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.
Bragulla H, Budras KD, Reilly JD.The fetal development of the white line (Zona alba) in the equine hoof is described. Its specific structure of lamellar and interlamellar horn, which in turn is composed of cap and terminal horn, is formed in the second half of the hoof's fetal development. In equine fetuses with a crown-rump length of less than 550 mm, the hoof capsule lacks a 'characteristic' white line since no borders between stratum medium, stratum internum and sole horn are discernible. In the hoof of an equine fetus with a crown-rump length of 550 mm, a narrow white line has taken shape. Its shallow lamellae are arrange...
Crossett B, Suire S, Herrler A, Allen WR, Stewart F.One of the major, progesterone-dependent proteins secreted into the uterine lumen of the mare is a 19-kDa lipocalin (P19). It associates strongly with the embryonic capsule that envelops the young horse conceptus in early gestation, suggesting that it may be involved in sustaining early development. However, it was not known whether the protein was transported through the capsule and/or trophoblast layer and into the yolk sac cavity. To address this question, polyclonal antisera were raised against a C-terminal peptide (based on the deduced amino acid sequence of P19) and a recombinant-derived...
Palmer E, Duchamp G, Cribiu EP, Mahla R, Boyazoglu S, Bézard J.The aim of this study was to test the possibility that ovulation can occur from a preovulatory follicle emptied of its follicular fluid. Transport of the oocyte into the oviduct and fertilisation in 29% of cases demonstrated that ovulation can occur in the absence of follicular fluid but the higher fertility achieved in control mares (62.5%) suggested that follicular fluid does serve a role during ovulation, fertilisation and oviductal transport. Injection of horse oocytes into preovulatory follicles in mules after removal of the follicular fluid, followed by insemination of the mules with hor...
Bruyas JF, Martins-Ferreira C, Fiéni F, Tainturier D.Seventeen horse embryos recovered on the sixth day after spontaneous ovulation were; 1) washed in PBS (n = 6), 2) treated with 1.5 M 1-2 propanediol (n = 6) or, 3) frozen and thawed using 1.5 M propanediol as the cryoprotectant (n = 5). After treatment, the embryos were incubated for 6 h in medium before they were fixed, serially sectioned and examined microscopically to count the total numbers of interphase, mitotic and pycnotic nuclei. Significant differences were measured only in the mean proportions of pycnotic cells (+/- s.d.), both between the control (9.2 +/- 7.3%) and frozen-thawed emb...
Juhász J, Nagy P, Huszenicza G, Szigeti G, Reiczigel J, Kulcsár M.The effect of long term administration of T-2 toxin was studied in 6 Trotter mares during the summer and early autumn. After one complete oestrous cycle (Cycle 1) each mare was given 7 mg purified T-2 toxin per os daily (1 mg/ml in ethyl alcohol) beginning on Day 10 after ovulation in Cycle 2. Exposure to toxin was continued for 32-40 days, until Day 7 of Cycle 4. During this period all the animals remained in good physical condition, but skin lesions were observed around the mouth in 3 cases. Toxin administration had no effect on the length of the interovulatory interval or on the lengths of ...
Camillo F, Cela M, Vannozzi I, Romagnoli S, Aria G.Fourteen normal, cyclic mares, treated to synchronise oestrus and ovulation and inseminated artificially with fresh semen, were assigned to a donor or a recipient group after ovulation, with the aim of obtaining a degree of synchrony of > or =2 days. Ten embryos, collected on Day 6 or 7 after ovulation (Day 0), were transferred nonsurgically to inseminated recipient mares (IRM) that had ovulated up to 5 days after the respective donors, or to pregnant recipient mares (PRM) that had ovulated 2-7 days before the donors. Embryonic size and development, as determined by ultrasound examination, wer...
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...
Kato H, Seidel GE, Squires EL, Wilson JM.In vitro matured horse oocytes with a first polar body (n = 68) were each injected with a single spermatozoon and divided into 2 groups: Group 1 oocytes were treated with 10 microM calcium ionophore A23187 for 5 min while Group 2 oocytes received no activation treatment. After culture in vitro for 2 days, significantly more oocytes treated with A23187 (5/24, 21%) cleaved than oocytes without activation treatment (2/44, 5%, P<0.05). All 7 cleaved zygotes from both treatment groups were transferred to recipient mares but no pregnancies resulted.
