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Topic:Blastocysts
Blastocysts in horses represent an early stage of embryonic development following fertilization and prior to implantation in the uterus. This stage is characterized by a fluid-filled cavity surrounded by a layer of cells, which will eventually develop into the embryo and placenta. Research on equine blastocysts focuses on their formation, development, and viability, as well as factors affecting successful implantation and pregnancy outcomes. Studies often explore the molecular and cellular mechanisms underlying blastocyst development, the impact of maternal health and environmental conditions, and techniques for improving reproductive success in horses. This page compiles peer-reviewed research studies and scholarly articles that examine the formation, development, and clinical implications of blastocysts in equine reproduction.
Trophoblast stem cell marker gene expression in inner cell mass-derived cells from parthenogenetic equine embryos. Although putative horse embryonic stem (ES)-like cell lines have been obtained recently from in vivo-derived embryos, it is currently not known whether it is possible to obtain ES cell (ESC) lines from somatic cell nuclear transfer (SCNT) and parthenogenetic (PA) embryos. Our aim is to establish culture conditions for the derivation of autologous ESC lines for cell therapy studies in an equine model. Our results indicate that both the use of early-stage blastocysts with a clearly visible inner cell mass (ICM) and the use of pronase to dissect the ICM allow the derivation of a higher proportion...
Viability of equine embryos after puncture of the capsule and biopsy for preimplantation genetic diagnosis. The equine embryo possesses a capsule that is considered essential for its survival. We assessed viability after breaching the capsule of early (Day 6) and expanded (Day 7 and 8) equine blastocysts by micromanipulation. The capsule was penetrated using a Piezo drill, and trophoblast biopsy samples were obtained for genetic analysis. Pregnancy rates for Day-6 embryos, which had intact zonae pellucidae at the time of recovery, were 3/3 for those biopsied immediately after recovery and 2/3 for those biopsied after being shipped overnight under warm (∼28 °C) conditions. The pregnancy rates for ...
The equine oocyte: factors affecting meiotic and developmental competence. There is currently much interest in assisted reproduction techniques in the horse, however, many aspects of oocyte maturation, fertilization, and embryo development in the horse differ from those in other species. Because of the close attachment of the equine oocyte to the follicle wall, scraping of the follicle is the most effective method for oocyte recovery. A notable feature of equine oocytes is that those with expanded cumuli (Ex oocytes), which originate from atretic follicles, have higher meiotic competence (ability to mature to metaphase II in vitro) than do oocytes with compact cumuli...
In vitro production of equine embryos: state of the art. In vitro embryo production is possible in the horse both clinically and for research applications. Oocytes may be collected from excised ovaries post-mortem, or from either immature follicles or stimulated pre-ovulatory follicles in the live mare. In vitro maturation of immature oocytes typically yields approximately 60% mature oocytes. As standard in vitro fertilization is not yet repeatable in the horse, fertilization is performed by intracytoplasmic sperm injection. Embryo culture requires medium with high glucose, at least during blastocyst development, and rates of blastocyst development ...
Heat shock protein 70 gene expression in equine blastocysts after exposure of oocytes to high temperatures in vitro or in vivo after exercise of donor mares. Heat above homeothermy can be detrimental to embryonic development, and cells may produce heat shock proteins to try to mitigate these effects. The authors examined the developmental competence of equine oocytes after a single heat exposure (42 degrees C, 2 or 4 h) during early or late stages of in vitro maturation. Rates of nuclear maturation, cleavage after intracytoplasmic sperm injection, and advanced embryonic development (morula or blastocyst) were compared to those for unexposed controls. Concentrations of heat shock protein 70 (HSPA1A) mRNA were determined by real-time RT-PCR in result...
