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Topic:Embryo
Equine embryo research focuses on the early developmental stages of horses, encompassing the formation, growth, and differentiation of the embryo. This area of study is significant for understanding reproductive biology, improving breeding programs, and advancing assisted reproductive technologies in equines. Key aspects include the processes of fertilization, embryonic development, and implantation. Researchers investigate factors influencing embryonic viability, such as genetic and environmental influences, to enhance reproductive success rates. This page aggregates peer-reviewed research studies and scholarly articles that explore the biological mechanisms, technological advancements, and applied methodologies related to equine embryos.
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...
Embryo production by ovum pick up from live donors. Embryo production by in vitro techniques has increased steadily over the years. For cattle where this technology is more advanced and is applied more, the number of in vitro produced embryos transferred to final recipients was over 30,000 in 1998. An increasing proportion of in vitro produced embryos are coming from oocytes collected from live donors by ultrasound-guided follicular aspiration (ovum pick up, OPU). This procedure allows the repeated production of embryos from live donors of particular value and is a serious alternative to superovulation. Ovum pick up is a very flexible technique...
Embryo development rates after transfer of oocytes matured in vivo, in vitro, or within oviducts of mares. Objectives of the present study were to use oocyte transfer: 1) to compare the developmental ability of oocytes collected from ovaries of live mares with those collected from slaughterhouse ovaries; and 2) to compare the viability of oocytes matured in vivo, in vitro, or within the oviduct. Oocytes were collected by transvaginal, ultrasound-guided follicular aspiration (TVA) from live mares or from slicing slaughterhouse ovaries. Four groups of oocytes were transferred into the oviducts of recipients that were inseminated: 1) oocytes matured in vivo and collected by TVA from preovulatory folli...
Cryopreservation of equine embryos by open pulled straw, cryoloop, or conventional slow cooling methods. Cryopreservation of equine embryos with conventional slow-cooling procedures has proven challenging. An alternative approach is vitrification, which can minimize chilling injuries by increasing the rates of cooling and warming. The open pulled straw (OPS) and cryoloop have been used for very rapid cooling and warming rates. The objective of this experiment was to compare efficacy of vitrification of embryos in OPS and the cryoloop to conventional slow cool procedures using 0.25 mL straws. Grade 1 or 2 morulae and early blastocysts (< or = 300 microm in diameter) were recovered from mares on Da...
Comparison of culture and insemination techniques for equine oocyte transfer. This study was designed to test 3 approaches for insemination and transfer of oocytes to recipient mares. Oocytes were recovered transvaginally from naturally cycling donor mares 24 to 26 h after an intravenous injection of 2500 IU of hCG when follicles reached 35 mm in diameter. Multiple oocytes (1 to 4) were transferred surgically into the oviducts of 4 or 5 recipient mares per group. Three groups of transfers were compared: 1) transfer of oocytes cultured in vitro for 12 to 14 h postcollection with insemination of the recipient 2 h postsurgery; 2) transfer of oocytes into the oviduct within...
Factors affecting pregnancy rates and early embryonic death after equine embryo transfer. In the present study, 638 embryo transfers conducted over 3 yr were retrospectively examined to determine which factors (recipient, embryo and transfer) significantly influenced pregnancy and embryo loss rates and to determine how rates could be improved. On Day 7 or 8 after ovulation, embryos (fresh or cooled/transported) were transferred by surgical or nonsurgical techniques into recipients ovulating from 5 to 9 d before transfer. At 12 and 50 d of gestation (Day 0 = day of ovulation), pregnancy rates were 65.7% (419 of 638) and 55.5% (354 of 638). Pregnancy rates on Day 50 were significantl...
Relationship between embryo recovery rate and uterine lavage fluid composition in postpartum mares. The aim of the study was to determine whether neutrophil numbers (PMN), trypsin-inhibitor capacity (TIC), lysozyme, N-acetyl-beta-D-glucosaminidase (NAGase), beta-glucuronidase (B-Gase), total protein, and plasmin in uterine lavage fluid of postpartum (p.p.) mares, either at the time of foal heat insemination or around the time of arrival of the embryo in the uterus, could be used in predicting conception. Fifteen mares were inseminated within 13 h after the first p.p. ovulation. Uterine lavage fluids were successfully collected from 9 out of 12 mares before insemination and from all 15 mares ...
