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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.
Body Condition, Metabolic Parameters and Semen Traits in Purebred Spanish Horses. The growing relevance of the Purebred Spanish horse has driven the extensive use of assisted reproductive technologies to preserve elite stallion genetics. Optimal reproductive performance is intrinsically linked to systemic health and high semen quality. This study aimed to evaluate the impact of body condition on the stallion's metabolic and endocrine profiles and their seminal parameters. Semen and blood samples were collected from 26 stallions (February-May in 2024-2025) whose body condition score (BCS) was assessed using the Henneke scale. They were classified into two groups, low BCS wit...
Efficiency of transvaginal oocyte aspiration (TVA), post-mortem ovary harvest (OH), and Intracytoplasmic Sperm Injection (ICSI) in a Quarter Horse mare population in North America. In a clinical setting, Intracytoplasmic Sperm Injection (ICSI)-derived equine blastocysts can be obtained either by transvaginal oocyte aspiration (TVA) or post-mortem ovary harvest (OH). Most peer-reviewed retrospective reports have described the suitability of these techniques for obtaining blastocysts, pregnancies, and live foals, particularly in Warmblood horse populations in Europe. In this study, retrospective data from Quarter Horse client-owned mares subjected to TVA-ICSI (n = 105; 151 cycles) or OH-ICSI (n = 42) at an academic institution in North America between 2023 and 2025...
Effects of equine sperm incubation under capacitation-supporting conditions on homologous and heterologous ICSI outcomes. Intracytoplasmic sperm injection (ICSI) in horses has become sufficiently efficient to be considered for clinical and commercial application; however, several factors influencing its efficiency remain poorly understood. This study evaluated whether incubating equine sperm under capacitating conditions induces capacitation-associated changes and affects embryo development following homologous equine ICSI, as well as oocyte activation, early cleavage, and 5-methylcytosine (5 mC) and 5-hydroxymethylcytosine (5hmC) levels following heterologous ICSI using bovine oocytes. Equine spermatozoa were ...
Isolation of high-quality stallion spermatozoa: a comparison of methods and their effects on sperm quality and yield, DNA damage, and functional competence by ICSI. Intracytoplasmic sperm injection (ICSI) is the primary method for equine in vitro embryo production, yet outcomes may be influenced by sperm selection techniques. Objective: This study compared four isolation methods: EquiPure™, Vetmotl™, Felix™, and Samson™, to assess effects on sperm quality and embryo development. Methods: Semen was collected from four fertile stallions, processed and evaluated for sperm motility, viability, DNA integrity, and yield of high-quality cells relative to input. Isolated samples were compared to non-isolated (centrifuged) samples. Spermatozoa were used fo...
Sperm Energy-restriction and Recovery (SER) enhances equine sperm function and embryo development after ICSI. Assisted reproductive technologies (ART) are extensively used in domestic animal reproduction; however, improving their efficiency and consistency remains a major challenge for achieving reliable reproductive outcomes. The equine industry faces unique challenges related to sperm capacitation, which limited the success of conventional in vitro fertilization (IVF) for many years. Consequently, intracytoplasmic sperm injection (ICSI) became the most widely used technique for commercial in vitro embryo production. Even though a successful IVF protocol was recently developed, substantial opportunit...
Factors Influencing Equine Embryo Outcomes in OPU‑ICSI Systems: Insights from a Large‑Scale Program in Brazil. Equine OPU‑ICSI supports commercial IVEP, yet substantial variability persists. We retrospectively analyzed a large dataset generated in Brazil spanning Quarter Horses, Warmbloods, and Mangalarga to evaluate whether predictors previously identified in the literature show comparable patterns under distinct ecological and breeding conditions. Overall oocyte and embryo outcomes were aligned with broadly reported trends from previous studies. Oocytes retrieved from estrous mares bearing a dominant follicle exhibited higher maturation and blastocyst rates. Stallion effects segregated into distinc...
Genetic Diversity in Domestic Horses: Temporal Patterns, the Influence of Assisted Reproductive Technologies, and Implications for the American Quarter Horse. Genetic diversity is the heritable variation within and among populations and is essential for the long-term health and selection potential of domestic horses. Previous studies show diversity across breeds is shaped by effective population size, founder events, artificial selection, and levels of inbreeding. While some closed-studbook breeds retain moderate genetic diversity, others have experienced rapid losses due to popular-sire effects and recent and extensive inbreeding. Even so, mare lines continue to help maintain genetic diversity within these populations. Assisted Reproductive Technol...
