Topic:Assisted Reproductive Techniques
Assisted Reproductive Techniques (ART) in horses encompass a range of technologies designed to aid in the breeding process. These techniques include artificial insemination, embryo transfer, intracytoplasmic sperm injection (ICSI), and oocyte transfer. ART is employed to enhance reproductive efficiency, manage genetic diversity, and support breeding programs for both commercial and conservation purposes. Artificial insemination involves the collection and deposition of semen into the mare's reproductive tract, while embryo transfer entails the collection of a fertilized embryo from a donor mare and its implantation into a recipient mare. ICSI involves the direct injection of a single sperm into an oocyte to achieve fertilization. Oocyte transfer involves the transfer of an oocyte from one mare to another for fertilization and gestation. This page compiles peer-reviewed research studies and scholarly articles that examine the methodologies, advancements, and applications of assisted reproductive techniques in equine reproduction.
Evaluation of Fertilizing Ability of Frozen Equine Sperm Using a Bovine Zona Pellucida Binding Assay. Frozen equine semen has lower fertility compared to cooled semen. Due to the difficulty to obtain equine oocytes, a heterologous zona pellucida binding assay (ZBA) is an alternative method to predict the fertilizing capability of equine frozen sperm. The rate of capacitated and hyperactivated sperm according to their motility characteristics were analyzed with a Computer Assisted Sperm Analyzer. We believe this report describes for the first time the in vitro hyperactivation induction and the heterologous ZBA to predict the fertilizing ability of frozen equine sperm. Objective: This work aimed...
A retrospective comparison of the efficiency of different assisted reproductive techniques in the horse, emphasizing the impact of maternal age. Advancing maternal age is known to negatively affect fertility in the horse. This age-related decrease in fertility has been linked primarily to reduced oocyte quality rather than to impaired uterine function. In the past decade, the use of ovum pick-up (OPU) and ICSI to produce foals has rapidly gaining popularity amongst sport horse breeders. However, it is not yet known how maternal age influences the efficiency of a commercial OPU-ICSI program and whether the age effect is similar to that observed for other ART in the horse. To answer this question, reproductive records of 289 mares bred b...
Enhancing Fertility in Mares: Recombinant Equine Gonadotropins. Advanced reproductive technologies have been developed to enhance fertility in mares and stallions. Some of these technologies in mares include superovulation, embryo transfer (ET), intracytoplasmic sperm injection (ICSI), oocyte transfer (OT), gamete intrafallopian transfer (GIFT), and cloning. Superovulation can provide multiple oocytes for these techniques. This review will focus on how recombinant equine follicle-stimulating hormone (reFSH) and recombinant equine luteinizing hormone (reLH) are important for superovulation and ET and may be useful for ICSI, OT, GIFT, and cloning. Superovula...
The Effect of Different Flushing Media Used to Aspirate Follicles on the Outcome of a Commercial Ovum Pickup-ICSI Program in Mares. The in vitro production of embryos by ovum pickup (OPU) and intracytoplasmic sperm injection (ICSI) is gaining popularity among horse breeders and veterinarians. Various collection media are available for flushing follicles during OPU. The objective of this study was to determine whether the type of flushing media used to aspirate follicles and collect oocytes influences the outcome of a commercial equine OPU-ICSI program. Two commercial embryo flushing media (EFM1 and EFM2) supplemented with heparin were compared with a flushing media designed specifically for the collection of oocytes (oocy...
Development of the central nervous system in equine twin fetuses derived by somatic cell nuclear transfer. Because of the growing importance of horses in leisure and several sports, somatic cell nuclear transfer (SCNT) is being used more frequently for cloning animals for performance and reproductive purposes. However, because of the need to perforate the zona pellucida during microsurgical reconstruction of the oocyte, it is possible that SCNT-derived embryos undergo premature hatching, resulting in embryo bisection and twinning. Therefore, because equine twin pregnancies often lead to abnormal embryo development and pregnancy failure, we performed a detailed comparative assessment of equine twin ...
Effect of breed and other animal-related factors on conception rate to artificial insemination with frozen semen in mares in Ethiopia. Equine reproduction is unique by having long behavioral estrus and differences in time of breeding between breeds and individuals of mares. An experimental study was conducted at the Balderas Sport Horses and Recreational Center, Addis Ababa, Ethiopia, from January to June, 2018, to evaluate conception rate to frozen semen in local and exotic crossbreed mares. Mares were teased to characterize estrus behavior and examined by ultrasound in determining imminent ovulation. Inseminations were done post ovulation within an average of 6-9 h using frozen-thawed semen. The overall conception rate to ...