Addition of ficoll and disaccharides to vitrification solutions improve in vitro viability of vitrified equine embryo. The aim of the present study was to evaluate the in vitro viability of equine embryos vitrified in three different solutions. Day 6 and 6.5 embryos were measured and morphologically evaluated. Only grade 1 or 2 morulae and early blastocysts were vitrified. Eighteen embryos were distributed in Group 1: 40 percent ethylene glycol in PBS, Group: 2 and 3: 40 percent ethylene glycol, 18 percent Ficoll, 0.3M sucrose or 0.3M trehalose in PBS, respectively. The vitrified embryos were loaded individually into 0.25 ml straws, which were cooled and immersed in liquid nitrogen. After warming at 20 degree ...
Recovery of mare oocytes on a fixed biweekly schedule, and resulting blastocyst formation after intracytoplasmic sperm injection. Oocytes may be collected from live mares from either the stimulated preovulatory follicle or from all visible immature follicles. We evaluated the yield of mature oocytes, and of blastocysts after intracytoplasmic sperm injection (ICSI), for both follicle types. In Experiment 1, mares were assigned to Progesterone (1.2g biorelease progesterone weekly) or Control treatments. Transvaginal aspiration of all follicles was performed every 14 d. Overall, 596 follicles were aspirated, with a 54% oocyte recovery rate. There was no difference between treatments in number of follicles punctured (9.0 to ...
Selection of reference genes for quantitative real-time PCR in equine in vivo and fresh and frozen-thawed in vitro blastocysts. Application of reverse transcription quantitative real-time polymerase chain reaction is very well suited to reveal differences in gene expression between in vivo and in vitro produced embryos. Ultimately, this may lead to optimized equine assisted reproductive techniques. However, for a correct interpretation of the real-time PCR results, all data must be normalized, which is most reliably achieved by calculating the geometric mean of the most stable reference genes. In this study a set of reliable reference genes was identified for equine in vivo and fresh and frozen-thawed in vitro embryos....
The uterine environment modulates trophectodermal POU5F1 levels in equine blastocysts. The reported patterns of trophectodermal expression of POU5F1 protein in blastocysts vary among species, and are possibly related to the differences in placental growth and function. This study investigated the pattern of embryonic POU5F1 expression in the horse, a species with delayed placental formation. Immature equine oocytes expressed POU5F1 protein in the cytoplasm and nucleus. Staining for POU5F1 protein in in vitro-produced (IVP) embryos decreased to day 5 of culture, then the nuclear staining increased to day 7. IVP day-7 to -11 blastocysts showed POU5F1 staining in nuclei throughout ...
Effect of sperm extract injection volume, injection of PLCzeta cRNA, and tissue cell line on efficiency of equine nuclear transfer. We evaluated the effect of different activation methods on blastocyst development after equine nuclear transfer. All activation treatments were followed by incubation in 2 mM 6-dimethylaminopurine for 4 h. In Experiment 1, reconstructed oocytes were injected with sperm extract for 0.1, 0.2, 0.4, 0.8, or 1.6 sec using a FemtoJet injection device, then treated with ionomycin. The blastocyst rate (9.8%) for 0.1-sec injection was significantly higher than that for 0.2 sec (0%) or 0.8 sec (1.4%). In Experiment 2, injection of murine PLCzeta cRNA before or after ionomycin treatment did not increase ...
Vitrification of early-stage bovine and equine embryos. The objectives of this study were to: (1) determine an optimal method and stage of development for vitrification of bovine zygotes or early embryos; and (2) use the optimal procedure for bovine embryos to establish equine pregnancies after vitrification and warming of early embryos. Initially, bovine embryos produced by in-vitro fertilization (IVF) were frozen and vitrified in 0.25mL straws with minimal success. A subsequent experiment was done using two vitrification methods and super open pulled straws (OPS) with 1- or 8-cell bovine embryos. In Method 1 (EG-O), embryos were exposed to 1.5M e...
