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.
Franciolli AL, Cordeiro BM, da Fonseca ET, Rodrigues MN, Sarmento CA, Ambrosio CE, de Carvalho AF, Miglino MA, Silva LA.In spite of numerous, substantial advances in equine reproduction, many stages of embryonic and fetal morphological development are poorly understood, with no apparent single source of comprehensive information. Hence, the objective of the present study was to provide a complete macroscopic and microscopic description of the equine embryo/fetus at various gestational ages. Thirty-four embryos/fetuses were aged based on their crown rump length (CRL), and submitted to macroscopic description, biometry, light and scanning microscopy, as well as the alizarin technique. All observed developmental c...
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...
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...
Choi YH, Velez IC, Riera FL, Roldán JE, Hartman DL, Bliss SB, Blanchard TL, Hayden SS, Hinrichs K.Effective cryopreservation of expanded equine blastocysts (> 300 μm in diameter) has been difficult, perhaps due to the volume of blastocoele fluid or the presence of the equine embryonic capsule. Recently, we reported normal viability of equine embryos after trophoblast biopsy, which resulted in blastocyst collapse. The present study addressed the effect of biopsy and resultant breach of the capsule and blastocyst collapse on survival of expanded equine blastocysts after vitrification. First, non-biopsied, small embryos (< 300 μm) were vitrified in fine-diameter microloader pipette ti...
Mattos MC, Bastos MR, Guardieiro MM, Carvalho JO, Franco MM, Mourão GB, Barros CM, Sartori R.The aim of this study was to evaluate the superovulatory (SOV) response of Sindhi (Bos indicus) donors submitted to an ovarian follicular superstimulatory protocol replacing the last two doses of pFSH by eCG. Forty-eight SOV treatments were performed in a crossover design in 19 nulliparous and primiparous females that were randomly divided into two groups: FSH (n=24), which consisted of eight pFSH injections, or FSH/eCG (n=24), which consisted of six pFSH injections followed by two eCG injections. Each female underwent two or three SOV treatments that consisted of an i.m. injection of 2mg estr...
Willmann C, Budik S, Walter I, Aurich C.A positive influence of altrenogest treatment on a retarded development of the conceptus around the beginning of placentation in mares older than 8 years could be recently demonstrated. In the present study, effects of altrenogest treatment in early-pregnant mares on conceptus development and expression of endometrial and embryonic genes were investigated. Genes were chosen according to a possible involvement in embryo-maternal interaction and embryonic development in the equine species. Mares were treated with altrenogest (0.044 mg/kg bodyweight) or sunflower oil (placebo) from day 5 to 11 af...
Klein C, Scoggin KE, Troedsson MH.Establishment of pregnancy is critically dependent upon a precisely orchestrated embryo-maternal interaction leading to a receptive uterine environment. The up-regulation of the interferon-stimulated protein 15 kDa (ISG15) during pregnancy has been described in various species and has been hypothesized to be part of the molecular repertoire that makes the uterus receptive to conceptus development. In the current study, the expression of ISG15 and enzymes involved in ISG15ylation was examined at the mRNA and protein level in equine endometrium at Day 14 of the luteal phase and at Day 14 and 50 ...
Paris DB, Kuijk EW, Roelen BA, Stout TA.Real-time quantitative PCR (qPCR) is invaluable for investigating changes in gene expression during early development, since it can be performed on the limited quantities of mRNA contained in individual embryos. However, the reliability of this method depends on the use of validated stably expressed reference genes for accurate data normalisation. The aim of the present study was to identify and validate a set of reference genes suitable for studying gene expression during equine embryo development. The stable expression of four carefully selected reference genes and one developmentally regula...
Smits K, Goossens K, Van Soom A, Govaere J, Hoogewijs M, Peelman LJ.In vitro-produced (IVP) equine blastocysts can give rise to successful pregnancies, but their morphology and developmental rate differ from those of in vivo-derived equine blastocysts. The aim of the present study was to evaluate this difference at the genetic level. Suppression subtractive hybridisation (SSH) was used to construct a cDNA library enriched for transcripts preferentially expressed in in vivo-derived equine blastocysts compared with IVP blastocysts. Of the 62 different genes identified in this way, six genes involved in embryonic development (BEX2, FABP3, HSP90AA1, MOBKL3, MCM7 a...
