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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.
Effect of sperm dosage transportation in stallions: Effect on sperm DNA fragmentation. Artificial insemination programs for horses usually involve ex vivo handling and transporting of sperm. The present experiment was designed to: (i) assess the effect of transportation on sperm DNA integrity at different time post semen collection, and (ii) evaluate if sperm DNA quality deteriorates rapidly beyond 24 h of cooled storage. After collection, the ejaculates were extended using INRA 96 and semen was prepared for prompt analysis (A0) or 24 h/48 h cooled-shipping (B24 and C48 respectively). Each sample was assessed for sperm DNA fragmentation index (SDFI) at time 0 and after inc...
Evaluation of Fertilizing Ability of Frozen Equine Sperm Using a Bovine Zona Pellucida Binding Assay. Frozen equine semen has lower fertility compared to cooled semen. Due to the difficulty to obtain equine oocytes, a heterologous zona pellucida binding assay (ZBA) is an alternative method to predict the fertilizing capability of equine frozen sperm. The rate of capacitated and hyperactivated sperm according to their motility characteristics were analyzed with a Computer Assisted Sperm Analyzer. We believe this report describes for the first time the in vitro hyperactivation induction and the heterologous ZBA to predict the fertilizing ability of frozen equine sperm. Objective: This work aimed...
Reduction of Trypanosoma equiperdum from equine semen by single layer centrifugation. Trypanosoma equiperdum (T. equiperdum) causes dourine, a venereally transmitted infection in horses. Purification of semen by single layer centrifugation (SLC) has been proven to be successful in reducing venereally transmitted diseases when dealing with other pathogens. The objective of this study was to evaluate the purification of T. equiperdum spiked semen by SLC. Semen was spiked using cryopreserved T. equiperdum stabilates (Dodola strain isolate 943). In total, 6 concentrations, varying from 10 to >5 × 10 trypanosomes, were added to semen samples. Subsequently, SLC was performed ...
Expression, localization, and concentration of A-kinase anchor protein 4 (AKAP4) and its precursor (proAKAP4) in equine semen: Promising marker correlated to the total and progressive motility in thawed spermatozoa. A-kinase anchor protein 4 (AKAP4) is playing a central role in flagellar structure, chemotaxis, capacitation and sperm motility. In mammals, AKAP4 is expressed during spermatogenesis. AKAP4 is synthesized as a precursor, proAKAP4, which is cleaved into mature AKAP4 during fibrous sheath assembly. The proAKAP4 is a good indicator of sperm quality in humans and boars. The aims of this work were to study the expression, the localization and the concentration of proAKAP4 and AKAP4 in equine semen, and to evaluate the possible correlation between the total and progressive motility and the concentra...
Effect of Mare Colostrum in Extenders for Freezing Stallion Semen. This study aimed to evaluate the addition of mare colostrum in stallion freezing extenders to improve sperm quality. First, colostrum samples were collected from four mares after the foal's birth and their composition was determined. Ejaculates were collected from nine fertile stallions. Sperm samples were pooled, diluted, and cryopreserved into three experimental extender groups: Lactose-based extender supplemented with mare colostrum (20%), lactose-based extender supplemented with egg yolk (20%), and BotuCrio. The quality of the post-thaw semen samples were evaluated assessing sperm motility...
Evaluation of semen characteristics, oxidative stress, and biochemical indices in Arabian horses of different ages during the hot summer season. Egypt is anticipated to be potentially influenced by the global climate warming. Therefore, the current study aimed to evaluate the influence of age on the fertility potential of Arabian stallions during summer breeding months. Arabian horses grouped according to their age into three groups, each involved six stallions: young (5-6 years), middle (11-12 years) and old (15-20 years) age groups, were weekly sampled during the months of July-August. Ejaculates were collected using artificial vagina, Missouri model, and examined for pH, volume, concentration, motility, livability and morphological ...
Vitrification of Large Volumes of Stallion Sperm in Comparison With Spheres and Conventional Freezing: Effect of Warming Procedures and Sperm Selection. Stallion sperm was vitrified using straws in comparison with spheres and conventional freezing. Vitrification was performed plunging 30 μL of sperm (spheres) or 0.5 mL straws into liquid nitrogen (LN) and conventional freezing using 0.5 mL straws frozen in LN vapors. Sperm vitrified in straws were submitted to different warming procedures (42°C/20 seconds; 60°C/15 seconds) and single-layer centrifugation (SLC). Total (TM, %) and progressive sperm motility (PM, %), plasma membrane (IMS, %) and acrosome integrity (AIS, %) were statistically compared between treatments (mean ± SEM). Signif...
