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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.
Reproductive characteristics of stallions during the breeding and non-breeding season in a tropical region. The objective of this study was to investigate reproductive characteristics of stallions at a tropical zone in the breeding and non-breeding seasons. The following parameters were assessed: testicular volume; semen quality; and serum concentrations of LH, FSH, and testosterone; in addition to the percentages of germ cells and proportions of germ cells/Sertoli cells by testicular cytology in stallions. Semen was collected from eight adult stallions twice a week during a 12-week period in both seasons (6 weeks before and 6 weeks after the summer and winter solstices). Jugular blood samples were ...
Post-thaw addition of seminal plasma reduces tyrosine phosphorylation on the surface of cryopreserved equine sperm, but does not reduce lipid peroxidation. The objective was to verify the relationship between equine semen cryopreservation and changes related to increased lipid peroxidation. Also, addition of autologous or homologous seminal plasma from a stallion with a good freezing response to post-thawed sperm was tested to determine whether it would confer protection. Frozen-thawed sperm were evaluated and allocated into three groups: without plasma addition, and supplemented with either homologous or autologous seminal plasma. All groups were evaluated at 0, 60 and 120 min after incubation at 37 °C. Cryopreservation did not increase plasma ...
Effects of components of semen extenders on the binding of stallion spermatozoa to bovine or equine zonae pellucidae. The effects of semen extender components on the ability of stallion sperm to bind to the zona pellucida (ZP) and the suitability of using bovine ZP for a ZP-binding assay for stallion sperm were investigated in a series of experiments. In Experiment I, binding of stallion sperm to both bovine and equine ZP was significantly increased when a skim milk-based extender (EZM) was used. In Experiment II, a threefold increase in sperm binding to ZP was observed when sperm were diluted in EZM compared with diluents, which contained no milk (TALP, LAC, and EmCare). In Experiment III, centrifuging the s...
Effect of daily semen centrifugation and resuspension on the longevity of equine sperm quality following cooled storage. An experiment was conducted to determine whether cooled semen quality could be maintained for a longer interval by conducting daily centrifugation of extended semen, with resuspension of the sperm pellet in fresh extender. Semen treatments included SP10NC and SP50NC which contained 10 and 50% seminal plasma, respectively, were not centrifuged (NC), and were stored at 4 to 7 °C for 96 h. Treatments SP10C and SP50C contained 10 and 50% seminal plasma, respectively, but were centrifuged (C) after 24, 48, and 72 h of cooled storage, with daily resuspension in fresh extender containing 10% seminal...
Dimethylformamide improves the in vitro characteristics of thawed stallion spermatozoa reducing sublethal damage. A total of 42 ejaculates were used in the experiment; six ejaculates per stallion, obtained from seven Pure Spanish stallions (PRE), were split and frozen in freezing media with different concentrations and combinations of cryoprotectant (CPA): (i) Cáceres (skim milk based extender) containing 2.5% glycerol (2.5GL), (ii) Cáceres containing 1.5% glycerol and 1.5% dimethylformamide (1.5%GL-1.5%DMFA), (iii) Cáceres extender supplemented with 1.5% glycerol and 2.5% dimethylformamide (1.5%GL-2.5%DMFA) and (iv) Cáceres extender supplemented with 4% dimethylformamide (4%DMFA). After at least 4 we...
Autophagy and apoptosis have a role in the survival or death of stallion spermatozoa during conservation in refrigeration. Apoptosis has been recognized as a cause of sperm death during cryopreservation and a cause of infertility in humans, however there is no data on its role in sperm death during conservation in refrigeration; autophagy has not been described to date in mature sperm. We investigated the role of apoptosis and autophagy during cooled storage of stallion spermatozoa. Samples from seven stallions were split; half of the ejaculate was processed by single layer centrifugation, while the other half was extended unprocessed, and stored at 5°C for five days. During the time of storage, sperm motility (C...
The impact of cushioned centrifugation protocols on semen quality of stallions. The objective was to determine if decreased cushion-fluid volume and increased sperm number during centrifugation, or if sperm concentration of extended semen following centrifugation, affected stallion sperm quality. Three ejaculates from each of three stallions were subjected to cushioned centrifugation (1,000g for 20 min). Cushion-fluid volume was set at 1 or 3.5 ml, and sperm number per centrifuge tube was set 1 billion or 3 billion. Following centrifugation, sperm pellets were resuspended in semen extender containing 20% seminal plasma (v/v) with sperm concentrations of 25 or 250 million/...
