{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T03:52:20Z","timestamp":1790653940166,"version":"4.1.0"},"reference-count":75,"publisher":"MDPI AG","issue":"12","license":[{"start":{"date-parts":[[2021,12,2]],"date-time":"2021-12-02T00:00:00Z","timestamp":1638403200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Veterinary Sciences"],"abstract":"<jats:p>In donkeys, the use of frozen-thawed sperm for artificial insemination (AI) leads to low fertility rates. Furthermore, donkey sperm produce a large amount of reactive oxygen species (ROS), and post-AI inflammation induces the formation of neutrophil extracellular traps (NETosis), which further generates many more ROS. These high ROS levels may induce lipid peroxidation in the sperm plasma membrane, thus affecting its integrity. Enzymatic and non-enzymatic antioxidants, mainly found in the seminal plasma (SP), are responsible for maintaining the redox balance. However, this fluid is removed prior to cryopreservation, thereby exposing sperm cells to further oxidative stress. The exogenous addition of antioxidants to the freezing medium can reduce the detrimental effects caused by ROS generation. Therefore, the aim of this study was to evaluate how the addition of different reduced glutathione (GSH) concentrations (control, 2 mM, 4 mM, 6 mM, 8 mM, and 10 mM) to fresh sperm affect their cryotolerance. Total and progressive motility, kinematic parameters and motile sperm subpopulations were significantly (p &lt; 0.05) different from the control in treatments containing 8 mM and 10 mM GSH, but not at lower concentrations. Plasma and acrosome membrane integrity, mitochondrial membrane potential (MMP) and intracellular superoxide levels (O2\u2212) were not affected (p &gt; 0.05) by any GSH concentration. Interestingly, however, the addition of 8 mM or 10 mM GSH reduced (p &lt; 0.05) the percentages of viable sperm with high overall ROS levels compared to the control. In conclusion, frozen-thawed donkey sperm are able to tolerate high GSH concentrations, which differs from what has been observed in other species. This antioxidant capacity suggests that ROS could be important during post-AI and that the impact of using exogenous antioxidants like GSH to improve the sperm resilience to freeze-thawing is limited in this species.<\/jats:p>","DOI":"10.3390\/vetsci8120302","type":"journal-article","created":{"date-parts":[[2021,12,2]],"date-time":"2021-12-02T02:56:14Z","timestamp":1638413774000},"page":"302","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":8,"title":["Addition of Reduced Glutathione (GSH) to Freezing Medium Reduces Intracellular ROS Levels in Donkey Sperm"],"prefix":"10.3390","volume":"8","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3613-2481","authenticated-orcid":false,"given":"Iv\u00e1n","family":"Y\u00e1nez-Ortiz","sequence":"first","affiliation":[{"name":"Equine Reproduction Service, Department of Animal Medicine and Surgery, Faculty of Veterinary Sciences, Autonomous University of Barcelona, ES-08193 Barcelona, Cerdanyola del Vall\u00e8s, Spain"},{"name":"Biotechnology of Animal and Human Reproduction (TechnoSperm), Institute of Food and Agricultural Technology, University of Girona, ES-17003 Girona, Spain"},{"name":"Unit of Cell Biology, Department of Biology, Faculty of Sciences, University of Girona, ES-17003 Girona, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0878-7819","authenticated-orcid":false,"given":"Jaime","family":"Catal\u00e1n","sequence":"additional","affiliation":[{"name":"Equine Reproduction Service, Department of Animal Medicine and Surgery, Faculty of Veterinary Sciences, Autonomous University of Barcelona, ES-08193 Barcelona, Cerdanyola del Vall\u00e8s, Spain"},{"name":"Biotechnology of Animal and Human Reproduction (TechnoSperm), Institute of Food and Agricultural Technology, University of Girona, ES-17003 Girona, Spain"},{"name":"Unit of Cell Biology, Department of Biology, Faculty of Sciences, University of Girona, ES-17003 