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.
Carnevale EM, Maclellan LJ, Stokes JAE.Establishment of optimal methods for equine embryo culture has been slow when compared to some domestic species. In part, this delay was caused by the failure of standard in vitro fertilization techniques in horses. However, the development of intracytoplasmic sperm injection (ICSI) for the assisted fertilization of equine oocytes has resulted in a renewed interest in establishing optimal methods for embryo culture. Currently, ICSI-produced equine embryos are cultured using media designed for other species or other cell cultures and, typically, with the addition of serum. Although systems spec...
Moros-Nicolás C, Douet C, Reigner F, Goudet G.In spite of many attempts to establish an in vitro fertilization (IVF) technique in the equine, no efficient conventional IVF technique is available. The presence of oviductal fluid or oviductal cells during IVF helps to improve embryo production in vitro but is not sufficient to reach high fertilization rates. Thus, our aim was to perform equine IVF either after sperm pre-incubation with oviductal fluid or in the presence of oviductal cells, and to evaluate the effect of cumulus removal from the oocyte or sperm pre-incubation with progesterone. In experiments 1 and 2, IVF was performed in the...
Martinez de Andino EV, Brom-de-Luna JG, Canesin HS, Rader K, Resende HL, Ripley AM, Love CC, Hinrichs K.To assess meiotic and developmental competence after transfer of immature cumulus-oocyte complexes (COCs) to the preovulatory follicles of mares (intrafollicular oocyte transfer (IFOT)). Methods: In Experiment 1, mares received an ovulatory stimulus at IFOT. Thirty hours later, COCs were recovered from the follicle, and mature oocytes underwent ICSI and embryo culture. In Experiments 2 and 3, autologous vs. allogeneic COCs were used. The mares were inseminated and embryos were recovered. In Experiment 3, the ovulatory stimulus was administered 9 h (autologous) and 15 h (allogeneic) before IF...
Marzano G, Mastrorocco A, Zianni R, Mangiacotti M, Chiaravalle AE, Lacalandra GM, Minervini F, Cardinali A, Macciocca M, Vicenti R, Fabbri R....Di-(2-ethylhexyl) phthalate (DEHP) is a commonly used plasticizer with endocrine-disrupting properties. In this study, we used an equine model to investigate DEHP concentrations in ovarian follicular fluid (FF), and to determine the effects of exposure of oocytes to potentially toxic concentrations of DEHP during in vitro maturation (IVM) on embryo development and quality. Embryo development was evaluated using time-lapse monitoring (TLM), a photomicroscopic tool that reveals abnormalities in cleavage kinetics unobservable by conventional morphology assessment. Blastocyst bioenergetic/oxidativ...
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...
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...
Aurich J, Köhne M, Wulf M, Nagel C, Beythien E, Gautier C, Zentek J, Aurich C.The importance of the amino acid L-arginine (ARG) for conceptus growth and litter size has been demonstrated in various species. L-arginine is part of embryo-derived polyamines, a substrate for nitric oxide synthase and stimulates protein synthesis by the embryo. In the present study, we have investigated whether dietary L-arginine supplementation stimulates early conceptus growth in mares. Warmblood mares with singleton pregnancies received either an arginine-supplemented diet (approximately 0.0125% of body weight, n = 12) or a control diet (n = 11) from days 15 to 45 after ovulation. Dia...
Klohonatz KM, Nulton LC, Hess AM, Bouma GJ, Bruemmer JE.Maternal recognition of pregnancy (MRP) in the mare is an unknown process. In a non-pregnant mare on day 14 post-ovulation (PO), prostaglandin F2α (PGF) is secreted by the endometrium causing regression of the corpus luteum. Prior to day 14, MRP must occur in order to attenuate secretion of PGF. The embryo is mobile throughout the uterus due to uterine contractions from day of entry to day 14. It is unknown what signaling is occurring. Literature stated that infusing oil or placing a glass marble into the equine uterus prolongs luteal lifespan and that in non-pregnant mares, serum exosomes co...
Cuervo-Arango J, Claes AN, Beitsma M, Stout TAE.The in vitro production of embryos by ovum pickup (OPU) and intracytoplasmic sperm injection (ICSI) is gaining popularity among horse breeders and veterinarians. Various collection media are available for flushing follicles during OPU. The objective of this study was to determine whether the type of flushing media used to aspirate follicles and collect oocytes influences the outcome of a commercial equine OPU-ICSI program. Two commercial embryo flushing media (EFM1 and EFM2) supplemented with heparin were compared with a flushing media designed specifically for the collection of oocytes (oocy...
