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.
Selected Metabolites Found in Equine Oviductal Fluid do not Modify the Parameters Associated to Capacitation of the Frozen-thawed Equine Spermatozoa In Vitro. In the horse, a repeatable protocol for in vitro fertilization has not been developed, possibly due to incomplete sperm capacitation. We have previously identified the metabolites present in equine oviductal fluid (OF). We aimed to test the effects of different metabolites found in equine oviductal fluid on quality parameters of frozen-thawed spermatozoa. Different concentrations of myoinositol (5-25 mM), lactate (6-60 mM), glycine (0.1-5 mM), β-alanine (1-6 mM), and histamine (0.05-0.4 mM) were added independently to modified Whitten's medium (pH = 7.25). Thawed equine spermatozoa (three s...
Past, present and future of ICSI in livestock species. During the past 2 decades, intracytoplasmic sperm injection (ICSI) has become a routine technique for clinical applications in humans. The widespread use among domestic species, however, has been limited to horses. In horses, ICSI is used to reproduce elite individuals and, as well as in humans, to mitigate or even circumvent reproductive barriers. Failures in superovulation and conventional in vitro fertilization (IVF) have been the main reason for the use of this technology in horses. In pigs, ICSI has been successfully used to produce transgenic animals. A series of factors have resulted in...
Advances in sperm cryopreservation in farm animals: Cattle, horse, pig and sheep. Sperm cryopreservation is one of the most important procedures in the development of biotechnologies for assisted reproduction. In some farm animals, the use of cryopreserved sperm has so many benefits for which relevance has become more evident in recent decades. Values for post-thaw sperm quality, however, are variable among species and within individuals of the same species. There is no standardized methodology for each of the stages of the cryopreservation procedure (andrological examination, semen collection, dilution, centrifugation, resuspension of the pellet with the freezing medium, p...
An Update on Semen Physiology, Technologies, and Selection Techniques for the Advancement of In Vitro Equine Embryo Production: Section II. As the use of assisted reproductive technologies (ART) and in vitro embryo production (IVP) expand in the equine industry, it has become necessary to further our understanding of available semen selection techniques. This segment of our two-section review will focus on the selection of spermatozoa based on quality and sex for equine intracytoplasmic sperm injection (ICSI), as well as current and future developments in sperm sorting technologies. Ultimately, novel methods of semen selection will be assessed based on their efficacy in other species and their relevance and future application towa...
Bicarbonate-Stimulated Membrane Reorganization in Stallion Spermatozoa. Classical in vitro fertilization (IVF) is still poorly successful in horses. This lack of success is thought to be due primarily to inadequate capacitation of stallion spermatozoa under in vitro conditions. In species in which IVF is successful, bicarbonate, calcium, and albumin are considered the key components that enable a gradual reorganization of the sperm plasma membrane that allows the spermatozoa to undergo an acrosome reaction and fertilize the oocyte. The aim of this work was to comprehensively examine contributors to stallion sperm capacitation by investigating bicarbonate-induced m...
An Update on Semen Physiology, Technologies, and Selection Techniques for the Advancement of In Vitro Equine Embryo Production: Section I. As the use of assisted reproductive technologies (ART) and in vitro embryo production (IVP) expand in the equine industry, it has become necessary to further our understanding of semen physiology as it applies to overall fertility. This segment of our two-section review will focus on normal sperm parameters, beginning with development and extending through the basic morphology of mature spermatozoa, as well as common issues with male factor infertility in IVP. Ultimately, the relevance of sperm parameters to overall male factor fertility in equine IVP will be assessed.
INRA96 Supplemented With Phospholipids Liposomes, A Promising Approach for Stallion Sperm Chilling. Among biotechnologies of reproduction in the equine species, artificial insemination remains the most used technology especially for cooled transported sperm. Although the use of INRA96 extender has demonstrated its efficiency for long-term sperm storage at 4°C or 15°C, some stallions ("bad coolers") are excluded from such technology. Some years ago, we demonstrated that liposomes produced from egg yolk (EY) phospholipids could be an alternative to egg yolk plasma in stallion freezing extenders. To develop a new extender for sperm chilling, we evaluated the protective effect of liposomes pr...
