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.
Dini P, Bogado Pascottini O, Ducheyne K, Hostens M, Daels P.In the present study, we examined the effect of holding equine oocytes in Syngro embryo holding medium (EHM) overnight at either 4 °C, 17 °C, or 22 °C to 25 °C, on the time to maturation and developmental competence. We also examined the effect of placing denuded oocyte without extruded polar body back in maturation condition on subsequent maturation rate. In experiment 1, cumulus-oocyte complexes (COCs) were recovered postmortem and placed in EHM at 22 °C to 25 °C for 18 to 20 hours (OH) or placed directly in maturation (DM). The maturation rate was assessed after 22, 24, or 28 hou...
Smits K, Nelis H, Van Steendam K, Govaere J, Roels K, Ververs C, Leemans B, Wydooghe E, Deforce D, Van Soom A.The equine oviduct plays a pivotal role in providing the optimal microenvironment for early embryonic development, but little is known about the protein composition of the oviducal fluid in the horse. The aim of the present study was to provide a large-scale identification of proteins in equine oviducal fluid and to determine the effects of ovulation and pregnancy. Four days after ovulation, the oviducts ipsilateral and contralateral to the ovulation side were collected from five pregnant and five non-pregnant mares. Identification and relative quantification of proteins in the oviducal fluid ...
Stout TA.The first month of equine pregnancy covers a period of rapid growth and development, during which the single-cell zygote metamorphoses into an embryo with a functional circulation and precursors of many important organs, enclosed within extraembryonic membranes responsible for nutrient uptake and gaseous exchange. After exiting the oviduct, the conceptus must influence uterine physiology to ensure adequate nutrition and preparation for implantation, while continued development results in the chorioallantois superseding the yolk sac as the primary interface for maternal interaction and exchange...
Klein C.Relaxin is a peptide hormone best known for its action during the latter half of pregnancy, in particular for its softening effect on pelvic ligaments that aids in preparation of the birth canal for the impending delivery of the fetus. The source of relaxin during early pregnancy varies across species, with the CL being the main source in a number of species. The main source of relaxin during late equine pregnancy is the placenta. In mares with impaired placental function, circulating relaxin levels decline before abortion. During early pregnancy, relaxin promotes endometrial angiogenesis thro...
Hinrichs K.A research study is a product of not only a question and its pursuit but also the people, places, and facilities available at the time. My work in equine assisted reproduction has progressed from embryo transfer to oocyte maturation, oocyte transfer, intracytoplasmic sperm injection, embryo biopsy, embryo vitrification, and cloning, as a result of collaborations with an array of remarkable people. This is a summary of some of the stories behind the studies.
Griffith OW, Brandley MC, Whittington CM, Belov K, Thompson MB.In oviparous amniotes (reptiles, birds, and mammals) the chorioallantoic membrane (CAM) lines the inside of the egg and acts as the living point of contact between the embryo and the outside world. In livebearing (viviparous) amniotes, communication during embryonic development occurs across placental tissues, which form between the uterine tissue of the mother and the CAM of the embryo. In both oviparous and viviparous taxa, the CAM is at the interface of the embryo and the external environment and can transfer signals from there to the embryo proper. To understand the evolution of placental ...
Gambini A, De Stéfano A, Jarazo J, Buemo C, Karlanian F, Salamone DF.The low efficiency of interspecies somatic cell nuclear transfer (iSCNT) makes it necessary to investigate new strategies to improve embryonic developmental competence. Embryo aggregation has been successfully applied to improve cloning efficiency in mammals, but it remains unclear whether it could also be beneficial for iSCNT. In this study, we first compared the effect of embryo aggregation over in vitro development and blastocyst quality of porcine, bovine, and feline zona-free (ZF) parthenogenetic (PA) embryos to test the effects of embryo aggregation on species that were later used as en...
Klein C."Maternal recognition of pregnancy" (MRP) is commonly used to describe the ongoing embryo-maternal communication during early pregnancy that culminates in prevention of luteolysis and ensures ongoing progestin support. The conceptus-derived pregnancy recognition signal has not yet been identified in the mare. Although equine conceptuses produce substantial amounts of estrogens, there is a lack of evidence that estrogens are the pregnancy recognition signal in mares. Conceptus mobility is integral to MRP and is driven by conceptus-derived prostaglandin production. Cessation of conceptus mobilit...
