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.
Martin-Pelaez S, Rabow Z, de la Fuente A, Draheim P, Loynachan A, Fiehn O, Meyers S, Lyman C, Dini P.Postmortem and pre-euthanasia oocyte retrieval provides the last opportunity to preserve the genetic material in mares. Pentobarbital (PB) is the most common euthanasia agent; however, its effect on the developmental competence of oocytes has not been determined. Here, we evaluated the concentration of PB in equine follicular fluid (FF) and investigated its effect on the developmental competence of oocytes using a bovine IVF model to overcome the low availability of equine oocytes. The concentration of PB was measured by gas-chromatography/mass-spectrometry in FF collected from mare ovaries im...
Cortez JV, Hardwicke K, Cuervo-Arango J, Grupen CG.The cloning of horses is a commercial reality, yet the availability of oocytes for cloned embryo production remains a major limitation. Immature oocytes collected from abattoir-sourced ovaries or from live mares by ovum pick-up (OPU) have both been used to generate cloned foals. However, the reported cloning efficiencies are difficult to compare due to the different somatic cell nuclear transfer (SCNT) techniques and conditions used. The objective of this retrospective study was to compare the in vitro and in vivo development of equine SCNT embryos produced using oocytes recovered from abatt...
Umair M, Beitsma M, de Ruijter-Villani M, Deelen C, Herrera C, Stout TAE, Claes A.The cryotolerance of equine blastocysts larger than 300 μm can be improved by aspirating blastocoele fluid prior to vitrification; however, it is not known whether blastocoele aspiration also enables successful slow-freezing. The aim of this study was therefore to determine whether slow-freezing of expanded equine embryos following blastocoele collapse was more or less damaging than vitrification. Grade 1 blastocysts recovered on day 7 or 8 after ovulation were measured (>300-550 μm, n = 14 and > 550 μm, n = 19) and blastocoele fluid was aspirated prior to slow-freezing in 10...
Lewis N, Canesin H, Choi YH, Foss R, Felix M, Rader K, Hinrichs K.Information on factors associated with developmental competence of equine in vitro -produced (IVP) blastocysts is lacking. Objective: To determine the relationships of stage, grade, day of development, and specific morphological parameters of equine IVP blastocysts, to pregnancy and foaling rates. Methods: Photomicrographs of 316 IVP embryos with known pregnancy outcomes were scrutinised individually by four observers. Inter-observer variation was assessed, and pregnancy outcome evaluated in relation to day of blastocyst development and assigned grade and stage. Individual component analysis w...
Rajabi-Toustani R, Tsogtgerel M, Gao Y, Li C, Sakato M, Haneda S, Cheong SH, Nambo Y.Japanese native horses, which consists of 8 breeds, are threatened with extinction. Embryo transfer (ET) is used to reproduce endangered animals in various mammalian species. We aimed to perform ET using native ponies from Kiso and Hokkaido as donors and recipients, respectively. ET operation included long-distance transport of non-cryopreserved embryos from Nagano Prefecture to Hokkaido. Embryos were transported 1500 km over 9 h in a container maintained at 22°C. After transferring two embryos to two recipients, one mare delivered a healthy live foal. These results demonstrated that reciproc...
Uchida M, Addai-Arhin S, Ishibashi H, Hirano M, Fukushima S, Ishibashi Y, Tominaga N, Arizono K.Equine estrogens (EQs) are steroidal hormones isolated from the urine of pregnant mares and are used in the formulation of human medications. This study initially investigated the embryonic developmental toxicity of equilin (Eq) and equilenin (Eqn) in medaka (Oryzias latipes). Malformations were observed in embryos exposed to nominal concentrations of 1 and 10 mg/L of Eq and Eqn. Delayed hatching was observed at 1 mg/L of Eq. To further investigate the molecular mechanism of developmental toxicity caused by Eq and Eqn, transcriptome and bioinformatics analyses were performed. Among 2016 and ...
