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.
Merlo B, Iacono E, Bucci D, Spinaci M, Galeati G, Mari G.In vitro embryo production in the horse is still not as efficient as in other species. Oxidative stress negatively affects oocyte and embryo culture. To attenuate/minimize the oxidative stress, antioxidants such as low-molecular thiol compounds can be added to culture media. Beta-mercaptoethanol (BME) has been shown to improve maturation and embryo development in different species. The aim of this study was to investigate whether the addition to maturation medium of BME at common (0.1 mM) and high (0.7 mM) concentration could improve oocyte maturation also in the horse. Equine oocytes recove...
Leemans B, Gadella BM, Stout TA, De Schauwer C, Nelis H, Hoogewijs M, Van Soom A.In contrast to man and many other mammalian species, conventional in vitro fertilization (IVF) with horse gametes is not reliably successful. The apparent inability of stallion spermatozoa to penetrate the zona pellucida in vitro is most likely due to incomplete activation of spermatozoa (capacitation) because of inadequate capacitating or fertilizing media. In vivo, the oviduct and its secretions provide a microenvironment that does reliably support and regulate interaction between the gametes. This review focuses on equine sperm-oviduct interaction. Equine sperm-oviduct binding appears to be...
Choi YH, Hinrichs K.There is a clinical demand for cryopreservation of both in vivo-recovered and in vitro-produced (IVP) equine embryos. We previously reported successful vitrification of expanded equine blastocysts in fine-diameter microloader pipette tips (MPTs) after blastocoel collapse, in a research setting. Here, we report the results of clinical application of the MPT vitrification technique for both in vivo-recovered and IVP blastocysts. In vivo-recovered blastocysts were obtained by referring veterinarians on Days 6 to 8 after ovulation, and shipped 1 to 10 hours to the laboratory before vitrificat...
Choi YH, Gibbons JR, Canesin HS, Hinrichs K.Prospective studies were conducted to help define procedural factors affecting in vitro embryo production via intracytoplasmic sperm injection (ICSI) of equine oocytes. In experiment 1, use of 10% fetal bovine serum as a protein source in embryo culture medium resulted in a higher blastocyst rate than did use of a combination of 3% fetal bovine serum, 3% equine preovulatory follicular fluid, and 4% human serum substitute (37% vs. 15%, respectively, P < 0.05). In experiment 2, the effect of zinc supplementation (0, 0.5, 1, or 1.5 μg/mL) during IVM was examined. There were no significant di...
Gibb Z, Grupen CG, Maxwell WM, Morris LH.The fertility of sex-sorted, cryopreserved stallion sperm must be improved for the sex-sorting technology to be applied commercially. Objective: To optimise the conditions used to liquid store stallion sperm prior to sex-sorting and assess the fertility of sperm following sex-sorting and cryopreservation. Methods: Both in vitro experiment and randomised controlled trial in healthy, client-owned mares. Methods: Stallion ejaculates (n = 9) were diluted in either a skimmed milk (KMT) or BSA (I-BSA) based media to 25 × 106 sperm/ml directly (+SP25) or washed to remove seminal plasma and diluted t...
Dini P, Bogado Pascottini O, Ducheyne K, Hostens M, Daels P.In the present study, we examined the effect of holding equine oocytes in Syngro embryo holding medium (EHM) overnight at either 4 °C, 17 °C, or 22 °C to 25 °C, on the time to maturation and developmental competence. We also examined the effect of placing denuded oocyte without extruded polar body back in maturation condition on subsequent maturation rate. In experiment 1, cumulus-oocyte complexes (COCs) were recovered postmortem and placed in EHM at 22 °C to 25 °C for 18 to 20 hours (OH) or placed directly in maturation (DM). The maturation rate was assessed after 22, 24, or 28 hou...
