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.
Blastocyst formation rates in vivo and in vitro of in vitro-matured equine oocytes fertilized by intracytoplasmic sperm injection. This study was conducted to evaluate in vivo and in vitro development of in vitro-matured equine oocytes fertilized by intracytoplasmic sperm injection. Oocytes were collected from slaughterhouse-derived ovaries, matured in vitro, and injected with frozen-thawed stallion sperm. In vivo development was assessed after transfer of injected oocytes to the oviducts of recipient mares. Mares were killed 7.5-8.5 days after transfer and the uterus and oviducts flushed for embryo recovery. Of 132 injected oocytes transferred, 69 (52%) were recovered; of these, 25 (36%) were blastocysts with a blastocoe...
Effects of in vitro production on horse embryo morphology, cytoskeletal characteristics, and blastocyst capsule formation. Blastocyst formation rates during horse embryo in vitro production (IVP) are disappointing, and embryos that blastulate in culture fail to produce the characteristic and vital glycoprotein capsule. The aim of this study was to evaluate the impact of IVP on horse embryo development and capsule formation. IVP embryos were produced by intracytoplasmic sperm injection of in vitro matured oocytes and either culture in synthetic oviduct fluid (SOF) or temporary transfer to the oviduct of a ewe. Control embryos were flushed from the uterus of mares 6-9 days after ovulation. Embryo morphology was eval...
Intracytoplasmic sperm injection of bovine oocytes with stallion spermatozoa. Five experiments were designed to study the fertilizability and development of bovine oocytes fertilized by intracytoplasmic sperm injection (ICSI) with stallion spermatozoa. Experiment 1 determined the time required for pronuclear formation after ICSI. Equine sperm head decondensation began 3 h after ICSI; 42% were decondensed 6 h after ICSI. Male pronuclei (MPN) began to form 12 h after ICSI. Female pronuclei (FPN), however, formed as early as 6 h after ICSI. In Experiment 2, ionomycin, ionomycin plus 6-dimethylaminopurine (DMAP), and thimerosal were used to activate ICSI ova. None of the IC...
Embryo technologies in the horse. Recent studies demonstrated that zwitterionic buffers could be used for satisfactory storage of equine embryos at 5 degrees C. The success of freezing embryos is dependent upon size and stage of development. Morulae and blastocysts <300 microm can be slowly cooled or vitrified with acceptable pregnancy rates after transfer. The majority of equine embryos are collected from single ovulating mares, as there is no commercially available product for superovulation in equine. However, pituitary extract, rich in FSH, can be used to increase embryo recovery three- to four-fold. Similar to human medic...
Isolation of embryonic stem-like cells from equine blastocysts and their differentiation in vitro. Embryonic stem (ES) cells are pluripotent cells with the potential capacity to generate any type of cell. We describe here the isolation of pluripotent ES-like cells from equine blastocysts that have been frozen and thawed. Our two lines of ES-like cells (E-1 and E-2) appear to maintain a normal diploid karyotype indefinitely in culture in vitro and to express markers that are characteristic of ES cells from mice, namely, alkaline phosphatase, stage-specific embryonic antigen-1, STAT-3 and Oct 4. After culture of equine ES-like cells in vitro for more than 17 passages, some ES-like cells diffe...
Production of nuclear transfer horse embryos by Piezo-driven injection of somatic cell nuclei and activation with stallion sperm cytosolic extract. We investigated the use of direct nuclear injection using the Piezo drill and activation by injection of stallion sperm cytosolic extract for production of cloned equine embryos. When metaphase II horse oocytes were injected with either of two dosages of sperm extract and cultured 20 h, similar activation rates (88% vs. 90%) and cleavage rates (49% vs. 46%) were obtained. The successful reconstruction rate of horse oocytes with horse somatic cell donor nuclei after direct injection using the Piezo drill was 82%. Four dosages of sperm extract (containing 59, 176, 293, or 1375 microg/ml protein)...