Shilton CA, Kahler A, Roach JM, Raudsepp T, de Mestre AM.Embryonic and foetal loss remain one of the greatest challenges in equine reproductive health with 5-10% of established day 15 pregnancies and a further 5-10% of day 70 pregnancies failing to produce a viable foal. The underlying reason for these losses is variable but ultimately most cases will be attributed to pathologies of the environment of the developing embryo and later foetus, or a defect intrinsic to the embryo itself that leads to lethality at any stage of gestation right up to birth. Historically, much research has focused on the maternal endometrium, endocrine and immune responses ...
Ashraf R, Rashid S, Rasheed I, Asif S.This paper includes the study of early embryonic death (EED), predisposing factors of EED and treatment. EED refers to the fetal mortality which varies in mare and camelids but most probably not later than 50 days of gestation. This duration may be divided into very early mortality, early mortality and late embryonic mortality. This also varies in mare and camelids. There are different embryonic, maternal, environmental/external, and infectious and noninfectious factors which lead to early embryonic loss. Diagnosis is very difficult as in most of the cases resorption of fetus occurs but it is ...
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...
Flores Bragulat AP, Ortiz I, Catalán J, Dorado J, Hidalgo M, Losinno L, Dordas-Perpinyà M, Yánez-Ortiz I, Martínez-Rodero I, Miró J, Gambini A.Equus members exhibit very divergent karyotype, genetic plasticity, and significant differences in their reproductive physiology. Despite the fact that somatic cell nuclear transfer and intracytoplasmic sperm injection (ICSI) has gained relevance in the last few years in horses, few reports have been published exploring ovum pick up (OPU) and in vitro maturation (IVM) of cumulus-oocyte complexes (COCs) in donkeys. Yet, some donkey species and breeds are considered endangered, and these assisted-reproductive technologies could help to preserve the genetic of valuable individuals. In this study...
Martinez Zuviria S, Ciurkiewicz M, Wohlsein P, Madariaga G, Zuccolilli G.Cystic hygroma (hygroma cysticum) is a malformation that has not yet been described as a cause of early pregnancy loss in equines. The condition is a congenital anomaly occurring during embryogenesis due to a failure in which the primitive lymphatic sac does not reach the venous system at the jugular vein, resulting in a lymphatic stasis that starts in the neck region and continues to the rest of the body. From 2015 to 2020, a total of 5,730 ultrasound examinations were performed in mares from 43 different horse farms and embryo transfer farms when sexing pregnancies. In 12 pregnant mares, a s...
Vegas AR, Podico G, Canisso IF, Bollwein H, Fröhlich T, Bauersachs S, Almiñana C.During initial maternal recognition of pregnancy (MRP), the equine embryo displays a series of unique events characterized by rapid blastocyst expansion, secretion of a diverse array of molecules, and transuterine migration to interact with the uterine surface. Up to date, the intricate transcriptome and proteome changes of the embryo underlying these events have not been critically studied in horses. Thus, the objective of this study was to perform an integrative transcriptomic (including mRNA, miRNAs, and other small non-coding RNAs) and proteomic analysis of embryos collected from days 10 t...
Ramírez-Agámez L, Hernández-Avilés C, Varner DD, Love CC.Intracytoplasmic Sperm Injection (ICSI) using frozen/thawed sperm is a common procedure to obtain embryos from fertile or subfertile mares and stallions. Stallion-associated factors that impact the efficiency of ICSI have been studied less than those associated with the mare. Three experiments were conducted: Experiment 1: the effect of freezing extender composition and cryoprotectant; Experiment 2: the effect of sperm exposure to seminal plasma prior to freezing (ejaculated vs. epididymal sperm; two-freeze/thaw cycles each); and Experiment 3: the effect of sperm morphologic feature used for f...
Lange-Consiglio A, Capra E, Giuliani D, Canesi S, Funghi F, Bosi G, Cretich M, Frigerio R, Galbiati V, Cremonesi F.Unlike humans and many other mammalian species, conventional in vitro fertilization (IVF) in equine species is not successful. To mimic in vitro equine spermatozoon-oviduct interaction as close as possible to that which occurs in vivo, extracellular vesicles (EVs) secreted by the female genital tract were used. Three female genital tracts were collected at slaughterhouse from mares in late estrus. Ipsilateral proximal and apical horn endometrial explants were digested with collagenase and trypsin and cells obtained were cultured on insert system to allow their polarization. Ipsilateral ovid...