Girona, Spain"},{"name":"Faculty of Veterinary Medicine, University of Teramo, Loc. Piano d\u2019Accio, IT-64100 Teramo, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5655-5003","authenticated-orcid":false,"given":"Ariadna","family":"Delgado-Berm\u00fadez","sequence":"additional","affiliation":[{"name":"Biotechnology of Animal and Human Reproduction (TechnoSperm), Institute of Food and Agricultural Technology, University of Girona, ES-17003 Girona, Spain"},{"name":"Unit of Cell Biology, Department of Biology, Faculty of Sciences, University of Girona, ES-17003 Girona, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2283-6146","authenticated-orcid":false,"given":"Augusto","family":"Carluccio","sequence":"additional","affiliation":[{"name":"Faculty of Veterinary Medicine, University of Teramo, Loc. Piano d\u2019Accio, IT-64100 Teramo, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1085-0039","authenticated-orcid":false,"given":"Jordi","family":"Mir\u00f3","sequence":"additional","affiliation":[{"name":"Equine Reproduction Service, Department of Animal Medicine and Surgery, Faculty of Veterinary Sciences, Autonomous University of Barcelona, ES-08193 Barcelona, Cerdanyola del Vall\u00e8s, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2209-340X","authenticated-orcid":false,"given":"Marc","family":"Yeste","sequence":"additional","affiliation":[{"name":"Biotechnology of Animal and Human Reproduction (TechnoSperm), Institute of Food and Agricultural Technology, University of Girona, ES-17003 Girona, Spain"},{"name":"Unit of Cell Biology, Department of Biology, Faculty of Sciences, University of Girona, ES-17003 Girona, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2021,12,2]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"103","DOI":"10.1016\/0011-2240(64)90002-1","article-title":"Low temperature research on spermatozoa and eggs","volume":"1","author":"Sherman","year":"1964","journal-title":"Cryobiology"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"663","DOI":"10.1016\/j.cveq.2006.07.002","article-title":"Advanced methods for handling and preparation of stallion semen","volume":"22","author":"Loomis","year":"2006","journal-title":"Vet. Clin. N. Am. Equine Pract."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"81","DOI":"10.1007\/s10344-007-0089-z","article-title":"Semen cryopreservation and the conservation of endangered species","volume":"53","author":"Fickel","year":"2007","journal-title":"Eur. J. Wildl. Res."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"44","DOI":"10.1016\/j.jevs.2017.11.008","article-title":"The current situation and trend of donkey industry in Europe","volume":"65","author":"Camillo","year":"2018","journal-title":"J. Equine Vet. Sci."},{"key":"ref_5","first-page":"1","article-title":"Genetic diversity in Spanish donkey breeds using microsatellite DNA markers","volume":"33","author":"Jordana","year":"2001","journal-title":"Genet. Sel. Evol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"793","DOI":"10.1016\/S0093-691X(98)00184-8","article-title":"A procedure for Poitou jackass sperm cryopreservation","volume":"50","author":"Trimeche","year":"1998","journal-title":"Theriogenology"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"22","DOI":"10.1016\/j.anireprosci.2008.03.016","article-title":"Differences in ability of jennies and mares to conceive with cooled and frozen semen containing glycerol or not","volume":"112","author":"Vidament","year":"2009","journal-title":"Anim. Reprod. Sci."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"503","DOI":"10.1016\/j.jevs.2012.06.075","article-title":"New procedures to freeze donkey semen and its influence on mares and jennies fertility","volume":"32","author":"Oliveira","year":"2012","journal-title":"J. Equine Vet. Sci."