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Topic:Equid Semen
Equid semen refers to the reproductive fluid produced by male horses, which contains sperm cells necessary for fertilization. The quality and viability of equid semen are important for successful breeding programs and can be influenced by various factors, including genetics, age, diet, and environmental conditions. Key parameters used to assess semen quality include volume, concentration, motility, and morphology of sperm cells. Techniques for collecting, evaluating, and preserving equid semen, such as cryopreservation, are critical for artificial insemination practices. This page compiles peer-reviewed research studies and scholarly articles that explore the physiology, assessment, and management of equid semen in the context of equine reproduction.
Questionnaire-Based Investigation into Stallion Management Practices in 28 Countries. Modern stud farm practices may limit stallion social contact, with potential welfare and safety implications. Objective: To describe housing, management and safety outcomes for stallions kept at horse breeding operations throughout the world and to assess regional differences. Methods: An online questionnaire was distributed via mailing lists and WhatsApp; 116 complete responses from breeding operations were collected and analysed. The focus was on management of the breeding facility, and stallion welfare indicated by management, handling and housing. Results: Respondents were from Europe (40....
The Effect of Different Extenders and Testicular Anaesthesia During Orchiectomy on Post-Thawed Spermatological Parameters of Stallion Epididymal Spermatozoa. Cryopreservation of epididymal stallion spermatozoa is a valuable tool for fertility preservation, particularly after orchiectomy. However, the potential interactions between local anaesthesia and extender composition on post-thaw spermatozoal quality remain poorly understood. This study investigated the effects of intratesticular lidocaine injection and the efficacy of various extender (egg yolk [EY] vs. lecithin [LC]) and cryoprotectant (glycerol [GLY] vs. dimethyl sulfoxide [DMSO]) combinations on stallion epididymal spermatological parameters. Using a paired factorial design, epididymal s...
Somatic cell nuclear transfer as a complementary tool for genetic conservation of the Peruvian Paso horse. The Peruvian Paso horse, a breed of national significance in Peru known for its unique gait, has been selectively bred for nearly 500 years and now faces potential risks due to reduced genetic diversity. To investigate the potential of somatic cell nuclear transfer to enhance genetic conservation strategies, a program was initiated to produce cloned Peruvian Paso foals. Skin biopsies from a highly awarded stallion with exceptional breed-defining traits were used as the source of donor somatic cells. Of the 15 cloned embryos reconstructed, seven blastocysts were produced, and five were transfer...
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...
Redefining stallion sperm bioenergetics: lessons from a decade of change in mammalian sperm metabolism. In brief: Sperm bioenergetics is now understood as an integrated system in which ATP production, redox homeostasis, substrate availability, and cellular heterogeneity jointly determine sperm function. This review uses the stallion as an oxidative-biased model to synthesize advances from 2016 to 2026 and proposes a metabolically plastic framework with direct implications for semen preservation and fertility assessment. Abstract: Over the last decade, mammalian sperm metabolism has moved from a binary view centered on glycolysis versus oxidative phosphorylation to an integrated model in which AT...
A multimodal approach to equine thoracolumbar myofascial pain: integrating clinical examination, AI-based gait analysis, and the ridden horse pain ethogram. Myofascial pain syndrome (MPS) is considered highly prevalent in horses, yet it is frequently underdiagnosed due to limited understanding of its clinical presentation and functional consequences, as well as the inherent challenges associated with its clinical assessment and objective measurement. In equines, the relationship between palpation-based MPS indicators, gait asymmetries, and pain-related behaviours during ridden work remains unclear. The primary aim of this study was to evaluate the association between thoracolumbar palpation pain scores and gait asymmetries measured in-hand and dur...
Nano-Encapsulated Vitamin E Enhances Cryo-Resistance of Stallion Spermatozoa: A Comprehensive Evaluation of Sperm Quality, Kinematic Parameters, Antioxidant Capacity, Microbiota and Ultrastructure. This experiment explores the comparative ability of vitamin E (VE) and its nano-emulsion (VEN) in enhancing cryo-resistance of stallion spermatozoa by examining semen quality, kinematic variables, acrosome integrity, apoptosis, antioxidant capacity, ultra-structural alterations and microbiota of cryopreserved stallion sperm. Semen from five stallions (8-10 years) was cryopreserved with 0.1 or 1 mM of VE or VEN; and a non-additive group served as the control. The VEN particles ranged in size from 72 to 172 nm, with a mean diameter of 287 nm. Supplementation with VE (1 mM) or VEN (0.1 ...