Achieving Success in Conventional Equine Embryo Transfer: A Practical Review. Equine embryo transfer is a well-established procedure in horse breeding programs and it's used to increase the number of offspring from valuable mares, obtain embryos from mares in competition and produce foals from mares unable to carry a pregnancy to term. The success of embryo recovery depends mainly on the day of flushing, management of insemination, and the fertility of donor mare and stallion. Other relevant factors include donor age and reproductive status, number of ovulations, operator experience, and flushing technique. Pregnancy rates after transfer are influenced by donor-recipien...
Follicular growth and atresia at the time of oocyte aspiration differentially affect in vitro embryo production in mares. Ovum pick-up (OPU) followed by intracytoplasmic sperm injection (ICSI) is increasingly used in horses, yet embryo production rates remain variable, potentially reflecting differences in oocyte developmental competence at the time of aspiration. We investigated whether the stage of the follicular wave, growth phase (GP) versus atresia phase (AP), and associated endocrine profiles in mares, influence oocyte developmental competence. Mares underwent follicular ablation to synchronize follicular waves, and oocytes were aspirated during GP or AP for in vitro production of embryos. Serum concentrati...
Cracks in the ice: biological challenges and incremental progress in equine oocyte vitrification. Equine oocyte vitrification could enhance the flexibility of in vitro embryo production, yet developmental competence remains reduced. Only vitrification of oocytes matured in vivo yields foaling rates comparable to fresh oocytes. Recent advances in vitrification at the germinal vesicle (GV) stage, supported by systematic comparisons of cryoprotective agents and exposure times, has enabled consistent blastocyst production and birth of several foals. Comparative studies of GV and metaphase II (MII) stages suggest biological advantages of vitrification after in vitro maturation (IVM), warranting...
Application of Anti-Müllerian Hormone in Regulation of Ovarian Function and Its Practical Relevance for Fertility and Embryo Production in Cows and Mares. In the female organism, Anti-Müllerian hormone (AMH) is produced exclusively by granulosa cells, particularly in secondary and early tertiary follicles. The amount of AMH released into the blood is proportional to the total number of follicles present in the ovaries and is therefore used to estimate the ovarian functional reserve. In both cattle and mares, a strong and highly repeatable association has also been demonstrated between AMH concentrations and antral follicle count. Therefore, AMH may serve as a biomarker with broad diagnostic utility in reproductive medicine in these species, as ...
Ovum pick-up in mares: Evidence review and best practice recommendations using a modified GRADE approach. Ovum pick-up (OPU) is a procedure that is now performed in mares worldwide to harvest oocytes for in vitro embryo production. There is a lack of consensus surrounding best practices for various procedural elements of the technique and recently there has been an increasing focus on the welfare aspects of the procedure. Our objective was to summarize current practice and to develop guidelines for OPU in the mare, using a modified GRADE approach to optimize efficiency and mare welfare. Published evidence on various aspects of the procedure was compiled, and to supplement the relative lack of publ...
Vitrification of in vivo produced equine embryos: Size really does matter! Vitrification rather than slow-freezing has become the method of choice for the cryopreservation of in-vivo produced embryos. It is a simple technique that does not require a programmable freezing machine and relies chiefly on placing the embryo on a surface, rather than within a straw, in a small volume of vitrification solution to achieve extremely rapid cooling rates. However, in the field of equine embryo vitrification size truly does matter. Methodologies for both vitrification and warming prior to transfer are largely dictated by the size of the embryo. Since embryo size is directly rela...
Embryo developmental kinetics and vitrification in relation to pregnancy outcome, foaling percentage and foal sex. While fast in vitro embryo development has been associated with improved outcomes, the relationships of slow in vitro embryo development and vitrified versus fresh in vitro produced (IVP) embryo transfer to foaling percentage and foal sex are less explored. Objective: To determine the relationships of (1) day of blastocyst formation (D7-11 after intracytoplasmic sperm injection (ICSI)) and (2) vitrification and warming method (one- and three-step) versus fresh transfer with pregnancy, early pregnancy loss (EPL), foaling percentage and foal sex. Methods: Retrospective clinical study. Methods: B...
Ovarian follicle dynamics in the plains zebra (Equus quagga). Understanding follicular dynamics during the estrous cycle is essential for developing breeding programs and assisted reproductive technologies (ARTs) in any species. In this context, knowledge of follicular dynamics and related uterine changes in the plains zebra (Equus quagga) is essential to increase the declining population of this vital African species and to provide comparative insights into more endangered zebra species. Likely due to the difficulty of handling these wild species, no detailed reproductive study using ultrasonography has been performed in any zebra. Therefore, the presen...
