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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.
Optimal concentrations of cryoprotective agents for semen from stallions that are classified ‘good’ or ‘poor’ for freezing. Cryopreserved stallion sperm displays a high degree of male-to-male variability with respect to cell viability after thawing. Animals that have semen with low viability after cryopreservation are classified as 'poor' freezers, and when post-thaw viability is high they are designated as 'good' freezers. Cryoprotective agents that are used for cryopreserving stallion sperm include glycerol, ethylene glycol, methyl formamide, and dimethylformamide, and are typically used in concentrations ranging from 1% to 4%. The aim of this study was to evaluate the osmotic stresses that stallion sperm is expo...
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...
Agreement between measures of total motility and membrane integrity in stallion sperm. Increasing seminal plasma concentrations in extended stallion semen were utilized to model decreasing sperm motility over time. Level of agreement was determined between flow cytometric measurement of sperm membrane integrity, using a combination of SYBR-14 and propidium iodide, and computer-assisted analysis of sperm motility. Values for total sperm motility (TMOT;%) and membrane integrity (SMI;%) were similar (∼80%) at Time 0 within all sperm treatments. However, TMOT was lower than SMI after 24 and 48 h of storage in treatments with >20% seminal plasma. At Time 0, agreement (bias and a...
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 ...
Influence of seminal plasma on fresh and post-thaw parameters of stallion epididymal spermatozoa. Fresh and post-thaw parameters (motility, morphology and viability) of stallion epididymal spermatozoa that have been and have not been exposed to seminal plasma were evaluated, and directly compared to fresh and post-thaw parameters of ejaculated spermatozoa. Six sperm categories of each stallion (n=4) were evaluated for motility, morphology and viability. These categories were fresh ejaculated spermatozoa (Fr-E), fresh epididymal spermatozoa that had been exposed to seminal plasma (Fr-SP+), fresh epididymal spermatozoa that had never been exposed to seminal plasma (Fr-SP-), frozen-thawed eja...
Fatty acids and plasmalogens of the phospholipids of the sperm membranes and their relation with the post-thaw quality of stallion spermatozoa. Fatty acids and plasmalogens were extracted from the phospholipids of the plasma membrane of stallion spermatozoa, to determine their relation with sperm quality after freezing and thawing. Sperm quality was rated using a quality index that combined the results of the analysis of sperm motility and velocity (CASA analysis), membrane status and mitochondrial membrane potential (flow cytometry) post thaw. Receiving operating system (ROC) curves were used to evaluate the value of specific lipid components of the sperm membrane herein studied as forecast of potential freezeability. From all parame...
Pregnancies following artificial insemination with spermatozoa from problem stallion ejaculates processed by single layer centrifugation with Androcoll-E. Some stallions produce ejaculates of low quality and/or low fertility when used for artificial insemination (AI). The purpose of these five case studies was to use Single Layer Centrifugation (SLC) to select the best spermatozoa from 'problem' ejaculates for subsequent use in AI. Sperm quality, in terms of motility, morphology and chromatin integrity, was improved in the SLC-selected samples compared to the corresponding uncentrifuged samples, with the exception of one stallion thought to have ampullary stasis. In this stallion, neither the incidence of spermatozoa with detached heads nor the ...
Validation and usefulness of the Sperm Quality Analyzer V equine for equine semen analysis. Routine semen analysis includes evaluation of concentration combined with seminal volume, morphology and motility. Subjective analysis of these parameters is known to be inaccurate, imprecise and subject to variability. Automated semen analysis could lead to an increased standardization in and between laboratories but for that to happen automated devices need to be validated. A new device, the sperm quality analyzer V equine (SQA-Ve) version 1.00.43, was evaluated for its repeatability and agreement with light microscopy (LM), for raw and extended equine semen. Results were compared with compu...
Single layer centrifugation of stallion spermatozoa consistently selects the most robust spermatozoa from the rest of the ejaculate in a large sample size. An improvement in sperm quality after single layer centrifugation (SLC) has been seen in previous studies using small sample sizes (for example, n = 10 stallions). There is a need to investigate whether this improvement is repeatable over several breeding seasons with a larger number of stallions (n ≥ 30 stallions). Objective: To make a retrospective analysis of the results of SLC performed on more than 250 sperm samples (176 ejaculates) from 31 stallions in 3 consecutive breeding seasons. Methods: Sperm quality (motility, proportion of morphologically normal spermatozoa and the proportion o...
