Topic:Blastocysts
Blastocysts in horses represent an early stage of embryonic development following fertilization and prior to implantation in the uterus. This stage is characterized by a fluid-filled cavity surrounded by a layer of cells, which will eventually develop into the embryo and placenta. Research on equine blastocysts focuses on their formation, development, and viability, as well as factors affecting successful implantation and pregnancy outcomes. Studies often explore the molecular and cellular mechanisms underlying blastocyst development, the impact of maternal health and environmental conditions, and techniques for improving reproductive success in horses. This page compiles peer-reviewed research studies and scholarly articles that examine the formation, development, and clinical implications of blastocysts in equine reproduction.
Nicotinic acid treatment improves the developmental potential of equine oocytes for cloned embryo production. Nicotinic acid (NA) treatment during in vitro maturation (IVM) has been shown to elevate nicotinamide adenine dinucleotide (NAD) levels and improve oocyte developmental competence. Suboptimal equine oocyte IVM systems currently limit the efficiency of viable embryo in vitro production. This study evaluated NA supplementation during IVM for cloned equine embryo production, using oocytes from abattoir-sourced ovaries and live mares via ovum pick-up (OPU). Abattoir-derived oocytes (n = 694) were treated without or with 50 or 200 μM NA during the 18 h holding period (Pre-IVM). Next, OPU-deriv...
Transcriptomic profiling reveals similarities between equine IVF and ICSI embryos. In vitro production of equine embryos has been performed using intracytoplasmic sperm injection (ICSI) for the last two decades. Since 2022, a repeatable protocol for conventional in vitro fertilization (IVF) provides a successful alternative. However, little is known about the influence of the fertilization method on embryo quality and the transcriptomic profile. In this study, we aimed to examine differentially expressed genes (DEGs) between ICSI and IVF embryos in the horse. Therefore, ten equine sibling blastocysts, produced in vitro by either ICSI or IVF from three different mares, were s...
Incubation of Frozen-Thawed Semen Under Capacitating Conditions Supports Successful In Vitro Fertilization and Improves Intracytoplasmic Sperm Injection-Results in Horses. In 2022, a repeatable protocol for in vitro fertilization (IVF) using fresh semen was established in horses. This facilitated successful capacitation of equine semen allowing to explore novel applications. Objective: We aimed to extend this technique to IVF with frozen-thawed semen and intracytoplasmic sperm injection (ICSI), and determine the outcome parameters such as blastocyst production and euploidy rates. Methods: A total of 221 oocytes were subjected to either IVF with frozen-thawed semen, ICSI with frozen-thawed semen incubated under capacitating conditions (ICSI cap) or control ICSI w...
Lipopolysaccharide reduces progesterone and cytokines in equine follicular fluid without affecting oocyte development in vitro. Lipopolysaccharide (LPS) in follicular fluid impairs steroid production and oocyte developmental competence in cows and mice. This study assessed LPS concentrations in equine follicular fluid and their association with steroid and some cytokine levels. Additionally, we evaluated whether LPS exposure during in vitro maturation (IVM) affects equine oocyte developmental competence. In experiment 1, follicular fluid from large follicles (>30 mm in diameter) was collected from 16 slaughterhouse mares, and concentrations of LPS, estradiol, progesterone, TNF-α, and IL-6 were measured. In experiment...
Studies on in vitro production of equine embryos by Intracytoplasmic Sperm Injection (ICSI) using non-sorted, or sex-sorted, frozen/thawed stallion sperm: Effects on post-thaw sperm quality, cleavage and blastocyst rates, and characterization of cellular events during the first 24 hours post-fertilization via confocal microscopy. Intracytoplasmic sperm injection (ICSI) using frozen/thawed (F/T) stallion sperm is a common procedure in the equine breeding industry. Historically, sex-sorted (SS) F/T stallion sperm has yielded lower cleavage (<30 %) and blastocyst rates (<5 %) after ICSI when compared to non-sorted (NS) F/T sperm. Recently, a new technology for sperm sex-sorting (Genesis III) has been validated by a commercial company. In Experiment 1, the post-thaw quality between NS-F/T and SS-F/T stallion sperm produced with this technology was compared. The post-thaw sperm motility was higher in NS-F/T (41 %) than i...