Conventional IVF in the horse past, present, and future: physiological and technical insights. For decades, conventional in vitro fertilization (IVF) in the horse was ineffective, leading to reliance on intracytoplasmic sperm injection (ICSI) for both research and clinical in vitro embryo production. However, the ability to achieve IVF is essential to the study of key reproductive processes, including sperm capacitation, fertilization, and early embryonic development. Recently we have established a repeatable and efficient IVF system for horses, using long-term (9-22 h) sperm pre-incubation. For clinical use, IVF has many features that may support higher embryo quality than does ICSI, a...
FGF2, LIF, and IGF1 supplementation during in vitro maturation or culture improves equine ICSI embryo development in an age-dependent manner. Ovum pickup (OPU) combined with intracytoplasmic sperm injection (ICSI) has reshaped equine assisted reproduction, enabling embryo production from mares and stallions with constrained breeding opportunities; however, limited efficiency of in vitro maturation (IVM) and in vitro culture (IVC) continues to hinder embryo production. Growth factor supplementation with fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), and insulin-like growth factor 1 (IGF1) in combination (FLI) has been shown to enhance oocyte and embryo developmental competence in several species. Objective: This...
Hyperactivation and Tyrosine Phosphorylation of Cryopreserved Stallion Spermatozoa in Relation to Conventional In Vitro Fertilization Blastocyst Production. Challenges in establishing clinical conventional in vitro fertilization (IVF) in horses include variation in sperm and oocyte quality, which can be affected by mare age. Extended preincubation of stallion spermatozoa has resulted in successful IVF. However, there is limited research on the impact of preincubation on sperm characteristics and IVF success. Objective: Responses of frozen-thawed stallion spermatozoa over time under IVF preincubation conditions were evaluated, and samples based on specific responses were tested for their ability to fertilize oocytes, including those from older mare...
Age, embryo donor status, and insemination with chilled semen all predispose to persistent breeding-induced endometritis in warmblood mares. Persistent breeding-induced endometritis (PBIE) is a common reason for mares failing to establish pregnancy. Objective: To examine the hypothesis that advancing mare age and embryo donor status predispose to PBIE and compromise fertility. Methods: Retrospective analysis of the impact of mare age, breeding system (embryo donor versus broodmare), and semen type (chilled versus frozen-thawed) on the likelihood of PBIE and subsequent pregnancy/embryo recovery. Methods: Warmblood mares (n = 769) inseminated during 1745 oestrous cycles were divided into three age categories (≤6, 7-13 and ≥14...
Equine Oocyte and Embryo Metabolism in the ART Setting: Current Knowledge and Clinical Significance. Equine in vitro embryo production has become very efficient and is used extensively worldwide for the clinical production of foals. What we know so far about offspring health and performance is promising; however, elements of the in vitro process remain unphysiological when compared to in vivo conditions. Studying the metabolism of oocytes and embryos can help inform the optimisation of culture systems and identify aberrations if they occur. Given the growing body of knowledge on the downstream effects of an inadequate peri-conception environment, both in vivo and in vitro, it is imperative th...
Holding immature equine oocytes for up to 3 nights prior to ICSI under practical shipping conditions (10-22 °C) is compatible with blastocyst development. In vitro embryo production via intracytoplasmic sperm injection (ICSI) is widely used in equine reproductive management. Shipping of immature oocytes allows oocyte recovery for ICSI to be performed by referring veterinarians. However, delays in oocyte shipment are common, and their effect on outcomes is unclear. Shipping is typically done at about 22 °C, but previous studies have shown that holding at 15 °C maintains equine oocyte developmental competence. We hypothesized that lower temperatures may better support oocyte viability if the case that delays occur. Oocytes (n = 664) were rec...
Impact of donor mare and stallion identity on the likelihood of pregnancy after transfer of in vitro produced embryos. The success of in vitro embryo production (IVEP) is influenced by donor mare and stallion. Objective: To determine whether donor mare and stallion influence the pregnancy rate after transfer of in vitro produced (IVP) blastocysts and to identify factors influencing the likelihood of obtaining one or more pregnancies from a single IVEP session. Methods: Retrospective clinical study. Methods: The influence of the following variables on pregnancy outcome was assessed in 2292 transfers of IVP blastocysts: speed of in vitro embryo development, embryo grade after thawing, age of the donor mare, blas...