Successful production of offspring after superovulation and in vitro culture of embryos from domestic ferrets (Mustela putorius furos). In an effort to expand the use of ferrets as models for genetic disease, several experimental parameters that are required for successful genetic manipulation in this species were investigated. Optimum superovulation (19.3 +/- 0.6 oocytes and embryos per female) was achieved after injections of 100 iu equine chorionic gonadotrophin (eCG) and 150 iu human chorionic gonadotrophin (hCG). The ovulation rate achieved by the treatment was more than double that induced by mating. Mating with a male immediately after hCG treatment did not significantly alter the number of oocytes ovulated or the numbe...
Developmental capacity of equine oocytes matured and cultured in equine trophoblast-conditioned media. The objective was to compare culture media for in vitro maturation of equine oocytes and for in vitro culture of zygotes produced from IVF of partially zona-removed oocytes. Cumulus-oocyte complexes from slaughterhouse-derived ovaries were washed in m-Dulbecco's PBS and cultured in TCM-199, F10-DMEM or c-F10-DMEM (50% F10-DMEM + 50% F10-DMEM conditioned medium from culture of an equine trophoblast monolayer for 3 or 4 days). All media included FSH, LH, E2, and 10% FCS. After 28 to 30 h maturation, cumulus expansion was scored from 0 (no expansion) to 4 (fully expanded). Oocytes with a 1st pola...
Influence of co-culture during maturation on the developmental potential of equine oocytes fertilized by intracytoplasmic sperm injection (ICSI). The influence of co-culture with either oviduct epithelial cells or fetal fibroblast cells on in vitro maturation of equine oocytes and their potential for development to blastocysts and fetuses after intracytoplasmic sperm injection (ICSI) was investigated. The oocytes were obtained from ovaries from abattoirs and were matured in vitro for 28-30 h in TCM-199 only, or in TCM-199 co-culture with oviduct epithelial cells or fetal fibroblast cells. Metaphase II oocytes were subjected to ICSI with an ionomycin-treated spermatozoon. The injected oocytes were cultured for 7-9 days in Dulbecco's modi...
Assessment of metabolism of equine morulae and blastocysts. Nutrient uptakes and metabolite production by equine morula and blastocyst stage embryos were determined by non-invasive microfluorometry. Equine morula took up equal amounts of both pyruvate and glucose. However, at the early blastocyst there was a small increase in glucose uptake and, by the expanded blastocyst stage, glucose was the predominant nutrient. Expanded blastocysts took up five times more glucose than pyruvate. Expanded blastocysts exhibited an exponential increase in glucose uptake and lactate production with respect to both diameter and surface area. As less than 50% of the gluc...
Tissue-specific localization of cytochrome P450 aromatase in the equine embryo by in situ hybridization and immunocytochemistry. Estrogen production by the preimplantation equine embryo is presumed to be important in maternal-conceptus communication in the mare. The synthesis of C(18) estrogens from C(19) androgens requires cytochrome P450 aromatase (P450(arom)) in the conceptus, but little information is available on the specific tissue location or potential developmental patterns of expression for the horse. The goal of this research was to localize P450(arom) in the equine conceptus by immunocytochemistry and in situ hybridization. Intact blastocyst-stage embryos were collected by nonsurgical flush on Days 12-15 of p...
Quantitative histological analysis of equine embryos at exactly 156 and 168 h after ovulation. Equine embryos were collected at exactly 156 +/- 0.5 (n=8) and 168 +/- 0.5 h (n=11) after ovulation. The embryos were fixed in glutaraldehyde, sectioned serially and observed using light microscopy. In the 156 h group, all embryos were early blastocysts except for one, which was a morula. The morula and one early blastocyst had no capsule. The capsules of the other embryos were thin. The mean +/- SD total number of cells was 275 +/- 105 (range 117-417). The mean +/- SD proportions of mitotic and pycnotic cells were 2.5 +/- 1.2 and 1.1 +/- 1.8%, respectively, and there were no differences betwe...
