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.
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 ...
Consuegra C, Crespo F, Dorado J, Diaz-Jimenez M, Pereira B, Sánchez-Calabuig MJ, Beltrán-Breña P, Pérez-Cerezales S, Rizos D, Hidalgo M.The aim of this study was to evaluate the fertilizing capacity of frozen or vitrified stallion sperm after assessing different warming procedures. In Experiment 1, different warming procedures were compared after sperm vitrification: immersion in extender at 43 °C (C), or in a water bath at 37 °C/30 s (W37), 43 °C/10 s (W43) or 60 °C/5 s (W60). With the W60 treatment, there were greater values (P < 0.05) for VCL (83.93 ± 3.6 μm/s) and ALH (3.00 ± 0.2 μm) than freezing and with the C group, and greater values (P < 0.001) for PM (35.33 ± 2.5 %) than with the W43 treatment. In Experiment...
Novello G, Segabinazzi LGTM, Lisboa FP, Canuto LE, Freitas-Dell'Aqua CP, Dell'Aqua JA, Canisso IF.This study compared the postthaw semen parameters of stallions with high and low body condition score (BCS) and evaluated associations between body morphometric parameters and postthaw semen parameters. Twenty stallions were split into Low BCS (BCS<7, n = 11) and High BCS (BCS ≥7, n = 9) groups, and underwent a complete morphometric analysis (e.g., neck scores and circumference, crest neck height, body weight, and height), and subcutaneous body fat thickness (SFT) at the tail head, withers, shoulders, and retroperitoneal space. A fasted oral sugar test (OST) was conducted on all stallio...
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...
Cazales N, Estradé MJ, Pereyra F, Fiala-Rechsteiner SM, Mattos RC.This study aimed to determine whether the insemination site and dose with cryopreserved sperm of reproductively normal mares affect the sperm population in uterine tubes and the intensity of endometrial inflammatory response. Experimental subjects were estrous mares inseminated, in the mid-uterine body (Body) or the tip of the uterine horn (Tip), ipsilateral to the dominant follicle, with one 0.5 mL straw with 50 × 106 sperm (50) or with eight straws with 50 × 106 sperm/straw (400). Mares were slaughtered 2 h, 4 h and 12 h after artificial insemination (AI) and randomly assigned to f...
Hernández-Avilés C, Love CC, Serafini R, Ramírez-Agámez L, Kelley DE, de Andino EM, Teague SR, LaCaze KA, Brinsko SP, Varner DD.In this study, the effectiveness of supplementing INRA-96® extender (INRA-Control; original antibiotic formulation: potassium penicillin G = 38 μg/mL; gentamicin sulfate = 105 μg/mL; amphotericin B = 0.315 μg/mL) with amikacin sulfate and potassium penicillin G (AP) was determined. In Exp. 1, two sources of amikacin (INRA-AP-Sigma or INRA-AP-GoldBio) in combination with penicillin G were compared with ticarcillin/clavulanate (INRA-Tim) or no-supplemental antibiotics (INRA-Control) to examine effects on sperm quality and commensal bacterial growth. No differences were detected in s...
Gambini A, Duque Rodríguez M, Rodríguez MB, Briski O, Flores Bragulat AP, Demergassi N, Losinno L, Salamone DF.Several equids have gone extinct and many extant equids are currently considered vulnerable to critically endangered. This work aimed to evaluate whether domestic horse oocytes support preimplantation development of zebra embryos obtained by intracytoplasmic sperm injection (ICSI, zebroid) and cloning, and to study the Hippo signaling pathway during the lineage specification of trophectoderm cells and inner cell mass cells. We first showed that zebra and horse sperm cells induce porcine oocyte activation and recruit maternal SMARCA4 during pronuclear formation. SMARCA4 recruitment showed to be...
Bubenickova F, Postlerova P, Simonik O, Sirohi J, Sichtar J.Seminal plasma (SP) is the natural environment for spermatozoa and contains a number of components, especially proteins important for successful sperm maturation and fertilization. Nevertheless, in standard frozen stallion insemination doses production, SP is completely removed and is replaced by a semen extender. In the present study, we analyzed the effects of the selected seminal plasma protein groups that might play an important role in reducing the detrimental effects on spermatozoa during the cryopreservation process. SP proteins were separated according to their ability to bind to hepar...