Abnormal cleavage patterns in equine in vitro-produced embryos lead to higher early pregnancy loss. Despite significant advances, in vitro production (IVP) of equine embryos continues to lack standardised embryo classification criteria and is associated with increased rates of early pregnancy loss compared with in vivo-derived blastocysts. Objective: To evaluate morphokinetic characteristics of the first mitotic division and early embryonic development in IVP blastocysts and their association with embryo development, as well as pregnancy rate and early pregnancy loss following embryo transfer. Methods: Retrospective analysis of archived material and clinical records. Methods: We retrospectiv...
Phenylbutazone treatment of mares does not reduce embryo production via intracytoplasmic sperm injection. Production of embryos via intracytoplasmic sperm injection (ICSI) is a common clinical procedure in equine practice. A recent study indicates that treatment of mares with phenylbutazone paste significantly lowers blastocyst production after ICSI of recovered oocytes. The current study was conducted to determine the effect of different methods of phenylbutazone administration on in vitro embryo production of recovered oocytes in mares. Unassigned: In study 1 (July 29 through September 18, 2024), 14 mares received either oral liquid phenylbutazone once daily for 10 days or no treatment. On the l...
Blastocyst production by conventional in vitro fertilization (cIVF) in horses: Effects of sperm storage method, incubation timing of cool-stored semen before gamete co-incubation, and comparisons between cIVF and intracytoplasmic sperm injection (ICSI). Currently, Intracytoplasmic Sperm Injection (ICSI) on in vitro-matured equine oocytes obtained by transvaginal oocyte aspiration (TVA) is the method of choice for in vitro production of equine blastocysts in a clinical setting. A protocol for conventional in vitro fertilization (cIVF) using either fresh or frozen/thawed stallion sperm incubated under capacitating conditions (Tyrode's Albumin Lactate Pyruvate medium + penicillamine, hypotaurine, epinephrine; FERT-PHE) has been recently reported. Several factors that may impact the clinical applicability of this cIVF protocol have yet to be st...
Effect of Pre-IVM Duration with cAMP Modulators on the Production of Cloned Equine Embryos and Foals. The asynchrony of cytoplasmic and nuclear maturation in cumulus-oocyte complexes (COCs) due to prematurely declining concentrations of cyclic adenosine monophosphate (cAMP) has been shown to result in reduced oocyte developmental competence. The objective of this study was to evaluate the effect of pre-IVM treatment with cAMP modulators for different durations on the developmental potential of equine oocytes used for cloned embryo production. Collected COCs were transferred to cryovials filled with transport medium at 20-22 °C. Within the cryovials, the COCs were either untreated (Control) fo...
Preimplantation genetic testing in horses: biopsy of Piezo-ICSI embryos for sex, coat color, and disease alleles. The introduction of ovum pick-up, Intracytoplasmic Sperm Injection (ICSI) and in vitro embryo production in equine reproduction offers significant advantages, including overcoming infertility, high blastocyst yields, embryo cryopreservation, and preimplantation genetic testing (PGT) by collecting few cells extruded spontaneously during embryo growth using a mouth pipette. PGT enables embryo selection before transfer and is particularly useful in breeds with a preference for specific offspring genders, coat color, and detecting genetic diseases. Here, we present the first large-scale validation...