Analysis of factors affecting the pregnancy rate of mares after inseminations with cooled transported stallion semen. Artificial insemination (AI) with cooled stallion semen has increased markedly during the last decades in all countries, but fertility is often lower than when fresh semen or natural mating is used. The objective of this study was to examine field data (1634 ,cycles 523 Standardbred (SB) mares, 575 Finnhorse (FH) mares, and 90 stallions) using multivariable logistic regression for factors influencing the pregnancy rate (PR) after AI with cooled transported semen from SB and FH stallions. The PR per cycle for the material was 47%: Finnhorses 42% and Standardbreds 53%. When assessed with multiva...
Evaluation of a portable device for assessment of motility in stallion semen. In horse breeding, quality assessment of semen before insemination is often requested. Non-laboratory-based techniques for objective analysis of sperm motility are thus of interest. The aim of this study was evaluating a portable device for semen analysis (Ongo sperm test) and its comparison with computer-assisted semen analysis (CASA). Semen was collected from 10 stallions, diluted to 100, 50 and 25 × 106 sperm/ml and analysed for total (TM) and progressive motility (PM). The final sperm concentration influenced total motility analysed by Ongo (p < 0.05) which was higher at 100 ×...
Effects of different concentration and combinations of cryoprotectants on sperm quality, functional integrity in three Indian horse breeds. Traditionally Glycerol (Gly) is being used as major cryoprotectant and its toxicity could be a reason for the variation on stallion sperm freezability and fertility. In an effort to minimize Gly toxicity alternative cryoprotective agents like Di-methyl Formamide (DMF) have been investigated. The effect of the cryoprotectant and dose of cryoprotective agent varies from breed to breed and also from stallion to stallion within the same breed. Considering these factors a study was designed to study the effects of Gly and DMF at different concentrations and combinations on the plasma membrane, acro...
Depletion of thiols leads to redox deregulation, production of 4-hydroxinonenal and sperm senescence: a possible role for GSH regulation in spermatozoa†. We hypothesized that thiols and particularly glutathione (GSH) are essential for the regulation of stallion sperm functionality. To test this hypothesis, we initially investigated the relationship between sperm function and GSH content, revealing highly significant correlations between GSH, sperm viability, motility, and velocity parameters (P < 0.001). Furthermore, the deleterious effects of GSH depletion using menadione and 1,3 dimethoxy 1,4, naphtoquinone (DMNQ) were able to be prevented by the addition of cysteine, but no other antioxidant. Pre-incubation with cysteine prevented menad...
Relationship of cysteine-rich secretory protein-3 gene and protein with semen quality in stallions. Cysteine-rich secretory protein-3 (CRISP-3) and some of its nonsynonymous polymorphism have been related to the fertility and freezability of stallion semen; however, the role of the CRISP-3 gene and its seminal plasma protein in the raw semen quality is still unknown. The aim of this study was to evaluate the relationship of CRISP-3 with semen quality in stallions. DNA was obtained from blood samples of 100 stallions, from which 30 stallions were randomly selected to obtain 60 ejaculates. Through PCR amplification and sequencing, the variation of four nonsynonymous SNPs from CRISP-3 was ident...
Seminal plasma does not aid in the transport of phenolsulfonphthalein across the uterotubal junction in mares. This study tested the hypothesis that the presence of prostaglandin E2 in seminal plasma would aid in the transport of phenolsulfonphthalein (PSP) across the uterotubal junction. Five mares in estrus were inseminated during estrus with PSP dissolved in phosphate-buffered saline and during the subsequent estrus with PSP added to a standard insemination dose. Serum and urine samples were obtained at hours 0, 1, 2, and 3 following treatment and examined for the presence of PSP. Phenolsulfonphthalein could not be detected in any of the urine samples collected from mares following either treatment....
Source and localization of ovulation-inducing factor/nerve growth factor in male reproductive tissues among mammalian species. The objectives of the study were to compare the presence and localization of ovulation-inducing factor (OIF)/nerve growth factor (NGF) in male reproductive organs and determine the abundance in ejaculates of species representative of both spontaneous and induced ovulators. We hypothesized that the protein is a widely conserved component of semen among mammals, but is most abundant in camelids. Immunohistochemical analysis was performed on tissues from the male reproductive system of llamas, rats, cattle, bison, elk, and white-tailed deer (n = 2 males/species), and the abundance of OIF/NGF in...
Effect of feeding pomegranate seed oil as a source of conjugated linolenic acid on Arabian stallion semen quality in cooled and postthawed condition. The objective was to assess the influence of pomegranate seed oil supplementation on the quality of fresh, cooled and frozen-thawed Arabian breed stallion semen. Eight stallions (n = 4 per group) received their normal diet (control group) or normal diet top dressed with 200 ml of pomegranate seed oil (PSO group). Semen was collected every fifteen days for 90 days. Stallions were reversed across the treatments after a sixty-day interval. In cooled and stored condition (2, 12 and 24 hr), spermatozoa motion characteristics, membrane integrity, viability, morphology and lipid peroxidation wer...
