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.
Quinn BA, Hayes MA, Waelchli RO, Kennedy MW, Betteridge KJ.During the third week of pregnancy, the equine conceptus is enclosed within a capsule, the glycan composition of which changes at around day 16 (ovulation = day 0) when the conceptus becomes immobilized (fixed) in the uterine lumen. Our objective was to characterize the process of fixation by identifying changes in major capsule-associated proteins. Individual equine conceptuses (n = 55) were collected transcervically by uterine lavage between days 13.5 and 26.5. Major proteins extracted from capsules were compared with those in fluids from the uterus and yolk sac by SDS-PAGE. Until day 14, a ...
Roasa LM, Choi YH, Love CC, Romo S, Varner DD, Hinrichs K.In vitro fertilization (IVF) was performed on in vitro-matured equine oocytes in three experiments. Frozen-thawed sperm were prepared using swim-up separation and heparin treatment. In Experiment 1, fertilization was achieved with sperm from only one frozen ejaculate of four obtained from the same stallion. Within this ejaculate, fertilization rates were higher with fresh media, as compared to media held for 6-8 days before use (39.6% versus 7.3%, respectively; P<0.001). The type of bovine serum albumin used affected fertilization rates (4% versus 39.6%; P<0.001). To determine if IVF rates wer...
Caillaud M, Dell'aquila ME, De Santis T, Nicassio M, Lacalandra GM, Goudet G, Gérard N.The interleukin-1 (IL-1) system is thought to be involved in periovulatory events in the mare. Previous in vivo studies have demonstrated that IL-1beta induces oocyte maturation, but depresses the pregnancy rate 14 days after ovulation. To better understand the role of IL-1 in oocyte maturation and fertilization, the effects of IL-1 on the in vitro maturation rate of equine oocytes in pure follicular fluid were evaluated and fertilization rate assessed following intracytoplasmic sperm injection (ICSI). Oocytes collected from slaughterhouse ovaries were cultured in four different media for 30 h...
Betteridge KJ.Carl Hartman's title of 47 years ago is invoked in tribute to his first recovery of a bovine embryo 30 years before that, and his legacy of an emphasis on the value of descriptive and comparative studies in reproductive biology. The horse's qualification as a farm animal has waned since those times but, in a conference understandably dominated by research in ruminants and pigs, there are lessons to be learned from some peculiarities of equine embryonic development. Morphological and physiological features of the conceptus and its interaction with its environment during the first month of pregn...
Zhou H, Liu C, Wang W.This study was conducted to reconstruct heterogeneous embryos using equine skin fibroblast cells as donor karyoplasts and the bovine oocytes as recipient cytoplast for investigating the reprogramming of equine somatic cell nuclear in bovine oocyte cytoplasm and the developmental potential of the reconstructed embryos. Adult horse skin fibroblast cells serum-starved were used as donor somatic cells. Bovine oocytes matured in vitro were employed as recipient cytoplasts. The fusion of fibroblast cells into recipient cytoplasm was induced by electofusion. The fused eggs were activated by inomycin ...
Matsukawa K, Akagi S, Adachi N, Sato F, Hasegawa T, Takahashi S.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...
Gündüz MC, Kaşikçi G, Kaya HH.In this study, the effects of oxytocin and an analog of prostaglandin (cloprostenol) on the uterine involution and pregnancy rates were investigated. Mares received 3 ml of 0.9% NaCl in Group C (n=10), 30 IU/mare of oxytocin in Group O (n=10) and 250 microg/mare of cloprostenol in Group P (n=10) within 12h after parturition. The gravid uterine horn's cross-sectional diameter was measured by ultrasonography. The mean uterine diameters did not differ significantly between the treatment (O and P) and the control (C) groups (p>0.05). The difference between the postpartum ovulation periods (Grou...
Arar S, Chan KH, Quinn BA, Waelchli RO, Hayes MA, Betteridge KJ, Monteiro MA.During the second and third weeks of pregnancy, the equine conceptus expands rapidly while it is enclosed within a glycan capsule. Around day 16 of gestation, the conceptus loses its mobility in the uterus by a process termed 'fixation', coinciding with various changes in the capsule. Here, we compared the structure of the carbohydrate moieties expressed by the capsule during pre- and post-fixation periods. The glycan structures were studied by chemical analyses in combination with mass spectrometry. Capsule material from conceptuses collected before fixation (days 13-16) was observed to carry...
