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Topic:Sperm
Equine sperm refers to the male reproductive cells produced by stallions, essential for the process of fertilization and successful breeding in horses. The study of equine sperm encompasses various aspects, including morphology, motility, viability, and genetic integrity. These parameters are critical for assessing stallion fertility and improving breeding outcomes. Research in this field often focuses on understanding the factors that influence sperm quality, such as age, nutrition, and environmental conditions. Additionally, advancements in assisted reproductive technologies, such as artificial insemination and cryopreservation, rely heavily on the detailed study of sperm characteristics. This page compiles peer-reviewed research studies and scholarly articles that explore the biology, evaluation, and technological applications related to equine sperm.
Phosphorylated AKT preserves stallion sperm viability and motility by inhibiting caspases 3 and 7. AKT, also referred to as protein kinase B (PKB or RAC), plays a critical role in controlling cell survival and apoptosis. To gain insights into the mechanisms regulating sperm survival after ejaculation, the role of AKT was investigated in stallion spermatozoa using a specific inhibitor and a phosphoflow approach. Stallion spermatozoa were washed and incubated in Biggers-Whitten-Whittingham medium, supplemented with 1% polyvinyl alcohol (PVA) in the presence of 0 (vehicle), 10, 20 or 30 μM SH5, an AKT inhibitor. SH5 treatment reduced the percentage of sperm displaying AKT phosphorylation, w...
Oviduct binding and elevated environmental ph induce protein tyrosine phosphorylation in stallion spermatozoa. Sperm-oviduct binding is an essential step in the capacitation process preparing the sperm for fertilization in several mammalian species. In many species, capacitation can be induced in vitro by exposing spermatozoa to bicarbonate, Ca(2+), and albumin; however, these conditions are insufficient in the horse. We hypothesized that binding to the oviduct epithelium is an essential requirement for the induction of capacitation in stallion spermatozoa. Sperm-oviduct binding was established by coincubating equine oviduct explants for 2 h with stallion spermatozoa (2 × 10(6) spermatozoa/ml), during...
Validation of a heterologous fertilization assay and comparison of fertilization rates of equine oocytes using in vitro fertilization, perivitelline, and intracytoplasmic sperm injections. IVF in horses is rarely successful. One reason for this could be the failure of sperm to fully capacitate or exhibit hyperactive motility. We hypothesized that the zona pellucida (ZP) of equine oocytes prevents fertilization in vitro, and bypassing the ZP would increase fertilization rates. Limited availability of equine oocytes for research has necessitated the use of heterologous oocyte binding assays using bovine oocytes. We sought to validate an assay using bovine oocytes and equine sperm and then to demonstrate that bypassing the ZP using perivitelline sperm injections (PVIs) with equine ...
In vitro equine embryo production using air-dried spermatozoa, with different activation protocols and culture systems. The aim of this work was to evaluate the use of air-dried spermatozoa for in vitro production of equine embryos and verify if sperm extract activation and in vivo culture improve in vitro embryo production. Cooled spermatozoa (control) and air-dried spermatozoa stored for 2, 14 or 28 days were used for ICSI sperm extract, or ionomycin was used for oocyte activation, and embryos were in vitro or in vivo (in mare's oviduct) cultured for 7 days. With in vitro culture, cleavage rate was higher when activating with sperm extract (P 0.05). Blastocysts were obtained with cooled spermatozoa, and ...
Stallion spermatozoa: putative target of estrogens; presence of the estrogen receptors ESR1, ESR2 and identification of the estrogen-membrane receptor GPER. Among mammals, the stallion produces the largest amount of testicular estrogens. These steroid hormones are produced mainly by Leydig and Sertoli cells in the testis and also in the epididymis. Their role in horse testicular physiology and their ability to act on spermatozoa are still unknown. In order to determine if spermatozoa are targets for estrogens, the presence of estrogen receptors in mature ejaculated spermatozoa has been investigated. The presence of a single isoform of ESR1 (66kDa) and ESR2 (61kDa) was found by Western-blot analysis in samples from seven stallions. Confocal analysi...
Use of mesenchymal stem cells or autologous conditioned serum to modulate the inflammatory response to spermatozoa in mares. Current treatments for Persistent mating-induced endometritis such as uterine lavage and oxytocin therapy focus on aiding the uterus in removal of inflammatory products, but these treatments do not modulate the inciting inflammatory response. Biological treatments, such as autologous conditioned serum (ACS) and mesenchymal stem cells (MSCs), have been used in human and veterinary medicine for immunomodulation for over 10 years. The objectives of this project were to evaluate the ability of ACS or MSCs to modulate the inflammatory response to spermatozoa after breeding. Two experiments were pe...
