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.
Alves NC, Freitas MM, Faria JRD, Horta CL, Martins-Filho OA, Araújo MSS, Costa GMJ, Costa EA, de Almeida FRL, Amaral PHR, Pérez JCG, Lana ÂQ....Niacin acts as an antioxidant that protects cells from oxidative damage. This study evaluated the effects of adding niacin to the equine semen freezing extender on sperm quality and gene expression after cryopreservation. Ejaculates from ten stallions were frozen using the INRA 96 extender (control) or extenders supplemented with 10- and 20-mM niacin. After thawing, sperm were analysed for motility, kinematics, viability, membrane integrity, mitochondrial potential, lipid peroxidation, nitrite, hydrogen peroxide, malondialdehyde and reactive oxygen species (ROS) concentrations, DNA integrity, ...
Ramírez-Agámez L, Crowley JB, Love CC, Hernández-Avilés C.Currently, Intracytoplasmic Sperm Injection (ICSI) on in vitro-matured equine oocytes obtained by transvaginal oocyte aspiration (TVA) is the method of choice for in vitro production of equine blastocysts in a clinical setting. A protocol for conventional in vitro fertilization (cIVF) using either fresh or frozen/thawed stallion sperm incubated under capacitating conditions (Tyrode's Albumin Lactate Pyruvate medium + penicillamine, hypotaurine, epinephrine; FERT-PHE) has been recently reported. Several factors that may impact the clinical applicability of this cIVF protocol have yet to be st...
Wen X, Bou G, He Q, Liu Q, Yi M, Ren H.The reproductive ability of equine species is a critical component of equine breeding programs, with sperm quality serving as a primary determinant of reproductive success. In this study, we perform an integrative analysis of proteomics and metabolomics in seminal plasma to identify proteins and metabolites associated with sperm quality and reproductive ability in equine species. Methods: We utilized the CEROS instrument to assess the morphology and motility of sperm samples from three horses and three donkeys. Additionally, we statistically analyzed the mating frequency and pregnancy rates in...
Hernández-Avilés C.Sperm quality analysis using computer-assisted sperm analysis (CASA) systems and fluorescence-based techniques has become common in the animal reproduction industry, particularly for large animals (i.e., bovine, porcine, equine). In this chapter, the methods commonly utilized in the author's laboratory to examine sperm motion characteristics via CASA and plasma membrane intactness by flow cytometry will be described. These include methods to properly dilute fresh (stallions, bulls, boars), cool-stored (stallions, boars), or frozen/thawed (stallions, bulls, boars) sperm for assessment of sperm ...
Bugno-Poniewierska M, Bielecka M, Pietras N, Kij-Mitka B, Podstawski Z, Długosz B.The Hucul horse is a Polish primitive breed with a small population size, which highlights the importance of preserving the genetic resources. The cryopreservation of semen is essential for creating gene banks, but its effect on the acrosome reaction in Hucul stallions has not yet been investigated. The acrosome reaction is one of the most important physiological events associated with the fertilization process. Therefore, our goal was to determine the level of acrosome reaction in chilled and frozen/thawed Hucul stallion semen using the FluoAcro test and the SCA semen analysis system. We foun...
Al-Kass Z, Morrell JM, Ntallaris T.Sperm quality is adversely affected by cryopreservation due to the increased production of reactive oxygen species, which affects the integrity of sperm membranes, motility, and DNA fragmentation. Three methods for removing seminal plasma, washing (centrifuging extended semen at 800× for 10 min) and Single Layer Centrifugation with high or low density Equicoll, were used to prepare 29 ejaculates from ten stallions for freezing. Sperm quality parameters (kinematics, plasma membrane integrity, superoxide and hydrogen peroxide production, mitochondrial membrane potential, and DNA fragmentation)...
Medica AJ, Swegen A, Seifi-Jamadi A, McIntosh K, Gibb Z.Oxidative stress plays a critical role in regulating sperm function, yet species-specific antioxidant mechanisms remain poorly understood. This study compared hydrogen peroxide (H2O2) tolerance in horse and human sperm and investigated the roles of catalase and glutathione peroxidase (GPx) in horses. A H2O2 dose-response assay (0-2000 µM) showed that horse sperm were significantly more resistant to oxidative damage, with an IC50 for progressive motility over 14-fold higher than that of human sperm (391.6 µM vs. 27.3 µM). Horse sperm also accumulated more intracellular H2O2 without loss of m...