Estimation of sodium and potassium concentrations in the uterine fluid of mares by microdialysis and ion chromatography. Equine blastocyst fluid has a lower [Na+], a higher [K+] and a lower osmolality than does normal blood serum. Based on the assumptions that the sodium pump is primarily responsible for fluid accumulation and that ions transported actively into the blastocyst increase blastocyst osmolality above that of the external medium, we hypothesized that the [Na+] and the osmolality of mare uterine fluid are lower than those of blastocyst fluid. Microdialysis and ion chromatography were used to estimate [Na+] and [K+] of uterine fluid. Mares (n=10) were used for in vivo measurements at different stages o...
Evidence for the presence of sodium- and potassium-dependent adenosine triphosphatase alpha1 and beta1 subunit isoforms and their probable role in blastocyst expansion in the preattachment horse conceptus. The unusual hypotonicity of equine blastocyst fluid has prompted us to investigate the role of sodium- and potassium-dependent adenosine triphosphatase (Na+,K+-ATPase) in the process of fluid accumulation in the horse conceptus. Nine mares were used for the experiments. Reverse transcriptase polymerase chain reaction was conducted on two sets of five conceptuses recovered between 12 and 28 days (+/- 1 day) after ovulation. Messenger RNAs encoding the alpha1 and beta1 subunit isoforms of Na+,K+-ATPase were detected in all embryonic tissues examined. Western blot analysis showed that alpha1 and ...
A developmental switch in expression from blastocyst to endometrial/placental-type cytochrome P450 aromatase genes in the pig and horse. Pig blastocysts exhibit a transient period of estrogen production at periimplantation, with a second, more sustained period of estrogen synthesis occurring in endometrium and placenta at later pregnancy. Previously we reported the isolation of cDNA clones encoding a novel isoform of cytochrome P450 aromatase (the terminal enzyme in the estrogen biosynthetic pathway) from porcine periimplantation blastocysts. The present study investigated pregnancy-associated expression, in blastocysts and maternal reproductive tract tissues of this and an additional mRNA transcript encoding a distinct P450 ar...
Trophectoderm projections: a potential means for locomotion, attachment and implantation of bovine, equine and human blastocysts. The behaviour of bovine, equine and human blastocysts was studied in vitro by time-lapse videomicrography and computer imaging. This study revealed that cytoplasmic extensions of the trophectoderm ['trophectoderm projections' (TEP)] were expressed by embryos of all three species, prior to or during zona escape. Bovine and human blastocysts escaped their zonae with a combination of blastocoele expansion, collapse and re-expansion coupled with the penetration of the zona pellucida by TEP. In equine embryos, after several cycles of blastocoele expansion and collapse, trophectoderm ruptured the zo...
Direct transfer of equine blastocysts frozen-thawed in the presence of ethylene glycol and sucrose. The present study was designed to examine the suitability of ethylene glycol as a cryoprotectant for equine embryos. Blastocysts recovered nonsurgically from Day 6 donor mares were cryopreserved by conventional 2-step freezing in the presence of 10% ethylene glycol (EG), 10% glycerol (Gly), or 10% ethylene glycol + 0.1M sucrose (EG + Suc). After thawing, the EG and Gly were removed by a 6-step manner, and the EG + Suc was diluted to one fourth in the freezing straw. The postthaw blastocysts were transferred nonsurgically into the uteri of recipient mares on Days 4 to 7 after ovulation. Pregnan...
