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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.
During cooled storage the extender influences processed autophagy marker light chain 3 (LC3B) of stallion spermatozoa. To investigate the role of the processed autophagy marker light chain 3 (LC3B) protein in sperm survival in stallion semen processing during cooled storage, split ejaculates were diluted in two different extenders, KMT and INRA 96, and LC3B processing and sperm quality evaluated during incubation at 5°C for five days. After 3 days of incubation there was a drop in total motility in both extenders, although the percentage of progressive motile sperm was greater (P<0.05) in samples extended in INRA96. On Day 5 of cooled storage all sperm parameters decreased significantly independent of the ...
Cryopreservation of epididymal stallion sperm. Any event that makes semen collection or mating impossible, such as death, castration, or injury, may terminate a stallion's breeding career. Fortunately, stallion sperm which are capable of fertilization can be harvested from the epididymis, and frozen for future use. However, the fertility of frozen-thawed epididymal sperm has been found to be lower than that of ejaculated sperm. Therefore, this study aimed to optimize the fertility of frozen epididymal stallion sperm by investigating the effects of different cryoprotectants and freezing protocols on sperm quality. Dimethylformamide was test...
Impact of using a fast-freezing technique and different thawing protocols on viability and fertility of frozen equine spermatozoa. The effects of freezing technique and thawing protocol on thawed semen viability and fertility were studied. Ejaculates from 5 stallions (n = 25) were frozen by conventional or a fast-freezing technique. Frozen semen was thawed by two thawing protocols (37 °C 30 s(-1) or 75 °C 7 s(-1) ). Thawed semen was evaluated by progressive motility, vigour, morphology and plasma membrane integrity. Mares (n = 25) were inseminated with 300 (n = 11) or 150 (n = 14) million spermatozoa. A greater (P < 0.05) vigour and progressively motile spermatozoa were detected, respectively, at thawing a...
Effect of single layer centrifugation using Androcoll-E-Large on the sperm quality parameters of cooled-stored donkey semen doses. The aim of this study was to determine the effect of single layer centrifugation (SLC) using Androcoll-E-Large on donkey sperm quality parameters after 24 h of cool-storage. Ejaculates were collected from Andalusian donkeys and then cooled at 5°C. SLC was carried out after 24 h of cool-storage using Androcoll-E-Large. In the first experiment, all sperm parameters assessed (total and progressive sperm motility, viability, sperm morphology and sperm kinematics VCL, VSL, VAP, LIN, STR, WOB, ALH and BCF) were statistically compared between semen samples processed or not with Androcoll-E-Large. Si...
First field results on the use of stallion sex-sorted semen in a large-scale embryo transfer program. Flow cytometry sex-sorting technology was developed in 1989. However, it is only the bovine species in which offspring of the desired sex are obtained at a commercial level. The aim of the present work was to evaluate efficiency parameters when using fresh sexed semen in a large-scale equine commercial embryo transfer program. During the 2009, 2010 and 2011 breeding seasons, 938 synchronized cycles were artificially inseminated. One hundred (10.6%) mares failed to ovulate, and for the remaining 838 useable cycles, 887 doses of sexed semen were used, representing 1.06 doses per cycle. In genera...
Use of cholesterol-loaded cyclodextrin: an alternative for bad cooler stallions. During the cooling process, sperm may suffer irreversible damage that compromises the fertility rate. Incorporating cholesterol-loaded cyclodextrin (CLC) represents a strategy to increase sperm resistance at low temperatures; however, high levels of cholesterol in the cell membrane can interfere with sperm capacitation. The goals of this study were to determine the CLC concentration and cooling temperature that produce optimal kinetic parameters and viability of sperm from stallions identified as bad coolers (BCs) and good coolers (GCs), as well as the effect of adding CLC on the occurrence of...
Effect of non-sperm cells removal with single-layer colloidal centrifugation on myeloperoxidase concentration in post-thaw equine semen. Myeloperoxidase (MPO) is a pro-oxidant enzyme contained in and released by neutrophils during degranulation or after lysis. Post-thaw semen contains MPO and its concentration is associated with decreased sperm motility. Recently, MPO concentration in post-thaw semen was shown to be associated with the presence of non-sperm cells (NSC). The objective of this study was to evaluate the effect of a single-layer colloidal centrifugation before cryopreservation on NSC and MPO concentrations in equine semen. The experimental design consisted of freezing semen with or without previous centrifugation t...