Smits K, Nelis H, Van Steendam K, Govaere J, Roels K, Ververs C, Leemans B, Wydooghe E, Deforce D, Van Soom A.The equine oviduct plays a pivotal role in providing the optimal microenvironment for early embryonic development, but little is known about the protein composition of the oviducal fluid in the horse. The aim of the present study was to provide a large-scale identification of proteins in equine oviducal fluid and to determine the effects of ovulation and pregnancy. Four days after ovulation, the oviducts ipsilateral and contralateral to the ovulation side were collected from five pregnant and five non-pregnant mares. Identification and relative quantification of proteins in the oviducal fluid ...
Stout TA.The first month of equine pregnancy covers a period of rapid growth and development, during which the single-cell zygote metamorphoses into an embryo with a functional circulation and precursors of many important organs, enclosed within extraembryonic membranes responsible for nutrient uptake and gaseous exchange. After exiting the oviduct, the conceptus must influence uterine physiology to ensure adequate nutrition and preparation for implantation, while continued development results in the chorioallantois superseding the yolk sac as the primary interface for maternal interaction and exchange...
Klein C.Relaxin is a peptide hormone best known for its action during the latter half of pregnancy, in particular for its softening effect on pelvic ligaments that aids in preparation of the birth canal for the impending delivery of the fetus. The source of relaxin during early pregnancy varies across species, with the CL being the main source in a number of species. The main source of relaxin during late equine pregnancy is the placenta. In mares with impaired placental function, circulating relaxin levels decline before abortion. During early pregnancy, relaxin promotes endometrial angiogenesis thro...
Hinrichs K.A research study is a product of not only a question and its pursuit but also the people, places, and facilities available at the time. My work in equine assisted reproduction has progressed from embryo transfer to oocyte maturation, oocyte transfer, intracytoplasmic sperm injection, embryo biopsy, embryo vitrification, and cloning, as a result of collaborations with an array of remarkable people. This is a summary of some of the stories behind the studies.
Griffith OW, Brandley MC, Whittington CM, Belov K, Thompson MB.In oviparous amniotes (reptiles, birds, and mammals) the chorioallantoic membrane (CAM) lines the inside of the egg and acts as the living point of contact between the embryo and the outside world. In livebearing (viviparous) amniotes, communication during embryonic development occurs across placental tissues, which form between the uterine tissue of the mother and the CAM of the embryo. In both oviparous and viviparous taxa, the CAM is at the interface of the embryo and the external environment and can transfer signals from there to the embryo proper. To understand the evolution of placental ...
Gambini A, De Stéfano A, Jarazo J, Buemo C, Karlanian F, Salamone DF.The low efficiency of interspecies somatic cell nuclear transfer (iSCNT) makes it necessary to investigate new strategies to improve embryonic developmental competence. Embryo aggregation has been successfully applied to improve cloning efficiency in mammals, but it remains unclear whether it could also be beneficial for iSCNT. In this study, we first compared the effect of embryo aggregation over in vitro development and blastocyst quality of porcine, bovine, and feline zona-free (ZF) parthenogenetic (PA) embryos to test the effects of embryo aggregation on species that were later used as en...
Klein C."Maternal recognition of pregnancy" (MRP) is commonly used to describe the ongoing embryo-maternal communication during early pregnancy that culminates in prevention of luteolysis and ensures ongoing progestin support. The conceptus-derived pregnancy recognition signal has not yet been identified in the mare. Although equine conceptuses produce substantial amounts of estrogens, there is a lack of evidence that estrogens are the pregnancy recognition signal in mares. Conceptus mobility is integral to MRP and is driven by conceptus-derived prostaglandin production. Cessation of conceptus mobilit...
Barreto Rda S, Rodrigues MN, Carvalho RC, De Oliveira E Silva FM, Rigoglio NN, Jacob JC, Gastal EL, Miglino MA.Musculoskeletal system development involves heterotypical inductive interactions between tendons, muscles, and cartilage and knowledge on organogenesis is required for clarification of its function. The aim of this study was to describe the organogenesis of horse musculoskeletal system between 21 and 105 days of gestation, using detailed macroscopic and histological analyses focusing on essential developmental steps. At day 21 of gestation the skin was translucid, but epithelial condensation and fibrocartilaginous tissues were observed on day 25 of pregnancy. Smooth muscle was seen in lymphat...