Somatic cell nuclear transfer in horses. The cloning of equids was achieved in 2003, several years after the birth of Dolly the sheep and also after the cloning of numerous other laboratory and farm animal species. The delay was because of the limited development in the horse of more classical-assisted reproductive techniques required for successful cloning, such as oocyte maturation and in vitro embryo production. When these technologies were developed, the application of cloning also became possible and cloned horse offspring were obtained. This review summarizes the main technical procedures that are required for cloning equids an...
Significance of aquaporins and sodium potassium ATPase subunits for expansion of the early equine conceptus. Expansion of the equine conceptus can be divided into blastocoel and yolk sac phases. The endodermal layer transforming the blastocoel into the yolk sac is completed around day 8 of pregnancy. From that time, the size of the spherical conceptus increases tremendously due mainly to the accumulation of fluid rather than cell multiplication. In this study, we have investigated the abundance and localisation of Na(+)/K(+)-ATPases and aquaporins (AQP) in the equine conceptus on days 8, 10, 12, 14 and 16 by multiplex reverse transcriptase PCR, Western blot and immunohistochemistry. During conceptus ...
Advancements in large animal embryo transfer and related biotechnologies. Embryo transfer has been an inherent part of cattle breeding for more than 35 years and has also gained remarkable interest from the equine industry after several breeds allowed registration of more than one foal per year. In both large animal species, non-surgical embryo recovery and transfer are well-established techniques. However, success rates after superovulation and cryopreservation of embryos in horses are still lagging behind those of cattle, and more research is needed to address these areas. To address the problem of freezing large equine embryos, we offer a preliminary demonstratio...
Expression of cell-surface antigens and embryonic stem cell pluripotency genes in equine blastocysts. Embryonic stem-like (ES-like) cells have now been derived from the inner cell mass (ICM) of horse embryos at the blastocyst stage. Because they have been shown to express cell-surface antigens found in both human and mouse ES cells, the present study investigated gene expression patterns in day-7 horse blastocysts from which the horse ES-like cells had been derived originally. The genes studied included Oct-4, stage-specific embryonic antigen-1 (SSEA-1), SSEA-3, SSEA-4, tumor rejection antigen-1-60 (TRA-1-60), TRA-1-81, and alkaline phosphatase activity, and whereas all three of the SSEA antig...
The presence of 19-norandrostenedione and its sulphate form in yolk-sac fluid of the early equine conceptus. C(18) neutral steroid formation by cytochrome P450 aromatase has been recorded for several equine and porcine tissues. High activity of P450 aromatase is reflected in the quantities of estrogens in yolk-sac (y-s) fluid of early equine conceptuses. In a previous study of y-s fluid we detected large amounts of androgens by radioimmunoassay (RIA), using an antiserum for androstenedione (A(4)). Here, we report that RIA, following chromatography, gave tentative identification of the major peak as norandrostenedione (19-norA) not as A(4). Furthermore, even greater quantities of 19-norA seemed to be ...
Transport of equine ovaries for assisted reproduction. Use of assisted reproduction to obtain foals from valuable mares post-mortem typically necessitates holding of ovaries during shipment to a laboratory. The present study evaluated whether holding ovaries briefly at a warm ( approximately 30 degrees C) temperature improves meiotic and developmental competence of oocytes, as determined after maturation in vitro and intracytoplasmic sperm injection. Ovaries were packaged in pairs in insulated containers, and held either at 24 or 25-35 degrees C for 4h, followed by cooling. Ovaries in both treatments were held for either a short (mean, 7-7.4h) or ...
Production of cloned horse foals using roscovitine-treated donor cells and activation with sperm extract and/or ionomycin. We evaluated the effect of different activation treatments on the production of blastocysts and foals by nuclear transfer. Donor cells were prepared using roscovitine treatment, which has previously been associated with increased production of viable offspring. All activation treatments were followed by culture in 6-dimethylaminopurine (6-DMAP) for 4 h. In experiment 1, blastocyst production after activation by injection of sperm extract followed by treatment with ionomycin was significantly higher than that for activation with a serial treatment of ionomycin, 6-DMAP, and ionomycin (12.5 vs 2....