Preliminary study of the contraceptive effect of a self-assembling intrauterine device (iUPODs) in mares maintained in a paddock with a fertile stallion. There is an urgent need for practical methods of population control (i.e., contraception and/or sterilization) for free-roaming (i.e., "wild" or "feral") horses and burros on Western Public Lands in the United States. The objective of this study was to evaluate the contraceptive efficacy of a novel self-assembling three-part polymer-coated magnetic intrauterine device termed as an intrauterine POD (self-assembling; iUPOD) when there are natural breeding conditions when iUPOD use was managed by veterinary professionals with no prior experience with the device. Six mares were administered an iUP...
Pregnancy and placental development in horses: an update. Horses have been domesticated by man and historical information mostly associates horses with men. Nowadays, however, horse riding is essentially by women. Women are also very much involved in equine sciences, with a large contribution to the understanding of fetoplacental development. While highlighting the work of female scientists, this review describes the recent advances in equine fetoplacental studies, focusing on data obtained by new generation sequencing and progress on the understanding of the role of placental progesterone metabolites throughout gestation. A second emphasis is made o...
Loading equine oocytes with cryoprotective agents captured with a finite element method model. Cryopreservation can be used to store equine oocytes for extended periods so that they can be used in artificial reproduction technologies at a desired time point. It requires use of cryoprotective agents (CPAs) to protect the oocytes against freezing injury. The intracellular introduction of CPAs, however, may cause irreversible osmotic damage. The response of cells exposed to CPA solutions is governed by the permeability of the cellular membrane towards water and the CPAs. In this study, a mathematical mass transport model describing the permeation of water and CPAs across an oocyte membrane...
A stallion spermatozoon’s journey through the mare’s genital tract: In vivo and in vitro aspects of sperm capacitation. Conventional in vitro fertilization is not efficacious when working with equine gametes. Although stallion spermatozoa bind to the zona pellucida in vitro, these gametes fail to initiate the acrosome reaction in the vicinity of the oocyte and cannot, therefore, penetrate into the perivitelline space. Failure of sperm penetration most likely relates to the absence of optimized in vitro fertilization media containing molecules essential to support stallion sperm capacitation. In vivo, the female reproductive tract, especially the oviductal lumen, provides an environmental milieu that appropriate...
Germ Cell Transplantation in Stallion Testes. The production of donor-derived sperm using spermatogonial stem cell transplantation has been studied in various animals including mice, rats, goats, boar, dogs, sheep, and monkeys. However, germ cell transplantation has not been applied in stallions. The objective of this study was to produce donor germ cell-derived sperm using germ cell transplantation in stallions. Donor germ cells were transplanted into the parenchyma of 3 recipient stallions that had been treated with busulfan IV injections of 15 mg/kg body weight. For the preparation of donor single germ cells, tissue (20 g) from each te...
The Mare: A Pertinent Model for Human Assisted Reproductive Technologies? Although there are large differences between horses and humans for reproductive anatomy, follicular dynamics, mono-ovulation, and embryo development kinetics until the blastocyst stage are similar. In contrast to humans, however, horses are seasonal animals and do not have a menstrual cycle. Moreover, horse implantation takes place 30 days later than in humans. In terms of artificial reproduction techniques (ART), oocytes are generally matured in vitro in horses because ovarian stimulation remains inefficient. This allows the collection of oocytes without hormonal treatments. In humans, in viv...
Evaluating the use of piezo manipulator, laser or their combination for blastocoel cavity puncture to improve cryopreservation outcomes of large equine embryos. 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.
Anti-Müllerian Hormone and OPU-ICSI Outcome in the Mare. Anti-Müllerian hormone (AMH) reflects the population of growing follicles and has been related to mammalian fertility. In the horse, clinical application of ovum pick-up and intracytoplasmic sperm injection (OPU-ICSI) is increasing, but results depend largely on the individuality of the mare. The aim of this study was to assess AMH as a predictor for the OPU-ICSI outcome in horses. Therefore, 103 mares with a total follicle count above 10 were included in a commercial OPU-ICSI session and serum AMH was determined using ELISA. Overall, the AMH level was significantly correlated with the number...
