# Enhancing sperm resilience: protective effects of ectoine on post-thaw bovine sperm quality under environmental stress conditions

## TL;DR

Ectoine, a natural osmolyte, protects bovine sperm from environmental stress without toxicity, showing promise for reproductive biotechnology.

## Contribution

This study is the first to demonstrate ectoine's protective effects on sperm under stress conditions.

## Key findings

- Ectoine at 0.5 and 5 mM maintained sperm motility and viability under heat stress.
- Ectoine improved sperm viability in hypoosmotic conditions for bulls with poor osmotic resistance.
- Sperm treated with 50 mM ectoine showed higher motility in cervicovaginal mucus.

## Abstract

Ectoine is a small, amino acid-derived osmolyte produced by extremophilic bacteria that acts as a compatible solute, protecting cellular macromolecules and structures from extreme environmental stress without disrupting essential cellular functions. The aim of this study was to evaluate the biocompatibility of ectoine with bull sperm and to assess the potential of ectoine to enhance the resilience of sperm under varying stress conditions. Thawed bovine sperm in the presence (0.5, 5, and 50 mM) or absence (control; 0 mM) of ectoine were subjected to a biocompatibility test (37 °C for 6 h; n = 8 bulls), heat stress (39 or 42 °C for 6 h; n = 8 bulls) or osmotic stress (150 or 400 mOsm for 15 min; n = 12 bulls), whereby motility and kinematic parameters, as well as viability, acrosome integrity, and membrane fluidity by flow cytometry were assessed. Sperm motility in cervicovaginal mucus (37 °C for 3 h; n = 6 bulls) was also assessed. All results are reported as mean ± SEM. Ectoine displayed a non-toxic effect across all motility and functional parameters (viability, acrosome integrity, and membrane fluidity). Nonetheless, a reduction in kinematic parameters, including straight line velocity (VSL), average path velocity (VAP) and straightness (STR) was observed at 50 mM ectoine (P < 0.05). Under heat stress at 39 and 42 °C, ectoine concentrations of 0.5 and 5 mM maintained motility and viability, comparable to controls across all time points (P > 0.05). In hypoosmotic conditions (150 mOsm), individual bulls displayed different degrees of osmotic resistance. In those bulls with poor osmotic resistance (n = 4), ectoine (0.5 and 5 mM) maintained sperm viability similar to the 0 mM control (P > 0.05). However, the viability of sperm incubated with 50 mM solute was 2-fold higher relative to the control (P < 0.001). In hyperosmotic conditions, addition of ectoine to sperm prior to exposure did not affect the total motility or viability compared to the no ectoine treatment (P > 0.05). When sperm were incubated in cervicovaginal mucus, there was an effect of ectoine treatment. Sperm treated with 50 mM ectoine exhibited higher motility throughout incubation compared to the control (0 mM) (P < 0.05). In conclusion, these findings establish ectoine as a promising candidate for improving sperm resilience and warrants further studies to assess additional protective effects of ectoine.

Ectoine, a naturally occurring osmolyte, has been shown to protect various cell types under extreme environmental stress, but has never been examined for the protection of sperm. This research showed ectoine’s potential to enhance sperm resilience under stress, highlighting its promise as a protective agent in reproductive biotechnology.