Equine Embryos Research Articles

Developmental Competence of equine MI oocytes following in vitro maturation culture

With an increasing demand for the in vitro production (IVP) of equine embryos over the past 10 years, the value of each oocyte recovered has also increased. Intracytoplasmic sperm injection (ICSI) is performed to fertilize the oocytes reaching MII stage of nuclear maturation, which is determined by microscopic evidence of an extruded polar body (PB). Cytoplasmic maturation, during which replication of organelles, proteins and RNA's necessary for postfertilization development occurs, is more difficult to evaluate. However, equine oocytes contain a large amount of cytoplasmic lipids and the migration and polarization of lipid granules can be microscopically visualized in mature oocytes. Furthermore, the distribution of lipids may reflect a level of cytoplasmic capacitation and developmental competence, although the cleavage rate of equine oocytes fertilized following maturation culture does not appear to be significantly different based on lipid polarization (Ambruosi et al. Therio 2009; 71:1093-1104). The present study evaluated the migration and polarization of cytoplasmic lipids (granularity) following in vitro maturation to determine if blastocyst development can occur in the absence of PB extrusion. Cumulus-oocyte-complexes (COC; n=411) obtained from ovarian follicles by transvaginal aspiration were stored in a holding medium overnight at 20°C before undergoing incubation at 38.5°C in 50ul drops of equilibrated maturation media as described by Foss. et al (EVJ Supp 2013;45:39-43). After 28.5 hours, oocytes were denuded followingexposure to 80IU hyaluronidase and evaluated for the presence of an extruded PB as well as lipid reorganization. The cytoplasm of MI oocytes that did not exhibit PB extrusion was graded according to the percentage with a dark granular appearance. All oocytes were fertilized by conventional ICSI with thawed spermatozoa from ICSI-fertile stallions. Fertilized oocytes were cultured as described by Foss et al (EVJ Suppl 2013;45:39-43). When examined under an inverted 1000X light microscope, 66% (N=273/411) of the denuded oocytes exhibited an intact membrane with an extruded polar body and were considered mature (MII), while 26% (N=106/411) of the oocytes without a visible polar body were classified as immature (MI). Of the MI oocytes injected, 23% (N=24/106) were deemed to have attained some level of cytoplasmic capacitation (Cc=40-70% granularity). A cleavage rate of 25.5% was recorded for all injected MI oocytes regardless of Cc status. Of the 24 MI oocytes with some Cc, nine (37%) cleaved and continued to develop, and seven of those developed to the blastocyst stage. Of the 82 MI oocytes that did not demonstrate Cc, 18 (22%) cleaved but no blastocysts (0%) were obtained. This study found a blastocyst developmental potential of 6.6% per total MI's injected and 29% per MI oocytes with at least a 40-70% Cc. Therefore, injecting oocytes that do not show nuclear maturation, yet have reached a level of cytoplasmic capacitation, may still result in blastocyst development.

Expression of Oxytocin/Neurophysin I and Oxytocinase in the Equine Conceptus from Day 8 to Day 21 Post-Ovulation.

Simple SummaryOxytocinase is an enzyme that breaks down oxytocin in serum and tissues. The presence of oxytocin and oxytocinase mRNA and protein and their relationship to other genes in the horse embryo/conceptus that regulate pregnancy recognition and embryo/conceptus fixation have not been investigated. Our objective was to characterize the expression of oxytocin, oxytocinase and other related genes in horse embryos. Oxytocinase was increased at D15 compared to D10 and was identified in the horse embryo membranes. Oxytocin expression was highest at D21, and lowest on D12 and D14; no protein was identified, while its receptor (OXTR) was highest on D14 and D21. Oxytocinase was highest at D15 compared to D10 and high expression of prostaglandin E synthase was found on D14, D15 and D21. Oxytocinase may have a role in embryonic fixation. Further investigation of the role of oxytocinase during pregnancy is warranted.Leucyl and cystinyl aminopeptidase (LNPEP/oxytocinase) is an enzyme that metabolizes oxytocin in serum and tissues. The presence of oxytocin/neurophysin I (OXT), oxytocin and LNPEP and their relationship to other genes is unknown in the equine conceptus. Our objective was to characterize gene expression of LNPEP and OXT on D8, 10, 12, 14, 15, 16 and 21 conceptuses in relationship to other genes. Immunohistochemistry, western blot and liquid chromatography with tandem mass spectrometry (LC-MS/MS) were used for identification of oxytocin and LNPEP in D15, 16 and 18 conceptuses. LNPEP was increased at D15 compared to D10, was immunolocalized in the equine trophectoderm and endoderm, and protein was confirmed by LC-MS/MS. Maximal abundance of OXT was at D21, and lowest on D12 and D14, but no protein was identified. OXTR abundance was highest on D14 and D21. LNPEP was correlated with PTGFR and PTGES on D12 and D14–D15, and high expression of PTGES, PTGS2 was found on D14, D15 and D21; PTGFR was found on D8 and D12–21. LNPEP may have a role in prostaglandin regulation and conceptus fixation by decreasing the availability of oxytocin. Further investigation on the role embryonic LNPEP during pregnancy is warranted.