Clinical pregnancy outcomes after transfer of fresh and vitrified-warmed conventional IVF equine embryos. In vitro fertilization (IVF) in horses has emerged as a valuable assisted reproductive technology, yet the impact of embryo cryopreservation on pregnancy outcomes when utilizing this technique remains unclear. This study reports pregnancy outcomes following transfer of conventional equine IVF embryos, with both fresh and vitrified-warmed embryos. Conventional IVF (cIVF) was used to produce 40 embryos, which were transferred to recipient mares, pregnancy diagnosis was performed at 6 days post-transfer followed by embryonic heartbeat detection between day 25 and 30 of gestation. Observed pregnan...
Embryonic vesicle diameter at first pregnancy diagnosis in in vivo- and in vitro-derived pregnancies and its relationship with pregnancy loss in the mare. This retrospective study compared the relationship between embryonic vesicle diameter at first pregnancy diagnosis and early embryonic loss (EEL) across three pregnancy types: artificially inseminated broodmares (AI; n = 263), recipients carrying in vivo-derived flushed embryos (IVD-F; n = 312), and recipients carrying in vitro-produced embryos by intracytoplasmic sperm injection (IVP-ICSI; n = 161). Vesicle diameter was recorded at Day 12 post-ovulation in AI, Day 4 post-embryo transfer (ET) for IVD-F, and Day 7 post-ET for IVP-ICSI pregnancies. Mean (±SD) vesicle diameters and E...
Comparative Evaluation of Sperm Quality Assessment Methods in Purebred Spanish Horses. Evaluating semen quality in Purebred Spanish Horses is essential to determine reproductive potential and optimize assisted reproductive techniques. Objective: This study compared several sperm analysis methods in Purebred Spanish Horses to standardize protocols suitable for both laboratory and field conditions. Methods: Fifteen stallions were evaluated through three ejaculates, analyzing sperm parameters using different techniques. Sperm kinematics was assessed by Sperm Computer Analyzer (SCA-CASA) or iSperm mCASA. Viability was analyzed by eosin-nigrosin-Giemsa (ENG) staining or Hoechst 33258...
Are antioxidants hindering stallion sperm capacitation? A perspective on hidden barriers to equine IVF success. Heavy use of antioxidants in equine in vitro fertilisation (IVF) media may suppress the reactive oxygen species (ROS) signals stallion sperm need for capacitation, which depends on high mitochondrial activity and a well-maintained endogenous redox homeostasis. A species-specific approach balancing oxidative protection with controlled ROS signalling could improve fertilisation efficiency and reduce prolonged incubation times.
Does the Reproductive Technique Affect Neonatal Health Parameters in Foals? This retrospective study evaluated whether different reproductive biotechnologies influence neonatal behaviour parameters in foals. Data from 102 foalings in two commercial breeding farms were analysed, including foals conceived by artificial insemination (AI), conventional embryo transfer (ET) and intracytoplasmic sperm injection (ICSI). Neonatal parameters recorded were birth weight, time to achieve sternal recumbency, time to stand, time to nurse and time to meconium elimination. Mixed statistical models were applied to assess the influence of reproductive technique, breed, sex and farm. Si...
Characterization of isolated late preantral and early antral equine ovarian follicles. Understanding how to effectively isolate a large number of healthy follicles is essential for increasing success rates in ARTs, such as in vitro culture and cryopreservation. Late secondary (preantral) and early tertiary (antral) follicles isolated from the intermediary portion of equine ovarian tissue sliced at two different thicknesses (0.25 mm vs. 0.50 mm) were characterized according to regional distribution (dorsal, medial, and ventral), morphofunctional features, and epigenetic profiles. After isolation via microdissection, follicles were evaluated for membrane integrity (trypan blue), v...
Temporal characterization of conditions that promote functional capacitation of stallion sperm. Equine in vitro fertilization (IVF) has historically been challenged due to lack of conditions that promote functional capacitation of stallion sperm. Capacitation success is often attributed to prolonged sperm incubation requirements. Advances to equine IVF are hindered by incomplete characterization of conditions that promote the functional capacitation of stallion sperm over time. Herein, we describe conditions that promote the functional capacitation of fresh, cooled, and frozen-thawed stallion sperm isolated by Percoll, Swim-Up, or Microfluidics (VetMotl). Fresh (diluted sperm) and previo...
Metabolomics reveals early predictors of blastocyst formation in equine ICSI-derived embryos. Equine in vitro embryo production (IVP) via ovum pick-up (OPU) and intracytoplasmic sperm injection (ICSI) has gained prominence in sport-horse breeding due to high success rates, efficient semen utilization, and year-round embryo production, including from mares with fertility challenges or postmortem. However, assessing viability of early-stage ICSI-produced equine embryos remains challenging due to subjective morphological evaluations and limited visibility of developmental features. Non-invasive metabolomic analysis of culture media offers a promising alternative to enhance embryo selectio...