Leisinger CA, Klein C, Markle ML, Premanandan C, Sones JL, Pinto CRF, Paccamonti DL.Aluteal cycles were induced in the mare to evaluate the effects of progesterone deprivation on the gene expression of embryos and endometrium collected eight days after ovulation. We hypothesized that the transcript expression would be altered during induced aluteal (AL) cycles (low progesterone <1 ng/mL) when compared with control cycles during diestrus (high progesterone; > 4 ng/mL) for 1) the embryonic expression of progesterone-mediated transcripts and those related to normal embryo growth and development and 2) the endometrial expression of progesterone-mediated transcripts and those ...
Rigoglio NN, Smith OE, Matias GSS, Miglino MA, Smith LC.Because of the growing importance of horses in leisure and several sports, somatic cell nuclear transfer (SCNT) is being used more frequently for cloning animals for performance and reproductive purposes. However, because of the need to perforate the zona pellucida during microsurgical reconstruction of the oocyte, it is possible that SCNT-derived embryos undergo premature hatching, resulting in embryo bisection and twinning. Therefore, because equine twin pregnancies often lead to abnormal embryo development and pregnancy failure, we performed a detailed comparative assessment of equine twin ...
Camargo Ferreira J, Linhares Boakari Y, Sousa Rocha N, Saules Ignácio F, Barbosa da Costa G, de Meira C.The present study characterized the luteal status and the dynamic of the conceptus during the first 20 days of gestation in mares with different ages and degrees of endometrial degeneration. Total area of the corpus luteum (CL), luteal vascularity, CL area with blood signals, progesterone concentrations (P4), embryonic vesicle diameter, number of embryonic location changes, embryonic fixation position and uterine contractility were evaluated. In Experiment 1, mares ≤6 years of age (Young group, 5.6 ± 0.2 years, n = 7 mares) and mares ≥15 years of age (Old group, 17.2 ± 0.9 yea...
Gonzalez-Castro RA, Carnevale EM.We determined if microfluidic sorting (MF) of frozen-thawed stallion sperm improves sperm population characteristics and results in embryo development after intracytoplasmic sperm injection (ICSI). The efficiency and efficacy of MF sperm separation was evaluated by comparing pre- and post-separation sperm population variables. Procedural comparisons were performed after sorting with MF, single-layer colloidal centrifugation (SLC) or swim-up (SU), and cleavage and embryo development were evaluated after ICSI using MF-sorted sperm. In Experiment 1, when compared to the original sperm sample, MF ...
Hannan MA, Haneda S, Itami Y, Wachi S, Saitoh T, Cheong SH, Nambo Y.There has been no report of equine embryo transfer in Japan for the last 24 years. Our objective was to establish an effective protocol for embryo transfer in domestic horse. A Hokkaido native pony was bred by deep-horn artificial insemination with frozen semen from a Connemara pony. Embryo collection was performed using a non-surgical method on day 7. Two embryos were obtained from three flushes (67% recovery) and were transferred fresh into crossbred recipient mares. Both recipient mares were diagnosed pregnant (100% pregnancy rate) 5 days after embryo transfer and had normal progesterone le...
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 ...
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...
Moro LN, Amin G, Furmento V, Waisman A, Garate X, Neiman G, La Greca A, Santín Velazque NL, Luzzani C, Sevlever GE, Vichera G, Miriuka SG.Cell reprogramming has been well described in mouse and human cells. The expression of specific microRNAs has demonstrated to be essential for pluripotent maintenance and cell differentiation, but not much information is available in domestic species. We aim to generate horse iPSCs, characterize them and evaluate the expression of different microRNAs (miR-302a,b,c,d, miR-205, miR-145, miR-9, miR-96, miR-125b and miR-296). Two equine iPSC lines (L2 and L3) were characterized after the reprogramming of equine fibroblasts with the four human Yamanaka's factors (OCT-4/SOX-2/c-MYC/KLF4). The plurip...