Use of microfluidics to sort stallion sperm for intracytoplasmic sperm injection. We determined if microfluidic sorting (MF) of frozen-thawed stallion sperm improves sperm population characteristics and results in embryo development after intracytoplasmic sperm injection (ICSI). The efficiency and efficacy of MF sperm separation was evaluated by comparing pre- and post-separation sperm population variables. Procedural comparisons were performed after sorting with MF, single-layer colloidal centrifugation (SLC) or swim-up (SU), and cleavage and embryo development were evaluated after ICSI using MF-sorted sperm. In Experiment 1, when compared to the original sperm sample, MF ...
Successful embryo transfer from Hokkaido native pony after artificial insemination with frozen semen. There has been no report of equine embryo transfer in Japan for the last 24 years. Our objective was to establish an effective protocol for embryo transfer in domestic horse. A Hokkaido native pony was bred by deep-horn artificial insemination with frozen semen from a Connemara pony. Embryo collection was performed using a non-surgical method on day 7. Two embryos were obtained from three flushes (67% recovery) and were transferred fresh into crossbred recipient mares. Both recipient mares were diagnosed pregnant (100% pregnancy rate) 5 days after embryo transfer and had normal progesterone le...
Vitrification of equine expanded blastocysts following puncture with or without aspiration of the blastocoele fluid. Historically, cryopreservation of equine embryos >300 μm gave poor pregnancy rates until researchers collapsed the blastocoele cavity and aspirated the blastocoele fluid prior to vitrification. Objective: To determine if aspiration of the blastocoele fluid prior to vitrification is essential for post warming survival. Methods: In vivo experiments. Methods: Fifty embryos were recovered on day 7-8 and washed in holding medium (HM; M-199HEPES + 20% FBS + antibiotics). Embryos were punctured using a micromanipulator mounted 30 μm biopsy needle; following this 28 had >90% of their bla...
Mitochondrial DNA replication is initiated at blastocyst formation in equine embryos. Intracytoplasmic sperm injection is the technique of choice for equine IVF and, in a research setting, 18-36% of injected oocytes develop to blastocysts. However, blastocyst development in clinical programs is lower, presumably due to a combination of variable oocyte quality (e.g. from old mares), suboptimal culture conditions and marginal fertility of some stallions. Furthermore, mitochondrial constitution appears to be critical to developmental competence, and both maternal aging and invitro embryo production (IVEP) negatively affect mitochondrial number and function in murine and bovine emb...
Freezing, Vitrification, and Freeze-Drying of Equine Spermatozoa: Impact on Mitochondrial Membrane Potential, Lipid Peroxidation, and DNA Integrity. Maintaining the integrity of equine sperm subjected to preservation protocols is essential for the successful development of assisted reproduction procedures. The aim of this study was to assess the mitochondrial membrane potential, lipid peroxidation, and DNA integrity of equine sperm subjected to freezing, vitrification, and freeze-drying. Eight ejaculates obtained from four Colombian Creole horses were subjected to programmable freezing, vitrification, and freeze-drying. After thawing or rehydration, sperm motility and kinetics were assessed through a CASA system. The mitochondrial membrane...
Effect of human tubal fluid medium and hyperactivation inducers on stallion sperm capacitation and hyperactivation. Conventional in vitro fertilization has not yet been implemented in the equine species. One of the main reasons has been the inability to develop a culture medium and incubation conditions supporting high levels of stallion sperm capacitation and hyperactivation in vitro. Although different culture media have been used for this purpose, human tubal fluid (HTF) medium, widely used in the manipulation of human and mice gametes, has not been reported so far in stallion sperm culture. The first part of this study aimed to compare HTF and Whitten's media on different stallion sperm quality and capa...
Assisted reproductive techniques in mares. A wide variety of assisted reproductive techniques (ARTs) are available to aid in managing aspects of equine reproduction. Embryo recovery and transfer can be used to obtain more than one foal per mare per year, and to obtain foals from mares that cannot carry a foal to term. Oocyte recovery and either transfer to the oviduct of an inseminated recipient mare (oocyte transfer), or intracytoplasmic sperm injection (ICSI) and embryo culture can be used to obtain foals from mares with some types of subfertility, such as problems of the tubular tract. ICSI can be used to obtain foals when sperm num...
Impact of Equine and Bovine Oocyte Maturation in Follicular Fluid From Young and Old Mares on Embryo Production in Vitro. Equine follicular fluid (FF) provides autocrine and paracrine factors from theca, granulosa, and cumulus cells, both reflecting and impacting oocyte and follicle maturation. We hypothesized that maturation of oocytes in FF from old versus young mares has a deleterious effect on oocyte maturation and their subsequent developmental potential. Follicular fluid was collected from the large, dominant follicle from young mares (4-13 years) or old mares (21-26 years) and classified as: (1) Noninduced follicular fluid (NFF), FF from noninduced follicle 33 ± 3 mm, or (2) Induced follicular fluid (I...