Desmarais JA, Demers SP, Suzuki J, Laflamme S, Vincent P, Laverty S, Smith LC.Although putative horse embryonic stem (ES)-like cell lines have been obtained recently from in vivo-derived embryos, it is currently not known whether it is possible to obtain ES cell (ESC) lines from somatic cell nuclear transfer (SCNT) and parthenogenetic (PA) embryos. Our aim is to establish culture conditions for the derivation of autologous ESC lines for cell therapy studies in an equine model. Our results indicate that both the use of early-stage blastocysts with a clearly visible inner cell mass (ICM) and the use of pronase to dissect the ICM allow the derivation of a higher proportion...
Klein C, Troedsson MH.Establishment and maintenance of pregnancy are critically dependent on embryo-maternal communication during the preimplantation period. The horse is one of the few domestic species in which the conceptus-derived pregnancy recognition signal has not been identified. To gain new insights into the factors released by the equine conceptus, transcriptional profiling analyses of conceptuses retrieved 8, 10, 12, and 14 days after ovulation were performed using a whole-genome microarray. Selected array data were confirmed using quantitative PCR, and the expression of proteins of interest was confirmed...
Newcombe JR, Cuervo-Arango J.One hundred and fifty-four mares were inseminated with fresh semen either during the pre- or post-ovulatory periods at different intervals relative to ovulation: 36-24 h (n = 17) and 24-0 h (n = 30) before ovulation; 0-8 h (n = 21), 8-16 h (n = 24), 16-24 h (n = 48) and 24-32 h (n = 14) h after ovulation. All mares received the same routine post-mating treatment consisting of an intrauterine infusion with 1 litre of saline and antibiotics followed 8 h later by an intravenous administration of oxytocin. Artificial inseminations (AI) from 36 h before ovulation up to 16 h post-ovulation were perf...
Newcombe JR, Paccamonti D, Cuervo-Arango J.Data were analysed retrospectively from fourteen breeding seasons at an Equine Fertility Clinic for the effect of interval between pre- and postovulatory examinations for immediate postovulatory insemination on pregnancy rate (PR) and embryo loss rate (ELR). Mares of various breeds and ages were examined at intervals which varied from 0.5 to 15h between the pre- and postovulatory period over 867 cycles. When ovulation was detected they were inseminated with a single dose of commercial frozen-thawed semen. All mares were treated in the post-insemination period with intrauterine antibiotics and ...
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.There is currently much interest in assisted reproduction techniques in the horse, however, many aspects of oocyte maturation, fertilization, and embryo development in the horse differ from those in other species. Because of the close attachment of the equine oocyte to the follicle wall, scraping of the follicle is the most effective method for oocyte recovery. A notable feature of equine oocytes is that those with expanded cumuli (Ex oocytes), which originate from atretic follicles, have higher meiotic competence (ability to mature to metaphase II in vitro) than do oocytes with compact cumuli...
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 ...
Scherzer J.Reproductive performance in mares can be enhanced by various techniques. Protocols hastening the onset of follicular development help establish pregnancy in mares and ensure that foals are born early in the year. The time spent breeding mares can be reduced by synchronizing estrus and inducing ovulation. After successful fertilization of the oocyte, the developing embryo can survive in the uterus only if postbreeding endometritis, if present, is treated.
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...
Lagares MA, Castanheira PN, Amaral DC, Vasconcelos AB, Veado JC, Arantes RM, Stahlberg R.The aim of the present study was to evaluate the in vitro viability of equine embryos vitrified in three different solutions. Day 6 and 6.5 embryos were measured and morphologically evaluated. Only grade 1 or 2 morulae and early blastocysts were vitrified. Eighteen embryos were distributed in Group 1: 40 percent ethylene glycol in PBS, Group: 2 and 3: 40 percent ethylene glycol, 18 percent Ficoll, 0.3M sucrose or 0.3M trehalose in PBS, respectively. The vitrified embryos were loaded individually into 0.25 ml straws, which were cooled and immersed in liquid nitrogen. After warming at 20 degree ...
Klein C, Scoggin KE, Ealy AD, Troedsson MH.Establishment and maintenance of pregnancy are critically dependent on embryo-maternal communication during the preimplantation period. To gain new insights into this complex process in the horse, transcriptional profiling of Day 13.5 pregnant and cyclic endometrial tissue samples was carried out using custom-designed microarrays. Selected array data were validated using quantitative RT-PCR, and proteins of interest were localized using immunohistochemistry. One hundred and six transcripts were up-regulated, whereas 47 transcripts showed lower expression levels in pregnant mares, that is, were...
