Zambrano Fabiola, Pezo Felipe, Furugen Cesar de Andrade André, Rivera-Concha Rodrigo, Uribe Pamela, Schulz Mabel, Zapparoli Henricco, Mendes de Oliveira Bezerra Luan, Hermosilla Carlos, Taubert Anja, Sánchez Raúl
Center of Excellence in Translational Medicine-Scientific and Technological Bioresource Nucleus (CEMT-BIOREN), Faculty of Medicine, Universidad de La Frontera, Temuco 4780000, Chile.
Department of Preclinical Sciences, Faculty of Medicine, Universidad de La Frontera, Temuco 4811230, Chile.
Antioxidants (Basel). 2025 Jun 24;14(7):778. doi: 10.3390/antiox14070778.
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 (PGE), 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 (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 ( < 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 (NETs and 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.
在猪的繁殖过程中，诸如中性粒细胞胞外陷阱（NET）形成等免疫介导机制会影响精子功能并降低繁殖成功率。本研究评估了抗氧化剂和生殖化合物——丁基羟基甲苯（BHT）、前列腺素E（PGE）、牛血清白蛋白（BSA）和精浆（SP）——在猪中性粒细胞与冷冻保存精子的共培养体系中调节NETosis的能力。通过核面积扩展对NET形成进行定量，并通过数字细胞计数法和免疫荧光法进行验证。BHT（0.5 mM）和PGE（10 µM）产生了最显著的抑制作用，将NETotic细胞百分比分别从34.5±2.7%（精子暴露对照组）降至12.2±1.3%和14.5±2.1%（P<0.01）。20%的SP将NETosis降至16.8±1.8%，而0.5%的BSA则适度降至21.3±2.5%。流式细胞术显示，经SP和BSA处理的精子中过氧亚硝酸盐水平降低。鉴定出两种NET表型（NETs和NETs）。BTS培养基增强了NET形成，而脱氧核糖核酸酶I有效地降解了NETs。这些发现表明猪的NETosis是一种体外可调节的氧化还原敏感途径，提示其在增强猪人工授精精子保存方面具有免疫作用。