Equine in vitro fertilization with frozen-thawed semen is associated with shortened pre-incubation time and modified capacitation-related changes. We recently reported successful equine IVF using fresh semen pre-incubated for a prolonged period (22 h) before co-culture with oocytes. In this study, we evaluated the feasibility of equine IVF with frozen-thawed sperm and evaluated capacitation-related changes in these sperm over the pre-incubation period. Sperm selected via a commercial sperm separation device (SSD) yielded significantly higher fertilization than did sperm selected by swim-up or by colloid centrifugation. Using the SSD method, fertilization rates with sperm pre-incubated for 15 min, 3 h, 6 h, and 9 h were 7.1, 22.2, 38...
Marked blastomere herniation is the only post-thaw morphological characteristic associated with a reduced pregnancy rate for in vitro produced equine blastocysts. It is not known whether the likelihood of pregnancy after transfer of cryopreserved equine in vitro produced (IVP) blastocysts is related to post-thaw morphological characteristics, or could be impacted by other factors such as embryo biopsy prior to cryopreservation. This study examined associations between post-thaw morphological appearance of equine IVP embryos, and biopsy prior to slow-freezing, on the likelihood of pregnancy. Biopsy of IVP blastocysts was performed by harvesting cells herniated through the intra-cytoplasmic sperm injection (ICSI)-derived hole in the zona pellucida (ZP). M...
Oocyte holding and in vitro maturation duration between 28 and 34 hours do not affect equine OPU-ICSI outcomes. Previous studies in the horse highlight the potential benefit of prolonged in vitro maturation (IVM) (34 h) compared to short IVM (24 h) with or without prior oocyte holding, but little is known about the optimal IVM duration within this interval. To determine the effect of oocyte holding and duration of IVM ranged between 28 and 34 h on nuclear maturation, cleavage, blastocyst formation, and pregnancy rates, a retrospective study was performed in an equine clinical OPU-ICSI setting. The study included data of 2114 aspirated oocytes from 201 OPU-ICSI sessions. Duration of IVM was divided in...
IVF with frozen-thawed sperm after prolonged capacitation yields comparable results to ICSI in horses: A morphokinetics study. Intracytoplasmic sperm injection (ICSI) is the current clinical practice for the in vitro production of equine embryos. The use of conventional fertilization methods such as in vitro fertilization (IVF), has historically been associated with poor success in horses. However, recent improvements have led to better outcomes with IVF, though only when using fresh semen, which limits its use in clinical practice. IVF remains in its infancy in equine reproduction, and several unknowns remain about the technique. One significant gap in knowledge concerns the morphokinetics of IVF embryos and how they...
A prospective study of the proteome of equine pre-implantation embryo. The present study was conducted to investigate the global proteome of 8-day-old equine blastocysts. Follicular dynamics of eight adult mares were monitored by ultrasonography and inseminated 24 h after the detection of a preovulatory follicle. Four expanded blastocysts were recovered, pooled, and subjected to protein extraction and mass spectrometry. Protein identification was conducted based on four database searches (PEAKS, Proteome Discoverer software, SearchGUI software, and PepExplorer). Enrichment analysis was performed using g:Profiler, Panther, and String platforms. After the elimina...
Early Embryonic Development in Agriculturally Important Species. The fertilization of oocytes ovulated by pigs, sheep, cows, and horses is not considered a limiting factor in successful establishment of pregnancy. Pig, sheep, and cow embryos undergo cleavage to the blastocyst stage, hatch from the zona pellucida, and undergo central-type implantation. Hatched blastocysts of pigs, sheep, and cows transition from tubular to long filamentous forms to establish surface area for exchange of nutrients and gases with the uterus. The equine blastocyst, surrounded by external membranes, does not elongate but migrates throughout the uterine lumen before attaching to ...
Successful equine in vitro embryo production by ICSI – effect of season, mares’ age, breed, and phase of the estrous cycle on embryo production. This retrospective study aimed at identifying factors that contribute to the success of equine in vitro embryo production by intracytoplasmic sperm injection (ICSI). A total of 7993 ovum pick-up (OPU) sessions were performed, totaling 2540 donor mares and semen from 396 stallions. Oocytes were aspirated at multiple sites in Brazil and were sent to the laboratory, within 6 h from OPU, in pre-maturation medium where they were in vitro matured (IVM) followed by ICSI and in vitro embryo culture for 7-8 days. The number of recovered oocytes, matured oocytes, cleaved embryos and blastocysts were us...