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1846","DOI":"10.1016\/j.theriogenology.2012.07.015","article-title":"Donkey jack (Equus asinus) semen cryopreservation: Studies of seminal parameters, post breeding inflammatory response, and fertility in donkey jennies","volume":"78","author":"Rota","year":"2012","journal-title":"Theriogenology"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"205","DOI":"10.1016\/j.cryobiol.2014.01.013","article-title":"Improved sperm cryosurvival in diluents containing amides versus glycerol in the Przewalski\u2019s horse (Equus ferus przewalskii)","volume":"68","author":"Pukazhenthi","year":"2014","journal-title":"Cryobiology"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"294","DOI":"10.1016\/j.theriogenology.2015.03.021","article-title":"Effect of single-layer centrifugation or washing on frozen\u2013thawed donkey semen quality: Do they have the same effect regardless of the quality of the sample?","volume":"84","author":"Ortiz","year":"2015","journal-title":"Theriogenology"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1990","DOI":"10.1071\/RD14449","article-title":"Freezability of Andalusian donkey (Equus asinus) spermatozoa: Effect of extenders and permeating cryoprotectants","volume":"28","author":"Acha","year":"2016","journal-title":"Reprod. Fertil. Dev."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1267","DOI":"10.1016\/j.theriogenology.2015.12.010","article-title":"Strategies to improve the fertility of fresh and frozen donkey semen","volume":"85","author":"Oliveira","year":"2016","journal-title":"Theriogenology"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"128","DOI":"10.1016\/j.anireprosci.2017.12.021","article-title":"Cryoprotective effect of glutamine, taurine, and proline on post-thaw semen quality and DNA integrity of donkey spermatozoa","volume":"189","author":"Bottrel","year":"2018","journal-title":"Anim. Reprod. Sci."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"19","DOI":"10.1016\/j.jevs.2018.03.002","article-title":"The effects of egg yolk concentration and particle size on donkey semen preservation","volume":"65","author":"Zhang","year":"2018","journal-title":"J. Equine Vet. Sci."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"102796","DOI":"10.1016\/j.jevs.2019.102796","article-title":"Ameliorative effect of ascorbic acid and glutathione in combating the cryoinjuries during cryopreservation of exotic jack semen","volume":"81","author":"Kumar","year":"2019","journal-title":"J. Equine Vet. Sci."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1777","DOI":"10.17582\/journal.pjz\/2019.51.5.1777.1781","article-title":"Effects of Vitamin C and Vitamin E on cryopreservation of Guanzhong donkey semen","volume":"51","author":"Yu","year":"2019","journal-title":"Pak. J. Zool."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"103393","DOI":"10.1016\/j.jevs.2021.103393","article-title":"L-proline: An effective agent for frozen and post-thawed donkey semen storage","volume":"101","author":"Li","year":"2021","journal-title":"J. Equine Vet. Sci."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"179","DOI":"10.1111\/j.2042-3306.2010.00130.x","article-title":"Seminal parameters and field fertility of cryopreserved donkey jack semen after insemination of horse mares","volume":"43","author":"Canisso","year":"2011","journal-title":"Equine Vet. J."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1019","DOI":"10.1016\/j.theriogenology.2013.01.006","article-title":"Effect of ketoprofen treatment on the uterine inflammatory response after AI of jennies with frozen semen","volume":"79","author":"Rabanal","year":"2013","journal-title":"Theriogenology"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"38","DOI":"10.1016\/j.jevs.2017.11.007","article-title":"Post\u2013Artificial Insemination endometrial inflammation and its control in donkeys","volume":"65","author":"Papas","year":"2018","journal-title":"J. Equine Vet. Sci."