Barreto Rda S, Rodrigues MN, Carvalho RC, De Oliveira E Silva FM, Rigoglio NN, Jacob JC, Gastal EL, Miglino MA.Musculoskeletal system development involves heterotypical inductive interactions between tendons, muscles, and cartilage and knowledge on organogenesis is required for clarification of its function. The aim of this study was to describe the organogenesis of horse musculoskeletal system between 21 and 105 days of gestation, using detailed macroscopic and histological analyses focusing on essential developmental steps. At day 21 of gestation the skin was translucid, but epithelial condensation and fibrocartilaginous tissues were observed on day 25 of pregnancy. Smooth muscle was seen in lymphat...
Hinrichs K, Choi YH.Embryo cryopreservation presents an essential method for banking of valuable genetics. However, in equine species the cryopreservation of embryos is complicated by three interacting factors: (1) the late entry of the embryo into the uterus (~6 days after ovulation); (2) the rapid expansion of the blastocyst; and (3) the formation of the equine embryonic capsule, a glycoprotein membrane that forms between the embryo and zona. Efforts to freeze or vitrify equine expanded blastocysts were initially met with little success. In addition, it was thought that breaching the capsule led to loss of embr...
Raudsepp T, Chowdhary BP.The association between chromosomal abnormalities and reduced fertility in domestic animals is well recorded and has been studied for decades. Chromosome aberrations directly affect meiosis, gametogenesis, and the viability of zygotes and embryos. In some instances, balanced structural rearrangements can be transmitted, causing fertility problems in subsequent generations. Here, we aim to give a comprehensive overview of the current status and future prospects of clinical cytogenetics of animal reproduction by focusing on the advances in molecular cytogenetics during the genomics era. We descr...
Smits K, De Coninck DI, Van Nieuwerburgh F, Govaere J, Van Poucke M, Peelman L, Deforce D, Van Soom A.Although the equine oviduct clearly affects early embryo development and the selective transport of equine embryos through the oviduct indicates a reciprocal interaction, the influence of the embryo on gene expression in the oviduct remains to be determined in the horse. The aim of this study was to examine this by means of RNA sequencing. Four days after ovulation, epithelial cells ipsilateral and contralateral to the ovulation side from five cyclic and five pregnant mares were collected from the oviduct. RNA was extracted, samples were sequenced, and data analysis was performed to determine ...
Choi YH, Velez IC, Macías-García B, Riera FL, Ballard CS, Hinrichs K.Equine intracytoplasmic sperm injection (ICSI) is being used clinically for foal production, but little information is available on factors affecting the efficiency of this procedure. We examined factors that may influence blastocyst development when ICSI is performed clinically, i.e., on oocytes recovered from live mares by transvaginal ultrasound-guided follicle aspiration (TVA), using sperm from the stallion of the client's choice. In a clinical setting, there may be a delay from the time of TVA to isolation of oocytes from the aspirated fluid. In a preliminary study, oocytes from fluid hel...
Franciosi F, Goudet G, Tessaro I, Papillier P, Dalbies-Tran R, Reigner F, Deleuze S, Douet C, Miclea I, Lodde V, Luciano AM.Implantation failure and genetic developmental disabilities in mammals are caused by errors in chromosome segregation originating mainly in the oocyte during meiosis I. Some conditions, like maternal ageing or in vitro maturation (IVM), increase the incidence of oocyte aneuploidy. Here oocytes from adult mares were used to investigate oocyte maturation in a monovulatory species. Experiments were conducted to compare: (1) the incidence of aneuploidy, (2) the morphology of the spindle, (3) the acetylation of lysine 16 on histone H4 (H4K16) and (4) the relative amount of histone acetyltransferase...