Bastos HBA, Martinez MN, Camozzato GC, Estradé MJ, Barros E, Vital CE, Vidigal PMP, Meikle A, Jobim MIM, Gregory RM, Mattos RC.There is a complex cascade involving proteins during early embryo development and maternal recognition, which is very important for maintenance of a conceptus. The aim of this study was to compare proteomic profile of uterine fluid after ovulation in pregnant and cyclic mares. In the first cycle, samples of uterine fluid of 30 cyclic mares were collected on days 7 (n = 10), 10 (n = 10) and 13 (n = 10) post ovulation and constituted the Cyclic group. In the second cycle, the same mares were bred to a fertile stallion. At days 7, 10 and 13 uterine fluid samples were collected. Immedi...
Hendriks WK, Colleoni S, Galli C, Paris DBBP, Colenbrander B, Stout TAE.Intracytoplasmic sperm injection is the technique of choice for equine IVF and, in a research setting, 18-36% of injected oocytes develop to blastocysts. However, blastocyst development in clinical programs is lower, presumably due to a combination of variable oocyte quality (e.g. from old mares), suboptimal culture conditions and marginal fertility of some stallions. Furthermore, mitochondrial constitution appears to be critical to developmental competence, and both maternal aging and invitro embryo production (IVEP) negatively affect mitochondrial number and function in murine and bovine emb...
Claes A, Cuervo-Arango J, van den Broek J, Galli C, Colleoni S, Lazzari G, Deelen C, Beitsma M, Stout TA.In vitro embryo production (IVEP) is increasingly popular but data assessing the outcome of transferred embryos are scarce. Objective: To determine the likelihood of pregnancy and embryonic loss after transfer of frozen-thawed IVP embryos and identify factors influencing success. Methods: Retrospective clinical study. Methods: Blastocysts (n = 261) were produced from immature oocytes of Warmblood mares (n = 116) by Intracytoplasmic Sperm Injection (ICSI) and in vitro culture, and cryopreserved. Thawed IVP embryos were transferred into recipient mares on day 4, 5 or 6 after ovulation. The ...
Merlo B, Mari G, Iacono E.The increase in demand for in vitro produced horse embryos is fostering the development of commercial laboratories for this purpose. Nevertheless, blastocyst production after intracytoplasmic sperm injection (ICSI) is still not as great as desired in most of these laboratories. In relation to horse oocyte classification, both expanded and compact cumulus-oocyte-complexes (COCs) are used for in vitro embryo production. The aim of this study was to compare in vitro embryo developmental capacity of COCs from horses including those with only the corona radiata, frequently collected after aspiratio...
Okada CTC, Andrade VP, Freitas-Dell'Aqua CP, Nichi M, Fernandes CB, Papa FO, Alvarenga MA.Embryo mobility occurs as a result of prostaglandin production by the embryo and endometrium, promoting uterine smooth muscle contractions, which propels the embryonic vesicle through the lumen. Non-steroidal anti-inflammatory drugs (NSAIDs), as flunixin meglumine, are routinely used in equine medicine and can alter the conceptus mobility if applied in early pregnancy, which may impair maternal recognition of pregnancy. The objective of this study was to evaluate and compare the effect of flunixin meglumine (FM; 1.1 mg/kg IV), firocoxib (FIRO; 0.2 mg/kg PO), and meloxicam (ML; 0.6 mg/kg,...
Hinrichs K.A wide variety of assisted reproductive techniques (ARTs) are available to aid in managing aspects of equine reproduction. Embryo recovery and transfer can be used to obtain more than one foal per mare per year, and to obtain foals from mares that cannot carry a foal to term. Oocyte recovery and either transfer to the oviduct of an inseminated recipient mare (oocyte transfer), or intracytoplasmic sperm injection (ICSI) and embryo culture can be used to obtain foals from mares with some types of subfertility, such as problems of the tubular tract. ICSI can be used to obtain foals when sperm num...
Camozzato GC, Martinez MN, Bastos HBA, Fiala-Rechsteiner S, Meikle A, Jobim MIM, Gregory RM, Mattos RC.The aim of this study was to evaluate ultrastructural and histological changes in the endometrium on days 7, 10 and 13 post-ovulation in pregnant and cyclic mares. Mares were routinely examined by transrectal palpation and ultrasonographic examination of the reproductive tract until estrus was detected. In the first cycle, endometrial biopsies from 30 cyclic mares (Cyclic group) were collected on days 7, 10 and 13 post-ovulation. In the second cycle, the same mares were bred by a fertile stallion. At days 7, 10 and 13 post-ovulation intrauterine biopsies were collected. Immediately after sampl...