Hinrichs K, Choi YH.Embryo cryopreservation presents an essential method for banking of valuable genetics. However, in equine species the cryopreservation of embryos is complicated by three interacting factors: (1) the late entry of the embryo into the uterus (~6 days after ovulation); (2) the rapid expansion of the blastocyst; and (3) the formation of the equine embryonic capsule, a glycoprotein membrane that forms between the embryo and zona. Efforts to freeze or vitrify equine expanded blastocysts were initially met with little success. In addition, it was thought that breaching the capsule led to loss of embr...
Raudsepp T, Chowdhary BP.The association between chromosomal abnormalities and reduced fertility in domestic animals is well recorded and has been studied for decades. Chromosome aberrations directly affect meiosis, gametogenesis, and the viability of zygotes and embryos. In some instances, balanced structural rearrangements can be transmitted, causing fertility problems in subsequent generations. Here, we aim to give a comprehensive overview of the current status and future prospects of clinical cytogenetics of animal reproduction by focusing on the advances in molecular cytogenetics during the genomics era. We descr...
Smits K, De Coninck DI, Van Nieuwerburgh F, Govaere J, Van Poucke M, Peelman L, Deforce D, Van Soom A.Although the equine oviduct clearly affects early embryo development and the selective transport of equine embryos through the oviduct indicates a reciprocal interaction, the influence of the embryo on gene expression in the oviduct remains to be determined in the horse. The aim of this study was to examine this by means of RNA sequencing. Four days after ovulation, epithelial cells ipsilateral and contralateral to the ovulation side from five cyclic and five pregnant mares were collected from the oviduct. RNA was extracted, samples were sequenced, and data analysis was performed to determine ...
Choi YH, Velez IC, Macías-García B, Riera FL, Ballard CS, Hinrichs K.Equine intracytoplasmic sperm injection (ICSI) is being used clinically for foal production, but little information is available on factors affecting the efficiency of this procedure. We examined factors that may influence blastocyst development when ICSI is performed clinically, i.e., on oocytes recovered from live mares by transvaginal ultrasound-guided follicle aspiration (TVA), using sperm from the stallion of the client's choice. In a clinical setting, there may be a delay from the time of TVA to isolation of oocytes from the aspirated fluid. In a preliminary study, oocytes from fluid hel...
Franciosi F, Goudet G, Tessaro I, Papillier P, Dalbies-Tran R, Reigner F, Deleuze S, Douet C, Miclea I, Lodde V, Luciano AM.Implantation failure and genetic developmental disabilities in mammals are caused by errors in chromosome segregation originating mainly in the oocyte during meiosis I. Some conditions, like maternal ageing or in vitro maturation (IVM), increase the incidence of oocyte aneuploidy. Here oocytes from adult mares were used to investigate oocyte maturation in a monovulatory species. Experiments were conducted to compare: (1) the incidence of aneuploidy, (2) the morphology of the spindle, (3) the acetylation of lysine 16 on histone H4 (H4K16) and (4) the relative amount of histone acetyltransferase...
Guignot F, Reigner F, Perreau C, Tartarin P, Babilliot JM, Bed'hom B, Vidament M, Mermillod P, Duchamp G.Preimplantation genetic diagnosis and embryo cryopreservation are important tools to improve genetic management in equine species with marked consequences on the economic value, health, biodiversity, and preservation of the animals. This study aimed to develop a biopsy method at the blastocyst stage that provides viable genotyped cryopreserved Welsh pony embryos. Embryos were collected at d 6.75 to 7 after ovulation. Biopsies were performed with either a microblade or a micropipette. After biopsy, embryos were cryopreserved. The survival rate of biopsied embryos was evaluated on fresh and cryo...
