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.
Hisey EA, Ross PJ, Meyers S.As standard in vitro fertilization is not a viable technique in horses yet, many different techniques have been used to create equine embryos for research purposes. One such method is parthenogenesis in which an oocyte is induced to mature into an embryo-like state without the introduction of a spermatozoon, and thus they are not considered true embryos. Another method is somatic cell nuclear transfer (SCNT), in which a somatic cell nucleus from an extant horse is inserted into an enucleated oocyte, creating a genetic clone of the donor horse. Due to limited availability of equine oocytes in t...
D'Fonseca NMM, Gibson CME, Hummel I, van Doorn DA, Roelfsema E, Stout TAE, van den Broek J, de Ruijter-Villani M.Obesity has been associated with altered reproductive activity in mares, and may negatively affect fertility. To examine the influence of long-term high-energy (HE) feeding on fertility, Shetland pony mares were fed a diet containing 200% of net energy (NE) requirements during a three-year study. The incidence of hemorrhagic anovulatory follicles (HAF) and annual duration of cyclicity were compared to those in control mares receiving a maintenance diet. Day-7 embryos were flushed and transferred between donor and recipient mares from both groups; the resulting conceptuses were collected 21 day...
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...
Wilsher S, Rigali F, Kovacsy S, Allen WT.Successful vitrification of equine expanded blastocysts requires collapse of the blastocoele cavity using a micromanipulator-mounted biopsy pipette on an inverted microscope. Such equipment is expensive and requires user skill. Objective: To develop a manual method of blastocoele collapse prior to vitrification using commercial products. Methods: In vivo experiment. Methods: Seventy-nine Day 7 or 8 embryos were measured and graded. Twenty were vitrified following micromanipulator-assisted puncture and aspiration before being used to validate commercial human vitrification and warming kits cont...
Agnieszka N, Joanna K, Wojciech W, Adam O.The aim of this study was determine the viability and developmental competence of equine oocytes after IVM and vitrification using the Rapid-I method, as part of an effort to develop an effective equine oocyte vitrification protocol. Equine oocytes were collected by scraping ovarian follicles of slaughtered mares. A total of 1052 ovaries were used in this study, from which 3135 oocytes were obtained. Of the 2853 oocytes retrieved, 2557 underwent in vitro maturation for approximately 36 h. After in vitro culture, 1202 oocytes (47%) had a first polar body. To evaluate the toxicity of the solu...
Hisey E, Ross PJ, Meyers SA.OCT4 is a core transcription factor involved in pluripotency maintenance in the early mammalian embryo. The POU5F1 gene that encodes the OCT4 protein is highly conserved across species, suggesting conserved function. However, studies in several species including mice, cattle, and pigs, suggest that there are differences in where and when OCT4 is expressed. Specifically, in the horse, several studies have shown that exposure to the uterine environment may be necessary to induce OCT4 expression restriction to the inner cell mass (ICM) of the developing embryo, suggesting that there may be equine...
Rodriguez J, Maserati M, Robilotta T, Augusto G, Alonso MA, Redoan M, Tibary A, Fleury P.Field collection of oocytes in mares using transvaginal follicular aspiration (TVA) for embryo production has the potential to revolutionate the equine industry. Protocols for TVA in specialized laboratory settings have been described in the scientific literature since the early 1980s. The objective of this study was to determine the success rate of TVA oocytes recovery under ambulatory conditions. A secondary goal of this study was to determine if TVA is associated with any health complications when performed by recently trained practitioners in the field. Follicles (n = 296) from 66 adult c...
Boakari YL, El-Sheikh Ali H, Schnobrich M, Lofrumento K, Scoggin C, Bradecamp E, Scoggin K, Esteller-Vico A, Claes A, Lawrence L, Ball B.High blood urea nitrogen (BUN) concentration is linked to low fertility in cows and ewes; however, this relationship has not been reported in mares. The study characterized the relationship between BUN and follicular fluid urea nitrogen (FUN) during follicle growth (Experiment 1) and the impact of BUN from embryo donors on the pregnancy outcome of recipient mares (Experiment 2). In experiment one, follicular fluid and blood samples were collected from mares during diestrus with growing follicles and during estrus with pre-ovulatory follicles (n = 16 and 10 mares, respectively). In experiment...