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"47","DOI":"10.1016\/j.theriogenology.2015.09.047","article-title":"Sperm cryopreservation update: Cryodamage, markers, and factors affecting the sperm freezability in pigs","volume":"85","author":"Yeste","year":"2016","journal-title":"Theriogenology"},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Aitken, R.J., De Iuliis, G.N., and Drevet, J.R. (2018). Role of oxidative stress in the etiology of male infertility and the potential therapeutic value of antioxidants. Oxidants, Antioxidants, and Impact of the Oxidative Status in Male Reproduction, Elsevier.","DOI":"10.1016\/B978-0-12-812501-4.00010-9"},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Pe\u00f1a, F.J., O\u2019Flaherty, C., Ortiz Rodr\u00edguez, J.M., Mart\u00edn Cano, F.E., Gaitskell-Phillips, G.L., Gil, M.C., and Ortega Ferrusola, C. (2019). Redox regulation and oxidative stress: The particular case of the stallion spermatozoa. Antioxidants, 8.","DOI":"10.3390\/antiox8110567"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"235","DOI":"10.1055\/s-2007-1016283","article-title":"Oxidation and the spermatozoa","volume":"16","author":"Kim","year":"1998","journal-title":"Semin. Reprod. Endocrinol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"180","DOI":"10.1016\/j.theriogenology.2019.08.032","article-title":"Activities of antioxidant seminal plasma enzymes (SOD, CAT, GPX and GSR) are higher in jackasses than in stallions and are correlated with sperm motility in jackasses","volume":"140","author":"Papas","year":"2019","journal-title":"Theriogenology"},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Papas, M., Catal\u00e1n, J., Recuero, S., Morrell, J.M., Yeste, M., and Mir\u00f3, J. (2020). Single layer centrifugation improves the quality of fresh donkey semen and modifies the sperm ability to interact with polymorphonuclear neutrophils. Animals, 10.","DOI":"10.3390\/ani10112128"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"87","DOI":"10.1080\/2090598X.2019.1599624","article-title":"Oxidative stress and sperm function: A systematic review on evaluation and management","volume":"17","author":"Dutta","year":"2019","journal-title":"Arab J. Urol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"929","DOI":"10.1016\/j.theriogenology.2013.01.008","article-title":"Good and bad freezability boar ejaculates differ in the integrity of nucleoprotein structure after freeze-thawing but not in ROS levels","volume":"79","author":"Yeste","year":"2013","journal-title":"Theriogenology"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"629","DOI":"10.1016\/0093-691X(94)90173-G","article-title":"Sperm-induced leukocytosis in the equine uterus","volume":"41","author":"Kotilainen","year":"1994","journal-title":"Theriogenology"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"273","DOI":"10.1016\/S0378-4320(01)00164-6","article-title":"Interaction between equine semen and the endometrium: The inflammatory response to semen","volume":"68","author":"Troedsson","year":"2001","journal-title":"Anim. Reprod. Sci."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"57","DOI":"10.1016\/j.jri.2009.10.007","article-title":"Spermadhesin PSP-I\/PSP-II heterodimer induces migration of polymorphonuclear neutrophils into the uterine cavity of the sow","volume":"84","author":"Saravia","year":"2010","journal-title":"J. Reprod. Immunol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1053","DOI":"10.1016\/j.fertnstert.2016.06.005","article-title":"Leukocytes coincubated with human sperm trigger classic neutrophil extracellular traps formation, reducing sperm motility","volume":"106","author":"Zambrano","year":"2016","journal-title":"Fertil. Steril."