Modulation of NETosis in Swine Neutrophil–Spermatozoa Co-Cultures In Vitro: Effects of Butylated Hydroxytoluene, Albumin, Prostaglandin E<sub>2</sub>, and Seminal Plasma
In swine reproduction, immune-mediated mechanisms such as neutrophil extracellular trap (NET) formation can affect sperm function and reduce fertility outcomes. This study evaluated the capacity of antioxidant and reproductive compounds—butylated hydroxytoluene (BHT), prostaglandin E<sub>2<...
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MDPI AG
2025-06-01
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| Series: | Antioxidants |
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| author | Fabiola Zambrano Felipe Pezo André Furugen Cesar de Andrade Rodrigo Rivera-Concha Pamela Uribe Mabel Schulz Henricco Zapparoli Luan Mendes de Oliveira Bezerra Carlos Hermosilla Anja Taubert Raúl Sánchez |
| author_facet | Fabiola Zambrano Felipe Pezo André Furugen Cesar de Andrade Rodrigo Rivera-Concha Pamela Uribe Mabel Schulz Henricco Zapparoli Luan Mendes de Oliveira Bezerra Carlos Hermosilla Anja Taubert Raúl Sánchez |
| author_sort | Fabiola Zambrano |
| collection | DOAJ |
| description | In swine reproduction, immune-mediated mechanisms such as neutrophil extracellular trap (NET) formation can affect sperm function and reduce fertility outcomes. This study evaluated the capacity of antioxidant and reproductive compounds—butylated hydroxytoluene (BHT), prostaglandin E<sub>2</sub> (PGE<sub>2</sub>), bovine serum albumin (BSA), and seminal plasma (SP)—to 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 PGE<sub>2</sub> (10 µM) produced the most significant inhibitory effects, reducing NETotic cell percentages from 34.5 ± 2.7% (sperm-exposed controls) to 12.2 ± 1.3% and 14.5 ± 2.1%, respectively (<i>p</i> < 0.01). SP at 20% decreased NETosis to 16.8 ± 1.8%, while BSA (0.5%) achieved a moderate reduction to 21.3 ± 2.5%. Flow cytometry revealed reduced peroxynitrite levels in sperm treated with SP and BSA. Two NET phenotypes (<i>agg</i>NETs and <i>spr</i>NETs) 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. |
| format | Article |
| id | doaj-art-acb7bf30f92849b1916dee4c0cf79f8d |
| institution | Kabale University |
| issn | 2076-3921 |
| language | English |
| publishDate | 2025-06-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Antioxidants |
| spelling | doaj-art-acb7bf30f92849b1916dee4c0cf79f8d2025-08-20T03:36:10ZengMDPI AGAntioxidants2076-39212025-06-0114777810.3390/antiox14070778Modulation of NETosis in Swine Neutrophil–Spermatozoa Co-Cultures In Vitro: Effects of Butylated Hydroxytoluene, Albumin, Prostaglandin E<sub>2</sub>, and Seminal PlasmaFabiola Zambrano0Felipe Pezo1André Furugen Cesar de Andrade2Rodrigo Rivera-Concha3Pamela Uribe4Mabel Schulz5Henricco Zapparoli6Luan Mendes de Oliveira Bezerra7Carlos Hermosilla8Anja Taubert9Raúl Sánchez10Center of Excellence in Translational Medicine—Scientific and Technological Bioresource Nucleus (CEMT—BIOREN), Faculty of Medicine, Universidad de La Frontera, Temuco 4780000, ChileFacultad de Ciencias Agropecuarias y Medioambiente, Universidad de La Frontera, Temuco 4780000, ChileDepartment of Animal Reproduction, School of Veterinary Medicine and Animal Science (FMVZ), University of São Paulo (USP), Pirassununga 13635-900, BrazilCenter of Excellence in Translational Medicine—Scientific and Technological Bioresource Nucleus (CEMT—BIOREN), Faculty of Medicine, Universidad de La Frontera, Temuco 4780000, ChileCenter of Excellence in Translational Medicine—Scientific and Technological Bioresource Nucleus (CEMT—BIOREN), Faculty of Medicine, Universidad de La