Huhtinen M, Lagneaux D, Koskinen E, Palmer E.Seventy-five embryos were collected 6 days after ovulation. Sixty embryos were frozen in straws using glycerol as the cryoprotectant in an automatic freezer. In Experiment 1 the freezing and thawing media were supplemented with 1.3 g/l PVP; in Experiment 2 the supplement was 5% FCS. The embryos were thawed for 30 s at +37 degrees C in a waterbath. In Experiment 1 glycerol was removed from 10 embryos in 6 steps. In 10 other embryos, glycerol and sucrose were both removed from the medium in 6 steps. After glycerol and sucrose removal, the embryos were stained with 4',6'-diamidino-2-phenylindole ...
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 ...
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...
Guèrand M, Mahla R, Lagneaux D, Amigues Y, Palmer E, Bézard J.Paternity analysis was performed on the DNA of 21 equine embryos collected nonsurgically 10 days after ovulation from known mares, but involving 3 possible sires. After extraction, the DNA of each embryo was typed by radioactive PCR amplification using 10 characterised microsatellites; HMS 1, 2, 5, 6, 7 and 8 (Guérin et al. 1994) and HTG 3, 4, 6 and 10 (Marklund et al. 1994). The 21 dams and 3 sires were genotyped using DNA extracted from blood and amplified by PCR. After electrophoresis and autoradiography of the PCR products of the embryo and parents, the alleles of the embryo were compared...
Brück I, Lehn-Jensen H, Yde G.A Warmblood mare was observed to ovulate spontaneously 12 follicles within 2 days, none of which exceeded 22 mm in diameter. On Days 13 and 17 after ovulation, 6 embryonic vesicles were identified in the uterus by ultrasonography but by Day 26, 5 of the vesicles had disappeared. Development of the surviving conceptus was monitored until Day 42. Plasma progesterone concentrations rose to 14 ng/ml on Day 7, decreased over the next 8 days and then plateaued to around 4-6 ng/ml until Day 70. The occurrence of multiple spontaneous ovulations was diagnosed repeatedly in this mare. However, the devel...
Lagneaux D, Huhtinen M, Koskinen E, Palmer E.Equine embryos recovered on Day 6 after ovulation were cooled to +4 degrees C, or frozen with AFP alone or together with glycerol. Twenty embryos (140-200 microm in diameter) were randomly assigned to 6 treatment groups. In the first 3 groups, the embryos were cooled from room temperature to +4 degrees C at a rate of 3 degrees C/min and warmed again at a rate of 32 degrees C/min in a programmable freezer. In the second 3 groups, the embryos were frozen using a standard protocol, stored in liquid nitrogen for 5-7 days and then thawed in a 37 degrees C waterbath. After cooling/warming or freezin...
Battut I, Colchen S, Fieni F, Tainturier D, Bruyas JF.The purpose of this study was to evaluate the exact age when the equine embryo reaches the uterus. The time of ovulation was determined by hourly ultrasound examinations starting 32 h after an injection of crude equine pituitary gonadotrophin or human chorionic gonadotrophin, or after the first of 4 injections of buserelin. Nonsurgical uterine flushings were carried out 144 h (Day 6), 156 h (Day 6.5) or 168 h (Day 7) after ovulation. Induction of ovulation was attempted in 101 oestrous cycles and 61 of 101 mares (60.4%) ovulated 32-44 h post injection. Sixty embryo collections were performed w...
Dell'Aquila ME, Cho YS, Minoia P, Traina V, Lacalandra GM, Maritato F.The aim of this study was to compare the effect of the addition of follicular fluid (FF) collected from preovulatory follicles with that of oestrous mare serum (EMS) (acting as the control) to TCM-199 medium on the in-vitro maturation, fertilization and development of equine cumulus-enclosed oocytes. Oocytes (<30 mm in diameter) were obtained from the ovaries of slaughtered mares. After in-vitro maturation in the presence of the two supplements, their fertilization, cleavage and developmental potential were compared after conventional in-vitro fertilization (IVF) or intracytoplasmic sperm inje...