Diagnosis and management of abnormal embryonic development characterized by formation of an embryonic vesicle without an embryo in mares. To determine the incidence, ultrasonographic characteristics, and risk factors associated with embryonic development characterized by formation of an embryonic vesicle without an embryo in mares. Methods: Prevalence survey. Methods: 159 pregnant mares. Methods: From 1994 to 1998, mares between 11 and 40 days after ovulation with normal and abnormal embryonic development were examined ultrasonographically, and characteristics of each conceptus were recorded. Results: The incidence of abnormal embryonic development in mares characterized by formation of an embryonic vesicle without an embryo was...
Effects of different activation treatments on fertilization of horse oocytes by intracytoplasmic sperm injection. The effects of four reagents on the activation and subsequent fertilization of equine oocytes, and the development of these after intracytoplasmic sperm injection, were investigated. Cumulus-oocyte complexes collected from equine ovaries obtained from an abattoir were matured in vitro for 40-44 h in TCM199 medium before being injected, when in metaphase II, with an immobilized stallion spermatozoon. The cumulus-oocyte complexes were then subjected to one of five activation treatments: (a) 10 micromol ionomycin l(-1) for 10 min; (b) 7% (v/v) ethanol for 10 min; (c) 100 micromol thimerosal l(-1)...
Comparative aspects of equine embryonic development. The developmental changes in the equine conceptus, its maternal environment and their interaction during the first 4 weeks following fertilization are reviewed. Attention is drawn to species-specific events to show why the horse is such a valuable model in which to study early pregnancy.
Insemination of mares with low numbers of either unsexed or sexed spermatozoa. Two experiments were conducted to determine pregnancy rates in mares inseminated 1) with 5, 25 and 500 x 10(6) progressively motile spermatozoa (pms), or 2) with 25 x 10(6) sex-sorted cells. In Experiment 1, mares were assigned to 1 of 3 treatments: Group 1 (n=20) was inseminated into the uterine body with 500 x 10(6) pms. Group 2 (n=21) and Group 3 (n=20) were inseminated into the tip of the uterine horn ipsilateral to the preovulatory follicle with 25 and 5 x 10(6) pms, respectively. Mares in all 3 groups were inseminated either 40 (n=32) or 34 h (n=29) after GnRH administration. More mares ...
Horse conceptuses secrete insulin-like growth factor-binding protein 3. Insulin-like growth factor-I (IGF-I) promotes early embryonic development in several species. In the rabbit, IGF-I binds to the embryonic coats from Day 3 of development onward by a 38-kDa protein that is probably insulin-like growth factor-binding protein 3 (IGFBP3). In the present study, ligand, Western, and Northern blot analyses were used to demonstrate the presence of IGF-I-binding activity, several immunoreactive IGFBP3 proteins, and IGFBP3 mRNA in horse conceptuses with particularly large amounts of immunoreactive IGFBP3 in the conceptus capsule. In addition, immunoprecipitation of radi...
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...
Effect of PGE2 on uterine contractility and tone in mares. A technique for transvaginal, ultrasound-guided intrauterine injection was developed. After preliminary study using different approaches, the procedure was successful in 24 of 25 (96%) mares, based on detecting fluid in the uterine lumen during and after the injection. The technique was used to study the effect of PGE2, reportedly produced by the embryonic vesicle, on uterine contractility on Day 12 (Day 0 = ovulation). Uterine contractility was scored (1 = minimal, 4 = maximal) every 10 min for 1 h and every 30 min for the next hour by a continuous 1-min ultrasound examination of a longitudin...
Effect of time during transport of excised mare ovaries on oocyte recovery rate and quality after in vitro maturation. In the mare only a limited number of oocytes can be successfully collected in vivo, so that when large numbers of oocytes are needed for experimentation, ovaries harvested from slaughtered mares must be used. The resulting temperature changes and time intervals mandated by handling and transport of ovaries from the slaughterhouse to the laboratory adversely affect the rate of oocyte recovery and their quality after IVF and maturation. We chose to study the effect of temperature and time in transit of excised ovaries by evaluating rate of oocyte recovery, nuclear maturation stage reached before...