Catalán J, Papas M, Gacem S, Mateo-Otero Y, Rodríguez-Gil JE, Miró J, Yeste M.Previous studies in other mammalian species have shown that stimulation of semen with red-light increases sperm motility, mitochondrial activity, and fertilizing capacity. This study sought to determine whether red-light stimulation using a light emitting diode (LED) at 620-630 nm affects sperm motility and structure of motile subpopulations, sperm viability, mitochondrial activity, intracellular ATP levels, rate of O consumption and DNA integrity of horse spermatozoa. For this purpose, nine ejaculates were collected from nine different adult stallions. Upon collection, semen was diluted in Ke...
Gaitskell-Phillips G, Martín-Cano FE, Ortiz-Rodríguez JM, Silva-Rodríguez A, Rodríguez-Martínez H, Gil MC, Ortega-Ferrusola C, Peña FJ.Some stallions yield ejaculates that do not tolerate conservation by refrigeration prior to artificial insemination (AI), showing improvement after removal of most of the seminal plasma (SP) by centrifugation. In this study, the SP-proteome of 10 different stallions was defined through high-performance liquid chromatography with tandem mass spectrometry and bioinformatic analysis in relation to the ability of the ejaculates to maintain semen quality when cooled and stored at 5°C. Stallions were classified into three groups, depending on this ability: those maintaining good quality after direc...
Kumar P, Mehta JS, Ravi SK, Dedar RK, Purohit GN, Legha RA, Tripathi BN, Talluri TR.The present study was conducted with the hypothesis that addition of cholesterol to the extender would stabilize the sperm membranes by increasing the cholesterol-to-phospholipid (C:P) ratio and would result in an improved post-thaw semen quality and reduce oxidative stress in the jack (Martina franca) semen. Forty-eight ejaculates from six jacks were collected and analyzed for the present study. The freshly collected semen sample of each jack stallion was divided into five equal fractions after addition of the primary extender without cholesterol-loaded cyclodextrin (CLC) (C) and with 1, 1.5,...
Bahrami A, Divar MR, Azari M, Kafi M.In the present study, we aimed to evaluate the possible protective effects of the nicotinic acid (NA) at three concentrations (10, 20, and 40 mM) on the equine cooled and frozen-thawed spermatozoa quality markers including viability, plasma membrane or acrosome integrity, DNA fragmentation, lipid peroxidation, and total oxidant levels. We also evaluated the effects of NA on preservation of the post-thaw sperm quality after 6 hours of cold storage before freezing. Five stallions were used for semen collections. The current experiment was repeated six times using pooled semen samples from two ...
Dominguez EM, Moreno-Irusta A, Rodriguez MB, Salamone DF, de Arruda RP, Losinno L, Giojalas LC.The successful use of assisted reproduction techniques (ART) depends in part on the sperm physiological status. Several sperm selection procedures have been applied to improve quality of sperm population when using the ART. There has previously been development of a Sperm Selection Assay (SSA) for humans which is based on the attraction of capacitated sperm by chemotaxis towards progesterone (P), resulting in an enriched sperm population with an optimal physiological status similar to capacitated spermatozoa, with these cells having very little DNA fragmentation and optimal concentrations of r...
Griffin RA, Swegen A, Baker M, Aitken RJ, Skerrett-Byrne DA, Silva Rodriguez A, Martín-Cano FE, Nixon B, Peña FJ, Delehedde M, Sergeant N, Gibb Z.The horse breeding industry relies upon optimal stallion fertility. Conventional sperm assessments provide limited information regarding ejaculate quality and are not individually predictive of fertilizing potential. The aim of this study was to harness mass spectrometry to compare the proteomic profiles of high- and low-quality stallion spermatozoa, with the ultimate goal of identifying fertility biomarker candidates. Extended stallion semen (n = 12) was fractionated using Percoll density gradients to isolate low-quality and high-quality sperm populations. Motility and morphological assessmen...