},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Mir\u00f3, J., Mar\u00edn, H., Catal\u00e1n, J., Papas, M., Gacem, S., and Yeste, M. (2020). Seminal plasma, sperm concentration, and sperm-PMN interaction in the donkey: An in vitro model to study endometrial inflammation at post-insemination. Int. J. Mol. Sci., 21.","DOI":"10.3390\/ijms21103478"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"1025","DOI":"10.1016\/S0093-691X(01)00710-5","article-title":"Generation of reactive oxygen species by equine neutrophils and their effect on motility of equine spermatozoa","volume":"57","author":"Baumber","year":"2002","journal-title":"Theriogenology"},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Y\u00e1nez-Ortiz, I., Catal\u00e1n, J., Mateo-Otero, Y., Dordas-Perpiny\u00e0, M., Gacem, S., Yeste, N., Bassols, A., Yeste, M., and Mir\u00f3, J. (2021). Extracellular reactive oxygen species (ROS) production in fresh donkey sperm exposed to reductive stress, oxidative stress and NETosis. Antioxidants, 10.","DOI":"10.3390\/antiox10091367"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"40","DOI":"10.1016\/j.cryobiol.2010.12.001","article-title":"Reduced glutathione content in human sperm is decreased after cryopreservation: Effect of the addition of reduced glutathione to the freezing and thawing extenders","volume":"62","author":"Gadea","year":"2011","journal-title":"Cryobiology"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"44","DOI":"10.1016\/j.smallrumres.2011.05.010","article-title":"Effects of reduced glutathione and catalase on the kinematics and membrane functionality of sperm during liquid storage of ram semen","volume":"100","author":"Pinto","year":"2011","journal-title":"Small Rumin. Res."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"271","DOI":"10.1016\/j.repbio.2012.10.001","article-title":"Glutathione-supplemented tris-citric acid extender improves the post-thaw quality and in vivo fertility of buffalo (Bubalus bubalis) bull spermatozoa","volume":"12","author":"Ansari","year":"2012","journal-title":"Reprod. Biol."},{"key":"ref_40","first-page":"104","article-title":"Glutathione for the freezing of cooled equine semen, using different protocols","volume":"11","author":"Oliveira","year":"2018","journal-title":"Anim. Reprod."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"81","DOI":"10.4995\/wrs.2021.14759","article-title":"Glutathione supplementation in semen extender improves rabbit sperm attributes during refrigeration","volume":"29","author":"Ahmad","year":"2021","journal-title":"World Rabbit Sci."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"396","DOI":"10.2164\/jandrol.04155","article-title":"Cooling and freezing of boar spermatozoa: Supplementation of the freezing media with reduced glutathione preserves sperm function","volume":"26","author":"Gadea","year":"2005","journal-title":"J. Androl."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"874","DOI":"10.1111\/j.1439-0531.2011.01758.x","article-title":"In vitro and in vivo evaluation of ram sperm frozen in tris egg-yolk and supplemented with superoxide dismutase and reduced glutathione","volume":"46","author":"Silva","year":"2011","journal-title":"Reprod. Domest. Anim."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"1148","DOI":"10.1016\/j.jevs.2013.05.001","article-title":"Addition of glutathione to an extender for frozen equine semen","volume":"33","author":"Oliveira","year":"2013","journal-title":"J. Equine Vet. Sci."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"116","DOI":"10.1262\/jrd.2014-130","article-title":"Glutathione supplementation to semen extender improves the quality of frozen-thawed canine spermatozoa for transcervical insemination","volume":"61","author":"Ogata","year":"2015","journal-title":"J. Reprod. Dev."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"171","DOI":"10.1016\/j.anireprosci.2018.03.005","article-title":"Effect of reduced glutathione supplementation on cryopreservation induced sperm cryoinjuries in Murrah bull semen","volume":"192","author":"Gangwar","year":"2018","journal-title":"Anim. Reprod. Sci."},{"key":"ref_47","first-page":"5","article-title":"The role of glutathione in mammalian gametes","volume":"5","author":"Luberda","year":"2005","journal-title":"Reprod. Biol."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"6","DOI":"10.1530\/ror.0.0010006","article-title":"Glutathione as a treatment for male infertility","volume":"1","author":"Irvine","year":"1996","journal-title":"Rev. Reprod."