Production of embryos by assisted reproduction in the horse. In vitro embryo production is not yet successful in the horse, largely due to low rates of fertilization in vitro. However, methods to produce embryos from isolated oocytes have been developed. Oocytes may be recovered from living mares by aspiration of the dominant preovulatory follicle by trans-abdominal puncture, and from both preovulatory and immature follicles by trans-vaginal ultrasound-guided puncture. Transfer of in vivo-matured oocytes to the oviducts of bred recipient mares has resulted in good pregnancy rates (75-85%). Little work has been done on transfer of horse oocytes matured i...
Modulation of oviductal transport in mares by local application of prostaglandin E2. Equine embryos enter the uterus 144-156 h after ovulation, before which time the passage of embryos through the oviduct is halted in the region of the ampullary-isthmic junction. It is thought that further onward movement of embryos to the uterus is facilitated by secretion of prostaglandin E2 (PGE2) by the embryos, which relaxes the smooth muscle of the isthmus. In the present study, the effect of a single local application of PGE2 on oviductal transport was examined in 22 Thoroughbred and Pony mares that were inseminated on alternate days during oestrus. On day 4 after ovulation, the ipsilat...
Identification of proteins in the equine embryonic capsule. An acellular embryonic capsule envelops equine conceptuses between day 6 and day 23 after ovulation. As all of the factors mediating embryo-mother signalling must pass through the capsule, it acts like a 'mailbox'. Therefore, we have started to map the proteins in this special extracellular matrix at the interface between mother and embryo. In the present study, one- and two-dimensional gel electrophoresis were used to examine a range of proteins. Use of western blotting identified three specific proteins in the capsules of equine conceptuses recovered on day 16 after ovulation: insulin-like g...
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...
Pregnancies produced from fertile and infertile stallions by intracytoplasmic sperm injection (ICSI) of single frozen-thawed spermatozoa into in vivo matured mare oocytes. The use of intracytoplasmic sperm injection (ICSI) for in vitro fertilization of equine oocytes and the developmental potential of these oocytes after transfer to the Fallopian tubes of synchronized mares were examined. Oocytes were aspirated from mature follicles 39 h after injection of a GnRH analogue and transported 190 km at 39 degrees C. Semen from a fertile and an infertile stallion was frozen and prepared for injection. Successfully injected oocytes were transferred surgically into the ampulla of the Fallopian tube either: (i) 4-8 h after semen injection; or (ii) after 24-48 h culture b...
Which insemination results in fertilization when several are performed before ovulation? The aim of the present study was to determine which artificial insemination results in fertilization when mares are inseminated several times before ovulation. Mares in oestrus were inseminated over 62 cycles with fresh semen at 48 h intervals from when a follicle > or =30 mm in diameter was detected until ovulation. The number of inseminations was limited to three. Three fertile stallions were used and a different stallion was used for each artificial insemination. The order of the three stallions was changed for each cycle. Embryos were collected between day 10 and day 12 after ovulation and...
Partial characterization of an equine conceptus prostaglandin inhibitory factor. Equine conceptuses are thought to produce antiluteolytic factors that inhibit endometrial PGF2alpha and, thus, prevent luteolysis in pregnant mares. The aim of the present study was to characterize partially the chemical nature of the prostaglandin inhibitory factor (PIF) produced by equine conceptuses in vitro. Embryos were collected from pregnant mares 13 +/- 0.5 days after ovulation and were cultured for 24 h. Harvested equine conceptus conditioned media (CCM) were assayed for antiluteolytic activity by determining the inhibition of endometrial PGF2alpha synthesis in vitro. Significant anti...
Production of live foals from sperm-injected oocytes harvested from pregnant mares. In vitro fertilization in horses has been less successful than anticipated owing to: (i) the inability to collect large numbers of good quality oocytes; (ii) alterations in the zona pellucida that occur during in vitro maturation of equine oocytes; and (iii) inadequate preparation of equine sperm cells. In addition, studies in humans, mice and cattle have indicated that high concentrations of glucose in culture media may inhibit embryonic development in vitro and this may also be a problem for development of equine embryos in vitro. The aims of the present study were: (i) to achieve fertilizat...