Nery IHAV, Araújo Silva RAJ, Souza HM, Arruda LCP, Monteiro MM, Seal DCM, Silva GR, Silva TMS, Carneiro GF, Batista AM, Câmara DR, Guerra MMP.l-Carnitine (LC) plays a key role in sperm metabolism, easily providing energy through β-oxidation, which positively affects motility. The objective of this study was to investigate the association between blood plasma and seminal plasma LC levels, as well as the effect of LC as an additive in a skimmed milk-based extender during sperm storage at 5°C. In the first experiment, semen and blood samples from 14 Quarter Horse stallions were used. The LC content in blood plasma and seminal plasma was determined by spectrophotometry and their relationships with seminal parameters were evaluated. In...
Treulen F, Aguila L, Arias ME, Jofré I, Felmer R.In vitro manipulation of spermatozoa leads to deleterious changes of structure and function that occur mainly due to oxidative stress, therefore, prevention or treatment is a strategy to improve the functions of processed sperm. In the present study, the aim was to evaluate the effects of MnTBAP supplementation, a compound with antioxidant activity, on in vitro capacitation conditions of thawed equine sperm. For this purpose, stallion spermatozoa (2 × 10 cells/mL) were incubated in the sperm-TLP base medium for 4 h in which there were three different conditions: non-capacitating, capacitating...
Catalán J, Llavanera M, Bonilla-Correal S, Papas M, Gacem S, Rodríguez-Gil JE, Yeste M, Miró J.The aim of this study was to evaluate whether red-light stimulation increases the longevity and resilience of cryopreserved stallion sperm to withstand post-thaw incubation for 120 min. Sixteen frozen straws of 0.5 mL from eight stallions were used. Samples were cryopreserved, thawed through incubation at 38 °C for 30 s and divided into the control and samples exposed to red-light using a triple LED photo-activation system (wavelength: 620-630 nm). Three irradiation protocols consisting of different light-dark-light intervals (1-1-1, 2-2-2 and 3-3-3 min) were tested. Sperm quality param...
Alamaary MS, Haron AW, Hiew MWH, Ali M.Present study aimed to investigate the effect of adding antioxidants, cysteine and ascorbic acid on the levels of glutamic oxaloacetic transaminase (GOT), glutamic-pyruvate (GPT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH) and γ-glutamyl transpeptidase (GGT) enzymes of post-thawed stallion sperm. Ten ejaculates were collected each from four healthy stallions and cryopreserved using HF-20 freezing extender containing either 0 mg/ml cysteine or ascorbic acid, 0.5 mg/ml cysteine and 0.5 mg/ml ascorbic acid. All samples in freezing extender containing cysteine or ascorbic acid or ...
Aitken RJ, Gregoratos D, Kutzera L, Towney E, Lin M, Wilkins A, Gibb Z.MTT is widely used in biology as a probe for cell viability by virtue of its ability to generate deposits of insoluble formazan at sites of intense oxidoreductase activity. This response is generally held to reflect mitochondrial redox activity; however, extra-mitochondrial MTT reduction has also been recorded in certain cell types. Given this background, we set out to determine the major sites of formazan deposition in mammalian spermatozoa. In the mouse, most MTT reduction took place within the extensive mitochondrial gyres, with a single minor site of formazan deposition on the sperm head. ...
Aurich J, Kuhl J, Tichy A, Aurich C.Differences in the cryotolerance of spermatozoa exist among stallions, but it remains to be determined to what extent such differences are affected by breed. In this study, post-thaw semen quality in stallions presented for semen cryopreservation was analysed retrospectively (1012 ejaculates from 134 stallions of 5 breeds). The percentage of frozen-thawed ejaculates acceptable for artificial insemination (AI) and the number of insemination doses per cryopreserved ejaculate was calculated. Logistic regression analysis revealed sperm motility in raw semen as the most important explanatory variab...
