Omneity® Pellets
All-In-One Vitamin & Mineral Pellet
Topic:Semen Analysis
Semen analysis involves the evaluation of semen quality to assess reproductive potential and health in horses. This process typically includes the examination of several parameters such as sperm concentration, motility, morphology, and volume. Semen analysis is used in breeding programs to determine the fertility of stallions and to identify any reproductive issues. The collected data can assist in making informed decisions about breeding management and interventions. This page compiles peer-reviewed research studies and scholarly articles that explore the methodologies, findings, and implications of semen analysis in equine reproduction.
Unraveling the genetic and physiological potential of donkeys: insights from genomics, proteomics, and metabolomics approaches. Donkeys (Equus asinus) have played a vital role in agriculture, transportation, and companionship, particularly in developing regions where they are indispensable working animals. The domestication of donkeys marked a significant turning point in human history, as they became essential for transportation, agriculture, and trade, especially in arid and semi-arid areas where their resilience and endurance were highly valued. In modern society, donkeys are indispensable due to their diversified applications, including meat, dairy, medicine, and functional bioproducts, supporting economic, cultura...
Spermatozoal acrosome dysfunction and its role in stallion subfertility. Cases of stallion subfertility due to acrosome dysfunction have been recognized since the 1990s. While some of these were observed in stallions with reduced sperm motility and morphology, a more severe form has been reported in stallions with normal-to-excellent sperm quality parameters, which is also uniquely observed in individuals of the Thoroughbred registry. These stallions carry a susceptibility genotype (A/A-A A in the gene FKBP6, exon 5) for Impaired Acrosomal Exocytosis (IAE). Current clinical observations from our group have identified a few highly subfertile stallions from other bre...
Prolonged incubation of frozen-thawed equine spermatozoa for in vitro fertilization: A preliminary study using low temperature and INRA96 medium. A protocol for conventional in vitro fertilization (IVF) in horses using fresh semen has been described, using a prolonged incubation in FERT-TALP medium (22 h) at 38.2°C in the presence of penicillamine, hypotaurine and epinephrine (PHE). Our work aimed to develop a protocol that maintains quality parameters in frozen-thawed equine spermatozoa incubated for 22 h in the presence of PHE using different media (FERT-TALP and INRA96) and incubation temperatures (30 and 38.2°C). Twelve frozen ejaculates from four stallions were thawed and then incubated in either FERT-TALP or INRA96 with PHE...
Effect of refreezing extender on stallion sperm quality and embryo production after intracytoplasmic sperm injection. Intracytoplasmic sperm injection (ICSI) is a valuable assisted reproduction technology in clinical practice, especially when semen availability is limited. Since the number of sperm required per ICSI cycle is much less than the number of sperm available in a standard straw of frozen semen, refreezing semen at lower sperm concentrations could yield multiple straws for ICSI use. However, there is little data on the effect of sperm refreezing on ICSI outcomes, especially on the effect of extender used for refreezing. The objective of the present study was to evaluate the effect of refreezing exte...
Effect of protein acetylation on capacitation of stallion sperm. Sperm capacitation is considered the main factor limiting conventional in vitro fertilization (IVF) in horses. A recent scientific breakthrough in sperm processing for IVF in horses has resulted in embryos and foals being produced; however, various aspects of the IVF process remain to be fully elucidated. Lysine acetylation has been shown to play a role in sperm capacitation in several species and the objective of this study was to detect and evaluate this process in the horse. Ejaculates of two stallions were collected and incubated in different conditions with deacetylase inhibitors to induc...
Pre- and Post-Thaw Addition of L-Carnitine and Pyruvate: Effect on Stallion Sperm Parameters. The addition of antioxidants to cryopreservation media reportedly improves sperm post-thaw quality and reproductive performance after artificial insemination. Therefore, the objectives of this study were to evaluate if the addition of L-carnitine and pyruvate to freezing media, or their addition to samples after thawing, improves the post-thaw quality of equine spermatozoa. Thus, in Experiment 1, stallion semen samples were cryopreserved in: (1) EDTA-glucose-based extender with 20% egg yolk and 5% dimethylformamide (EDTA control); (2) skim milk-based extender with 20% egg yolk and 5% dimethylf...
