# Crocin Supplementation of Semen Extender Preserves Chilled Ram Semen Quality During Extended Storage

## TL;DR

Adding crocin to ram semen storage solutions helps protect sperm quality during cold storage by reducing oxidative stress and preventing cell damage.

## Contribution

The study demonstrates that crocin, a natural antioxidant, can preserve chilled ram semen quality during extended storage by modulating stress response and apoptosis pathways.

## Key findings

- Crocin at 1 mM concentration preserved sperm motility, viability, and kinematic parameters up to three days of cold storage.
- Crocin enhanced glutathione and Hsp70 levels while inhibiting apoptosis in a dose-dependent manner.
- Higher crocin concentration (1 mM) showed superior antioxidant and cytoprotective effects compared to 0.5 mM.

## Abstract

Ram semen is particularly vulnerable to damage from temperature fluctuations, as chilling or freezing semen during storage induces the production of reactive oxygen species (ROS). ROS are highly reactive molecules that induce oxidative stress and thus can inflict damage to cell molecules and structures, like membranes, all of which culminate in reduced sperm function. This has rather important implications for artificial insemination (AI), a practice that is applied for the genetic improvement of herds. In the present study, we investigated the molecular mechanisms implicated in cold shock of ram spermatozoa and the potential of crocin, a potent antioxidant, to inhibit the ROS-mediated damage and modulate the implicated pathways. We have identified that crocin protects spermatozoa from cold shock by inhibiting the induction of regulated sperm death (apoptosis) and fortifying cell survival.

Ram semen is highly susceptible to cold shock, which induces irreversible damage to the integrity and fluidity of membranes. Chilled semen is commonly used within 24 h of collection. However, while its storage at 5 °C extends semen lifespan, it is often accompanied by quality deterioration due to accumulation of reactive oxygen species (ROS). This study evaluated the potential of crocin, a carotenoid with antioxidant properties, to improve the quality of chilled ram semen stored at 5 °C for up to three days in a soybean lecithin–based extender supplemented with two crocin concentrations (0.5 and 1 mM). Sperm motility, viability, glutathione levels, the expression of proteins involved in the heat stress response (HSR), and apoptosis were assessed at 24 h intervals. Crocin preserved motility (up to Day 1), viability (up to Day 2,) and kinematic parameters (up to Day 3). In addition, crocin enhanced intracellular glutathione and Hsp70 levels and inhibited apoptotic levels dose-dependently, indicating the antioxidant and cytoprotective role of crocin. Despite 0.5 mM being effective up to Day 1, 1 mM crocin augmented antioxidant capacity, modulated stress response mechanisms, and preserved sperm quality during chilled storage up to Day 3, highlighting its potential as a valuable additive of ram semen extenders.