Osmolality of equine blastocyst fluid from day 11 to day 25 of pregnancy. Horse conceptuses collected between Day 11 and Day 18 of pregnancy float in isotonic media. To investigate this phenomenon, blastocyst fluids from 30 conceptuses from 13 mares were analysed for osmolality and for concentrations of Na+, Cl-, K+, glucose, urea and creatinine. In conceptuses from Group A, samples from Day 11 to Day 16 yielded the following results (mean +/- s.e.m.): osmolality, 121.4 +/- 1.5 mOsm kg-1; Na+, 11.0 +/- 2.2 mM; Cl-, 29.3 +/- 2.5 mM; K+, 26.2 +/- 2.6 mM; glucose, 0.6 +/- 0.1 mM; urea, 6.0 +/- 0.6 mM; creatinine, 9.6 +/- 1.1 microM. Between Day 16 and Day 25, the osmol...
Large equine blastocysts are damaged by vitrification procedures. Viability following vitrification of equine blastocysts with different sizes was investigated in vitro. Twenty-four blastocysts were classified into three groups according to their diameters ( 300 microns; n = 8 each). The solution used for vitrification was defined as EFS and contained 40% ethylene glycol, 18% Ficoll and 0.3 M sucrose in modified-phosphate-buffered saline (m-PBS). During pretreatment with 20% ethylene glycol in m-PBS for 20 min, the larger blastocysts responded to the osmotic pressure caused by 20% ethylene glycol more slowly than the smaller blastocysts. Single blastocysts w...
In vitro development of day 2 embryos obtained from young, fertile mares and aged, subfertile mares. This study was designed to investigate the development of day 2 embryos obtained from young and aged mares, co-cultured with oviductal epithelial cells obtained from mares in each age group in a 2 x 2 crossover design. Young, fertile mares (n = 19; 2-7 years of age) and aged, subfertile, mares (n = 16; 17-24 years of age) were used as embryo and oviductal epithelial cell donors. Embryos (n = 37) were collected from the oviducts 2 days after ovulation and were paired (embryos obtained from young mares with embryos obtained from aged mares) so that eight pairs were co-cultured with young mare ov...
In vitro viability of cryopreserved equine embryos following different freezing protocols. The main objective of this study was to evaluate two freezing protocols and the effect of agar embedding on survival of day 6.5 equine embryos. A total of 133 embryos were used, in one group (n = 51), embryos were first embedded in agar before the freezing protocol was started. A freezing protocol to -30 degrees C or -33 degrees C was used before plunging embryos into liquid nitrogen (LN2). The embryos were thawed in water at 37 degrees C, evaluated and placed in culture. After 24 h culture, the embryos were evaluated for their morphology and development. No differences were observed between e...
The equine embryonic capsule practical implications of recent research. In most domestic animals, the zona pellucida is the outermost extracellular layer that covers the blastocyst before implantation. However, in the horse, an acellular membrane, the capsule, replaces the zona pellucida and envelops the developing conceptus during the 2nd and 3rd weeks of gestation. Although this structure was first described by Bonnet in 1889, it received little attention until the 1970s when its rediscovery by Marrable and Flood (1975) led to a series of reports (see review by Betteridge 1989). Nevertheless, until recently the structure, origin, and function of the capsule have...
Culture of equine embryos in media containing egg yolk, mare’s milk and saline: Preliminary results. A medium containing egg yolk, mare's milk and/or modified PBS was used to culture Day-8 to 8.5 equine blastocysts. Twenty-one variants of the medium containing different concentrations of the 3 components were prepared. Embryos were recovered nonsurgically and placed into the media at 37 degrees C for 24 h. A total of 45 embryos was cultured; of these 7 died in culture and 13 showed inadequate development at the onset, while 25 continued to grow in the media. It was established that embryos grew best in media containing 20 to 60% yolk, 20 to 60% mare's milk and/or 20 to 60% PBS. It was found e...
Developmentally regulated changes in the glycoproteins of the equine embryonic capsule. The embryonic capsule, which covers the equine blastocyst after it loses its zona pellucida, is composed of mucin-like glycoproteins. In the present study, we investigated both macroscopic and molecular changes in the capsule during development. The weight of the capsule increased from day 11-12 of pregnancy and reached a maximum at about day 18, coinciding with the time during which the conceptus migrates extensively throughout the uterus. The sialic acid content of the capsule declined markedly from about day 16, the time of conceptus 'fixation' in the uterus, which suggests a unique develop...