The Induced Expression of BPV E4 Gene in Equine Adult Dermal Fibroblast Cells as a Potential Model of Skin Sarcoid-like Neoplasia

The equine sarcoid is one of the most common neoplasias in the Equidae family. Despite the association of this tumor with the presence of bovine papillomavirus (BPV), the molecular mechanism of this lesion has not been fully understood. The transgenization of equine adult cutaneous fibroblast cells (ACFCs) was accomplished by nucleofection, followed by detection of molecular modifications using high-throughput NGS transcriptome sequencing. The results of the present study confirm that BPV-E4- and BPV-E1^E4-mediated nucleofection strategy significantly affected the transcriptomic alterations, leading to sarcoid-like neoplastic transformation of equine ACFCs. Furthermore, the results of the current investigation might contribute to the creation of in vitro biomedical models suitable for estimating the fates of molecular dedifferentiability and the epigenomic reprogrammability of BPV-E4 and BPV-E4^E1 transgenic equine ACFC-derived sarcoid-like cell nuclei in equine somatic cell-cloned embryos. Additionally, these in vitro models seem to be reliable for thoroughly recognizing molecular mechanisms that underlie not only oncogenic alterations in transcriptomic signatures, but also the etiopathogenesis of epidermal and dermal sarcoid-dependent neoplastic transformations in horses and other equids. For those reasons, the aforementioned transgenic models might be useful for devising clinical treatments in horses afflicted with sarcoid-related neoplasia of cutaneous and subcutaneous tissues.

Local embryo-mediated changes in endometrial gene expression during embryo mobility in mares

Prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α) are involved in equine embryo mobility throughout the uterus on Days 11–15 (ovulation = Day 0). On a day (Day 12) of maximal embryo mobility in pregnant mares (n = 13) and before luteolysis in nonbred mares (n = 10), gene expressions were compared between the uterine horns that did and did not contain the mobile embryo and between pregnant and nonbred mares. A cytobrush was used to collect an endometrial sample from the middle of each uterine horn. In nonbred mares, there was no difference for any of the considered gene expressions between the uterine horn ipsilateral and contralateral to the CL or for side (left vs right). For endometrial estrogen receptors, ESR1 was lower (P < 0.03) and ESR2 was greater (P < 0.04) for pregnant than nonbred mares. The mRNA abundance for PGE2 synthase (PTGES) was greater (P < 0.05) in the horn with (1.40 ± 0.10) than without (0.89 ± 0.10) the embryo and was greater (P < 0.05) in the horn with the embryo than in the combined horns of nonbred mares (1.06 ± 0.10). The hypothesis that the embryo locally upregulates PGE2 and PGF2α synthesis in the endometrium adjacent to the embryo in the pregnant group but not in the uterine horns of the nonbred group, was partially supported; only PGE2 synthase (PTGES) was locally upregulated in the endometrium adjacent to the mobile embryo.

Colabamento manual da blastocele não foi efetivo para aumentar a viabilidade de embriões equinos vitrificados

Embryo cryopreservation methods have been used for commercialization and formation of genetic banks. Cryopreservation of equine embryos &lt;300 μm in diameter, collected at days 6-6.5 after ovulation, allows satisfactory pregnancy rates. However, higher embryo collection rates in mares are obtained when uterine flush is performed between days 7 and 8 after ovulation when embryos are &gt;300 μm in diameter, needing blastocoel collapse for satisfactory resistance to cryopreservation by vitrification. To evaluate the viability of simplified blastocoel collapse by embryo puncture with low technology and low-cost equipment, 22 embryos, collected at day 8 post-ovulation (D8), were allocated to the following groups: (1) micropuncture with a 30 G needle, assisted by a mechanical micromanipulator, before vitrification (n=4); (2) manual blade microsection before vitrification (n=6); (3) no manipulation prior to vitrification (n=8); and (4) freshly inovulated embryos (n=4). Despite the high re-expansion rates observed after vitrification, embryos manipulated prior to vitrification (groups MP and MS) did not result in pregnancy 25 days after transfer. On the other hand, embryos from groups NM (non-micromanipulated) and FR (freshly inovulated) resulted in pregnancies at 25 days. Under the conditions of the present study, manual blastocoel collapse was not efficient in increasing cryotolerance to vitrification among large embryos, requiring improvements to obtain pregnancies.