Waelchli RO, MacPhee DJ, Kidder GM, Betteridge KJ.The unusual hypotonicity of equine blastocyst fluid has prompted us to investigate the role of sodium- and potassium-dependent adenosine triphosphatase (Na+,K+-ATPase) in the process of fluid accumulation in the horse conceptus. Nine mares were used for the experiments. Reverse transcriptase polymerase chain reaction was conducted on two sets of five conceptuses recovered between 12 and 28 days (+/- 1 day) after ovulation. Messenger RNAs encoding the alpha1 and beta1 subunit isoforms of Na+,K+-ATPase were detected in all embryonic tissues examined. Western blot analysis showed that alpha1 and ...
Willink DL, Smeenk LA, van Oyen PW, de Kruif A.Data from the literature and own data for 67 twin pregnancies were used to establish the factors essential to the decision on how to treat twins at different gestational ages. Spontaneous (natural) reduction was compared with manual embryo reduction. Manual embryo reduction is always indicated when a twin is diagnosed before day 16 after ovulation. Thereafter, the type of fixation is the main determinant. Manual embryo reduction is always first choice for bilateral and unilateral non adjacent embryos and must be applied as early as possible. The change of natural reduction up to day 30, is hig...
Cottrill CM, Ho SY, O'Connor WN.The research examines the embryological development of the equine (horse) heart, aiming to understand its formation, function, and performance abilities. The study observes the developing morphology of the equine heart, […]
Dell'Aquila ME, Cho YS, Minoia P, Traina V, Fusco S, Lacalandra GM, Maritato F.Conventional IVF as well as several assisted microfertilization techniques have shown limited success in the horse. After recent positive results achieved with intracytoplasmic injection of a single spermatozoon (ICSI) in human IVF, we chose to try the method in the horse. We compared conventional IVF to ICSI by fertilization rates of oocytes with compact and expanded cumuli and by developmental potential of the resulting embryos. Cumulus-oocyte complexes (COCs) were obtained by aspirating the follicular fluid from the ovaries of slaughtered mares. Complexes showing complete cumulus investment...
Chu JW, Sharom FJ, Oriol JG, Betteridge KJ, Cleaver BD, Sharp DC.The equine embryonic capsule, an acellular covering that envelops the conceptus during the second and third weeks of pregnancy, is composed of mucin-like glycoproteins. Its structure is consistent with a dual role during early pregnancy: protection of the conceptus, and communication between the embryo and the mother. Loss of sialic acid from the capsular glycoproteins at day 16 correlates with the time of "fixation," or loss of conceptus mobility throughout the uterine horns. This study investigated how the structure of the capsule is linked to the maintenance of pregnancy. Six pregnancies, c...
Choi I, Collante WR, Simmen RC, Simmen FA.Pig blastocysts exhibit a transient period of estrogen production at periimplantation, with a second, more sustained period of estrogen synthesis occurring in endometrium and placenta at later pregnancy. Previously we reported the isolation of cDNA clones encoding a novel isoform of cytochrome P450 aromatase (the terminal enzyme in the estrogen biosynthetic pathway) from porcine periimplantation blastocysts. The present study investigated pregnancy-associated expression, in blastocysts and maternal reproductive tract tissues of this and an additional mRNA transcript encoding a distinct P450 ar...
Brück I, Anderson GA, Hyland JH.The influence of different maternal plasma progesterone concentrations on embryonic glucose metabolism was studied. Uterine flushes were obtained after treating ovariectomized mares (n = 3) with 0 (control), 100 or 200 mg progesterone daily for 7 d. A group of progesterone-induced proteins (PIP) of Mr approximately 20,000 were identified in flushes from progesterone treatments by SDS-PAGE but were not observed in control flushes. Progesterone-induced proteins were removed from half the pooled flush in each treatment group by Sepharose blue CL-6B. In a 3 x 2 factorial (progesterone treatments, ...
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...
