus, this study aimed to compare the protein composition of: (1) amniotic and allantoic fluids of healthy mares obtained immediately after euthanasia and (2) allantoic fluid harvested via centesis before and after experimental induction of placentitis via transcervical inoculation of Streptococcus equi ssp zooepidemicus in healthy mares. Fetal fluids were analysed with a high-throughput proteomic technique after in-gel digestion. Statistical comparisons were performed following normalisation of peptide spectral match. Global normalisation was performed to calculate relative expression. There were 112 unique proteins present in both allantoic and amniotic fluids. There were 13 and 29 proteins defined as amniotic- or allantoic-specific respectively that were present in at least two fluid samples. Another 26 proteins were present in both amniotic and allantoic fluids. Panther DB functional classification grouped fetal-fluid proteins as transfer carriers, signalling molecules, receptors, immunity, hydrolase, enzymes, membrane traffic, cytoskeleton, cell adhesion, calcium binding and extracellular matrix. Experimentally induced placentitis resulted in 10 proteins being upregulated and 10 downregulated in allantoic fluid. Newly identified proteins and changes in the fetal-fluid proteome provide clues about the physiology of pregnancy and pathogenesis of placentitis.

Small RNA (sRNA) expression in the chorioallantois, endometrium and serum of mares following experimental induction of placentitis.

Intrauterine infection and inflammation remain a major cause of preterm labour in women and mares, with little known about small RNA (sRNA) expression in tissue or circulation. To better characterise placental inflammation (placentitis), we examined sRNA expression in the endometrium, chorioallantois and serum of mares with and without placentitis. Disease was induced in 10 mares via intracervical inoculation of Streptococcus equi ssp. zooepidemicus, either with moderate or high levels of inoculum; three uninoculated gestationally matched mares were used as controls. Matched chorioallantois and endometrium were sampled in two locations: Region 1, gross inflammation near cervical star with placental separation and Region 2, gross inflammation without placental separation. In Region 1, 26 sRNAs were altered in chorioallantois, while 20 were altered in endometrium. Within Region 2, changes were more subdued in both chorioallantois (10 sRNAs) and endometrium (two sRNAs). Within serum, we identified nine significantly altered sRNAs. In summary, we have characterised the expression of sRNA in the chorioallantois, the endometrium and the serum of mares with experimentally induced placentitis using next-generation sequencing, identifying significant changes within each tissue examined. These data should provide valuable information about the physiology of placental inflammation to clinicians and researchers alike.

Equine Arteritis Virus Has Specific Tropism for Stromal Cells and CD8+ T and CD21+ B Lymphocytes but Not for Glandular Epithelium at the Primary Site of Persistent Infection in the Stallion Reproductive Tract.

Equine arteritis virus (EAV) has a global impact on the equine industry as the causative agent of equine viral arteritis (EVA), a respiratory, systemic, and reproductive disease of equids. A distinctive feature of EAV infection is that it establishes long-term persistent infection in 10 to 70% of infected stallions (carriers). In these stallions, EAV is detectable only in the reproductive tract, and viral persistence occurs despite the presence of high serum neutralizing antibody titers. Carrier stallions constitute the natural reservoir of the virus as they continuously shed EAV in their semen. Although the accessory sex glands have been implicated as the primary sites of EAV persistence, the viral host cell tropism and whether viral replication in carrier stallions occurs in the presence or absence of host inflammatory responses remain unknown. In this study, dual immunohistochemical and immunofluorescence techniques were employed to unequivocally demonstrate that the ampulla is the main EAV tissue reservoir rather than immunologically privileged tissues (i.e., testes). Furthermore, we demonstrate that EAV has specific tropism for stromal cells (fibrocytes and possibly tissue macrophages) and CD8+ T and CD21+ B lymphocytes but not glandular epithelium. Persistent EAV infection is associated with moderate, multifocal lymphoplasmacytic ampullitis comprising clusters of B (CD21+) lymphocytes and significant infiltration of T (CD3+, CD4+, CD8+, and CD25+) lymphocytes, tissue macrophages, and dendritic cells (Iba-1+ and CD83+), with a small number of tissue macrophages expressing CD163 and CD204 scavenger receptors. This study suggests that EAV employs complex immune evasion mechanisms that warrant further investigation.IMPORTANCE The major challenge for the worldwide control of EAV is that this virus has the distinctive ability to establish persistent infection in the stallion's reproductive tract as a mechanism to ensure its maintenance in equid populations. Therefore, the precise identification of tissue and cellular tropism of EAV is critical for understanding the molecular basis of viral persistence and for development of improved prophylactic or treatment strategies. This study significantly enhances our understanding of the EAV carrier state in stallions by unequivocally identifying the ampullae as the primary sites of viral persistence, combined with the fact that persistence involves continuous viral replication in fibrocytes (possibly including tissue macrophages) and T and B lymphocytes in the presence of detectable inflammatory responses, suggesting the involvement of complex viral mechanisms of immune evasion. Therefore, EAV persistence provides a powerful new natural animal model to study RNA virus persistence in the male reproductive tract.