Influence of storage at 5°C and vitrification on apoptotic changes in equine blastocysts

The aim of the study was to determine the extent of nuclear DNA fragmentation in equine embryos at the blastocyst stage stored at 5° C for 6 and 24 h or vitrified in straws or in the Rapid-i system. An additional aim was to determine the relationship between the age of fresh and stored embryos and the intensity of apoptosis. In this study, 42 embryos were analyzed by the TUNEL method and allocated to the following groups: fresh (control -14 embryos), stored at 5° C for 6 h (7 embryos), stored at 5° C for 24 h (7 embryos), vitrified in straws (7 embryos), and vitrified in the ultra-fast Rapid-i system (7 embryos). It was found that 36.7% of fresh equine embryos had apoptotic nuclei, and the mean dead cell index (DCI) was 0.3%. This was evident as an increase in DNA fragmentation in equine embryos after vitrification in straws (2.3%) compared to fresh embryos (0.3%) and those vitrified in the Rapid-i system (0.3%). Embryo storage at 5° C did not affect the apoptosis frequency in nuclei regardless of the time of storage. DCI for embryos stored for 6 h was 0.44%, and after 24 h of incubation it was 0.64%. The intensity of apoptotic changes observed in equine embryos depended on their age and size. The apoptosis frequency was negatively correlated with the age and diameter of fresh embryos, embryos stored at 5° C, and embryos vitrified in Rapid-I, whereas for embryos vitrified in straws the correlation was positive.

Spatiotemporal endometrial transcriptome analysis revealed the luminal epithelium as key player during initial maternal recognition of pregnancy in the mare

During the period of maternal recognition of pregnancy (MRP) in the mare, the embryo needs to signal its presence to the endometrium to prevent regression of the corpus luteum and prepare for establishment of pregnancy. This is achieved by mechanical stimuli and release of various signaling molecules by the equine embryo while migrating through the uterus. We hypothesized that embryo’s signals induce changes in the endometrial gene expression in a highly cell type-specific manner. A spatiotemporal transcriptomics approach was applied combining laser capture microdissection and low-input-RNA sequencing of luminal and glandular epithelium (LE, GE), and stroma of biopsy samples collected from days 10–13 of pregnancy and the estrous cycle. Two comparisons were performed, samples derived from pregnancies with conceptuses ≥ 8 mm in diameter (comparison 1) and conceptuses ≤ 8 mm (comparison 2) versus samples from cyclic controls. The majority of gene expression changes was identified in LE and much lower numbers of differentially expressed genes (DEGs) in GE and stroma. While 1253 DEGs were found for LE in comparison 1, only 248 were found in comparison 2. Data mining mainly focused on DEGs in LE and revealed regulation of genes related to prostaglandin transport, metabolism, and signaling, as well as transcription factor families that could be involved in MRP. In comparison to other mammalian species, differences in regulation of genes involved in epithelial barrier formation and conceptus attachment and implantation reflected the unique features of equine reproduction at the time of MRP at the molecular level.

Allele-specific expression analysis reveals conserved and unique features of preimplantation development in equine ICSI embryos†.

Embryonic genome activation and dosage compensation are major genetic events in early development. Combined analysis of single embryo RNA-seq data and parental genome sequencing was used to evaluate parental contributions to early development and investigate X-chromosome dynamics. In addition, we evaluated dimorphism in gene expression between male and female embryos. Evaluation of parent-specific gene expression revealed a minor increase in paternal expression at the 4-cell stage that increased at the 8-cell stage. We also detected eight genes with allelic expression bias that may have an important role in early development, notably NANOGNB. The main actor in X-chromosome inactivation, XIST, was significantly upregulated at the 8-cell, morula, and blastocyst stages in female embryos, with high expression at the latter. Sexual dimorphism in gene expression was identified at all stages, with strong representation of the X-chromosome in females from the 16-cell to the blastocyst stage. Female embryos showed biparental X-chromosome expression at all stages after the 4-cell stage, demonstrating the absence of imprinted X-inactivation at the embryo level. The analysis of gene dosage showed incomplete dosage compensation (0.5 < X:A < 1) in MII oocytes and embryos up to the 4-cell stage, an increase of the X:A ratio at the 16-cell and morula stages after genome activation, and a decrease of the X:A ratio at the blastocyst stage, which might be associated with the beginning of X-chromosome inactivation. This study represents the first critical analysis of parent- and sex-specific gene expression in early equine embryos produced in vitro.