Membrane and mitochondrial antioxidants improve post-thaw functional quality of stallion spermatozoa. Cryopreservation of stallion semen induces oxidative stress that compromises sperm motility, membrane integrity, and mitochondrial function. Given the high mitochondrial reliance of equine spermatozoa and their susceptibility to oxidative damage, compartment-specific antioxidant strategies may improve post-thaw functional resilience. This study evaluated the effects of astaxanthin, a membrane-active carotenoid, and MitoTEMPO, a mitochondria-targeted superoxide scavenger, on the functional and oxidative status of frozen-thawed stallion spermatozoa. Ejaculates from nine stallions were cryopreser...
Application of 3D scanning for modeling and morphometric assessment of Mangalarga Marchador Horses. Morphometric traits are essential for assessing conformation, locomotion, and selection in equine breeding programs. However, manual measurements are laborious, require physical contact, and may cause stress to animals, highlighting the need for alternative technologies. Objective: This study evaluated the use of a portable 3D scanner for three-dimensional modeling and morphometric assessment of Mangalarga Marchador horses. Methods: Fifty-two adult horses were measured using manual and 3D scanning methods to assess ten morphometric traits: withers height (WH), hip height (HH), head length (HL)...
ProAKAP4 as a predictor of semen freezability in Asturcon ponies. Cryopreservation is a valuable tool in equine reproduction, although its success remains highly variable among stallions. The proAKAP4 protein has been proposed as a molecular marker of sperm quality and cryotolerance. Objective: This study aimed to evaluate the relationship between proAKAP4 concentration and sperm cryotolerance in ejaculates from Asturcon ponies, and to assess whether proAKAP4 levels in fresh semen can predict post-thaw sperm quality parameters before and after cryopreservation. Methods: Twenty-four ejaculates from eight different stallions were used (three ejaculates per ani...
Progressive structural and functional change in horses: a conceptual framework for systemic equine (patho-)physiology. A wide range of locomotor, postural, and behavioral pathophysiology in horses-often grouped under terms such as Topline Syndrome, Myofascial Dysfunction, or Poor Posture Syndrome-lack a coherent systemic explanation. These presentations share reduced performance, stability, and resilience, yet are commonly managed through isolated symptom-focused interventions. To address this gap, we propose progressive structural and functional change (PSF) as a systemic framework describing progressive reorganization processes within the equine body that link fragmented pathological domains into a coherent ...
Conditional Antioxidant Protection of Stallion Spermatozoa by Butylated Hydroxytoluene Under Induced Oxidative Stress. Despite advances in the method, equine semen cryopreservation still leads to a marked decline in sperm quality, partly attributed to oxidative stress, which motivates the search for lipophilic antioxidants capable of stabilizing the plasma membrane during cooling and freezing. This study performed three experiments to evaluate butylated hydroxytoluene (BHT) as an additive to commercial skim milk-based (Botu-Sêmen) and egg yolk-based (Botu-Crio) extenders. In Experiment 1, a dose-response curve (0.5, 1.0, and 1.5 mM) was performed using stallion semen to assess toxicity and establish a safe ...
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...
Niacin Improves Cryopreserved Equine Sperm Quality and Gene Expression: An Artificial Intelligence Assisted Evaluation. Niacin acts as an antioxidant that protects cells from oxidative damage. This study evaluated the effects of adding niacin to the equine semen freezing extender on sperm quality and gene expression after cryopreservation. Ejaculates from ten stallions were frozen using the INRA 96 extender (control) or extenders supplemented with 10- and 20-mM niacin. After thawing, sperm were analysed for motility, kinematics, viability, membrane integrity, mitochondrial potential, lipid peroxidation, nitrite, hydrogen peroxide, malondialdehyde and reactive oxygen species (ROS) concentrations, DNA integrity, ...
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 ...
Addition of cholesterol linked to cyclodextrin in processing of cryopreserved equine spermatozoa and addition of motility stimulants post-thaw. This study investigated the effects of cholesterol-loaded cyclodextrin (CLC) before cryopreservation and the use of FertTalp (FT) after thawing on the structural and functional quality of equine spermatozoa. Methods: Two ejaculates from twelve stallions were divided into four groups: G1 (0 mg CLC), G2 (1.0 mg), G3 (1.5 mg), and G4 (2.0 mg). Post-thaw semen quality was evaluated through computer-assisted sperm analysis and flow cytometry. Results: In Experiment I, supplementation with 1.5 mg (G3) significantly improved total motility (64.9 ± 10.1 %) and progressive motility (40.5 ± 8.8 %) com...