Clérico G, Taminelli G, Veronesi JC, Polola J, Pagura N, Pinto C, Sansinena M.Oocyte vitrification is considered experimental in the horse with only three live foals reported. The oxidative conditions induced by vitrification could in part explain the poor results and melatonin, a powerful antioxidant, could stimulate ROS metabolization and restore mitochondrial function in these oocytes. Our objective was to determine the oxidative status of vitrified equine oocytes and to analyze the effect of melatonin on mitochondrial-specific ROS (mROS), oocyte maturation, ICSI embryo development and viability. Immature, abattoir-derived oocytes were held for 15 h and vitrified in...
Boakari YL, El-Sheikh Ali H, Dini P, Loux S, Fernandes CB, Scoggin K, Esteller-Vico A, Lawrence L, Ball B.High blood urea nitrogen (BUN) in cows and ewes has a negative effect on embryo development; however, no comparable studies have been published in mares. The aims of the present study were to evaluate the effects of high BUN on blastocoele fluid, systemic progesterone and Day 14 equine embryos. When a follicle with a mean (±s.e.m.) diameter of 25±3mm was detected, mares were administered urea (0.4g kg-1) with sweet feed and molasses (n=9) or sweet feed and molasses alone (control; n=10). Blood samples were collected every other day. Mares were subjected to AI and the day ovulation was detect...
Consuegra C, Crespo F, Dorado J, Diaz-Jimenez M, Pereira B, Sánchez-Calabuig MJ, Beltrán-Breña P, Pérez-Cerezales S, Rizos D, Hidalgo M.The aim of this study was to evaluate the fertilizing capacity of frozen or vitrified stallion sperm after assessing different warming procedures. In Experiment 1, different warming procedures were compared after sperm vitrification: immersion in extender at 43 °C (C), or in a water bath at 37 °C/30 s (W37), 43 °C/10 s (W43) or 60 °C/5 s (W60). With the W60 treatment, there were greater values (P < 0.05) for VCL (83.93 ± 3.6 μm/s) and ALH (3.00 ± 0.2 μm) than freezing and with the C group, and greater values (P < 0.001) for PM (35.33 ± 2.5 %) than with the W43 treatment. In Experiment...
Checura CM, Momont HW, Castañeira C, Flores-Bragulat A, Losinno L.In horses, prostaglandin E (PGE) is produced by embryos around Day 5 post-ovulation; PGE functions directly at the oviduct promoting embryo transport into the uterus. Non-surgical collection of horse embryos for cryopreservation is recommended at Day 6.5-7 post-ovulation. It was proposed that misoprostol administered orally will hasten oviductal transport of horse embryos. In Experiment 1 (n = 15) there was comparison of time of embryo recovery (Day 6 and 6.5 post-ovulation) from mares administered misoprostol (Day 5 and 5.5) orally to that of untreated mares. On Day 6, embryo collections were...
Sánchez-Calabuig MJ, Fernández-González R, Hamdi M, Smits K, López-Cardona AP, Serres C, Macías-García B, Gutiérrez-Adán A.Equine embryos exhibit an unusual pattern of glucose tolerance in vitro and are currently cultured in hyperglycaemic conditions. Objective: Our main objective was to analyse the effect of different glucose concentrations on in vitro-produced equine embryo development and quality. Methods: Experiments comparing in vitro and in vivo produced embryos. Methods: Oocytes (n = 641) were collected from post-mortem ovaries, matured in vitro and fertilised by intracytoplasmic sperm injection (ICSI). Embryo culture was divided from Day 0 to Day 4 and from Day 4 to Day 9 in three groups: 5-10 (5 and 10...
Moro LN, Viale DL, Bastón JI, Arnold V, Suvá M, Wiedenmann E, Olguín M, Miriuka S, Vichera G.The application of new technologies for gene editing in horses may allow the generation of improved sportive individuals. Here, we aimed to knock out the myostatin gene (MSTN), a negative regulator of muscle mass development, using CRISPR/Cas9 and to generate edited embryos for the first time in horses. We nucleofected horse fetal fibroblasts with 1, 2 or 5 µg of 2 different gRNA/Cas9 plasmids targeting the first exon of MSTN. We observed that increasing plasmid concentrations improved mutation efficiency. The average efficiency was 63.6% for gRNA1 (14/22 edited clonal cell lines) and 96.2% ...