Effect of Overfeeding Shetland Pony Mares on Embryonic Glucose and Lipid Accumulation, and Expression of Imprinted Genes

Simple SummaryIn pregnant individuals, maternal overnutrition is associated with disturbances in the expression of specific genes and nutrient transporters in the early embryo, which can affect both fetal and placental development and have lasting effects on the health of resulting offspring. To examine how maternal overfeeding affects the equine embryo, Shetland pony mares were fed either a high-energy (HE: 200% of net energy requirements) or maintenance (control) diet. Mares from both groups were inseminated, and day-seven embryos were recovered and transferred to recipients from the same or the alternate group. The expression of several genes, nutrient transporters and DNA methyltransferases (DNMTs; play an important role in regulating gene expression) were determined in extra-embryonic membranes after recovery on day 28 of gestation. The expression of nutrient transporters was also assessed in endometrium recovered from recipient mares immediately after embryo removal. In addition, glucose uptake by day-28 extra-embryonic membranes, and lipid droplet accumulation in day-seven embryos were assessed. Maternal overfeeding resulted in elevated expression of several genes, DNMTs and nutrient transporters following embryo transfer from an HE to a control mare. The expression of two amino acid transporters was also elevated in the endometrium after embryo transfer from HE to control. Maternal overfeeding did not affect lipid droplet accumulation in day-seven embryos, or glucose uptake by membranes of day-28 embryos. It remains to be seen whether the alterations in gene expression are maintained throughout gestation and into postnatal life.Maternal overfeeding is associated with disturbances in early embryonic epigenetic reprogramming, leading to altered expression of imprinted genes and nutrient transporters, which can affect both fetal and placental development and have lasting effects on the health of resulting offspring. To examine how maternal overfeeding affects the equine embryo, Shetland pony mares were fed either a high-energy (HE: 200% of net energy requirements) or maintenance (control) diet. Mares from both groups were inseminated, and day-seven embryos were recovered and transferred to recipients from the same or the alternate group. The expression of a panel of imprinted genes, glucose and amino acid transporters, and DNA methyltransferases (DNMTs) were determined in conceptus membranes after recovery on day 28 of gestation (late pre-implantation phase). The expression of nutrient transporters was also assessed in endometrium recovered from recipient mares immediately after conceptus removal. In addition, glucose uptake by day-28 extra-embryonic membranes, and lipid droplet accumulation in day-seven blastocysts were assessed. Maternal overfeeding resulted in elevated expression of imprinted genes (IGF2, IGF2R, H19, GRB10, PEG10 and SNRPN), DNMTs (DNMT1 and DNMT3B), glucose (SLC2A1), fructose (SLC2A5) and amino acid (SLC7A2) transporters following ET from an HE to a control mare. Expression of amino acid transporters (SLC1A5 and SLC7A1) was also elevated in the endometrium after ET from HE to control. Maternal overfeeding did not affect lipid droplet accumulation in blastocysts, or glucose uptake by day-28 membranes. It remains to be seen whether the alterations in gene expression are maintained throughout gestation and into postnatal life.

Evaluating the use of piezo manipulator, laser or their combination for blastocoel cavity puncture to improve cryopreservation outcomes of large equine embryos.

The main difficulty of large equine embryo cryopreservation is the replacement of blastocoel fluid with cryoprotectant solution. The objective of this study was to improve the cryopreservation of large equine embryos with PMAP and/or LAP. Embryos were collected via the non-surgical transcervical procedure and divided into three groups based on their size (A≤300µm, 300µm<B<700µm and C≥700µm). Six embryos 233-1360µm in diameter were punctured via piezo manipulator and/or laser pulse before cryopreservation. All embryos were cryopreserved on a Cryotop®. Frozen-thawed embryos were cultured for 3h and transferred to the recipient mares. After one week, pregnancy was diagnosed by ultrasonography. Two of six embryos resulted in a positive pregnancy, the result of pregnancy in group A and B was positive, but in group C was negative, and further investigation is necessary for ≥700µm embryos. The results showed laser-assisted puncture could be helpful to extract blastocoel fluid and replace it with cryoprotectant. This is the first positive pregnancy report in laser puncture-assisted frozen-thawed equine embryo (>300µm). However, more research is required to find the best method for embryos ≥700µm.

Anti-Müllerian Hormone and OPU-ICSI Outcome in the Mare.

Simple SummaryThe in vitro production of equine embryos, using ovum pick-up (OPU) and intracytoplasmic sperm injection (ICSI), is gaining popularity for breeding sport horses. However, the results of this complicated procedure are variable and hard to predict. Therefore, the aim of this study was to evaluate anti-Müllerian hormone (AMH), a factor which has been linked to reproductive success in human and cattle, as an indicator of the OPU-ICSI outcome in horses. Therefore, for 103 mares subjected to commercial OPU-ICSI, the AMH level was measured in the blood at the moment of OPU and linked to the number of oocytes and embryos produced by ICSI. We found that mares with a high level of AMH gave rise to a better oocyte collection and a higher number of embryos. However, since mares with a low AMH value could also produce embryos, a single measurement of the AMH in the blood is not sufficient as an independent predictor of the OPU-ICSI outcome in the horse.Anti-Müllerian hormone (AMH) reflects the population of growing follicles and has been related to mammalian fertility. In the horse, clinical application of ovum pick-up and intracytoplasmic sperm injection (OPU-ICSI) is increasing, but results depend largely on the individuality of the mare. The aim of this study was to assess AMH as a predictor for the OPU-ICSI outcome in horses. Therefore, 103 mares with a total follicle count above 10 were included in a commercial OPU-ICSI session and serum AMH was determined using ELISA. Overall, the AMH level was significantly correlated with the number of aspirated follicles and the number of recovered oocytes (p < 0.001). Mares with a high AMH level (≥2.5 µg/L) yielded significantly greater numbers of follicles (22.9 ± 1.2), oocytes (13.5 ± 0.8), and blastocysts (2.1 ± 0.4) per OPU-ICSI session compared to mares with medium (1.5–2.5 µg/L) or low AMH levels (<1.5 µg/L), but no significant differences in blastocyst rates were observed. Yet, AMH levels were variable and 58% of the mares with low AMH also produced an embryo. In conclusion, measurement of serum AMH can be used to identify mares with higher chances of producing multiple in vitro embryos, but not as an independent predictor of successful OPU-ICSI in horses.