Endocrine changes, fetal growth, and uterine artery hemodynamics after chronic estrogen suppression during the last trimester of equine pregnancy.

Equine pregnancy is characterized by very high circulating concentrations of estrogens. The physiological roles of estrogens during equine gestation are largely unknown, although some studies suggest a role in the regulation of uterine artery hemodynamics and a relationship between low circulating estrogen concentrations and late pregnancy loss. The objectives of this experiment were to evaluate the effects of estrogen suppression on uterine artery hemodynamics and on pregnancy outcome. Estrogen synthesis was suppressed using letrozole, a potent aromatase inhibitor. Twelve pregnant mares were randomly assigned to a control (n = 6) or treatment (n = 6; 500 mg letrozole orally every 4 days) group with treatment starting at 240 days of gestation and continuing until parturition. Weekly serum samples were analyzed to determine testosterone, dehydroepiandrosterone sulfate, estradiol, estrone sulfate, progestins, and prostaglandin F2α metabolite concentrations. Ultrasonographic examinations were performed biweekly and measurements included uterine artery hemodynamics (diameter, pulsatility, and resistance indices), fetal growth using the diameter of the fetal eye, and placental evaluation using the combined thickness of the uterus and placenta. At parturition, gestational length, foal weight, and neonatal viability were determined. Letrozole suppressed estrogen synthesis during gestation by approximately 90% compared to control values. This large reduction in circulating estrogens had no effect on uterine artery hemodynamics, normal placental development, maintenance of pregnancy, or neonatal viability. However, neonates from letrozole-treated mares had lower (P < 0.05) birth weights than controls, suggesting that estrogens may play a role in fetal growth that is not mediated through regulation of uterine blood flow.

Breakthroughs in Equine Embryo Cryopreservation.

Most equine embryos are collected from the donor mare and transferred immediately as fresh embryos or shipped cooled to a recipient station for transfer within 24 hours. Very few equine embryos are frozen despite the numerous advantages of embryo cryopreservation. There are 2 major hurdles: Only the small embryos (<300 µm) provide good pregnancy rates after freezing/thawing and transfer. Also there is no good procedure for superovulating mares; thus, extra embryos for freezing are not readily available. Using either a slow cool or a vitrification method, pregnancy rates of small equine embryos after freezing/thawing are 50% to 70%.

Effect of testicular degeneration on expression of sperm protein at 22 kDa in stallions.