Gambini A, Duque Rodríguez M, Rodríguez MB, Briski O, Flores Bragulat AP, Demergassi N, Losinno L, Salamone DF.Several equids have gone extinct and many extant equids are currently considered vulnerable to critically endangered. This work aimed to evaluate whether domestic horse oocytes support preimplantation development of zebra embryos obtained by intracytoplasmic sperm injection (ICSI, zebroid) and cloning, and to study the Hippo signaling pathway during the lineage specification of trophectoderm cells and inner cell mass cells. We first showed that zebra and horse sperm cells induce porcine oocyte activation and recruit maternal SMARCA4 during pronuclear formation. SMARCA4 recruitment showed to be...
Herrera C.Embryo cryopreservation is normally performed with great success in species like humans and cattle. The large size of in vivo-derived equine embryos and the presence of a capsule-impermeable to cryoprotectants-have complicated the use of embryo cryopreservation in equine reproduction. A breakthrough for this technique was obtained when large equine embryos could be successfully cryopreserved after collapsing the blastocoel cavity using a micromanipulation system. High pregnancy rates have been obtained when vitrification is used in combination with embryo collapse.
Wilsher S, Rigali F, Kovacsy S, Allen WT.Vitrification of embryos >300 µm in diameter requires puncture of the glycoprotein capsule, although the size of the hole compatible with embryo survival is unknown. Forty-five day-7 or -8 embryos were punctured using a 30-µm glass biopsy pipette mounted on a micromanipulator (n = 20) or manually with either an acupuncture needle (∼100-µm diameter -hole; n = 10) or a microneedle with a <1 µm tip to produce a ∼30-µm diameter hole (n = 15) before transferring to recipient mares; further 12 embryos were punctured with either the acupuncture needle or microneedle before being cultured ...
Panzani D, Fanelli D, Camillo F, Rota A.In industrialized countries, the donkey population had a dramatic decrease during the last century and this has brought almost all European donkey breeds to risk. Embryo technologies have already been employed as a tool for the conservation of endangered equid species (e.g. Equus Przewalskii). Today it is possible to obtain pregnancies after the transfer of donkey embryos in synchronized recipients, even though embryo transfer is not widely used as a reproductive technique in this species. So far, very few foals are born after transfer of cryopreserved embryos. To date, no pregnancies have bee...
Aguiar FLN, Gastal GDA, Alves KA, Alves BG, Figueiredo JR, Gastal EL.During the reproductive lifespan of a female, only a limited quantity of oocytes are naturally ovulated; therefore, the mammalian ovary possesses a substantial population of preantral follicles available to be handled and explored in vitro. Hence, the manipulation of preantral follicles enclosed in ovarian tissue aims to recover a considerable population of oocytes of high-value animals for potential application in profitable assisted reproductive technologies (ARTs). For this purpose, the technique of preantral follicle in vitro culture (IVC) has been the most common research tool, achievin...
Camacho CA, Estradé MJ, Cazales N, Caballeros JE, Fiala-Rechsteiner SM, Neves AP, Mattos RC.This experiment aimed to verify if the proteins present in a 13 day conceptus induce changes in the equine endometrial ultra-structure, histology, and vascularization, two days after its infusion. Ten healthy cyclic mares were used. Once estrus was confirmed, mares were examined daily to detect ovulation (day 0). After ovulation, mares were examined daily until day seven by transrectal palpation and B-mode and Doppler ultrasonography. In this first cycle, intrauterine biopsies were collected at day seven after ovulation, constituting the Cyclic group (n = 10). In the second cycle, the same mar...
Jones CJP, Aplin JD, Allen WRT, Wilsher S.From Day 6.5-7 post-conception until its loss around Day 22, the equine embryo is enclosed in a mucinous capsule that prevents direct intercellular interaction between the trophectoderm and uterine epithelium. The embryo is, however, bathed in glycoprotein-rich secretions. In this study, lectin histochemistry was used to characterise the distribution and glycan composition of uterine glycoproteins destined for secretion, and to ascertain the local effect of an embryo on glycosylation in the endometrium. Endometrial biopsies were taken from mares in estrus, on Days 5, 8, 12 and 15 of diestrus, ...