Guignot F, Reigner F, Perreau C, Tartarin P, Babilliot JM, Bed'hom B, Vidament M, Mermillod P, Duchamp G.Preimplantation genetic diagnosis and embryo cryopreservation are important tools to improve genetic management in equine species with marked consequences on the economic value, health, biodiversity, and preservation of the animals. This study aimed to develop a biopsy method at the blastocyst stage that provides viable genotyped cryopreserved Welsh pony embryos. Embryos were collected at d 6.75 to 7 after ovulation. Biopsies were performed with either a microblade or a micropipette. After biopsy, embryos were cryopreserved. The survival rate of biopsied embryos was evaluated on fresh and cryo...
Aurich C, Budik S.Early equine pregnancy shares many features with that of more intensively assessed domestic animals species, but there are also characteristic differences. Some of those are poorly understood. Descent of the equine conceptus into the uterine lumen occurs at day 5 to 6 after ovulation but is only possible when the embryo secretes prostaglandin E2. Although maintenance of equine pregnancy probably involves secretion of a conceptus derived anti-luteolytic factor, this agent has not been identified. Rapid growth, conceptus mobility and presence of an acellular capsule at the time of maternal recog...
Ezashi T, Yuan Y, Roberts RM.This review deals with the latest advances in the study of embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) from domesticated species, with a focus on pigs, cattle, sheep, goats, horses, cats, and dogs. Whereas the derivation of fully pluripotent ESC from these species has proved slow, reprogramming of somatic cells to iPSC has been more straightforward. However, most of these iPSC depend on the continued expression of the introduced transgenes, a major drawback to their utility. The persistent failure in generating ESC and the dependency of iPSC on ectopic genes probably s...
Diaz F, Bondiolli K, Paccamonti D, Gentry GT.Pregnancy rates after cryopreservation of large equine blastocyst stage embryos have remained lower than other domesticated livestock species. It is generally accepted that the embryonic capsule is the primary barrier to cryoprotectant entry into the embryo proper and techniques need to be developed to circumvent this obstacle. Therefore, the objective of this study was to develop an efficient Day 8 equine embryo cryopreservation protocol through blastocyst micromanipulation and vitrification. Grade 1 and 2 embryos recovered from mares (n = 15) 8 days after ovulation were used in these experim...
Ghosh S, Das PJ, Avila F, Thwaits BK, Chowdhary BP, Raudsepp T.Balanced autosomal translocations are a known cause for repeated early embryonic loss (REEL) in horses. In most cases, carriers of such translocations are phenotypically normal, but the chromosomal aberration negatively affects gametogenesis giving rise to both genetically balanced and unbalanced gametes. The latter, if involved in fertilization, result in REEL, whereas gametes with the balanced form of translocation will pass the defect into next generation. Therefore, in order to reduce the incidence of REEL, identification of translocation carriers is critical. Here, we report about a pheno...
González-Fernández L, Macedo S, Lopes JS, Rocha A, Macías-García B.Equine in vitro fertilization (IVF) is still inconsistent. In the present work, we studied how modified Whitten's (MW) medium and Tissue Culture Medium 199 (TCM) added with Foetal Bovine Serum (FBS; 10% v/v) or Bovine Serum Albumin (BSA; 7 mg/ml) affected equine gametes to subsequently run IVF trials. Compact (Cp) and expanded (Ex) cumuli equine oocytes were matured and placed in TCM or MW supplemented with BSA or FBS for 18-20 h (no sperm added). In Ex oocytes, TCM-199 added with FBS or BSA resulted in higher metaphase II (MII) rates (75.7% and 62.7%, respectively) than MW added with BSA (54%...
Geisert RD.Establishment and maintenance of pregnancy in a number of mammalian species depends upon a tightly regulated interaction between the semiallogeneic conceptus and the maternal uterine endometrium.The term "Maternal Recognition of Pregnancy" is attributed to Roger V. Short's paper titled "Implantation and the Maternal Recognition of Pregnancy" which was published in proceedings from the 1969 Symposium on Foetal Autonomy.Professor Short's landmark paper stimulated increased interest in elucidating how the conceptus signals its presence to assure maintenance of the corpus luteum beyond the normal ...