The Combination of hCG and GnRH Analog to Hasten Ovulation in Mares Does not Change Luteal Function and Pregnancy Outcome in Embryo Recipient Mares. Equine practitioners often prescribe the combined use of hCG and GnRH to hasten ovulation due to presumed synergistic effects. Therefore, this study aimed to test whether the combination of hCG and deslorelin acetate to hasten ovulation in mares would show any effect in inducing ovulation more efficiently than when either drug is used by itself, and to verify whether this association would affect progesterone concentrations; corpus luteum (CL) diameter and blood flow; and pregnancy outcome in recipient mares after embryo transfer (ET). Seventeen mares had the ovulation hastened (≥35 mm folli...
Sperm migration, selection, survival, and fertilizing ability in the mammalian oviduct†. In vitro fertilization (IVF) gives rise to embryos in a number of mammalian species and is currently widely used for assisted reproduction in humans and for genetic purposes in cattle. However, the rate of polyspermy is generally higher in vitro than in vivo and IVF remains ineffective in some domestic species like pigs and horses, highlighting the importance of the female reproductive tract for gamete quality and fertilization. In this review, the way the female environment modulates sperm selective migration, survival, and acquisition of fertilizing ability in the oviduct is being considered...
Deslorelin Slow-Release Implants Delay Ovulation and Increase Plasma AMH Concentration and Small Antral Follicles in Haflinger Mares. There is an increasing interest in the manipulation of ovarian follicular populations in large domestic animals because this could prove beneficial for assisted reproductive techniques such as ovum pick-up (OPU). The aim of the present study was to evaluate the effects of deslorelin slow-release implants (SRI) on the interovulatory interval, antral follicle count (AFC), number of follicles of different size ranges and plasma anti-Muellerian hormone (AMH) concentration in mares. To synchronize their estrous cycles, Haflinger mares (n = 12) were treated twice with a PGF2α analogue. One day afte...
Development of a suitable manufacturing process for production of a bioactive recombinant equine chorionic gonadotropin (reCG) in CHO-K1 cells. Equine chorionic gonadotropin (eCG) is a heterodimeric glycoprotein hormone produced by pregnant mares that has been used to improve reproductive performance in different domestic species. Several strategies to produce the hormone in a recombinant way have been reported; nevertheless, no approach has been able to produce a recombinant eCG (reCG) with significant in vivo bioactivity or in sufficient quantities for commercial purposes. For this reason, the only current product available on the market consists of partially purified preparations from serum of pregnant mares (PMSG). Herein, we des...
Epigenetic Changes in Equine Embryos after Short-Term Storage at Different Temperatures. 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...
Antioxidants in assisted reproductive technologies: An overview on dog, cat, and horse. Assisted reproductive technologies (ARTs) are widely used as a tool to improve reproductive performance in both humans and animals. In particular, in the veterinary field, ARTs are used to improve animal genetics, recover endangered animals, and produce offspring in the event of subfertility or infertility in males or females. However, the use of ARTs did not improve the fertilization rate in some animals due to various factors such as the difficulty in reproducing an anatomical and humoral substrate typical of the natural condition or due to the increase in catabolites and their difficult eli...
Successful vitrification of manually punctured equine embryos. Successful vitrification of equine expanded blastocysts requires collapse of the blastocoele cavity using a micromanipulator-mounted biopsy pipette on an inverted microscope. Such equipment is expensive and requires user skill. Objective: To develop a manual method of blastocoele collapse prior to vitrification using commercial products. Methods: In vivo experiment. Methods: Seventy-nine Day 7 or 8 embryos were measured and graded. Twenty were vitrified following micromanipulator-assisted puncture and aspiration before being used to validate commercial human vitrification and warming kits cont...
Effects of In Vitro Interactions of Oviduct Epithelial Cells with Frozen-Thawed Stallion Spermatozoa on Their Motility, Viability and Capacitation Status. Cryopreservation by negatively affecting sperm quality decreases the efficiency of assisted reproduction techniques (ARTs). Thus, we first evaluated sperm motility at different conditions for the manipulation of equine cryopreserved spermatozoa. Higher motility was observed when spermatozoa were incubated for 30 min at 30 × 106/mL compared to lower concentrations (p < 0.05) and when a short centrifugation at 200× g was performed (p < 0.05). Moreover, because sperm suitable for oocyte fertilization is released from oviduct epithelial cells (OECs), in response to the capacitation process, we e...