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...
Effect on equine sperm of post-thaw glycerol dilution using two different semen extenders. Glycerol, a penetrating cryoprotectant, is most employed for deep freezing spermatozoa. However, it can induce toxic, chemical, and osmotic effects, altering the lipid structure of the sperm membrane. Rapid glycerol addition has been demonstrated to have fewer toxic effects than its removal. Objective: To minimize both the toxic and osmotic effects of glycerol on equine sperm through post-thaw dilution using two different extenders. Methods: Samples of equine semen frozen with 5% glycerol from nine stallions were thawed, re-diluted 1:2, and incubated for 30 minutes at 22°C in Tyrodes (Tyr) an...
Quantitative holographic analysis in stallion spermatozoa following cryopreservation. This study employs Holographic tomography (HT) to examine structural and biophysical changes occurring during the cryopreservation of stallion sperm. HT is an advanced imaging technique that integrates digital holography with tomography to achieve three-dimensional, quantitative reconstructions of objects without the need for treatment or reporter dyes. By using refractive index (RI) intervals to represent specific structural regions of sperm cells, variations in optical density, surface area, volume, and dry mass across different cryopreservation extenders and donors have been quantified. Thr...
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...
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...
Evaluation of a Chemically Defined, Long-Term Extender for Liquid Storage of Stallion Semen. Efficient use of stallion semen in liquid state is limited by its relatively short shelf-life. A chemically defined extender (Beyond) is now available for long-term liquid semen preservation. The objectives of the present study were to compare Beyond with milk extenders for the preservation of semen at two temperatures, and to evaluate fertility of semen cooled for 4-8 days before artificial insemination. Semen was processed using different extenders: milk, cholesterol (BotuSemen Special); milk-based (INRA 96); and Beyond. Sperm motility, membrane and acrosome integrity, and chromatin struct...
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...
Developing a greenhouse gas emission model for sport horses: case studies in dressage and jumping. Sustainability is gaining growing attention within the equine sector. Sustainability consists of various subjects including feed and nutrition, resource efficiency, animal welfare, carbon footprint (CFP) and biodiversity, for example. Studies in horse research focus mainly on behaviour and welfare. Little information is available about the environmental impact of equids. The few studies that can be found are principally concerned with local impact, such as nitrogen leaching or ammonia concentrations in bedding. Little is known about greenhouse gas emissions (GHG) for horses, although GHG emiss...
Butylated hydroxytoluene (BHT) improved semen quality and sperm DNA of frozen-thawed Arabian stallions preserved in modified INRA-82 extender. Alpha-tocopherol is one of the non-enzymatic lipophilic antioxidants. Butylated hydroxytoluene (BHT) is a synthetic analog with similar modes of action in protecting the cryopreserved sperms. This study hypothesized that a certain concentration of any antioxidant is suitable for improving the post-thaw semen quality of stallions. To determine the optimum BHT concentration, a synthetic antioxidant similar to vitamin E in potency and scavenging oxidative stress power in concentrations of 0.0, 0.25, 0.50, 1.0, 2.0, and 4.0 mM/ml were added to semen extender. The post-thaw sperm progressive moti...
Influence of Cryopreservation on the Acrosome Reaction in Hucul Stallion Spermatozoa. The Hucul horse is a Polish primitive breed with a small population size, which highlights the importance of preserving the genetic resources. The cryopreservation of semen is essential for creating gene banks, but its effect on the acrosome reaction in Hucul stallions has not yet been investigated. The acrosome reaction is one of the most important physiological events associated with the fertilization process. Therefore, our goal was to determine the level of acrosome reaction in chilled and frozen/thawed Hucul stallion semen using the FluoAcro test and the SCA semen analysis system. We foun...
Effect of Centrifugation of Stallion Semen Through a Low Density Colloid Prior to Freezing on Sperm Cryosurvival. Sperm quality is adversely affected by cryopreservation due to the increased production of reactive oxygen species, which affects the integrity of sperm membranes, motility, and DNA fragmentation. Three methods for removing seminal plasma, washing (centrifuging extended semen at 800× for 10 min) and Single Layer Centrifugation with high or low density Equicoll, were used to prepare 29 ejaculates from ten stallions for freezing. Sperm quality parameters (kinematics, plasma membrane integrity, superoxide and hydrogen peroxide production, mitochondrial membrane potential, and DNA fragmentation)...