Merlo B, Del Prete C, Mari G, Iacono E.The demand for equine in vitro produced embryos has increased over the last decade. The aim of this study was to compare the effects of an extended IVM or a prolonged period before fertilization, including holding time, on equine immature oocyte developmental competence. Oocytes, collected from abattoir-derived ovaries, were divided into 4 groups: H0/24 (n = 165) 0 h holding + standard 24-26 h IVM; H8/36 (n = 160) 8 h holding + 36 h IVM; H20/24 (n = 187) 20 h holding + 24 h IVM; H0/44 (n = 164) 0 h holding + 44 h IVM. Oocytes matured to MII were fertilized by intracytoplasm...
Felix MR, Turner RM, Dobbie T, Hinrichs K.Methods for standard in vitro fertilization have been difficult to establish in the horse. We evaluated whether prolonged sperm pre-incubation would support subsequent fertilization. Fresh sperm were pre-incubated with penicillamine, hypotaurine, and epinephrine (PHE) for 22 h. Co-incubation of cumulus-oocyte complexes (COCs) for 6 h yielded 43% fertilization; culture of presumptive embryos yielded 21% blastocysts. Sperm incubated similarly, but without PHE, did not fertilize oocytes. Use of extended semen in the system yielded 54% blastocysts and was applied in subsequent experiments. Trans...
Tasma Z, Hou W, Damani T, Seddon K, Kang M, Ge Y, Hanlon D, Hollinshead F, Hisey CL, Chamley LW.Mesenchymal stromal cell (MSC)-derived extracellular vesicles (EVs) have shown promise as off-the-shelf therapeutics; however, producing them in sufficient quantities can be challenging. In this study, MSCs were isolated from preimplantation equine embryos and used to produce EVs in two commercially available bioreactor designs. Mesenchymal stromal cells (MSC) have recently been explored for their potential use as therapeutics in human and veterinary medicine applications, such as the treatment of endometrial inflammation and infertility. Allogeneic MSC-derived extracellular vesicles (EVs) may...
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...
Walbornn SR, Felix M, Schnobrich MR, Bradecamp EA, Scoggin CF, Stefanovski D, Hinrichs K.To determine the effect of stage of estrus cycle (day after ovulation) at the time of transvaginal ultrasound-guided follicle aspiration (TVA) on parameters related to the success of in vitro equine embryo production. 14 healthy mares were used; 11 completed the study and were included for analysis. Mares underwent TVA of all follicles ≥ 5 mm diameter at each of 3 timepoints: 7 days after ovulation, 14 days after ovulation, and S-DSF (subordinate to a dominant stimulated follicle), during estrus at 24 hours after gonadotropin administration. The 3 treatments were assigned to each mare in ran...
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...
Amorim GB, Segabinazzi LG, Oliveira OM, Perecmanis S, Arruda R, Canisso IF.Misoprostol, a synthetic PGE1, is becoming a common therapy for mares with suspected uterine tube obstruction. Recently, there have been concerns that uterine administration of misoprostol induces exacerbated uterine inflammation; however, this has not been critically evaluated. This study aimed to assess the inflammatory response and potential systemic reactions after uterine administration of misoprostol, either during prebreeding or immediately after postembryo flushing. Privately owned embryo donor mares (n = 11) were randomly assigned in a crossover design to receive misoprostol (3 mL +...
Thompson RE, Meyers MA, Veeramachaneni DNR, Pukazhenthi BS, Hollinshead FK.Organoids are a type of three-dimensional (3D) cell culture that more closely mimic the in vivo environment and can be maintained in the long term. To date, oviductal organoids have only been reported in laboratory mice, women, and cattle. Equine oviductal organoids were generated and cultured for 42 days (including 3 passages and freeze-thawing at passage 1). Consistent with the reports in mouse and human oviductal organoids, the equine oviductal organoids revealed round cell clusters with a central lumen. Developing a 3D model of the mare oviduct may allow for an increased understanding of t...