Epigenetic Changes in Equine Embryos after Short-Term Storage at Different Temperatures.

Simple SummaryIn embryos subjected to assisted reproductive techniques, epigenetic modifications may occur that can influence embryonic development and establishment of pregnancy. In horses, the storage temperature during transport of fresh embryos before transfer is a major concern. The aim of this study was, therefore, to determine the effects of two storage temperatures (5 °C and 20 °C) on equine embryos, collected at day seven after ovulation and stored for 24 h, concerning morphological development, expression of candidate genes associated with embryo growth and development, maternal recognition of pregnancy, methylation, apoptosis and gene-specific and global DNA methylation. Temperature during storage did not affect embryo size. There were no changes in pH and lipid peroxidation of the medium irrespective of group. mRNA expression and gene-specific DNA methylation of genes related to growth and development, maternal recognition of pregnancy, DNA methylation and apoptosis in stored embryos (5 °C and 20 °C) were altered when compared to fresh embryos. Therefore, our study demonstrates for the first time the gene-specific and global DNA methylation status of fresh equine embryos collected on days seven and eight after ovulation. Short-term storage, regardless of temperature, may compromise embryo development after transfer.In embryos subjected to assisted reproductive techniques, epigenetic modifications may occur that can influence embryonic development and the establishment of pregnancy. In horses, the storage temperature during transport of fresh embryos before transfer is a major concern. The aim of this study was, therefore, to determine the effects of two storage temperatures (5 °C and 20 °C) on equine embryos, collected at day seven after ovulation and stored for 24 h, on: (i) morphological development; (ii) expression of candidate genes associated with embryo growth and development, maternal recognition of pregnancy, methylation and apoptosis, and (iii) gene-specific and global DNA methylation. Embryos (n = 80) were collected on day seven or day eight after ovulation and assigned to four groups: day seven control (E7F, fresh); day seven, stored for 24 h at 5 °C (E5C); day seven, stored for 24 h at 20 °C (E20C) and day eight control (E8F, fresh 24h time control). The embryos and the storage medium (EquiHold, holding medium, Minitube, Tiefenbach, Germany) from all treatment groups were analyzed for (i) medium temperature, pH, and lipid peroxidation (malondialdehyde; MDA) and (ii) embryo morphology, mRNA expression and DNA methylation (immunohistochemistry and gene-specific DNA methylation). The size of embryos stored at 5 °C was larger (p < 0.01), whereas embryos stored at 20 °C were smaller (p < 0.05) after 24 h. There were no changes in pH and MDA accumulation irrespective of the group. The mRNA expression of specific genes related to growth and development (POU5F1, SOX2, NANOG), maternal recognition of pregnancy (CYP19A1, PTGES2), DNA methylation (DNMT1, DNMT3A, DNMT3B) and apoptosis (BAX) in the E5C and E20C were either up or downregulated (p < 0.05) when compared to controls (E7F and E8F). The immune expression of 5mC and 5hmC was similar among treatment groups. Percentage of methylation in the CpG islands was lower in the specific genes ESR1, NANOG and DNMT1 (p < 0.001) in E20C embryos when compared to E8F (advanced embryo stage). Therefore, our study demonstrates for the first time the gene-specific and global DNA methylation status of fresh equine embryos collected on days seven and eight after ovulation. Although our results suggest some beneficial effects of storage at 20 °C in comparison to 5 °C, the short-term storage, regardless of temperature, modified gene expression and methylation of genes involved in embryo development and may compromise embryo viability and development after transfer.

