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.
Brogna R, Fan J, Sieme H, Wolkers WF, Oldenhof H.Even though dried sperm is not viable, it can be used for fertilization as long as its chromatin remains intact. In this study, we investigated drying- and temperature-induced conformational changes of nucleic acids and stallion sperm chromatin. Sperm was diluted in preservation formulations with and without sugar/albumin and subjected to convective drying at elevated temperatures on glass substrates. Accumulation of reactive oxygen species was studied during storage at different temperatures, and the sperm chromatin structure assay was used to assess DNA damage. Fourier transform infrared spe...
Sampaio B, Ortiz I, Resende H, Felix M, Varner D, Hinrichs K.Exposure to the calcium ionophore A23187 may present a "universal" sperm treatment for IVF, as it bypasses capacitation pathways. However, success in utilizing A23187 is variable, especially in equine spermatozoa. Notably, albumin is used during A23187 treatment but paradoxically is thought to suppress A23187 action. Essentially no critical data are available on the effects of A23187 and albumin concentrations, ratios, or addition protocols on changes in intracellular calcium ([Ca] ) in any cell type. To determine factors that affect the action of A23187 on [Ca] in equine and murine spermatozo...
Oddi S, Carluccio A, Ciaramellano F, Mascini M, Bucci R, Maccarrone M, Robbe D, Dainese E.Sperm cryopreservation represents a powerful tool for horse breeding. To improve the efficiency of artificial insemination in the horse using cryopreserved spermatozoa, an adequate understanding of the underlying biophysical properties that affect sperm cryosurvival needs to be reached yet. In this pilot study, we described isolation and analysis of the main fatty acids from sperms of stallions classified as good and poor freezers (7 GF and 5 PF, according to sperm motility and viability, before and after cryopreservation). Fatty acid profiles were only assessed in pre-thaw sperms. Eight mai...
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...
Pruß D, Yang H, Luo X, Liu D, Hegermann J, Wolkers WF, Sieme H, Oldenhof H.Stallion sperm is typically cryopreserved using low cooling rates and low concentrations of cryoprotective agents (CPAs). The inevitable water-to-ice phase transition during cryopreservation is damaging and can be prevented using vitrification. Vitrification requires high cooling rates and high CPA concentrations. In this study, the feasibility of stallion sperm vitrification was investigated. A dual-syringe pump system was used to mix sperm equilibrated in a solution with a low concentration of CPAs, with a solution containing a high CPA concentration, and to generate droplets of a defined si...
Ortiz I, Felix M, Resende H, Ramírez-Agámez L, Love CC, Hinrichs K.To define the effect of sperm agglutination, associated with incubation under capacitating conditions, on accuracy of membrane assessment via flow cytometry and to develop methods to mitigate that effect. Methods: Sperm motility was measured by CASA. Sperm were stained with PI-PSA or a novel method, LD-PSA, using fixable live/dead stain and cell dissociation treatment, before flow-cytometric analysis. Using LD-PSA, acrosome reaction and plasma membrane status were determined in equine sperm treated with 10 μm A23187 for 10 min, followed by 0, 1, or 2 h incubation in capacitating conditions. R...
Miró J, Catalán J, Marín H, Yánez-Ortiz I, Yeste M.While artificial insemination (AI) with frozen-thawed sperm results in low fertility rates in donkeys, the addition of seminal plasma, removed during cryopreservation, partially counteracts that reduction. Related to this, an apparent inflammatory reaction in jennies is induced following AI with frozen-thawed sperm, as a high amount of polymorphonuclear neutrophils (PMN) are observed within the donkey uterus six hours after AI. While PMN appear to select the sperm that ultimately reach the oviduct, two mechanisms, phagocytosis and NETosis, have been purported to be involved in that clearance. ...
Ortiz-Rodríguez JM, Martín-Cano FE, Gaitskell-Phillips GL, Silva A, Ortega-Ferrusola C, Gil MC, Peña FJ.Energy metabolism in spermatozoa is complex and involves the metabolism of carbohydrate fatty acids and amino acids. The ATP produced in the electron transport chain in the mitochondria appears to be crucial for both sperm motility and maintaining viability, whereas glycolytic enzymes in the flagella may contribute to ATP production to sustain motility and velocity. Stallion spermatozoa seemingly use diverse metabolic strategies, and in this regard, a study of the metabolic proteome showed that Gene Ontology terms and Reactome pathways related to pyruvate metabolism and the Krebs cycle were pr...