Gonzales DS, Jones JM, Pinyopummintr T, Carnevale EM, Ginther OJ, Shapiro SS, Bavister BD.The behaviour of bovine, equine and human blastocysts was studied in vitro by time-lapse videomicrography and computer imaging. This study revealed that cytoplasmic extensions of the trophectoderm ['trophectoderm projections' (TEP)] were expressed by embryos of all three species, prior to or during zona escape. Bovine and human blastocysts escaped their zonae with a combination of blastocoele expansion, collapse and re-expansion coupled with the penetration of the zona pellucida by TEP. In equine embryos, after several cycles of blastocoele expansion and collapse, trophectoderm ruptured the zo...
Gastal MO, Gastal EL, Kot K, Ginther OJ.The temporal relationships among day of conceptus fixation (cessation of mobility), conceptus diameter, uterine tone, uterine contractility, and myometrial and endometrial thickness of the middle and caudal segments of the uterine horns were assessed in 13 pony mares with fixation in the caudal segment of a uterine horn. The mean day of fixation (14.9 +/- 0.3) was established by 2-h mobility trials. Uterine tone increased (P < 0.0001) gradually over Days 11 to 21, whereas uterine contractility decreased (P < 0.0001) between Days 14 and 18. The diameter of the spherical embryonic vesicle ...
Grøndahl C, Hyttel P.The nucleolus is believed to be the active site of rRNA synthesis in all eukaryotic cells. In preimplantation embryos, the embryonic genome is apparently more or less silent up to a species-specific developmental stage at which a major burst of transcription occurs. Here we report on nucleologenesis and some ultrastructural aspects of the onset of RNA synthesis in equine embryos during in vivo development. The zygotes and embryos up to blastocyst stages were surgically recovered from normally cycling mares. Mares were induced to ovulate by treatment with 3000 IU hCG and inseminated 20 and 34 h...
Falge R, Ehling C, Niemann H.The conservation of endangered breeds as live animals is at present the main national strategy of the government and breeding organizations to maintain genetic diversity. Fourty-three breeds and some old strains of cattle, pig, sheep, goat and horses are currently involved. Cryopreservation and banks for sperm, embryos or DNA are another type of genetic material which could subsequently be used for breeding and production in agriculture. Present semen banks involve 9 endangered cattle breeds and also a small amount of deep-frozen sperm of some endangered sheep and horse breeds. Only 2 embryo b...
Seidel GE.Principles and procedures for cryopreservation of equine embryos are described. Embryos less than 250 microM in diameter can be cryopreserved successfully if glycerol is used as the cryoprotectant. Cooling is takes place in such a way that most of the water leaves the cells before intracellular ice forms, and glycerol is removed after thawing without undue osmotic swelling of cells. Vitrification procedures also show promise for small embryos. Satisfactory procedures for cryopreserving embryos of more than 250 microM in diameter are not yet available.
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...
Wilsher S, Rigali F, Kovacsy S, Allen WT.Vitrification of embryos >300 µm in diameter requires puncture of the glycoprotein capsule, although the size of the hole compatible with embryo survival is unknown. Forty-five day-7 or -8 embryos were punctured using a 30-µm glass biopsy pipette mounted on a micromanipulator (n = 20) or manually with either an acupuncture needle (∼100-µm diameter -hole; n = 10) or a microneedle with a <1 µm tip to produce a ∼30-µm diameter hole (n = 15) before transferring to recipient mares; further 12 embryos were punctured with either the acupuncture needle or microneedle before being cultured ...
Sirois J, Betteridge KJ.To recover intact Day-10.5 to Day-16.5 equine conceptuses (Day 0 = ovulation), a rigid catheter was used for 131 collections from donor mares diagnosed pregnant by ultrasonography. A total of 139 conceptuses were recovered, comprising 124 singletons, six pairs of twins and one set of triplets. Of these, 120 (86%) were intact after the collection, 14 (10%) had collapsed, and in five cases (4%), collapsed trophoblastic membranes were surrounded by an intact capsule. The recovery rate of intact conceptuses ranged from 99% on Days 10.5 to 12.5 to 40% on Day 16.5. More uterine flushes per recovery ...