Influence of counting chamber type on CASA outcomes of equine semen analysis. Sperm motility is considered to be one of the key features of semen analysis. Assessment of motility is frequently performed using computer-assisted sperm analysis (CASA). Nevertheless, no uniform standards are present to analyse a semen sample using CASA. Objective: We hypothesised that the type of counting chamber used might influence the results of analysis and aimed to study the effect of chamber type on estimated concentration and motility of an equine semen sample assessed using CASA. Methods: Commonly used disposable Leja chambers of different depths were compared with disposable and re...
Effect of centrifugal fractionation protocols on quality and recovery rate of equine sperm. Centrifugal fractionation of semen is commonly done to improve quality of human semen in assisted-reproduction laboratories, allowing sperm separation based on their isopycnic points. Sperm with morphologic abnormalities are often more buoyant, promoting their retention above defined density media, with structurally normal sperm passing through the media following centrifugation. Three experiments were conducted to evaluate the effects of density-medium type, centrifuge-tube size, sperm number, and density-medium volume (column height) on stallion sperm quality and recovery rate in sperm pelle...
Purchase examinations and importation requirements for European performance horses and their semen entering the United States. A comprehensive purchase examination is expected by American clients intent on importing a horse from a foreign country. American veterinarians may be involved in performing purchase examinations in foreign countries or, more often, interpreting findings from foreign veterinarians for their American clients. Exportation and importation requirements for horses and semen vary from country to country. Detailed knowledge of the requirements by all involved veterinarians is essential for efficient and successful international equine travel.
Effect of staining and freezing media on sortability of stallion spermatozoa and their post-thaw viability after sex-sorting and cryopreservation. Sex-sorted, frozen-thawed stallion spermatozoa remain out of reach of commercial horse breeders because of the low efficiency of the sex-sorting process and unacceptable fertility rates after insemination. Two experiments were designed to test the effects of alternative staining and freezing media to improve the viability of sex-sorted frozen-thawed stallion spermatozoa. Experiment 1 compared two freezing media, INRA 82(®) and a modified lactose-ethylenediaminetetraacetic acid (EDTA), for the cryopreservation of sex-sorted stallion spermatozoa. No significant differences between the two freez...
Liposomes as an alternative to egg yolk in stallion freezing extender. Egg yolk is normally used as a protective agent to freeze semen of equine and other species. However, addition of egg yolk in extenders is not without disadvantages and the demand to find cryoprotective alternatives is strong. The objective of this study was to test the cryoprotective capacities of liposomes composed of egg yolk phospholipids. Two experiments were conducted: 1) the first to determine the optimal composition and concentration of liposomes to preserve post-thaw motility and membrane integrity of spermatozoa; 2) the second to assess in vivo the cryoprotective capacities of these ...
Dissecting the molecular damage to stallion spermatozoa: the way to improve current cryopreservation protocols? We review recent developments in the technology of freezing stallion sperm, paying special attention to the molecular lesions that spermatozoa suffer during freezing and thawing, such as osmotic stress, oxidative damage, and apoptotic changes. We also discuss the applicability of colloidal centrifugation in stallion sperm cryobiology. Increased knowledge about the molecular injuries that occur during cryopreservation may lead to improved protective techniques and thus to further improvements in fertility in the current decade.
Evaluation of two magnetic-bead-based viral nucleic acid purification kits and three real-time reverse transcription-PCR reagent systems in two TaqMan assays for equine arteritis virus detection in semen. This study showed that under specifically defined conditions with respect to nucleic acid extraction method and testing reagents, a previously described real-time reverse transcription-PCR (rRT-PCR) assay (T1 assay) provides sensitivity equal to or higher than that of virus isolation for the detection of equine arteritis virus in semen.
Freezing stallion semen with the new Cáceres extender improves post thaw sperm quality and diminishes stallion-to-stallion variability. Ejaculates from 7 stallions were split and simultaneously frozen in three different extenders, INRA 96 egg yolk glycerol, Ghent and the newly developed extender Caceres. After thawing, samples were evaluated for motility (CASA system) sperm membrane integrity and early membrane changes (YoPro-1/Eth staining), acrosome integrity (FICT-PNA), and mitochondrial membrane potential (JC-1) (flow cytometry). Samples frozen in Caceres extender consistently showed the best results in post-thaw motility (increases ranging from 11 to 17%, p<0.05) and velocity (p<0.05), membrane integrity (increases ...