Characterization of preovulatory follicular fluid secretome and its effects on equine oocytes during in vitro maturation. In vitro maturation (IVM) of oocytes is clinically used in horses to produce blastocysts but current conditions used for horses are suboptimal. We analyzed the composition of equine preovulatory follicular fluid (FF) secretome and tested its effects on meiotic competence and gene expression in oocytes subjected to IVM. Preovulatory FF was obtained, concentrated using ultrafiltration with cut-off of 10 kDa, and stored at -80 °C. The metabolic and proteomic composition was analyzed, and its ultrastructural composition was assessed by cryo-transmission microscopy. Oocytes obtained post-mortem ...
The ability of donkey sperm to induce oocyte activation and mule embryo development after ICSI. Members of the Equus genus exhibit a fascinating capacity for hybridization, giving rise to healthy offspring. Mules, resulting from the mating of a mare with a jack, represent the most prevalent equid hybrid, serving diverse roles in our society. While in vitro embryo production, particularly through Intracytoplasmic Sperm Injection (ICSI), has rapidly gained significance in domestic horses, the in vitro production in other equids remains largely unexplored. Utilizing donkey sperm for fertilizing horse oocytes not only addresses this gap but also provides an opportunity to investigate donkey ...
Cloned Foal Born from Postmortem-Obtained Ear Sample Refrigerated for 5 Days Before Fibroblast Isolation and Decontamination of the Infected Monolayer Culture. A 6-year-old mare, a valuable polo horse, died of complications following postcolic surgery. To preserve its genetics, ear skin samples were collected immediately after death and stored in an equine embryo transfer medium at 4°C for 5 days. After trypsin digestion, monolayer fibroblast cultures were established, but signs of massive bacterial infection were found in all of them. As an ultimate attempt for rescue, rigorously and repeatedly washed cells were individually cultured in all wells of four 96-well dishes. New monolayers were established from the few wells without contamination and us...
Genome-wide equine preimplantation genetic testing enabled by simultaneous haplotyping and copy number detection. In different species, embryonic aneuploidies and genome-wide errors are a major cause of developmental failure. The increasing number of equine embryos being produced worldwide provides the opportunity to characterize and rank or select embryos based on their genetic profile prior to transfer. Here, we explored the possibility of generic, genome-wide preimplantation genetic testing concurrently for aneuploidies (PGT-A) and monogenic (PGT-M) traits and diseases in the horse, meanwhile assessing the incidence and spectrum of chromosomal and genome-wide errors in in vitro-produced equine embryos....
Oxidative stress in donor mares for ovum pick-up delays embryonic development. The in vitro production of equine embryos via ovum pick-up (OPU) and intracytoplasmic sperm injection (ICSI) has increased rapidly. There is a marked effect of the individual mare on the outcome of OPU-ICSI, but little is known about the influence of the mare's health condition. This study aimed to investigate the potential associations between the concentrations of interleukin-6 (IL-6), reactive oxygen metabolites (d-ROMs), and biological antioxidant potential (BAP) in serum of oocytes' donor mares and the subsequent embryonic development. Just before OPU, a blood sample was collected from 28...
Use of confocal microscopy and intracytoplasmic sperm injection (ICSI) to assess viability of equine oocytes from young and old mares after vitrification. The impact of vitrification on oocyte developmental competence as a function of donor age remains an important issue in assisted reproductive technologies (ARTs). Methods: Equine germinal vesicle (GV) or metaphase II (M(II) oocytes were vitrified using the Cryotop® method. Spindle organization and chromosome alignment were evaluated from confocal imaging data sets of in vivo (IVO) or in vitro (IVM) matured oocytes subjected to vitrification or not. Intracytoplasmic sperm injection (ICSI) from the same groups was used to assess developmental potential. Results: An increase in chromosome misali...
