{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T19:00:58Z","timestamp":1785438058274,"version":"3.56.0"},"reference-count":44,"publisher":"MDPI AG","issue":"14","license":[{"start":{"date-parts":[[2026,7,14]],"date-time":"2026-07-14T00:00:00Z","timestamp":1783987200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Ministry of Science, Innovation and Universities, Spain [MICIU]","award":["JDC2022-049684-I"],"award-info":[{"award-number":["JDC2022-049684-I"]}]},{"award":["JDC2022-049684-I"],"award-info":[{"award-number":["JDC2022-049684-I"]}],"id":[{"id":"https:\/\/ror.org\/05r0vyz12","id-type":"ROR","asserted-by":"publisher"}]},{"name":"Catalan Agency for the Management of University and Research Grants","award":["2021-SGR-00900"],"award-info":[{"award-number":["2021-SGR-00900"]}]},{"award":["2021-SGR-00900"],"award-info":[{"award-number":["2021-SGR-00900"]}],"id":[{"id":"https:\/\/ror.org\/01n4pqe45","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Animals"],"abstract":"<jats:p>A deeper understanding of the donkey\u2019s reproductive strategy is essential to improve reproductive efficiency in this species. In the jenny\u2019s uterus, sperm selection relies on the post-insemination endometrial reaction, characterized by the influx of polymorphonuclear neutrophils (PMNs), which form extracellular traps (NETs) that capture spermatozoa. Of these, some are liberated through the action of a deoxyribonuclease (DNase) present in the seminal plasma. This study aimed to develop an in vitro model to evaluate the effects of NETs and DNase action on donkey sperm viability and motility. For this purpose, donkey ejaculates were collected, PMNs were isolated from the jenny\u2019s blood and free spermatozoa were selected using a Silica gel filtration (EquiPure\u00ae, EP, Nidacon International AB, M\u00f6lndal, Sweden) to form five experimental groups: control (semen), SPZ-PMN, SPZ-PMN-DNase, SPZ-PMN-EP, SPZ-PMN-DNase-EP. Sperm viability and motility were assessed in all experimental groups using eosin-nigrosin staining and CASA sperm analysis system, respectively, at 1, 2 and 3 h of incubation. Sperm viability remained stable in the groups containing PMN, while it significantly (p &lt; 0.05) decreased in the control group, suggesting a protective effect of PMN on sperm viability. Regarding kinetic parameters, spermatozoa initially captured by NETs and later liberated by DNase exhibited higher velocity (VCL, VSL, VAP), linearity (LIN), straightness (STR) and oscillation (WOB). These changes in sperm motility may be related to the physiological changes occurring before sperm capacitation. This study can improve the understanding of the unique reproductive strategy of donkeys and could be useful to improve in vitro sperm capacitation and embryo production.<\/jats:p>","DOI":"10.3390\/ani16142185","type":"journal-article","created":{"date-parts":[[2026,7,14]],"date-time":"2026-07-14T08:04:31Z","timestamp":1784016271000},"page":"2185","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["DNase Promotes the Release of Sperm Trapped by NETs and Induces Important Changes in Donkey Sperm Motility Patterns"],"prefix":"10.3390","volume":"16","author":[{"given":"Sandra","family":"Llorens","sequence":"first","affiliation":[{"name":"Equine Reproduction Service, Department of Animal Medicine and Surgery, Faculty of Veterinary Sciences, Autonomous University of Barcelona, ES-08193 Cerdanyola del Vall\u00e8s, Spain"},{"name":"Department of Veterinary Medicine, University of Teramo, Localit\u00e0 Piano d\u2019Accio, IT-64100 Teramo, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-8589-6523","authenticated-orcid":false,"given":"Sebasti\u00e0","family":"Company","sequence":"additional","affiliation":[{"name":"Equine Reproduction Service, Department of Animal Medicine and Surgery, Faculty of Veterinary Sciences, Autonomous University of Barcelona, ES-08193 Cerdanyola del Vall\u00e8s, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Luisina","family":"Chapero","sequence":"additional","affiliation":[{"name":"Consejo Nacional de Investigaciones Cient\u00edficas y T\u00e9cnicas (CONICET), Centro Cient\u00edfico Tecnol\u00f3gico (CCT) Patagonia Confluencia, Neuqu\u00e9n 8300, Argentina"},{"name":"Faculty of Veterinary Sciences, National University of La Pampa, General Pico 