Jacobson CC, Choi YH, Hayden SS, Hinrichs K.Oocytes may be collected from live mares from either the stimulated preovulatory follicle or from all visible immature follicles. We evaluated the yield of mature oocytes, and of blastocysts after intracytoplasmic sperm injection (ICSI), for both follicle types. In Experiment 1, mares were assigned to Progesterone (1.2g biorelease progesterone weekly) or Control treatments. Transvaginal aspiration of all follicles was performed every 14 d. Overall, 596 follicles were aspirated, with a 54% oocyte recovery rate. There was no difference between treatments in number of follicles punctured (9.0 to ...
Walter I, Tschulenk W, Budik S, Aurich C.The present study gives a detailed ultrastructural description of equine conceptuses at Day 14 (n = 2) and Day 16 (n = 3) after ovulation. Whereas on Day 14 only primitive structures were seen, on Day 16 neurulation and formation of mesodermal somites had taken place. The ectoderm of the embryo itself and the surrounding trophoblast ectodermal cells were characterised by specific cell surface differentiations. At the embryonic ectodermal cell surface (14 and 16 days) remarkable protruded and fused cytoplasmic projections were seen, typically associated with macropinocytotic events involved in ...
BMC research notesDecember 11, 2009
Volume 2 246 doi: 10.1186/1756-0500-2-246
Smits K, Goossens K, Van Soom A, Govaere J, Hoogewijs M, Vanhaesebrouck E, Galli C, Colleoni S, Vandesompele J, Peelman L.Application of reverse transcription quantitative real-time polymerase chain reaction is very well suited to reveal differences in gene expression between in vivo and in vitro produced embryos. Ultimately, this may lead to optimized equine assisted reproductive techniques. However, for a correct interpretation of the real-time PCR results, all data must be normalized, which is most reliably achieved by calculating the geometric mean of the most stable reference genes. In this study a set of reliable reference genes was identified for equine in vivo and fresh and frozen-thawed in vitro embryos....
Lange Consiglio A, Dell'Aquila ME, Fiandanese N, Ambruosi B, Cho YS, Bosi G, Arrighi S, Lacalandra GM, Cremonesi F.The identification of the adipocyte-derived obesity gene product, leptin (Ob), and subsequently its association with reproduction in rodents and humans led to speculations that leptin may be involved in the regulation of oocyte and preimplantation embryo development. In mice and pigs, in vitro leptin addition significantly increased meiotic resumption and promoted preimplantation embryo development in a dose-dependent manner. This study was conducted to determine whether leptin supplementation during in vitro maturation (IVM) to horse oocytes could have effects on their developmental capacity ...
Sharma S, Davies Morel MC, Dhaliwal GS, Dadarwal D.Ultrasonographic pregnancy records of 195 mares from six Thoroughbred stud farms, over a period of 7 years were retrospectively analysed to assess the effect of various factors on embryonic vesicle (EV) fixation pattern and pregnancy outcome. Of the total of 746 pregnancies analysed, significantly (p < 0.01) more EV fixations were evident in the right uterine horn than in the left (53.35% vs 46.65% respectively). There was no significant effect of either, the side of ovulation, or age of the mare, on the side of EV fixation. However, EV fixation, was significantly (p < 0.001) more likely...
Raeside JI, Christie HL, Waelchli RO, Betteridge KJ.Estrogen production by the trophoblast is considered important in early equine pregnancy and leads to high concentrations in yolk-sac (Y-S) fluid. The embryo proper is a potential site for their action. We examined estrogen metabolism in the embryo proper because some actions of estrogens are derived from locally formed metabolites. The embryo proper, as well as separated extraembryonic tissues, of conceptuses collected about day 25 of pregnancy, were incubated with (3)[H]-estrone (E(1)) and (3)[H]-estradiol (E(2)). Steroids were recovered from media by solid-phase extraction and eluted separa...