Novello G, Podico G, Segabinazzi LGTM, Lima FS, Canisso IF.The objective of this study was to compare semen parameters and embryo recovery rates of cooled stallion semen extended with INRA 96 or BotuSemen Gold. In experiment 1, 45 ejaculates from nine mature stallions were collected, assessed, and equally split between both extenders and then extended to 50 million sperm/mL. Then, the extended semen was stored in three passive cooling containers (Equitainer, Equine Express II, and BotuFlex) for 48 hours. In experiment 2, the same ejaculates extended in experiment 1 were cushion-centrifuged, the supernatant was discarded, and the pellets were resuspend...
Stout TAE.The first reports of in vitro embryo production (IVEP) by conventional in vitro fertilization and intracytoplasmic sperm injection in horses date respectively from approximately 30 and 25 years ago. However, IVEP has only become established in clinical practice during the last decade. The initial slow uptake of IVEP was largely because the likelihood of success was too low to make it an economically viable means of breeding horses. During the last decade, the balance has shifted, primarily because of significant improvements in the efficiency of recovering immature oocytes from live donor m...
Totorikaguena L, Olabarrieta E, Lolicato F, Romero-Aguirregomezcorta J, Smitz J, Agirregoitia N, Agirregoitia E.The cannabinoid (CB) system has been involved in many aspects of reproduction and it is known that the systemic chronic use of exogenous CBs are deleterious to reproductive processes. Even so, it is not known what happens in relation to the physiology of the ovary when CB receptors are absent. The present study investigated the effect of the lack of CB1 and CB2 receptors in mice ovarian morphology, folliculogenesis, oocyte retrieval, and oocyte maturation and evaluated the use of Δ9-tetrahydrocannabinol (THC) on oocyte in vitro maturation (IVM) by comparing classical IVM and two-step IVM by a...
De Coster T, Velez DA, Van Soom A, Woelders H, Smits K.Invitro embryo production has evolved rapidly in the horse over the past decade, but blastocyst rates from vitrified equine oocytes remain quite poor and further research is needed to warrant application. Oocyte vitrification is affected by several technical and biological factors. In the horse, short exposure of immature oocytes to the combination of permeating and non-permeating cryoprotective agents has been associated with the best results so far. High cooling and warming rates are also crucial and can be obtained by using minimal volumes and open cryodevices. Vitrification of invivo-matur...
Cook NL, Masterson KR, Battaglia D, Beck R, Metcalf ES.Numerous variables affect invitro blastocyst development following intracytoplasmic sperm injection (ICSI). The paternal factor is affected by initial semen quality, processing techniques and final selection of individual spermatozoon for injection. This study investigated whether there was an effect of sperm cryoprotectant agent (CPA) on equine invitro blastocyst production, and reviews recent developments examining how processing equine semen affects ICSI outcomes. Single ejaculates from five stallions were collected and processed in a freezing extender containing either 1M dimethyl sulfoxid...
Claes A, Cuervo-Arango J, Colleoni S, Lazzari G, Galli C, Stout TA.The success of invitro embryo production (IVEP) in horses has increased considerably during recent years, but little is known about the effect of the speed of invitro embryo development. Blastocysts (n=390) were produced by intracytoplasmic sperm injection of IVM oocytes from warmblood mares, cryopreserved, thawed and transferred into recipient mares on Days 3, 4, 5 or 6 after ovulation. The time required for invitro-produced (IVP) embryos to reach the blastocyst stage was recorded (Day 7 vs Day 8). The likelihood of foaling was affected by the speed of invitro embryo development and recipient...
Hinrichs K.Methods for holding of oocytes and embryos during shipment as well as for their cryopreservation can greatly aid equine reproductive management. Oocytes can be held at room temperature overnight or at cooler temperatures for two nights without affecting maturation or embryo development after intracytoplasmic sperm injection. In contrast, methods for cryopreservation of equine oocytes that support high rates of embryo development have not yet been established. Equine embryos may be held overnight at temperatures from 5°C to 19°C without reduction in viability, but longer holding periods, or h...
Souza JRM, Gonçalves PBD, Bertolin K, Ferreira R, Ribeiro ASS, Ribeiro DB, Rechsteiner SMDEF, Araújo RR, Pimentel CA, Pimentel AMH.The aim of the present study was to evaluate the correlation of age and heat cycle to determine reproductive efficiency in young and aged Thoroughbred mares bred on foal heat (FH) or on second heat (SH) after foaling. Embryo mortality (EmbM) was determined every time a mare was found open after a positive pregnancy diagnosis. Parturition to breeding interval, pregnancy rate (PregR) and EmbM rate were the dependent variables and the treatments were breeding on the FH or on SH. The cutoff age to obtain above-average probability for the EmbM was 10 years old. PregR in mares bred on FH was lower ...