BACKGROUND: Sperm protein at 22 kDa has been associated with fertility. OBJECTIVES: The objectives of this study were to determine (1) the localization pattern of SP22 on ejaculated and caudal epididymal equine spermatozoa and in epididymal fluid, and to (2) characterize SP22 protein and mRNA expression in testicular and epididymal tissues in response to heat-induced testicular degeneration. MATERIALS AND METHODS: Semen was collected before and after hemi-castration, as well as prior to and following insulation of the remaining testes, and tissue specimens were collected for analysis. RESULTS: Histopathology confirmed degeneration in insulated testes. Ejaculated and epididymal spermatozoa from samples collected prior to insulation of the testicles had a predominant staining pattern of SP22 over the equatorial region. However, the equatorial pattern in the pre-insulation epididymal semen samples was significantly lower than in the pre-insulation ejaculated semen samples (68 ± 3, 81 ± 2.6, respectively). Ejaculated and epididymal samples collected after insulation of the testicles showed a complete loss of staining as the predominant pattern. Western blot analysis verified the presence of SP22 on fresh ejaculated spermatozoa prior to and following heat-induced degeneration, on epididymal spermatozoa after testicular insulation, and in testicular and epididymal tissues. Heat insulation significantly reduced messenger RNA expression in the head of the epididymis and testicular tissues. Immunohistochemistry of the testicular and epididymal tissues pre-heating showed considerably weaker staining than the same tissues post-heating. DISCUSSION AND CONCLUSION: It was concluded that heat-induced testicular damage causes both loss and relocation of SP22 on the sperm membrane. Future studies are warranted to determine the diagnostic value of these findings.

Effects of components of semen extenders on the binding of stallion spermatozoa to bovine or equine zonae pellucidae.

The effects of semen extender components on the ability of stallion sperm to bind to the zona pellucida (ZP) and the suitability of using bovine ZP for a ZP-binding assay for stallion sperm were investigated in a series of experiments. In Experiment I, binding of stallion sperm to both bovine and equine ZP was significantly increased when a skim milk-based extender (EZM) was used. In Experiment II, a threefold increase in sperm binding to ZP was observed when sperm were diluted in EZM compared with diluents, which contained no milk (TALP, LAC, and EmCare). In Experiment III, centrifuging the sperm through Percoll did not increase sperm binding to the ZP but did remove any positive effect of EZM on sperm-ZP binding. In Experiment IV, exposure of either sperm or ZP to EZM before co-incubation did not increase sperm binding to ZP. In Experiment V, sperm diluted in TALP containing skim milk, EZM, or INRA96 bound more efficiently to the ZP than sperm diluted in TALP without milk proteins. In Experiment VI, sodium caseinate, native phosphocaseinate, and caseinoglycopeptide increased sperm binding to the ZP. In conclusion, diluents containing milk or milk proteins markedly enhanced the number of sperm bound to both equine and bovine ZP.

Gene profiling of inflammatory genes in day 18 endometria from pregnant and non-pregnant mares.

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

Alterations of Circulating Biomarkers During Late Term Pregnancy Complications in the Horse Part II: Steroid Hormones and Alpha-Fetoprotein.

Preterm labor and/or abortion causes considerable economic impact on the equine industry. Unfortunately, few experimental models exist for the induction of various pregnancy-related complications, and therefore extrapolations are made from the experimental model for ascending placentits, although inferences may be minimal. Certain steroid hormones (progestogens, estrogens) and fetal proteins (alpha-fetoprotein; AFP) might improve the diagnostics for abnormal pregnancy, but the utility of these markers in the field is unknown. To assess this, thoroughbred mares (n = 702) were bled weekly beginning in December 2013 until parturition/abortion. Following parturition, fetal membranes were assessed histopathologically and classified as either ascending placentitis (n = 6), focal mucoid placentitis (n = 6), idiopathic abortion (n = 6) or no disease (n = 20). Weekly serum samples were analyzed for concentrations of progesterone, estradiol-17ß, and AFP. Samples were analyzed retrospectively from the week of parturition/abortion in addition to the preceding four weeks. For both ascending and focal mucoid placentitis, a significant increase in progesterone and AFP was noted, alongside a significant decrease in estradiol-17ß and the ratio of estradiol-17ß to progesterone in comparison to controls. In contrast, idiopathic abortions experienced a decrease in progesterone concentrations alongside an increase in AFP, and this was only noted in the week preceding parturition/abortion. In conclusion, spontaneous placental infection in the horse altered both endocrine and feto-secretory markers in maternal circulation, while minimal changes were noted preceding noninfectious idiopathic abortion. Additionally, this is the first study to report an alteration in steroid hormones and AFP during the disease process of focal mucoid placentitis, the etiology of which includes Nocardioform placentitis.