Effect of pentoxifylline treatment on testicular perfusion and semen quality in Miniature horse stallions. The objective was to investigate the effects of pentoxifylline (PTX) on testicular perfusion and sperm production in stallions. In a preliminary study, six mature Miniature horse stallions were given 0, 8.5, or 17.0 mg/kg of PTX orally, twice daily, for 3 d. Total Arterial Blood Flow Rate (TABFR) was higher (P < 0.05) in all treated versus control stallions during and after treatment. Two months later (during the fall and winter), the same stallions received either 0 or 17 mg/kg of PTX orally, twice daily for 60 d. Resistance and pulsatility indices (RI and PI, respectively) decreased in PT...
Sources of spermatozoa loss during collection and artificial insemination of horses. During artificial insemination of horses, it is important to accurately estimate the number of spermatozoa in each insemination dose. However, little research exists regarding sources of spermatozoa loss during collection and artificial insemination. Therefore, spermatozoal losses were quantified in the dismount loss (187.6×10(6)±62.5×10(6)spermatozoa), gel fraction (179.8×10(6)±61.7×10(6)spermatozoa), and the collection receptacle (136.1×10(6)±26.9×10(6)spermatozoa). Spermatozoal losses were examined in the centrifuge tube (25.8×10(6)±2.1×10(6)spermatozoa), AI pipette during the a...
Effect of method and clinician on stallion sperm morphology evaluation. The objective of this study was to determine the effects of method and clinician on stallion sperm morphology evaluation. Five clinicians evaluated 60 semen samples using wet-mount preparations with phase-contrast, eosin/nigrosin-stained semen smears, and Papanicolaou-stained semen smears. There were significant differences among methods for all sperm morphology categories and most intra-class correlation coefficients were only fair to moderate. The use of wet-mount preparations facilitated detection of acrosome defects, nuclear vacuoles, and cytoplasmic droplets when compared to stained smear...
Presence and distribution of fungi and bacteria in the reproductive tract of healthy stallions. A saprophytic bacterial flora is present on the penis and the distal part of the urethra of stallions. Little is known about the fungal flora of their reproductive tract. As micro organisms play an important role in mares fertility, the aim of the study was to describe the distribution of fungi and bacteria in the normal genital apparatus of stallions. The microbic flora of the reproductive tract of 11 healthy, fertile stallions was evaluated, collecting samples from 5 different locations: urethral fossa, penis/internal lamina of the prepuce, urethra pre- and post-ejaculation, and semen. For f...
Density gradient centrifugation of sperm from a subfertile stallion and effect of seminal plasma addition on fertility. Stallions are not selected for fertility but for other criteria (pedigree, conformation, performances, progeny), therefore valuable but subfertile stallions with poor semen quality are frequently used in commercial breeding programs. The object of this study was to evaluate whether sperm selection through a silane-coated silica colloid gradient centrifugation, with or without the addition of seminal plasma of a high fertile stallion, could improve the pregnancy rates of an oligospermic valuable stallion in a commercial breeding program. In 2008 breeding season (experiment 1, n=104 mares), simp...
The association of the presence of seminal plasma and its components with sperm longevity in fractionated stallion ejaculates. Semen jets in the stallion's ejaculate differ in both the biochemical composition of seminal plasma (SP) and in sperm concentration, forming sperm-rich fractions (HIGH) and sperm-poor fractions (LOW). This study examined (i) the association of the levels of selected components of SP [alkaline phosphatase (AP), acid phosphatase (ACP), Na(+), Cl(-), K(+), Ca, Mg and prostaglandin E(2)] with semen quality, sperm longevity and fertility; (ii) the effects of the presence of SP on sperm survival during cooled storage; (iii) the differences in the composition of SP between sperm-rich and sperm-poor e...
Relationship between sperm motility, morphology and the fertility of stallions. Sperm quality has an important role in determining fertility. Although there have been numerous studies to document the relationship between sperm quality and fertility, the methods of determining this association and conclusions vary. In the present study, computer-assisted sperm analysis (CASA) was used for evaluation of sperm motility, and differential interference contrast (DIC) microscopy was used for evaluating sperm morphologic features of breeding stallions. Fertility was measured using three endpoints: seasonal pregnancy rate (PR), percent pregnant/cycle (PC), and percent pregnant/fir...
Osmotic properties of stallion sperm subpopulations determined by simultaneous assessment of cell volume and viability. The aim of this study was to determine the osmotic tolerance limits of stallion sperm as well as the osmotic behavior of different sperm subpopulations, including viable and non-viable cells as well as viable cells of different average sizes. A flow cytometric approach was used for simultaneous assessment of cell volume and permeability of the plasma membrane for the fluorescent dye propidium iodide while exposing the cells to media with different solute concentrations. Equine spermatozoa have limited osmotic tolerance limits: exposure to hypotonic conditions below approximately 240 mOsm kg(-1...