Kölle S, Dubois CS, Caillaud M, Lahuec C, Sinowatz F, Goudet G.In the equine, the zona pellucida (ZP) is the major barrier to successful in vitro fertilization. Therefore the aim of our studies was to analyze species-specific features of the equine ZP in regard to structure and glycoprotein ZPB and ZPC expression sites during oocyte development and embryogenesis. The equine ZP revealed high immunological cross-reactivity to porcine ZPB and ZPC. In the ovary, the distribution of ZPB and ZPC was co-localized and correlated with the developmental stage of the follicle. ZPB and ZPC expression started in the oocyte of the late primordial and primary follicle. ...
Carnevale EM.Vitrification can be used successfully to cryopreserve equine embryos. Embryos for vitrification should be collected from donor mares' uteri when they are 300 mm or less in diameter, however,and at the morula or early blastocyst stage of development. No special equipment is required for vitrification; the straw containing the embryo is exposed to vapor for 1 minute before plunging it into liquid nitrogen. Warming of the straw requires no special equipment,and the embryo can be transferred directly from the straw into a recipient's uterus. Vitrification has been repeatedly successful when the p...
Tharasanit T, Colleoni S, Lazzari G, Colenbrander B, Galli C, Stout TA.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....
Galli C, Colleoni S, Duchi R, Lagutina I, Lazzari G.Development of assisted reproductive technologies in horses has been relatively slow compared to other domestic species, namely ruminants and pigs. The scarce availability of abattoir ovaries and the lack of interest from horse breeders and breed associations have been the main reasons for this delay. Progressively though, the technology of oocyte maturation in vitro has been established followed by the application of ICSI to achieve fertilization in vitro. Embryo culture was initially performed in vivo, in the mare oviduct or in the surrogate sheep oviduct, to achieve the highest embryo devel...
Saito S, Yokoyama K, Tamagawa T, Ishiwata I.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 ...
Choi YH, Love LB, Varner DD, Hinrichs K.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...
Logan NL, McCue PM, Alonso MA, Squires EL.Superovulation could potentially increase embryo recovery for immediate transfer or cryopreservation. The objectives were to evaluate the effect of pretreatment with progesterone and estradiol (P+E) on follicular response to eFSH and compare doses of eFSH and ovulatory agents on follicular development and ovulation in mares. In Experiment 1, 40 mares were assigned to one of four treatment groups. Group 1 consisted of untreated controls. Group 2 mares were administered eFSH without pretreatment with P+E. Group 3 mares were administered P+E for 10 days starting in mid-diestrus followed by eFSH t...
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...
Li X, Zhou SG, Imreh MP, Ahrlund-Richter L, Allen WR.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 ...
Saito S, Sawai K, Minamihashi A, Ugai H, Murata T, Yokoyama KK.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...
Squires EL, McCue PM.Embryo recovery from single ovulating mares is approximately 50 per cent per estrous cycle. Superovulation could be used to increase embryo recovery and provide extra embryos for embryo freezing. This review addresses some historical approaches to superovulation, as well as examines factors that affect the response of mares to equine FSH. eCG, GnRH and inhibin vaccines have been of limited success in stimulating multiple ovulation. Numerous studies have shown that injection of equine pituitary extract (EPE) will result in three to four ovulations per estrous cycle and two embryos. A purified, ...
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 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...
Choi YH, Love LB, Varner DD, Hinrichs K.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...
Luciano AM, Goudet G, Perazzoli F, Lahuec C, Gérard N.The in vitro developmental competence of equine oocytes is still low in comparison with other domestic mammals. A major factor affecting the viability of cells during in vitro culture is the increased oxidative stress. Oxidative modifications could be responsible for oocyte incompetence for in vitro maturation (IVM). Cysteamine, a glutathione (GSH) synthesis enhancer, has been shown to increase intracellular GSH content and to improve embryo development when added during IVM of bovine, porcine, and ovine oocytes. The aim of the present study was (1) to determine whether equine cumulus-oocyte c...