Dell'Aquila ME, Masterson M, Maritato F, Hinrichs K.There is a great variability in the success of horse oocyte maturation and fertilization among laboratories. This study was conducted to determine if the meiotic and developmental competence of horse oocytes could be dependent on the method of oocyte collection, i.e., aspiration of follicular fluid with a vacuum apparatus, or opening follicles and scraping the granulosa layer. Horse oocytes were recovered from abattoir ovaries by aspiration or scraping and classified as having compact (Cp), expanded (Ex), or partial (P) cumuli. In Experiment 1 (Part A in May and Part B in October), oocytes wer...
Crossett B, Suire S, Herrler A, Allen WR, Stewart F.One of the major, progesterone-dependent proteins secreted into the uterine lumen of the mare is a 19-kDa lipocalin (P19). It associates strongly with the embryonic capsule that envelops the young horse conceptus in early gestation, suggesting that it may be involved in sustaining early development. However, it was not known whether the protein was transported through the capsule and/or trophoblast layer and into the yolk sac cavity. To address this question, polyclonal antisera were raised against a C-terminal peptide (based on the deduced amino acid sequence of P19) and a recombinant-derived...
Klein C.Relaxin is a peptide hormone best known for its action during the latter half of pregnancy, in particular for its softening effect on pelvic ligaments that aids in preparation of the birth canal for the impending delivery of the fetus. The source of relaxin during early pregnancy varies across species, with the CL being the main source in a number of species. The main source of relaxin during late equine pregnancy is the placenta. In mares with impaired placental function, circulating relaxin levels decline before abortion. During early pregnancy, relaxin promotes endometrial angiogenesis thro...
Choi YH, Love CC, Love LB, Varner DD, Brinsko S, Hinrichs K.This study was undertaken to evaluate the development of equine oocytes in vitro and in vivo after intracytoplasmic sperm injection (ICSI) with either fresh or frozen-thawed spermatozoa, without the use of additional activation treatments. Oocytes were collected from ovaries obtained from an abattoir and oocytes classified as having expanded cumulus cells were matured in M199 with 10% fetal bovine serum and 5 microU FSH ml(-1). After 24-26 h of in vitro maturation, oocytes with a first polar body were selected for manipulation. Fresh ejaculated stallion spermatozoa were used for the experiment...
Choi YH, Love CC, Chung YG, Varner DD, Westhusin ME, Burghardt RC, Hinrichs K.We investigated the use of direct nuclear injection using the Piezo drill and activation by injection of stallion sperm cytosolic extract for production of cloned equine embryos. When metaphase II horse oocytes were injected with either of two dosages of sperm extract and cultured 20 h, similar activation rates (88% vs. 90%) and cleavage rates (49% vs. 46%) were obtained. The successful reconstruction rate of horse oocytes with horse somatic cell donor nuclei after direct injection using the Piezo drill was 82%. Four dosages of sperm extract (containing 59, 176, 293, or 1375 microg/ml protein)...
Lagneaux D, Huhtinen M, Koskinen E, Palmer E.Equine embryos recovered on Day 6 after ovulation were cooled to +4 degrees C, or frozen with AFP alone or together with glycerol. Twenty embryos (140-200 microm in diameter) were randomly assigned to 6 treatment groups. In the first 3 groups, the embryos were cooled from room temperature to +4 degrees C at a rate of 3 degrees C/min and warmed again at a rate of 32 degrees C/min in a programmable freezer. In the second 3 groups, the embryos were frozen using a standard protocol, stored in liquid nitrogen for 5-7 days and then thawed in a 37 degrees C waterbath. After cooling/warming or freezin...
Leith GS, Ginther OJ.Intrauterine mobility patterns of the embryonic vesicle were characterized on Days 9 to 17 after ovulation in pony mares using real-time ultrasonography (n=5 or 7 mares per day). The location of the vesicle was determined by dividing the uterus into right horn, left horn, and body. Each uterine horn was further divided into three approximately equal portions (cranial third, middle third, caudal third), yielding seven segments (body plus three portions of each horn). Location of the vesicle within the uterus was recorded every five minutes for two consecutive hours (25 location determinations p...