Medica A, Aitken RJ, Swegen A, Gibb Z.Context Equine reproductive technologies are crucial for overcoming challenges in natural fertilisation, particularly in sub-fertile stallions and breeding programs focused on genetic conservation and performance enhancement. Assisted reproductive technologies (ARTs), such as artificial insemination (AI), intracytoplasmic sperm injection (ICSI), and in vitro fertilisation (IVF), improve fertility outcomes and enable breeding across geographical distances. Aims This review examines sperm isolation techniques used in ART, evaluating their efficacy, limitations, and potential to enhance reproduct...
Wen X, Ren H, He Q, Yi M, Ulaangerel T, Bou G.Horses and donkeys, as integral members of the equine family, exhibit distinct reproductive capabilities and characteristics. Seminal plasma, the fluid component of semen, contains a variety of proteins that play critical roles in sperm function and fertility. This study aimed to systematically compare the protein profiles in the seminal plasma of horses and donkeys, thereby elucidating the molecular differences between these two species. The study utilized 4D-DIA proteomics technology to analyze seminal plasma from horses and donkeys and further validated key proteins through Western blot. Ou...
Hernández-Avilés C, Ramírez-Agámez L, Love CC.In vitro production of equine embryos by Intracytoplasmic Sperm Injection (ICSI) is commonly utilized to maximize the availability of stallion sperm, particularly from stallions with limited semen supply. The relationship between standard measures of sperm quality in frozen/thawed semen (i.e., sperm motility, normal morphology, DNA quality) and embryo production after ICSI of in vitro-matured equine oocytes has not been thoroughly studied. In this study, frozen/thawed semen from 47 stallions utilized in a commercial ICSI program was analyzed for post-thaw sperm total and progressive motility (...
Usuga A, Vergara AK, Tobón MC, Vargas S, Rojano B, Restrepo G.Maintaining sperm energy homeostasis in vitro is very important to improve the efficacy of stallion sperm preservation. Equine spermatozoa preferentially utilize oxidative phosphorylation over glycolysis to generate ATP. Metformin and rosiglitazone are antidiabetic compounds that enhances metabolic flexibility and glucose utilization. The aim of this study was to evaluate metformin and rosiglitazone supplementation of the freezing medium on quality and oxidative status of thawed stallion semen. A total of 15 ejaculates from five horses were collected and supplemented before freezing with metfo...
Hidalgo M, Ortiz I.Sperm vitrification is a novel-assisted reproductive technique that is increasingly gaining relevance in the last years. This technique allows to cryopreserve sperm from valuable stallions and donkeys without the exposure to permeable cryoprotectants, particularly toxic for the gametes of these species.This chapter aims to describe the current range of methodologies available that are key to ensure sperm quality after vitrification and warming of stallion and donkey sperm.
Miró J, Martínez-Rodero I, Yeste M, Catalán J.Cryopreservation is currently the only strategy for long-term conservation of equine sperm. To get optimal post-thaw sperm survival, carefully following each step of the freezing protocol is crucial. First, one needs to obtain and exhaustively analyze an ejaculate of good sperm quality. Then, the seminal plasma is removed by centrifugation, and the resulting pellet is resuspended in a certain volume of the freezing medium to reach the right sperm concentration. Finally, sperm samples are packaged into 0.5-mL straws, cooled, and frozen using an automatic, controlled-rate freezer. Once the tempe...
Abah KO, Ligocka-Kowalczyk Z, Itodo JI, Ameh G, Partyka A, Nizanski W.Accurately predicting male fertility is crucial for the animal breeding industry due to its significant economic implications. Existing literature suggests that mammalian fertility is partially dependent on sperm DNA integrity. However, routine semen analysis often fails to detect DNA damage and does not consistently correlate with field fertility outcomes. While assessing sperm DNA integrity provides valuable biological insights, its role in diagnosing animal infertility remains uncertain. Results: This meta-analysis evaluated the association between sperm DNA fragmentation (SDF) and fertilit...
Brito LFC.Semen analysis is an important laboratory diagnostic test for stallions. Evaluation of sperm motility is integral to basic semen analysis and results are important for breeding management and clinical practice. Computer-assisted sperm analysis (CASA) allows objective sperm motility evaluation and increases analytical precision. The objective of the present study was to validate a portable CASA system (AndroScope). Fresh/cooled semen samples (33 ejaculates, 18 stallions) and frozen semen (40 ejaculates and one epididymal flush, 27 stallions) were evaluated using the AndroScope and the IVOS II C...