Biochemical characterization of stallion prostasomes and comparison to their human counterparts. Release of nanometer-sized prostasomes into human and equine semen suggests essential functions in their relationships with sperm cells and the fertilization process. The two types of prostasomes displayed ultrastructural similarities, albeit the human prostasomes were somewhat larger than the stallion prostasomes. A high ratio of saturated fatty acids was characteristic for the two prostasome types. Electrophoretic separation systems revealed an equine prostasomal pattern different from that of human. The 21 distinctive low molecular weight protein spots in the 2D-gel (with no counterparts in...
Effect of sex sorting on stallion spermatozoa: Heterologous oocyte binding, tyrosine phosphorylation and acrosome reaction assay. The interest on sex sorting by flow cytometry on the equine industry has been increasing over the years. In this work, three different tests were performed in order to evaluate the membrane status of sorted stallion spermatozoa: assessment of binding ability to porcine oocytes, evaluation of acrosome integrity after stimulation with A23187, and detection of tyrosine phosphorylation. These evaluations were made after incubation for 0h, 1.5h and 3h in a capacitating medium. Sorted stallion spermatozoa attached similarly to the porcine oocytes, when compared with control samples. Sorted spermatoz...
Trypan blue/giemsa staining to assess sperm membrane integrity in salernitano stallions and its relationship to pregnancy rates. Aim of this study was to test the reliability of Trypan blue/Giemsa staining to evaluate sperm membrane integrity, acrosomal intactness and morphology in stallion to verify whether it could be applied in vitro as useful tool for sperm fertilizing ability. Fertility data on inseminated mares were collected to evaluate the relationship of sperm quality to pregnancy rates. Forty-one ejaculates were collected from 3 stallions of Salernitano Horse Breed and evaluated for gross appearance, volume, visual motility and membrane integrity with Trypan blue/Giemsa staining and thirty-five mares were inse...
Cushioned versus noncushioned centrifugation: sperm recovery rate and integrity. It was hypothesized that optimal sperm recovery rate (RR) without damage to the sperm would be obtained after centrifugation without a cushion solution. Semen collected three times from six light breed stallions was extended to 25 × 10(6) sperm/mL and centrifuged at CON (noncentrifuged), 900NC (no-cushion), 900C (cushion), 1800NC, and 1800C × g for 10 minutes. Sperm concentration, motility (TM and PM), and intact plasma membranes (PLM) and acrosomes (ACR) pre- and postcentrifugation (D0) and after 24 hours (D1) of cooling were evaluated. The RR in the CON (100 ± 0.0), 900NC (93.7 ± 2.9), a...
Proteomic characterization and cross species comparison of mammalian seminal plasma. Seminal plasma contains a large protein component which has been implicated in the function, transit and survival of spermatozoa within the female reproductive tract. However, the identity of the majority of these proteins remains unknown and a direct comparison between the major domestic mammalian species has yet to be made. As such, the present study characterized and compared the seminal plasma proteomes of cattle, horse, sheep, pig, goat, camel and alpaca. GeLC-MS/MS and shotgun proteomic analysis by 2D-LC-MS/MS identified a total of 302 proteins in the seminal plasma of the chosen mammali...
Reactive oxygen species in stallion semen can be affected by season and colloid centrifugation. There are anecdotal reports that equine fertility may decline towards the end of the breeding season. Previous studies have examined differences in sperm quality between the breeding season and non-breeding season but few studies have investigated the proportions of superoxide or peroxide containing spermatozoa at different times during the breeding season. The purpose of this study was to measure the content of these reactive oxygen species (ROS) at the beginning and end of the Swedish breeding season, using flow cytometric analysis of the fluorescence produced after staining with hydroethidi...
Influence of semen collection on salivary cortisol release, heart rate, and heart rate variability in stallions. The aim of this study was to evaluate the stress response of stallions (n = 16) aged 3-13 years with previous sexual experience to semen collection by determination of heart rate, heart rate variability, and cortisol in saliva. Recordings were done on two consecutive days. The time intervals from leaving the box until arrival in the collection barn and from first exposure to the teaser mare until ejaculation as well as the number of mounts until ejaculation were neither affected by collection day nor by age, sexual experience (i.e., the number of breeding seasons the stallion experienced), or ...
Pre-selection by double layer density gradient centrifugation improves the fertilising capacity of frozen-thawed, capacitated stallion sperm. The effect of combining double layer density gradient centrifugation (DL-DGC) with different capacitation treatments on the fertilising capacity of frozen-thawed stallion sperm was examined via a heterologous assay involving in vitro-matured, zona pellucida-free bovine oocytes. In a first experiment, aliquots of frozen-thawed stallion sperm were subjected to one of five capacitation treatments without DL-DGC - ionomycin at 1.0μM, 0.1μM, 0.05μM or 0.01μM, or caffeine at 200μg/mL. The fertilising capacity of the semen was then assessed at 18h by staining the above oocytes with 4,6-diamidino...