106 Maternal recognition of pregnancy (MRP) and mares: Is the epithelial sodium channel (ENaC) involved?

Maternal recognition of pregnancy (MRP) describes the prevention of luteolysis via the early conceptus. By prolonging the presence of the corpus luteum (CL), the conceptus ensures an environment capable of supporting pregnancy. In mares, MRP is enigmatic; the horse is one of few domesticated species wherein the MRP signal has yet to be discovered. Equine conceptus mobility throughout the uterine horns, termed migration, is known to be essential for pregnancy establishment. However, while the MRP signal in several other species is conceptus-derived, such a signal has yet to be observed from the equine embryo. We hypothesize that the migration of the equine conceptus itself activates MRP, potentially through epithelial sodium channel (ENaC). ENaC has been previously detected in multiple equine organs, observed in the uteri of various mammalian species, and also found to be upregulated during preimplantation in mice. However, neither the presence of ENaC within the mare uterus nor its potential involvement with the equine pregnancy has been researched. The aim of this study was 1) to determine if ENaC was present within the endometrium of the mare uterus and 2) to describe the location of ENaC if observed. Estrus synchronization was completed in mares (n = 14) during peak breeding season. Mares were subsequently blocked by age and breed and then placed into one of 2 groups: control mares (C; n = 7) and treatment mares (T; n = 7). Transrectal ultrasonography (US) was completed in all mares daily when one or more ovarian follicles reached 25 mm. When a mare had a 35 mm or larger follicle present, she received 2,000 international units of human chorionic gonadotropin intramuscularly to induce ovulation (OV) and US continued daily. Additionally, T mares were artificially inseminated (AI) with fresh, extended semen every day until the day after OV and were subsequently checked for pregnancy 12 d post-OV. C mares had the cervix manually manipulated the day after OV to replicate physical stimulation that occurs to the cervix when AI; however, no semen was deposited in C mare reproductive tracts. Endometrial biopsies were collected from all mares before OV, after OV, and during early pregnancy (when applicable) and samples were then processed for ENaC immunohistochemistry analysis. ENaC protein was immunodetected in all C and T mare endometrial samples and was localized to both uterine luminal epithelium and uterine glands. While further analysis will be conducted to quantify endometrial ENaC expression in C versus T (open versus pregnant) mares, the present histological data confirms that ENaC is present within the equine uterus.

105 Sexual dimorphism in equine D8 blastocysts

In mammals, male embryos grow faster. Equine embryo size is highly variable for same developmental stage, with no reported relation to embryo sex nor gene expression. Our objective was to evaluate gene expression sexual dimorphism on equine D8 blastocysts. Non-nursing, Saddlebred mares located on 2 experimental farms were inseminated with the semen of one unique stallion per farm. At 8 d post ovulation, 17 and 11 embryos from each farm, each from a different dam, were recovered by uterine flushing and bissected to obtain samples of pure (TE) or inner cell mass enriched (TE-ICM) trophoblast. RNA expression was analyzed separately in the TE-ICM and the TE of 16 female and 12 male embryos using paired end, non-oriented RNA sequencing (Illumina, NextSeq500). To discriminate gene expression in the ICM from that in the TE, deconvolution (DeMixT R package) was used on the TE-ICM data. Differential expression was analyzed (DESeq2) with farm and embryo size as cofactors using a false discovery rate (FDR) < 0.05 cutoff. Gene Set Enrichment Analysis was performed using the KEGG and GO BP databases. Out of the 13,786 and 13,210 genes expressed in ICM and TE, respectively, only 184 (51.1% on X chromosome) in ICM and 415 (39.0% on X chromosome) in TE were differentially expressed, with 111 genes in common. Overall, 145 in ICM and 312 in TE genes were significantly upregulated in female. As reported in bovine embryos, glucose-6-phosphate dehydrogenase (G6PD) was upregulated in female (log2 Fold Change = 0.96 in ICM and 0.83 in TE; both FDR <0.001). G6PD is required for detoxification of oxygen radicals that have both cellular damage and growth-stimulant effects. Reduced levels of oxygen radicals have been hypothesized to be responsible for the growth delay in female bovine embryos. In ICM cells, 16 and 1 gene sets were enriched in male embryos in GO BP and KEGG, respectively while 3 KEGG pathways were enriched in female ICM. In the TE part, 27 and 6 gene sets were enriched in male embryos in GO BP and KEGG, respectively, while no pathway was enriched in female embryos. Biological processes enriched in male embryos are mostly common between ICM and TE and are related to translation and spliceosome, ribosome biogenesis, mitochondrial function, cytoskeleton organization and amino acid metabolism. Pathways that were only enriched in male ICM relate to potassium ion homeostasis while in TE, they relate to protein export. In females, enriched pathways in ICM were related to Krebs cycle, glycosaminoglycan degradation and lysosome. Thus, although no growth retardation has been observed in equine female embryo, sexual dimorphism in gene expression of genes unrelated to the sex chromosomes is observed.

Expression of Enzymes Associated with Prostaglandin Synthesis in Equine Conceptuses.