Staigmiller RB, Bellows RA, Anderson GB, Seidel GE, Foote WD, Menino AR, Wright RW.Superovulation has been practiced in cattle for more than 50 years but the results have been highly variable. Scientists at six locations compared a horse pituitary extract (HAP) with a single batch of porcine FSH (pFSH) to determine the efficacy of these hormones to induce superovulation and to test for variability in the superovulatory response. Acetone-dried equine pituitaries were suspended in 40% ethanol containing 6% ammonium acetate, and the supernatant was mixed with 2.5 volumes of cold ethanol. The resulting precipitate was washed with cold ether and dried. Total doses of 18 mg of HAP...
Gastal GDA, Scarlet D, Melchert M, Ertl R, Aurich C.In embryos subjected to assisted reproductive techniques, epigenetic modifications may occur that can influence embryonic development and the establishment of pregnancy. In horses, the storage temperature during transport of fresh embryos before transfer is a major concern. The aim of this study was, therefore, to determine the effects of two storage temperatures (5 °C and 20 °C) on equine embryos, collected at day seven after ovulation and stored for 24 h, on: (i) morphological development; (ii) expression of candidate genes associated with embryo growth and development, maternal recognitio...
Brockschmidt LD, Loch WE, Sikes JD.An experiment was conducted to test the effect of repeated transcervical (non-surgical) uterine flushing and a prostaglandin analogue (PG) on the estrous cycle of pony mares. Uteri in group A were trancervically flushed for embryos 7 to 9 days post ovulation. In addition, group B mares were given 5 ml of PG by intramuscular injection on the day of flushing. Group C served as controls and were not flushed or given PG but were allowed to cycle normally. All mares (except controls) were bred A.I. every other day during estrus. There was no effect on embryo recovery rate from repeated flushing or ...
Brom-de-Luna JG, Salgado RM, Felix MR, Canesin HS, Stefanovski D, Diaw M, Hinrichs K.In vitro production of horse embryos via intracytoplasmic sperm injection (ICSI) is a useful clinical and research technique. Current rates of blastocyst production are typically sub-optimal, and few methods to increase the rate of equine blastocyst development have been reported. Factors that might improve blastocyst production in a horse embryo culture system were explored. Myo-inositol is found in the horse oviduct and improves blastocyst development in other species, thus Experiment 1 was conducted to assess the effect of 10 mM myo-inositol added to Day 0-5 embryo culture medium, using hor...
Gündüz MC, Kaşikçi G, Kaya HH.In this study, the effects of oxytocin and an analog of prostaglandin (cloprostenol) on the uterine involution and pregnancy rates were investigated. Mares received 3 ml of 0.9% NaCl in Group C (n=10), 30 IU/mare of oxytocin in Group O (n=10) and 250 microg/mare of cloprostenol in Group P (n=10) within 12h after parturition. The gravid uterine horn's cross-sectional diameter was measured by ultrasonography. The mean uterine diameters did not differ significantly between the treatment (O and P) and the control (C) groups (p>0.05). The difference between the postpartum ovulation periods (Grou...
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 ...
McKinnon AO, Lacham-Kaplan O, Trounson AO.The use of intracytoplasmic sperm injection (ICSI) for in vitro fertilization of equine oocytes and the developmental potential of these oocytes after transfer to the Fallopian tubes of synchronized mares were examined. Oocytes were aspirated from mature follicles 39 h after injection of a GnRH analogue and transported 190 km at 39 degrees C. Semen from a fertile and an infertile stallion was frozen and prepared for injection. Successfully injected oocytes were transferred surgically into the ampulla of the Fallopian tube either: (i) 4-8 h after semen injection; or (ii) after 24-48 h culture b...
Boakari YL, El-Sheikh Ali H, Dini P, Loux S, Fernandes CB, Scoggin K, Esteller-Vico A, Lawrence L, Ball B.High blood urea nitrogen (BUN) in cows and ewes has a negative effect on embryo development; however, no comparable studies have been published in mares. The aims of the present study were to evaluate the effects of high BUN on blastocoele fluid, systemic progesterone and Day 14 equine embryos. When a follicle with a mean (±s.e.m.) diameter of 25±3mm was detected, mares were administered urea (0.4g kg-1) with sweet feed and molasses (n=9) or sweet feed and molasses alone (control; n=10). Blood samples were collected every other day. Mares were subjected to AI and the day ovulation was detect...