Aurich C, Budik S.Early equine pregnancy shares many features with that of more intensively assessed domestic animals species, but there are also characteristic differences. Some of those are poorly understood. Descent of the equine conceptus into the uterine lumen occurs at day 5 to 6 after ovulation but is only possible when the embryo secretes prostaglandin E2. Although maintenance of equine pregnancy probably involves secretion of a conceptus derived anti-luteolytic factor, this agent has not been identified. Rapid growth, conceptus mobility and presence of an acellular capsule at the time of maternal recog...
Ezashi T, Yuan Y, Roberts RM.This review deals with the latest advances in the study of embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) from domesticated species, with a focus on pigs, cattle, sheep, goats, horses, cats, and dogs. Whereas the derivation of fully pluripotent ESC from these species has proved slow, reprogramming of somatic cells to iPSC has been more straightforward. However, most of these iPSC depend on the continued expression of the introduced transgenes, a major drawback to their utility. The persistent failure in generating ESC and the dependency of iPSC on ectopic genes probably s...
Diaz F, Bondiolli K, Paccamonti D, Gentry GT.Pregnancy rates after cryopreservation of large equine blastocyst stage embryos have remained lower than other domesticated livestock species. It is generally accepted that the embryonic capsule is the primary barrier to cryoprotectant entry into the embryo proper and techniques need to be developed to circumvent this obstacle. Therefore, the objective of this study was to develop an efficient Day 8 equine embryo cryopreservation protocol through blastocyst micromanipulation and vitrification. Grade 1 and 2 embryos recovered from mares (n = 15) 8 days after ovulation were used in these experim...
Ghosh S, Das PJ, Avila F, Thwaits BK, Chowdhary BP, Raudsepp T.Balanced autosomal translocations are a known cause for repeated early embryonic loss (REEL) in horses. In most cases, carriers of such translocations are phenotypically normal, but the chromosomal aberration negatively affects gametogenesis giving rise to both genetically balanced and unbalanced gametes. The latter, if involved in fertilization, result in REEL, whereas gametes with the balanced form of translocation will pass the defect into next generation. Therefore, in order to reduce the incidence of REEL, identification of translocation carriers is critical. Here, we report about a pheno...
González-Fernández L, Macedo S, Lopes JS, Rocha A, Macías-García B.Equine in vitro fertilization (IVF) is still inconsistent. In the present work, we studied how modified Whitten's (MW) medium and Tissue Culture Medium 199 (TCM) added with Foetal Bovine Serum (FBS; 10% v/v) or Bovine Serum Albumin (BSA; 7 mg/ml) affected equine gametes to subsequently run IVF trials. Compact (Cp) and expanded (Ex) cumuli equine oocytes were matured and placed in TCM or MW supplemented with BSA or FBS for 18-20 h (no sperm added). In Ex oocytes, TCM-199 added with FBS or BSA resulted in higher metaphase II (MII) rates (75.7% and 62.7%, respectively) than MW added with BSA (54%...
Geisert RD.Establishment and maintenance of pregnancy in a number of mammalian species depends upon a tightly regulated interaction between the semiallogeneic conceptus and the maternal uterine endometrium.The term "Maternal Recognition of Pregnancy" is attributed to Roger V. Short's paper titled "Implantation and the Maternal Recognition of Pregnancy" which was published in proceedings from the 1969 Symposium on Foetal Autonomy.Professor Short's landmark paper stimulated increased interest in elucidating how the conceptus signals its presence to assure maintenance of the corpus luteum beyond the normal ...
Klein C.Few, if any, biological processes are as diverse among domestic species as establishment of early pregnancy, in particular maternal recognition of pregnancy. Following fertilization and initial development in the mare oviduct, selective transport of the embryo through the uterotubal junction driven by embryo-derived PGE2 occurs. Upon arrival in the uterus, an acellular glycoprotein capsule is formed that covers the embryo, blastocyst, and conceptus (embryo and associated extraembryonic membranes) between the second and third weeks of pregnancy. Between Days 9 and 15/16 of pregnancy, the concep...