Comparison of the cryoprotectant properties of glycerol and ethylene glycol for early (day 6) equine embryos. Early (day 6) equine embryos (n=23) were assigned to four treatment groups to assess the cryoprotectant properties of glycerol and ethylene glycol and the effect of adding sucrose during removal of the cryoprotectant: (i) group GG (n=5) embryos were frozen and thawed using 1.5 mol glycerol l(-1) as the cryoprotectant, which was added at 22 degrees C in four steps (0.375, 0.75, 1.125 and 1.5 mol glycerol l(-1)), and removed after thawing in five steps (1.5, 1.125, 0.75, 0.375 and 0.0 mol glycerol l(-1)); (ii) group GS (n=6) embryos were frozen and thawed using 1.5 mol glycerol l(-1) as for grou...
The effect of dietary protein on reproduction in the mare. VII. Embryonic development, early embryonic death, foetal losses and their relationship with serum progestagen. Sixty-four Thoroughbred and Anglo-Arab mares aged 6-12 years were randomly allocated to 4 dietary groups and fed diets that differed in the total protein content and quality (essential amino-acids). Forty mares were non-lactating and 24 lactating. Eight mares were withdrawn from the investigation owing to injuries or gynaecological pathology. An overall conception rate of 94.6% and a foaling rate of 80% was achieved. Five of 14 (35.7%) mares (Group 1) fed a low-quality protein diet suffered from early embryonic loss before 90 days of pregnancy compared to 3 of 41 (7.3%) mares in the remaining ...
Preliminary observations in in vitro development of equine embryo after ICSI. The objective of this study was to perform intracytoplasmic sperm injection (ICSI) on in vitro matured equine oocytes and to improve in vitro embryonic development on Vero cells after activation of the microinjected oocytes with calcium ionophore. After maturation (23 or 40 h, 38.5 degrees C, 5% CO2), the cumulus-oocyte complexes were denuded, centrifuged and all oocytes exhibiting the first polar body were microinjected. ICSI was performed using fresh semen from three fertile stallions. Microinjected oocytes were activated with calcium ionophore A23187 (10 min, 10 microM) and cultured individ...
Fetal development of the white line (Zona alba) of the equine hoof. The fetal development of the white line (Zona alba) in the equine hoof is described. Its specific structure of lamellar and interlamellar horn, which in turn is composed of cap and terminal horn, is formed in the second half of the hoof's fetal development. In equine fetuses with a crown-rump length of less than 550 mm, the hoof capsule lacks a 'characteristic' white line since no borders between stratum medium, stratum internum and sole horn are discernible. In the hoof of an equine fetus with a crown-rump length of 550 mm, a narrow white line has taken shape. Its shallow lamellae are arrange...
Transfer of a uterine lipocalin from the endometrium of the mare to the developing equine conceptus. One of the major, progesterone-dependent proteins secreted into the uterine lumen of the mare is a 19-kDa lipocalin (P19). It associates strongly with the embryonic capsule that envelops the young horse conceptus in early gestation, suggesting that it may be involved in sustaining early development. However, it was not known whether the protein was transported through the capsule and/or trophoblast layer and into the yolk sac cavity. To address this question, polyclonal antisera were raised against a C-terminal peptide (based on the deduced amino acid sequence of P19) and a recombinant-derived...
Follicular fluid is not a compulsory carrier of the oocyte at ovulation in the mare. The aim of this study was to test the possibility that ovulation can occur from a preovulatory follicle emptied of its follicular fluid. Transport of the oocyte into the oviduct and fertilisation in 29% of cases demonstrated that ovulation can occur in the absence of follicular fluid but the higher fertility achieved in control mares (62.5%) suggested that follicular fluid does serve a role during ovulation, fertilisation and oviductal transport. Injection of horse oocytes into preovulatory follicles in mules after removal of the follicular fluid, followed by insemination of the mules with hor...
The effect of propanediol on the morphology of fresh and frozen equine embryos. Seventeen horse embryos recovered on the sixth day after spontaneous ovulation were; 1) washed in PBS (n = 6), 2) treated with 1.5 M 1-2 propanediol (n = 6) or, 3) frozen and thawed using 1.5 M propanediol as the cryoprotectant (n = 5). After treatment, the embryos were incubated for 6 h in medium before they were fixed, serially sectioned and examined microscopically to count the total numbers of interphase, mitotic and pycnotic nuclei. Significant differences were measured only in the mean proportions of pycnotic cells (+/- s.d.), both between the control (9.2 +/- 7.3%) and frozen-thawed emb...