Raz T, Amorim MD, Stover BC, Card CE.The objective of this study was to compare the efficacy of purified equine- and porcine-FSH treatment regimes in mares in early vernal transition. Mares (n = 22) kept under ambient light were examined ultrasonographically per-rectum, starting January 30th. They were assigned to one of two treatment groups using a sequential alternating treatment design when a follicle >or= 25 mm was detected. In the eFSH group, mares were treated twice daily with equine-FSH, and in the pFSH group mares were treated twice daily with porcine-FSH; treatments were continued until follicle(s) >or= 35 mm, and 24 h l...
Kingma SE, Thibault ME, Betteridge KJ, Schlaf M, Gartley CJ, Chenier TS.Poor survival of cryopreservation by equine expanded blastocysts may involve low penetration of the embryonic capsule by cryoprotective agents (CPAs). This study characterized the permeation and accumulation rates of the CPAs ethylene glycol (EG) and glycerol (GLY) across isolated capsule in vitro, using a dual-chambered Valia-Chien permeation apparatus. Pieces of Days 14 to 18 ± 1 capsules separated media in the "donor" chamber containing either 1.5 M EG (n = 6), 0.74 M EG (n = 5), 0.87 M GLY (n = 7), or 0.15 M NaCl (saline, SAL) (n = 6), from the "recipient" chamber. Concentrations of CPA, ...
Camacho CA, Estradé MJ, Cazales N, Caballeros JE, Fiala-Rechsteiner SM, Neves AP, Mattos RC.This experiment aimed to verify if the proteins present in a 13 day conceptus induce changes in the equine endometrial ultra-structure, histology, and vascularization, two days after its infusion. Ten healthy cyclic mares were used. Once estrus was confirmed, mares were examined daily to detect ovulation (day 0). After ovulation, mares were examined daily until day seven by transrectal palpation and B-mode and Doppler ultrasonography. In this first cycle, intrauterine biopsies were collected at day seven after ovulation, constituting the Cyclic group (n = 10). In the second cycle, the same mar...
Brück I, Hyland JH.The contributions of 2 biochemical pathways to the total metabolism of glucose (the Embden-Meyerhof pathway [EMP] and the pentose phosphate pathway [PPP]), were assessed for equine embryos recovered on Day 4.5, 7.5 and 11.5 post ovulation. At all developmental stages studied, glucose was metabolized through both pathways. Through the EMP, the amounts of glucose metabolized per nl embryo volume per hour were 4.0, 9.9 and 3.1 pmol, whereas via the PPP, amounts were 0.9, 1.7 and 0.07 pmol for Day-4.5, -7.5 and -11.5 embryos, respectively. The ratio of EMP:PPP with age was 9.7 for Day -4.5 embryos...
Martin-Pelaez S, Rabow Z, de la Fuente A, Draheim P, Loynachan A, Fiehn O, Meyers S, Lyman C, Dini P.Postmortem and pre-euthanasia oocyte retrieval provides the last opportunity to preserve the genetic material in mares. Pentobarbital (PB) is the most common euthanasia agent; however, its effect on the developmental competence of oocytes has not been determined. Here, we evaluated the concentration of PB in equine follicular fluid (FF) and investigated its effect on the developmental competence of oocytes using a bovine IVF model to overcome the low availability of equine oocytes. The concentration of PB was measured by gas-chromatography/mass-spectrometry in FF collected from mare ovaries im...
Ginther OJ.After the cessation of equine embryo mobility (fixation) on mean Day 16, the embryonic vesicle is rotated or oriented so that the pole with the embryo proper is opposite to the mesometrial attachment. Orientation involves massage of the vesicle by contractions of the turgid uterine horn and greater thickening of the vesicle at the pole with the embryo proper. Thickening of the dorsal endometrium (encroachment) especially on each side of the mesometrial attachment accounts for a guitar-pick shape of the vesicle when viewed in cross section of the uterine horn. On Days 21-40, the allantoic sac e...