Rizzo M, Ducheyne KD, Deelen C, Beitsma M, Cristarella S, Quartuccio M, Stout TAE, de Ruijter-Villani M.Advanced mare age is associated with declining fertility and an increased risk of early pregnancy loss. Compromised oocyte quality is probably the primary reason for reduced fertility, but the defects predisposing to embryonic death are unknown. In women, advanced age predisposes to chromosome segregation errors during meiosis, which lead to embryonic aneuploidy and a heightened risk of miscarriage. Objective: To evaluate the effect of advanced mare age on chromosome alignment and meiotic spindle morphology in in vitro-matured (IVM) oocytes. Methods: Morphometric and morphological analysis. M...
Spacek SG, Carnevale EM.Equine follicular fluid (FF) provides autocrine and paracrine factors from theca, granulosa, and cumulus cells, both reflecting and impacting oocyte and follicle maturation. We hypothesized that maturation of oocytes in FF from old versus young mares has a deleterious effect on oocyte maturation and their subsequent developmental potential. Follicular fluid was collected from the large, dominant follicle from young mares (4-13 years) or old mares (21-26 years) and classified as: (1) Noninduced follicular fluid (NFF), FF from noninduced follicle 33 ± 3 mm, or (2) Induced follicular fluid (I...
Gibson C, de Ruijter-Villani M, Rietveld J, Stout TAE.Maternally derived amino acids (AA) are essential for early conceptus development, and specific transporters enhance histotrophic AA content during early ruminant pregnancy. In the present study we investigated AA transporter expression in early equine conceptuses and endometrium, during normal pregnancy and after induction of embryo-uterus asynchrony. 'Normal' conceptuses and endometrium were recovered on Days 7, 14, 21 and 28 after ovulation. To investigate asynchrony, Day 8 embryos were transferred to recipient mares on Day 8 or Day 3, and conceptuses were recovered 6 or 11 days later. Endo...
Morris LHA.The development of techniques to produce equine embryos in vitro is reviewed with specific reference to intracytoplasmic sperm injection (ICSI). Unexplored 50 years ago, this technology has progressed rapidly in the last 20 years to become a commercial reality for the equine breeding industry. Improvements in our understanding of oocyte and embryo competence in the horse have been key factors in overcoming some of the initial problems associated with ICSI. It is now possible to obtain high nuclear maturation and cleavage rates in vitro and the most limiting factor, presently, is the low rate o...
Sessions-Bresnahan DR, Heuberger AL, Carnevale EM.Maternal body composition can be an important determinant for development of obesity and metabolic syndrome in adult offspring. Obesity-related outcomes in offspring may include epigenetic alterations; however, mechanisms of fetal programming remain to be fully elucidated. This study was conducted to determine the impact of maternal obesity in the absence of a high fat diet on equine endometrium and preimplantation embryos. Embryos were collected from normal and obese mares at 8 and 16 days and a uterine biopsy at 16 days (0 day = ovulation). With the exception of 8 day embryos, each sample ...
Berglund LA, Sharp DC, Vernon MW, Thatcher WW.Uterine flushings were obtained through the cervix (Method A) and through the wall of the uterus after hysterectomy (Method B) of ovariectomized Pony mares after s.c. injection of oestrogen for 1 week and progesterone for 2 weeks (Exp. 1). Non-pregnant and pregnant mares were flushed by Method A on Day 14 after ovulation and the flushings compared with those of non-pregnant mares injected i.v. with flunixen meglumine, a prostaglandin synthetase inhibitor, shortly before flushing (Exp. 3). Uterine flushings were also collected by Methods A and B from non-pregnant and pregnant Pony mares on Day ...
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...
Hayes MA, Quinn BA, Keirstead ND, Katavolos P, Waelchli RO, Betteridge KJ.A critical period of early gestation in the mare involves the immobilization (fixation) of the encapsulated conceptus at around days 16-17. We compared the major proteins in the normal equine embryonic capsule and endometrial secretions around the period of fixation with those from pregnancies in the process of termination induced by administration of an analogue of prostaglandin F(2 alpha) (PGF(2 alpha)). Uterocalin and beta(2)-microglobulin (beta(2)M) associated with the embryonic capsule were proteolytically converted to smaller forms during the fixation period. These conversions were simil...