Simple SummaryThe mobile preimplantative phase of equine gestation, taking place between day 9 and 16 after ovulation, is characterized by peristaltic contractions of the uterus caused by secretion of prostaglandins by the spheric equine conceptus. This mobility is necessary for maternal recognition of pregnancy in equids, taking place around day 14 after ovulation. The presented study investigated the spatial and temporal abundance of prostaglandin synthesis enzymes of the equine conceptus, elucidating a basal and an inducible system for prostaglandin E2. Prostaglandin F2α synthesis is restricted to the “periembryonic”pole area and relies on enzymatic conversion of prostaglandin E2. This scenario led to a model able to explain the embryonic forward motion driven by the peristaltic contractions of the uterus. In vitro incubation of primary trophoblast cell cultures with oxytocin showed no influence of this hormone on prostaglandin synthesis.In the horse, mobility of the conceptus is required for maternal recognition of pregnancy depending on secretion of prostaglandins by the conceptus. The aim of this study was to determine the expression and localization of key enzymes of the different pathways leading to synthesis of prostaglandin E2 and F2α in the equine conceptus during the mobility phase. Enzyme expression was analyzed via quantitative RT-PCR in total RNA samples of equine conceptuses collected on days 10 (n = 5), 12 (n = 12), 14 (n = 5) and 16 (n = 7) from healthy mares. Relative abundance of cyclooxygenase (COX)-2 mRNA was higher (p < 0.05) than of COX-1 irrespective of conceptus age and for phospholipase A2 on day 16 in comparison to all other days (p < 0.01). Abundance of mRNA of cytosolic and microsomal prostaglandin E synthase (PGES) and of carbonyl reductase (CBR) 1 was not influenced by conceptus age. Immunohistochemically, COX-1, COX-2, as well as cytosolic and microsomal PGES were present in both the ectodermal and endodermal layer of the yolk sac wall. CBR-1 was restricted to periembryonic disc area. The localisation of the key enzymes explains the mechanism of embryo mobility. In vitro incubation of primary trophoblast cell cultures with oxytocin had no effect on key enzyme synthesis.

Successful vitrification of manually punctured equine embryos.

Successful vitrification of equine expanded blastocysts requires collapse of the blastocoele cavity using a micromanipulator-mounted biopsy pipette on an inverted microscope. Such equipment is expensive and requires user skill. To develop a manual method of blastocoele collapse prior to vitrification using commercial products. In vivo experiment. Seventy-nine Day 7 or 8 embryos were measured and graded. Twenty were vitrified following micromanipulator-assisted puncture and aspiration before being used to validate commercial human vitrification and warming kits containing, respectively, 2-step concentrations of DMSO and ethylene glycol (7.5%-15% v:v) and decreasing concentrations of sucrose. After warming, embryos were transferred to recipient mares. Once validated, the commercial kits were used to vitrify and warm a further 39 embryos which were punctured manually using a microneedle, 2 (5%) were damaged during puncture and excluded; 20 more embryos were vitrified without puncture. Embryos were grouped as follows: non-punctured≤300µm (n=10) and>300 to≤560µm (n=10), punctured small (>300 to≤560µm; n=17) and large (>560µm; n=10) and exposed to the equilibration solution (ES) in the kit for 6min. An additional group of punctured large embryos was exposed to ES for 8min (n=10). For the initial warming step, embryos were exposed for 1min to the thawing solution at 42°C, before being moved to a dilution solution at room temperature. Vitrified, manually punctured embryos≤560µm exposed to ES for 6min resulted in a pregnancy rate of 82% (14/17). Unpunctured embryos≤300µm gave an 80% (8/10) pregnancy rate. Larger unpunctured embryos, punctured embryos>560µm and embryos exposed to ES for 8min gave significantly reduced pregnancy rates. Limited group sizes. High pregnancy rates can be achieved by manually puncturing≤560µm equine embryos prior to their vitrification and subsequent warming in commercial media.

Equine early pregnancy endocrine profiles and ipsilateral endometrial immune cell, gene expression and protein localisation response.

We investigated the early effects of the equine embryo on maternal serum concentrations of insulin-like growth factor 1 (IGF1), leptin and adiponectin, uterine immune cells and genes and proteins related to embryo development and the maintenance of pregnancy. Ipsilateral endometrial expression was assessed on Days 7 and 13 after ovulation for the following transcripts: oestrogen receptor ERα (ESR1), progesterone receptor (PGR), progestin and adipoQ receptor family member 5 (PAQR5), oxytocin receptor (OXTR), prostaglandin-endoperoxide synthase 2 (PTGS2), raf-1 proto-oncogene serine/threonine kinase (RAF1), p21-activated kinase 6 (PAK6), fibroblast growth factor family member 9 (FGF9), IGF1 and its receptor (IGF1R), mucin 1 (MUC1), osteopontin (OPN), leptin receptor (LEPR) and adiponectin receptors 1 and 2 (ADIPOR1 and ADIPOR2). Ipsilateral endometrial immunological cell infiltration and immunohistochemical protein localisation were evaluated on Days 7, 10 and 13 after ovulation for ERα, PGR, OXTR, PTGS2, IGF1, IGF1R, IGF2 and MUC1. Serum hormone concentrations were not affected by reproductive status. Pregnancy downregulated ESR1 and PGR mRNA levels, upregulated the expression of all other genes and affected the expression of all genes, except PGR, on Day 7 (compared with eight genes affected at Day 13). Proteins were affected by pregnancy or by its interaction with other variables (day of extraction and endometrial compartment). Pregnant mares had a higher lymphocyte count, which decreased towards Day 13. The effect of pregnancy on leucocytes and proteins was more evident in superficial endometrial compartments. The results of this study suggest that the equine embryo exerts prompt paracrine regulation of critical biological processes.

