# Cytotoxic Oxidative Stress Effects of Neutrophil Extracellular Traps’ Components on Cattle Spermatozoa

## TL;DR

This study shows that components of neutrophil extracellular traps cause oxidative stress and damage to cattle sperm cells in vitro.

## Contribution

The first investigation of cytotoxic oxidative stress effects from individual NETs components on bull spermatozoa.

## Key findings

- H2A, NE, and LL-37 caused membrane disruption in bull sperm within 15 minutes.
- MPO and Cat-G induced oxidative stress after 1 hour of exposure.
- NET formation occurred within 15 minutes in PMN/sperm co-cultures.

## Abstract

Bovine spermatozoa are highly susceptible to oxidative stress (OS), and it is known to affect their cellular functions. The main leukocyte producers of reactive oxygen species (ROS) in mammalian semen are polymorphonuclear neutrophils (PMN). PMN activation can result in the formation of neutrophil extracellular traps (NETs), which have been shown to affect the motility and function of spermatozoa. However, OS effects on bull spermatozoa derived from individual NETs components have not been investigated. The hypothesis of this study was that specific NETs components might generate OS on bull spermatozoa. Bovine sperm cells were incubated with five NETs-associated molecules, including 30 μg/mL histone 2A (H2A), neutrophil elastase (NE), 1 μg/mL myeloperoxidase (MPO), cathepsin G (Cat-G), and cathelicidin LL37 (LL-37), for a time course ranging from 15 to 240 min. Fluorescence microscopy was used to evaluate the coincubation of bovine PMN and sperm cells. Within 15 min, H2A, NE, and LL-37 caused membrane disruption, while MPO and Cat-G caused OS on bull spermatozoa after 1 h of coincubation. NET formation was observed within 15 min of coincubation in co-cultures of bovine PMN/sperm cells. This study is the first to report on the role of cytotoxic OS effects caused by NETs-derived components in bovine sperm in vitro.