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

Area of Science:

• Reproductive Biology
• Genomics
• Animal Science

Background:

• Equine germplasm banks are vital for conserving stallion semen.
• Assessing stallion semen quality post-thaw is crucial for reproductive success.
• Gene expression analysis requires reliable reference genes, but RNA degrades rapidly in cryopreserved sperm.

Purpose of the Study:

• To identify stable reference genes in equine cryopreserved sperm for future gene expression studies.
• To evaluate candidate genes for their suitability as internal controls in stallion semen quality research.

Main Methods:

• Selected live spermatozoa from cryopreserved semen of 20 stallions using a concentration gradient.
• Purified RNA using combined organic and column extraction with DNase treatment.
• Amplified nine candidate genes via one-step reverse transcription and real-time quantitative polymerase chain reaction (qRT-PCR).
• Assessed gene specificity via melting curves, gel electrophoresis, and sequencing; calculated gene stability.

Main Results:

• Five of nine candidate genes (β-Actin, ATP synthase subunit beta, Protamine 1, L32 ribosomal protein, Ubiquitin B) showed adequate amplification.
• β-Actin and L32 ribosomal protein exhibited the highest stability.
• ATP synthase subunit beta and Ubiquitin B also demonstrated good stability.

Conclusions:

• β-Actin and L32 ribosomal protein are highly suitable reference genes for equine cryopreserved sperm studies.
• These validated genes will enhance the accuracy of gene expression analysis in stallion semen quality research.
• This research supports improved management and reproductive applications of equine germplasm.