Rieger D, Bruyas JF, Lagneaux D, Bézard J, Palmer E.The decrease in embryo viability caused by cryopreservation may be due, in part, to metabolic disturbances. To determine the effect of cryopreservation on metabolism, Day -6.5 horse embryos were either frozen and thawed using glycerol as the cryoprotectant, given only the glycerol treatment or washed an equal number of times in phosphate buffered saline (PBS). Before and after treatment, individual embryos were incubated with L-[14C(U)]-glutamine, to measure Krebs cycle activity, and D-[5-3H]-glucose, to measure Embden-Meyerhof pathway activity. Before treatment, glucose metabolism ranged from...
Willink DL, Smeenk LA, van Oyen PW, de Kruif A.Data from the literature and own data for 67 twin pregnancies were used to establish the factors essential to the decision on how to treat twins at different gestational ages. Spontaneous (natural) reduction was compared with manual embryo reduction. Manual embryo reduction is always indicated when a twin is diagnosed before day 16 after ovulation. Thereafter, the type of fixation is the main determinant. Manual embryo reduction is always first choice for bilateral and unilateral non adjacent embryos and must be applied as early as possible. The change of natural reduction up to day 30, is hig...
Guèrand M, Mahla R, Lagneaux D, Amigues Y, Palmer E, Bézard J.Paternity analysis was performed on the DNA of 21 equine embryos collected nonsurgically 10 days after ovulation from known mares, but involving 3 possible sires. After extraction, the DNA of each embryo was typed by radioactive PCR amplification using 10 characterised microsatellites; HMS 1, 2, 5, 6, 7 and 8 (Guérin et al. 1994) and HTG 3, 4, 6 and 10 (Marklund et al. 1994). The 21 dams and 3 sires were genotyped using DNA extracted from blood and amplified by PCR. After electrophoresis and autoradiography of the PCR products of the embryo and parents, the alleles of the embryo were compared...
Brück I, Lehn-Jensen H, Yde G.A Warmblood mare was observed to ovulate spontaneously 12 follicles within 2 days, none of which exceeded 22 mm in diameter. On Days 13 and 17 after ovulation, 6 embryonic vesicles were identified in the uterus by ultrasonography but by Day 26, 5 of the vesicles had disappeared. Development of the surviving conceptus was monitored until Day 42. Plasma progesterone concentrations rose to 14 ng/ml on Day 7, decreased over the next 8 days and then plateaued to around 4-6 ng/ml until Day 70. The occurrence of multiple spontaneous ovulations was diagnosed repeatedly in this mare. However, the devel...
Clément F, Vincent P, Mahla R, Meriaux JC, Palmer E.The aim of the present study was to determine which artificial insemination results in fertilization when mares are inseminated several times before ovulation. Mares in oestrus were inseminated over 62 cycles with fresh semen at 48 h intervals from when a follicle > or =30 mm in diameter was detected until ovulation. The number of inseminations was limited to three. Three fertile stallions were used and a different stallion was used for each artificial insemination. The order of the three stallions was changed for each cycle. Embryos were collected between day 10 and day 12 after ovulation and...
Barfield JP, McCue PM, Squires EL, Seidel GE.Cryopreservation of equine embryos>300microm in diameter results in low survival rates using protocols that work well for smaller equine embryos. These experiments tested the potential benefit of incorporating a dehydration step prior to standard cryopreservation procedures. Forty-six, day 7-8, grade 1, equine embryos 300-1350microm in diameter were subjected to one of the following treatments: (A) 2 min in 0.6M galactose, 10min in 1.5M glycerol, slow freeze (n=21); (B) 10min in 1.5M glycerol, slow freeze (n=15); (C) 2min in 0.6M galactose, 10min in 1.5M glycerol, followed by exposure to thaw ...
