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.
Heras S, Smits K, Leemans B, Van Soom A.Hoechst staining has traditionally been used to evaluate fertilization and parental origin of pronuclei. However, prevalence of parthenogenetic activation cannot be distinguished accurately by this protocol, and variation of relative pronuclear size and position makes it impossible to determine parental origin. We demonstrate that in equine zygotes, the epigenetic modification histone 3 lysine 9 trimethylation (H3K9me3) shows an asymmetric pattern between maternal and paternal pronuclei. H3K9me3 immunostaining appears to be a robust technique to identify the parent of origin of equine pronucle...
Hendriks WK, Roelen BA, Colenbrander B, Stout TA.Equine embryos are cryopreserved by slow-freezing or vitrification. While small embryos (<300 μm) survive cryopreservation reasonably well, larger embryos do not. It is not clear if slow-freezing or vitrification is less damaging to horse embryos. Objective: To compare the type and extent of cellular damage suffered by small and large embryos during cryopreservation by slow-freezing vs. vitrification. Methods: Sixty-three Day 6.5-7 embryos were subdivided by size and assigned to one of 5 treatments: control, exposure to slow-freezing or vitrification cryoprotectants (CPs), and cryopreser...
Herrera C, Morikawa MI, Castex CB, Pinto MR, Ortega N, Fanti T, Garaguso R, Franco MJ, Castañares M, Castañeira C, Losinno L, Miragaya MH, Mutto AA.Normal mammalian early embryonic development involves apoptosis of blastomeres as a remodeling process during differentiation, starting at the blastocyst stage. Genomic DNA has been recently detected in the blastocele fluid of human embryos and has been amplified by real-time polymerase chain reaction (PCR) to diagnose the sex of in vitro-produced human embryos. This new approach varies from conventional preimplantation genetic diagnosis in that no cells are extracted from the embryo and only the blastocele fluid is aspirated and used as a DNA sample for diagnosis. In the present work, we inv...
Martino NA, Dell'Aquila ME, Filioli Uranio M, Rutigliano L, Nicassio M, Lacalandra GM, Hinrichs K.Evaluation of mitochondrial function offers an alternative to evaluate embryo development for assessment of oocyte viability, but little information is available on the relationship between mitochondrial and chromatin status in equine oocytes. We evaluated these parameters in immature equine oocytes either fixed immediately (IMM) or held overnight in an Earle's/Hank's' M199-based medium in the absence of meiotic inhibitors (EH treatment), and in mature oocytes. We hypothesized that EH holding may affect mitochondrial function and that holding temperature may affect the efficiency of meiotic su...
Gil L, Olaciregui M, Luño V, Malo C, González N, Martínez F.In recent years, there has been an increased interest in new preservation techniques that facilitate sperm storage and distribution, with freeze-drying (FD) having been proposed as an alternative method for sperm preservation and maintenance of genetic resources in different animal species. FD is a method in which frozen material is dried by sublimation of ice, thereby involving a direct transition from a solid (ice) to a vapour (gas) phase. One of the main advantages of FD is that nitrogen and dry ice are no longer required for the storage and shipment of frozen sperm, which can be stored at ...
Jenner F, van Osch GJ, Weninger W, Geyer S, Stout T, van Weeren R, Brama P.Articular cartilage regeneration is the focus and goal of considerable research effort. Since articular chondrocytes descend from a distinct cohort of progenitor cells located in embryonic nascent joints (interzones), establishing the timing of equine interzone formation is an essential first step towards understanding equine joint and articular cartilage development. Objective: To establish the time frame during which the equine femorotibial interzone forms. Methods: Descriptive anatomical study. Methods: Equine embryos were harvested at 37 (E37), 40, 42, 45, 50 and 65 days' gestation. The fe...
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 ...
Morrell JM, Richter J, Martinsson G, Stuhtmann G, Hoogewijs M, Roels K, Dalin AM.A successful outcome after artificial insemination with cooled semen is dependent on many factors, the sperm quality of the ejaculate being one. Previous studies have shown that spermatozoa with good motility, normal morphology, and good chromatin integrity can be selected by means of colloid centrifugation, particularly single layer centrifugation (SLC) using species-specific colloids. The purpose of the present study was to conduct an insemination trial with spermatozoa from "normal" ejaculates, i.e., from stallions with no known fertility problem, to determine whether the improvements in sp...
