# Correlation Analysis of Sperm Cryopreservation Quality with Serum Testosterone and Sperm gDNA Methylation Levels in Xiaoshan Chickens

## TL;DR

This study finds that serum testosterone and sperm DNA methylation are linked to better sperm cryopreservation in Xiaoshan chickens, offering insights for improving poultry breeding.

## Contribution

The study identifies serum testosterone and specific DNA methylation patterns as novel indicators for predicting and improving chicken sperm cryopreservation quality.

## Key findings

- Serum testosterone levels are significantly correlated with post-thaw sperm motility in Xiaoshan chickens.
- Differentially methylated genes like PRKACB and ACSL1 are associated with sperm motility after cryopreservation.
- Whole-genome bisulfite sequencing reveals 217 DMRs and 116 DMGs linked to semen cryopreservation outcomes.

## Abstract

Semen cryopreservation refers to the method of long-term storage of sperm through ultra-low temperature technology, aiming to maintain the biological activity and fertilization ability of the sperm for subsequent use. Chicken semen cryopreservation is crucial for utilizing high-quality genetics, but the outcomes are limited. This study explored semen cryopreservation outcomes with the perspective of serum indicators and sperm DNA epigenetic modification. Through serum detection, semen quality testing, WGBS (whole-genome bisulfite sequencing), and BSP (bisulfite sequencing PCR), a significant correlation between serum testosterone levels and semen cryopreservation quality was established, and key genes linked to cryopreserved sperm motility were sequenced and verified as well. These findings provide insights into improving semen preservation and have potential applications in poultry breeding and cryopreservation technologies.

Chicken semen cryopreservation is crucial for utilizing high-quality cockerel genetics, but semen is highly sensitive to cryoinjury, leading to poor preservation outcomes. This study aimed to establish a theoretical foundation for selecting cockerels for semen cryopreservation through serum testing and to improve semen quality via DNA methylation editing. Semen and serum samples were collected from 102 Xiaoshan cockerels, with semen cryopreserved and thawed following standardized protocols. Post-thaw semen quality and serum testosterone (T) levels were assessed. Eight cockerels were selected based on motile sperm quality, and whole-genome bisulfite sequencing (WGBS) was used to analyze sperm DNA methylation. The results showed a significant positive correlation between serum T levels and sperm motility. There were notable differences in sperm motility and serum T levels between high-quality and low-quality semen groups but no differences in estradiol (E2), superoxide dismutase (SOD), or glutathione peroxidase (GSH-Px) levels. A total of 217 differentially methylated regions (DMRs) and 116 differentially methylated genes (DMGs) were identified. Key genes such as PRKACB (protein kinase, cAMP-dependent, catalytic, beta) and ACSL1 (long-chain-fatty-acid--CoA ligase 1) were associated with sperm motility. These findings provide important insights for improving semen cryopreservation and contribute to breeding practices and the development of cryoprotectants.