Egg yolk plasma can replace egg yolk in stallion freezing extenders. Hen egg yolk is normally used as a cryoprotective agent in semen freezing extenders, but its use has sanitary and practical disadvantages. Moreover the protection afforded by egg yolk has not yet been completely elucidated. The objective of this study was to compare the egg yolk plasma fraction to whole egg yolk in stallion freezing extender. Plasma contains mainly Low Density Lipoproteins (LDL), which are widely presumed to be the cryoprotective agent in egg yolk. Plasma can be produced on an industrial scale, sterilised by gamma-irradiation and incorporated in a ready-to-use extender (our ul...
Redox status of equine seminal plasma reflects the pattern and magnitude of DNA damage in sperm cells. Antioxidant status of seminal plasma from 23 stallions was evaluated. We found a negative correlation between total antioxidant capacity (ABTS(•+) decolorization assay) and thiol content of seminal plasma, and sperm DNA damage (8-oxoG immunostaining, TUNEL reaction, comet assay). Low seminal redox status was the strongest correlated with 8-oxoG level which may indicate that seminal total antioxidant capacity influences mainly the formation of single strand DNA breaks in sperm cells. Since inter-individual differences in seminal antioxidant status were reported, we postulated that the redox s...
Comparative studies on bull and stallion seminal DNase activity and interaction with semen extender and spermatozoa. We performed a series of comparative studies of bull and stallion seminal plasma (SP) and its role on sperm-neutrophil binding as well as the interaction between semen extender and seminal DNase. Because of contrasting roles of SP on sperm-neutrophil binding between horses and cattle, it was suspected there were some species-specific differences on sperm interaction with SP proteins due to the variations in the natural location of semen deposition (uterus compared to vagina). Bull frozen-thawed sperm removed from egg yolk extender showed similar results to fresh sperm, but this also caused ext...
Biomarkers of in vivo fertility in sperm and seminal plasma of fertile stallions. The global proteome of sperm and seminal plasma of fertile stallions was investigated to determine whether associations with relative in vivo fertility exist. Seven stallions at stud in a commercial breeding station were collected throughout the breeding season and bred to a total of 164 mares to determine conception rates. On three occasions during the breeding season, raw semen was obtained from a regular collection for proteomic analysis using two-dimensional electrophoresis and also assessed for routine semen quality end points. First cycle conception rate was negatively related to ejacula...
Protein composition of seminal plasma in fractionated stallion ejaculates. Seminal plasma (SP) contains several types of compounds derived from the epididymides and accessory glands. The aim of this study was to examine the protein composition of different ejaculate fractions. Trial I: fractionated ejaculates were collected from two normal and two subfertile stallions. Samples containing pre-sperm fluid and the first sperm-rich jets (HIGH-1), the main sperm-rich portion (HIGH-2), the jets with low sperm concentrations (LOW), and a combined whole-ejaculate (WE) sample was centrifuged, and the SP was filtered and frozen. A part of each SP sample was stored (5°C, 24 h)...
Toxic effects induced by mycotoxin fumonisin B1 on equine spermatozoa: assessment of viability, sperm chromatin structure stability, ROS production and motility. Fumonisin B1 (FB1) is a mycotoxin produced by Fusarium species that exerts its toxic effect through interference with the sphingolipid pathway by inhibiting ceramide synthase. A FB1-dependent sperm toxicity was reported in boars. No information on FB1-related reproductive toxicity in stallions, the most sensitive animal species, has been reported. The aim of the present study was to assess the in vitro toxicity of FB1 on fresh and frozen-thawed equine spermatozoa by analyzing sperm viability, chromatin stability (SCSA) and reactive oxygen species (ROS) production by flow cytometry and sperm mo...
