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.
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...
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...
Comparative Evaluation of Follicular Flushing Frequency and Scraping Time During Ovum Pick-Up in Mares: Effects on Oocyte Recovery Rate and Technical Considerations. Transvaginal ultrasound-guided follicular aspiration or ovum pick-up (OPU) has become the standard technique for oocyte collection in mares for intracytoplasmic sperm injection (ICSI). Although repeated follicular flushes and wall scraping are commonly used to improve oocyte recovery rate (ORR), the relative contribution of each remains unclear. This study aimed to compare the effects of multiple flushes versus controlled scraping time on ORR in mares. A controlled trial was conducted in two phases: (1) an ex vivo phase using slaughterhouse ovaries (n = 32), and (2) an in vivo phase in c...
Cryoprotective Efficacy of Omega-3 Nano-Emulsion on Kinematic Parameters, Acrosome Status, Subcellular Ultrastructure, and Oxidative/Antioxidant Markers in Cryopreserved Stallion Semen. This study aims to investigate the cryoprotective effect of Omega-3 nano-emulsion (Omega-3 NE) on stallion sperm quality, kinematic parameters, acrosome status, subcellular ultrastructure, oxidative/antioxidant markers, and semen microbiota. Forty ejaculates were collected, extended, and cryopreserved from 5 fertile Pure Egyptian stallions (Equus caballus). The ejaculates were divided into five groups: a control group (without additive) and four groups supplemented with 25, 50, 100, and 200 μg of Omega-3 NE/mL. The Omega-3 NE exhibited an average particle size of 51-146 nm, a PDI of 0.58,...
Assessing the Sperm Head DNA Damage in Frozen/Thawed Horse Spermatozoa via Xenogeneic ICSI. In the mouse, spermatozoa are highly resistant to DNA damage, even when frozen without cryoprotectants, and can produce offspring when subsequently used for ICSI (intracytoplasmic sperm injection). It is not known whether the same applies to other mammals as well. For example, in the horse, even conventional sperm freezing is still very problematic and frequently leads to sperm immobility. It has, however, never been tested whether sperm immobility also mirrors sperm head DNA damage, and if so, to what extent. In our study, we evaluated the damage to DNA in horse frozen and thawed motile and i...
Myo-Inositol Nano-Emulsion for Enhancing Stallion Chilled Semen: Insights from In Vivo and In Silico Analyses of Sperm Quality, Oxidative Stress, Ultrastructure, Mitochondrial Response, and Semen Bacteriology. Oxidative stress is a major contributor to male infertility. Therefore, fortifying assisted reproductive technology with nanotechnology could enhance sperm preservation. Objective: This study aimed to examine the impact of myo-inositol nano-emulsion (MINE) supplementation in semen extender on sperm quality, redox balance, semen bacteriology, apoptosis, ultrastructure, and acrosome status of chilled stallion semen. Methods: Semen samples were collected and preserved with 0 (MINE0), 1 (MINE1), and 2 (MINE2) mg of MINE/mL of extender. Results: Results revealed that extender fortified with 1 or 2 ...
Histomorphometric Analysis of the Endometrium of Jennies (Equus asinus) and Mares (Equus caballus) in Estrus: Anatomical Differences and Possible Reproductive Implications. Assisted reproductive techniques are often extrapolated from horses to donkeys, despite poorer fertility outcomes in jennies. This issue has been attributed to unknown uterine species-specific differences. This study compared, through histomorphometry, the endometrium of jennies and mares. Endometrial biopsies (N = 12) were taken from reproductively sound jennies (n = 6) and mares (n = 6) in estrus. Histomorphometric analysis evaluated luminal (LE, µm) and glandular epithelium height (GE, µm), glandular lumen diameter (LD, µm), glandular area (GA, µm), the number of glands (#G), and glandu...