Tharasanit T, Colenbrander B, Stout TA.Oocyte cryopreservation is a potentially valuable technique for salvaging the germ-line when a valuable mare dies, but facilities for in vitro embryo production or oocyte transfer are not immediately available. This study examined the influence of maturation stage and freezing technique on the cryopreservability of equine oocytes. Cumulus oocyte complexes were frozen at the immature stage (GV) or after maturation in vitro for 30 hr (MII), using either conventional slow freezing (CF) or open pulled straw vitrification (OPS); cryoprotectant-exposed and untreated nonfrozen oocytes served as contr...
Vanderwall DK, Woods GL, Roser JF, Schlafer DH, Sellon DC, Tester DF, White KL.Cloning is one of several new assisted reproductive techniques being developed for clinical use in the equine industry. Potential uses of equine cloning include: (1) the preservation of genetics from individual animals that would otherwise not be able to reproduce, such as geldings; (2) the preservation of genetic material of endangered and/or exotic species, such as the Mongolian wild horse (Przewalski's horse); and (3) because of the companion animal role that horses fill for some individuals, it is likely that some horse owners will have individual animals cloned for emotional fulfillment. ...
Silva LA, Ginther OJ.The spherical equine embryonic vesicle is mobile throughout the uterine lumen for several days before becoming fixed in the caudal segment of a uterine horn on Day 16 (ovulation = Day 0). Orientation refers to the position of the embryo proper at the periphery of the vesicle relative to the position of the mesometrial attachment. In mares, the embryonic pole of the vesicle is antimesometrial after completion of orientation. Day of vesicle fixation, differential thickening of the endometrium near the mesometrial attachment, and orientation of the embryonic vesicle were studied in 30 ponies, usi...
Lagutina I, Lazzari G, Duchi R, Colleoni S, Ponderato N, Turini P, Crotti G, Galli C.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 nuclear...
Veronesi MC, Faustini M, Villani M, Kindahl H, Galeati G, Battocchio M.The aim of the present study was to highlight the effect of two different techniques of one embryo crushing on some hormonal changes. Ten twinning mares were submitted to the mobile or fixed manual crushing of one blastocyst within day 19 after the last mating. Blood sample was collected from 20 min before to 90 min, 24 and 72 h after the procedure was performed to analyse 15-ketodihydro-PGF(2alpha), cortisol and progesterone plasma concentrations. Singleton pregnancy diagnosis was checked 72 h after crushing and at term of pregnancy. Because the unwanted crushing of both embryos occurred in o...
Tharasanit T, Colenbrander B, Bevers MM, Stout TA.The only gonadotrophin preparation shown to stimulate commercially useful multiple ovulation in mares is equine pituitary extract (EPE); even then, the low and inconsistent ovulatory response has been ascribed to the variable, but high, LH content. This study investigated the effects of an LH-free FSH preparation, recombinant human follicle stimulating hormone (rhFSH), on follicle development, ovulation and embryo production in mares. Five mares were treated twice-daily with 450 i.u. rhFSH starting on day 6 after ovulation, coincident with PGF(2alpha) analogue administration; five control mare...
Choi YH, Love CC, Varner DD, Hinrichs K.This study was conducted to evaluate the effects of thawing, division into aliquots and refreezing on fertilizing capacity (ability to support embryo development after intracytoplasmic sperm injection; ICSI) of frozen stallion semen. Frozen semen from a fertile stallion was thawed, diluted 1:100 with freezing extender, and refrozen (2F treatment). Control semen was frozen only once. In vitro matured equine oocytes were injected with: (1) motile control spermatozoa; (2) motile 2F spermatozoa; (3) non-motile 2F spermatozoa; or (4) non-motile 2F spermatozoa, followed by injection of sperm extract...
Rigoglio NN, Matias GSS, Miglino MA, Mess AM, Jacob JCF, Smith LC.Hybrids between species are often infertile and extremely rare among mammals. Mules, i.e. crossing between the horse and the donkey, on the other hand are very common in agricultural and leisure practices due to their enhanced post-natal physical characteristics that is believed to occur for outbreeding or hybrid vigor. Since no reports are availableon the effects of hybrid vigor during early development, this study focused on characterizing the intrauterine development of mule conceptuses during critical embryo-to-fetus transition period. Nine embryos and fetuses of early gestation, obtained ...