Influences of intrauterine semen administration on regulatory T lymphocytes in the oestrous mare (Equus caballus). In the mare, early pregnancy loss is common, but involvement of the maternal immune system in the pathogenesis of this condition has not been investigated in detail so far. In the present study, we assessed effects of exposure of the endometrium to semen or seminal plasma in oestrous mares on the response of regulatory T lymphocytes (Tregs) in the peripheral circulation as well as in the endometrium. Raw semen, seminal plasma or PBS (control) were introduced into the uterus of oestrous mares (n = 12). Blood was collected immediately before insemination or PBS infusion (time 0), and 12, 24 ...
Equine sperm-bound antisperm antibodies are associated with poor semen quality. Antisperm antibodies (ASAs) have been associated with infertility in stallions. The objectives of this study were to investigate the frequency of ASA-positive semen samples in satisfactory and non-satisfactory breeder stallions, the association between ASA binding and semen quality, and factors that may affect the diagnosis. Breeding soundness examinations were performed in 21 stallions and the percentage of IgG- and IgA-bound spermatozoa was evaluated using flow cytometry. Median IgG and IgA binding did not differ between the first and second ejaculates. The percentage of IgA-bound spermatozo...
Effects of implants containing the GnRH agonist deslorelin on testosterone release and semen characteristics in Shetland stallions. The hypothesis in this study was continuous treatment of stallions with the GnRH agonist deslorelin inhibits reproductive functions. A 2-week pre-experimental period was followed by an 11-week deslorelin implant treatment. Stallions received 4.7 (D1, n = 7), or 18.8 mg deslorelin (D2, n = 5) or remained untreated (C, n = 5). Libido, sperm motility, membrane integrity, DNA fragmentation, estrogen receptors, basal plasma testosterone and Anti Muellerian hormone (AMH) concentrations were evaluated once weekly during the treatment period. The testosterone response to the GnRH agonist buserelin a...
Quercetin in equine frozen semen. BACKGROUND: Supplementation of sperm diluents to reduce the damage caused by the freeze-thaw cycle is broadly used in equine semen cryopreservation. Objective: The present study aimed at determining the most appropriate quercetin supplementation in equine freezing extender. Methods: Quercetin at four different concentrations (0.25, 0.5, 0.75 or 1 mM) was added in the sperm freezing diluent before the freeze-thaw cycle. The spermatozoa population was analyzed by flow cytometry and a statistical analysis was conducted to detect significant differences between control and treated samples. R...
Variable Inter-assay Estimation of Sperm DNA Fragmentation In Stallions Classified as Good and Bad Semen Freezers. Semen cryopreservation causes DNA damage, thus requiring continuous monitoring. Objective: To compare two assays for sperm DNA fragmentation (SDF) from stallions with contrasting semen freezability. Methods: Thirteen stallions were classified as good semen freezers (GSF) or bad semen freezers (BSF). Ejaculates were cryopreserved with three diluents. Semen was subject to SDF evaluation using the sperm chromatin structure assay (SCSA) and Halomax after thawing (0 h) and after a 4 h thermoresistance test. Results: On semen of BSF, analysis by SCSA was similar between evaluations, but Halomax show...
Seminal plasma influences the fertilizing potential of cryopreserved stallion sperm. Seminal plasma (SP) contains proteins that may influence cryosurvival and prevent capacitation-like changes due to freezing and thawing. The objective of this study was to investigate the effect of adding pooled SP from "good" (GF) or "bad" (BF) freezer stallions on sperm cells' fertilizing ability. "Good freezers" refers to stallions that usually produce ejaculates which can withstand cryopreservation, whilst "bad freezer" stallions produce ejaculates which cannot tolerate the freezing process. A heterologous zona binding assay with in vitro matured bovine oocytes was used to assess the bind...
Sperm quality assays: How good are they? The horse perspective. Sperm quality assays have increased in number in the last 10 years. Most of these assays are flow cytometry based in application and are modified from assays that have been developed to measure somatic cell function. The goal of any sperm quality assay should be to advance the clinicians/researchers understanding of sperm cell function and the relationship to fertility. While these assays appear to measure somatic cell-like functions in sperm there tends to be little understanding how the results of these assays relate to fertility.