Choi YH, Harding HD, Hartman DL, Obermiller AD, Kurosaka S, McLaughlin KJ, Hinrichs K.The reported patterns of trophectodermal expression of POU5F1 protein in blastocysts vary among species, and are possibly related to the differences in placental growth and function. This study investigated the pattern of embryonic POU5F1 expression in the horse, a species with delayed placental formation. Immature equine oocytes expressed POU5F1 protein in the cytoplasm and nucleus. Staining for POU5F1 protein in in vitro-produced (IVP) embryos decreased to day 5 of culture, then the nuclear staining increased to day 7. IVP day-7 to -11 blastocysts showed POU5F1 staining in nuclei throughout ...
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...
Bugno M, Jabłońska Z, Słota E.The aim of the study was to optimize hybridization conditions of molecular probes specific for X sex chromosomes of the domestic horse in mare oocyte chromosomes. Mare oocytes, recovered from slaughterhouse ovaries by scraping the granulosa layer, were cultured in vitro. Metaphase II mature oocytes were treated with hypotonic solution and fixed, followed by hybridization of the molecular probe specific for the X chromosome ofthe domestic horse. Hybridization of probes specific for mouse heterosomes on mouse oocytes and early embryos was performed to verify the FISH technique. Of 438 oocytes an...
Sharma S, Dhaliwal GS, Dadarwal D.Service records of 253 mares (1181 mare-years) spanning over 7 consecutive years, from nine organized Thoroughbred stud farms, situated in the subtropical northwestern India were retrospectively analyzed to assess their reproductive performance. The overall per cycle pregnancy rate at Day 16 and overall foaling rates were 50.30% and 68.95%, respectively, and were significantly higher in mares aged 3-7 years than > or =18 years old mares. The late embryonic losses (9.86%) that occurred between Days 16 and 39 post-ovulation contributed more than 50% of the overall detected pregnancy losses (19.1...
Tharasanit T, Colenbrander B, Bevers MM, Stout TA.The only gonadotrophin preparation shown to stimulate commercially useful multiple ovulation in mares is equine pituitary extract (EPE); even then, the low and inconsistent ovulatory response has been ascribed to the variable, but high, LH content. This study investigated the effects of an LH-free FSH preparation, recombinant human follicle stimulating hormone (rhFSH), on follicle development, ovulation and embryo production in mares. Five mares were treated twice-daily with 450 i.u. rhFSH starting on day 6 after ovulation, coincident with PGF(2alpha) analogue administration; five control mare...
Flores Bragulat AP, Ortiz I, Catalán J, Dorado J, Hidalgo M, Losinno L, Dordas-Perpinyà M, Yánez-Ortiz I, Martínez-Rodero I, Miró J, Gambini A.Equus members exhibit very divergent karyotype, genetic plasticity, and significant differences in their reproductive physiology. Despite the fact that somatic cell nuclear transfer and intracytoplasmic sperm injection (ICSI) has gained relevance in the last few years in horses, few reports have been published exploring ovum pick up (OPU) and in vitro maturation (IVM) of cumulus-oocyte complexes (COCs) in donkeys. Yet, some donkey species and breeds are considered endangered, and these assisted-reproductive technologies could help to preserve the genetic of valuable individuals. In this study...
Li X, Morris LH, Allen WR.This study investigated the basic conditions required for the production of horse embryos by the transfer of the nuclei of fetal and adult fibroblast cells to enucleated oocytes. Cumulus-oocyte complexes were recovered from abattoir ovaries and matured in vitro in groups of 20-30 for 28-30 h in tissue culture medium 199 containing 20% v:v fetal bovine serum in coculture with equine oviduct epithelial cells. Fetal fibroblast cells (FFC) were derived from a 32-day-old Thoroughbred x Pony fetus, and adult skin fibroblast cells (SFC) were obtained from subdermal biopsies recovered from a 4-yr-old ...
Moussa M, Tremoleda JL, Duchamp G, Bruyas JF, Colenbrander B, Bevers MM, Daels PF.The aim of this study was to evaluate the viability (percentage of dead cells) and the incidence of DNA fragmentation of horse embryos after storage in three different media at 5 degrees C for 6 and 24 h. Forty embryos were stored in Emcare Holding Solution for 6 and 24 h, in Hams'F10 or Vigro Holding Plus for 24 h at 5 degrees C (n = 9-10 per group) and 10 embryos were evaluated immediately after collection. First, embryos were stained, immediately after collection or following storage, to detect dead cells (DAPI) and, subsequently, DAPI-stained embryos were fixed and stained to detect DNA fr...