Sultan T, Chaudhary AK, Mehta JS, Mehra R, Manuja A, Talluri TR, Bhattacharya TK.Sperm cryopreservation is one of the most important procedures in the development of biotechnologies for assisted reproduction. Cryopreservation of stallion spermatozoa tends to cause plasma membrane damage due to the low ratio of cholesterol to phospholipids. Different substances and compounds can be added to semen extenders to improve sperm quality. Objective: To investigate the effect of supplementing semen extender with zinc nanoparticles (ZnONPs) and gold nanoparticles (AuNPs) on cooled and frozen-thawed spermatozoa of Marwari stallion. Methods: A total of 20 ejaculates from four Marwari ...
Felix MR, Dobbie T, Woodward E, Linardi R, Okada C, Santos R, Hinrichs K.We recently reported successful equine IVF using fresh semen pre-incubated for a prolonged period (22 h) before co-culture with oocytes. In this study, we evaluated the feasibility of equine IVF with frozen-thawed sperm and evaluated capacitation-related changes in these sperm over the pre-incubation period. Sperm selected via a commercial sperm separation device (SSD) yielded significantly higher fertilization than did sperm selected by swim-up or by colloid centrifugation. Using the SSD method, fertilization rates with sperm pre-incubated for 15 min, 3 h, 6 h, and 9 h were 7.1, 22.2, 38...
Alim S, Cheppali SK, Pawar SS, Swamy MJ.Fibronectin type-II (FnII) proteins are major constituents in the seminal plasma of many mammals and play a crucial role in sperm capacitation. Additionally, the seminal FnII proteins from bull and horse exhibit chaperone-like activity (CLA), by acting as small heat shock proteins (shsps). The present work demonstrates that the major FnII protein of donkey seminal plasma, DSP-1 exhibits CLA with broad specificity and protects various client proteins such as alcohol dehydrogenase, lactate dehydrogenase and enolase against thermal and oxidative stress. Binding of phosphorylcholine (PrC) - the he...
Vitoria A, Barrachina L, Romero A, Fuente S, de Blas I, Gil L, Vázquez FJ.A new surgical technique using a polyether ether ketone (PEEK) anchoring device for testicle-sparing laparoscopic inguinal hernioplasty in stallions was described in 2023 and is known as the PEEK harpoon technique (PHT). In breeding stallions, it is essential that the surgery is effective in preventing inguinal hernia but also that it does not impair the testicular function. This study aims to evaluate whether the PHT may affect testicular function. To achieve that, changes in the testicular blood flow, sperm production and motility characteristics were assessed 28 days after use of the PHT. S...
Hidalgo M.Sperm vitrification is an alternative freezing method, which includes high cooling rates and non-permeable cryoprotectants agents. The first attempt in equids was using the spheres technique by directly dropping small volumes of the sperm into liquid nitrogen. Later, vitrification was developed using 0.25 mL straws with outer covers, which resulted in similar progressive motility when compared to conventional freezing in donkeys (44.3 ± 15.0 % vs. 44.7 ± 18.2 %) or even higher in horses (48.2 ± 2.3 % vs. 37.3 ± 2.2 %). Subsequently, the vitrification of larger volumes of sperm in 0.5 mL st...
Samper JC, Hernandez Aviles JC, Ramirez-Agamez LF, Love CC, Gonzalez-Marin C, Fleury P, Dini P, De La Fuente A, Foss R, Campos FL, Ross PJ.The application of sex-sorted semen in horses has historically lagged the bovine industry due to differences in the reproductive physiology and grater variability in equine semen quality. Recent advancements, including SuperGen™ sorters and Ultraplus™ semen processing methods developed by STgenetics® (Navasota, Texas), have significantly improved the efficiency of the sex-sorting process and reduced the sperm damage previously reported. To facilitate the broader use sex-sorted semen in the equine industry, it is essential to address challenges such as shipping cooled semen to a central so...
Ortiz-Rodriguez JM, Bucci D, Tovar-Pascual L, Granata S, Spinaci M, Nesci S.Sperm metabolism consists of a sophisticated network of biochemical reactions and varies between species, resulting in different metabolic strategies for ATP production to maintain sperm functionality. ATP can be produced through glycolysis or in the mitochondria by oxidative phosphorylation (OXPHOS). Since OXPHOS is the predominant metabolic pathway in horses spermatozoa, various assessments of mitochondrial activity are used to evaluate fertility, utilizing techniques such as fluorescent probes analysed via microscopy or flow cytometry, and polarographic electrode assays to measure current f...