Klein C.Few, if any, biological processes are as diverse among domestic species as establishment of early pregnancy, in particular maternal recognition of pregnancy. Following fertilization and initial development in the mare oviduct, selective transport of the embryo through the uterotubal junction driven by embryo-derived PGE2 occurs. Upon arrival in the uterus, an acellular glycoprotein capsule is formed that covers the embryo, blastocyst, and conceptus (embryo and associated extraembryonic membranes) between the second and third weeks of pregnancy. Between Days 9 and 15/16 of pregnancy, the concep...
Crișan MI, Damian A, Morar I, Páll E, Peștean C, Groza IȘ.The ability to choose the sex of the offspring is of upmost economic importance for horse breeders. Unlike other species, horses present several reproductive peculiarities that interfere with assisted reproductive technologies used in other large animals (such as bovine) and make them difficult to apply. Thus, there is a great interest to determine the sex of the offspring as soon as possible. This has led to the development of several technologies to serve this purpose, which can be classified into two categories. One is equine embryo sexing by either non-invasive biotechnological methods, su...
Roberts MA, London K, Campos-Chillón LF, Altermatt JL.An equine embryo produced by intracytoplasmic sperm injection (ICSI) was trans-cervically transferred to a recipient mare and pregnancy was confirmed via ultrasound examination on days 11, 12 and 15. On days 20 and 22, a single embryonic proper with a heartbeat was observed. On day 29, two embryos proper appeared during ultrasound examination, each possessing a heartbeat. Subsequent examinations on days 35 and 39 revealed continued viability and development of both embryos proper. On day 49, demise of both fetuses was present. Although no DNA analysis or post-partum examinations were performed...
Love CC, Noble JK, Standridge SA, Bearden CT, Blanchard TL, Varner DD, Cavinder CA.The relationship between the quality of cool-shipped stallion semen and fertility has not been adequately described. This study evaluated sperm quality of cool-shipped semen from 459 ejaculates (N = 130 stallions) that were used for insemination of 196 embryo donor mares (n = 496 estrous cycles). Embryo recovery rate (ERR; %) increased, as all sperm measures (e.g., motility, viability, DNA quality, morphology, concentration, and total number) increased. Threshold values are reported for each sperm quality measure (e.g., total sperm motility ≥ 65%) that separate two ERR groups (e.g., av...
Raeside JI, Christie HL, Betteridge KJ.Steroid production and metabolism by early conceptuses are very important for the establishment and maintenance of pregnancy in horses. Our earlier work suggested the possible formation of 5alpha-reduced steroids in equine conceptuses. We have now demonstrated the formation of 5alpha-reduced metabolites of androstenedione, testosterone, and progesterone by the embryo and its membranes. A total of 44 conceptuses were collected from 26 mares between 20 and 31 days of pregnancy. Tissues from the embryo proper and from the separated components of the conceptus (bilaminar and trilaminar trophoblast...
Lee W, Song K, Lee I, Shin H, Lee BC, Yeon S, Jang G.Transvaginal ultrasound-guided follicle aspiration is one method of obtaining recipient oocytes for equine somatic cell nuclear transfer (SCNT). This study was conducted: (1) to evaluate the possibility of oocyte aspiration from pre-ovulatory follicles using a short disposable needle system (14-G) by comparing the oocyte recovery rate with that of a long double lumen needle (12-G); (2) to investigate the developmental competence of recovered oocytes after SCNT and embryo transfer. The recovery rates with the short disposable needle vs. the long needle were not significantly different (47.5% an...
Leemans B, Gadella BM, Stout TA, Heras S, Smits K, Ferrer-Buitrago M, Claes E, Heindryckx B, De Vos WH, Nelis H, Hoogewijs M, Van Soom A.Coincubating equine gametes in the presence of procaine has been reported to facilitate in vitro fertilization, with cleavage rates exceeding 60%. We report that while procaine does trigger sperm hyperactivation, it independently induces cleavage of equine oocytes. First, we found that procaine (1-5 mM) did not facilitate stallion sperm penetration of equine oocytes but instead induced sperm-independent oocyte cytokinesis in the absence of the second polar body extrusion. Indeed, 56 ± 4% of oocytes cleaved within 2.5 days of exposure to 2.5 mM procaine regardless of sperm presence. However, t...