Lançoni R, Celeghini ECC, Giuli V, de Carvalho CPT, Zoca GB, Garcia-Oliveros LN, Batissaco L, Oliveira LZ, de Arruda RP.Coenzyme Q-10 (CoQ-10) is a cofactor for mitochondrial electron transport chain and may be an alternative to improve sperm quality of cryopreserved equine semen. This work aimed to improve stallion semen quality after freezing by adding CoQ-10 to the cryopreservation protocol. Seven saddle stallions were utilized. Each animal was submitted to five semen collections and freezing procedures. For cryopreservation, each ejaculate was divided in three treatments: 1) Botucrio® diluent (control); 2) 50 μmol CoQ-10 added to Botucrio® diluent; 3) 1 mmol CoQ-10 added to Botucrio® diluent. Semen batc...
Tongu EAO, Segabinazzi LGTM, Alvarenga ML, Monteiro A, Papa FO, Alvarenga MA.This study aimed to evaluate the effects of mesenchymal stem cell-conditioned medium (MSC-CM) on sperm parameters, intrauterine polymorphonuclear neutrophils (PMN), intrauterine fluid accumulation (IUF), and fertility in mares. In experiment 1, two ejaculates from ten stallions were extended to 50 million sperm/mL using a milk-based extender. Thereafter, 20 mL of extended semen was added of MSC-CM as follows: 0, 5, 10, 15, and 20 mL. Sperm kinetics and plasma membrane integrity were evaluated immediately after dilution (T0) and 2 h post-incubation at 37 °C (T2). In experiment 2, mares cha...
Podico G, Lago-Alvarez Y, Carossino M, Ferrer MS, Arnold CE, Canisso IF.This study aimed to describe and compare semen parameters (pre-freeze and post-freezing) and antisperm antibodies (ASA) of donkeys with epididymal sperm granuloma and healthy controls. Feral donkeys (n = 10) castrated in a concurrent study were enrolled in the present experiment. Three donkeys had unilateral granulomas, two donkeys had bilateral granulomas, whereas the remaining five had grossly normal epididymides. The granulomas were either single or multiple, firm, well-circumscribed, tan to red, and 1-5 mm in size. Upon incision, abundant, thick, tan to white-yellow fluid was recovered. ...
Al-Kass Z, Brown A, Johannisson A, Ntallaris T, Morrell JM.Although single layer centrifugation (SLC) selects robust spermatozoa from stallion semen, the effect of individual variation has not been studied in detail. The objective of this study was to determine the variation among stallions in the effects of SLC on sperm quality during cooled storage for up to 48 hr. Semen samples from seven stallions (18 ejaculates) were split, with one portion being used for SLC and the other serving as a control (CON). Sperm quality (kinematics, reactive oxygen species (ROS) production, membrane integrity (MI) and chromatin integrity) were analysed at 0, 24 and 48...
Ortiz-Rodriguez JM, Nerozzi C, Bucci D, Mislei B, Mari G, Tamanini C, Peña FJ, Spinaci M, Galeati G.Sperm are redox-regulated cells, and deregulation of their redox status is considered to affect male fertility and to reduce their fertilizing ability following biotechnological procedures, such as cryopreservation. Cystine (CysS), after incorporation in sperm via SLC7A11 antiporter, has been demonstrated to increase intracellular GSH content, the most important non enzymatic antioxidant. This study was aimed at investigating the role of SLC7A11 antiporter on frozen-thawed stallion sperm ability to respond to in vitro capacitating environment after post-thaw incubation with CysS and/or Sulfas...
Donnelly CG, Bellone RR, Hales EN, Nguyen A, Katzman SA, Dujovne GA, Knickelbein KE, Avila F, Kalbfleisch TS, Giulotto E, Kingsley NB, Tanaka J....Following the successful creation of a biobank from two adult Thoroughbred mares, this study aimed to recapitulate sample collection in two adult Thoroughbred stallions as part of the Functional Annotation of the Animal Genome (FAANG) initiative. Both stallions underwent thorough physical, lameness, neurologic, and ophthalmic (including electroretinography) examinations prior to humane euthanasia. Epididymal sperm was recovered from both stallions immediately postmortem and cryopreserved. Aseptically collected full thickness skin biopsies were used to isolate, culture and cryopreserve dermal f...