Vogelsang MM, Vogelsang SG, Lindsey BR, Massey JM.Mares were subjected to frequent examination by diagnostic ultrasound and data were compiled with respect to reproductive efficiency. The data were collected over a 3-yr period on 1032 light horse mares. The cummulative pregnancy rate at 35 d post-ovulation was 96.8% and the pregnancy rate per cycle was 76.0% as determined by ultrasound examination. The average number of cycles per conception was 1.43, with an average of 2.29 inseminations per cycle. The incidence of early embryonic death was 7.8%. Mares were subjected to an average of 5.04 scans during the follicular phase of the cycle. The a...
Klein C, Troedsson MH.Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine expressed by a wide range of tissues, which has been implicated to be involved in reproduction. Relative abundance of MIF mRNA in conceptus and endometrial tissue was assessed using real-time RT-PCR. Western blot analysis and immunohistochemistry were used to detect MIF protein expression. MIF transcript abundance was lowest in conceptuses obtained 16 days after ovulation, while the remaining stages of conceptus development that were analysed showed relatively constant expression levels. Migration inhibitory factor expressi...
Scoggin CF, Meira C, McCue PM, Carnevale EM, Nett TM, Squires EL.Equine pituitary extract (EPE) has been reported to induce heightened follicular development in mares, but the response is inconsistent and lower than results obtained in ruminants undergoing standard superovulatory protocols. Three separate experiments were conducted to improve the ovarian response to EPE by evaluating: (1) effect of increasing the frequency or dose of EPE treatment; (2) use of a potent gonadotropin-releasing hormone agonist (GnRH-a) prior to EPE stimulation; (3) administration of EPE twice daily in successively decreasing doses. In the first experiment, 50 mares were randoml...
Douet C, Parodi O, Martino NA, Lacalandra GM, Nicassio M, Reigner F, Deleuze S, Dell'Aquila ME, Goudet G.Most wild equids and many domestic horse breeds are at risk of extinction, so there is an urgent need for genome resource banking. Embryos cryopreservation allows the preservation of genetics from male and female and is the fastest method to restore a breed. In the equine, embryo production in vitro would allow the production of several embryos per cycle. Intracytoplasmic sperm injection (ICSI) is used to generate horse embryos, but it requires expensive equipment and expertise in micromanipulation, and blastocyst development rates remain low. No conventional in vitro fertilization (IVF) techn...
Walters KW, Corbin CJ, Anderson GB, Roser JF, Conley AJ.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...
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...
Karasiewicz J, Andrzej-Modlinski J.Our Department of Experimental Embryology originated from The Laboratory of Embryo Biotechnology, which was organized and directed by Dr. Maria Czlonkowska until her premature death in 1991. Proving successful international transfer of frozen equine embryos and generation of an embryonic sheep-goat chimaera surviving ten years were outstanding achievements of her term. In the 1990s, we produced advanced fetuses of mice after reconstructing enucleated oocytes with embryonic stem (ES) cells, as well as mice originating entirely from ES cells by substitution of the inner cell mass with ES cells. ...
Segabinazzi LGTM, Andrade LRP, Alvarenga MA, Dell'Aqua JA, Canisso IF.The objectives of this study were: (1) to assess uterine features and serum progesterone concentrations of acyclic mares synchronized and resynchronized with intravaginal progesterone release device (IPRD), and (2) to compare pregnancy rates and losses of cyclic and acyclic embryo recipient mares treated with different synchronization protocols. In Experiment 1, mares (n = 12) received estradiol for 3 days (E2-3d), and then 24 h after the last injection, an IPRD was inserted and kept in place for 9 days. Three days after IPRD removal, mares were treated with E2-3d, and then a new IPRD was inse...
Miller A, Woods GL.Reproductive Physiology 1. Twin pregnancies result in high rates of abortion, stillbirth, and neonatal mortality. 2. Twins develop subsequent to multiple ovulations. Multiple ovulations are related to breed, parity, and mare history. Multiple ovulations are most frequently seen in Thoroughbred and Draft mares. Multiple ovulations are more common in barren and perhaps maiden mares than in lactating mares, and they are more common in certain individual mares. 3. Equine embryos are motile in the uterus from the time of first detection (Days 9 to 10) until fixation (Day 16). They are frequently lo...