Malschitzky E, Pimentel AM, Garbade P, Jobim M, Gregory RM, Mattos RC.The objective of this retrospective study was to evaluate the effect of management strategies aiming to improve animal well-being on pregnancy and embryonic death (ED) rates. Breeding records of a cohort of 1206 Thoroughbred mares brought to a stallion station facility, to be bred with the stallions housed there, were evaluated during ten breeding seasons. Mares were blocked according to management strategies in two groups: Stress and Relax. Strategies used to improve animal well-being (Relax group) were as follows: stopping the teasing routine, reducing or eliminating stall confinement, reduc...
Agnieszka N, Joanna K, Wojciech W, Adam O.The aim of this study was determine the viability and developmental competence of equine oocytes after IVM and vitrification using the Rapid-I method, as part of an effort to develop an effective equine oocyte vitrification protocol. Equine oocytes were collected by scraping ovarian follicles of slaughtered mares. A total of 1052 ovaries were used in this study, from which 3135 oocytes were obtained. Of the 2853 oocytes retrieved, 2557 underwent in vitro maturation for approximately 36 h. After in vitro culture, 1202 oocytes (47%) had a first polar body. To evaluate the toxicity of the solu...
Ball BA, Sabeur K, Allen WR.Sperm-mediated gene transfer has been reported as a method for production of transgenic animals in a variety of species, and this technique represents a possible method for production of transgenic equids. Objective: To evaluate the uptake of exogenous DNA (enhanced green fluorescent protein; pEGFP) by equine spermatozoa and to assess the ability of transfected spermatozoa to introduce this transgene into early equine embryos. Methods: To evaluate incorporation of pEGFP into equine spermatozoa, washed spermatozoa were incubated with 32P-pEGFP, with or without lipofection. Spermatozoa were also...
Enders AC, Lantz KC, Liu IK, Schlafke S.Twelve blastocysts, collected 7-12 days after ovulation (Day 0), were examined by light and electron microscopy to investigate the nature of the relationship of the polar trophoblast (Rauber's layer) to the inner cell mass. On Day 7, the polar trophoblast was intact and formed a flattened layer overlying the epiblast cells of the inner cell mass. As blastocysts enlarged to greater than 1 mm in diameter, small discontinuities appeared in the polar trophoblast, where epiblast cells intruded onto the surface. At this time, trophoblast cells adhered closely to adjacent and underlying epiblast cell...
De Coster T, Velez DA, Van Soom A, Woelders H, Smits K.Invitro embryo production has evolved rapidly in the horse over the past decade, but blastocyst rates from vitrified equine oocytes remain quite poor and further research is needed to warrant application. Oocyte vitrification is affected by several technical and biological factors. In the horse, short exposure of immature oocytes to the combination of permeating and non-permeating cryoprotective agents has been associated with the best results so far. High cooling and warming rates are also crucial and can be obtained by using minimal volumes and open cryodevices. Vitrification of invivo-matur...
Budik S, Walter I, Tschulenk W, Helmreich M, Deichsel K, Pittner F, Aurich C.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 ...
Budik S, Walter I, Leitner MC, Ertl R, Aurich C.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 ()-2 mRNA was ...
Lewis N, Schnauffer K, Hinrichs K, Morganti M, Troup S, Argo C.The use of time-lapse imaging (TLI) in the evaluation of morphokinetics associated with invitro developmental competence is well described for human, cattle and pig embryos. It is generally accepted that embryos that complete early cleavage sooner are more likely to form blastocysts and that timing of later events, such as blastocyst formation and expansion, are predictive of implantation potential and euploid status. In the horse, morphokinetics as a predictor of developmental competence has received little attention. In this study we evaluated the morphokinetics of early equine embryo develo...
Smits K, Govaere J, Hoogewijs M, Piepers S, Van Soom A.Intracytoplasmic sperm injection (ICSI) is the method of choice for the in vitro production (IVP) of equine embryos. However, conventional ICSI has been associated with mechanical damage to the oocyte caused by the deformation of the zona pellucida (ZP) and exposure of the oolemma to negative pressure during injection. Introduction of the less traumatic and more efficient piezo drill-assisted ICSI (PDAI) yielded higher cleavage rates and more consistent results. Nevertheless, PDAI is also associated with disadvantages such as the use of mercury and possible DNA damage. This led us to explore a...