Lear TL, Lundquist J, Zent WW, Fishback WD, Clark A.Repeated early embryonic loss (REEL) represents a considerable economic loss to the horse industry. Mares that experience REEL may be overlooked as potential carriers of a chromosome abnormality. Here we report three different autosomal translocations in Thoroughbred mares presented for chromosome analysis because of REEL. The karyotypes were 64,XX,t(1;21), 64,XX,t(16;22), and 64,XX,t(4;13), respectively. In order to confirm the chromosomes involved in the translocations, to map the breakpoints, and to determine if the translocations were reciprocal, genes surrounding the breakpoints were iden...
Battut I, Colchen S, Fieni F, Tainturier D, Bruyas JF.The purpose of this study was to evaluate the exact age when the equine embryo reaches the uterus. The time of ovulation was determined by hourly ultrasound examinations starting 32 h after an injection of crude equine pituitary gonadotrophin or human chorionic gonadotrophin, or after the first of 4 injections of buserelin. Nonsurgical uterine flushings were carried out 144 h (Day 6), 156 h (Day 6.5) or 168 h (Day 7) after ovulation. Induction of ovulation was attempted in 101 oestrous cycles and 61 of 101 mares (60.4%) ovulated 32-44 h post injection. Sixty embryo collections were performed w...
Esteves CL, Sharma R, Dawson L, Taylor SE, Pearson G, Keen JA, McDonald K, Aurich C, Donadeu FX.Expression of several putative markers of pluripotency (OCT4, SOX2, NANOG, LIN28A, REX1, DNMT3B and TERT) was examined in a range of equine tissues, including early embryos, induced pluripotent stem cells (iPSCs), testis, adipose- and bone marrow-derived mesenchymal stromal cells (MSCs), and keratinocytes. Transcript levels of all markers were highest in embryos and iPSCs and, except for SOX2, were very low or undetectable in keratinocytes. Mean expression levels of all markers were lower in testis than in embryos or iPSCs and, except for DNMT3B, were higher in testis than in MSCs. Expression ...
Campos-Chillòn LF, Suh TK, Barcelo-Fimbres M, Seidel GE, Carnevale EM.The objectives of this study were to: (1) determine an optimal method and stage of development for vitrification of bovine zygotes or early embryos; and (2) use the optimal procedure for bovine embryos to establish equine pregnancies after vitrification and warming of early embryos. Initially, bovine embryos produced by in-vitro fertilization (IVF) were frozen and vitrified in 0.25mL straws with minimal success. A subsequent experiment was done using two vitrification methods and super open pulled straws (OPS) with 1- or 8-cell bovine embryos. In Method 1 (EG-O), embryos were exposed to 1.5M e...
Gaivão MM, Rambags BP, Stout TA.Experimental studies and field surveys suggest that embryonic loss during the first 6 weeks of gestation is a common occurrence in the mare. During the first 2 weeks of development, a number of important cell differentiation events must occur to yield a viable embryo proper containing all three major germ layers (ectoderm, mesoderm, and endoderm). Because formation of the mesoderm and primitive streak are critical to the development of the embryo proper, but have not been described extensively in the horse, we examined tissue development and differentiation in early horse conceptuses using a c...
Goszczynski DE, Tinetti PS, Choi YH, Hinrichs K, Ross PJ.Embryonic genome activation is a critical event in embryo development, in which the transcriptional program of the embryo is initiated. The timing and regulation of this process are species-specific. In vitro embryo production is becoming an important clinical and research tool in the horse; however, very little is known about genome activation in this species. The objective of this work was to identify the timing of genome activation, and the transcriptional networks involved, in in vitro-produced horse embryos. RNA-Seq was performed on oocytes and embryos at eight stages of development (MII,...
Hackett CH, Fortier LA.The field of regenerative medicine research is rapidly expanding. One area of interest to equine researchers is the possibility of isolating or generating pluripotent cells, capable of producing differentiated cell types derived from all 3 primary germ layers. Reports of equine embryonic stem-like (ES) cell isolation can be found in the literature. Other groups are working to produce equine-induced pluripotent stem (iPS) cells. This article summarizes the essential features needed to characterize a cell type as pluripotent, specific challenges in using the horse as a model organism for pluripo...