6360, Argentina"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3613-2481","authenticated-orcid":false,"given":"Iv\u00e1n","family":"Y\u00e1nez-Ortiz","sequence":"additional","affiliation":[{"name":"School of Veterinary Medicine, Faculty of Medical, Health and Life Sciences, International University of Ecuador, Quito 170411, Ecuador"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2283-6146","authenticated-orcid":false,"given":"Augusto","family":"Carluccio","sequence":"additional","affiliation":[{"name":"Department of Veterinary Medicine, University of Teramo, Localit\u00e0 Piano d\u2019Accio, IT-64100 Teramo, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2819-4455","authenticated-orcid":false,"given":"Ippolito De","family":"Amicis","sequence":"additional","affiliation":[{"name":"Department of Veterinary Medicine, University of Teramo, Localit\u00e0 Piano d\u2019Accio, IT-64100 Teramo, Italy"}],"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":"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\/0000-0002-1085-0039","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 Cerdanyola del Vall\u00e8s, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,7,14]]},"reference":[{"key":"ref_1","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_2","doi-asserted-by":"crossref","first-page":"249","DOI":"10.1016\/j.anireprosci.2010.06.003","article-title":"Comparative Studies on Bull and Stallion Seminal DNase Activity and Interaction with Semen Extender and Spermatozoa","volume":"121","author":"Alghamdi","year":"2010","journal-title":"Anim. Reprod. Sci."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"711","DOI":"10.1093\/humupd\/dmv042","article-title":"Sperm Selection in Natural Conception: What Can We Learn from Mother Nature to Improve Assisted Reproduction Outcomes?","volume":"21","author":"Sakkas","year":"2015","journal-title":"Hum. Reprod. Update"},{"key":"ref_4","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_5","doi-asserted-by":"crossref","unstructured":"Angeletti, A., Volpi, S., Bruschi, M., Lugani, F., Vaglio, A., Prunotto, M., Gattorno, M., Schena, F., Verrina, E., and Ravelli, A. (2021). Neutrophil Extracellular Traps-DNase Balance and Autoimmunity. Cells, 10.","DOI":"10.3390\/cells10102667"},{"key":"ref_6","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_7","doi-asserted-by":"crossref","first-page":"415","DOI":"10.1111\/evj.13457","article-title":"Seminal Plasma, and Not Sperm, Induces Time and Concentration-dependent Neutrophil Extracellular Trap Release in Donkeys","volume":"54","author":"Zambrano","year":"2022","journal-title":"Equine Vet. J."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"331","DOI":"10.1016\/j.anireprosci.2008.10.015","article-title":"Species-Specific Interaction of Seminal Plasma on Sperm\u2013Neutrophil Binding","volume":"114","author":"Alghamdi","year":"2009","journal-title":"Anim. Reprod. Sci."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"157","DOI":"10.1095\/biolreprod.110.084491","article-title":"Equine CRISP3 Modulates Interaction Between Spermatozoa and Polymorphonuclear Neutrophils1","volume":"85","author":"Doty","year":"2011","journal-title":"Biol. Reprod."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1174","DOI":"10.1095\/biolreprod.105.045666","article-title":"Seminal DNase Frees Spermatozoa Entangled in Neutrophil Extracellular Traps","volume":"73","author":"Alghamdi","year":"2005","journal-title":"Biol. Reprod."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"929","DOI":"10.1042\/bj20040046","article-title":"Expression Pattern of the Deoxyribonuclease 1 Gene: Lessons from the Dnase1 Knockout Mouse","volume":"380","author":"Napirei","year":"2004","journal-title":"Biochem. J."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1059","DOI":"10.1111\/j.1742-4658.2008.06849.x","article-title":"Murine Serum Nucleases\u2013Contrasting Effects of Plasmin and Heparin on the Activities of DNase1 and DNase1-like 3 (DNase1l3)","volume":"276","author":"Napirei","year":"2009","journal-title":"FEBS J."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"13114","DOI":"10.1016\/S0021-9258(18)33630-5","article-title":"Purification and Properties of Bull Seminal Plasma Ca2+,Mg2+-Dependent Endonuclease","volume":"257","author":"Hashida","year":"1982","journal-title":"J. Biol. Chem."