Cuervo-Arango J, Mateu-Sánchez S, Aguilar JJ, Nielsen JM, Etcharren V, Vettorazzi ML, Newcombe JR.The objective of this study was to determine the effect of the interval from induced luteolysis to ovulation on fertility of mares from two different farms. At farm 1, 215 mares were inseminated with frozen/thawed semen during 513 estrous cycles over seven consecutive breeding seasons. Estrus was induced with analogues of PGF2α in 179 cycles. At farm 2, 375 embryo flushings were performed in 65 donor mares inseminated with fresh semen; of which, 327 were performed following artificial insemination after PGF-induced luteolysis. In both farms, the intervals from PGF treatment to ovulation (ITO)...
Silva ESM, Cuervo-Arango J, de Ruijter-Villani M, Klose K, Oquendo PS, Stout TAE.Field data indicate that a longer period of estrus prior to ovulation correlates positively with fertility. To test the hypothesis that the duration of exposure to estrogens prior to progesterone dominance influences endometrial function, we used anestrous mares to simulate varying durations of estrus (3 groups of 5 mares): long (LE), short (SE), and no estrus (NE), as determined by the duration of estradiol priming prior to progesterone treatment: 7, 2 and 0 days for the LE, SE and NE, respectively. Endometrial biopsies were recovered 4 days after progesterone administration in all groups for...
Caballeros JE, Camacho C, Cazales N, Estradé MJ, Fiala-Rechsteiner S, Jobim MIM, Mattos RC.Rapid endometrial adaptations occur with the embryo entering the uterus to create a receptive uterine environment, which is essential for the conceptus' development. The aim of this study was to demonstrate ultrastructural and histological changes of the endometrium at day 5 after ovulation in cyclic and inseminated mares. Mares were daily examined by transrectal palpation and ultrasonographic examination of the reproductive tract until ovulation was detected. In the first cycle, endometrial biopsies from 10 cyclic mares (Cyclic group) were collected on day 5 post-ovulation. In the second cycl...
Curran S, Ginther OJ.Embryonic heart rate was studied during 87 examinations in 56 mares on Days 25 to 174 post ovulation. Real-time B-mode using a stopwatch and M-mode ultrasonography were compared. For analyses of heart rates, data were grouped into weeks or months. For the stopwatch and M-mode techniques, respectively, heart rates averaged 123 and 133 beats per minute during Month 1, peaked during Month 3 at 172 or 196 beats, then gradually decreased over the duration of the study to 126 and 130 beats during Month 6. There was a significant increase in heart rate from Months 1 to 2 and Months 2 to 3 and a signi...
Ghosh S, Das PJ, Avila F, Thwaits BK, Chowdhary BP, Raudsepp T.Balanced autosomal translocations are a known cause for repeated early embryonic loss (REEL) in horses. In most cases, carriers of such translocations are phenotypically normal, but the chromosomal aberration negatively affects gametogenesis giving rise to both genetically balanced and unbalanced gametes. The latter, if involved in fertilization, result in REEL, whereas gametes with the balanced form of translocation will pass the defect into next generation. Therefore, in order to reduce the incidence of REEL, identification of translocation carriers is critical. Here, we report about a pheno...
Novello G, Podico G, Segabinazzi LGTM, Lima FS, Canisso IF.The objective of this study was to compare semen parameters and embryo recovery rates of cooled stallion semen extended with INRA 96 or BotuSemen Gold. In experiment 1, 45 ejaculates from nine mature stallions were collected, assessed, and equally split between both extenders and then extended to 50 million sperm/mL. Then, the extended semen was stored in three passive cooling containers (Equitainer, Equine Express II, and BotuFlex) for 48 hours. In experiment 2, the same ejaculates extended in experiment 1 were cushion-centrifuged, the supernatant was discarded, and the pellets were resuspend...