Crișan MI, Damian A, Morar I, Páll E, Peștean C, Groza IȘ.The ability to choose the sex of the offspring is of upmost economic importance for horse breeders. Unlike other species, horses present several reproductive peculiarities that interfere with assisted reproductive technologies used in other large animals (such as bovine) and make them difficult to apply. Thus, there is a great interest to determine the sex of the offspring as soon as possible. This has led to the development of several technologies to serve this purpose, which can be classified into two categories. One is equine embryo sexing by either non-invasive biotechnological methods, su...
Roberts MA, London K, Campos-Chillón LF, Altermatt JL.An equine embryo produced by intracytoplasmic sperm injection (ICSI) was trans-cervically transferred to a recipient mare and pregnancy was confirmed via ultrasound examination on days 11, 12 and 15. On days 20 and 22, a single embryonic proper with a heartbeat was observed. On day 29, two embryos proper appeared during ultrasound examination, each possessing a heartbeat. Subsequent examinations on days 35 and 39 revealed continued viability and development of both embryos proper. On day 49, demise of both fetuses was present. Although no DNA analysis or post-partum examinations were performed...
Love CC, Noble JK, Standridge SA, Bearden CT, Blanchard TL, Varner DD, Cavinder CA.The relationship between the quality of cool-shipped stallion semen and fertility has not been adequately described. This study evaluated sperm quality of cool-shipped semen from 459 ejaculates (N = 130 stallions) that were used for insemination of 196 embryo donor mares (n = 496 estrous cycles). Embryo recovery rate (ERR; %) increased, as all sperm measures (e.g., motility, viability, DNA quality, morphology, concentration, and total number) increased. Threshold values are reported for each sperm quality measure (e.g., total sperm motility ≥ 65%) that separate two ERR groups (e.g., av...
Panzani D, Rota A, Pacini M, Vannozzi I, Camillo F.One year old fillies are able to conceive but, usually, not to give birth to a living foal. Although embryo transfer allows the production of foals from mature mares with repeated pregnancy losses, no reports are available on the use of one year old fillies as embryo donors. To evaluate this possibility, eleven 12-16 months old Haflinger mares were inseminated with fresh semen and subjected to embryo recovery. Some of the recovered embryos were non-surgically transferred into synchronized mature recipients. Pregnancies were terminated using PGF2alpha at day 25. Fillies' embryo recovery rate an...
Lodde V, Colleoni S, Tessaro I, Corbani D, Lazzari G, Luciano AM, Galli C, Franciosi F.Several studies report that a two-step culture where mammalian oocytes are first kept under meiosis-arresting conditions (prematuration) followed by IVM is beneficial to embryo development. The most promising results were obtained by stratifying the oocyte population using morphological criteria and allocating them to different culture conditions to best meet their metabolic needs. In this study, horse oocytes were characterised to identify subpopulations that may benefit from prematuration. We investigated gap-junction (GJ) coupling, large-scale chromatin configuration and meiotic competence ...
Ginther OJ.Recent findings on the development and natural outcome of twins from Day 17 (immediately after fixation) to Day 40 are reviewed. Incidence of embryo reduction was increased significantly when the vesicles became fixed unilaterally, rather than bilaterally, and when the vesicles were unequal in diameter. Of 68 mares with twins on the day of fixation, post fixation embryo reduction occurred in 41 (60 per cent). The incidence of reduction was 41 of 48 (85 per cent) following unilateral fixation; reduction occurred in all of 22 mares with vesicles of dissimilar size (4 mm or more difference in dia...
Choi YH, Love CC, Varner DD, Hinrichs K.This study was conducted to evaluate the effects of thawing, division into aliquots and refreezing on fertilizing capacity (ability to support embryo development after intracytoplasmic sperm injection; ICSI) of frozen stallion semen. Frozen semen from a fertile stallion was thawed, diluted 1:100 with freezing extender, and refrozen (2F treatment). Control semen was frozen only once. In vitro matured equine oocytes were injected with: (1) motile control spermatozoa; (2) motile 2F spermatozoa; (3) non-motile 2F spermatozoa; or (4) non-motile 2F spermatozoa, followed by injection of sperm extract...