Choi YH, Ritthaler J, Hinrichs K.Cloned animals possess mitochondria derived from the host ooplast, which typically differ genetically from those of the donor. This is of special concern to horse breeders, as maternal lines are prized and athletic performance is a key factor in genetic value. To evaluate the feasibility of producing mitochondrial-identical cloned foals, we collected oocytes from immature follicles of two mares, BL and SM, maternally related to the donor stallion. In vitro matured, enucleated oocytes were treated with roscovitine-synchronized donor cells and blastocysts were transferred transcervically to reci...
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...
Bostedt H, Sieme H, Bartmann CP, Handler J, Sobiraj A, Wehrend A.This review describes stepwise the recto-manual and transrectal ultrasonographic evidence of early pregnancy detection in the horse. The morphological and physiological conditions in the individual phases of early pregnancy are presented in correlation to the potential clinical findings. The importance of embryonic and early foetal losses is presented. Communication and documentation of findings are also addressed. The final section is devoted to the evaluation of the examination effort. In this regard, it is emphasized that the gynaecological examination for the evaluation of the pregnancy st...
Sessions-Bresnahan DR, Graham JK, Carnevale EM.IVF in horses is rarely successful. One reason for this could be the failure of sperm to fully capacitate or exhibit hyperactive motility. We hypothesized that the zona pellucida (ZP) of equine oocytes prevents fertilization in vitro, and bypassing the ZP would increase fertilization rates. Limited availability of equine oocytes for research has necessitated the use of heterologous oocyte binding assays using bovine oocytes. We sought to validate an assay using bovine oocytes and equine sperm and then to demonstrate that bypassing the ZP using perivitelline sperm injections (PVIs) with equine ...
Alonso A, Baca Castex C, Ferrante A, Pinto M, Castañeira C, Trasorras V, Gambarotta MC, Losinno L, Miragaya M.The aim of this work was to evaluate the use of air-dried spermatozoa for in vitro production of equine embryos and verify if sperm extract activation and in vivo culture improve in vitro embryo production. Cooled spermatozoa (control) and air-dried spermatozoa stored for 2, 14 or 28 days were used for ICSI sperm extract, or ionomycin was used for oocyte activation, and embryos were in vitro or in vivo (in mare's oviduct) cultured for 7 days. With in vitro culture, cleavage rate was higher when activating with sperm extract (P 0.05). Blastocysts were obtained with cooled spermatozoa, and ...
Iqbal K, Chitwood JL, Meyers-Brown GA, Roser JF, Ross PJ.Formation of the inner cell mass (ICM) and trophectoderm (TE) marks the first differentiation event in mammalian development. These two cell types have completely divergent fates for the remainder of the developmental process. The molecular mechanisms that regulate ICM and TE formation are poorly characterized in horses. The objective of this study was to establish the transcriptome profiles of ICM and TE cells from horse blastocysts using RNA sequencing (RNA-seq). A total of 12 270 genes were found to be expressed in either lineage. Global analysis of the transcriptome profiles by unsupervi...
Gambini A, Jarazo J, Karlanian F, De Stéfano A, Salamone DF.The current limitations for obtaining ovaries from slaughterhouses and the low efficiency of in vivo follicular aspiration necessitate a complete understanding of the variables that affect oocyte developmental competence in the equine. For this reason, we assessed the effect on equine oocyte meiotic competence and the subsequent in vitro cloned embryo development of 1) the time interval between ovary collection and the onset of oocyte in vitro maturation (collection-maturation interval time) and 2) the pregnancy status of the donor mares. To define the collection-maturation interval time, coll...
Herrera C, Morikawa MI, Bello MB, von Meyeren M, Centeno JE, Dufourq P, Martinez MM, Llorente J.Preimplantation genetic diagnosis (PGD) allows identifying genetic traits in early embryos. Because in some equine breeds, like Polo Argentino, females are preferred to males for competition, PGD can be used to determine the gender of the embryo before transfer and thus allow the production of only female pregnancies. This procedure could have a great impact on commercial embryo production programs. The present study was conducted to adapt gender selection by PGD to a large-scale equine embryo transfer program. To achieve this, we studied (i) the effect on pregnancy rates of holding biopsied e...