Sperm abnormalities in post-thawed semen of Tunisian Arab stallions. The study was undertaken in order to evaluate sperm morphology features of post-thawed semen of Tunisian Arab stallions. Forty two ejaculates was collected and frozen, during years 2009 and 2010, from 9 stallions aged between 9 to 24 years. After thawing, sperm morphology was studied after eosin-nigrosin stain. The percentages of abnormal head, mid piece, flagella, sperm with droplets and the total abnormal sperm were determined. Analysis of variance was carried out using SAS software. The results showed that all sperm morphology features varied among ejaculates within stallion and among stall...
Cholesterol-loaded-cyclodextrins improve the post-thaw quality of stallion sperm. An unacceptable proportion of stallion sperm do not survive the freeze-thaw process. The hypothesis of this study was that adding cholesterol to a stallion semen extender would stabilise the sperm membrane, resulting in an improved post-thaw semen quality in terms of increased sperm viability, membrane integrity and fluidity, and reduced oxidative stress. Semen was collected from three stallions and diluted in four extenders: TALP; TALP+0.75mg methyl-β-cyclodextrin-cholesterol (MβCD)/mL (MβCD0.75); TALP+1.5mg MβCD-cholesterol/mL (MβCD1.5); and Equipro. Following 15min incubation, samples ...
Concentration, activity and biochemical characterization of myeloperoxidase in fresh and post-thaw equine semen and their implication on freezability. Myeloperoxidase (MPO) is a pro-oxidant enzyme associated with decreased motility in thawed equine semen. This study aimed to describe MPO concentration, activity and subunits in raw and thawed semen and to correlate these data with motilities in raw and thawed semen. Semen samples from five stallions were collected four times. Motilities were assessed in raw and thawed semen. MPO assays were performed in raw seminal plasma, raw sperm-rich pellet and thawed semen. Total and active MPO concentrations were, respectively, assayed by enzyme-linked immunosorbent assay and specific immunological extr...
Influences of a diet supplemented with linseed oil and antioxidants on quality of equine semen after cooling and cryopreservation during winter. Seasonal changes in the reproductive physiology of stallions contribute to a decrease in the quality of frozen-thawed semen during late winter. Changes in the lipid composition of the sperm plasma membrane may contribute to this phenomenon. In the present study, we have, therefore, investigated the effects of adding linseed oil (LO) in combination with antioxidants to the diet of breeding stallions on the motility and membrane integrity of cooled-stored and cryopreserved semen. Starting in November, the diet of LO stallions (n = 6) but not control (C) stallions (n = 5) was supplemented with LO...
Sperm yield after single layer centrifugation with Androcoll-E is related to the potential fertility of the original ejaculate. Many attempts have been made to identify laboratory tests that are predictive of sperm fertility, both to improve the quality of stallion semen doses for artificial insemination (AI) and to identify potential breeding sires if no fertility data are available. Sperm quality at the stud is mostly evaluated by assessing subjective motility, although this parameter can be poorly indicative of fertility. Sperm morphology and chromatin integrity in Swedish stallions are correlated to pregnancy rate after AI. Because single layer centrifugation (SLC) selects for spermatozoa with normal morphology and...
Removal of bacteria from stallion semen by colloid centrifugation. Bacteria (environmental contaminants and occasionally potential pathogens) are found in most stallion ejaculates and may negatively affect sperm quality during storage. Since the use of antibiotics can lead to the development of resistance, an alternative means of microbial control is desirable. The removal of bacteria from stallion semen using Single Layer Centrifugation through Androcoll-E was investigated. Known doses of cultured bacteria were added to freshly collected ejaculates (15mL aliquots) before processing by Single Layer Centrifugation. The resulting sperm pellets and controls (not...
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...
Thiol-disulfide proteins of stallion epididymal spermatozoa. Thiol groups of cysteine residues represent redox centers involved in multiple biological functions. It has been postulated that changes in the redox status of mammalian epididymal spermatozoa contribute to the sperm maturation process. The present work shows the thiol-disulfide protein profile of stallion epididymal spermatozoa achieved by two-dimension electrophoresis and MALDI-TOF/TOF mass spectrometry of proteins labeled with a thiol-reactive fluorescent tag, monobromobimane. Our results have shown the formation of disulfide bonds in several sperm protein fractions during the epididymal ma...