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...
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...
Li X, Morris LH, Allen WR.The effects of four reagents on the activation and subsequent fertilization of equine oocytes, and the development of these after intracytoplasmic sperm injection, were investigated. Cumulus-oocyte complexes collected from equine ovaries obtained from an abattoir were matured in vitro for 40-44 h in TCM199 medium before being injected, when in metaphase II, with an immobilized stallion spermatozoon. The cumulus-oocyte complexes were then subjected to one of five activation treatments: (a) 10 micromol ionomycin l(-1) for 10 min; (b) 7% (v/v) ethanol for 10 min; (c) 100 micromol thimerosal l(-1)...
Cuervo-Arango J, Aguilar J, Newcombe JR.Embryonic vesicle growth in the mare is easily monitored by ultrasound. Apart from pregnancy diagnosis, assessment of the embryonic vesicle in practice is also useful to evaluate its viability. Although subject to individual variation, embryo growth rate follows a constant pattern in the early stages of development in relation to embryonic age. Previous studies have shown a significant effect of some factors routinely used in practice, such as post-ovulation insemination and embryo transfer, on embryonic growth and the time in which the vesicle is first detected. This study attempts to confirm...
Leisinger CA, Medina V, Markle ML, Paccamonti DL, Pinto CRF.A novel in vivo model utilizing serial administrations of PGF2α to induce aluteal cycles in the mare was used to evaluate the effects of progesterone-deprivation on the morphology of in vivo preimplantation embryos. We hypothesized that equine embryos produced during induced aluteal cycles (AL) would be developmentally affected, characterized by earlier embryo stage at collection, smaller embryo diameter, and lower quality grade, compared with those collected on the same day post-ovulation from control cycles during diestrus (high progesterone; > 4 ng/mL). Seven cyclic mares with a median ...
Zhao G, Wu K, Cui L, Zhao L, Liu Y, Tan X, Zhou H.Three media were evaluated for their ability to support in vitro maturation of donkey (Equus asinus) oocytes and their development after parthenogenetic activation. The basal medium for Medium 1 (M1) and Medium 2 (M2) was M199 and DMEM/F12 respectively, whereas, Medium 3 (M3) consisted of equal parts (v/v) of M199 and DMEM/F12. All three media were supplemented with 10% (v/v) fetal calf serum, 0.01 units/mL porcine FSH, 0.01 units/mL equine LH, 200 ng/mL insulin-like growth factor 1(IGF-I), 10 μl/mL insulin-transferrin-selenium (ITS), 0.1 mg/mL taurine, 0.1 mg/mL L-cysteine, 0.05 mg/mL L-glut...
Goncalves GR, Morotti F, Colombo AHB, Bonato DV, Bizarro-Silva C, Rosa CO, Cavalieri FLB, Seneda MM.Age and antral follicle count (AFC) are related to fertility in cattle, but this information remains limited in mares. Methods: To verify the influence of age and AFC on the reproductive characteristics of mares, 15 Quarter Horse donors, with 5-15 antral follicles, ranging from three to 17 years old, healthy and in good nutritional status, were divided into groups with low AFC (≤9 follicles) or high AFC (≥10 follicles) and mares considered young (≤9 years) or old (≥10 years). Mares were submitted to ultrasonography to determine the dominant follicle diameter, follicular growth rate, de...
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...
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,...
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...
Colchen S, Battut I, Fiéni F, Tainturier D, Siliart B, Bruyas JF.Equine embryos were collected at exactly 156 +/- 0.5 (n=8) and 168 +/- 0.5 h (n=11) after ovulation. The embryos were fixed in glutaraldehyde, sectioned serially and observed using light microscopy. In the 156 h group, all embryos were early blastocysts except for one, which was a morula. The morula and one early blastocyst had no capsule. The capsules of the other embryos were thin. The mean +/- SD total number of cells was 275 +/- 105 (range 117-417). The mean +/- SD proportions of mitotic and pycnotic cells were 2.5 +/- 1.2 and 1.1 +/- 1.8%, respectively, and there were no differences betwe...