In vitro-produced equine embryos: production of foals after transfer, assessment by differential staining and effect of medium calcium concentrations during culture. Viability of equine embryos produced by oocyte maturation, intracytoplasmic sperm injection and embryo culture to the blastocyst stage in vitro was evaluated after transfer of embryos to recipient mares. No pregnancies were produced after transfer of five blastocysts that had been cultured in G media. Transfer of 10 blastocysts cultured in modified DMEM/F-12 medium produced five pregnancies and three live foals; the two lost pregnancies developed only trophoblast (based on transrectal ultrasonography). To evaluate the status of the inner cell mass, equine blastocysts produced in vivo and in vi...
In vitro development of equine oocytes from preserved ovaries after intracytoplasmic sperm injection. In this study, we evaluated the meiotic competence of equine oocytes from ovaries preserved for one day. We also investigated fertilization, cleavage rate, developmental competence and freezability of equine embryos after intracytoplasmic sperm injection (ICSI). After collection from ovaries, the oocytes were classified into two groups comprised of those having compact cumulus layers (Cp) or those having expanded cumulus layers (Ex). Oocytes with a first polar body were subjected to fertilization by ICSI using frozen-thawed stallion spermatozoa and were then cultured in CR1aa medium. The rates...
Comparative aspects of somatic cell nuclear transfer with conventional and zona-free method in cattle, horse, pig and sheep. Nuclear transfer (NT) is a complex procedure that requires considerable technical skills. Over the years attempts have been made to simplify the micromanipulations involved and to make the procedure more user-friendly. A significant step forwards has been the development of the zona-free NT methods. We have used zona-free NT with mechanical aspiration of the metaphase plate as a mean of enucleation, in a comparative approach with the conventional nuclear transfer zona-enclosed method in cattle, horse, sheep and pig. The absence of the zona considerably facilitates the enucleation step and sign...
Effect of cumulus morphology and maturation stage on the cryopreservability of equine oocytes. Oocyte cryopreservation is a potentially valuable way of preserving the female germ line. However, the developmental competence of cryopreserved oocytes is presently poor. This study investigated whether the morphology of the cumulus complex surrounding an immature equine oocyte and/or the oocyte's stage of maturation affect its cryopreservability. Compact (Cp) and expanded (Ex) cumulus oocyte complexes (COCs) were vitrified either shortly after recovery (germinal vesicle stage, GV) or after maturation in vitro (IVM); cryoprotectant-treated and -untreated non-frozen oocytes served as controls....
Derivation and induction of the differentiation of animal ES cells as well as human pluripotent stem cells derived from fetal membrane. We succeeded in the derivation and maintenance of pluripotent embryonic stem (ES) cells from equine and bovine blastocysts. These cells expressed markers that are characteristics of mouse ES cells, namely, alkaline phosphatase, stage-specific embryonic antigen 1, STAT 3 and Oct 4. We confirmed the pluripotential ability of these cells, which were able to undergo somatic differentiation in vitro to neural progenitors and to endothelial or hematopoietic lineages. We were able to use bovine ES cells as a source of nuclei for nuclear transfer and we generated cloned cattle with a higher frequency ...
Effect of holding technique and culture drop size in individual or group culture on blastocyst development after ICSI of equine oocytes with low meiotic competence. The effect of medium-to-embryo ratio on blastocyst development of equine embryos from oocytes with compact cumuli was evaluated in the present experiment. In addition, two methods for holding oocytes before in vitro maturation were compared. In Experiment 1, oocytes cultured with roscovitine for 16-18h before maturation were fertilized by intracytoplasmic sperm injection and cultured individually in 2.5, 5, 10 or 50microl droplets. In Experiment 2, oocytes were either cultured with roscovitine or held in a modified M199 with 20% serum at room temperature (EH treatment) for 16-18h, then matured...