Choi YH, Love LB, Varner DD, Hinrichs K.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...
Li ZY, Jiang QS, Zhang YL, Liu XM, Engelhardt JF.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...
Guignot F, Bezard J, Palmer E.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...
Waelchli RO, MacPhee DJ, Kidder GM, Betteridge KJ.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 ...
Choi I, Collante WR, Simmen RC, Simmen FA.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...
McClellan A, Paterson YZ, Paillot R, Guest DJ.In horses and humans, tendon injuries are a significant problem. Not only can they occur in both athletes and nonathletes, they require lengthy periods of recuperation and undergo poor natural regeneration, which leads to high reinjury rates. Embryonic stem cells (ESCs) may provide a renewable source of allogeneic cells to use in clinical applications to aid tissue regeneration. Equine ESCs can undergo tenocyte differentiation in vivo and in vitro, but the immune properties of tenocytes isolated from either ESCs or tissues have not previously been characterized. Here, we demonstrate that equin...
Carnevale EM, Maclellan LJ, Coutinho da Silva MA, Scott TJ, Squires EL.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...
Weber JA, Woods GL, Freeman DA, Vanderwall DK.Prostaglandin E2 (PGE2) secreted by Day-6, Day-7, Day-8 and Day-9 equine embryos (ovulation = Day 0) during in vitro incubation was measured by radioimmunoassay. Embryonic PGE2 secretion (ng/embryo/24 hr) was detectable on Day 6 (0.27 +/- 0.39), tended to increase (P less than 0.1) on Day 7 (0.57 +/- 0.88), and increased significantly (P less than 0.05) on Day 8 (2.23 +/- 0.86) and Day 9 (4.13 +/- 0.71). Embryo diameter at the start of the incubation period was linearly correlated (P less than 0.01) to embryonic PGE2 secretion.
Palmer E, Duchamp G, Cribiu EP, Mahla R, Boyazoglu S, Bézard J.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...
Welsford GE, Munk R, Villagómez DA, Hyttel P, King WA, Revay T.Testicular feminization, an earlier term coined for describing a syndrome resulting from failure of masculinization of target organs by androgen secretions during embryo development, has been well documented not only in humans but also in the domestic horse. The pathology, actually referred to as androgen insensitivity syndrome (AIS), has been proposed to follow an X-linked recessive pattern of inheritance in some horse breeds already investigated. Affected individuals are characterized by a female phenotype but with a stallion genotype of 64,XY SRY+ constitution. We identified a Warmblood hor...
Li X, Qin Y, Wilsher S, Allen WR.Various types of cell cycle organization occur in mammals. In this study, centrosome changes during meiosis in horse oocytes, and first cell cycle organization following fertilization, parthenogenesis and nuclear transfer, were monitored. Cumulus oocyte complexes harvested from horse ovaries obtained from slaughtered mares were cultured in vitro. Meiotic oocytes of germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I and II (MI and MII) stages were selected at various set times during in vitro maturation. Embryos at the first cell cycle stage were generated by subjecting MII s...
Panzani D, Fanelli D, Camillo F, Rota A.In industrialized countries, the donkey population had a dramatic decrease during the last century and this has brought almost all European donkey breeds to risk. Embryo technologies have already been employed as a tool for the conservation of endangered equid species (e.g. Equus Przewalskii). Today it is possible to obtain pregnancies after the transfer of donkey embryos in synchronized recipients, even though embryo transfer is not widely used as a reproductive technique in this species. So far, very few foals are born after transfer of cryopreserved embryos. To date, no pregnancies have bee...
Panzani D, Rota A, Pacini M, Vannozzi I, Camillo F.One year old fillies are able to conceive but, usually, not to give birth to a living foal. Although embryo transfer allows the production of foals from mature mares with repeated pregnancy losses, no reports are available on the use of one year old fillies as embryo donors. To evaluate this possibility, eleven 12-16 months old Haflinger mares were inseminated with fresh semen and subjected to embryo recovery. Some of the recovered embryos were non-surgically transferred into synchronized mature recipients. Pregnancies were terminated using PGF2alpha at day 25. Fillies' embryo recovery rate an...