Gonzalez-Castro RA, Amoroso-Sanches F, Stokes JE, Graham JK, Carnevale EM.Oocyte activation is initiated when a fertilising spermatozoon delivers sperm-borne oocyte-activating factor(s) into the oocyte cytoplasm. Candidates for oocyte activation include two proteins, phospholipase Cζ1 (PLCZ1) and postacrosomal WW-binding protein (PAWP; also known as WBP2 N-terminal like (WBP2NL)). We localised PLCZ1 and WBP2NL/PAWP in stallion spermatozoa and investigated the PLCZ1 content and sperm parameters as well as cleavage after intracytoplasmic sperm injection (ICSI). PLCZ1 was identified as 71-kDa protein in the acrosomal and postacrosomal regions, midpiece and principal p...
Poitras P, Guay P, Vaillancourt D, Zidane N, Bigras-Poulin M.The main objective of this study was to evaluate two freezing protocols and the effect of agar embedding on survival of day 6.5 equine embryos. A total of 133 embryos were used, in one group (n = 51), embryos were first embedded in agar before the freezing protocol was started. A freezing protocol to -30 degrees C or -33 degrees C was used before plunging embryos into liquid nitrogen (LN2). The embryos were thawed in water at 37 degrees C, evaluated and placed in culture. After 24 h culture, the embryos were evaluated for their morphology and development. No differences were observed between e...
Kooistra LH, Ginther OJ.At day 24 of gestation, pregnant mares were allotted to 1 of 5 treatment groups (3 to 5 mares/group): group A--nontreated controls; group B--intraembryonic injection of 4 mg of colchicine on day 24; group C--removal of embryo on day 24; group D--subcutaneous injection of 1.25 mg of prostaglandin F2alpha (PGF2alpha) on day 32; and group E--removal of embryo on day 24 and subcutaneous injection of PGF2alpha on day 32. In all mares treated with colchicine (group B), the fetal bulge was absent within 2 days. The interval from injection of colchicine to onset of estrus was very short (mean, 4 days)...
Braun J.Although foals born after embryo transfer are eligible for registration in the majority of horse breeds, application of embryo transfer is still rare. This is mainly due to the lack of a possibility for superovulation. Uterine stage embryos can be recovered by a non-surgical flushing technique. Transfer can be accomplished by non-surgical as well as surgical methods. In contrast to the situation in cattle, most related technologies are scarcely available. Methods of cryopreservation as well as bisection of embryos are hampered by the fact that suitable embryos (morula) can be collected from th...
Checura CM, Momont HW, Castañeira C, Flores-Bragulat A, Losinno L.In horses, prostaglandin E (PGE) is produced by embryos around Day 5 post-ovulation; PGE functions directly at the oviduct promoting embryo transport into the uterus. Non-surgical collection of horse embryos for cryopreservation is recommended at Day 6.5-7 post-ovulation. It was proposed that misoprostol administered orally will hasten oviductal transport of horse embryos. In Experiment 1 (n = 15) there was comparison of time of embryo recovery (Day 6 and 6.5 post-ovulation) from mares administered misoprostol (Day 5 and 5.5) orally to that of untreated mares. On Day 6, embryo collections were...
Rodriguez J, Maserati M, Robilotta T, Augusto G, Alonso MA, Redoan M, Tibary A, Fleury P.Field collection of oocytes in mares using transvaginal follicular aspiration (TVA) for embryo production has the potential to revolutionate the equine industry. Protocols for TVA in specialized laboratory settings have been described in the scientific literature since the early 1980s. The objective of this study was to determine the success rate of TVA oocytes recovery under ambulatory conditions. A secondary goal of this study was to determine if TVA is associated with any health complications when performed by recently trained practitioners in the field. Follicles (n = 296) from 66 adult c...
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...
Choi YH, Roasa LM, Love CC, Varner DD, Brinsko SP, Hinrichs K.This study was conducted to evaluate in vivo and in vitro development of in vitro-matured equine oocytes fertilized by intracytoplasmic sperm injection. Oocytes were collected from slaughterhouse-derived ovaries, matured in vitro, and injected with frozen-thawed stallion sperm. In vivo development was assessed after transfer of injected oocytes to the oviducts of recipient mares. Mares were killed 7.5-8.5 days after transfer and the uterus and oviducts flushed for embryo recovery. Of 132 injected oocytes transferred, 69 (52%) were recovered; of these, 25 (36%) were blastocysts with a blastocoe...