Horse embryonic stem cell lines from the proliferation of inner cell mass cells. Inner cell mass (ICM) cells were isolated immunosurgically from day 7-8 horse blastocysts and, after proliferation in vitro for 15-28 passages, three lines of cells were confirmed to be embryonic stem (ES) cells by their continued expression of alkaline phosphatase activity and their ability to bind antisera specific for the recognized stem cell markers, SSEA-1, TRA-1-60, TRA-1-81, and the key embryonic gene Oct-4. When maintained under feeder cell-free conditions in vitro, the three lines of cells differentiated into cells of ectodermal, endodermal, and mesodermal lineages. However, they did ...
Derivation, maintenance, and induction of the differentiation in vitro of equine embryonic stem cells. We describe here the isolation and maintenance of pluripotent embryonic stem (ES) cells from equine blastocysts that have been frozen and thawed. Equine ES cells appear to maintain a normal diploid karyotype in culture. These cells express markers that are characteristic of mouse ES cells, namely, alkaline phosphatase, stage-specific-embryonic antigen 1, STAT3, and Oct4. We also describe protocols for the induction of differentiation in vitro to neural precursor cells in the presence of basic fibroblast growth factor (bFGF), epidermal growth factor, and platelet-derived growth factor and to he...
Production of horse foals via direct injection of roscovitine-treated donor cells and activation by injection of sperm extract. We evaluated the effects of different donor cell treatments and activation methods on production of blastocysts after equine nuclear transfer. Nuclear transfer was performed by direct injection of donor cells, using a piezo drill, and standard activation was by injection of sperm factor followed by culture with 6-dimethylaminopurine. There was no difference in blastocyst development between embryos produced with roscovitine-treated or confluent donor cells (3.6% for either treatment). Addition of injection of roscovitine or culture with cycloheximide at the time of activation did not affect bl...
Blastocyst development in equine oocytes with low meiotic competence after suppression of meiosis with roscovitine prior to in vitro maturation. This study was conducted to evaluate the in vitro development of equine oocytes with compact cumuli that had been subjected to a period of meiotic suppression with roscovitine before in vitro maturation. In experiment 1, oocytes were recovered from slaughterhouse-derived ovaries and held in M199 + 10% fetal bovine serum containing 66 microM roscovitine with or without an overlay of mineral oil in 5% CO2 in air at 38.2 degrees C for 16-18 or 24 h. No oocytes treated with roscovitine in the absence of an oil overlay for 16-18 h were maturing, compared with 2-4% of oocytes in other treatments. In...
Holding immature equine oocytes in the absence of meiotic inhibitors: effect on germinal vesicle chromatin and blastocyst development after intracytoplasmic sperm injection. Holding immature oocytes before the onset of maturation simplifies oocyte transport and aids in scheduling later manipulations. We report here a method for holding equine oocytes in the absence of meiotic inhibitors. In Experiment 1, immature oocytes with expanded cumuli were cultured at 38.2 degrees C in medium containing cycloheximide, or were held at room-temperature in M199 with Hanks' salts, for 16-18 h before maturation. Control oocytes were matured immediately after recovery. Oocytes were fertilized by intracytoplasmic sperm injection and cultured for 4d. Embryo development was not diff...
Somatic cell nuclear transfer in horses: effect of oocyte morphology, embryo reconstruction method and donor cell type. The objective of the present work was to investigate and clarify the factors affecting the efficiency of somatic cell nuclear transfer (NT) in the horse, including embryo reconstruction, in vitro culture to the blastocyst stage, embryo transfer, pregnancy monitoring and production of offspring. Matured oocytes, with zona pellucida or after zona removal, were fused to cumulus cells, granulosa cells, and fetal and adult fibroblasts, and fused couplets were cultured in vitro. Blastocyst development to Day 8 varied significantly among donor cells (from 1.3% to 16%, P < 0.05). In total, 137 nucl...
