Modulation of NETosis in Swine Neutrophil–Spermatozoa Co-Cultures In Vitro: Effects of Butylated Hydroxytoluene, Albumin, Prostaglandin E<sub>2</sub>, and Seminal Plasma

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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Main Authors: 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
Format: Article
Language:English
Published: MDPI AG 2025-06-01
Series:Antioxidants
Subjects:
PMN
NETosis
spermatozoa
oxidative stress
antioxidants
Online Access:https://www.mdpi.com/2076-3921/14/7/778
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Summary: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.
ISSN:2076-3921

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