Gaitskell-Phillips G, Martín-Cano FE, Ortiz-Rodríguez JM, Silva-Rodríguez A, Gil MC, Ortega-Ferrusola C, Peña FJ.Although cryopreservation is widely used in animal breeding, the technique is still suboptimal. The population of spermatozoa surviving the procedure experiences changes attributed to alteration in their redox regulation. In order to expand our knowledge regarding this particular aspect, the proteome in fresh and frozen thawed aliquots of equine spermatozoa was studied to identify the proteins most severely affected by the procedure. If alteration of redox regulation is a major factor explaining cryodamage, proteins participating in redox regulation should be principally affected. Using a spli...
Magalhaes HB, Canuto LEF, Zhao F, Li M, Li X, Ji C, Canisso IF.This study aimed to assess the sexual behavior of jacks mounting jennies in estrus and determine the daily sperm output (DSO) and spermatogenic efficiency using two equations to calculate testicular volume (TV). Eight sexually rested mature jacks, aging 5 to 10 years old, had semen collected once a day for 10 consecutive days using jennies in good standing estrus for mounting. Sexual behavior and semen parameters were assessed during each collection. Testicular measurements of height, width, and length were taken immediately before the first semen collection, and these measurements were used t...
D'Anza E, Albarella S, Galdiero G, Tafuri S, Del Prete C, Cocchia N, Ciani F, Mastellone V, Pasolini MP, Carotenuto D, Selvaggi M, Ciotola F....The reproductive performances of livestock play an essential role in the economic management of the farm. The improvement of semen quantity and quality through the use of food supplements that lack substances which are forbidden in animal feeding, or that may have detrimental effects, is an important goal. Maca (Lepidium meyenii) is a plant that has been used for centuries in the Andes for nutrition and fertility enhancement in humans and animals. The aim of this study was to evaluate the effects of food supplementation of stallions with maca during the breeding season on spermatozoa parameter...
Li N, Yu J, Yang F, Shao Y, Wu S, Liu B, Li M, Wang T, Li J, Zeng S.The aim of this study was to evaluate the effects of L-proline on the extender quality of frozen and post-thawed jackass semen. Jackass (n = 6) semen samples were collected and cryopreserved in gradient concentrations (0-80 mM) of L-proline in extenders; post-thawed semen samples were cultured in L-proline medium for 10 hours at 37°C. For cryopreservation experiment I, the motile parameters, mitochondrial membrane potential (MMP), and plasma membrane, acrosome, and chromatin structure integrities of post-thawed semen were assessed. For culture experiment II, additional ROS contents were ana...
Kareskoski M, Vakkamäki J, Laukkanen K, Palviainen M, Johannisson A, Katila T.Matrix metalloproteinase (MMP)-2 and MMP-9 are gelatinases that take part in several reproductive processes. The aim of this study was to measure levels of MMP-2 and MMP-9 in fractionated stallion ejaculates, and to evaluate the association between these components and semen quality, and sperm longevity during cooled storage. Semen quality were assessed separately for sperm-rich fractions (HIGH), sperm-poor fractions (LOW), and whole ejaculate samples (WE) from 33 stallions. After cooled storage with SP either present or removed, sperm motility and DFI were determined. The relative activity of...
Rossetto L, Farcey MF, Bilbao MG, Bartolomé JA, Gallelli MF, Miragaya MH.The stress associated with training may reduce reproductive efficiency in Criollo stallions. The objective of this study was to compare semen quality and hormone concentrations in Criollo stallions under training or under regular field conditions. Criollo breed stallions (n = 18) were evaluated during the spring. The exercise group (n = 9) performed 1 hour of exercise per day and participated in competitions during the experimental period. The control group (n = 9) neither performed exercise nor participated in competitions. Serum and semen samples were obtained every 15 days (two separat...
Catalán J, Yánez-Ortiz I, Gacem S, Papas M, Bonet S, Rodríguez-Gil JE, Yeste M, Miró J.Previous research has determined that irradiation of mammalian sperm with red light increases motility, mitochondrial activity, and fertilization capacity. In spite of this, no study has considered the potential influence of the color of the straw and the extender used. Therefore, this study tests the hypothesis that the response of mammalian sperm to red light is influenced by the color of the straw and the turbidity/composition of the extender. Using the horse as a model, 13 ejaculates from 13 stallions were split into two equal fractions, diluted with Kenney or Equiplus extender, and stored...
Gimeno BF, Bariani MV, Laiz-Quiroga L, Martínez-León E, Von-Meyeren M, Rey O, Mutto AÁ, Osycka-Salut CE.Cryopreservation by negatively affecting sperm quality decreases the efficiency of assisted reproduction techniques (ARTs). Thus, we first evaluated sperm motility at different conditions for the manipulation of equine cryopreserved spermatozoa. Higher motility was observed when spermatozoa were incubated for 30 min at 30 × 106/mL compared to lower concentrations (p < 0.05) and when a short centrifugation at 200× g was performed (p < 0.05). Moreover, because sperm suitable for oocyte fertilization is released from oviduct epithelial cells (OECs), in response to the capacitation process, we e...