Spatial distribution of preantral follicles in the equine ovary. Comprehensive studies on spatial distribution of preantral follicles in the ovary are scarce. Considering that preantral follicles represent the main ovarian reserve, harvesting of these follicles is crucial for the development/use of assisted reproductive techniques. Therefore, knowledge on follicle spatial distribution can be helpful for targeting areas with richer number of preantral follicles through biopsy procedures. The aim of this study was to assess the distribution and localization of equine preantral follicles according to: (i) age, (ii) ovarian portion (lateral and intermediary) an...
The development of in vitro embryo production in the horse. The development of techniques to produce equine embryos in vitro is reviewed with specific reference to intracytoplasmic sperm injection (ICSI). Unexplored 50 years ago, this technology has progressed rapidly in the last 20 years to become a commercial reality for the equine breeding industry. Improvements in our understanding of oocyte and embryo competence in the horse have been key factors in overcoming some of the initial problems associated with ICSI. It is now possible to obtain high nuclear maturation and cleavage rates in vitro and the most limiting factor, presently, is the low rate o...
Effect of embryo transfer technique on the likelihood of pregnancy in the mare: a comparison of conventional and Wilsher’s forceps-assisted transfer. The aim of this study was to compare the success of embryo transfer (ET) performed by veterinarians with different degrees of experience using one of two ET techniques. Over three years, 179 embryos were transferred by three operators with moderate to high experience using a 'conventional' manual technique, and 170 embryos were transferred by four operators with little or no previous ET experience using a 'Wilsher' technique (Polansky speculum and Wilsher's cervical forceps). The pregnancy status of recipient mares at the first pregnancy diagnosis and the embryo loss by the last examination we...
Identification of serotoninergic system components in stallion sperm. Assisted reproductive techniques have been used on several domestic animals to preserve desirable traits in strains of high genetic and commercial value; however in equines its efficiency rate is relatively low. To increase the conception ratio in stallions, some research groups have used pharmacological treatments which promote sperm hyperactivation in order to increase male's fertility rates. In this way, our previous work suggests that serotonin (5-HT) could be a good pharmacological candidate that facilitates conception rate in domestic horses. 5-HT is a neurohormone involved in several re...
Lower blastocyst quality after conventional vs. Piezo ICSI in the horse reflects delayed sperm component remodeling and oocyte activation. The aim of this study was to evaluate the differential effects of conventional and Piezo-driven ICSI on blastocyst development, and on sperm component remodeling and oocyte activation, in an equine model. Methods: In vitro-matured equine oocytes underwent conventional (Conv) or Piezo ICSI, the latter utilizing fluorocarbon ballast. Blastocyst development was compared between treatments to validate the model. Then, oocytes were fixed at 0, 6, or 18 h after injection, and stained for the sperm tail, acrosome, oocyte cortical granules, and chromatin. These parameters were compared between inject...
Blastocyst production after intracytoplasmic sperm injection with semen from a stallion with testicular degeneration. In horse breeding, intracytoplasmic sperm injection (ICSI) has gained interest to obtain offspring from subfertile individuals. This paper presents a case report of a stallion with severe testicular degeneration. Semen analysis showed very low motility and 83.5% of detached heads. Histology of a testicular biopsy showed severely decreased spermatogenesis, while transmission electron microscopy of the sperm cells revealed no significant abnormalities. A total of 39 oocytes were fertilized by ICSI with frozen-thawed spermatozoa of this stallion: 25 oocytes with intact spermatozoa and 24 with det...
Review: The potential of seminal fluid mediated paternal-maternal communication to optimise pregnancy success. Artificial insemination has been a landmark procedure in improving animal agriculture over the past 150 years. The utility of artificial insemination has facilitated a rapid improvement in animal genetics across agricultural species, leading to improvements of growth, health and productivity in poultry, swine, equine and cattle species. The utility of artificial insemination, as with all assisted reproductive technologies side-steps thousands of years of evolution that has led to the development of physiological systems to ensure the transmission of genetics from generation to generation. The ...
Effect of different shipping temperatures (∼22 °C vs. ∼7 °C) and holding media on blastocyst development after overnight holding of immature equine cumulus-oocyte complexes. Intracytoplasmic sperm injection (ICSI) is an important tool for equine embryo production in both clinical and research settings. In clinical ICSI programs, immature equine cumulus-oocyte complexes (COCs) are often collected at the mare's location and shipped to the ICSI laboratory. To simplify shipment and aid scheduling of subsequent procedures, COCs can be held overnight at room temperature (∼22 °C) before placement into maturation culture, with no detrimental effect on meiotic or developmental competence. A recent study indicated that it might be possible to hold COCs overnight at col...