Albrizio M, Lacalandra GM, Volpe S, Nicassio M, Cinone M.Heat Shock Proteins are chaperones primary involved in the repair of cellular damages induced by temperature. The harmful effect of temperature on the male gonad is well known, on the contrary knowledge on the effects of the environment on semen quality are still insufficient. The aim of this paper was to learn more about the role of HSPs and the environment in modulating the physiology of equine male gonads. We showed a detailed analysis of equine semen characteristic and the expression level of three HSPs (60-70-90) over a one-year period analyzing the effects of temperature and humidity and...
Rubessa M, Feugang JM, Kandel ME, Schreiber S, Hessee J, Salerno F, Meyers S, Chu I, Popescu G, Wheeler MB.The capacity for microscopic evaluation of sperm is useful for assisted reproductive technologies (ART), because this can allow for specific selection of sperm cells for in vitro fertilization (IVF). The objective of this study was to analyze the same sperm samples using two high-resolution methods: spatial light interference microscopy (SLIM) and atomic force microscopy (AFM) to determine if with one method there was more timely and different information obtained than the other. To address this objective, there was evaluation of sperm populations from boars and stallions. To the best of our k...
Miró J, Marín H, Catalán J, Papas M, Gacem S, Yeste M.In the donkey, artificial insemination (AI) with frozen-thawed semen is associated with low fertility rates, which could be partially augmented through adding seminal plasma (SP) and increasing sperm concentration. On the other hand, post-AI endometrial inflammation in the jenny is significantly higher than in the mare. While previous studies analyzed this response through recovering Polymorphonuclear Neutrophils (PMN) from uterine washings, successive lavages can detrimentally impact the endometrium, leading to fertility issues. For this reason, the first set of experiments in this work inten...
Suliman Y, Becker F, Tuchscherer A, Wimmers K.Horses are seasonal breeders with a natural breeding season beginning in spring and extending through midsummer. In this study, quantitative and qualitative parameters of chilled stallion semen were compared between fertile and subfertile stallions and between the breeding and the non-breeding season. Semen quality parameters compared included ejaculate volume, sperm concentration, total sperm number, sperm morphology, and computer-assisted semen analysis (CASA)-derived sperm movement characteristics obtained from two groups of warmblood stallions ( ; four fertile stallions and four subfert...
Novello G, Podico G, Segabinazzi LGTM, Lima FS, Canisso IF.The objective of this study was to compare semen parameters and embryo recovery rates of cooled stallion semen extended with INRA 96 or BotuSemen Gold. In experiment 1, 45 ejaculates from nine mature stallions were collected, assessed, and equally split between both extenders and then extended to 50 million sperm/mL. Then, the extended semen was stored in three passive cooling containers (Equitainer, Equine Express II, and BotuFlex) for 48 hours. In experiment 2, the same ejaculates extended in experiment 1 were cushion-centrifuged, the supernatant was discarded, and the pellets were resuspend...
Ing NH, Konganti K, Ghaffari N, Johnson CD, Forrest DW, Love CC, Varner DD.It is not unusual for stallions to have fertility problems. For many, artificial insemination with more dense spermatozoa (isolated by density gradient centrifugation) results in greater pregnancy rates compared with the rates when using unfractionated spermatozoa. RNAs in spermatozoa delivered to the oocyte at conception are required for embryo development. Novel molecular assays of spermatozoa that reflect function are needed to predict the fertility of stallions. To describe and compare the RNA populations in more dense and less dense spermatozoa from stallions. Spermatozoa from five stalli...
Köhne M, Mönnig F, Papin J, Schöniger S, Tönissen A, Rohn K, Martinsson G, Schoon HA, Sieme H.The effects of standard uterine body and hysteroscopic insemination on endometrial health were investigated. For this purpose, 33 mares were assigned to five different protocols: control (no insemination; n = 7), sham AI (sham uterine body insemination; n = 6), sham HysAI (sham hysteroscopic insemination; n = 7), standard AI (standard uterine body insemination, 300 × 106 progressively motile sperms (PMS); n = 7) and HysAI (hysteroscopic insemination, 100 × 106 PMS; n = 6). Sampling included uterine swabbing for microbiological examination, cytology for determination of polymorphonucle...
