Comparative transcript profiling of gene expression of fresh and frozen–thawed bull sperm

Abstract

Although frozen semen is widely used commercially in the cattle breeding industry, the resultant pregnancy rate is lower than that produced using fresh semen. Cryodamage is a major problem in semen cryopreservation; it causes changes to sperm transcripts that may influence sperm function and motility. We used suppression subtractive hybridization technology to establish a complementary DNA subtractive library, and combined microarray technology and sequence homology analysis to screen and analyze differentially expressed genes in the library, comparing fresh sperm with the frozen–thawed sperm of nine bulls. Overall, 19 positive differentially expressed unigenes were identified using microarray data and Significance Analysis of Microarrays software (|score (d)| ≥ 2, fold change > 1, and false discovery rate < 0.05). Of 15 differentially expressed unigenes exhibited high sequence homology (E-value ≤ 1 × 10−3), 12 were upregulated in frozen–thawed sperm, the remaining 3 were upregulated in fresh sperm, and 4 other clones were identified as unknown because of incomplete sequences or because there was no significant sequence homology (E-value > 1E−03) and were considered novel genes. The expression of five of these genes—RPL31, PRKCE, PAPSS2, PLP1, and R1G7—was verified by quantitative real-time reverse transcription–polymerase chain reaction. There was a significant differential expression of the RPL31 gene (P < 0.05). Our preliminary results provide an overview of differentially expressed transcripts between fresh and frozen–thawed sperm of Holstein bulls.