Factors affecting pregnancy rates in mares bred with cryopreserved semen. Historically, 8 × 0.5 ml straws, containing approximately 800 million sperm and 250 million progressively motile sperm were provided as a single 'breeding dose' of cryopreserved stallion semen. With the use of deep horn artificial insemination, there is a trend to reduce the number of 0.5 ml straws sold as a breeding dose, sometimes down to as little as one straw. Our aims were to determine if the number of straws provided as a breeding dose, as well as other mare, stallion and management factors, have an impact on pregnancy outcome in mares inseminated with cryopreserved semen. Unexpectedly,...
Effect of Mitoquinone on sperm quality of cryopreserved stallion semen. This study aimed to investigate the effect of mitochondria-targeted antioxidants (Mitoquinone, MitoQ) on the quality of frozen-thawed stallion semen. Semen samples collected from three fertile stallions aged 10 - 13 years, were filtered, centrifuged in a skimmed milk-based extender, and diluted to a final concentration of 50 × 106 sperm/mL in freezing medium. Diluted semen was divided into five experimental groups supplemented with MitoQ at concentrations of 0 (control), 25, 50, 100, and 200 nM and then subjected to freezing after cooling and equilibration. After thawing, semen was evaluated ...
Comparative Study on the Sperm Proteomes of Horses and Donkeys. The reproductive performance of horse sperm and donkey sperm has been reported to differ. Sperm proteins play a crucial role in sperm viability and fertility. Although differences between species are known, no prior study has investigated disparities in the sperm proteome between horses and donkeys. Therefore, this study characterized and compared the sperm proteomes of horses and donkeys using 4D-DIA mass spectrometry technology. We identified 3436 proteins in horse sperm and 3404 proteins in donkey sperm. Of these, 3363 proteins were expressed in both horse and donkey sperm, with 73 proteins...
Impact of low-dose ozone supplementation on motility parameters and bacterial growth in horse cryopreserved semen. Two studies were conducted to evaluate the use of medical ozone (O) in commercial extenders for equine semen cryopreservation. In the first study (Study 1), 0, 5, and 15 µg/mL of O were added to diluents of refrigerated or frozen semen. Samples were evaluated for sperm kinematics at different time points for the chilled samples and after a thermoresistence test for the frozen/thawed samples. In the second study (Study 2), 0, 5, and 10 µg/mL of O were added to an antibiotic-free diluent for refrigerated semen for comparison with the control group in which semen was diluted in the same diluent...
Specific microRNAs in stallion spermatozoa are potential biomarkers of high functionality. Males of some species, from horses to humans, require medical help for subfertility problems. There is an urgent need for novel molecular assays that reflect spermatozoal function. In the last 25 years, studies examined RNAs in spermatozoa as a window into gene expression during their development and, more recently, for their functions in early embryo development. In clinics, more dense spermatozoa are isolated by density gradient centrifugation before use in artificial insemination to increase pregnancy rates. The objectives of the current study were to discover and quantify the microRNAs i...
The Effects of Supplementation of the Freezing Extender with Silymarin on the Quality Parameters of Frozen-Thawed Arabian Stallion Sperm: A Preliminary Evaluation. This study evaluated the effects of supplementation of the freezing extender with different concentrations of silymarin on the quality of frozen-thawed Arabian stallion spermatozoa. Semen samples from three stallions (1, 2, and 3) were suspended in the freezing extender without or with silymarin (0, 25 μg/mL, 50 μg/mL, 75 μg/mL, and 100 μg/mL) and cryopreserved in 0.5 mL straws. After 1 month of storage, the frozen semen samples in straws were thawed and evaluated in terms of viability, mitochondrial membrane potential, kinematic parameters, total and progressive motility, plasma membrane ...
Effect of exogenous sperm capacitation inducers on stallion sperm. Although under appropriate laboratory conditions, sperm from different mammalian species can be capacitated in vitro, the optimal conditions for sperm capacitation in the stallion have been elusive. This study evaluated the effect of different capacitating inducers in Whitten and Tyrode media and assessed their impact on capacitation-related factors. Stallion sperm were incubated with different combinations of capacitating inducers at 38.5 °C in an air atmosphere. Sperm quality variables such as motility, mitochondrial membrane potential, and lipid peroxidation were assessed. Membrane fluidi...
The future of equine semen analysis. We are currently experiencing a period of rapid advancement in various areas of science and technology. The integration of high throughput 'omics' techniques with advanced biostatistics, and the help of artificial intelligence, is significantly impacting our understanding of sperm biology. These advances will have an appreciable impact on the practice of reproductive medicine in horses. This article provides a brief overview of recent advances in the field of spermatology and how they are changing assessment of sperm quality. This article is written from the authors' perspective, using the sta...