Steroid synthesis by equine conceptuses between days 7 and 14 and endometrial steroid metabolism. The objective of this study was to determine if changes in steroid synthesis occurred in the horse blastocyst about the time of maternal recognition of pregnancy. Embryos collected between days 7.5 and 14.5 were incubated for 8 hr in vitro in HAM's F10 containing radiolabelled pregnenolone. The steroid metabolites in the incubation medium were separated by reverse phase HPLC and the major peaks expressed as a percentage of total metabolites. It was found that there were no major changes in the profile of metabolites throughout the period of study, although there was increased conversion as the...
Development to blastocysts of one- to two-cell equine embryos after coculture with uterine tubal epithelial cells. Development of 1- to 2-cell in vivo fertilized equine embryos cultured with or without uterine tubal epithelial cells (UTEC) was studied. One- to 2-cell embryos (n = 26) were collected surgically from the uterine tubes of pony mares 1 day after ovulation. Four- to 8-cell embryos (n = 9) were collected 2 days after ovulation. Presumptive zygotes and 2-cell embryos were cultured with (n = 17) or without (n = 9) UTEC, and all 4- to 8-cell embryos were cocultured with UTEC as positive controls. Uterine tubal epithelial cells were used as cell suspensions within 2 weeks after initiation of cultures...
Influence of exogenous progesterone on early embryonic development in the mare. The influence of exogenous progesterone on the development of equine oviductal embryos was determined based upon the recovery of Day-7 uterine blastocysts from treated mares (n=13) that were given 450 mg progesterone daily between Days 0 and 6 and from untreated control mares (n=13). Daily administration of 450 mg progesterone in oil significantly (P<0.02) increased serum progesterone concentrations in the treated mares. There was no significant difference in the recovery rate of Day-7 embryos between treated and control mares (8/13 versus 6/13, respectively). Embryonic development, assesse...
Survival of equine embryos co-cultured with equine oviductal epithelium from the four- to eight-cell to the blastocyst stage after transfer to synchronous recipient mares. In this study we examined the ability of equine oviductal epithelial cells (OEC) to support the development of four- to eight-cell equine embryos in vitro and investigated the ability of co-cultured embryos to continue normal development after transfer to synchronous recipient mares. Equine embryos obtained at Day 2 after ovulation were cultured with or without OEC for 5 days. Those OEC co-cultured embryos that reached the blastocyst stage and embryos recovered from the uterus at Day 7 were surgically transferred to synchronous recipient mares. Co-culture with OEC improved (P < 0.01) develo...
Regulation of mitogen- and TCGF-induced lymphocyte blastogenesis by prostaglandins and supernatant from equine embryos and endometrium. Immunosuppressive substances which interfere with lymphocyte blastogenesis are released in vitro by embryos and endometrium from mares in early pregnancy. Immunosuppression was not evident when tissues were cultured in the presence of indomethacin (a prostaglandin-synthesis inhibitor). Various prostaglandins (PGs) were added to equine lymphocytes and lymphocyte proliferation was measured after the addition of concanavalin A (Con A) or phytohaemagglutinin A (PHA). PGE2 and PGF2 alpha inhibited Con A-induced blastogenesis down to final concentrations of 1.8 x 10(-9) M and 1.3 x 10(-6) M, respect...
Lodgement of the equine blastocyst in the uterus from fixation through endometrial cup formation. The equine blastocyst becomes fixed in position in the uterus on approximately Day 16 of gestation, but allantochorionic villi are not formed until about Day 50. The purpose of this study was to examine evidence that the blastocyst is orientated during this time period, and to determine what morphological features might assist retention of the position of the blastocyst within the uterus. Implantation sites were collected on Days 10-42 of gestation, and the reproductive tracts perfused with fixative for light and electron microscopic examination. The conceptus is found at the bend of a uterine...