Martin-Pelaez S, Fuente A, Takahashi K, Perez IT, Orozco J, Okada CTC, Neto CR, Meyers S, Dini P.Intracytoplasmic sperm injection (ICSI) is the current clinical practice for the in vitro production of equine embryos. The use of conventional fertilization methods such as in vitro fertilization (IVF), has historically been associated with poor success in horses. However, recent improvements have led to better outcomes with IVF, though only when using fresh semen, which limits its use in clinical practice. IVF remains in its infancy in equine reproduction, and several unknowns remain about the technique. One significant gap in knowledge concerns the morphokinetics of IVF embryos and how they...
Brito LFC, Felix MR, Linardi RL, Martinez de Andino EV, Balamurugan NS, Hernández-Avilés C, Hinrichs K.Intracytoplasmic sperm injection (ICSI) is a valuable assisted reproduction technology in clinical practice, especially when semen availability is limited. Since the number of sperm required per ICSI cycle is much less than the number of sperm available in a standard straw of frozen semen, refreezing semen at lower sperm concentrations could yield multiple straws for ICSI use. However, there is little data on the effect of sperm refreezing on ICSI outcomes, especially on the effect of extender used for refreezing. The objective of the present study was to evaluate the effect of refreezing exte...
Navarrete-López P, Asselstine V, Maroto M, Lombó M, Cánovas Á, Gutiérrez-Adán A.RNA molecules within ejaculated sperm can be characterized through whole-transcriptome sequencing, enabling the identification of pivotal transcripts that may influence reproductive success. However, the profiling of sperm transcriptomes through next-generation sequencing has several limitations impairing the identification of functional transcripts. In this study, we explored the nature of the RNA sequences present in the sperm transcriptome of two livestock species, cattle and horses, using RNA sequencing (RNA-seq) technology. Through processing of transcriptomic data derived from bovine and...
Aguiar LH, Pinto CRF.Sperm capacitation is considered the main factor limiting conventional in vitro fertilization (IVF) in horses. A recent scientific breakthrough in sperm processing for IVF in horses has resulted in embryos and foals being produced; however, various aspects of the IVF process remain to be fully elucidated. Lysine acetylation has been shown to play a role in sperm capacitation in several species and the objective of this study was to detect and evaluate this process in the horse. Ejaculates of two stallions were collected and incubated in different conditions with deacetylase inhibitors to induc...
Becerro-Rey L, Martín-Cano FE, Fabres Robaina Sancler-Silva Y, Gil MC, Ortega-Ferrusola C, Aparicio IM, Gaitskell-Phillips G, da Silva-Álvarez E....Conservation of equine semen in the liquid state is a central procedure in horse breeding and constitutes the basis of associated reproductive technologies. The intense mitochondrial activity of the stallion spermatozoa increases oxidative stress along the storage period, leading to sperm demise within 24-48 h of storage, particularly when maintained at room temperature. Recently, the relationship between metabolism and oxidative stress has been revealed. The study aimed to extend the period of conservation of equine semen, at room temperature through modification of the metabolites present i...
Ren H, Wen X, He Q, Yi M, Dugarjaviin M, Bou G.The reproductive performance of horse sperm and donkey sperm has been reported to differ. Sperm proteins play a crucial role in sperm viability and fertility. Although differences between species are known, no prior study has investigated disparities in the sperm proteome between horses and donkeys. Therefore, this study characterized and compared the sperm proteomes of horses and donkeys using 4D-DIA mass spectrometry technology. We identified 3436 proteins in horse sperm and 3404 proteins in donkey sperm. Of these, 3363 proteins were expressed in both horse and donkey sperm, with 73 proteins...
Ing NH, Konganti K, Ghaffar N, Johnson CD, Forrest DW, Love CC, Varner DD.Males of some species, from horses to humans, require medical help for subfertility problems. There is an urgent need for novel molecular assays that reflect spermatozoal function. In the last 25 years, studies examined RNAs in spermatozoa as a window into gene expression during their development and, more recently, for their functions in early embryo development. In clinics, more dense spermatozoa are isolated by density gradient centrifugation before use in artificial insemination to increase pregnancy rates. The objectives of the current study were to discover and quantify the microRNAs i...
Hernández-Avilés C, Ramírez-Agámez L, Weintraub ST, Scoggin CF, Davis BW, Raudsepp T, Varner DD, Love CC.Thoroughbred stallions that carry a double-homozygous genotype A/A-A/A for SNPs rs397316122 and rs69101140 in exon 5 of the FKBP6 gene (chr13; EquCab3.0) are uniquely subfertile due to impaired acrosomal exocytosis (IAE). In this study, the sperm proteome in frozen/thawed semen from subfertile Thoroughbred stallions was studied and compared to that of frozen/thawed sperm from fertile Thoroughbred stallions. A total of 2,220 proteins was identified, of which 140 proteins were found to be differentially abundant in sperm from the subfertile stallions compared to that of fertile stallions (83 les...