Journal of proteomicsApril 2, 2020
Volume 221 103765 doi: 10.1016/j.jprot.2020.103765
Martín-Cano FE, Gaitskell-Phillips G, Ortiz-Rodríguez JM, Silva-Rodríguez A, Román Á, Rojo-Domínguez P, Alonso-Rodríguez E, Tapia JA, Gil MC....Proteomic technologies allow the detection of thousands of proteins at the same time, being a powerful technique to reveal molecular regulatory mechanisms in spermatozoa and also sperm damage linked to low fertility or specific biotechnologies. Modifications induced by the cryopreservation in the stallion sperm proteome were studied using UHPLC/MS/MS. Ejaculates from fertile stallions were collected and split in two subsamples, one was investigated as fresh (control) samples, and the other aliquot frozen and thawed using standard procedures and investigated as frozen thawed subsamples. UHPLC/M...
Elkhawagah AR, Nervo T, Poletto M, Martino NA, Gallo D, Bertero A, Vincenti L.The aim of the study was to ascertain effects of different concentrations of relaxin added to extender medium during the pre-freezing incubation periods on quality variables of stallion frozen-thawed spermatozoa. Semen samples collected from three stallions were filtered, diluted with skim milk, and centrifuged at 600g for 10 min. Sperm pellets were suspended in BotuCrio freezing medium to a final concentration of 50 × 10 sperm/mL. The diluted semen was divided into five experimental groups supplemented with 0 (control), 12.5, 25, 50, or 100 ng/mL of relaxin. The semen samples were transferre...
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...
Al-Essawe EM, Johannisson A, Wulf M, Aurich C, Morrell JM.Addition of seminal plasma (SP) prior to cryopreservation may influence stallion sperm cryosurvival. The objective of this study was to investigate the addition of pooled SP from "good" or "bad" freezer stallions to spermatozoa selected by single layer centrifugation (SLC) prior to cryopreservation on post-thaw sperm quality. Semen from 12 stallions was collected; 5 mL was frozen as control (C) and the remainder was processed by SLC to remove SP and was divided into three aliquots: i) SLC sample without SP (SLC); ii) SLC plus pooled SP from "good freezer" stallions (SLC-GF); iii) SLC plus po...
Dias GM, López ML, Ferreira AT, Chapeaurouge DA, Rodrigues A, Perales J, Retamal CA.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...
Delgado-Bermúdez A, Noto F, Bonilla-Correal S, Garcia-Bonavila E, Catalán J, Papas M, Bonet S, Miró J, Yeste M.Aquaporins (AQPs), a family of ubiquitous water channels divided into orthodox AQPs, aquaglyceroporins (GLPs), and superAQPs, are present in stallion spermatozoa. The aim of this study was to elucidate the functional relevance of each group of AQPs during stallion sperm cryopreservation through the use of three different inhibitors: acetazolamide (AC), phloretin (PHL) and propanediol (PDO). Sperm quality and function parameters were evaluated in the presence or absence of each inhibitor in fresh and frozen-thawed samples. In the presence of AC, different parameters were altered ( < 0.05), b...
Ortiz-Rodriguez JM, Martín-Cano FE, Ortega-Ferrusola C, Masot J, Redondo E, Gázquez A, Gil MC, Aparicio IM, Rojo-Domínguez P, Tapia JA....Oxidative stress is considered a major mechanism causing sperm damage during cryopreservation and storage, and underlies male factor infertility. Currently, oxidative stress is no longer believed to be caused only by the overproduction of reactive oxygen species, but rather by the deregulation of redox signaling and control mechanisms. With this concept in mind, here, we describe for the first time the presence of the soluble carrier family 7 member 11 (SLC7A11) antiporter, which exchanges extracellular cystine (Cyss) for intracellular glutamate, in stallion spermatozoa, as well as its impact ...