A Simple Microaspiration Technique for Isolating Somatic Cells from Cryopreserved Equine Semen as Nuclear Donors for Cloning. Semen is a complex fluid that, in addition to spermatozoa, contains other cell populations, including immune cells, immature male germ cells, epithelial cells, and fibroblasts. These cells share the diploid condition, making them suitable candidates as nuclear donors for somatic cell nuclear transfer (SCNT) cloning. The generation of viable embryos and offspring has been demonstrated using these cells. Effective methods for isolating them from semen include centrifugation and osmotic gradient techniques; however, prolonged in vitro culture periods are necessary to establish primary cultures fr...
Equine oviduct-specific glycoprotein is modulated by hormones and sperm cells. Oviduct glycoprotein 1 (OVGP1) is a key protein involved in oviductal functions. β-estradiol (E) and progesterone (P), oxytocin (OXT) and tumor necrosis factor-α (TNFα) modulate the equine oviduct function, through prostaglandin regulation. The objective was to evaluate OVGP1 expression within each equine oviduct segment (infundibulum, ampulla isthmus), throughout the estrous cycle. The in vitro effect of (i) E, P, OXT, TNFα; and (ii) spermatozoa, on oviduct OVGP1 transcription and secretion was studied. Gene transcription was assessed by real-time PCR; protein expression by western blot; ...
Efficacy of butylated hydroxytoluene nanoparticles in enhancing the quality and preservation of stallion chilled semen. Encapsulating natural antioxidants presents a robust strategy to neutralize oxidative stress, thereby improving semen preservation. Therefore, this study aimed to evaluate the efficacy of butylated hydroxytoluene nanoparticles (BHT-NPs) in improving stallion semen preservation by investigating sperm quality, redox balance, semen bacteriology, apoptosis, ultrastructure, and acrosome status of chilled stallion semen preserved at 4 °C for 72 h. This in vitro experiment was evidenced by molecular docking analysis. Twenty-five ejaculates from five stallions were collected and extended with 0 (BH...
Naloxone supplementation during vitrification of equine in vitro matured oocytes after overnight holding: insights from a comparative study with the bovine model. Cryopreservation of equine mature oocytes remains a major challenge in assisted reproduction, mainly due to the limited availability of material and logistical constraints requiring a holding phase. To address these issues, this study evaluated whether naloxone (NX), an opioid receptor antagonist with reported antioxidant properties, could improve vitrification outcomes and whether bovine oocytes could serve as a suitable preliminary and supportive model. Two experiments were conducted. In experiment 1, immature (bGV) and mature (bMII) bovine oocytes were vitrified without (VIT) or with NX (VI...
From clinic to lab: Sperm parameters for equine in vitro embryo production. Equine in vitro embryo production (IVP) offers a valuable opportunity to enhance the efficient utilization of genetically and economically valuable horses. In cases of subfertility, sudden death, emergency euthanasia, or limited sperm availability, IVP becomes the only viable option to produce offspring. Due to steadily improving success rates, IVP has become one of the most sought-after assisted reproductive techniques. The rising global demand has created a clear need for veterinarians and stallion owners to provide IVP-suitable sperm, where the selection of intact sperm for intracytoplasmic...
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...
Optimising Stallion Semen Cryopreservation: Preliminary Insights Into Pre-Centrifugation Extender Effects. This study evaluated the effects of cholesterol, pentoxifylline and casein, with or without skim milk, added to extenders during pre-centrifugation on equine sperm cryosurvival. Seven ejaculates from four stallions (n = 28) were divided into four groups: SM (skim milk), SMP (SM + pentoxifylline), SMCho (SM + cholesterol) and ChoCa (cholesterol + casein). After centrifugation and freezing, sperm kinematics and plasma membrane integrity were assessed immediately and 30 min post-thaw. SMCho and ChoCa showed superior results compared with SM and SMP. These findings indicate that chol...
Comparison of Oocyte Collection Yield Between Conventional Follicle Scraping and Ultrasound-Guided Follicle Aspiration in Postmortem Excised Horse Ovaries. Post-mortem oocyte collection serves to preserve the genetic material of valuable mares and to obtain recipient oocytes for cloning purposes. Therefore, the number of oocytes retrieved per ovary is a critical factor in increasing the likelihood of obtaining a viable embryo. This study evaluated the efficiency of two post-mortem oocyte retrieval techniques: follicular scraping (Scraping) and ultrasound-guided follicular aspiration (OPU). The comparison was based on several parameters, including the number of follicles aspirated per ovary, oocyte recovery rate (ORR), number of oocytes obtained p...