Baumber J, Ball BA, Gravance CG, Medina V, Davies-Morel MC.The objective of this study was to examine the influence of reactive oxygen species (ROS), generated through the use of the xanthine (X)-xanthine oxidase (XO) system, on equine sperm motility, viability, acrosomal integrity, mitochondrial membrane potential, and membrane lipid peroxidation. Equine spermatozoa were separated from seminal plasma on a discontinuous Percoll gradient, and spermatozoa were incubated with 0.6 mM X and 0.05 U/mL XO for 30 minutes. Catalase (150 U/mL), superoxide dismutase (SOD, 150 U/mL), or glutathione (GSH, 1.5 mM) were evaluated for their ability to preserve sperm ...
Parks JE, Lynch DV.Composition and thermotropic phase behavior of sperm membrane lipids from species ranging in sensitivity to cold shock were determined. Lipids from whole sperm and sperm plasma membrane were fractionated into neutral lipid, glycolipid, and phospholipid fractions. Compositional analyses were completed for free sterols, phospholipids and phospholipid-bound fatty acids. Phase transition temperatures were determined for phospholipid and glycolipid fractions using differential scanning calorimetry. Cholesterol was the major sterol in sperm lipids of all species. Cholesterol to phospholipid molar ra...
Hahn S, Giaglis S, Hoesli I, Hasler P.The intention of this review is to provide an overview of the potential role of neutrophil extracellular traps (NETs) in mammalian reproduction. Neutrophil NETs appear to be involved in various stages of the reproductive cycle, starting with fertility and possibly ending with fetal loss. The first suggestion that NETs may play a role in pregnancy-related disorders was in preeclampsia, where vast numbers were detected in the intervillous space of affected placentae. The induction of NETosis involved an auto-inflammatory component, mediated by the increased release of placental micro-debris in p...
Katila T.This review attempts to summarize the current knowledge on uterine inflammatory response after mating in horses, pigs and cattle. Post-mating endometritis has been extensively studied in horses as it has been considered to cause infertility. The inflammation is known to occur also in cattle, but it has not been investigated to a similar extent. There are a number of publications about mechanisms of post-mating uterine inflammation in pigs, which seem to resemble those in horses. The major focus of this review is the horse, but relevant literature is presented also on swine and cattle. Spermato...
Plaza Davila M, Martin Muñoz P, Tapia JA, Ortega Ferrusola C, Balao da Silva C C, Peña FJ.Mitochondria have been proposed as the major source of reactive oxygen species in somatic cells and human spermatozoa. However, no data regarding the role of mitochondrial ROS production in stallion spermatozoa are available. To shed light on the role of the mitochondrial electron transport chain in the origin of oxidative stress in stallion spermatozoa, specific inhibitors of complex I (rotenone) and III (antimycin-A) were used. Ejaculates from seven Andalusian stallions were collected and incubated in BWW media at 37 °C in the presence of rotenone, antimycin-A or control vehicle. Incubation...
LeBlanc MM, Causey RC.Endometritis, a major cause of mare infertility arising from failure to remove bacteria, spermatozoa and inflammatory exudate post-breeding, is often undiagnosed. Defects in genital anatomy, myometrial contractions, lymphatic drainage, mucociliary clearance, cervical function, plus vascular degeneration and inflamm-ageing underlie susceptibility to endometritis. Diagnosis is made through detecting uterine fluid, vaginitis, vaginal discharge, short inter-oestrous intervals, inflammatory uterine cytology and positive uterine culture. However, these signs may be absent in subclinical cases. Hyper...
Westhoff D, Kamp G.Evidence is provided that the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase is covalently linked to the fibrous sheath. The fibrous sheath is a typical structure of mammalian spermatozoa surrounding the axoneme in the principal piece of the flagellum. More than 90% of boar sperm glyceraldehyde 3-phosphate dehydrogenase activity is sedimented after cell disintegration by centrifugation. Detergents, different salt concentrations or short term incubation with chymotrypsin do not solubilize the enzyme, whereas digestion with trypsin or elastase does. Short term incubation with trypsin...
Aitken RJ, Gibb Z, Mitchell LA, Lambourne SR, Connaughton HS, De Iuliis GN.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 ...