Curran S, Urven L, Ginther OJ.Eighteen equine embryos, 3 each on Days 20, 22, 24, 26, 28 and 30 post ovulation, were collected transcervically by uterine lavage, fixed in 4% paraformaldehyde and embedded in paraffin wax. Ten micron serial sections were stained to determine alkaline phosphatase (AP) activity in the cells. Positive cells were counted and their approximate location determined. The cells were approximately 8 microm in diameter and the entire cell, except the nucleus, stained strongly with many small round areas of intense staining in the cytoplasm. The cells varied from round to elongated in shape and pseudopo...
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...
Ginther OJ.The equine embryo or embryonic vesicle on Days 11-15 postovulation travels with profound physiologic purpose throughout the lumen of the two uterine horns and uterine body making 12 to 22 trips between the two uterine horns per day. This phenomenon is termed embryo mobility and is unique in equids among domestic species. Apparently, the embryo first reaches the uterine body on Days 8 or 9. Mobility increases to maximum by Days 11 or 12 and continues until an abrupt cessation of mobility (fixation) on Days 15 (ponies) or 16 (horses and donkeys). The embryo is propelled by uterine contractions i...
do Nascimento AD, Marques JCC, Cezar ARR, Batista AM, Kastelic JP, Câmara DR.Although equine blastocysts ≤ 300 µm in diameter can be successfully vitrified, larger equine blastocysts are not good candidates for cryopreservation. As Na, K-ATPase is involved in maintaining blastocyst expansion, perhaps inhibition of this enzyme would be a viable method of reducing blastocyst diameter prior to cryopreservation. Objectives were to evaluate effects of ouabain-induced inhibition of Na, K-ATPase in equine blastocysts. Sixteen mares were ultrasonographically monitored, given deslorelin acetate to induce ovulation, and inseminated. Embryos (D7 and D9) were harvested and Na, ...
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...
Vanderwall DK.Tremendous progress has been made in the development of assisted reproductive techniques that may enhance the reproductive efficiency of the horse. However, techniques that involve the manipulation of oocytes and/or embryos may themselves be detrimental to embryo viability and subsequent development. Therefore, an objective method of assessing viability of embryos before and/or after oocyte/embryo manipulation is desirable. At this time, morphologic evaluation is the most widely used method of determining the viability of equine embryos. Although morphologic assessment of embryo quality will n...
van Niekerk FE, van Niekerk CH.Sixty-four Thoroughbred and Anglo-Arab mares aged 6-12 years were randomly allocated to 4 dietary groups and fed diets that differed in the total protein content and quality (essential amino-acids). Forty mares were non-lactating and 24 lactating. Eight mares were withdrawn from the investigation owing to injuries or gynaecological pathology. An overall conception rate of 94.6% and a foaling rate of 80% was achieved. Five of 14 (35.7%) mares (Group 1) fed a low-quality protein diet suffered from early embryonic loss before 90 days of pregnancy compared to 3 of 41 (7.3%) mares in the remaining ...
D' Fonseca NMM, Gibson CME, van Doorn DA, Roelfsema E, de Ruijter-Villani M, Stout TAE.Maternal overfeeding is associated with disturbances in early embryonic epigenetic reprogramming, leading to altered expression of imprinted genes and nutrient transporters, which can affect both fetal and placental development and have lasting effects on the health of resulting offspring. To examine how maternal overfeeding affects the equine embryo, Shetland pony mares were fed either a high-energy (HE: 200% of net energy requirements) or maintenance (control) diet. Mares from both groups were inseminated, and day-seven embryos were recovered and transferred to recipients from the same or th...
Silva ESM, Vaz IS, Rodrigues TS, Brandão FZ, Oquendo PS, Oquendo FMG, Beletti ME, Cuervo-Arango J.Uterocalin (P19) is a lipocalin protein that has binding activity for the provisioning of the embryo with lipids, including those essential for morphogenesis and pattern formation. Therefore, it is thought that P19 plays an important role in supporting the survival of the early embryo. Previous research has shown that the endometrium from acyclic mares expressed more P19 following a simulated long estrus: treatment of 8 mg of estradiol benzoate (EB) split over five consecutive days, followed by long-acting progesterone administration (LA-P4) 72 hours later, when compared to a single 2.5 mg EB ...
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...