Choi YH, Velez IC, Riera FL, Roldán JE, Hartman DL, Bliss SB, Blanchard TL, Hayden SS, Hinrichs K.Effective cryopreservation of expanded equine blastocysts (> 300 μm in diameter) has been difficult, perhaps due to the volume of blastocoele fluid or the presence of the equine embryonic capsule. Recently, we reported normal viability of equine embryos after trophoblast biopsy, which resulted in blastocyst collapse. The present study addressed the effect of biopsy and resultant breach of the capsule and blastocyst collapse on survival of expanded equine blastocysts after vitrification. First, non-biopsied, small embryos (< 300 μm) were vitrified in fine-diameter microloader pipette ti...
Buchanan BR, Seidel GE, McCue PM, Schenk JL, Herickhoff LA, Squires EL.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 ...
Choi YH, Ross P, Velez IC, Macías-García B, Riera FL, Hinrichs K.Equine embryos develop in vitro in the presence of high glucose concentrations, but little is known about their requirements for development. We evaluated the effect of glucose concentrations in medium on blastocyst development after ICSI. In experiment 1, there were no significant differences in rates of blastocyst formation among embryos cultured in our standard medium (DMEM/F-12), which contained >16 mM glucose, and those cultured in a minimal-glucose embryo culture medium (<1 mM; Global medium, GB), with either 0 added glucose for the first 5 days, then 20 mM (0-20) or 20 mM for th...
Oriol JG, Betteridge KJ, Clarke AJ, Sharom FJ.The equine embryonic capsule replaces the zona pellucida and envelopes the conceptus during the second and third weeks of pregnancy. Although this capsule was described more than 100 years ago, its molecular structure has not been characterized. Here we present evidence that the glycoprotein(s) of the equine capsule resembles those of the mucin glycoprotein family. The resistance of the capsule to chemical and enzymatic solubilization was confirmed, and, as in mucins, protein constituted only 35-40% of its total dry mass. Determination of the sugar composition of the capsule using colorimetric...
Beckelmann J, Budik S, Helmreich M, Palm F, Walter I, Aurich C.An adjustment of sex ratio of offspring to the conditions present at conception is seen in many mammals including horses. This depends on preferential survival of male embryos under conditions of high energy intake. In several species, growth factors including insulin like growth factor (IGF)-1 have been shown to promote embryonic development by decreasing apoptosis and increasing cell proliferation. We hypothesized that sex-related differences in IGF-1 expression in equine embryos during the phase of maternal recognition of pregnancy might exist and thus contribute to preferential survival of...
Choi YH, Norris JD, Velez IC, Jacobson CC, Hartman DL, Hinrichs K.The presence of heterogenous mitochondria from the host ooplast affects the acceptance of offspring obtained by somatic cell nuclear transfer. This might be avoided by obtaining oocytes from selected females, but is then complicated by low numbers of available oocytes. We examined the efficiency of equine somatic cell nuclear transfer using oocytes recovered by transvaginal aspiration of immature follicles from 11 mares. Use of metaphase I oocytes as cytoplasts and of scriptaid (a histone deacetylase inhibitor) treatment during oocyte activation were evaluated to determine if these approaches ...
Grøndahl C, Hyttel P.The nucleolus is believed to be the active site of rRNA synthesis in all eukaryotic cells. In preimplantation embryos, the embryonic genome is apparently more or less silent up to a species-specific developmental stage at which a major burst of transcription occurs. Here we report on nucleologenesis and some ultrastructural aspects of the onset of RNA synthesis in equine embryos during in vivo development. The zygotes and embryos up to blastocyst stages were surgically recovered from normally cycling mares. Mares were induced to ovulate by treatment with 3000 IU hCG and inseminated 20 and 34 h...
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...
Lane M, O'Donovan MK, Squires EL, Seidel GE, Gardner DK.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...
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...
Bedford SJ, Kurokawa M, Hinrichs K, Fissore RA.In all species studied, fertilization induces intracellular Ca2+ ([Ca2+]i) oscillations required for oocyte activation and embryonic development. This species-specific pattern has not been studied in the equine, partly due to the difficulties linked to in vitro fertilization in this species. Therefore, the objective of this study was to use intracytoplasmic sperm injection (ICSI) to investigate fertilization-induced [Ca2+]i signaling and, possibly, ascertain problems linked to the success of this technology in the horse. In vivo- and in vitro-matured mare oocytes were injected with a single mo...