Alves NC, Diniz SA, Viegas RN, Cortes SF, Costa ED, Freitas MM, Martins-Filho OA, Araújo MSS, Lana ÂMQ, Wenceslau RR, Lagares MA.The aim of this study was to improve the quality of frozen-thawed equine sperm by the addition of caffeine to it. Semen from nine stallions was frozen and different concentrations of caffeine (3, 5 and 7.5 mM) were added to frozen-thawed semen. The sperm kinetic parameters, membrane functionality and integrity, and acrosome integrity and spontaneous acrosome reacted sperm were evaluated with a computer-assisted sperm analysis, a hypoosmotic swelling test and epifluorescent microscopy, respectively. Nitrite and hydroperoxide concentrations of frozen-thawed semen were measured using spectrophot...
Diaz-Jimenez M, Rota A, Dorado J, Consuegra C, Pereira B, Camillo F, Panzani D, Fanelli D, Tesi M, Monaco D, Hidalgo M.Sperm vitrification has been recently developed, but fertility trials have not been performed yet in equine species. In this study, a new warming technique for vitrified donkey semen was developed and the uterine inflammatory response and fertility were compared to conventional freezing. In Experiment 1, sperm was vitrified in straws and warmed in 3 ml of extender or in a water bath at: 37 °C/30 s; 43 °C/10 s; and 60 °C/5 s. Sperm motility, plasma and acrosome membranes and DNA integrity were compared between treatments. In Experiment 2, jennies were inseminated twice (500 ...
Hernández-Avilés C, Ramírez-Agámez L, Love CC, Friedrich M, Pearson M, Kelley DE, Beckham AMN, Teague SR, LaCaze KA, Brinsko SP, Varner DD.Under in vitro conditions, stallion sperm might preferentially use energy substrates that primarily undergo mitochondrial metabolism. The present study sought to determine the effects of glucose, pyruvate, lactate, or their combinations on the quality of stallion sperm subjected to cooled storage at different temperatures, when using a skim milk-based semen extender. In Experiment 1, no substrate (Control), glucose (40 mM; Glu-40), pyruvate (2 mM, 19.8 mM; Pyr-2, Pyr-19), lactate (2 mM, 19.8 mM; Lac-2, Lac-19, respectively), or their combinations (G/P/L-2 or G/P/L-19, respectively) were ...
Vieira LA, Matás C, Torrecillas A, Saez F, Gadea J.Seminal plasma (SP) plays a crucial role in sperm protection and functionality. However, the effect of SP on the sperm cryopreservation is dependent on the stallion and SP composition. The use of epididymal spermatozoa incubated in the presence of SP could help the identification of the components of SP that are able to confer protection upon the spermatozoa during freezing. The aims of this study were (i) to identify SP components involved in the potential protection of epididymal spermatozoa during the freeze-thawing process and (ii) to identify and evaluate the proteins likely related to sp...
Gacem S, Bompart D, Valverde A, Catalán J, Miró J, Soler C.This study was conducted to determine optimum image capture frame rates (FRO) when there was evaluation of different types of counting chambers used for CASA-Mot determinations of stallion sperm motility. Sperm VCL was determined at frame rates of 25-250 f/s in: 1) Spermtrack® (Spk) 10 and 20 chambers (drop displacement-type chambers 10 and 20 μm-deep respectively; and 2) ISAS®D4C10, ISAS®D4C20 (10 and 20 μm-deep respectively) and ISAS®D4C20 L (20 μm-deep) capillary loaded chambers. Values for different sperm kinematic variables were determined using each chamber at 250 f/s, whi...
Crespo F, Wilson R, Díaz-Jimenez M, Consuegra C, Dorado J, Barrado BG, Gosálvez J, Smit RL, Hidalgo M, Johnston S.Horses are long-day seasonal breeding animals, however, with modern stallion reproductive management it is important for collection of semen during periods that are not part of the traditional breeding season. This study was conducted to examine variation in the seminal characteristics of individual stallions in Avila, Spain during 1 year with a particular emphasis on sperm DNA fragmentation. Semen was collected twice per season from a total of 20 stallions. There was a marked seasonal effect on all seminal characteristics, with the greatest on progressive motility, % membrane integrity and le...