Kisspeptin has an independent and direct effect on the pituitary gland in the mare.

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.

The Effect of Mycobacterium Cell Wall Fraction on Histologic, Immunologic, and Clinical Parameters of Postpartum Involution in the Mare.

Maintaining yearly foal production is important for the economic success of the broodmare, and this requires breeding to occur as quickly postpartum as possible. The initial postpartum estrus occurs within 5-20 days postpartum, whereas the uterus is still undergoing repair from tissue alterations during pregnancy and parturition, a process known as involution. Attempts have been made to hasten this process, but with minimal success. Mycobacterium cell wall fraction (MCWF) is an immunomodulator that has been shown to reduce bacterial growth and alter aspects of the immune response to breeding, but it is unknown if MCWF hastens the process of involution. Therefore, the objectives of this study were to (1) investigate the effect of MCWF on tissue remodeling, (2) assess the effect of MCWF on the local immune system of the uterus, and (3) determine the optimal treatment interval needed for these processes to occur. We hypothesize that repeated treatments of MCWF postpartum will hasten the process of involution. To study this, 16 pregnant mares of mixed breeds were evaluated postpartum. Control mares (n = 4) received 1.5 mL lactated Ringer's solution intravenously on Day 1 (Day 0 = day of parturition) postpartum and again on Day 7, whereas treated mares either received 1.5 mL Settle intravenously on Day 1 and Day 7 (TX1; n = 6) or 1.5 mL Settle intravenously on Day 1 and then every 3 days until ovulation was detected (TX2; n = 6) and then evaluated until 15 days postpartum. Mares were assessed every 3 days for clinical, immunologic, and histologic parameters. Clinical parameters were assessed with transrectal ultrasonography and included ovarian activity, uterine fluid retention, and measurement of the uterine diameter, in addition to endometrial culture. Immunologic parameters included endometrial biopsies for quantitative polymerase chain reaction for expression of various cytokines (interleukin [IL]-1ß, IL-1RN, IL-4, IL-6, IL-8, IL-10, tumor necrosis factor [TNF], interferon [IFN]-γ, and granulocyte-macrophage colony-stimulating factor) in addition to endometrial cytology. Formalin-fixed endometrial biopsies were histologically assessed for the retention of microcaruncles, dilation of endometrial glands, and inflammation of the mucosa, stratum compactum, and spongiosum. Statistics were performed using SAS 9.4, using a mixed model for repeated measures with mare and treatment as a random effect. All post-hoc analysis was done using a Tukey's honestly significant difference test. Involution was considered complete by Day 15 postpartum in all mares, and the day postpartum had a significant effect on almost all parameters investigated, indicating the immunologic process of involution. Treatment with MCWF decreased the magnitude of bacterial growth in addition to time to negative culture. In addition, MCWF increased the expression of IL-1ß, IFNγ, and TNF. Although minimal treatment effect was noted histologically, a decrease in mucosal inflammation was seen in MCWF-treated mares. In conclusion, involution appears to be influenced by the immune system. In addition, MCWF appears to have a bactericidal effect on the postpartum mare, and this may be because of an increase in proinflammatory cytokines. It is unknown if this bactericidal property will improve fertility on the first estrous cycle postpartum, and future studies are needed to determine this.

Osmotic tolerance limits and membrane permeability characteristics of stallion spermatozoa treated with cholesterol.