Young CA, Squires EL, Seidel GE, Kato H, McCue PM.Larger grade 1 or 2 (1 = excellent,.... 4 = degenerate) equine embryos that ranged in diameter from 300 to 680 microm and were recovered from mares on Day 7 or 8 after ovulation, were randomly assigned to 3 widely divergent cryopreservation treatments. Treatment 1 consisted of cooling from -6 degrees C to -35 degrees C at 0.5 degrees C per min followed by plunging into liquid nitrogen, with a one-step addition and a 4-step removal of 1.0 M glycerol. Treatment 2 (step-down equilibration) consisted of a 2-step addition of glycerol to 4.0 M followed by a decrease to 2.0 M prior to freezing, with ...
Pruitt JA, Forrest DW, Burghardt RC, Evans JW, Kraemer DC.The viability and ultrastructure of equine embryos were assessed following culture in a static or perifusion system. The percentage change in diameter was greater (P less than 0.025) for embryos in the static treatment (71%) than in the perifusion treatment (33%). Fluorescein diacetate (FD) scores, the percentage of fluorescing cells (FC) and fluorescent intensity (FI), also were greater (P less than 0.05 and P less than 0.01) following static culture than for embryos cultured in the perifusion system. Four of 9 control embryos resulted in pregnancies but no embryos cultured in either system p...
Choi YH, Love LB, Varner DD, Hinrichs K.This study was conducted to evaluate the effect of initial cumulus morphology (expanded or compact) and duration of in vitro maturation (24, 30 or 42 h) on the developmental competence of equine oocytes after intracytoplasmic sperm injection (ICSI). The effect of manipulation temperature (room temperature vs 37 degrees C) at the time of ICSI and concentration of glucose (0.55 vs 5.5 mM) during embryo culture was also investigated. The nuclear maturation rates of expanded (Ex) oocytes were significantly (P < 0.001) higher than those of compact (Cp) oocytes at all maturation times (61-72 vs 23-2...
Ferreira JC, Meira C, Papa FO, Landin e Alvarenga FC, Alvarenga MA, Buratini J.Six or 7-day-old equine embryos were divided into 4 groups; Group 1, n = 15, Day 7 embryos destined for immediate transfer; Group 2, n = 15, Day 6 embryos destined for deep-freezing with glycerol plus sucrose as cryoprotectant; Group 3, n = 10, Day 6 embryos destined for deep-freezing with glycerol plus 1,2-propanediol as cryoprotectant and Group 4, n = 3, fresh embryos destined for ultrastructural analysis. All the frozen/thawed embryos were transferred to recipient mares, except 3 embryos in Group 3 that were subjected to ultrastructural analysis. After thawing the cryoprotectants were remov...
Derisoud E, Jouneau L, Dubois C, Archilla C, Jaszczyszyn Y, Legendre R, Daniel N, Peynot N, Dahirel M, Auclair-Ronzaud J, Wimel L, Duranthon V....Breeding a mare until she is not fertile or even until her death is common in equine industry but the fertility decreases as the mare age increases. Embryo loss due to reduced embryo quality is partly accountable for this observation. Here, the effect of mare's age on blastocysts' gene expression was explored. Day 8 post-ovulation embryos were collected from multiparous young (YM, 6-year-old, N = 5) and older (OM, > 10-year-old, N = 6) non-nursing Saddlebred mares, inseminated with the semen of one stallion. Pure or inner cell mass (ICM) enriched trophoblast, obtained by embryo bise...
Choi YH, Chung YG, Walker SC, Westhusin ME, Hinrichs K.This study was conducted to evaluate the effects of insulin-like growth factor I (IGF-I) and other media factors during oocyte maturation, and the presence of different compositions of amino acids in embryo culture medium, on the development of equine embryos. Oocytes recovered from slaughterhouse-derived ovaries were matured in vitro for 24 h and those with a polar body were subjected to intracytoplasmic sperm injection (ICSI) or nuclear transfer with adult fibroblasts (NT). For ICSI embryos, there were no significant differences in rates of morphological cleavage, cleavage with normal nuclei...
Tachibana Y, Sakurai T, Bai H, Shiota K, Nambo Y, Nagaoka K, Imakawa K.Extensive studies have been conducted to characterize the unique phenomena of equine pregnancy. Most studies have focused on embryo transmigration when the embryo is covered with a mucin-like glycoprotein capsule and on the characterization of the chorionic girdle and chorionic gonadotropin (CG) secretion. However, the events preceding and following capsule disappearance have not been well studied. In this study, the mRNA expression in conceptus membranes at days 19, 21, and 25 (day 0 = day of ovulation) was analyzed by RNA-seq (SOLiD3), and transcript levels on these three days and day 13...