Derisoud E, Jouneau L, Dubois C, Archilla C, Jaszczyszyn Y, Legendre R, Daniel N, Peynot N, Dahirel M, Auclair-Ronzaud J, Wimel L, Duranthon V....Breeding a mare until she is not fertile or even until her death is common in equine industry but the fertility decreases as the mare age increases. Embryo loss due to reduced embryo quality is partly accountable for this observation. Here, the effect of mare's age on blastocysts' gene expression was explored. Day 8 post-ovulation embryos were collected from multiparous young (YM, 6-year-old, N = 5) and older (OM, > 10-year-old, N = 6) non-nursing Saddlebred mares, inseminated with the semen of one stallion. Pure or inner cell mass (ICM) enriched trophoblast, obtained by embryo bise...
Leal GR, Monteiro CAS, Carvalheira LR, Souza-Fabjan JMG.Simulated Physiological Oocyte Maturation (SPOM) mimics in vitro the physiological events of oocyte maturation. The system uses cAMP modulators in two steps (pre IVM and IVM) and has presented promising results that are arousing the curiosity of IVF programs in different animal species, generating several papers, adaptations, and controversies worldwide. This study systematically analyses the data in the literature on the use of SPOM and compares the outcomes to the original paper (Albuz et al. Hum. Rep., 25: 2999-3011 2010), classifying them into success or failure. The PubMed, Scopus, and ...
Claes A, Stout TAE.In vitro embryo production (IVEP) via Ovum Pick-Up (OPU) and Intracytoplasmic Sperm Injection (ICSI) has become a popular breeding technique in Warmblood mares because of the high success rate and several practical advantages. IVEP offers a solution for a variety of reproductive issues including, but not limited to, sub-fertility in stallions or mares, poor quality or scarce frozen semen, difficulty in synchronizing donor and recipient mares, and inefficient use of recipient mares. In 515 OPU-ICSI sessions performed in our facility in 2021, a mean of 25.9 antral follicles were aspirated yield...
Diel de Amorim M, Klein C, Foster R, Dong L, Lopez-Rodriguez MF, Card C.Leucyl and cystinyl aminopeptidase (LNPEP/oxytocinase) is an enzyme that metabolizes oxytocin in serum and tissues. The presence of oxytocin/neurophysin I (OXT), oxytocin and LNPEP and their relationship to other genes is unknown in the equine conceptus. Our objective was to characterize gene expression of LNPEP and OXT on D8, 10, 12, 14, 15, 16 and 21 conceptuses in relationship to other genes. Immunohistochemistry, western blot and liquid chromatography with tandem mass spectrometry (LC-MS/MS) were used for identification of oxytocin and LNPEP in D15, 16 and 18 conceptuses. LNPEP was increas...
Derisoud E, Jouneau L, Margat A, Gourtay C, Dubois C, Archilla C, Jaszczyszyn Y, Dahirel M, Daniel N, Peynot N, Briot L, De Geoffroy F, Wimel L....No abstract available
Derisoud E, Auclair-Ronzaud J, Palmer E, Robles M, Chavatte-Palmer P.Although puberty can occur as early as 14-15months of age, depending on breed and use, the reproductive career of mares may continue to advanced ages. Once mares are used as broodmares, they will usually produce foals once a year until they become unfertile, and their productivity can be enhanced and/or prolonged through embryo technologies. There is a general consensus that old mares are less fertile, but maternal age and parity are confounding factors because nulliparous mares are usually younger and older mares are multiparous in most studies. This review shows that age critically affects c...
Foss R, Ortis H, Hinrichs K.Intracytoplasmic sperm injection (ICSI) is used to produce foals from otherwise infertile mares and from stallions with limited sperm stores, but requires expensive equipment and is technically demanding. Methods to transport oocytes to ICSI laboratories would allow collection of oocytes by the referring veterinarian and enable greater application of this technique. Objective: This study was conducted to evaluate protocols that could be used to transport immature and maturing oocytes for ICSI. Methods: In vitro experiment. Methods: Oocytes were recovered by transvaginal ultrasound-guided folli...
