Determining ACTB, ATP5B and RPL32 as optimal reference genes for quantitative RT-PCR studies of cryopreserved stallion semen.

This research explores optimal genes to study in cryopreserved stallion semen as a measure of quality, focusing on ACTB, ATP5B, and RPL32. The study provides insights into which genes are expressed, stable, and reliable to use as reference points in future investigations about equine semen preservation and quality.

Research context and Purpose

• Studying the cryopreservation of stallion semen helps in the management of equine germplasm banks and improves understanding of semen quality, post-thaw semen properties, spermatogenesis, and sperm quality maturation.
• Analysing genes expressed in this preserved sperm could provide a measure of quality, but the rapid degradation of sperm post-thaw creates challenges. This study aims to identify genes that have the potential to be used as reference points in future studies due to their stability and amplification properties.

Methodology

• The team used live sperm from cryopreserved semen straws from 20 stallions.
• RNA purification involved organic and column extraction methods alongside deoxyribonuclease treatment.
• Nine candidate genes underwent selective amplification via reverse transcription and real-time polymerase chain reaction (qRT-PCR).
• Gene specificities were analysed through melting curves, agarose gel electrophoresis, and sequencing. The team also calculated gene stabilities.

Findings

• Out of the nine genes tested, five (β-Actin, ATP synthase subunit beta, Protamine 1, L32 ribosomal protein, and Ubiquitin B) amplified as desired.
• β-Actin and the L32 Ribosomal protein demonstrated the highest stability, making them the most suitable genes for use as reference points in future studies of equine cryopreserved sperm.
• The ATP synthase subunit beta and Ubiquitin B ranked next in line.

Implications

• The findings provide valuable insights for future research into the cryopreservation of equine semen, shedding light on the best genes to analyse as measures of quality.
• The identified reference genes can be used in further studies to provide consistent and reliable results.
• This research contributes to the broader understanding and enhancement of semen cryopreservation techniques, aiding in the optimization of stallion semen quality and equine germplasm bank management.