Capacitation in the presence of methyl-β-cyclodextrin results in enhanced zona pellucida-binding ability of stallion spermatozoa. While IVF has been widely successful in many domesticated species, the development of a robust IVF system for the horse remains an elusive and highly valued goal. A major impediment to the development of equine IVF is the fact that optimised conditions for the capacitation of equine spermatozoa are yet to be developed. Conversely, it is known that stallion spermatozoa are particularly susceptible to damage arising as a consequence of capacitation-like changes induced prematurely in response to semen handling and transport conditions. To address these limitations, this study sought to develop a...
Effect of Cholesterol and Equex-STM Addition to an Egg Yolk Extender on Pure Spanish Stallion Cryopreserved Sperm. Cholesterol and Equex-STM are frequently added to different commercial and experimental extenders improving postthawing sperm quality. Doses of 125-150 mM of cholesterol from pig liver and 0.5-0.7% of Equex-STM were evaluated in a standard eggyolk extender (Martin et al., 1979). Six ejaculates per stallion from six pure Spanish stallions (6-8 years old) were collected in Martin's extender (B) and different mixtures of 125 mM-0.5% (I), 125 mM-0.7% (II), 150 mM-0.5% (III), and 150 mM-0.7% (IV) were added to original Martin's extender. Samples were frozen in 0.5 mL straws (100 × 10(6...
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 thawin...
The effects of ergot alkaloids on the breeding stallion reproductive system. Ergot alkaloids cause a range of pathological conditions in mares. There is no evaluation of the effects of ergot alkaloids from endophyte-infected tall fescue on the stallion breeding soundness examination spermiogram. Objective: The objective of this study was to investigate the effect of ergot alkaloids from endophyte-infected tall fescue on the stallion's reproductive functions. Methods: Crossover toxicology experiment. Methods: Six stallions were fed either toxic endophyte-infected tall fescue seed or a nontoxic endophyte tall fescue seed (Flecha AR-542, MaxQ). The fescue seed content was...
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...
CatSper and the relationship of hyperactivated motility to intracellular calcium and pH kinetics in equine sperm. In vitro fertilization does not occur readily in the horse. This may be related to failure of equine sperm to initiate hyperactivated motility, as treating with procaine to induce hyperactivation increases fertilization rates. In mice, hyperactivated motility requires a sperm-specific pH-gated calcium channel (CatSper); therefore, we investigated this channel in equine sperm. Motility was assessed by computer-assisted sperm motility analysis and changes in intracellular pH and calcium were assessed using fluorescent probes. Increasing intracellular pH induced a rise in intracellular calcium, w...
Use of two conventional staining methods to assess the acrosomal status of stallion spermatozoa. The acrosome is a highly specialised region of the spermatozoon that is essential for fertilisation. Defects or dysfunction of this structure have been associated with fertility problems in man and various domestic species including stallions. Current methods of evaluating the acrosome of stallion spermatozoa are time consuming and require specialised equipment, which is cost prohibitive to the average practitioner. Objective: To evaluate 2 conventional stains (Dip Quick and Spermac) and determine their usefulness in assessing acrosome integrity in stallions as compared with specific acrosomal...
Localization of alkali-labile sites in donkey (Equus asinus) and stallion (Equus caballus) spermatozoa. The presence of constitutive alkali-labile sites (ALS) has been investigated using a protocol of DNA breakage detection-fluorescence in situ hybridization and comet assay in spermatozoa of donkey (Equus asinus) and stallion (Equus caballus). These results were compared with those obtained using a similar experimental approach using somatic cells. The relative abundance of ALS was of the order of four times more in spermatozoa than in somatic cells. Alkali-labile sites showed a tendency to cluster localized at the equatorial-distal regions of the sperm. The amount of hybridized signal in the AL...
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...
Focal adhesion kinases and calcium/calmodulin-dependent protein kinases regulate protein tyrosine phosphorylation in stallion sperm. Protein tyrosine phosphorylation (PY) is a hallmark of sperm capacitation. In stallion sperm, calcium inhibits PY at pH 7.8). These results show for the first time a role for calcium/calmodulin-dependent kinases in PRKA-dependent sperm PY; a non-PRKA-dependent pathway regulating sperm PY; and the apparent involvement of the FAK family of protein tyrosine kinases downstream in both pathways.