Stallion spermatozoa exhibit osmotic damage during the cryopreservation process. Recent studies have shown that the addition of cholesterol to spermatozoal membranes increases the cryosurvival of bull, ram and stallion spermatozoa, but the exact mechanism by which added cholesterol improves cryosurvival is not understood. The objectives of this study were to determine if adding cholesterol to stallion sperm membranes alters the osmotic tolerance limits and membrane permeability characteristics of the spermatozoa. In experiment one, stallion spermatozoa were treated with cholesterol-loaded cyclodextrin (CLC), subjected to anisotonic solutions and spermatozoal motility analyzed. The spermatozoa were then returned to isotonic conditions and the percentages of motile spermatozoa again determined. CLC treatment increased the osmotic tolerance limit of stallion spermatozoa in anisotonic solutions and when returned to isotonic conditions. The second and third experiments utilized an electronic particle counter to determine the plasma membrane characteristics of stallion spermatozoa. In experiment two, stallion spermatozoa were determined to behave as linear osmometers. In experiment three, spermatozoa were treated with CLC, incubated with different cryoprotectants (glycerol, ethylene glycol or dimethyl formamide) and their volume excursions measured during cryoprotectant removal at 5 degrees and 22 degrees C. Stallion spermatozoa were less permeable to the cryoprotectants at 5 degrees C than 22 degrees C. Glycerol was the least permeable cryoprotectant in control cells. The addition of CLC's to spermatozoa increased the permeability of stallion spermatozoa to the cryoprotectants. Therefore, adding cholesterol to spermatozoal membranes reduces the amount of osmotic stress endured by stallion spermatozoa during cryopreservation.

Lipidomics of equine amniotic fluid: Identification of amphiphilic (O-acyl)-ω-hydroxy-fatty acids.

Amniotic fluid is essential for the growth and maturation of the fetus prior to parturition. While our knowledge of human amniotic fluid is extensive, current data for equine amniotic fluid is limited. We therefore undertook a detailed lipidomics analysis of equine amniotic fluid. Using a non-targeted high-resolution mass spectrometric lipidomics analysis of equine amniotic fluid, we were able to characterize a diverse array of individual lipids. This non-biased analytical approach detected, for the first time, the presence of (O-acyl)-ω-hydroxy-fatty acids (OAHFA) with up to 52 carbon chain lengths in amniotic fluid. The identities of these lipid amphiphiles were validated both by high-resolution mass spectrometry and by tandem mass spectrometry (<2 ppm mass error) which identified the fatty acid and hydroxy-fatty acid components of individual OAHFAs. The only previous identification of OAHFAs has been in sperm and meibomian glands, and their sebaceous secretions, suggesting that these lipids may have unique functional roles in highly specialized compartments. The amphiphilic and surfactant properties of these unique lipids could provide an interface between amniotic lipids and fetal skin and/or lungs. The potential roles of OAHFAs as well as their source in amniotic fluid remain to be explored based upon these novel lipidomics findings but our study of the developmental time course of amniotic OAHFAs suggest that they may act as lubricants in delivery and/or a role in the development of fetal lung function around parturition.

Identification of Reference Genes for Analysis of microRNA Expression Patterns in Equine Chorioallantoic Membrane and Serum.

MicroRNAs (miRNAs) have important posttranscriptional regulatory abilities, and there is considerable interest in evaluating their expression patterns in different pathophysiological states. The most common method of quantifying miRNA expression is quantitative reverse transcription PCR; however, the identification of tissue-specific and species-specific reference miRNA is a prerequisite for miRNA expression analysis. Currently, no reference genes have been described for evaluating miRNA expression in equine serum and chorioallantoic membrane (CAM) during pregnancy. The aim of the present study was to characterize reference genes for normalization of miRNA expression in CAM and serum in the pregnant equine. To identify the most stable miRNAs in serum, expression of potential candidates was evaluated in serum samples from diestrous mares, pregnant mares and geldings. To identify the most stable miRNAs in CAM, expression of potential candidates was evaluated in CAM, collected from mares at 4, 6 and 10 months of pregnancy and immediately postpartum. From a previously generated miRNA sequencing dataset, two separate lists of potential reference miRNAs were identified (serum and CAM) using the NormFinder program, in addition to the commonly used small RNA normalizers, 5S rRNA and U6 snRNA. The putative reference miRNAs were selected using geNorm and NormFinder. In case of a nonsignificant correlation between the results of ranking and stability value between these two programs, ranking from BestKeeper was also included. NormFinder and geNorm consistently identified eca-miR-21-5p, eca-let-7a-5p and eca-miR-10a-5p as the three most stable reference genes for the normalization of serum miRNAs. Within CAM samples, the average ranking obtained from the ranking of NormFinder, geNorm and BestKeeper identified eca-miR-8908a-1-5p, eca-miR-369-5p and eca-miR-106a-5p as the three most stable miRNAs. These observations provide information about equine-specific reference genes that can be used for normalizing miRNAs expression patterns in CAM and serum during the equine pregnancy.