Embryo Pulsing: Repeated Expansion and Contraction of In Vivo and In Vitro Equine Blastocysts. Morphokinetic evaluation of embryo development has allowed the discovery of events occurring during blastulation. Here, we describe equine embryo pulsing, determined as continued expansion and contraction of both in vivo and in vitro produced blastocysts. Using time-lapse imaging, we demonstrated that pulsing starts during early blastocyst development of in vitro-produced embryos in horses. The median time for a complete contraction was 0.22h (0.08h-2h; min-max) where embryos reduced their sizes around 12.0% (median; 2.3%-27.0%) and the median time for an expansion was 3.3h (0.75-9.0h) where e...
Horse Somatic Cell Nuclear Transfer Using Zona Pellucida-Enclosed and Zona-Free Oocytes. Horse cloning by somatic cell nuclear transfer (SCNT) is an attractive scientific and commercial endeavor. Moreover, SCNT allows generating genetically identical animals from elite, aged, castrated, or deceased equine donors. Several variations in the horse SCNT method have been described, which may be useful for specific applications. This chapter describes a detailed protocol for horse cloning, thus including SCNT protocols using zona pellucida (ZP)-enclosed or ZP-free oocytes for enucleation. These SCNT protocols are under routine use for commercial equine cloning.
Cloning horses by somatic cell nuclear transfer: Effects of oocyte source on development to foaling. The cloning of horses is a commercial reality, yet the availability of oocytes for cloned embryo production remains a major limitation. Immature oocytes collected from abattoir-sourced ovaries or from live mares by ovum pick-up (OPU) have both been used to generate cloned foals. However, the reported cloning efficiencies are difficult to compare due to the different somatic cell nuclear transfer (SCNT) techniques and conditions used. The objective of this retrospective study was to compare the in vitro and in vivo development of equine SCNT embryos produced using oocytes recovered from abatt...
Vitrifying expanded equine embryos collapsed by blastocoel aspiration is less damaging than slow-freezing. The cryotolerance of equine blastocysts larger than 300 μm can be improved by aspirating blastocoele fluid prior to vitrification; however, it is not known whether blastocoele aspiration also enables successful slow-freezing. The aim of this study was therefore to determine whether slow-freezing of expanded equine embryos following blastocoele collapse was more or less damaging than vitrification. Grade 1 blastocysts recovered on day 7 or 8 after ovulation were measured (>300-550 μm, n = 14 and > 550 μm, n = 19) and blastocoele fluid was aspirated prior to slow-freezing in 10...
Equine in vitro produced blastocysts: relationship of embryo morphology, stage and speed of development to foaling rate. Information on factors associated with developmental competence of equine in vitro -produced (IVP) blastocysts is lacking. Objective: To determine the relationships of stage, grade, day of development, and specific morphological parameters of equine IVP blastocysts, to pregnancy and foaling rates. Methods: Photomicrographs of 316 IVP embryos with known pregnancy outcomes were scrutinised individually by four observers. Inter-observer variation was assessed, and pregnancy outcome evaluated in relation to day of blastocyst development and assigned grade and stage. Individual component analysis w...
First sex modification case in equine cloning. Somatic cell nuclear transfer (SCNT) is an asexual reproductive technique where cloned offspring contain the same genetic material as the original donor. Although this technique preserves the sex of the original animal, the birth of sex-reversed offspring has been reported in some species. Here, we report for the first time the birth of a female foal generated by SCNT of a male nuclear donor. After a single SCNT procedure, 16 blastocysts were obtained and transferred to eight recipient mares, resulting in the birth of two clones: one male and one female. Both animals had identical genetic prof...