Okada CTC, Kaps M, Reichart U, Walter I, Gautier C, Aurich J, Aurich C.While detrimental effects of reduced plasma progesterone concentration in the early luteal phase on conceptus development in horses have recently been demonstrated, there is no information on associated effects on the endometrium, allantochorion (AC), and chorionic girdle (CG) in this species. We hypothesised that reduced early postovulatory progesterone concentration in pregnant horses is detrimental to endometrial function and development of the embryonic membranes and is an underlying cause of delayed conceptus development. After insemination and ovulation, mares (n = 11) were assigned to...
Ruggeri E, DeLuca KF, Galli C, Lazzari G, DeLuca JG, Stokes JE, Carnevale EM.Confocal microscopy was used to image stages of equine zygote development, at timed intervals, after intracytoplasmic sperm injection (ICSI) of oocytes that were matured in vivo or in vitro. After fixation for 4, 6, 8, 12, or 16 h after ICSI, zygotes were incubated with α/β tubulin antibodies and human anticentromere antibody (CREST/ACA), washed, incubated in secondary antibodies, conjugated to either Alexa 488 or Alexa 647, and incubated with 561-Phalloidin and Hoechst 33258. An Olympus IX81 spinning disk confocal microscope was used for imaging. Data were analyzed using χ 2 and Fisher's e...
Kooistra LH, Ginther OJ.At day 24 of gestation, pregnant mares were allotted to 1 of 5 treatment groups (3 to 5 mares/group): group A--nontreated controls; group B--intraembryonic injection of 4 mg of colchicine on day 24; group C--removal of embryo on day 24; group D--subcutaneous injection of 1.25 mg of prostaglandin F2alpha (PGF2alpha) on day 32; and group E--removal of embryo on day 24 and subcutaneous injection of PGF2alpha on day 32. In all mares treated with colchicine (group B), the fetal bulge was absent within 2 days. The interval from injection of colchicine to onset of estrus was very short (mean, 4 days)...
Bass LD, Denniston DJ, Maclellan LJ, McCue PM, Seidel GE, Squires EL.Equine embryos (n=43) were recovered nonsurgically 7-8 days after ovulation and randomly assigned to be cryopreserved in one of two cryoprotectants: 48% (15M) methanol (n=22) or 10% (136 M) glycerol (n=21). Embryos (300-1000 microm) were measured at five intervals after exposure to glycerol (0, 2, 5, 10 and 15 min) or methanol (0, 15, 35, 75 and 10 min) to determine changes (%) in diameter over time (+/-S.D.). Embryos were loaded into 0.25-ml plastic straws, sealed, placed in a programmable cell freezer and cooled from room temperature (22 degrees C) to -6 degrees C. Straws were then seeded, h...
Hochi S, Fujimoto T, Oguri N.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...
Dippert KD, Jasko DJ, Seidel GE, Squires EL.Embryo recovery per ovulation has been shown to be lower in superovulated mares than in untreated controls. The objectives of this study were to 1) determine whether follicles stimulated with superovulatory treatment ovulate or luteinize without ovulation, 2) determine fertilization rates of oocytes in oviducts of superovulated and control mares, and 3) evaluate viability of early stage embryos from superovulated and control mares when cultured in equine oviductal cell-conditioned medium. Cyclic mares were randomly assigned to 1 of 2 groups (n=14 per group) on the day of ovulation (Day 0): Gro...
Ball BA, Altschul M, Freeman KP, Hillman RB.Trophoblastic vesicles have been used to study early embryonic development and maternal recognition of pregnancy in domestic animals. The purpose of this study was to characterize the formation of trophoblastic vesicles from Day-12 to Day-16 equine conceptuses. Conceptuses (n = 19) were collected nonsurgically from mares, the capsule was removed, and the conceptus (trophoblast and inner cell mass) was dissected into 2- to 4-mm fragments. Conceptus fragments were cultured in either Ham's F10 (HF10) or Minimum Essential Media (MEM) with 10% fetal bovine serum (FBS) in 24-well plates. Plates were...
Gastal MO, Gastal EL, Kot K, Ginther OJ.The temporal relationships among day of conceptus fixation (cessation of mobility), conceptus diameter, uterine tone, uterine contractility, and myometrial and endometrial thickness of the middle and caudal segments of the uterine horns were assessed in 13 pony mares with fixation in the caudal segment of a uterine horn. The mean day of fixation (14.9 +/- 0.3) was established by 2-h mobility trials. Uterine tone increased (P < 0.0001) gradually over Days 11 to 21, whereas uterine contractility decreased (P < 0.0001) between Days 14 and 18. The diameter of the spherical embryonic vesicle ...