Development of the genital microflora in stallions used for artificial insemination throughout the breeding season. An important factor influencing stallion fertility is the microbial contamination of semen. Aims of this study were to investigate changes in the microbiological population of the genital mucosa and semen in artificial insemination stallions (n=16) from before to after one breeding season (February-August). MALDI-TOF-MS (matrix assisted laser desorption/ionization time-of-flight mass spectrometry) was used for identification of microbial agents. For bacteriology, swabs from the urethral opening, urethral fossa and penile sheath as well as semen were collected at 4-week-intervals. For semen mot...
Assessment of extent of apoptosis and DNA defragmentation in chilled semen of stallions during the breeding season. The objective of the study was to assess apoptosis and DNA defragmentation in equine semen diluted and chilled to +4°C. Semen was collected from nine fertile stallions, including four Arabian thoroughbreds and five coldbloods. Examinations were carried out immediately after semen collection (0) and at five storage times (24, 48, 72, 96 and 120 h). The basic semen evaluation was performed in terms of volume, sperm concentration, viable sperm percentage, progressive motility and morphology. Using flow cytometry, DNA defragmentation and cell membrane integrity of spermatozoa were determined. Th...
Sex sorting increases the permeability of the membrane of stallion spermatozoa. At present, the only repeatable means of selecting the sex of offspring is the Beltsville semen sorting technology using flow cytometry (FC). This technology has reached commercial status in the bovine industry and substantial advances have occurred recently in swine and ovine species. In the equine species, however, the technology is not as well developed. To better understand the changes induced in stallion spermatozoa during the sorting procedure, pooled sperm samples were sorted: sperm motility and kinematics were assessed using computer assisted sperm analysis, sperm membrane integrity wa...
Effect of surgical technique for unilateral orchiectomy on subsequent testicular function in Miniature Horse stallions. Management decisions on unilateral orchiectomy are often influenced by the potential for post operative return to successful breeding. The effects of 2 surgical methods (first intention [FI] vs. second intention [SI] incision healing) for unilateral orchiectomy on resulting semen quality and scrotal temperature were evaluated. Objective: To evaluate the effects of 2 surgical unilateral orchiectomy techniques on scrotal healing, size of the remaining testis and post operative sperm quality. Methods: Unilateral orchiectomy was performed on mature Miniature Horse stallions. Semen was collected pr...
Effect of ketoprofen treatment on the uterine inflammatory response after AI of jennies with frozen semen. Artificial insemination (AI) involving the placing of frozen-thawed semen directly into the jenny uterine body is associated with very low pregnancy rates. This might be because of an exacerbation of the acute response of the endometrium to sperm, as seen in mares with persistent induced mating endometritis. Pregnancy rates can be increased in such mares, however, by including anti-inflammatory treatments in the insemination protocol (Bucca S, Carli A, Buckley T, Dolci G, Fogarty U. The use of dexamethasone administered to mares at breeding time in the modulation of persistent mating induced e...
Quercetin improves the postthaw characteristics of cryopreserved sex-sorted and nonsorted stallion sperm. Excessive reactive oxygen species generation during sex sorting and cryopreservation of stallion sperm leads to DNA fragmentation, lipid peroxidation, and motility loss. In this study we investigated whether antioxidant supplementation during sex sorting and cryopreservation could ameliorate the effects of reactive oxygen species on stallion sperm. In experiment 1, the postthaw characteristics of stallion sperm (N = 9) cryopreserved in the presence or absence of catalase (200 U/mL), cysteine (0.2 mg/mL), or quercetin (0.15 mM) was examined. Motility and acrosome integrity were assessed at 0, 1...
The presence and gene expression of bovine papillomavirus in the peripheral blood and semen of healthy horses. Papillomavirus (PV) are double-stranded DNA viruses that can cause both benignant and malignant tumours in mammals. Twelve genotypes of bovine papillomavirus (BPV1-12) have been identified so far. The presence of BPV1 and 2 has been found in the body fluids of cattle and horses. The aim of this study is to investigate the presence of BPV DNA and the expression of viral genes in the blood and sperm cells of healthy horses using PCR and RT-PCR. BPV-1 or 2 was detected in 14 of 70 blood samples (20%) and in 11 of 31 semen samples (35%). In five of fourteen blood samples, the E5 expression tested ...