A comparison of progesterone assays for determination of peripheral pregnane concentrations in the late pregnant mare.

During the latter half of gestation in mares, there is a complex milieu of pregnanes in peripheral blood. Progesterone concentrations are often assessed by immunoassay during late gestation as a measure of pregnancy well-being; however, interpretation of results is complicated by the numerous cross-reacting pregnanes present in high concentrations during late gestation. Further, many mares are supplemented with an exogenous progestin, altrenogest, which may also cross-react with existing assays and further confound interpretation. The objectives of this study were: 1) to compare differences in pregnane concentrations determined with four immunoassays compared to LC-MS/MS and 2) to assess cross-reactivity observed with the same immunoassays, specifically considering pregnenolone (P5), progesterone (P4), 5α-dihydroprogesterone (DHP), allopregnanolone, and altrenogest. Blood samples from four healthy mares in late gestation were evaluated by immunoassay and by LC-MS/MS. Measured immuno-reactive progesterone (ir-progesterone) concentrations differed (p < 0.0001) between immunoassays, although results were highly correlated (r = 0.85-1.0; p < 0.001). Measured ir-progesterone concentrations by immunoassay were linearly associated (r2 = 0.68-0.76; p < 0.001) with concentrations of P5, P4, DHP, and allopregnanolone determined by LC-MS/MS. There was no detectable cross-reaction of altrenogest in any immunoassay, but varying degrees of cross-reactivity was observed with other pregnanes analyzed. These data confirm ir-progesterone concentrations during late gestation vary depending upon the assay used and the cross-reactivity to other pregnanes present in late gestation, although the synthetic progestin altrenogest did not affect the results of any immunoassay tested.

Aspiration of oocytes from transitional, cycling, and pregnant mares.

The aim of this study was to compare the efficacy of three approaches for recovering equine oocytes via transvaginal ultrasound-guided follicular aspiration. Fourteen mares were used as oocyte donors during the spring transition period and physiologic breeding season, and 11 mares were bred for use as oocyte donors during early gestation. In all mares, large (>20 mm) and small (10-20 mm) follicles were aspirated in eight rounds every 10-11 days. In each of the four rounds during the transition period, half the mares received 12.5 mg eFSH once daily for 4 days prior to aspiration. For each of the four rounds during the cycling season, half the mares received 12.5 mg eFSH twice daily for 3 days prior to aspiration. Pregnant mares were aspirated on days 25, 40 and 55 of gestation and received no eFSH. There were more large (>20 mm) follicles in cycling controls (2.25+/-0.27) and cycling FSH-treated (2.64+/-0.27) mares than in transitional FSH-treated mares (1.18+/-0.27). The number of oocytes recovered from small (10-20 mm) follicles varied by mare (P<0.05) and averaged 1.08+/-0.22 per aspiration for transitional mares and 1.23+/-0.22 per aspiration for cycling mares (P>0.1). The number of oocytes per aspiration from large follicles was greater in cycling FSH-treated mares (0.46+/-0.09) than in transitional control mares (0.11+/-0.09). In pregnant mares, more large follicles were present at day 25 than at any other time, and the number of oocytes per aspiration from large follicles was greater at day 25 (0.73+/-0.16) than at day 55 (0.04+/-0.18). When compared across all seasons and treatments, the day 25 pregnant mares yielded the greatest number of oocytes per aspiration (2.91+/-0.66 per mare).