Ball BA, Altschul M, Ellington JE.This study compared the in vitro development of Day-2 equine embryos co-cultured with either trophoblastic vesicles or oviductal explants. Embryos were collected surgically from the oviducts of pony mares 2 d after ovulation and assessed for stage of development. Culture medium was Ham's F12 and Dulbecco's Modified Eagle's Medium (50:50 v/v) in a humidified atmosphere of 5% CO(2) in air at 38.5 degrees C with either trophoblastic vesicles or oviductal explants. The quality score of embryos was assessed daily. After 4 d in culture, embryos were stained (Hoechst 33342) and evaluated with epifluo...
Hanada M, Maeda Y, Oikawa MA.The secretions of the equine endometrial glands are essential for the survival, growth, and development of the conceptus in early pregnancy, and endometrial gland density is directly related to successful pregnancy outcome. Endometrial biopsy is routinely used to assess the reproductive potential of broodmares. Some previous studies have shown that equine endometrial glands are uniformly distributed throughout the uterus; however, other work has shown variation of the endometrial architecture between biopsy sites, suggesting that a single biopsy is not representative of the entire endometrium....
Waelchli RO, Betteridge KJ.Twin ovulations are common in horses, but twin pregnancies are rarely carried to term. Theories of how one or both twins is/are naturally eliminated in early pregnancy, termed 'embryo reduction', have been based on ultrasonographic, not morphological, studies. Here we describe conceptuses recovered transcervically between Days 15 and 28 from 31 twin and two triplet pregnancies. Signs of contact between conceptuses were deduced from those seen in one pair that remained attached by their capsules on Day 18. Signs were found on capsules in two of 10 pairs before or during fixation (immobilisation...
Schlindwein X, Werneburg I.We compared embryogenesis of five species of domesticated even-toed and one odd-toed ungulate and used a phylogenetic framework to contextualize such comparison. Organ systems that occur relatively earlier in embryogenesis generally have more time to develop and therefore are found to be more mature at birth when compared to structures that appear later in development. We hypothesized that the less mature the animals' organs are at birth, the more they are susceptible to artificial selection. The horse had the most mature organs at birth, followed by cattle, reindeer, sheep/goat, and pig. This...
Bruyas JF, Sanson JP, Battut I, Fiéni F, Tainturier D.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...
Alvarenga MA, McCue PM, Bruemmer J, Neves Neto JR, Squire EL.Equine pituitary extract (EPE), has been reported to induce multiple ovulation in mares, however ovulation rates are poor in comparison to those obtained in other species. Attempts to improve the effectiveness of EPE for induction of superovulation in cyclic mares has focused on daily frequency of EPE treatment. Two experiments were performed to compare the ovarian response of cyclic mares given EPE once or twice-daily. Mares were assigned to one of two treatment groups 6 to 8 days after ovulation: prostaglandin was given once and EPE (25 mg) was given once daily (Group 1) or twice daily (Grou...
Wilsher S, Rigali F, Couto G, Camargo S, Allen WR.Historically, cryopreservation of equine embryos >300 μm gave poor pregnancy rates until researchers collapsed the blastocoele cavity and aspirated the blastocoele fluid prior to vitrification. Objective: To determine if aspiration of the blastocoele fluid prior to vitrification is essential for post warming survival. Methods: In vivo experiments. Methods: Fifty embryos were recovered on day 7-8 and washed in holding medium (HM; M-199HEPES + 20% FBS + antibiotics). Embryos were punctured using a micromanipulator mounted 30 μm biopsy needle; following this 28 had >90% of their bla...
Freeman DA, Butler JE, Weber JA, Geary RT, Woods GL.Oviductal and uterine embryos were collected from mares at 5 to 7 days following ovulation 1) to evaluate the effects of oviductal tissue explants on in vitro growth and development of equine embryos and 2) to study the morphologic development of equine embryos in culture. Embryos were incubated for 5 days in a medium (control group) or in medium supplemented with oviductal tissue explants (co-culture group). Embryos were evaluated and the media changed daily. Following 5 days in culture, 10 10 (100%) control embryos and 27 29 (93%) co-cultured embryos had doubled in diameter. All embryos that...
Oriol JG.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...