Hinrichs K, DiGiorgio LM.A technique was developed in which immature horse oocytes, obtained from slaughterhouse specimens, were transferred to the pre-ovulatory follicle of a mare in vivo, with resulting oocyte maturation, ovulation, fertilization and embryo development. Oocytes were collected from all follicles greater than 3 mm, and were classified as immature, maturing, expanded or denuded. The transfers were performed in the standing, tranquilized mare. The ovary containing the pre-ovulatory follicle was grasped per rectum. A trochar and cannula were placed through the abdominal wall in the flank area, ipsilatera...
Diaz F, Bondiolli K, Paccamonti D, Gentry GT.Pregnancy rates after cryopreservation of large equine blastocyst stage embryos have remained lower than other domesticated livestock species. It is generally accepted that the embryonic capsule is the primary barrier to cryoprotectant entry into the embryo proper and techniques need to be developed to circumvent this obstacle. Therefore, the objective of this study was to develop an efficient Day 8 equine embryo cryopreservation protocol through blastocyst micromanipulation and vitrification. Grade 1 and 2 embryos recovered from mares (n = 15) 8 days after ovulation were used in these experim...
Roasa LM, Choi YH, Love CC, Romo S, Varner DD, Hinrichs K.In vitro fertilization (IVF) was performed on in vitro-matured equine oocytes in three experiments. Frozen-thawed sperm were prepared using swim-up separation and heparin treatment. In Experiment 1, fertilization was achieved with sperm from only one frozen ejaculate of four obtained from the same stallion. Within this ejaculate, fertilization rates were higher with fresh media, as compared to media held for 6-8 days before use (39.6% versus 7.3%, respectively; P<0.001). The type of bovine serum albumin used affected fertilization rates (4% versus 39.6%; P<0.001). To determine if IVF rates wer...
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...
Wilson JM, Caceci T, Potter GD, Kraemer DC.Embryos were recovered non-surgically at about Day 6 after ovulation from 15 Quarter horse-type mares and were evaluated for morphological changes which may occur because of exposure to the cryoprotectant and/or cryopreservation. Electron microscopy was used to elucidate the fine structure of intracellular organelles which, if damaged, could cause cellular death. The horse embryo does not totally re-expand in the 10% glycerol freezing medium, nor will it completely re-expand in the isotonic holding medium following glycerol removal whether or not the embryo has been frozen. Embryos in this stu...
The present experiment aimed at determining whether the timing of the maternal recognition of pregnancy (MRP) was specific to individual mares by determining when luteostasis, a failure to return to oestrus, reliably occurred in individuals following embryo reduction. Singleton (n = 150) and synchronous twin pregnancies (n = 9) were reduced in 10 individuals (5-29 reductions/mare) at pre-determined time points within days 10 (n = 20), 11 (n = 65), 12 (n = 47), 13 (n = 12) or 14 (n = 15) of pregnancy. Prior to embryo reduction, the vesicle diameter was measured in 71% (106/150) of the singleton...
Diaw M, Salgado RM, Canesin HS, Gridley N, Hinrichs K.Intracytoplasmic sperm injection (ICSI) is an important tool for equine embryo production in both clinical and research settings. In clinical ICSI programs, immature equine cumulus-oocyte complexes (COCs) are often collected at the mare's location and shipped to the ICSI laboratory. To simplify shipment and aid scheduling of subsequent procedures, COCs can be held overnight at room temperature (∼22 °C) before placement into maturation culture, with no detrimental effect on meiotic or developmental competence. A recent study indicated that it might be possible to hold COCs overnight at col...
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...