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"5","DOI":"10.1016\/0009-8981(93)90217-R","article-title":"Human Seminal Deoxyribonuclease I (DNase I): Purification, Enzymological and Immunological Characterization and Origin","volume":"218","author":"Yasuda","year":"1993","journal-title":"Clin. Chim. Acta"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"145","DOI":"10.1002\/(SICI)1098-2795(199910)54:2<145::AID-MRD6>3.0.CO;2-6","article-title":"Purification and Characterization of Fertility-Associated Antigen (FAA) in Bovine Seminal Fluid","volume":"54","author":"McCauley","year":"1999","journal-title":"Mol. Reprod. Dev."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"104","DOI":"10.1006\/excr.2000.5079","article-title":"Regulation of Foreign DNA Uptake by Mouse Spermatozoa","volume":"262","author":"Carballada","year":"2001","journal-title":"Exp. Cell Res."},{"key":"ref_17","first-page":"582","article-title":"Purification and Characteristics of Ca2+, Mg2+ and Ca2+, Mn2+ Dependent and Acid DNases from Spermatozoa of the Sea Urchin Strongylocentrotus intermedius","volume":"68","author":"Shastina","year":"2003","journal-title":"Biochemistry"},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Company, S., Sancler-Silva, Y.F.R., Y\u00e1nez-Ortiz, I., Mart\u00ednez-Hern\u00e1ndez, J., Costa, M., Catal\u00e1n, J., and Mir\u00f3, J. (2025). Eosinophils from Jennies Form Extracellular Traps (EETs) in Vitro in Response to Seminal Plasma: A New Perspective on Uterine Immune Defense in Donkeys. Anim. Reprod. Sci., 282.","DOI":"10.1016\/j.anireprosci.2025.108017"},{"key":"ref_19","first-page":"327","article-title":"Minimal Contamination Techniques for Breeding Mares: Technique and Preliminary Findings","volume":"21","author":"Kenney","year":"1975","journal-title":"Proc. Am. Assoc. Equine Pract."},{"key":"ref_20","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_21","doi-asserted-by":"crossref","unstructured":"Wei, Z., Hong, H., Liu, W., Jiang, L., Xu, J., Gao, X., Qian, Y., Jiang, Y., Jin, Z., and Jin, Q. (2024). DNase I Rescues Goat Sperm Entrapped by Neutrophil Extracellular Traps. Dev. Comp. Immunol., 153.","DOI":"10.1016\/j.dci.2023.105107"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"308","DOI":"10.1017\/S1751731113002097","article-title":"Effect of Single Layer Centrifugation Using Androcoll-E-Large on the Sperm Quality Parameters of Cooled-Stored Donkey Semen Doses","volume":"8","author":"Ortiz","year":"2014","journal-title":"Animal"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"332","DOI":"10.1071\/RD13246","article-title":"Colloid Single-Layer Centrifugation Improves Post-Thaw Donkey (Equus asinus) Sperm Quality and Is Related to Ejaculate Freezability","volume":"27","author":"Ortiz","year":"2015","journal-title":"Reprod. Fertil. Dev."},{"key":"ref_24","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_25","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_26","doi-asserted-by":"crossref","first-page":"1017","DOI":"10.1016\/j.theriogenology.2009.06.012","article-title":"Effects of Dilution and Centrifugation on the Survival of Spermatozoa and the Structure of Motile Sperm Cell Subpopulations in Refrigerated Catalonian Donkey Semen","volume":"72","author":"Taberner","year":"2009","journal-title":"Theriogenology"},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Catal\u00e1n, J., Papas, M., Trujillo-Rojas, L., Blanco-Prieto, O., Bonilla-Correal, S., Rodr\u00edguez-Gil, J.E., Mir\u00f3, J., and Yeste, M. (2020). Red LED Light Acts on the Mitochondrial Electron Chain of Donkey Sperm and Its Effects Depend on the Time of Exposure to Light. Front. Cell Dev. Biol., 8.","DOI":"10.3389\/fcell.2020.588621"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"32","DOI":"10.1095\/biolreprod60.1.32","article-title":"Sperm Subpopulations in Boar (Sus scrofa) and Gazelle (Gazella dama mhorr) Semen as Revealed by Pattern Analysis of Computer-Assisted Motility Assessments","volume":"60","author":"Abaigar","year":"1999","journal-title":"Biol. Reprod."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"801","DOI":"10.1016\/S0093-691X(01)00609-4","article-title":"Effects of Glucose and Fructose on Motility Patterns of Dog Spermatozoa from Fresh Ejaculates","volume":"56","author":"Rigau","year":"2001","journal-title":"Theriogenology"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1973","DOI":"10.1016\/S0093-691X(02)01297-9","article-title":"Identification of Sperm Subpopulations with Specific Motility Characteristics in Stallion Ejaculates","volume":"59","year":"2003","journal-title":"Theriogenology"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"316","DOI":"10.1095\/biolreprod.104.032730","article-title":"Sperm Subpopulations in Iberian Red Deer Epididymal Sperm and Their Changes Through the Cryopreservation Process","volume":"72","author":"Alvarez","year":"2005","journal-title":"Biol. Reprod."