Ortiz-Rodríguez JM, Martín-Cano FE, Gaitskell-Phillips G, Silva A, Tapia JA, Gil MC, Redondo E, Masot J, Ortega-Ferrusola C, Peña FJ.Spermatozoa are redox-regulated cells, and stallion spermatozoa, in particular, present an intense mitochondrial activity in which large amounts of reactive oxygen species (ROS) are produced. To maintain the redox potential under physiological conditions, sophisticated mechanisms ought to be present, particularly in the mitochondria. In the present study, we investigated the role of the SLC7A11 antiporter. This antiporter exchanges intracellular glutamate for extracellular cystine. In the spermatozoa, cystine is reduced to cysteine and used for GSH synthesis. The importance of the antiporter f...
Gacem S, Catalán J, Valverde A, Soler C, Miró J.In order to optimize the donkey sperm motility analysis by the CASA (Computer Assisted Sperm Analysis)-Mot system, twelve ejaculates were collected from six jackasses. Capillary loaded chamber (CLC), ISASD4C depths 10 and 20 µm, ISASD4C Leja 20 and drop displacement chamber (DDC), Spermtrack (Spk) depths 10 and 20 µm were used. Sperm kinematic variables were evaluated using each chamber and a high-resolution camera capable of capturing a maximum of 500 frames/second (fps). The optimum frame rate (OFR) (defined according to curvilinear velocity-VCL) was dependent on chamber type. The highest ...
Serafini R, Longobardi V, Spadetta M, Neri D, Ariota B, Gasparrini B, Di Palo R.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...
Dobrinski I, Suarez SS, Ball BA.Interaction of spermatozoa with oviductal epithelial cells (OEC) in the oviductal isthmus prolongs the life span of spermatozoa. The hypothesis that the interaction of equine spermatozoa with OEC affects their intracellular calcium concentration ([Ca2+]i) was tested in a sperm-OEC coculture model. Changes in [Ca2+]i in spermatozoa loaded with the fluorescent calcium indicator indo-1 acetoxymethylester (AM) were determined for spermatozoa attached to OEC or to Matrigel, as well as for free-swimming spermatozoa incubated without oviductal epithelium. [Ca2+]i was determined before incubation and ...
Gacem S, Papas M, Catalan J, Miró J.The accessory sex glands play a major role in the production of seminal plasma, and testicular artery blood flow seems to strongly influence testicular function. However, very little ultrasound imaging of these organs has been undertaken in donkeys. The present work reports the results of such examinations in five jackasses along the year. The accessory glands were inspected by B-mode ultrasound while the testicular artery blood flow was assessed by colour pulsed-wave Doppler ultrasound. The testicular artery was examined at pampiniform plexus (PPT), supratesticular area (ST) and capsular arte...
Olaciregui M, Gil L, Montón A, Luño V, Jerez RA, Martí JI.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...
Blommaert D, Sergeant N, Delehedde M, Donnay I, Lejeune JP, Franck T, Serteyn D.The quality of fresh or thawed sperm in stallions has been generally determined by the viability and total and progressive motility of the sperm. Today, the expression of ProAKAP4, a protein present in the flagellum of spermatozoa, appears to be an innovative and relevant functional marker to assess semen quality and male fertility. This study aims to compare the concentration of ProAKAP4 in the semen from 5 stallions frozen with two different extenders immediately after thawing (T0) and 4 h post-thawing (T4). Viability, total and progressive motility were measured in parallel. Significant di...
Akbarinejad V, Fathi R, Shahverdi A, Esmaeili V, Rezagholizadeh A, Ghaleno LR.Equine sperm possesses a unique physiology because its energy supply is mostly dependent on oxidative phosphorylation of mitochondria as an aerobic source of adenosine triphosphate (ATP) generation. The present study was, therefore, conducted to investigate the relationship between sperm kinematic and functional variables in stallions. Semen samples were collected from five warmblood stallions (three ejaculates from each stallion), diluted with INRA96 and transferred to the laboratory. Next, sperm motility, mitochondrial membrane potential (MMP), production of superoxide anion (as a reactive o...
Dobrinski I, Ignotz GG, Thomas PG, Ball BA.To test the hypotheses that the attachment of equine spermatozoa to uterine tubal (oviductal) epithelial cells (OEC) in vitro is mediated by glycoproteins, and that proteins with carbohydrate-binding properties are present in the periacrosomal plasma membrane of equine spermatozoa. Methods: 4 reproductively sound stallions, and 1 mare in estrus. Methods: In experiment 1a, fluorescent-labeled spermatozoa were cocultured with monolayers of OEC in the presence of 50 mM glucose, fructose, galactose, mannose, N-acetyl glucosamine, N-acetyl galactosamine, or N-acetyl neuraminic acid, or 10 mg of fet...