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"1036","DOI":"10.1071\/RD12230","article-title":"Reduced glutathione and procaine hydrochloride protect the nucleoprotein structure of boar spermatozoa during freeze-thawing by stabilising disulfide bonds","volume":"25","author":"Yeste","year":"2013","journal-title":"Reprod. Fertil. Dev."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"1706","DOI":"10.1016\/j.theriogenology.2004.07.022","article-title":"Sperm motility patterns and metabolism in Catalonian donkey semen","volume":"63","author":"Lobo","year":"2005","journal-title":"Theriogenology"},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"607","DOI":"10.1016\/j.cveq.2019.08.014","article-title":"Key aspects of donkey and mule reproduction","volume":"35","author":"Canisso","year":"2019","journal-title":"Vet. Clin. N. Am. Equine Pract."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"181","DOI":"10.1111\/rda.13604","article-title":"Examination of jackass (Equus asinus) accessory sex glands by B-mode ultrasound and of testicular artery blood flow by colour pulsed-wave Doppler ultrasound: Correlations with semen production","volume":"55","author":"Gacem","year":"2020","journal-title":"Reprod. Domest. Anim."},{"key":"ref_53","unstructured":"Kenney, M.R. (1975). Minimal contamination techniques for breeding mares: Techniques and priliminary findings. Proc. Am. Assoc. Equine Pract., 327\u2013336."},{"key":"ref_54","doi-asserted-by":"crossref","unstructured":"Gacem, S., Catal\u00e1n, J., Valverde, A., Soler, C., and Mir\u00f3, J. (2020). Optimization of CASA-mot analysis of donkey sperm: Optimum frame rate and values of kinematic variables for different counting chamber and fields. Animals, 10.","DOI":"10.3390\/ani10111993"},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"1245","DOI":"10.1016\/0093-691X(88)90004-0","article-title":"Evaluation of acrosomal integrity of boar spermatozoa by bright field microscopy using an eosin-nigrosin stain","volume":"29","author":"Bamba","year":"1988","journal-title":"Theriogenology"},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"926","DOI":"10.1002\/cyto.a.20623","article-title":"MIFlowCyt: The minimum information about a flow cytometry experiment","volume":"73","author":"Lee","year":"2008","journal-title":"Cytometry"},{"key":"ref_57","doi-asserted-by":"crossref","first-page":"276","DOI":"10.1095\/biolreprod53.2.276","article-title":"Viability assessment of mammalian sperm using SYBR-14 and propidium iodide","volume":"53","author":"Garner","year":"1995","journal-title":"Biol. Reprod."},{"key":"ref_58","doi-asserted-by":"crossref","first-page":"462","DOI":"10.1095\/biolreprod65.2.462","article-title":"Evaluation of in vitro capacitation of stallion spermatozoa","volume":"65","author":"Rathi","year":"2001","journal-title":"Biol. Reprod."},{"key":"ref_59","doi-asserted-by":"crossref","first-page":"213","DOI":"10.2164\/jandrol.107.003640","article-title":"Detection of \u201capoptosis-like\u201d changes during the cryopreservation process in equine sperm","volume":"29","author":"Muriel","year":"2008","journal-title":"J. Androl."},{"key":"ref_60","doi-asserted-by":"crossref","first-page":"62","DOI":"10.1016\/j.anireprosci.2013.05.006","article-title":"Reactive oxygen species in stallion semen can be affected by season and colloid centrifugation","volume":"140","author":"Morrell","year":"2013","journal-title":"Anim. Reprod. Sci."},{"key":"ref_61","doi-asserted-by":"crossref","first-page":"2089","DOI":"10.2527\/jas.2005-766","article-title":"Determination of intracellular reactive oxygen species and high mitochondrial membrane potential in Percoll-treated viable boar sperm using fluorescence-activated flow cytometry","volume":"84","author":"Guthrie","year":"2006","journal-title":"J. Anim. Sci."