Baumber J, Ball BA, Linfor JJ, Meyers SA.The objective of this study was to examine the effect of reactive oxygen species (ROS) and cryopreservation on DNA fragmentation of equine spermatozoa. In experiment 1, equine spermatozoa were incubated (1 hour, 38 degrees C) according to the following treatments: 1) sperm alone; 2) sperm + xanthine (X, 0.3 mM)-xanthine oxidase (XO, 0.025 U/mL); 3) sperm + X (0.6 mM)-XO (0.05 U/mL); and 4) sperm + X (1 mM)-XO (0.1 U/mL). In experiment 2, spermatozoa were incubated (1 hour, 38 degrees C) with X (1 mM)-XO (0.1 U/mL) and either catalase (200 U/mL), superoxide dismutase (SOD, 200 U/mL), or reduced...
Su TW, Choi I, Feng J, Huang K, McLeod E, Ozcan A.We report the discovery of an entirely new three-dimensional (3D) swimming pattern observed in human and horse sperms. This motion is in the form of 'chiral ribbons', where the planar swing of the sperm head occurs on an osculating plane creating in some cases a helical ribbon and in some others a twisted ribbon. The latter, i.e., the twisted ribbon trajectory, also defines a minimal surface, exhibiting zero mean curvature for all the points on its surface. These chiral ribbon swimming patterns cannot be represented or understood by already known patterns of sperms or other micro-swimmers. The...
Ortega-Ferrusola C, Sotillo-Galán Y, Varela-Fernández E, Gallardo-Bolaños JM, Muriel A, González-Fernández L, Tapia JA, Peña FJ.The kinematics of the appearance of apoptotic markers was studied by flow cytometry and immunoblot assays in equine spermatozoa subjected to freezing and thawing. Caspase activity, low mitochondrial membrane potential, and increases in sperm membrane permeability were observed in all of the phases of the cryopreservation procedure. Freezing and thawing caused an increase in membrane permeability and changes in the pattern of caspase activity; decreases in mitochondrial membrane potential were observed after centrifugation and cooling to 4 degrees C and after freezing and thawing. It is propose...
Rathi R, Colenbrander B, Bevers MM, Gadella BM.The primary aim of this study was to establish a flow cytometric technique for determining the capacitation status of stallion spermatozoa. To this end, a flow cytometric technique that demonstrates changes in plasma membrane fluidity; namely, merocyanine 540 staining, was compared with the more conventional Ca(2+)-dependent fluorescence microscopic technique, chlortetracycline (CTC) staining, for assessing capacitation status. In addition, the effect of bicarbonate/CO(2) on the progress of capacitation and the acrosome reaction (AR) and on temporal changes in sperm motility, with particular r...
Loux SC, Crawford KR, Ing NH, González-Fernández L, Macías-García B, Love CC, Varner DD, Velez IC, Choi YH, Hinrichs K.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...
Darr CR, Varner DD, Teague S, Cortopassi GA, Datta S, Meyers SA.Stallion sperm rely primarily on oxidative phosphorylation for production of ATP used in sperm motility and metabolism. The objective of the study was to identify which substrates included in Biggers, Whitten, and Whittingham (BWW) media are key to optimal mitochondrial function through measurements of sperm motility parameters, mitochondrial oxygen consumption, and cellular reactive oxygen species (ROS) production. It was expected that mitochondrial substrates, pyruvate and lactate, would support sperm motility and mitochondrial function better than the glycolytic substrate, glucose, due to d...
Ortega Ferrusola C, González Fernández L, Morrell JM, Salazar Sandoval C, Macías García B, Rodríguez-Martinez H, Tapia JA, Peña FJ.Lipid peroxidation (LPO) of stallion spermatozoa was assessed in fresh semen and in samples of the same ejaculates after freezing and thawing. Particular attention was paid to individual differences in the susceptibility to LPO and its possible relationship with freezability. Innate levels of LPO were very low in fresh spermatozoa but increased after thawing, a change that was largely stallion-dependent. The level of LPO in fresh spermatozoa was not correlated with that of the thawed spermatozoa. Negative correlations existed between LPO and intact membranes post-thaw (r=-0.789, P<0.001), a...
McPartlin LA, Suarez SS, Czaya CA, Hinrichs K, Bedford-Guaus SJ.Capacitation is a complex and not well-understood process that encompasses all the molecular changes sperm must undergo to successfully fertilize an oocyte. In vitro fertilization has remained elusive in the horse, as evidenced by low in vitro fertilization (IVF) rates (0%-33%); moreover, only two foals have ever been produced using IVF. Incubation of stallion sperm in modified Whittens supplemented with bovine serum albumin and sodium bicarbonate yielded significant rates of time-dependent protein tyrosine phosphorylation and induced acrosomal exocytosis, consistent with capacitation. The obj...