Single-layer centrifugation reduces equine arteritis virus titre in the semen of shedding stallions. Several countries have adopted strategies for preventing and/or controlling equine viral arteritis based on vaccination and restricting the breeding activities of carrier stallions. However, in some cases, carrier stallions are only identified after they have transmitted virus to a mare. Therefore, a mechanism for separating virus from spermatozoa in the semen of carrier stallions would facilitate control measures for preventing disease transmission. In this study, the use of several modifications of single-layer centrifugation (SLC, SLC with an inner tube and double SLC) through Androcoll-E, ...
Sperm motility is lost in vitro as a consequence of mitochondrial free radical production and the generation of electrophilic aldehydes but can be significantly rescued by the presence of nucleophilic thiols. The prolonged incubation of human spermatozoa in vitro was found to induce a loss of motility associated with the activation of mitochondrial reactive oxygen species generation in the absence of any change in mitochondrial membrane potential. The increase in mitochondrial free radical production was paralleled by a loss of protein thiols and a concomitant rise in the formation of 4-hydroxynonenal, an electrophilic product of lipid peroxidation that was found to directly suppress sperm movement. These results prompted a search for nucleophiles that could counteract the action of such cytotoxic ...
Characteristics of stallion epididymal spermatozoa at collection and effect of two refrigeration protocols on the quality of the frozen/thawed sperm cells. Cryopreservation of epididymal spermatozoa is a useful tool to preserve genetic material of valuable stallions after emergency castration or unexpected death. For that, testicles and epididymides are generally sent refrigerated to the laboratory. Collection of epididymal spermatozoa is a simple procedure that reduces the volume of the material to be shipped, and may improve the quality of the chilled epididymal sperm cells. In the present study we compared the characteristics of frozen/thawed epididymal spermatozoa after refrigeration of the epididymis or after direct refrigeration of the exte...
Equine spermatozoa stored in the epididymis for up to 96h at 4°C can be successfully cryopreserved and maintain their fertilization capacity. After injury or death of a valuable male, recovery of epididymal spermatozoa may be the last chance to ensure preservation of its genetic material. The objective of this research was to study the effect of sperm storage, at 4°C up to 96h, in the epididymides obtained from castrated horses and its effect on different functional sperm parameters. Aims were to study the effect of (1) sperm storage on viability and chromatin condensation; (2) pre-incubation of recovered epididymal sperm in the freezing extender, prior cryopreservation, on viability and chromatin condensation; and (3) freezing-tha...
Ovulation-inducing factor in seminal plasma: a review. Ovulation in mammals involves pulsatile release of GnRH from the hypothalamus into the hypophyseal portal system with subsequent release of LH from the anterior pituitary into systemic circulation. Elevated circulating concentrations of LH induce a cascade of events within the mature follicle, culminating in follicle rupture and evacuation. The broad classification of species as either spontaneous or induced ovulators is based on the type of stimulus responsible for eliciting GnRH release from the hypothalamus. In spontaneously ovulating species (e.g., human, sheep, cattle, horse, pigs), relea...
Effect of antimicrobial-containing semen extender on risk of dissemination of contagious equine metritis. To determine the impact of antimicrobial-containing semen extender on the growth of Taylorella equigenitalis in semen culture-positive for contagious equine metritis (CEM) and the development of CEM after artificial insemination with CEM-positive semen extended with antimicrobial-containing semen extender. Methods: Prospective study. Methods: 21 mature mares free of CEM, 1 mature stallion experimentally infected with CEM, and semen from a stallion naturally infected with CEM. Methods: CEM-positive semen was incubated in semen extender with and without antimicrobials (amikacin [final concentrat...
Sex-sorting sperm using flow cytometry/cell sorting. The sex of mammalian offspring can be predetermined by flow sorting relatively pure living populations of X- and Y-chromosome-bearing sperm. This method is based on precise staining of the DNA of sperm with the nucleic acid-specific fluorophore, Hoechst 33342, to differentiate between the subpopulations of X- and Y-sperm. The fluorescently stained sperm are then sex-sorted using a specialized high speed sorter, MoFlo(®) SX XDP, and collected into biologically supportive media prior to reconcentration and cryopreservation in numbers adequate for use with artificial insemination for some specie...
Venereal shedding of equid herpesvirus-1 (EHV-1) in naturally infected stallions. Equid herpesvirus 1 (EHV-1) is a highly prevalent pathogen in horse populations worldwide. Oronasal infection represents the classic route of disease transmission. Venereal shedding of EHV-1 is not regarded relevant in terms of virus spreading, which is in contrast to the close relatives of EHV-1, bovine and suid alphaherpesvirus, for which artificial insemination is a well-documented and accepted means of virus spread. Objective: Documentation of venereal EHV-1 shedding in 3 naturally infected stallions. Methods: Three stallions were infected during an acute outbreak by an EHV-1 strain with t...