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"936","DOI":"10.1016\/j.theriogenology.2008.05.056","article-title":"Effects of Freezing\/Thawing on Motile Sperm Subpopulations of Boar and Donkey Ejaculates","volume":"70","author":"Flores","year":"2008","journal-title":"Theriogenology"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1067","DOI":"10.1016\/j.theriogenology.2014.01.033","article-title":"Evaluation of Sperm Subpopulation Structure in Relation to in Vitro Sperm\u2013Oocyte Interaction of Frozen-Thawed Semen from Holstein Bulls","volume":"81","author":"Ferraz","year":"2014","journal-title":"Theriogenology"},{"key":"ref_34","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_35","doi-asserted-by":"crossref","first-page":"420","DOI":"10.1071\/RD18087","article-title":"Melatonin Reduces cAMP-Stimulated Capacitation of Ram Spermatozoa","volume":"31","author":"Casao","year":"2019","journal-title":"Reprod. Fertil. Dev."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"22","DOI":"10.1016\/j.theriogenology.2024.02.014","article-title":"The Role of Equine Seminal Plasma Derived Cysteine Rich Secretory Protein 3 (CRISP3) in the Interaction between Polymorphonuclear Neutrophils (PMNs) and Populations of Viable or Dead Spermatozoa, and Bactera","volume":"219","author":"Doty","year":"2024","journal-title":"Theriogenology"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"119","DOI":"10.1046\/j.1439-0531.2003.00397.x","article-title":"Hyperactivated Motility in Sperm","volume":"38","author":"Suarez","year":"2003","journal-title":"Reprod. Domest. Anim."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"199","DOI":"10.1095\/biolreprod.108.074880","article-title":"Hyperactivation of Stallion Sperm Is Required for Successful In Vitro Fertilization of Equine Oocytes","volume":"81","author":"McPartlin","year":"2009","journal-title":"Biol. Reprod."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"75","DOI":"10.1002\/j.1939-4640.1992.tb01631.x","article-title":"Movement Characteristics of Boar Sperm Obtained from the Oviduct or Hyperactivated In Vitro","volume":"13","author":"Suarez","year":"1992","journal-title":"J. Androl."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"195","DOI":"10.1002\/j.1939-4640.1990.tb03228.x","article-title":"Kinematics of Human Spermatozoa Incubated Under Capacitating Conditions","volume":"11","author":"Mortimer","year":"1990","journal-title":"J. Androl."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"1626","DOI":"10.1095\/biolreprod.103.026476","article-title":"Different Signaling Pathways in Bovine Sperm Regulate Capacitation and Hyperactivation1","volume":"70","author":"Marquez","year":"2004","journal-title":"Biol. Reprod."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"6446","DOI":"10.1038\/s41598-022-10390-x","article-title":"Hyperactivation Is Sufficient to Release Porcine Sperm from Immobilized Oviduct Glycans","volume":"12","author":"Sharif","year":"2022","journal-title":"Sci. Rep."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"647","DOI":"10.1093\/humupd\/dmn029","article-title":"Control of Hyperactivation in Sperm","volume":"14","author":"Suarez","year":"2008","journal-title":"Hum. Reprod. Update"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"779","DOI":"10.1095\/biolreprod46.5.779","article-title":"Hyperactivated Sperm Progress in the Mouse Oviduct1","volume":"46","author":"Demott","year":"1992","journal-title":"Biol. Reprod."}],"container-title":["Animals"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-2615\/16\/14\/2185\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,7,14]],"date-time":"2026-07-14T08:14:57Z","timestamp":1784016897000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-2615\/16\/14\/2185"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,7,14]]},"references-count":44,"journal-issue":{"issue":"14","published-online":{"date-parts":[[2026,7]]}},"alternative-id":["ani16142185"],"URL":"https:\/\/doi.org\/10.3390\/ani16142185","relation":{},"ISSN":["2076-2615"],"issn-type":[{"value":"2076-2615","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,7,14]]}}}