Pascottini OB, Aurich C, England G, Grahofer A.Endometritis is a leading cause of sub- and infertility in domestic animal species. The healthy uterus is colonized by commensal bacteria, viruses and yeast/fungi that represent the nonpathogenic microbiota. A shift in the number or type of organisms accompanied by immune dysfunction, however, may trigger uterine infection and inflammation. Metritis is associated with inflammation of all uterine layers (endometrium, myometrium and perimetrium), whereas endometritis is a more superficial inflammation involving solely the endometrium. Endometritis generally occurs at two time points in domestic ...
Ortiz-Escribano N, Bogado Pascottini O, Woelders H, Vandenberghe L, De Schauwer C, Govaere J, Van den Abbeel E, Vullers T, Ververs C, Roels K....The success rate for vitrification of immature equine oocytes is low. Although vitrified-warmed oocytes are able to mature, further embryonic development appears to be compromised. Objective: The aim of this study was to compare two vitrification protocols, and to examine the effect of the number of layers of cumulus cells surrounding the oocyte during vitrification of immature equine oocytes. Methods: Experimental in vitro and in vivo trials. Methods: Immature equine oocytes were vitrified after a short exposure to high concentrations of cryoprotective agents (CPAs), or a long exposure to l...
Hinrichs K.This paper reviews current equine assisted reproduction techniques. Embryo transfer is the most common equine ART, but is still limited by the inability to superovulate mares effectively. Immature oocytes may be recovered by transvaginal ultrasound-guided aspiration of immature follicles, or from ovaries postmortem, and can be effectively matured in vitro. Notably, the in vivo-matured oocyte may be easily recovered from the stimulated preovulatory follicle. Standard IVF is still not repeatable in the horse; however, embryos and foals can be produced by surgical transfer of mature oocytes to th...
Hinrichs K, Love CC, Brinsko SP, Choi YH, Varner DD.Three experiments were conducted to evaluate the effect of oocyte and sperm treatments on rates of in vitro fertilization (IVF) in the horse and to determine the capacity of in vitro-matured horse oocytes to be fertilized in vivo. There was no effect of duration of oocyte maturation (24 vs. 42 h) or calcium ionophore concentration during sperm capacitation (3 microM vs. 7.14 microM) on in vitro fertilization rates. Oocytes matured in 100% follicular fluid had significantly higher fertilization (13% to 24%) than did oocytes matured in maturation medium or in 20% follicular fluid (0% to 12%; P <...
Almiñana C, Rudolf Vegas A, Tekin M, Hassan M, Uzbekov R, Fröhlich T, Bollwein H, Bauersachs S.Extracellular vesicles (EVs) have been identified in the uterine fluid in different species and have been pointed as key players in the embryo-maternal dialogue, maternal recognition of pregnancy and establishment of pregnancy. However, little is known about the uterine EVs in the mare. Therefore, the present study aimed at characterizing EVs from uterine lavage of cyclic mares by comparing five EVs isolation methods and the combination of them: (1) ultracentrifugation (UC); (2) concentration of lavage volume by Centricon ultrafiltration (CE); (3) the use of CE with different washing steps (ph...
Pereyra-Bonnet F, Fernández-Martín R, Olivera R, Jarazo J, Vichera G, Gibbons A, Salamone D.Transgenesis is an essential tool in many biotechnological applications. Intracytoplasmic sperm injection (ICSI)-mediated gene transfer is a powerful technique to obtain transgenic pups; however, most domestic animal embryos do not develop properly after ICSI. An additional step in the protocol, namely assistance by haploid chemical activation, permits the use of ICSI-mediated gene transfer to generate transgenic preimplantation embryos in a wide range of domestic species, including ovine, porcine, feline, equine and bovine. In the present study, spermatozoa from five species were coincubated ...
