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.
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...
Cook NL, Masterson KR, Battaglia D, Beck R, Metcalf ES.Numerous variables affect invitro blastocyst development following intracytoplasmic sperm injection (ICSI). The paternal factor is affected by initial semen quality, processing techniques and final selection of individual spermatozoon for injection. This study investigated whether there was an effect of sperm cryoprotectant agent (CPA) on equine invitro blastocyst production, and reviews recent developments examining how processing equine semen affects ICSI outcomes. Single ejaculates from five stallions were collected and processed in a freezing extender containing either 1M dimethyl sulfoxid...
Squires E.Numerous reproductive technologies have been developed in the past several decades, which have dramatically changed the way mares are bred. This review will focus on embryo recovery and transfer, cooled-shipped embryos, embryo freezing, oocyte freezing, oocyte collection and transfer, intracytoplasmic sperm injection (ICSI), and sexed semen. Embryo transfer procedures have been constant for many years and the costs have not changed. The major change has been the ability to store embryos at 5 C for 12-24 hours and transport them to recipient stations. Embryo freezing has become more common us...
Hemeida NA, Sack WO, McEntee K.Dutculi efferentes were studied by microdissection and histologic methods in 2 boar, 6 goat, 5 ram, 25 bull, and 19 stallion epididymides. On the basis of the present findings and the data of others and relating them to the total number of specimens examined in each species, an estimate (narrow range) was made of the minimal-maximal numbers of efferent ductules as follows: dog 13 to 15; cat 14 to 17; boar 14 to 16; goat 18 to 19; ram 17 to 20; bull 13 to 16; and stallion 14 to 17. The histologic appearance of the efferent ductules of the bull, including measurements of their diameter, lumen, a...
Berlinguer F, Pasciu V, Succu S, Cossu I, Caggiu S, Addis D, Castagna A, Fontani V, Rinaldi S, Passino ES.REAC technology (acronym for Radio Electric Asymmetric Conveyor) is a technology platform for neuro and bio modulation. It has already proven to optimize the ions fluxes at the molecular level and the molecular mechanisms driving cellular asymmetry and polarization. Methods: This study was designed to verify whether this technology could extend spermatozoa life-span during liquid storage, while preserving their functions, DNA integrity and oxidative status. At 0, 24, 48, and 72 h. of storage at 4 °C, a battery of analyses was performed to assess spermatozoa viability, motility parameters, a...
Catalán J, Yánez-Ortiz I, Tvarijonaviciute A, González-Aróstegui LG, Rubio CP, Barranco I, Yeste M, Miró J.The objective of this study was to determine the relationship of enzymatic (superoxide dismutase, SOD; glutathione peroxidase, GPX; catalase, CAT; and paraoxonase type 1, PON1) and non-enzymatic antioxidants (measured in terms of: Trolox equivalent antioxidant capacity, TEAC; cupric-reducing antioxidant capacity, CUPRAC; and ferric-reducing ability of plasma, FRAP), as well as the oxidative stress index (OSI) in seminal plasma (SP) with the resilience of horse sperm to freeze-thawing. Twenty-one ejaculates (one per individual) were collected and split into two aliquots: the first was used to h...
Wach-Gygax L, Burger D, Malama E, Bollwein H, Fleisch A, Jeannerat E, Thomas S, Schuler G, Janett F.In this study annual fluctuations of DNA fragmentation and quality of cold-stored equine sperm were evaluated. Ejaculates were collected weekly during one year from 15 stallions. Ejaculate volume, sperm concentration and total sperm count were determined and semen was then extended and cold-stored for 48 h. Sperm motility was evaluated by CASA before and after 24 as well as 48 h of cold storage. In addition, the percentages of sperm with intact plasma membrane and acrosome (PMAI %) and with low intracellular Ca2+ level were determined in cold-stored semen (24 h, 48 h). SCSA™ was performe...
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...
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...