Frontera, Temuco 4780000, ChileCenter of Excellence in Translational Medicine—Scientific and Technological Bioresource Nucleus (CEMT—BIOREN), Faculty of Medicine, Universidad de La Frontera, Temuco 4780000, ChileDepartment of Animal Reproduction, School of Veterinary Medicine and Animal Science (FMVZ), University of São Paulo (USP), Pirassununga 13635-900, BrazilDepartment of Animal Reproduction, School of Veterinary Medicine and Animal Science (FMVZ), University of São Paulo (USP), Pirassununga 13635-900, BrazilInstitute of Parasitology, Justus Liebig University Giessen, 35392 Giessen, GermanyInstitute of Parasitology, Justus Liebig University Giessen, 35392 Giessen, GermanyCenter of Excellence in Translational Medicine—Scientific and Technological Bioresource Nucleus (CEMT—BIOREN), Faculty of Medicine, Universidad de La Frontera, Temuco 4780000, ChileIn swine reproduction, immune-mediated mechanisms such as neutrophil extracellular trap (NET) formation can affect sperm function and reduce fertility outcomes. This study evaluated the capacity of antioxidant and reproductive compounds—butylated hydroxytoluene (BHT), prostaglandin E<sub>2</sub> (PGE<sub>2</sub>), bovine serum albumin (BSA), and seminal plasma (SP)—to 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 PGE<sub>2</sub> (10 µM) produced the most significant inhibitory effects, reducing NETotic cell percentages from 34.5 ± 2.7% (sperm-exposed controls) to 12.2 ± 1.3% and 14.5 ± 2.1%, respectively (<i>p</i> < 0.01). SP at 20% decreased NETosis to 16.8 ± 1.8%, while BSA (0.5%) achieved a moderate reduction to 21.3 ± 2.5%. Flow cytometry revealed reduced peroxynitrite levels in sperm treated with SP and BSA. Two NET phenotypes (<i>agg</i>NETs and <i>spr</i>NETs) 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.https://www.mdpi.com/2076-3921/14/7/778PMNNETosisspermatozoaoxidative stressantioxidants |
| spellingShingle | Fabiola Zambrano Felipe Pezo André Furugen Cesar de Andrade Rodrigo Rivera-Concha Pamela Uribe Mabel Schulz Henricco Zapparoli Luan Mendes de Oliveira Bezerra Carlos Hermosilla Anja Taubert Raúl Sánchez Modulation of NETosis in Swine Neutrophil–Spermatozoa Co-Cultures In Vitro: Effects of Butylated Hydroxytoluene, Albumin, Prostaglandin E<sub>2</sub>, and Seminal Plasma Antioxidants PMN NETosis spermatozoa oxidative stress antioxidants |
| title | Modulation of NETosis in Swine Neutrophil–Spermatozoa Co-Cultures In Vitro: Effects of Butylated Hydroxytoluene, Albumin, Prostaglandin E<sub>2</sub>, and Seminal Plasma |
| title_full | Modulation of NETosis in Swine Neutrophil–Spermatozoa Co-Cultures In Vitro: Effects of Butylated Hydroxytoluene, Albumin, Prostaglandin E<sub>2</sub>, and Seminal Plasma |
| title_fullStr | Modulation of NETosis in Swine Neutrophil–Spermatozoa Co-Cultures In Vitro: Effects of Butylated Hydroxytoluene, Albumin, Prostaglandin E<sub>2</sub>, and Seminal Plasma |
| title_full_unstemmed | Modulation of NETosis in Swine Neutrophil–Spermatozoa Co-Cultures In Vitro: Effects of Butylated Hydroxytoluene, Albumin, Prostaglandin E<sub>2</sub>, and Seminal Plasma |
| title_short | Modulation of NETosis in Swine Neutrophil–Spermatozoa Co-Cultures In Vitro: Effects of Butylated Hydroxytoluene, Albumin, Prostaglandin E<sub>2</sub>, and Seminal Plasma |
| title_sort | modulation of netosis in swine neutrophil spermatozoa co cultures in vitro effects of butylated hydroxytoluene albumin prostaglandin e sub 2 sub and seminal plasma |
| topic | PMN NETosis spermatozoa oxidative stress antioxidants |
| url | https://www.mdpi.com/2076-3921/14/7/778 |
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