Yánez-Ortiz I, Catalán J, Mateo-Otero Y, Dordas-Perpinyà M, Gacem S, Yeste N, Bassols A, Yeste M, Miró J.Jenny shows a large endometrial reaction after semen influx to the uterus with a large amount of polymorphonuclear neutrophils (PMN) migrating into the uterine lumen. PMN act as a sperm selection mechanism through phagocytosis and NETosis (DNA extrudes and, together with proteins, trap spermatozoa). While a reduced percentage of spermatozoa are phagocytosed by PMN, most are found to be attached to neutrophil extracellular traps (NETs). This selection process together with sperm metabolism produces a large amount of reactive oxygen species (ROS) that influence the reproductive success. The pres...
Gravance CG, Liu IK, Davis RO, Hughes JP, Casey PJ.The heads of stallion spermatozoa were analysed by computer automated sperm head morphometry and the morphometric values of the major subpopulations of sperm heads were assessed. The criteria for normal dimensions of stallion sperm heads are proposed based on the analysis of these measurements. Semen samples were collected from 10 fertile and 10 subfertile stallions, processed by a standard method, smeared onto microscope slides and stained using haematoxylin. At least 200 properly digitized sperm heads were analysed from each stallion. The measurements for length, width, area, perimeter and w...
Martins HS, da Silva GC, Cortes SF, Paes FO, Martins Filho OA, Araujo M, Stahlberg R, Lagares MA.During cryopreservation, sperm was submitted to an increase in reactive oxygen species generation. This work aimed to improve the quality of frozen equine sperm after the addition of antioxidants lactoferrin (Lf) and catalase (Cat) to a freezing extender. Semen from six stallions was frozen with the extenders: F1) control, INRA 82 freezing extender, F2) F1 + 500 μg/ml Lf and F3) F1 + 200 IU/ml Cat. After thawing, sperm motility parameters, membrane functionality and integrity, and acrosome integrity and spontaneous acrosome-reacted sperm were evaluated with a computer-assisted sperm anal...
Schober D, Aurich C, Nohl H, Gille L.Cryopreservation of spermatozoa is of essential importance for artificial insemination and breeding programs in horses. Besides other factors, spermatozoal motility depends on mitochondrial energy metabolism. Based on changes of single mitochondrial functions it has been suggested that mitochondrial damage during cryopreservation could be a major reason for diminished post thaw semen quality. However, it is still unclear to which extent this influences the whole bioenergetic performance of mitochondria and whether this plays a role during routine cryopreservation procedures. Therefore, it was ...
Macías-García B, González-Fernández L, Loux SC, Rocha AM, Guimarães T, Peña FJ, Varner DD, Hinrichs K.Repeatable methods for IVF have not been established in the horse, reflecting the failure of standard capacitating media to induce changes required for fertilization capacity in equine sperm. One important step in capacitation is membrane cholesterol efflux, which in other species is triggered by cholesterol oxidation and is typically enhanced using albumin as a sterol acceptor. We incubated equine sperm in the presence of calcium, BSA, and bicarbonate, alone or in combination. Bicarbonate induced an increase in reactive oxygen species (ROS) that was abolished by the addition of calcium or BSA...
Restrepo G, Varela E, Duque JE, Gómez JE, Rojas M.Maintaining the integrity of equine sperm subjected to preservation protocols is essential for the successful development of assisted reproduction procedures. The aim of this study was to assess the mitochondrial membrane potential, lipid peroxidation, and DNA integrity of equine sperm subjected to freezing, vitrification, and freeze-drying. Eight ejaculates obtained from four Colombian Creole horses were subjected to programmable freezing, vitrification, and freeze-drying. After thawing or rehydration, sperm motility and kinetics were assessed through a CASA system. The mitochondrial membrane...
Varner DD, Blanchard TL, Love CL, Garcia MC, Kenney RM.Two experiments were conducted to examine the effects of semen fractionation and dilution ratio on motility parameters of stallion spermatozoa. In Experiment 1, three ejaculates from each of three stallions were divided into sperm-rich (SR) and sperm-poor (SP) fractions to determine the difference in sperm concentration. Mean sperm concentration in SR fractions (349.5 x 10(6)/ml) was greater (P < 0.001) than that of SP fractions (96.9 x 10(6)/ml). In Experiment 2, three ejaculates from each of two stallions were divided into SR and SP fractions. Fifty percent of the original volume of SR fract...