Kelley D, LeBlanc MM, Warren LK, Mortensen CJ.Supplementation with L-arginine can increase uterine arterial blood flow and vascular perfusion of the preovulatory follicle in mares. Increased vascular perfusion of the preovulatory follicle has been correlated with successful pregnancy in mares. The objective of this study was to determine if supplemental L-arginine would increase ovarian arterial blood flow, vascular perfusion of the preovulatory follicle, and embryo recovery rates in mares. Mares were blocked by age and breed and assigned at random within block to L-arginine supplementation or control groups. Mares were fed L-arginine beg...
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...
Ginther OJ.Gray-scale ultrasonic imaging (UI) was introduced in 1980 and initially was used to examine clinically the reproductive tract of mares. By 1983 in mares and 1984 in heifers/cows, UI had become a tool for basic research. In each species, transrectal gray-scale UI has been used extensively to characterize follicle dynamics and investigate the gonadotropic control and hormonal role of the follicles. However, the use of transrectal UI has also disclosed and characterized many other aspects of reproduction in each species, including (1) endometrial echotexture as a biological indicator of circulati...
de Ruijter-Villani M, van Boxtel PR, Stout TA.Uterine-derived growth factors and cytokines play essential roles in regulating preimplantation conceptus development. In several species, fibroblast growth factor-2 (FGF2) promotes embryogenesis, trophoblast cell migration, and adhesion. This study investigated mRNA expression for FGF2, its receptors (FGFR1-4), the activating factor FGF binding protein (FGF-BP) in equine endometrium and trophectoderm during early pregnancy and the estrous cycle, and localized FGF2 protein in both endometrium and conceptus tissues. FGF2, FGFRs1-4, and FGFBP mRNAs were expressed in endometrium throughout the es...
Jenner F, Närväinen J, de Ruijter-Villani M, Stout TA, van Weeren PR, Brama P.Equine embryogenesis post implantation is not well studied, and only two-dimensional illustrations are available. A thorough appreciation of the complex three-dimensional relationship between tissues and organs and their development is, however, crucial for understanding physiological and pathological mechanisms. Objective: The goals were 2-fold: 1) to establish a freely accessible online atlas as a reference tool for the scientific and pedagogic communities; and 2) to create a framework for integration of data with known spatiotemporal distribution, such as gene expression or cell lineage. Me...
Nelis H, D'Herde K, Goossens K, Vandenberghe L, Leemans B, Forier K, Smits K, Braeckmans K, Peelman L, Van Soom A.Equine embryos remain for 6 days in the oviduct and thus there is a need for an in vitro model to study embryo-oviductal interactions in the horse, since this subtle way of communication is very difficult to analyse in vivo. Until now, no equine oviduct explant culture model has been characterised both morphologically and functionally. Therefore, we established a culture system for equine oviduct explants that maintained epithelial morphology during 6 days of culture, as revealed by light microscopy and transmission electron microscopy. We demonstrated the presence of highly differentiated, ta...
Choi YH, Norris JD, Velez IC, Jacobson CC, Hartman DL, Hinrichs K.The presence of heterogenous mitochondria from the host ooplast affects the acceptance of offspring obtained by somatic cell nuclear transfer. This might be avoided by obtaining oocytes from selected females, but is then complicated by low numbers of available oocytes. We examined the efficiency of equine somatic cell nuclear transfer using oocytes recovered by transvaginal aspiration of immature follicles from 11 mares. Use of metaphase I oocytes as cytoplasts and of scriptaid (a histone deacetylase inhibitor) treatment during oocyte activation were evaluated to determine if these approaches ...
Zaniboni A, Merlo B, Zannoni A, Bernardini C, Lavitrano M, Forni M, Mari G, Bacci ML.Sperm mediated gene transfer (SMGT) has been reported to be a powerful tool for producing transgenic livestock with applications in biomedicine and agriculture. To date, two studies have reported the production of transgenic equine embryo with, however, low efficiency in blastocyst production and transgene expression. The aim of the present study was to develop a method which allowed the efficient production of transgene-expressing embryos of the equine species through SMGT. To overcome problems due to in vitro fertilization (IVF) in horses, the ICSI procedure was associated with SMGT. The upt...