Choi YH, Okada Y, Hochi S, Braun J, Sato K, Oguri N.Frozen-thawed ejaculated stallion spermatozoa were preincubated for 3 h in BO medium containing 5 mM caffeine and then treated with 0.1 micro M calcium ionophore A23187 for 60 sec. Aliquots of the sperm suspension (final concentration 1-2 x 10(7)/ml) were added to the oocytes which had been matured in vitro for 32 h. In Experiment 1, there were 3 groups of oocytes; cumulus intact, denuded zona-intact, and zona-free. Cumulus cells were removed with 0.5% hyaluronidase and the zona pellucida with 0.1% protease. The oocytes were fixed 20 h after insemination with acetic acid:ethanol (1:3) and stai...
Bergfelt DR, Woods JA, Ginther OJ.Characteristics of spontaneous embryonic loss in 21 mares were compared with those of 52 contemporary mares that maintained pregnancy. Embryonic losses were, in retrospect, grouped according to day of loss and length of the interovulatory interval, respectively, as follows: group 1, less than or equal to day 20 and less than or equal to 30 days (n = 10); group 2, less than or equal to day 20 and greater than 30 days (n = 3); and group 3, greater than day 20 and greater than 30 days (n = 8); ovulation was day 0. Mean diameter of the embryonic vesicle in group 1 was smaller (P less than 0.05) on...
Choi YH, Ritthaler J, Hinrichs K.Cloned animals possess mitochondria derived from the host ooplast, which typically differ genetically from those of the donor. This is of special concern to horse breeders, as maternal lines are prized and athletic performance is a key factor in genetic value. To evaluate the feasibility of producing mitochondrial-identical cloned foals, we collected oocytes from immature follicles of two mares, BL and SM, maternally related to the donor stallion. In vitro matured, enucleated oocytes were treated with roscovitine-synchronized donor cells and blastocysts were transferred transcervically to reci...
Ball BA.Fertilization rates were similar for normal and subfertile mares, and much of the difference in fertility between normal and subfertile mares was due to embryonic loss. Fertilization rate estimates for mares ranged from 71 to 96 per cent. The incidence of embryonic loss detected by ultrasonography between Days 11 and 50 was approximately 9 per cent for normal mares, and the estimated incidence of embryonic loss before Day 14 was also 9 per cent. Therefore, the estimated incidence of embryonic loss in normal mares between fertilization and Day 50 is approximately 18 per cent (Fig. 1). In subfer...
Bastos HBA, Martinez MN, Camozzato GC, Estradé MJ, Barros E, Vital CE, Vidigal PMP, Meikle A, Jobim MIM, Gregory RM, Mattos RC.There is a complex cascade involving proteins during early embryo development and maternal recognition, which is very important for maintenance of a conceptus. The aim of this study was to compare proteomic profile of uterine fluid after ovulation in pregnant and cyclic mares. In the first cycle, samples of uterine fluid of 30 cyclic mares were collected on days 7 (n = 10), 10 (n = 10) and 13 (n = 10) post ovulation and constituted the Cyclic group. In the second cycle, the same mares were bred to a fertile stallion. At days 7, 10 and 13 uterine fluid samples were collected. Immedi...
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...
Addis A, Canciani E, Campagnol M, Colombo M, Frigerio C, Recupero D, Dellavia C, Morroni M.Different xenogeneic inorganic bone substitutes are currently used as bone grafting materials in oral and maxillo-facial surgery. The aim of the present study was to determine the physicochemical properties and the in vivo performance of an anorganic equine bone (AEB) substitute. AEB is manufactured by applying a process involving heating at >300 °C with the aim of removing all the antigens and the organic components. AEB was structurally characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), X-ray fluorescence (XRF), and Fourier-transformed infrared (FT-IR) spectros...
Albihn A, Waelchli RO, Samper J, Oriol JG, Croy BA, Betteridge KJ.A novel xenogeneic transplantation approach was used to determine whether it is embryonic or maternal tissue that produces the material that gives rise to the mucin-like glycoprotein of the equine embryonic capsule. Endometrial biopsy samples and conceptuses from six mares at days 13-15 after ovulation were prepared as 1 mm(3) grafts of endometrium, trophoblast and capsule for transplantation, alone or in combination, into various sites in 88 immunodeficient (severe combined immunodeficient or RAG2/gamma(c) double mutant) mice. The overall recovery rate of grafts was over 50%, reaching 100% wi...