Bang FB.The titration curve for the virus of Eastern equine encephalomyelitis inoculated into the 10 day old chick embryo shows that the maximum increase in virus content continues until shortly before the generalized destruction of the embryo is apparent. This is followed by a stationary phase. Histological studies of infected embryos fail to demonstrate selective tissue destruction, and titrations show the virus to be distributed throughout the egg, although concentrated in the embryo. The chorioallantoic membrane gradually becomes increasingly resistant with age to both the Eastern and Western viru...
Drews B, Milojevic V, Giller K, Ulbrich SE.During early pregnancy, the secretome of both oviduct and uterus serves as exchange medium for signaling factors between embryo and mother and provides the embryo with nutrients. The preimplantation embryo can utilize the fatty acids (FA) therein via direct incorporation into cell membrane lipid bilayers and for energy production via β-oxidation. The FA concentration and composition of the oviduct (OF) and uterine fluid (UF) might be regulated by ovarian hormones to meet the changing needs of the growing embryo. In our study, we analyzed the FA profile of blood plasma (BP) and reproductive fl...
Angel-Velez D, De Coster T, Azari-Dolatabad N, Fernandez-Montoro A, Benedetti C, Bogado Pascottini O, Woelders H, Van Soom A, Smits K.Equine oocyte vitrification would benefit the growing in vitro embryo production programs, but further optimization of the protocol is necessary to reach clinical efficiency. Therefore, we aimed to perform a direct comparison of non-permeating and permeating cryoprotective agents (CPAs) during the vitrification and warming of equine immature oocytes. In the first experiment, cumulus oocytes complexes (COCs) were vitrified comparing sucrose, trehalose, and galactose in combination with ethylene glycol (EG) and dimethyl sulfoxide (DMSO). In the second experiment, the COCs were vitrified using th...
Rudolf Vegas A, Podico G, Canisso IF, Bollwein H, Almiñana C, Bauersachs S.During the period of maternal recognition of pregnancy (MRP) in the mare, the embryo needs to signal its presence to the endometrium to prevent regression of the corpus luteum and prepare for establishment of pregnancy. This is achieved by mechanical stimuli and release of various signaling molecules by the equine embryo while migrating through the uterus. We hypothesized that embryo's signals induce changes in the endometrial gene expression in a highly cell type-specific manner. A spatiotemporal transcriptomics approach was applied combining laser capture microdissection and low-input-RNA se...
Herrler A, Pell JM, Allen WR, Beier HM, Stewart F.Insulin-like growth factor-I (IGF-I) promotes early embryonic development in several species. In the rabbit, IGF-I binds to the embryonic coats from Day 3 of development onward by a 38-kDa protein that is probably insulin-like growth factor-binding protein 3 (IGFBP3). In the present study, ligand, Western, and Northern blot analyses were used to demonstrate the presence of IGF-I-binding activity, several immunoreactive IGFBP3 proteins, and IGFBP3 mRNA in horse conceptuses with particularly large amounts of immunoreactive IGFBP3 in the conceptus capsule. In addition, immunoprecipitation of radi...
Mortensen CJ, Choi YH, Ing NH, Kraemer DC, Vogelsang MM, Hinrichs K.Heat above homeothermy can be detrimental to embryonic development, and cells may produce heat shock proteins to try to mitigate these effects. The authors examined the developmental competence of equine oocytes after a single heat exposure (42 degrees C, 2 or 4 h) during early or late stages of in vitro maturation. Rates of nuclear maturation, cleavage after intracytoplasmic sperm injection, and advanced embryonic development (morula or blastocyst) were compared to those for unexposed controls. Concentrations of heat shock protein 70 (HSPA1A) mRNA were determined by real-time RT-PCR in result...
Choi YH, Gustafson-Seabury A, Velez IC, Hartman DL, Bliss S, Riera FL, Roldán JE, Chowdhary B, Hinrichs K.The equine embryo possesses a capsule that is considered essential for its survival. We assessed viability after breaching the capsule of early (Day 6) and expanded (Day 7 and 8) equine blastocysts by micromanipulation. The capsule was penetrated using a Piezo drill, and trophoblast biopsy samples were obtained for genetic analysis. Pregnancy rates for Day-6 embryos, which had intact zonae pellucidae at the time of recovery, were 3/3 for those biopsied immediately after recovery and 2/3 for those biopsied after being shipped overnight under warm (∼28 °C) conditions. The pregnancy rates for ...