Ball BA, Gravance CG, Medina V, Baumber J, Liu IK.To characterize the activity of catalase in equine semen. Methods: 15 stallions of known and unknown reproductive history. Methods: Seminal plasma was collected from raw equine semen by centrifugation, and samples of seminal plasma were frozen prior to assay for catalase activity. Tissue samples (n = 3 stallions) from the bulbourethral gland, prostate gland, vesicular gland, and testis were homogenized, and cauda epididymal fluid was collected for determination of catalase activity. Catalase activity was determined as an enzyme kinetic assay by the disappearance of H2O2 as measured by ultravio...
Hayden SS, Blanchard TL, Brinsko SP, Varner DD, Hinrichs K, Love CC.Dilution of semen to less than 20 × 10(6) sperm/mL has been reported to decrease sperm quality in multiple species, a phenomenon known as the semen "dilution effect." Critical evaluation of stallion semen diluted to these concentrations, however, has not been reported. This study evaluated sperm motion characteristics (percent total motility [TMOT], percent progressive motility [PMOT], curvilinear velocity [μm/s], and percent straightness) and plasma membrane integrity (percent plasma membrane intact [PMI]) in semen samples diluted to 2.5 × 10(6) sperm/mL with the addition of 0%, 7.5%, ...
Ortiz-Rodriguez JM, Balao da Silva C, Masot J, Redondo E, Gazquez A, Tapia JA, Gil C, Ortega-Ferrusola C, Peña FJ.The population of stallion spermatozoa that survive thawing experience compromised mitochondrial functionality and accelerated senescence, among other changes. It is known that stallion spermatozoa show very active oxidative phosphorylation that may accelerate sperm senescence through increased production of reactive oxygen species. Rosiglitazone has been proven to enhance the glycolytic capability of stallion spermatozoa maintained at ambient temperature. Thus, we hypothesized that thawed sperm may also benefit from rosiglitazone supplementation. Thawed sperm were washed and resuspended in Ty...
Squires EL, Keith SL, Graham JK.Although use of cryopreserved stallion spermatozoa is currently accepted by many breed registries, utilization of this technique remains limited due to poor fertility for some stallions. One reason for these results is osmotic stress that spermatozoa experiences when the cryoprotectant (glycerol) is added to the cells prior to freezing and removal from the cells after thawing. In an effort to minimize osmotic damage, alternative cryoprotectants, having lower molecular weights and greater membrane permeability than glycerol, were evaluated to determine their effectiveness for cryopreserving sta...
Mugnier S, Kervella M, Douet C, Canepa S, Pascal G, Deleuze S, Duchamp G, Monget P, Goudet G.Oviduct epithelial cells (OEC) co-culture promotes in vitro fertilization (IVF) in human, bovine and porcine species, but no data are available from equine species. Yet, despite numerous attempts, equine IVF rates remain low. Our first aim was to verify a beneficial effect of the OEC on equine IVF. In mammals, oviductal proteins have been shown to interact with gametes and play a role in fertilization. Thus, our second aim was to identify the proteins involved in fertilization in the horse. Results: In the first experiment, we co-incubated fresh equine spermatozoa treated with calcium ionophor...
Mugnier S, Dell'Aquila ME, Pelaez J, Douet C, Ambruosi B, De Santis T, Lacalandra GM, Lebos C, Sizaret PY, Delaleu B, Monget P, Mermillod P....The mechanism of fertilization remains largely enigmatic in mammals. Most studies exploring the molecular mechanism underlying fertilization have been restricted to a single species, generally the mouse, without a comparative approach. However, the identification of divergences between species could allow us to highlight key components in the mechanism of fertilization. In the pig, in vitro fertilization (IVF) and polyspermy rates are high, and spermatozoa penetrate easily through the zona pellucida (ZP). In contrast, IVF rates are low in the horse, and polyspermy is scarce. Our objective was ...
Doty A, Buhi WC, Benson S, Scoggin KE, Pozor M, Macpherson M, Mutz M, Troedsson MH.Equine spermatozoa induce a uterine inflammatory response characterized by a rapid, transient influx of polymorphonuclear neutrophils (PMNs). Seminal plasma proteins have been shown to modulate the interaction between spermatozoa and PMNs, but a specific protein responsible for this function has not been identified. The objective of this study was to isolate and identify a protein in equine seminal plasma that suppresses binding between spermatozoa and PMNs. Seminal plasma was pooled from five stallions, and proteins were precipitated in 60% (w/v) ammonium sulfate and dialyzed (3500 MW cutoff)...