Meyers S, Burruel V, Kato M, de la Fuente A, Orellana D, Renaudin C, Dujovne G.In this study we examined the timeline of mitotic events of invitro-produced equine embryos that progressed to blastocyst stage using non-invasive time-lapse microscopy (TLM). Intracytoplasmic sperm injection (ICSI) embryos were cultured using a self-contained imaging incubator system (Miri®TL; Esco Technologies) that captured brightfield images at 5-min intervals that were then generated into video for retrospective analysis. For all embryos that progressed to the blastocyst stage, the initial event of extrusion of acellular debris preceded all first cleavages and occurred at mean (±s.e.m.)...
Betteridge KJ, Eaglesome MD, Flood PF.Two experiments were conducted using 14 mares. In Exp. 1, mares were inseminated with semen treated with TEPA, which, in other species, has been shown to lead to an arrest in ovum cleavage at 2--4 cells. The oviducts and/or uterus were then flushed 7--10 days after ovulation in 6 mares (Group A) or 2--6 days after ovulation in 5 mares (Group B). Fresh eggs were found in the oviduct flushes of 5 Group A and 5 Group B mares: 9 of the 10 eggs appeared to have cleaved, but none had developed beyond 16-cells. Seven eggs contained spermatozoa and 3 of 4 eggs from each group showed evidence of fertil...
Handschuh S, Okada CTC, Walter I, Aurich C, Glösmann M.Here, we describe a workflow for high-detail microCT imaging of formalin-fixed and paraffin-embedded (FFPE) equine embryos recovered on Day 34 of pregnancy (E34), a period just before placenta formation. The presented imaging methods are suitable for large animals' embryos with intention to study morphological and developmental aspects, but more generally can be adopted for all kinds of FFPE tissue specimens. Microscopic 3D imaging techniques such as microCT are important tools for detecting and studying normal embryogenesis and developmental disorders. To date, microCT imaging of vertebrate e...
Lagares MA, Castanheira PN, Amaral DC, Vasconcelos AB, Veado JC, Arantes RM, Stahlberg R.The aim of the present study was to evaluate the in vitro viability of equine embryos vitrified in three different solutions. Day 6 and 6.5 embryos were measured and morphologically evaluated. Only grade 1 or 2 morulae and early blastocysts were vitrified. Eighteen embryos were distributed in Group 1: 40 percent ethylene glycol in PBS, Group: 2 and 3: 40 percent ethylene glycol, 18 percent Ficoll, 0.3M sucrose or 0.3M trehalose in PBS, respectively. The vitrified embryos were loaded individually into 0.25 ml straws, which were cooled and immersed in liquid nitrogen. After warming at 20 degree ...
Deleuze S, Goudet G, Caillaud M, Lahuec C, Duchamp G.In vivo techniques, such as intraoviductal oocyte transfer (OT) and intrafollicular oocyte transfer (IFOT), can be considered as alternatives to bypass the lack of efficient superovulation treatments and the inadequacy of conventional in vitro fertilization techniques in the horse. We compared embryo production after transfer of in vivo recovered oocytes (1) into a recipient's oviduct or (2) into her preovulatory follicle either immediately after ovum pick-up or (3) after in vitro maturation (IVM). Recipients were inseminated with fresh semen of a stallion with a known normal fertility. Ten da...
Moros-Nicolás C, Douet C, Reigner F, Goudet G.In spite of many attempts to establish an in vitro fertilization (IVF) technique in the equine, no efficient conventional IVF technique is available. The presence of oviductal fluid or oviductal cells during IVF helps to improve embryo production in vitro but is not sufficient to reach high fertilization rates. Thus, our aim was to perform equine IVF either after sperm pre-incubation with oviductal fluid or in the presence of oviductal cells, and to evaluate the effect of cumulus removal from the oocyte or sperm pre-incubation with progesterone. In experiments 1 and 2, IVF was performed in the...
Sharma S, Dhaliwal GS, Dadarwal D.Service records of 253 mares (1181 mare-years) spanning over 7 consecutive years, from nine organized Thoroughbred stud farms, situated in the subtropical northwestern India were retrospectively analyzed to assess their reproductive performance. The overall per cycle pregnancy rate at Day 16 and overall foaling rates were 50.30% and 68.95%, respectively, and were significantly higher in mares aged 3-7 years than > or =18 years old mares. The late embryonic losses (9.86%) that occurred between Days 16 and 39 post-ovulation contributed more than 50% of the overall detected pregnancy losses (19.1...