Tremoleda JL, Stout TA, Lagutina I, Lazzari G, Bevers MM, Colenbrander B, Galli C.Blastocyst formation rates during horse embryo in vitro production (IVP) are disappointing, and embryos that blastulate in culture fail to produce the characteristic and vital glycoprotein capsule. The aim of this study was to evaluate the impact of IVP on horse embryo development and capsule formation. IVP embryos were produced by intracytoplasmic sperm injection of in vitro matured oocytes and either culture in synthetic oviduct fluid (SOF) or temporary transfer to the oviduct of a ewe. Control embryos were flushed from the uterus of mares 6-9 days after ovulation. Embryo morphology was eval...
Swegen A, Grupen CG, Gibb Z, Baker MA, de Ruijter-Villani M, Smith ND, Stout TAE, Aitken RJ.Early pregnancy in the mare is a poorly understood, high risk period during which the embryo communicates its presence to the maternal endometrium. Remarkably, the maternal recognition of pregnancy signal is unknown in the horse. This study aimed to profile the proteins secreted by equine blastocysts into their immediate environment, along with proteins contained in the blastocoel and within the acellular embryo capsule. Embryos were recovered on day 8 after ovulation and cultured for 48 hours. Secretomes of day 9 and day 10 embryos were analyzed by LC-MS/MS and supported by analysis of blast...
Caillaud M, Duchamp G, Gérard N.A growing body of evidence suggests that the interleukin-1 system is involved in periovulatory events. Previous work from our lab demonstrated that in the mare, interleukin-1beta (IL-1beta) increases the ovulatory rate of metaphase II oocytes. The present study was conducted to analyze in vivo the effect of IL-1 on oocyte cytoplasmic maturation, ovulation and pregnancy rate. In the present work, IL-1beta (experiment 1, n = 13; experiment 2, n = 25) and interleukin-1RA (IL-1RA; experiment 1, n = 25) were injected intrafollicularly by using the transvaginal ultrasound-guided injection method. In...
Claes A, Stout TAE.In vitro embryo production (IVEP) via Ovum Pick-Up (OPU) and Intracytoplasmic Sperm Injection (ICSI) has become a popular breeding technique in Warmblood mares because of the high success rate and several practical advantages. IVEP offers a solution for a variety of reproductive issues including, but not limited to, sub-fertility in stallions or mares, poor quality or scarce frozen semen, difficulty in synchronizing donor and recipient mares, and inefficient use of recipient mares. In 515 OPU-ICSI sessions performed in our facility in 2021, a mean of 25.9 antral follicles were aspirated yield...
Jacobson CC, Choi YH, Hayden SS, Hinrichs K.Oocytes may be collected from live mares from either the stimulated preovulatory follicle or from all visible immature follicles. We evaluated the yield of mature oocytes, and of blastocysts after intracytoplasmic sperm injection (ICSI), for both follicle types. In Experiment 1, mares were assigned to Progesterone (1.2g biorelease progesterone weekly) or Control treatments. Transvaginal aspiration of all follicles was performed every 14 d. Overall, 596 follicles were aspirated, with a 54% oocyte recovery rate. There was no difference between treatments in number of follicles punctured (9.0 to ...
Sandra O, Charpigny G, Galio L, Hue I.In mammalian species, endometrial receptivity is driven by maternal factors independently of embryo signals. When pregnancy initiates, paracrine secretions of the preattachment embryo are essential both for maternal recognition and endometrium preparation for implantation and for coordinating development of embryonic and extraembryonic tissues of the conceptus. This review mainly focuses on domestic large animal species. We first illustrate the major steps of preattachment embryo development, including elongation in bovine, ovine, porcine, and equine species. We next highlight conceptus secret...
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...
Ortiz-Rodriguez JM, Ortega-Ferrusola C, Gil MC, Martín-Cano FE, Gaitskell-Phillips G, Rodríguez-Martínez H, Hinrichs K, Álvarez-Barrientos A....Artificial insemination with cryopreserved spermatozoa is a major assisted reproductive technology in many species. In horses, as in humans, insemination with cryopreserved sperm is associated with lower pregnancy rates than those for fresh sperm, however, direct effects of sperm cryopreservation on the development of resulting embryos are largely unexplored. The aim of this study was to investigate differences in gene expression between embryos resulting from fertilization with fresh or cryopreserved sperm. Embryos were obtained at 8, 10 or 12 days after ovulation from mares inseminated post-...