Oldenhof H, Friedel K, Sieme H, Glasmacher B, Wolkers WF.Cellular membranes are one of the primary sites of injury during freezing and thawing for cryopreservation of cells. Fourier transform infrared spectroscopy (FTIR) was used to monitor membrane phase behavior and ice formation during freezing of stallion sperm. At high subzero ice nucleation temperatures which result in cellular dehydration, membranes undergo a profound transition to a highly ordered gel phase. By contrast, low subzero nucleation temperatures, that are likely to result in intracellular ice formation, leave membrane lipids in a relatively hydrated fluid state. The extent of free...
Macías García B, González Fernández L, Morrell JM, Ortega Ferrusola C, Tapia JA, Rodriguez Martínez H, Peña FJ.The present study attempted to select the subpopulation of stallion spermatozoa that best survived a conventional freezing and thawing procedure, using centrifugation of post-thawed semen samples through a single layer of a glycidoxypropyltrimethoxysilane-coated silica colloid with a species-specific formulation (Androcoll-E). After freezing and thawing, four sperm subpopulations were identified, listed as FT1 to FT4. While subpopulations FT1 and FT2 were characterized by low sperm velocity, high velocities characterized the ones called FT3 and FT4. The single-layer centrifugation (SLC)-handle...
Leemans B, Gadella BM, Sostaric E, Nelis H, Stout TA, Hoogewijs M, Van Soom A.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...
Crespo F, Quiñones-Pérez C, Ortiz I, Diaz-Jimenez M, Consuegra C, Pereira B, Dorado J, Hidalgo M.The aim of this study was to assess seasonal variations during different periods of the breeding season (spring and summer) on stallion sperm DNA fragmentation and in vivo fertility associated with cooled-stored semen samples. Ejaculates were collected from eleven stallions and assessed for sperm motility (assessed by computer-assisted sperm analysis) and plasma membrane integrity (evaluated under fluorescence microscopy). Sperm DNA fragmentation (evaluated by the Sperm Chromatin Dispersion test) was assessed in cooled-stored semen at 5 °C for up to 24 h. Artificial insemination was perfor...
Macías-García B, González-Fernández L, Gallardo-Bolaños JM, Peña FJ, Johannisson A, Morrell JM.The aim of this study was to elucidate if SLC after 24 h storage selects the subpopulation of spermatozoa that better withstands osmotic shock. To test this hypothesis, viability, mitochondrial membrane potential (MMP) and superoxide anion (O(2)(·-)) production of uncentrifuged (UC) and single layer centrifugation (SLC) - selected spermatozoa were analyzed following SLC after storage of the semen. An aliquot of the extended ejaculate (100×10(6) spermatozoa/mL) was centrifuged through a single layer of a silane-coated silica based colloid formulation optimized for equine spermatozoa (Androcol...
Balao da Silva CM, Ortega Ferrusola C, Morillo Rodriguez A, Gallardo Bolaños JM, Plaza Dávila M, Morrell JM, Rodriguez Martínez H, Tapia JA....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...
Gaitskell-Phillips G, Martín-Cano FE, Ortiz-Rodríguez JM, Silva-Rodríguez A, Gil MC, Ortega-Ferrusola C, Peña FJ.Although cryopreservation is widely used in animal breeding, the technique is still suboptimal. The population of spermatozoa surviving the procedure experiences changes attributed to alteration in their redox regulation. In order to expand our knowledge regarding this particular aspect, the proteome in fresh and frozen thawed aliquots of equine spermatozoa was studied to identify the proteins most severely affected by the procedure. If alteration of redox regulation is a major factor explaining cryodamage, proteins participating in redox regulation should be principally affected. Using a spli...
Minervini F, Lacalandra GM, Filannino A, Garbetta A, Nicassio M, Dell'aquila ME, Visconti A.Fumonisin B1 (FB1) is a mycotoxin produced by Fusarium species that exerts its toxic effect through interference with the sphingolipid pathway by inhibiting ceramide synthase. A FB1-dependent sperm toxicity was reported in boars. No information on FB1-related reproductive toxicity in stallions, the most sensitive animal species, has been reported. The aim of the present study was to assess the in vitro toxicity of FB1 on fresh and frozen-thawed equine spermatozoa by analyzing sperm viability, chromatin stability (SCSA) and reactive oxygen species (ROS) production by flow cytometry and sperm mo...