Sex-steroid receptors, prostaglandin E2 receptors, and cyclooxygenase in the equine cervix during estrus, diestrus and pregnancy: Gene expression and cellular localization.

The cervix is a dynamic structure that undergoes dramatic changes during the estrous cycle, pregnancy and parturition. It is well established that hormonal changes, including estrogens, progestogens and prostaglandins, regulate the expression of key proteins involved in cervical function. The arachidonic acid cascade is important in the remodeling and relaxation of the cervix in the days preceding parturition. Despite the complexity of this mechanism, regulation of cervical function has received little study in the mare. Therefore, the objective of this study was to compare the expression of estrogen receptor α (ESR1) and ß (ESR2), progesterone receptor (PGR), prostaglandin E2 type 2 (PTGER2) and type 4 (PTGER4) receptors as well as cyclooxygenase-1 (PTGS1) and -2 (PTGS2) in the equine cervical mucosa and stroma during estrus, diestrus and late pregnancy using qPCR. Immunohistochemistry was used to localize ESR1, ESR2, PGR, PTGER2 and PTGER4 receptors in these regions of the cervix. Relative mRNA expression of ESR1 and PGR was greater during estrus and diestrus than in late pregnancy in both the mucosa and stroma of the cervix. Expression of PTGER2 was highest in the cervical stroma during late pregnancy compared to either estrus or diestrus. Moreover, PTGS1 expression in mucosa and PTGS2 in stroma was greater during late pregnancy compared with estrus, but not diestrus. Immunostaining for ESR1, ESR2, PGR, PTGER2 and PTGER4 was consistently detected in the nucleus and cytoplasm of epithelium of the endocervix as well as the smooth muscle cytoplasm of the cervix in all stages evaluated. Immunolabeling in smooth muscle nuclei was detected for ESR1 and PGR in estrus, diestrus and late pregnancy, and for ESR2 in estrus and late pregnancy stages. The changes noted in late gestation likely reflect preparation of the equine cervix for subsequent parturition.

The influence of age, antral follicle count and diestrous ovulations on estrous cycle characteristics of mares.

Reproductive aging must be well understood to optimize the reproductive management of older mares and to predict their reproductive life-span. The objectives of this study were to: 1) examine age-related differences in follicular dynamics, endocrine profiles, and primordial follicle counts, 2) evaluate the influence of antral follicle count (AFC) on age-related changes in follicular parameters, and 3) determine the influence of diestrous ovulations on the estrous cycle. Young (3-8yr; n = 10), middle-aged (9-18 yr; n = 16), and old (>18 yr; n = 19) light horse mares were examined with transrectal ultrasonography to monitor follicular growth over two consecutive estrous cycles. Jugular blood samples were taken and plasma progesterone and FSH concentrations were determined by an enzyme immunoassay and radioimmunoassay, respectively. Both interovulatory intervals and follicular phases were longer and the day of follicle deviation occurred later in aged mares. Furthermore, older mares had a tendency to ovulate smaller follicles. Neither follicular growth rate, the number of ovulations nor the length of luteal phase was influenced by mare age. Interestingly, as mare age increased, mares with low AFC had longer interovulatory intervals and follicular phases than mares with medium or high AFC. In addition, the number of primordial follicles declined with an increase in mare age but varied considerably between mares of the same age. Progesterone concentrations were positively influenced by age, whereas FSH concentrations were not, despite that FSH concentrations appeared higher in aged mares during the follicular phase. Estrous cycles with a diestrous ovulation had a longer interovulatory interval as well as a longer follicular and luteal phase while day of deviation occurred later. Progesterone concentrations were significantly higher on day 14 and 16 in estrous cycles with a diestrous ovulation than without a diestrous ovulation. In conclusion, aging in mares is associated with changes in follicular parameters which in turn are closely linked to differences in antral follicle count suggesting a relationship with ovarian reserve. Therefore, determination of antral follicle counts in aged mares can provide valuable information about the reproductive aging process. Finally, diestrous ovulations have a significant influence on different estrous cycle parameters.