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This study evaluated the capacity of antioxidant and reproductive compounds\u2014butylated hydroxytoluene (BHT), prostaglandin E2 (PGE2), bovine serum albumin (BSA), and seminal plasma (SP)\u2014to modulate NETosis in co-cultures of swine neutrophils and cryopreserved spermatozoa. NET formation was quantified by nuclear area expansion and validated by digital cytometry and immunofluorescence. BHT (0.5 mM) and PGE2 (10 \u00b5M) produced the most significant inhibitory effects, reducing NETotic cell percentages from 34.5 \u00b1 2.7% (sperm-exposed controls) to 12.2 \u00b1 1.3% and 14.5 \u00b1 2.1%, respectively (p &lt; 0.01). SP at 20% decreased NETosis to 16.8 \u00b1 1.8%, while BSA (0.5%) achieved a moderate reduction to 21.3 \u00b1 2.5%. Flow cytometry revealed reduced peroxynitrite levels in sperm treated with SP and BSA. Two NET phenotypes (aggNETs and sprNETs) were identified. BTS medium enhanced NET formation, whereas DNase I degraded NETs effectively. These findings identify porcine NETosis as a redox-sensitive pathway modulated in vitro, suggesting an immunological role in enhancing sperm preservation for swine artificial insemination.<\/jats:p>","DOI":"10.3390\/antiox14070778","type":"journal-article","created":{"date-parts":[[2025,6,24]],"date-time":"2025-06-24T09:24:18Z","timestamp":1750757058000},"page":"778","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Modulation of NETosis in Swine Neutrophil\u2013Spermatozoa Co-Cultures In Vitro: Effects of Butylated Hydroxytoluene, Albumin, Prostaglandin E2, and Seminal Plasma"],"prefix":"10.3390","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-4840-0508","authenticated-orcid":false,"given":"Fabiola","family":"Zambrano","sequence":"first","affiliation":[{"name":"Center of Excellence in Translational Medicine\u2014Scientific and Technological Bioresource Nucleus (CEMT\u2014BIOREN), Faculty of Medicine, Universidad de La Frontera, Temuco 4780000, Chile"},{"name":"Department of Preclinical Sciences, Faculty of Medicine, Universidad de La Frontera, Temuco 4811230, Chile"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Felipe","family":"Pezo","sequence":"additional","affiliation":[{"name":"Facultad de Ciencias Agropecuarias y Medioambiente, Universidad de La Frontera, Temuco 4780000, Chile"},{"name":"Center of Excellence in Reproductive Biotechnology (CEBIOR-BIOREN), Faculty of Medicine, Universidad de La Frontera, Temuco 4780000, Chile"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0148-4372","authenticated-orcid":false,"given":"Andr\u00e9","family":"Furugen Cesar de Andrade","sequence":"additional","affiliation":[{"name":"Department of Animal Reproduction, School of Veterinary Medicine and Animal Science (FMVZ), University of S\u00e3o Paulo (USP), Pirassununga 13635-900, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4916-5079","authenticated-orcid":false,"given":"Rodrigo","family":"Rivera-Concha","sequence":"additional","affiliation":[{"name":"Center of Excellence in Translational Medicine\u2014Scientific and Technological Bioresource Nucleus (CEMT\u2014BIOREN), Faculty of Medicine, Universidad de La Frontera, Temuco 4780000, Chile"},{"name":"Ph.D. Program in Medical Sciences, Faculty of Medicine, Universidad de La Frontera, Temuco 4780000, Chile"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7358-7541","authenticated-orcid":false,"given":"Pamela","family":"Uribe","sequence":"additional","affiliation":[{"name":"Center of Excellence in Translational Medicine\u2014Scientific and Technological Bioresource Nucleus (CEMT\u2014BIOREN), Faculty of Medicine, Universidad de La Frontera, Temuco 4780000, Chile"},{"name":"Department of Internal Medicine, Faculty of Medicine, Universidad de La Frontera, Temuco 4780000, Chile"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mabel","family":"Schulz","sequence":"additional","affiliation":[{"name":"Center of Excellence in Translational Medicine\u2014Scientific and Technological Bioresource Nucleus (CEMT\u2014BIOREN), Faculty of Medicine, Universidad de La Frontera, Temuco 4780000, Chile"},{"name":"Department of Preclinical Sciences, Faculty of Medicine, Universidad de La Frontera, Temuco 4811230, Chile"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0006-9947-3626","authenticated-orcid":false,"given":"Henricco","family":"Zapparoli","sequence":"additional","affiliation":[{"name":"Department of Animal Reproduction, School of Veterinary Medicine and Animal Science (FMVZ), University of S\u00e3o Paulo (USP), Pirassununga 13635-900, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0003-7408-3670","authenticated-orcid":false,"given":"Luan","family":"Mendes de Oliveira Bezerra","sequence":"additional","affiliation":[{"name":"Department of Animal Reproduction, School of Veterinary Medicine and Animal Science (FMVZ), University of S\u00e3o Paulo (USP), Pirassununga 13635-900, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4550-8492","authenticated-orcid":false,"given":"Carlos","family":"Hermosilla","sequence":"additional","affiliation":[{"name":"Institute of Parasitology, Justus Liebig University Giessen, 35392 Giessen, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8649-1020","authenticated-orcid":false,"given":"Anja","family":"Taubert","sequence":"additional","affiliation":[{"name":"Institute of Parasitology, Justus Liebig University Giessen, 35392 Giessen, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4853-4282","authenticated-orcid":false,"given":"Ra\u00fal","family":"S\u00e1nchez","sequence":"additional","affiliation":[{"name":"Center of Excellence in Translational Medicine\u2014Scientific and Technological Bioresource Nucleus (CEMT\u2014BIOREN), Faculty of Medicine, Universidad de La Frontera, Temuco 4780000, Chile"},{"name":"Department of Preclinical Sciences, Faculty of Medicine, Universidad de La Frontera, Temuco 4811230, Chile"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,6,24]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"166","DOI":"10.1111\/j.1439-0531.2007.00872.x","article-title":"Interaction of Intact Porcine Spermatozoa with Epithelial Cells and Neutrophilic Granulocytes during Uterine Passage","volume":"43","author":"Taylor","year":"2008","journal-title":"Reprod. 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