Pickett BW, Sullivan JJ, Byers WW, Pace MM, Remmenga EE.The effect of centrifugation of diluted and undiluted semen on equine and bovine spermatozoan motility and fertility was examined, as was the effect of seminal plasma and dilution on stallion spermatozoa during incubation before and after freezing. Centrifugation at 370 g or 829 g was not detrimental (P greater than 0.05) to prefreeze or postfreeze motility if a final concentration of 10% seminal plasma was present. A reduction of seminal plasma from 10% to 2% significantly (P smaller than 0.05) reduced motility. A centrifugal force of 956 g significantly reduced prefreeze but not postfreeze m...
Macías García B, González Fernández L, Ortega Ferrusola C, Morillo Rodríguez A, Gallardo Bolaños JM, Rodríguez Martinez H, Tapia JA....Fatty acids and plasmalogens were extracted from the phospholipids of the plasma membrane of stallion spermatozoa, to determine their relation with sperm quality after freezing and thawing. Sperm quality was rated using a quality index that combined the results of the analysis of sperm motility and velocity (CASA analysis), membrane status and mitochondrial membrane potential (flow cytometry) post thaw. Receiving operating system (ROC) curves were used to evaluate the value of specific lipid components of the sperm membrane herein studied as forecast of potential freezeability. From all parame...
Gonzalez-Castro RA, Carnevale EM.We determined if microfluidic sorting (MF) of frozen-thawed stallion sperm improves sperm population characteristics and results in embryo development after intracytoplasmic sperm injection (ICSI). The efficiency and efficacy of MF sperm separation was evaluated by comparing pre- and post-separation sperm population variables. Procedural comparisons were performed after sorting with MF, single-layer colloidal centrifugation (SLC) or swim-up (SU), and cleavage and embryo development were evaluated after ICSI using MF-sorted sperm. In Experiment 1, when compared to the original sperm sample, MF ...
Dini P, Troch L, Lemahieu I, Deblende P, Daels P.The objective of this study was to evaluate the accuracy of a novel, portable device (iSperm Equine for assessing concentration and motility of stallion semen). In the first experiment, semen concentration was determined by the iSperm Equine (Aidmics Biotechnology), Androvision (Minitube) and NucleoCounter SP-100™ (ChemoMetec). The total motility and progressive motility were determined by the iSperm Equine and the Androvision using the manufacturer's guidelines. Frozen/thawed semen samples (n = 33) at various dilutions were analysed for concentration and motility with the above-mentioned ...
Catalán J, Yánez-Ortiz I, Tvarijonaviciute A, González-Arostegui LG, Rubio CP, Yeste M, Miró J, Barranco I.This study investigated whether the activities of the antioxidant components of donkey seminal plasma (SP)-both enzymatic (superoxide dismutase (SOD), catalase-like (CAT), glutathione peroxidase-like (GPX), and paraoxonase type 1 (PON1)) and non-enzymatic (measured in terms of total thiol, copper-reducing antioxidant capacity (CUPRAC), ferric-reducing ability of plasma (FRAP), and Trolox equivalent antioxidant capacity (TEAC))-and oxidative stress index (OSI) are related to sperm cryotolerance. For this purpose, 15 ejaculates from jackasses (one per individual) were collected and split into tw...
Thomas PG, Ball BA, Brinsko SP.Regulation of attachment of equine spermatozoa to homologous oviduct epithelium was investigated by co-culture of spermatozoa with oviductal epithelial cell explants. Stallion spermatozoa were incubated with explants derived from the isthmus and ampulla of follicular, postovulatory, and diestrous mares. Steroid treatments (estradiol, progesterone, or control) were applied across all explant groups. Estimates of motility and total numbers of attached spermatozoa were made 0.5, 24, and 48 h after initiation of co-culture. Equine spermatozoa attached by their rostral acrosomal region to both cili...
Pommer AC, Rutllant J, Meyers SA.Cryopreservation requires exposure of sperm to extreme variations in temperature and osmolality. The goal of this experiment was to determine the osmotic tolerance levels of equine sperm by analyzing motility, viability, mitochondrial membrane potential (MMP), and mean cell volume (MCV). Spermatozoa were incubated at 22 degrees C for 10 min in isosmolal TALP (300 mOsm/kg), or a range of anisosmolal TALP solutions (75-900 mOsm/kg), for initial analysis, and then returned to isosmolal conditions for 10 min for further analysis. Total sperm motility was lower (P < 0.05) in anisosmolal conditio...