Carnevale EM, Ramirez RJ, Squires EL, Alvarenga MA, Vanderwall DK, McCue PM.In the present study, 638 embryo transfers conducted over 3 yr were retrospectively examined to determine which factors (recipient, embryo and transfer) significantly influenced pregnancy and embryo loss rates and to determine how rates could be improved. On Day 7 or 8 after ovulation, embryos (fresh or cooled/transported) were transferred by surgical or nonsurgical techniques into recipients ovulating from 5 to 9 d before transfer. At 12 and 50 d of gestation (Day 0 = day of ovulation), pregnancy rates were 65.7% (419 of 638) and 55.5% (354 of 638). Pregnancy rates on Day 50 were significantl...
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...
McDowell KJ, Sharp DC, Grubaugh W, Thatcher WW, Wilcox CJ.Cycling pony mares were bred and used to test the effect of restricted conceptus mobility on luteal maintenance (i.e. maternal recognition of pregnancy). In Experiment 1, uterine horns were ligated to restrict conceptus mobility to one uterine horn, Group 1; one horn plus the uterine body, Group 2; or one horn, the body and approximately 80% of the second horn, Group 3. Pregnancies were monitored with real-time ultrasonography. Four of five mares in Group 1 and two of four mares in Group 2 returned to estrus (Day 16.0 +/- 1.9 and 14.5 +/- 0.7, respectively) and subsequently lost the embryonic ...
Bavin EP, Atkinson F, Barsby T, Guest DJ.The transcription factor scleraxis is required for tendon development and is upregulated during embryonic stem cell (ESC) differentiation into tenocytes. However, its role beyond early embryonic development is not defined. We utilized a short hairpin RNA to knock down scleraxis expression in ESCs and adult and fetal tenocytes. No effect on growth or morphology was observed in two-dimensional cultures. However, scleraxis knockdown in fetal tenocytes significantly reduced COL1A1, COMP, and SOX9 gene expression. Scleraxis knockdown in adult tenocytes had no effect on the expression of these genes...
Dell'Aquila ME, Cho YS, Minoia P, Traina V, Lacalandra GM, Maritato F.The aim of this study was to compare the effect of the addition of follicular fluid (FF) collected from preovulatory follicles with that of oestrous mare serum (EMS) (acting as the control) to TCM-199 medium on the in-vitro maturation, fertilization and development of equine cumulus-enclosed oocytes. Oocytes (<30 mm in diameter) were obtained from the ovaries of slaughtered mares. After in-vitro maturation in the presence of the two supplements, their fertilization, cleavage and developmental potential were compared after conventional in-vitro fertilization (IVF) or intracytoplasmic sperm inje...
Thompson RE, Meyers MA, Veeramachaneni DNR, Pukazhenthi BS, Hollinshead FK.Organoids are a type of three-dimensional (3D) cell culture that more closely mimic the in vivo environment and can be maintained in the long term. To date, oviductal organoids have only been reported in laboratory mice, women, and cattle. Equine oviductal organoids were generated and cultured for 42 days (including 3 passages and freeze-thawing at passage 1). Consistent with the reports in mouse and human oviductal organoids, the equine oviductal organoids revealed round cell clusters with a central lumen. Developing a 3D model of the mare oviduct may allow for an increased understanding of t...
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...
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 ...
Silva LA, Gastal EL, Beg MA, Ginther OJ.The equine embryonic vesicle is mobile on Days 12-14 (Day 0 = ovulation), when it is approximately 9-15 mm in diameter. Movement from one uterine horn to another occurs, on average, approximately 0.5 times per hour. Mobility ceases (fixation) on Days 15-17. Transrectal color Doppler ultrasonography was used to study the relationship of embryo mobility (experiment 1) and fixation (experiment 2) to endometrial vascular perfusion. In experiment 1, mares were bred and examined daily from Day 1 to Day 16 and were assigned, retrospectively, to a group in which an embryo was detected (pregnant mares;...