Two Novel Cases of Autosomal Translocations in the Horse: Warmblood Family Segregating t(4;30) and a Cloned Arabian with a de novo t(12;25). We report 2 novel autosomal translocations in the horse. In Case 1, a breeding stallion with a balanced t(4p;30) had produced normal foals and those with congenital abnormalities. Of his 9 phenotypically normal offspring, 4 had normal karyotypes, 4 had balanced t(4p;30), and 1 carried an unbalanced translocation with tertiary trisomy of 4p. We argue that unbalanced forms of t(4p;30) are more tolerated and result in viable congenital abnormalities, without causing embryonic death like all other known equine autosomal translocations. In Case 2, two stallions produced by somatic cell nuclear tran...
Case Report: Suppression of Harem Stallion Behavior and Fertility Following Anti-Gonadotropin-Releasing Hormone Vaccination of a Captive Wild Przewalski’s Horse (Equus ferus przewalskii). This report describes an option to modulate the testicular function of wild horses and field methods to assess it. Non-surgical castration of a captive wild Przewalski's stallion with anti-gonadotropin-releasing hormone (GnRH) immunization was performed by sub-cutaneous injection of two doses of 450 μg (3 ml) of GnRH conjugated to diphtheria toxin, further repeated every 6 months. Semen quality was assessed after collection by electro-ejaculation under general anesthesia. Endocrine and behavioral consequences were studied during a 2-year follow-up period. The procedure of electro-ejaculation ...
Analysis of Genetic Variability in the Argentine Polo Horse With a Panel of Microsatellite Markers. Argentine Polo (AP) is a young horse breed with a fast expansion because of an open registry policy and the application of assisted reproduction technologies. In the last years, AP showed a remarkable progress associated with the use of embryo transfer technology and intensive selection based on sport performance. However, these practices could have affected the genetic variability of the breed. To monitor these potential changes, a parentage panel of 11-15 microsatellites was investigated for changes in allele frequencies, heterozygosity, and exclusion probability over a 16 year period. Frequ...
Mitochondrial function, blastocyst development and live foals born after ICSI of immature vitrified/warmed equine oocytes matured with or without melatonin. Oocyte vitrification is considered experimental in the horse with only three live foals reported. The oxidative conditions induced by vitrification could in part explain the poor results and melatonin, a powerful antioxidant, could stimulate ROS metabolization and restore mitochondrial function in these oocytes. Our objective was to determine the oxidative status of vitrified equine oocytes and to analyze the effect of melatonin on mitochondrial-specific ROS (mROS), oocyte maturation, ICSI embryo development and viability. Immature, abattoir-derived oocytes were held for 15 h and vitrified in...
Factors Affecting Embryo Recovery Rate, Quality, and Diameter in Andalusian Donkey Jennies. Embryo transfer and the vitrification of embryos could be used for the conservation and recovery of endangered donkey breeds. It is important to develop techniques that optimize recovery rates and the cryotolerance of donkey embryos. This study evaluates factors affecting the recovery rate, quality, and diameter of embryos obtained from donor jennies as a starting point for the use of vitrification and embryo transfer in the conservation of the Andalusian donkey. A total of 100 embryos were recovered out of 124 estrous cycles (80.6%). The donor jenny affected the rates of positive flushings (P...
Fertilizing capacity of vitrified stallion sperm assessed utilizing heterologous IVF after different semen warming procedures. The aim of this study was to evaluate the fertilizing capacity of frozen or vitrified stallion sperm after assessing different warming procedures. In Experiment 1, different warming procedures were compared after sperm vitrification: immersion in extender at 43 °C (C), or in a water bath at 37 °C/30 s (W37), 43 °C/10 s (W43) or 60 °C/5 s (W60). With the W60 treatment, there were greater values (P < 0.05) for VCL (83.93 ± 3.6 μm/s) and ALH (3.00 ± 0.2 μm) than freezing and with the C group, and greater values (P < 0.001) for PM (35.33 ± 2.5 %) than with the W43 treatment. In Experiment...