Identification of serotoninergic system components in stallion sperm. Assisted reproductive techniques have been used on several domestic animals to preserve desirable traits in strains of high genetic and commercial value; however in equines its efficiency rate is relatively low. To increase the conception ratio in stallions, some research groups have used pharmacological treatments which promote sperm hyperactivation in order to increase male's fertility rates. In this way, our previous work suggests that serotonin (5-HT) could be a good pharmacological candidate that facilitates conception rate in domestic horses. 5-HT is a neurohormone involved in several re...
Downregulation of MicroRNA eca-mir-128 in Seminal Exosomes and Enhanced Expression of CXCL16 in the Stallion Reproductive Tract Are Associated with Long-Term Persistence of Equine Arteritis Virus. Equine arteritis virus (EAV) can establish long-term persistent infection in the reproductive tract of stallions and is shed in the semen. Previous studies showed that long-term persistence is associated with a specific allele of the CXCL16 gene (CXCL16S) and that persistent infection is maintained despite the presence of a local inflammatory and humoral and mucosal antibody responses. In this study, we demonstrated that equine seminal exosomes (SEs) are enriched in a small subset of microRNAs (miRNAs). Most importantly, we demonstrated that long-term EAV persistence is associated with the dow...
Simultaneous evaluation of superoxide content and mitochondrial membrane potential in stallion semen samples provides additional information about sperm quality. An improved fertility prediction for stallions is of importance for equine breeding. Here, we investigate the potential of a combined staining of stallion spermatozoa for superoxide and mitochondrial membrane potential (MMP) for this purpose. Semen samples were analysed immediately after arrival at the laboratory, as well as after 24 h. Superoxide was measured by MitoSOXRed, while MMP was measured with JC-1. Menadione was used to stimulate superoxide production. In addition, other parameters of sperm quality, namely motility, membrane integrity, chromatin integrity, sperm kinematics and Hoec...
How does the microbial load affect the quality of equine cool-stored semen? Contaminating bacteria present in stallion ejaculates may compromise sperm quality during storage. Different procedures have been used to reduce the load of microorganisms in semen and avoid bacterial growth during storage. The aims of this study were: 1) to evaluate different techniques to eliminate bacteria in semen 2) to study the relationship between total microflora load (TML) and ROS production; and 3) to determine if TML affects the functionality of cool-stored sperm. Ejaculates from 11 stallions were split and processed in 3 ways: A. extended semen; B. conventional centrifuged semen, a...
Blastocyst production after intracytoplasmic sperm injection with semen from a stallion with testicular degeneration. In horse breeding, intracytoplasmic sperm injection (ICSI) has gained interest to obtain offspring from subfertile individuals. This paper presents a case report of a stallion with severe testicular degeneration. Semen analysis showed very low motility and 83.5% of detached heads. Histology of a testicular biopsy showed severely decreased spermatogenesis, while transmission electron microscopy of the sperm cells revealed no significant abnormalities. A total of 39 oocytes were fertilized by ICSI with frozen-thawed spermatozoa of this stallion: 25 oocytes with intact spermatozoa and 24 with det...
Stallion semen quality depends on major histocompatibility complex matching to teaser mare. The major histocompatibility complex (MHC) has repeatedly been found to influence mate choice of vertebrates, with MHC-dissimilar mates typically being preferred over MHC-similar mates. We used horses (Equus caballus) to test whether MHC matching also affects male investment into ejaculates after short exposure to a female. Semen characteristics varied much among stallions. Controlling for this variance with a full-factorial within-subject experimental design, we found that a short exposure to an MHC-dissimilar mare enhanced male plasma testosterone and led to ejaculates with elevated sperm nu...
Short-term tolerance of equine spermatozoa to various abiotic factors. The aim of this study was to determine the effects of various abiotic factors, such as light, physical stress (pipetting) and thermal shock, on the quality of fresh and cooled equine sperm. In experiment I, four sperm aliquots were subjected to different light exposures: (i) protected control samples (CTRL), (ii) exposed to UV light at 10 cm (UV10), (iii) exposed to UV light at 20 cm (UV20) and (iv) exposed to laboratory lighting (LAB). In experiment II, four semen aliquots were subjected to repeated pipetting for 0, 10, 20 and 30 times (CTRL, P10, P20 and P30, respectively). In experiment I...
Influences of dietary supplementation with Lepidium meyenii (Maca) on stallion sperm production and on preservation of sperm quality during storage at 5 °C. Stallion semen is damaged by oxidative stress during cooling and transport. Semen processing and extenders have been tested to improve the fertilizing capacity of semen and to preserve semen during transport. Dietary supplementation with natural antioxidants has been proposed to prevent oxidative damages. In this study, for the first time, the effect of dietary supplementation with Lepidium meyenii (Maca) on the characteristics of fresh and chilled stallion semen was evaluated. Maca is a traditional Andean crop used as a nutraceutical for the fertility-enhancing properties that are linked with...