Placentation in Equids.

This chapter focuses on the early stages of placental development in horses and their relatives in the genus Equus and highlights unique features of equid reproductive biology. The equine placenta is classified as a noninvasive, epitheliochorial type. However, equids have evolved a minor component of invasive trophoblast, the chorionic girdle and endometrial cups, which links the equine placenta with the highly invasive hemochorial placentae of rodents and, particularly, with the primate placenta. Two types of fetus-to-mother signaling in equine pregnancy are mediated by the invasive equine trophoblast cells. First, endocrinological signaling mediated by equine chorionic gonadotrophin (eCG) drives maternal progesterone production to support the equine conceptus between days 40 and 100 of gestation. Only in primates and equids does the placenta produce a gonadotrophin, but the evolutionary paths taken by these two groups of mammals to produce this placental signal were very different. Second, florid expression of paternal major histocompatibility complex (MHC) class I molecules by invading chorionic girdle cells stimulates strong maternal anti-fetal antibody responses that may play a role in the development of immunological tolerance that protects the conceptus from destruction by the maternal immune system. In humans, invasive extravillous trophoblasts also express MHC class I molecules, but the loci involved, and their likely function, are different from those of the horse. Comparison of the cellular and molecular events in these disparate species provides outstanding examples of convergent evolution and co-option in mammalian pregnancy and highlights how studies of the equine placenta have produced new insights into reproductive strategies.

Studies on Equine Embryo Transfer: Effect of Flushing Media and Environment in Maiden and Adult Arabian Mares

The advances in equine embryo transfer (ET) allowed its application in small facilities with promising results. ET has been carried in 27 donor mares where the procedures were applied in 2 facilities; 1) farm group (n=18) where the ET was carried out in stable confined group. 2) procedures were applied in the lab (lab group, n=9). Two solutions, phosphate buffered saline (PS) or ringer lactate (RL), were used as flushing media. ET was applied by directing the pipette with finger through the cervix. In farm group 39 flushes resulted in 17 embryos with 43.6% recovery rat with 63.2% pregnancy rate. In lab group 16 flushes resulted in 14 embryos with recovery rat 87.5% and pregnancy rate 71%. In conclusion, ET could be conducted in equine farms without affecting recovery or pregnancy rate, RL could be used as flushing media without decreasing pregnancy rate, and cervical penetration dose not decreasing pregnancy rate.

Kinetics of placenta-specific 8 (PLAC8) in equine placenta during pregnancy and placentitis

Placenta-specific 8 (PLAC8) is one of the placenta-regulatory genes which is highly conserved among eutherian mammals. However, little is known about its expression in equine placenta (chorioallantois; CA and endometrium; EN) during normal and abnormal pregnancy. Therefore, the current study was designed to 1) elucidate the expression of PLAC8 in equine embryonic membranes during the preimplantation period, 2) characterize the expression profile of PLAC8 in equine CA (45d, 4mo, 6mo, 10 mo, 11 mo and postpartum) and EN (14d, 4mo, 6mo, 10 mo, and 11 mo) obtained from pregnant mares (n = 4/timepoint), as well as, d14 non-pregnant EN (n = 4), and 3) investigate the expression profile of PLAC8 in ascending placentitis (n = 5) and in nocardioform placentitis (n = 6) in comparison to normal CA. In the preimplantation period, PLAC8 mRNA was not abundant in the trophectoderm of d8 equine embryo and d14 conceptus, while it was abundant later in d 30, 31, 34, and 45 chorion. In normal pregnancy, PLAC8 mRNA expression in CA at 45 d gradually decline to reach nadir at 6mo before gradually increasing to its peak at 11mo and postpartum CA. The mRNA expression of PLAC8 was significantly upregulated in CA from mares with ascending and nocardioform placentitis compared to control mares. Immunohistochemistry revealed that PLAC8 is localized in equine chorionic epithelium and immune cells. Our results revealed that PLAC8 expression in equine chorion is dynamic during pregnancy and is regulated in an implantation-dependent manner. Moreover, PLAC8 is implicated in the immune response in CA during equine ascending placentitis and nocardioform placentitis.