Leemans B, Gadella BM, Stout TA, De Schauwer C, Nelis H, Hoogewijs M, Van Soom A.In contrast to man and many other mammalian species, conventional in vitro fertilization (IVF) with horse gametes is not reliably successful. The apparent inability of stallion spermatozoa to penetrate the zona pellucida in vitro is most likely due to incomplete activation of spermatozoa (capacitation) because of inadequate capacitating or fertilizing media. In vivo, the oviduct and its secretions provide a microenvironment that does reliably support and regulate interaction between the gametes. This review focuses on equine sperm-oviduct interaction. Equine sperm-oviduct binding appears to be...
Das PJ, McCarthy F, Vishnoi M, Paria N, Gresham C, Li G, Kachroo P, Sudderth AK, Teague S, Love CC, Varner DD, Chowdhary BP, Raudsepp T.Mature mammalian sperm contain a complex population of RNAs some of which might regulate spermatogenesis while others probably play a role in fertilization and early development. Due to this limited knowledge, the biological functions of sperm RNAs remain enigmatic. Here we report the first characterization of the global transcriptome of the sperm of fertile stallions. The findings improved understanding of the biological significance of sperm RNAs which in turn will allow the discovery of sperm-based biomarkers for stallion fertility. The stallion sperm transcriptome was interrogated by analy...
Kotilainen T, Huhtinen M, Katila T.The objective of this study was to investigate the inflammatory reaction induced in the equine uterus by insemination with fresh and frozen semen. Eleven groups (6 to 8 mares per group) were studied during 2 breeding seasons. The mares were inseminated using raw semen, frozen semen, extended fresh and frozen semen, concentrated fresh semen, seminal plasma and seminal extenders only. One group was bred naturally. Six hours after insemination, the uteri were flushed with 50 ml of phosphate-buffered saline (PBS). Seventeen out of 104 samples (16%) exhibited slight bacterial growth. Neutrophil con...
Alghamdi AS, Lovaas BJ, Bird SL, Lamb GC, Rendahl AK, Taube PC, Foster DN.Bovine semen is naturally deposited in the vagina and spermatozoa migrate through the cervix into the uterus leaving the bulk of seminal plasma (SP) behind. In equine, both spermatozoa and SP are deposited directly in the uterus and SP reduces sperm binding to neutrophils and prevents the formation of DNA-based neutrophil extracellular traps (NETs). We investigated the role of bovine SP on sperm-neutrophil binding using the four most common bovine semen extenders. Contrary to equine, bovine spermatozoa removed from SP had low binding to neutrophils for up to 3h, but as little as 10% SP increas...
Cheng FP, Fazeli A, Voorhout WF, Marks A, Bevers MM, Colenbrander B.Peanut agglutinin (PNA) was used to assess the sperm acrosomal status and the acrosome reaction during gamete interaction in the equine species. PNA exclusively binds to the outer acrosomal membrane of stallion spermatozoa, as was established by transmission electron microscopy. Fluorescein isothiocyanate-PNA (FITC-PNA) labeling was used to monitor sperm acrosomal changes during a prolonged incubation period of 24 hours and during a 2-hours incubation in the presence of 5 microM calcium ionophore A23187. In addition, after a 4-hours preincubation in SP-TALP medium, sperm samples were incubated...
Wood PL, Scoggin K, Ball BA, Troedsson MH, Squires EL.Using a nontargeted lipidomics analysis of equine sperm and seminal plasma, we were able to characterize a diverse array of individual lipids including ethanolamine and choline ether lipids and seminolipids essential to membrane raft function. We also detected, for the first time in sperm, the presence of (O-acyl)-ω-hydroxy-fatty acids (OAHFA) with up to 52 carbon chain lengths, which were localized to the head and not the tail of sperm. The only previous identification of OAHFAs has been in meibomian glands and their sebaceous secretions. The identities of these lipid amphiphiles were valida...
Choi YH, Varner DD, Love CC, Hartman DL, Hinrichs K.Work with lyophilized sperm helps delineate the factors required for successful fertilization. We investigated the use of lyophilized sperm in equine embryo production. In Experiment 1, sperm DNA fragmentation index was not affected by three freeze/thaw or lyophilization cycles. In Experiment 2, oocytes injected with lyophilized sperm or with sperm from a treatment in which lyophilized sperm were suspended in sperm cytoplasmic extract (SE) yielded blastocyst development rates of 0 and 28% respectively (P < 0.05). In Experiment 3, blastocyst development rate was 6-11% after injection of sper...