Ababneh MM, Troedsson MH, Michelson JR, Seguin BE.Equine conceptuses are thought to produce antiluteolytic factors that inhibit endometrial PGF2alpha and, thus, prevent luteolysis in pregnant mares. The aim of the present study was to characterize partially the chemical nature of the prostaglandin inhibitory factor (PIF) produced by equine conceptuses in vitro. Embryos were collected from pregnant mares 13 +/- 0.5 days after ovulation and were cultured for 24 h. Harvested equine conceptus conditioned media (CCM) were assayed for antiluteolytic activity by determining the inhibition of endometrial PGF2alpha synthesis in vitro. Significant anti...
Vanderwall DK, Woods GL, Aston KI, Bunch TD, Li G, Meerdo LN, White KL.The objectives of the present study were to: (1) clone horses using adult cumulus cells; and (2) determine whether the cumulus cell donor affected the outcome. In vivo-matured cumulus-oocyte complexes were obtained using transvaginal ultrasound-guided follicle aspiration; oocytes were used as cytoplasts, whereas cumulus cells (from one of three different mares) were used as donor cells. Immediately following nuclear transfer and activation procedures, cloned embryos were transferred surgically to the oviduct of recipient mares (n = 2-5 embryos per recipient) that had ovulated within 24 h prior...
Ginther OJ.The equine embryonic vesicle has been shown to be highly mobile prior to day 15, moving from one horn to the other many times per day. In Experiment 1, intrauterine mobility patterns of the vesicle were compared between barren and postpartum mares on days 12, 13, or 14, using an ultrasound instrument. Location of the vesicle (left horn, right horn, body) was determined every five minutes during six two-hour trials in each group. Averaged over all trials, the vesicle moved from one horn to the other 1.1 times per two-hour trial. There were no significant differences between barren and postpartu...
Brinsko SP, Ball BA, Miller PG, Thomas PG, Ellington JE.This study was designed to investigate the development of day 2 embryos obtained from young and aged mares, co-cultured with oviductal epithelial cells obtained from mares in each age group in a 2 x 2 crossover design. Young, fertile mares (n = 19; 2-7 years of age) and aged, subfertile, mares (n = 16; 17-24 years of age) were used as embryo and oviductal epithelial cell donors. Embryos (n = 37) were collected from the oviducts 2 days after ovulation and were paired (embryos obtained from young mares with embryos obtained from aged mares) so that eight pairs were co-cultured with young mare ov...
Catandi GD, Obeidat YM, Broeckling CD, Chen TW, Chicco AJ, Carnevale EM.Advanced maternal age is associated with a decline in fertility and oocyte quality. We used novel metabolic microsensors to assess effects of mare age on single oocyte and embryo metabolic function, which has not yet been similarly investigated in mammalian species. We hypothesized that equine maternal aging affects the metabolic function of oocytes and in vitro-produced early embryos, oocyte mitochondrial DNA (mtDNA) copy number, and relative abundance of metabolites involved in energy metabolism in oocytes and cumulus cells. Samples were collected from preovulatory follicles from young (≤1...
Gregg K, Polejaeva I.Prevention and regulation of equine infectious anemia virus (EIAV) disease transmission solely depend on identification, isolation, and elimination of infected animals because of lack of an effective vaccine. Embryo production via the somatic cell nuclear transfer (SCNT) technology uses oocytes collected mainly from untested animals, which creates a potential risk of EIAV transmission through infected embryos. The current review examines the risk of EIAV disease transmission through SCNT embryo production and transfer. Equine infectious anemia virus is a lentivirus from the family Retroviridae...
Choi SJ, Anderson GB, Roser JF.In vitro production of free estrogens and estrogen conjugates by intact Day 12.5, 13.5 and 14.5 equine embryos was measured at 2-h intervals over a 24-h culture period. Production of free estrogens was higher for Day 14.5 than Day 12.5 embryos. Differences in production of conjugated estrogens were not significant, but a trend toward increased production with increased age of embryo was apparent. No trend toward increased free and conjugated estrogen production per cell was observed with age. Embryo diameter and number of cells increased with age but varied considerably within groups. The amou...