Application of sperm motion kinematics and motility-related proteins for prediction of male fertility. The selection of superior sires is paramount for enhancing the efficiency of animal production in the livestock industry. However, semen quality assessment still relies on conventional semen analysis techniques in both animals and humans. Despite extensive efforts to develop various biomarkers for more accurate and precise predictions of male fertility potential, more effective physiological indicators and advance potential biomarkers are needed. Herein, we aimed to develop new potential biomarkers related to sperm motion kinematics for male fertility prediction. We first evaluated sperm motio...
Freezing of equine semen is influenced by exposure time and concentration of the cryoprotectant glycerol. Glycerol is a cryoprotectant widely used in the freezing of mammalian semen, but no study has demonstrated its optimum concentration and the appropriate exposure time for equine species. Objective: To demonstrate that the exposure time (15, 30, 45, 60, 75 and 90 min) versus concentration (2, 3, 4 and 5%) of the cryoprotectant glycerol influences the freezing success of equine semen. Methods: The ejaculate of 12 stallions were frozen in different glycerol concentrations following different exposure times. The thawed sperm was evaluated for kinetic parameters using a Computer Assisted Semen Anal...
Effect of nutraceutical supplementation on semen quality in stallions. The use of reproductive biotechnologies in equine practice has shown that some stallions are subfertile, so ways to improve fertility have been sought. This study aimed to evaluate the effect of nutraceutical supplementation on improving semen quality in Quarter Horse stallions. Semen from six Quarter Horse stallions was assessed for 4 months every 20 days using the computer-assisted semen analysis system. They were evaluated for 60 days before supplementation; then, the same stallions were re-evaluated for 60 days with nutraceutical supplementation (30 g/day). Volume showed no significant...
Sperm Quality Assessment in Stallions: How to Choose Relevant Assays to Answer Clinical Questions. Stallion sperm analysis is indicated for infertility diagnosis, pre-sale expertise, production of fresh or frozen doses, and frozen straw quality control. Various collection methods are described, and numerous assays can be performed on semen. Determining an approach for each of these cases is challenging. This review aims to discuss how to obtain relevant clinical results, answering stallion owners' concerns. Semen can be collected with an artificial vagina on a phantom or a mare, by electro-ejaculation under anesthesia, or after pharmacological induction. The collection method influences the...
Multiparametric Flow Cytometry for Determination of Viability, Caspase 3 and 7 Activity, and Lipid Peroxidation Adduct (4-Hydroxynonenal) in Equine Spermatozoa. Flow cytometry is a powerful tool for the analysis of cell samples formed of multipopulations, such as spermatozoa. In recent years, multiparametric cytometers have evolved, allowing the study of different cellular characteristics, such as protein expression, DNA analysis, or mitochondrial activity. Whether using traditional fluorescent dyes or fluorophore-conjugated antibodies, each cell or cellular component is individually stained, the sample is analyzed at high velocities, and then is displayed and interpreted in a dot-plot. We hereby describe the procedure to perform a multiparametric flo...
Flow cytometric detection and identification of different leukocyte subpopulations in stallion semen. To improve accuracy in evaluating stallion ejaculates, an antibody-based, flow cytometric assay for the detection and identification of leukocyte subpopulations (CD4-, CD8-, CD21-, CD172a-positive cells) in stallion semen (n = 12) was established. For establishment of the assay, native semen was supplemented with blood leukocytes (control: 20% leukocytes, 80% sperm cells) and analysed by flow cytometry. Adding antioxidants (ascorbic acid and butylated hydroxytoluol) to semen immediately after collection inhibited rapid death of lymphoid cells in sperm leukocyte mixtures. In control set-ups...
Standardization of a Sex-Sorting Protocol for Stallion Spermatozoa by Means of Absolute RT-qPCR. Sperm sexing is a technology that can generate great economic benefits in the animal production sector. Techniques such as sex-sorting promise over 90% accuracy in sperm sexing. However, for the correct standardization of the technique, some laboratory methodologies are required. The present manuscript describes in detail a standardized equine sperm sex-sorting protocol using an absolute qPCR-based methodology. Furthermore, the results of absolute qPCR were implemented and validated by generating equine/bovine heterologous embryos by intracytoplasmic sperm injection (ICSI) of presumably sexed ...