Choi YH, Penedo MC, Daftari P, Velez IC, Hinrichs K.Preimplantation genetic diagnosis has great potential in the horse, but information on evaluation of equine embryo biopsy samples is limited. Blastocysts were biopsied using a Piezo drill and methods for whole-genome amplification (WGA) investigated. Results for 33 genetic loci were then compared between biopsy samples from in vitro-produced (IVP) and in vivo-recovered (VIV) blastocysts. Under the experimental conditions described, WGA using the Qiagen Repli-g Midi kit was more accurate than that using the Illustra Genomiphi V2 kit (98.2% vs 25.8%, respectively). Using WGA with the Qiagen kit,...
Rajabi-Toustani R, Watanabe H, Tsogtgerel M, Gao Y, Canbo L, Haneda S, Cheong SH, Nambo Y.The main difficulty of large equine embryo cryopreservation is the replacement of blastocoel fluid with cryoprotectant solution. The objective of this study was to improve the cryopreservation of large equine embryos with PMAP and/or LAP. Embryos were collected via the non-surgical transcervical procedure and divided into three groups based on their size (A ≤ 300 µm, 300 µm<B 300 µm). However, more research is required to find the best method for embryos ≥700 µm.
Walter I, Tschulenk W, Budik S, Aurich C.The present study gives a detailed ultrastructural description of equine conceptuses at Day 14 (n = 2) and Day 16 (n = 3) after ovulation. Whereas on Day 14 only primitive structures were seen, on Day 16 neurulation and formation of mesodermal somites had taken place. The ectoderm of the embryo itself and the surrounding trophoblast ectodermal cells were characterised by specific cell surface differentiations. At the embryonic ectodermal cell surface (14 and 16 days) remarkable protruded and fused cytoplasmic projections were seen, typically associated with macropinocytotic events involved in ...
Deleuze S, Dubois CS, Caillaud M, Bruneau B, Goudet G, Duchamp G.Contents The effect of cysteamine on in vitro nuclear and cytoplasmic maturation of equine oocytes collected by transvaginal ultrasound guided follicular aspiration was assessed. Oocytes were matured in vitro with (cysteamine group) or without (control group) cysteamine. The nuclear stage after DNA Hoechst staining, penetration rates after two different in vitro fertilization (IVF) techniques (IVF media with ionophore and Hepes buffer with heparin) and the embryo yield following oocyte intra-oviductal transfer were used as a criterion for assessing nuclear and cytoplasmic maturation, respectiv...
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 ...
Shilton CA, Kahler A, Roach JM, Raudsepp T, de Mestre AM.Embryonic and foetal loss remain one of the greatest challenges in equine reproductive health with 5-10% of established day 15 pregnancies and a further 5-10% of day 70 pregnancies failing to produce a viable foal. The underlying reason for these losses is variable but ultimately most cases will be attributed to pathologies of the environment of the developing embryo and later foetus, or a defect intrinsic to the embryo itself that leads to lethality at any stage of gestation right up to birth. Historically, much research has focused on the maternal endometrium, endocrine and immune responses ...
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...
Leisinger CA, Klein C, Markle ML, Premanandan C, Sones JL, Pinto CRF, Paccamonti DL.Aluteal cycles were induced in the mare to evaluate the effects of progesterone deprivation on the gene expression of embryos and endometrium collected eight days after ovulation. We hypothesized that the transcript expression would be altered during induced aluteal (AL) cycles (low progesterone <1 ng/mL) when compared with control cycles during diestrus (high progesterone; > 4 ng/mL) for 1) the embryonic expression of progesterone-mediated transcripts and those related to normal embryo growth and development and 2) the endometrial expression of progesterone-mediated transcripts and those ...
Squires EL.Equine oocytes obtained either by transvaginal ultrasound-guided follicular aspiration or from slaughterhouse ovaries can be matured in vitro. This generally requires culture in TCM-199 containing serum and hormones for 30 to 36 hours. With this protocol, approximately 50% to 60% of the oocytes are at metaphase-II at the end of the culture period. At least some of these oocytes appear viable based on production of fertilized eggs either through in vitro fertilization or fertilization in vivo of a recipient mare. The success of producing equine embryos in vitro is still extremely low. More than...