Selenium in blood, semen, seminal plasma and spermatozoa of stallions and its relationship to sperm quality. The essential trace element selenium is indispensable for male fertility in mammals. Until now, little data existed regarding the relationship between selenium and sperm quality in the stallion. Selenium, or selenium-dependent glutathione peroxidase activity, was determined in red blood cells, semen, seminal plasma and spermatozoa, and the percentages of spermatozoa with progressive motility (PMS), intact membranes (PMI), altered (positive) acrosomal status (PAS) and detectable DNA damage, determined by the sperm chromatin structure assay, were evaluated in 41 healthy stallions (three samples ...
Seasonal variation of plasminogen activator activity in spermatozoa and seminal plasma of boar, buck, bull and stallion. Plasminogen activators (PA) are proteolytic enzymes present in the spermatozoa and seminal plasma of various species. They play a role in the binding of the spermatozoon and its penetration through the layers surrounding the oocyte. Plasminogen activator activity (PAA) is modulated by hormones that have a seasonal variation, such as testosterone and melatonin. The present study investigates the seasonal variation of PA activity in sperm extracts and seminal plasma of four farm animal species: boar, buck, bull and stallion. Semen samples were collected every second week during a 12-month period...
Metabolic evaluation of cooled equine spermatozoa. Microscopy has been used in the routine evaluation of sperm metabolism. Nevertheless, it has limited capacity to preview male fertility. As calorimetry may be used to evaluate directly the metabolic activity of a biological system, the aim of this study was to use microcalorimetry as an additive method for sperm metabolism evaluation of cooled equine semen. Two ejaculates of four stallions were collected and motility, viability (eosin 3%) and membrane functional integrity (hyposmotic swelling test) of spermatozoa were evaluated. Sperm samples were processed following different protocols and th...
Single layer centrifugation of stallion spermatozoa improves sperm quality compared with sperm washing. This study compared the effect on semen quality of different handling methods used in the preparation of stallion semen doses for artificial insemination. The three methods were (i) extending the ejaculate to 30-50 x 10(6)/ml, (ii) single layer centrifugation (SLC) and (iii) sperm washing (centrifugation without a colloid). An additional treatment was to add seminal plasma (SP) in various proportions to some SLC preparations. The resulting samples were evaluated for sperm motility by computer assisted sperm analysis, membrane integrity using the Nucleocounter SP-100 and chromatin integrity by ...
Influence of different centrifugation protocols on equine semen preservation. Three experiments were conducted to evaluate the impact of centrifugation on cooled and frozen preservation of equine semen. A standard centrifugation protocol (600 x g for 10 min=CP1) was compared to four protocols with increasing g-force and decreased time period (600 x g, 1200 x g, 1800 x g and 2400 x g for 5 min for CP2, 3, 4, and 5, respectively) and to an uncentrifuged negative control. In experiment 1, the influence of the different CPs on sperm loss was evaluated by calculating the total number of sperm cells in 90% of the supernatant. Moreover, the effect on semen quality following ce...
Lectin-binding sites on ejaculated stallion sperm during breeding and non-breeding periods. Stallion sperm from semen collected in Southern Italy during the breeding (June-July) and non-breeding (December-January) periods were analyzed by means of twelve lectins to evaluate the glycoconjugate pattern and to verify whether there are any seasonal differences in the glycosylation pattern of the sperm glycocalyx. The acrosomal cap showed reactivity for Maackia amurensis (MAL II), Sambucus nigra (SNA), Arachis hypogaea (PNA), Glycine max (SBA), Helix pomatia (HPA), Canavalia ensiformis (Con A) Triticum vulgaris (WGA), and Griffonia simplicifolia isolectin II (GSA II) in breeding and non-b...
Effect of season on fresh and cryopreserved stallion semen. The objective of this study was to determine the effect of season on sperm quality variables, expression of the fertility-related protein SP22 and selected mRNA transcripts in fresh and cryopreserved stallion sperm. Four stallions were collected in each of the four seasons: summer, fall, winter and spring. Ejaculates were divided and then evaluated for motility, morphology, SP22 staining and expression of selected mRNAs as either fresh semen samples or cryopreserved samples. A significant interaction between season and cryopreservation status was found for total and progressive sperm motility....
Seasonal functional relevance of sperm characteristics in equine spermatozoa. A group of stallions with different reproductive indexes were used to study seasonal variations in sperm quality (Equus caballus). Semen samples were collected from late September to July and analyzed according to four seasonal periods: late September-December, January-March, late March-May, and June-July. Parameters monitored included sperm concentration, sperm motility, sperm morphology, sperm viability, acrosomal status, plasma membrane stability, and sperm mitochondrial membrane potential. Overall, seminal parameters monitored are affected mostly by time period, followed by animal and last...