Aparicio IM, Martin Muñoz P, Salido GM, Peña FJ, Tapia JA.Use of cooled and frozen semen is becoming increasingly prevalent in the equine industry. However, these procedures cause harmful effects in the sperm cell resulting in reduced cell lifespan and fertility rates. Apoptosis and necrosis-related events are increased during semen cryopreservation. However, a third type of cell death, named autophagy, has not been studied during equine semen storage. Light chain (LC)3 protein is a key component of the autophagy pathway. Under autophagy activation, LC3-I is lipidated and converted to LC3-II. The ratio of LC3-II/LC3-I is widely used as a marker of au...
Mahé C, Zlotkowska AM, Reynaud K, Tsikis G, Mermillod P, Druart X, Schoen J, Saint-Dizier M.In vitro fertilization (IVF) gives rise to embryos in a number of mammalian species and is currently widely used for assisted reproduction in humans and for genetic purposes in cattle. However, the rate of polyspermy is generally higher in vitro than in vivo and IVF remains ineffective in some domestic species like pigs and horses, highlighting the importance of the female reproductive tract for gamete quality and fertilization. In this review, the way the female environment modulates sperm selective migration, survival, and acquisition of fertilizing ability in the oviduct is being considered...
Peña FJ, Ortiz-Rodríguez JM, Gaitskell-Phillips GL, Gil MC, Ortega-Ferrusola C, Martín-Cano FE.An overview of the sperm metabolism is presented; using the stallion as a model we review glycolysis, Krebs Cycle and oxidative phosphorylation, paying special attention to the interactions among them. In addition, metabolism implies a series of coordinated oxidation-reduction reactions and in the course of these reactions reactive oxygen species (ROS) and reactive oxoaldehydes are produced ; the electron transport chain (ETC) in the mitochondria is the main source of the anion superoxide and hydrogen peroxide, while glycolysis produces 2-oxoaldehydes such as methylglyoxal as byproducts; due t...
Leemans B, Stout TAE, De Schauwer C, Heras S, Nelis H, Hoogewijs M, Van Soom A, Gadella BM.In contrast to various other mammalian species, conventional in vitro fertilization (IVF) with horse gametes is not reliably successful. In particular, stallion spermatozoa fails to penetrate the zona pellucida, most likely due to incomplete activation of stallion spermatozoa (capacitation) under in vitro conditions. In other mammalian species, specific capacitation triggers have been described; unfortunately, none of these is able to induce full capacitation in stallion spermatozoa. Nevertheless, knowledge of capacitation pathways and their molecular triggers might improve our understanding o...
Gravance CG, Garner DL, Baumber J, Ball BA.The fluorescent carbocyanine dye, JC-1, labels mitochondria with high membrane potential orange and mitochondria with low membrane potential green. Evaluation of mitochondrial membrane potential with JC-1 has been used in a variety of cell types, including bull spermatozoa; however, JC-1 staining has not yet been reported for equine spermatozoa. The aim of this study was to apply JC-1 staining and assessment by flow cytometry or a fluorescence microplate reader for evaluation of mitochondrial function of equine spermatozoa. Six ejaculates from four stallions were collected and centrifuged thro...
Sailer BL, Jost LK, Evenson DP.Sperm from four mammalian species were analyzed by the sperm chromatin structure assay (SCSA) and the terminal deoxynucleotidyl transferase assay (TdTA) using flow cytometry. The SCSA quantitates the susceptibility of sperm nuclear DNA to in situ acid denaturation, while the TdTA quantitates the presence of endogenous DNA strand breaks in sperm nuclear chromatin. Correlations were seen between the percentage of sperm cells showing susceptibility to in situ acid denaturation and the percentage of cells showing the presence of DNA strand breaks for humans (r = 0.56, P = 0.004), rams (r = 0.84, P...
Ball BA, Vo AT, Baumber J.To characterize generation of reactive oxygen species (ROS) by equine spermatozoa. Methods: Multiple semen samples collected from 9 stallions. Methods: Equine spermatozoa were separated from seminal plasma on a discontinuous polyvinylpyrrolidone (PVP)-coated silica gradient and resuspended in a modified Tyrode albumin-lactate-pyruvate medium. Amount of hydrogen peroxide (H2O2) generated was assayed by use of a 1-step fluorometric assay, using 10-acetyl-3,7-dihydroxyphenoxazine as a probe for detection of H2O2 in a microplate assay format. Concentration of H2O2 was determined by use of a fluore...