Effect of cryopreservation protocol on postthaw characteristics of stallion sperm. Three ejaculates from each of eight stallions were subjected to cryopreservation in a milk/egg yolk-based freezing extender or an egg yolk-based freezing extender. Semen was exposed to a fast prefreeze cooling rate (FAST; semen immediately subjected to cryopreservation) or a slow prefreeze cooling rate (SLOW; semen pre-cooled at a controlled rate for 80 min prior to cryopreservation). Postthaw semen was diluted in initial freezing medium (FM) or INRA 96 (IMV Technologies, L'Aigle, France) prior to analysis of 10 experimental end points: total motility (MOT; %), progressive motility (PMOT; %), ...
Comparison of methods for assessing integrity of equine sperm membranes. Sperm membrane integrity (SMI) is thought to be an important measure of stallion sperm quality. The objective was to compare three methods for evaluating SMI: flow cytometry using SYBR-14/propidium iodide (PI) stain; an automated cell counting device using PI stain; and eosin-nigrosin stain. Raw equine semen was subjected to various treatments containing 20 to 80% seminal plasma in extender, with differing sperm concentrations, to simulate spontaneous loss of SMI. The SMI was assessed immediately, and after 1 and 2 d of cooled storage. Agreement between methods was determined according to Blan...
Improved cryopreservability of stallion sperm using a sorbitol-based freezing extender. Cryopreservation of stallion semen is often associated with poor post-thaw sperm quality. Sugars are among the important components of a freezing extender and act as non-permeating cryoprotectants. This study aimed to compare the quality of stallion sperm frozen with glucose, fructose or sorbitol-containing freezing extenders. Semen was collected from six stallions of proven fertility and cryopreserved using a freezing extender containing different types of monosaccharide sugars (glucose, fructose or sorbitol). After thawing, the semen was examined for sperm motility, viability, acrosome integ...
Simple and economic colloidal centrifugation protocols may be incorporated into the clinical equine sperm processing procedure. For many years in human assisted-reproduction procedures there have been special protocols to prepare and improve sperm quality. Colloidal centrifugation (CC) is a useful technique that has been proved to enhance semen quality by selection of the best spermatozoa for different species. Its use is recommended to improve fertility of subfertile stallions but current CC protocols are clinically complicated in the equine sperm processing technique due to economic and technical difficulties. The aim of this study was to determine the optimal processing procedures to adapt the use of a CC product (E...
Effect of dietary antioxidant supplementation on fresh semen quality in stallion. In this study, the effect of dietary supplementation of organic selenium, vitamin E, and zinc on raw semen characteristics was evaluated. Ten stallions with normal fertility were divided into two groups: a control group (CG), in which standard diet was provided, and a treated group (TG), in which the standard diet was supplemented with 1500 mg of α-tocopherol acetate, 360 mg of zinc, and 2.5 mg of organic selenium on a daily basis. Semen parameters on fresh semen were evaluated three times in all stallions before antioxidant supplementation (T0) and 30 (T1), 60 (T2), and 90 (T3) d after suppl...
Effect of sod (superoxide dismutase) protein supplementation in semen extenders on motility, viability, acrosome status and ERK (extracellular signal-regulated kinase) protein phosphorylation of chilled stallion spermatozoa. New studies are underway to find new methods for supporting longer storage of cooled stallion semen. It is known that high concentrations of reactive oxygen species (ROS) cause sperm pathology. The metalloprotein superoxide dismutase (SOD) is responsible for H(2)O(2) and O(2) production, by dismutation of superoxide radicals. The aim of this study is to assess the quality of chilled stallion semen processed with extenders containing SOD at different concentrations as antioxidant additives. A total of 80 ejaculates collected from 5 standardbred stallions was divided into 5 aliquots treated as: ...
Quality and fertility of cooled-shipped stallion semen at the time of insemination. Stallion semen processing is far from standardized and differs substantially between AI centers. Suboptimal pregnancy rates in equine AI may primarily result from breeding with low quality semen not adequately processed for shipment. It was the aim of the study to evaluate quality and fertility of cooled-shipped equine semen provided for breeding of client mares by commercial semen collection centers in Europe. Cooled shipped semen (n = 201 doses) from 67 stallions and 36 different EU-approved semen collection centers was evaluated. At arrival, semen temperature was 9.8 ± 0.2 °C, mean sperm ...