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...
Beckelmann J, Budik S, Helmreich M, Palm F, Walter I, Aurich C.An adjustment of sex ratio of offspring to the conditions present at conception is seen in many mammals including horses. This depends on preferential survival of male embryos under conditions of high energy intake. In several species, growth factors including insulin like growth factor (IGF)-1 have been shown to promote embryonic development by decreasing apoptosis and increasing cell proliferation. We hypothesized that sex-related differences in IGF-1 expression in equine embryos during the phase of maternal recognition of pregnancy might exist and thus contribute to preferential survival of...
Nagao JF, Neves Neto JR, Papa FO, Alvarenga MA, Freitas-Dell'Aqua CP, Dell'Aqua JA.This study aimed to determine whether deslorelin acetate could induce double ovulation in mares. In Experiment 1, eight mares were treated with prostaglandin on Day 8 (D8) after ovulation, then treated with saline or with 100 μg of a controlled-release formulation of deslorelin acetate vehicle intramuscularly (IM) every 12h from D8 after ovulation until at least two follicles reached 33 mm. At this time, ovulation was induced with 2500 IU of hCG. Artificial insemination was performed 24h after induction, and embryos were collected on the eighth day after ovulation was first detected. In Exper...
Hanada M, Maeda Y, Oikawa MA.The secretions of the equine endometrial glands are essential for the survival, growth, and development of the conceptus in early pregnancy, and endometrial gland density is directly related to successful pregnancy outcome. Endometrial biopsy is routinely used to assess the reproductive potential of broodmares. Some previous studies have shown that equine endometrial glands are uniformly distributed throughout the uterus; however, other work has shown variation of the endometrial architecture between biopsy sites, suggesting that a single biopsy is not representative of the entire endometrium....
Raeside JI, Christie HL, Waelchli RO, Betteridge KJ.The embryo proper in early equine pregnancy has recently been shown to have a remarkable capacity for metabolism of oestrogens. High concentrations of oestrogens in yolk-sac fluid could provide substrate for local metabolism in tissues of the embryo proper and this activity could have significance for early development. Due to the high level of oestrogen metabolism in the embryo proper we examined the possibility that it could also biosynthesise oestrogens. Conceptuses were collected in the fourth week of pregnancy (n=23) and the embryo was separated from extraembryonic tissues for incubation ...
Waelchli RO, Betteridge KJ.Twin ovulations are common in horses, but twin pregnancies are rarely carried to term. Theories of how one or both twins is/are naturally eliminated in early pregnancy, termed 'embryo reduction', have been based on ultrasonographic, not morphological, studies. Here we describe conceptuses recovered transcervically between Days 15 and 28 from 31 twin and two triplet pregnancies. Signs of contact between conceptuses were deduced from those seen in one pair that remained attached by their capsules on Day 18. Signs were found on capsules in two of 10 pairs before or during fixation (immobilisation...
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...
Rader K, Choi YH, Hinrichs K.Intracytoplasmic sperm injection is becoming a common clinical procedure in the horse, but little information is available on techniques for its performance. Each laboratory uses different procedures and different media for the steps involved with in vitro embryo production. This article outlines the procedures used in the Clinical Equine Intracytoplasmic Sperm Injection Program at Texas A&M University for in vitro blastocyst production during the past 3 years.
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...
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 ...
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...
Marsico TV, Valente RS, Annes K, Oliveira AM, Silva MV, Sudano MJ.A wide-ranging review study regarding the molecular characterization of the first cell lineages of the developmental embryo is lacking, especially for the primary events during earliest differentiation which leads to the determination of cellular fate. Here, a systematic review and meta-analysis were conducted according to PRISMA guidelines. MEDLINE-PubMed was searched based on an established search strategy through April 2021. Thirty-six studies fulfilling the inclusion criteria were subjected to qualitative and quantitative analysis. Among the studies, 50 % (18/36) used mice as an animal mod...
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...
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...
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...