Applicability of 2,4-dinitrophenylhydrazine (DNPH) method of protein oxidative damage measurement in the seminal plasma of canine ( Canis lupus familiaris) and stallion ( Equus caballus). Seminal plasma (SP) proteins are responsible for sperm functional quality. Developing a reliable method to determine the degree of oxidative damage of these proteins is important for establishing semen fertilizing ability. The main aim of the study was to verify the applicability of protein carbonyl derivatives measurement in the SP of canine and stallion, using a method with 2,4-dinitrophenylhydrazine (DNPH). The research material consisted of ejaculates obtained from eight English Springer Spaniels, and from seven half-blood stallions during the breeding and non-breeding season. The content ...
Impact of inbreeding and genetic parameter estimates for seminal traits in Lusitano horses. The objectives of this study were to establish baseline information for seminal traits in Lusitano stallions, to assess the impact of inbreeding, interval between collections and age on semen quality during the breeding and non-breeding seasons, and to estimate the corresponding genetic parameters. A total of 2129 ejaculates by 146 Lusitano stallions used for artificial insemination, obtained from four equine reproduction centers distributed throughout Portugal, over a period of 14 years (2008-2021), were included in the study. The seminal traits analyzed, and the corresponding means and stand...
Effect of testicular degeneration on expression of sperm protein at 22 kDa in stallions. Sperm protein at 22 kDa has been associated with fertility. Objective: The objectives of this study were to determine (1) the localization pattern of SP22 on ejaculated and caudal epididymal equine spermatozoa and in epididymal fluid, and to (2) characterize SP22 protein and mRNA expression in testicular and epididymal tissues in response to heat-induced testicular degeneration. Methods: Semen was collected before and after hemi-castration, as well as prior to and following insulation of the remaining testes, and tissue specimens were collected for analysis. Results: Histopathology confirmed ...
Predicting the Outcome of Equine Artificial Inseminations Using Chilled Semen. This study aimed to determine whether an analysis of stallion ejaculate could accurately predict the likelihood of pregnancy resulting from artificial insemination in mares. This study involved 46 inseminations of 41 mares, using 7 standardbred stallions over a 5-week period at an Australian pacing stud. Semen quality was assessed immediately after collection and again after chilling at ~5 °C for 24 h. The assessment involved evaluating ejaculate volume, sperm concentration, and motility parameters using an iSperm® Equine portable device. After the initial evaluation, a subpopulation of cell...
Case report: Long term follow-up of a large unilateral epididymal cyst in a stallion used for teaching: Is this condition associated with infertility? A 30-year-old Lusitano stallion presented with an enlarged right epididymis. The ultrasound scan revealed a cyst-like formation and the histopathological examination was compatible with epididymal cyst located at the body/tail transition, epididymal spermatocele and sperm granuloma and epididymitis. However, these conditions did not seem to affect the animal's reproductive performance, nor did the semen parameters analyzed over the 8 years after the diagnosis show significant changes. Nevertheless, since the ejaculate contains mostly sperm cells from the tail of the epididymis, where fertile s...
Freezability and Fertility Rates of Stallion Semen Supplemented With Trehalose in Lactose Extender. Cryopreservation of stallion semen is often associated with poor post-thaw sperm quality. One of the reason for this diminished quality is osmotic stress that spermatozoa experiences during freezing and thawing process. The aim of the present study was to evaluate the cryoprotective effect of trehalose on stallion sperm quality and field fertility rates subjected to cooling and freeze-thawing process. Semen samples were collected from six Marwari breed stallions, divided into three different treatments in a final concentration of 150 × 106 sperm/mL by using Lactose based extender containing...
Dual use of breeding stallions is possible without affecting the sperm quality. Artificial insemination (AI) is commonly used in the equine industry to enhance the genetic value in breeding programs and to effectively utilize ejaculates. Many stallions are used as breeding stallions as well as in high-level sports competitions to improve their market value. The goal of the present study was to investigate whether this dual use of stallions influences the animals´ stress levels and/or the quality of their ejaculates. For this purpose, 18 stallions were grouped into two categories: breeding stallions with (BSC = breeding stallion competition), and breeding stallions with...
A matter of agreement: The effect of the technique and evaluator on the analysis of morphologic defects in stallion sperm. Analysis of sperm morphology is an important part of the stallion breeding soundness evaluation since it provides an objective measure of a stallion's sperm quality and is one of many factors that estimate a stallion's fertility potential. To describe the effect of sperm quality level on the technique (Differential Interference Contrast - DIC; Phase-contrast - PH; Dip-Quick staining - DQ; and eosin-nigrosin staining - EN; semen samples fixed in buffered-formal saline) and evaluator (three evaluators; using only DIC), stallions were categorized based on sperm quality into three categories: High...