},{"key":"ref_62","doi-asserted-by":"crossref","first-page":"97","DOI":"10.1016\/j.theriogenology.2011.01.022","article-title":"Sperm morphometric subpopulations are differentially distributed in rams with different maturity age in cryopreserved ejaculates","volume":"76","author":"Aparicio","year":"2011","journal-title":"Theriogenology"},{"key":"ref_63","unstructured":"Samper, J.C. (2009). Sperm physiology. Equine Breeding Management and Artificial Insemination, Saunders Elsevier."},{"key":"ref_64","doi-asserted-by":"crossref","first-page":"11","DOI":"10.1111\/j.1600-0897.2011.01033.x","article-title":"Seminal plasma proteins: What role do they play?","volume":"66","author":"Kvist","year":"2011","journal-title":"Am. J. Reprod. Immunol."},{"key":"ref_65","doi-asserted-by":"crossref","first-page":"690","DOI":"10.1016\/j.theriogenology.2003.11.013","article-title":"Decrease in glutathione content in boar sperm after cryopreservation: Effect of the addition of reduced glutathione to the freezing and thawing extenders","volume":"62","author":"Gadea","year":"2004","journal-title":"Theriogenology"},{"key":"ref_66","doi-asserted-by":"crossref","first-page":"1090","DOI":"10.1093\/biolre\/ioy241","article-title":"Depletion of thiols leads to redox deregulation, production of 4-hydroxinonenal and sperm senescence: A possible role for GSH regulation in spermatozoa","volume":"100","author":"Tapia","year":"2019","journal-title":"Biol. Reprod."},{"key":"ref_67","doi-asserted-by":"crossref","first-page":"588621","DOI":"10.3389\/fcell.2020.588621","article-title":"Red LED light acts on the mitochondrial electron chain of donkey sperm and its effects depend on the time of exposure to light","volume":"8","author":"Papas","year":"2020","journal-title":"Front. Cell Dev. Biol."},{"key":"ref_68","doi-asserted-by":"crossref","first-page":"56","DOI":"10.1016\/j.cryobiol.2017.07.002","article-title":"The addition of reduced glutathione to cryopreservation media induces changes in the structure of motile subpopulations of frozen-thawed boar sperm","volume":"78","author":"Estrada","year":"2017","journal-title":"Cryobiology"},{"key":"ref_69","doi-asserted-by":"crossref","first-page":"88","DOI":"10.1111\/j.2047-2927.2013.00144.x","article-title":"Supplementing cryopreservation media with reduced glutathione increases fertility and prolificacy of sows inseminated with frozen-thawed boar semen","volume":"2","author":"Estrada","year":"2014","journal-title":"Andrology"},{"key":"ref_70","doi-asserted-by":"crossref","first-page":"508","DOI":"10.2460\/ajvr.2001.62.508","article-title":"Generation of reactive oxygen species by equine spermatozoa","volume":"62","author":"Ball","year":"2001","journal-title":"Am. J. Vet. Res."},{"key":"ref_71","doi-asserted-by":"crossref","first-page":"275","DOI":"10.1016\/S0093-691X(01)00562-3","article-title":"Thiols prevent H2O2-mediated loss of sperm motility in cryopreserved bull semen","volume":"56","author":"Bilodeau","year":"2001","journal-title":"Theriogenology"},{"key":"ref_72","doi-asserted-by":"crossref","first-page":"97","DOI":"10.1146\/annurev.bi.64.070195.000525","article-title":"Superoxide radical and superoxide dismutases","volume":"64","author":"Fridovich","year":"1995","journal-title":"Annu. Rev. Biochem."},{"key":"ref_73","first-page":"287","article-title":"First line defence antioxidants-superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX): Their fundamental role in the entire antioxidant defence grid","volume":"54","author":"Ighodaro","year":"2018","journal-title":"Alex. J. Med."},{"key":"ref_74","doi-asserted-by":"crossref","first-page":"675","DOI":"10.1071\/RD9930675","article-title":"Maintenance of bioenergetic balance in sperm and prevention of lipid peroxidation: A review of the effect on design of storage preservation systems","volume":"5","author":"Hammerstedt","year":"1993","journal-title":"Reprod. Fertil. Dev."},{"key":"ref_75","doi-asserted-by":"crossref","first-page":"68","DOI":"10.1111\/j.1439-0531.2011.01862.x","article-title":"Mechanisms of sperm storage in the female reproductive tract: An interspecies comparison","volume":"46","author":"Holt","year":"2011","journal-title":"Reprod. Domest. 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