Hinrichs K.In vitro embryo production is possible in the horse both clinically and for research applications. Oocytes may be collected from excised ovaries post-mortem, or from either immature follicles or stimulated pre-ovulatory follicles in the live mare. In vitro maturation of immature oocytes typically yields approximately 60% mature oocytes. As standard in vitro fertilization is not yet repeatable in the horse, fertilization is performed by intracytoplasmic sperm injection. Embryo culture requires medium with high glucose, at least during blastocyst development, and rates of blastocyst development ...
Canesin HS, Brom-de-Luna JG, Choi YH, Ortiz I, Diaw M, Hinrichs K.We evaluated the meiotic and developmental competence of GV-stage equine oocytes vitrified under different conditions. In a preliminary study, using dimethyl sulfoxide (D), ethylene glycol (EG) and sucrose (S) as cryoprotectants, the maturation rate was higher for cumulus-oocyte complexes (COCs) held overnight before vitrification (37%) than for those vitrified immediately (14%; P < 0.05). Thereafter, all COCs were held overnight before vitrification. In Experiment 1 we compared 1 min (1m) and 4 min (4m) exposure to vitrification and warming solutions; oocytes that subsequently matured wer...
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 ...
Canesin HS, Brom-de-Luna JG, Choi YH, Pereira AM, Macedo GG, Hinrichs K.Previous studies have found low rates of blastocyst development (0-11%) after vitrification of germinal vesicle (GV)-stage equine oocytes. In this study, we systematically evaluated a short (non-equilibrating) system for GV-stage oocyte vitrification. In Exp. 1, we assessed oocyte volume in cumulus-oocyte complexes (COCs) exposed to components of a short protocol, using 2% each of ethylene glycol and propylene glycol in the first solution (VS1); 17.5% of each plus 0.3 M trehalose in the second solution (VS2); and fetal bovine serum as the base medium. Based on the time to oocyte minimum volu...
Heras S, Smits K, De Schauwer C, Van Soom A.Global epigenetic reprogramming is considered to be essential during embryo development to establish totipotency. In the classic model first described in the mouse, the genome-wide DNA demethylation is asymmetric between the paternal and the maternal genome. The paternal genome undergoes ten-eleven translocation (TET)-mediated active DNA demethylation, which is completed before the end of the first cell cycle. Since TET enzymes oxidize 5-methylcytosine to 5-hydroxymethylcytosine, the latter is postulated to be an intermediate stage toward DNA demethylation. The maternal genome, on the other ha...
Angel D, Canesin HS, Brom-de-Luna JG, Morado S, Dalvit G, Gomez D, Posada N, Pascottini OB, Urrego R, Hinrichs K, Velez IC.Effects of meiotic stage and cumulus status on development of equine oocytes after vitrification was evaluated. Immature oocytes with corona radiata (IMM); in vitro-matured oocytes with corona radiata (MAT CR+); and in vitro-matured oocytes denuded of cumulus (MAT CR-) were vitrified using the Cryotech® method. Warming medium was equilibrated either in 5% CO2 or Air. IMM oocytes underwent in vitro maturation after warming. Recovery, survival, and maturation rates, and cleavage and blastocyst rates after ICSI, were evaluated. Recovery was higher for oocytes warmed in CO2- than Air-equilibrated...
Enders AC, Liu IK.The equine blastocyst becomes fixed in position in the uterus on approximately Day 16 of gestation, but allantochorionic villi are not formed until about Day 50. The purpose of this study was to examine evidence that the blastocyst is orientated during this time period, and to determine what morphological features might assist retention of the position of the blastocyst within the uterus. Implantation sites were collected on Days 10-42 of gestation, and the reproductive tracts perfused with fixative for light and electron microscopic examination. The conceptus is found at the bend of a uterine...
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...
Morel MC, Newcombe JR, Swindlehurst JC.Numerous and conflicting reports exist regarding factors that may effect mare reproductive performance, in particular multiple ovulation (MO) and its consequences. Sequential ultrasonic examination was used to monitor 3075 ovulations in 1581 mainly Thoroughbred mares to ascertain: whether increasing age is associated with an increase in MO; whether this is counteracted by an increase in embryo mortality (EM) prior to Day 13; and whether this embryonic loss may be associated with small-for-age embryonic vesicles (Days 13/14). Overall ovulation rate was 1.31, MO occurring in 29.3% of cycles. MO ...