Alkaline and acid phosphatase, β-glucuronidase and electrolyte levels in fractionated stallion ejaculates. Seminal plasma (SP) is a mixture of contents from the testes, epididymides and accessory sex glands. The sperm concentration is highest in the first few jets, or fractions, of the ejaculate, and the composition of SP varies between these fractions because accessory gland secretions are released in a specific order. The aim of this study was to compare the levels of Na, Cl, K, Mg, Ca, inorganic phosphate (Pi) and the enzymes alkaline phosphatase (AP), acid phosphatase (ACP) and β-glucuronidase (BG) in the different fractions of the ejaculate and in different stallions. All semen collections we...
Effects of in vitro exposure to natural levels of zearalenone and its derivatives on chromatin structure stability in equine spermatozoa. The purpose of this study was to assess the natural exposure of male horses (Equus caballus) to the mycotoxin zearalenone (ZEA) by using the ELISA test and to evaluate the effects of in vitro exposure of sperm cells to mycotoxin-containing urine extracts on sperm chromatin structure stability. Because of their occurrence in urine samples, ZEA and its derivatives were tested by sperm chromatin structure assay (SCSA) at natural levels detected by ELISA. Thirty-eight urine extracts of Italian (n = 11) and northeastern European (n = 27) horses were tested on frozen-thawed spermatozoa to evaluate t...
Sexual behavior and ejaculate characteristics in Pêga donkeys (Equus asinus) mounting estrous horse mares (Equus caballus). The objectives were to (i) characterize sexual behavior of donkey stallions (jacks; Equus asinus) during on-farm semen collection using estrous horse mares (mares; Equus caballus); (ii) compare behavior of young (less experienced) versus older (more experienced) jacks; (iii) determine whether semen suitable for artificial insemination (AI) could be collected using mares; and (iv) determine the suitability of using mares in field collection of semen from jacks. Six Pêga jacks (3.5 to 16 yr old), previously conditioned to breed mares, were used. Mount mares were confirmed in estrus by a teaser ...
Purification and partial characterization of proteinase inhibitors of equine seminal plasma. The aims of the study were: 1/ to isolate and identify equine seminal plasma proteinase inhibitors, 2/ to evaluate their inhibitory potential, and 3/ to test a correlation between protein concentration in seminal plasma supernatant (obtained after precipitation with 36% ammonium sulfate) and stallion sexual maturity. Seminal plasma proteins obtained from six stallions were chromatographed in a Superose 12 (FPLC system) column followed by C(18) HPLC reverse-phase. Inhibition of trypsin amidase activity was evaluated in the collected fractions. Active proteins with a molecular mass of 6.3-7.0 KD...
Cholesterol addition protects membrane intactness during cryopreservation of stallion sperm. Addition of cholesterol to sperm membranes improved equine sperm stability during semen cryopreservation; however, it also reduced in vivo fertility. The objective of the present study was to determine the effects of adding cholesterol to stallion sperm prior to freezing, and subsequently removing it from frozen-thawed sperm. Semen from 12 stallions was subjected to four treatments: (T1) control, semen was diluted with Kenney extender, centrifuged, and resuspended to 100 x 10(6)spermatozoa/mL in INRA 82 freezing extender, packaged into 0.5-mL straws, cooled to 5 degrees C, and cryopreserved in...
Does the microbial flora in the ejaculate affect the freezeability of stallion sperm? In an attempt to evaluate the possible relationship between the microbial flora in the stallion ejaculate and its ability to freeze,three ejaculates from five stallions were frozen using a standard protocol. Before freezing, an aliquot was removed for bacteriological analysis. Bacterial growth was observed in all the ejaculates studied. The isolated microorganisms were:Staphylococcus spp. and Micrococcus spp. (in all the stallions), beta-haemolytic Streptococcus (in stallions 3 and 4), Corynebacterium spp. (in stallions 1, 3-5), Rhodococcus spp. (in stallion number 2), Pseudomonas spp. (in sta...