Intravenous clomiphene citrate pharmacokinetics in healthy mares as a first step toward use in reproduction. To investigate the IV pharmacokinetic parameters of clomiphene citrate (CC), a selective estrogen receptor modulator, in mares. Unassigned: This was an experimental study to assess the pharmacokinetic parameters of CC in the mare. Eight mares received a single 2,000-mg dose of IV CC to identify plasma concentrations over 336 hours. Following this, 6 mares received a multidose regimen of IV CC (2,000 mg, q 24 h) for 5 days to identify trough plasma concentration each day. Unassigned: CC was well tolerated, demonstrated a short elimination half-life of 4.95 ± 1.10 hours and a mean residence tim...
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...
The efficacy of embryo recovery on consecutive days from Day 6.5 to obtain small embryos for vitrification. Vitrified embryos ≤300 μm give better pregnancy rates following warming and transfer than larger ones. Embryo recovery undertaken close to when the embryo enters the uterus (Day 6-6.5) helps in the recovery of embryos ≤300 μm. However, flushing early can mean missing an embryo not yet in the uterus, whereas later can result in embryos >300 μm. Objective: To evaluate if repeated embryo flushing on consecutive days from Day 6.5 would increase the number of embryos ≤300μm recovered. Methods: Retrospective case series. Methods: Four hundred and ninety-six inseminations with cooled ...
Ultra-rapid freezing in spheres yields a higher cryoresistance than in straws but remains inferior to conventional slow freezing of stallion sperm. This study evaluated the cryoresistance of stallion sperm frozen by ultra-rapid (UR) methods using microspheres and straws or by the conventionally-slow (CS) method. Sixteen ejaculates from four stallions were each divided into three aliquots according to the freezing method: UR freezing in 30-μL spheres (UR-Spheres) by direct immersion in liquid nitrogen (LN); UR freezing in 0.25-mL straws (UR-Straws) by direct horizontal submersion in LN; and CS freezing in LN vapors. Ultra-rapid freezing medium included 100 mM trehalose +1 % BSA, and the CS freezing medium contained 5 % dimethylformamid...
Sexual arousal intensity modulates copulatory behavior and semen quality in Arabian stallions: Effects of age, origin, and collection method. Stallion sexual behavior during semen collection can be influenced by multiple factors, yet the role of sexual arousal intensity remains underexplored. Understanding how arousal modulates behavioral and physiological reproductive traits is essential for improving artificial insemination (AI) efficiency and semen quality. This study aimed to evaluate the effects of sexual arousal intensity, age, origin, and semen collection method on stallion sexual behavior and the quality of fresh and frozen semen. Unassigned: Thirteen Arabian stallions (7 Tunisian, 6 foreign parentage) aged 6-20 years (total...
Retrospective Evaluation of Dystocia in Miniature Equids: 78 Cases (2002-2023). To describe patient characteristics, laboratory data, fetal orientation, methods of correction, survival, and treatment in miniature equids with dystocia. Methods: Retrospective study conducted from January 2002 to June 2023. Methods: Equine referral hospital and field service. Methods: Seventy animals with a total of 78 instances of dystocia. Methods: None. Results: Recorded variables included signalment, clinicopathologic data, fetal presentation, correction method, survival to discharge, and complications. The survival of miniature equid mares was 94% (73/78), while survival of foals was 17...
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...
Endocrine profile and OPU-ICSI outcomes in mares: a comparative study. Despite recent improvements in equine ovum pick-up (OPU) combined with intracytoplasmic sperm injection (ICSI), there is still significant inter-individual variability. In this study, serum concentrations of the oocyte-secreted factors growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), as well as the GDF9/BMP15 complex, anti-Müllerian hormone (AMH), progesterone (P4), and free and conjugated estradiol-17β (E2), were determined in 59 mares (3-24 years) at the time of OPU. Mares were classified retrospectively, based on the number of embryos obtained, into low or...