Comparison of different cryopreservation methods for horse and donkey embryos. Few studies have been published about cryopreservation and embryo assessment in horses and donkeys. Objective: To evaluate the viability of embryos collected from mares and jennies that were cryopreserved by slow freezing or by vitrification. Methods: Randomised controlled experiment. Methods: Horse (n=19) and donkey (n=16) embryos (≤300 μm) were recovered on days 6.5-7.5 post-ovulation and assigned to control or cryopreservation protocols of slow freezing or vitrification. For slow freezing, 1.5 mol/L ethylene glycol (EG) was used. For vitrification, horse embryos were exposed to 1.4 mol/L...
Use of Confocal Microscopy to Evaluate Equine Zygote Development After Sperm Injection of Oocytes Matured In Vivo or In Vitro. Confocal microscopy was used to image stages of equine zygote development, at timed intervals, after intracytoplasmic sperm injection (ICSI) of oocytes that were matured in vivo or in vitro. After fixation for 4, 6, 8, 12, or 16 h after ICSI, zygotes were incubated with α/β tubulin antibodies and human anticentromere antibody (CREST/ACA), washed, incubated in secondary antibodies, conjugated to either Alexa 488 or Alexa 647, and incubated with 561-Phalloidin and Hoechst 33258. An Olympus IX81 spinning disk confocal microscope was used for imaging. Data were analyzed using χ 2 and Fisher's e...
Intracytoplasmic sperm injection in domestic and wild mammals. Intracytoplasmic sperm injection (ICSI) has become a useful technique for clinical applications in the horse-breeding industry. However, both ICSI blastocyst and offspring production continues to be limited for most farm and wild species. This article reviews technical differences of ICSI performance among species, possible biological and methodological reasons for the variable efficiency and potential strategies to improve the outcomes. One of the major applications of ICSI in animal production is the reproduction of high-value specimens. Unfortunately, some domestic species like the bovine s...
Exposure to follicular fluid during oocyte maturation and oviductal fluid during post-maturation does not improve in vitro embryo production in the horse. Most wild equids and many domestic horse breeds are at risk of extinction, so there is an urgent need for genome resource banking. Embryos cryopreservation allows the preservation of genetics from male and female and is the fastest method to restore a breed. In the equine, embryo production in vitro would allow the production of several embryos per cycle. Intracytoplasmic sperm injection (ICSI) is used to generate horse embryos, but it requires expensive equipment and expertise in micromanipulation, and blastocyst development rates remain low. No conventional in vitro fertilization (IVF) techn...
Impact of equine assisted reproductive technologies (standard embryo transfer or intracytoplasmic sperm injection (ICSI) with in vitro culture and embryo transfer) on placenta and foal morphometry and placental gene expression. Assisted reproductive technologies (ARTs) such as intracytoplasmic sperm injection (ICSI), in vitro embryo culture and embryo transfer (ET) may be associated with alterations in fetal and placental development. In horses, ET has been used for decades. More recently, in vitro embryo production by ICSI and in vitro culture, followed by embryo transfer (ICSI-C) has become an accepted method for clinical foal production. However, no information is available on the effects of ICSI-C or even of standard ET itself on placental and neonatal parameters in horses. We therefore evaluated placental and ne...
Molecular characterization, modeling, in silico analysis of equine pituitary gonadotropin alpha subunit and docking interaction studies with ganirelix. Equine pituitary gonadotropins (eLH, eFSH, eCG) are heterodimeric glycoprotein hormones with alpha (α) and beta (β) subunits. It is responsible for maintenance of pregnancy in mares during early gestation and fairly valuable for inducing superovulation in animals other than equines. The alpha subunit is common, while beta subunit is species-specific in all glycoprotein hormones. In the present investigation, molecular cloning and in silico characterization including homology modeling and molecular docking analysis of the equine chorionic gonadotropin (eCG) alpha subunit was carried out for g...
Analysis of Chromosome Segregation, Histone Acetylation, and Spindle Morphology in Horse Oocytes. The field of assisted reproduction has been developed to treat infertility in women, companion animals, and endangered species. In the horse, assisted reproduction also allows for the production of embryos from high performers without interrupting their sports career and contributes to an increase in the number of foals from mares of high genetic value. The present manuscript describes the procedures used for collecting immature and mature oocytes from horse ovaries using ovum pick-up (OPU). These oocytes were then used to investigate the incidence of aneuploidy by adapting a protocol previous...