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

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.