Smits K, Gansemans Y, Tilleman L, Van Nieuwerburgh F, Van De Velde M, Gerits I, Ververs C, Roels K, Govaere J, Peelman L, Deforce D, Van Soom A.The signal for maternal recognition of pregnancy (MRP) has still not been identified in the horse. High-throughput molecular biology at the embryo-maternal interface has substantially contributed to the knowledge on pathways affected during MRP, but an integrated study in which proteomics, transcriptomics and miRNA expression can be linked directly is currently lacking. The aim of this study was to provide such analysis. Endometrial biopsies, uterine fluid, embryonic tissues, and yolk sac fluid were collected 13 days after ovulation during pregnant and control cycles from the same mares. Micro...
Felix MR, Turner RM, Dobbie T, Hinrichs K.Methods for standard in vitro fertilization have been difficult to establish in the horse. We evaluated whether prolonged sperm pre-incubation would support subsequent fertilization. Fresh sperm were pre-incubated with penicillamine, hypotaurine, and epinephrine (PHE) for 22 h. Co-incubation of cumulus-oocyte complexes (COCs) for 6 h yielded 43% fertilization; culture of presumptive embryos yielded 21% blastocysts. Sperm incubated similarly, but without PHE, did not fertilize oocytes. Use of extended semen in the system yielded 54% blastocysts and was applied in subsequent experiments. Trans...
Hendriks WK, Colleoni S, Galli C, Paris DB, Colenbrander B, Roelen BA, Stout TA.Advanced maternal age and in vitro embryo production (IVP) predispose to pregnancy loss in horses. We investigated whether mare age and IVP were associated with alterations in mitochondrial (mt) DNA copy number or function that could compromise oocyte and embryo development. Effects of mare age (<12 vs ≥12 years) on mtDNA copy number, ATP content and expression of genes involved in mitochondrial replication (mitochondrial transcription factor (TFAM), mtDNA polymerase γ subunit B (mtPOLB) and mitochondrial single-stranded DNA-binding protein (SSB)), energy production (ATP synthase-couplin...
Weber JA, Freeman DA, Vanderwall DK, Woods GL.The hypothesis that treatment of pregnant mares with prostaglandin E2 (PGE2) hastens the oviductal transport of equine embryos was tested by treating bred mares with PGE2 on Day 3 after ovulation and subsequently measuring the rate of hastened oviductal transport (estimated by the uterine embryo recovery rate on Day 4 after ovulation). In a preliminary, noncontrolled experiment, oviductal transport was apparently not hastened after intramuscular, intrauterine, or intraperitoneal PGE2 administration to bred mares (0/6, 0/3, and 0/3 mares, respectively). Oviductal transport appeared to be hasten...
Altermatt JL, Suh TK, Stokes JE, Carnevale EM.Young (4 to 9 yr) and old (>or=20 yr) mares were treated with equine follicle-stimulating hormone (eFSH), and oocytes were collected for intracytoplasmic sperm injections (ICSI). Objectives were to compare: (1) number, morphology and developmental potential of oocytes collected from young v. old mares from cycles with or without exogenous eFSH and (2) oocyte morphology parameters with developmental competence. Oocytes were collected from preovulatory follicles 20 to 24 h after administration of recombinant equine LH and imaged before ICSI for morphological measurements. After ICSI, embryo d...
Watson ED, Sertich PL.Embryos, endometrial biopsies, and uterine lavage fluid were collected from pregnant and non-pregnant mares 14 days after ovulation. Embryos were cultured for 20.5 h with and without endometrial tissue from pregnant mares, and endometrial tissue was cultured alone. Endometrial content of PGF tended to be higher (P = 0.06) in non-pregnant than in pregnant mares, but the amount of PGF released from tissue during culture was similar for pregnant and non-pregnant mares. Lavage fluid from non-pregnant mares also tended (P = 0.08) to contain higher concentrations of PGF. Coincubation of embryos with...