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...
Whitesell K, Stefanovski D, McDonnell S, Turner R.The stallion Breeding Soundness Examination (BSE), as proposed by the Society for Theriogenology, recommends that a stallion produce a minimum of one billion progressively motile, morphologically normal sperm (PMMNS) in the second of two ejaculates collected 1 h apart to be classified as a Satisfactory Prospective Breeder. With this in mind, the first objective of this study was to determine if the classification outcome of the traditional BSE differs depending on the methods used to evaluate sperm motility, morphology and concentration. We hypothesized that application of Computer Assisted ...
Catalán J, Llavanera M, Bonilla-Correal S, Papas M, Gacem S, Rodríguez-Gil JE, Yeste M, Miró J.The aim of this study was to evaluate whether red-light stimulation increases the longevity and resilience of cryopreserved stallion sperm to withstand post-thaw incubation for 120 min. Sixteen frozen straws of 0.5 mL from eight stallions were used. Samples were cryopreserved, thawed through incubation at 38 °C for 30 s and divided into the control and samples exposed to red-light using a triple LED photo-activation system (wavelength: 620-630 nm). Three irradiation protocols consisting of different light-dark-light intervals (1-1-1, 2-2-2 and 3-3-3 min) were tested. Sperm quality param...
Gamboa S, Ramalho-Santos J.Proteins implicated in the "SNARE hypothesis" for membrane fusion have been characterized in the acrosome of several mammalian species, and a functional role for these proteins during the acrosome reaction has been proposed. We have investigated the presence of SNAREs in equine sperm, using semen samples obtained from stallions with varying fertility. Immunocytochemical analysis revealed that members of different SNARE families can be detected on the acrosome of equine sperm, notably in the acrosomal cap and equatorial segment. These proteins include the t-SNARE syntaxin, the v-SNARE synaptobr...
Katila T.The uterus of domestic animals, including the horse, has a dual role in the interaction of the uterus and sperm. On one hand, uterine contractions carry sperm toward the oviduct, and on the other hand the uterus eliminates excessive sperm. The selection of sperm for the small numbers of "good" cells that gain access to the oviduct and for the majority of sperm that will be destroyed takes place in the uterus. The sperm-uterine interaction works both ways; sperm and seminal plasma also have several effects on the uterus. Sperm and seminal plasma probably provoke uterine contractions. Sperm indu...
Ball BA, Vo A.Osmotic stress attributed to differences in the relative permeability of cryoprotectants, such as glycerol and water, appears to be an important factor in cryodamage. The objective of this study was to characterize the osmotic tolerance of equine spermatozoa, and to evaluate the effects of addition and removal of cryoprotectants from equine spermatozoa on their motility, and membrane and acrosomal integrity, as well as their mitochondrial membrane potential. Equine spermatozoa had a limited osmotic tolerance to anisosmotic conditions. Although the addition of increasing concentrations of glyce...
Murcia-Robayo RY, Jouanisson E, Beauchamp G, Diaw M.Evaluation of sperm morphology is part of the assessment of fertility in human and animal reproduction. Analyses can be performed using different techniques, including the use of staining methods In our prospective study, the morphology of equine sperm was evaluated using 3 staining methods Diff-Quik, eosin-nigrosin, and SpermBlue, the latter being a relatively new stain commonly used for human sperm. Our hypotheses were that (1) SpermBlue allows easier assessment of horse sperm morphology and facilitates better identification of sperm abnormalities, and (2) sperm morphology classification dif...
Hoogewijs M, Morrell J, Van Soom A, Govaere J, Johannisson A, Piepers S, De Schauwer C, De Kruif A, De Vliegher S.The increasing use of modern reproductive techniques in human medicine has led to a higher demand for isolation of motile sperm. Several of these isolation techniques have been adapted for veterinary use and can be applied for the selection of a superior sperm sample from stallion semen. Until recently a major disadvantage of such isolation techniques was the limitation in sperm volume that could be handled. Androcoll-E had been shown to be successful for processing large volumes of equine semen but there are few data to substantiate the potential beneficial effect of freezing an Androcoll-E s...
Ellington JE, Samper JC, Jones AE, Oliver SA, Burnett KM, Wright RW.Formation of a spermatozoa ('sperm') reservoir in the mare is thought to occur through lectin-mediated sperm attachment to the oviductal epithelium. Once attached, prefertilization sperm survival is supported by oviductal factors. Cryopreservation of stallion sperm decreases the number of sperm attaching to oviduct epithelial cells (OEC) and the length of time these sperm survive. Quantification of in vitro interactions between sperm and OEC in a co-culture system may provide an assay for functional integrity of cryopreserved or fresh sperm samples. Additionally, superior additives for in vitr...
Usuga A, Rojano BA, Restrepo G.Contribution of seminal plasma proteins to semen freezability has been reported in several species, suggesting these proteins as genetic markers. The aim of this study was to evaluate the relationship between cysteine-rich secretory protein-3 (CRISP-3) and some of its single-nucleotide polymorphisms (SNPs) with post-thawing semen quality in stallions. DNA was obtained from 100 stallions, regions of interest were amplified by polymerase chain reaction and sequenced. Evaluated SNPs within the equine CRISP-3 gene were CRISP3c.+199A>G (SNP1), CRISP3c.+566C>A (SNP2), CRISP3c.+622G>A (SNP3)...
Hinrichs K.Intracytoplasmic sperm injection (ICSI) has recently become efficient enough to be considered for clinical use. With ICSI, one spermatozoa is injected into a mature oocyte. Harvesting of an oocyte ex vivo, followed by ICSI and transfer of the fertilized oocyte to the oviduct, may be applicable when semen quality is insufficient for standard insemination. Sperm injection, followed by in vitro embryo culture to the blastocyst stage, may be used in cases where multiple oocytes are to be fertilized (e.g. when oocytes are collected post-mortem). Nuclear transfer (cloning) of horses is possible but ...
Morrell JM, Rodriguez-Martinez H, Johannisson A.An improvement in sperm quality after single layer centrifugation (SLC) has been seen in previous studies using small sample sizes (for example, n = 10 stallions). There is a need to investigate whether this improvement is repeatable over several breeding seasons with a larger number of stallions (n ≥ 30 stallions). Objective: To make a retrospective analysis of the results of SLC performed on more than 250 sperm samples (176 ejaculates) from 31 stallions in 3 consecutive breeding seasons. Methods: Sperm quality (motility, proportion of morphologically normal spermatozoa and the proportion o...
Contreras MJ, Arias ME, Fuentes F, Muñoz E, Bernecic N, Fair S, Felmer R.Cryopreservation of stallion semen does not achieve the post-thaw quality or fertility results observed in other species like cattle. There are many reasons for this, but the membrane composition and intracellular changes in stallion sperm predispose them to low resistance to the cooling, freezing, and subsequent thawing process. Damage to the sperm results from different processes activated during cryopreservation, including oxidative stress, apoptosis, and structural modifications in the sperm membrane that increase the deleterious effect on sperm. In addition, significant individual variabi...
Love CC.The sperm chromatin structure assay (SCSA) was introduced by as a method to determine the susceptibility of sperm DNA to denaturation and how those results related to fertility. This initial study used human sperm and was followed by studies in bulls and boars . This assay was one of the first to introduce the technique of flow cytometry, which has the ability to evaluate specific sperm compartments of large numbers of sperm in a short time, as a methodology to evaluate sperm quality and further define the relationship of sperm quality to fertility. For any assay to be of use clinically, it mu...
Morrell JM, Richter J, Martinsson G, Stuhtmann G, Hoogewijs M, Roels K, Dalin AM.A successful outcome after artificial insemination with cooled semen is dependent on many factors, the sperm quality of the ejaculate being one. Previous studies have shown that spermatozoa with good motility, normal morphology, and good chromatin integrity can be selected by means of colloid centrifugation, particularly single layer centrifugation (SLC) using species-specific colloids. The purpose of the present study was to conduct an insemination trial with spermatozoa from "normal" ejaculates, i.e., from stallions with no known fertility problem, to determine whether the improvements in sp...
