151 COMPARATIVE ANALYSIS OF FRESH AND CRYOPRESERVED BOAR SPERMATOZOA USING RNA SEQUENCING

Fertility of cryopreserved spermatozoa is significantly reduced compared with that of their fresh counterparts, which is certainly due to the inflicted sublethal damage to spermatozoa that is observed at various molecular and cellular levels. The identification and characterisation of this damage will help us better understand sperm cryobiology and therefore develop suitable media and procedures to improve sperm cryopreservation and fertility outcomes, especially in swine. Here, we present our preliminary assessment of RNA pools of fresh and frozen‐thawed spermatozoa using RNA-sequencing technology. Semen ejaculates of 8 fertile boars were harvested and divided into 2 fractions for each ejaculate. Fraction 1 was freshly extended in commercial diluent (FD) and fraction 2 was frozen in 5-mL plastic straws (FT). Both specimens were shipped to our laboratory for analyses. The samples were purified through Percoll gradient centrifugation and resulting motile spermatozoa were washed in cold PBS. Pelleted spermatozoa were used for total RNA extraction, followed by an in-column DNase digestion. Purity and integrity of RNA samples were checked and rRNA depleted. After random priming, 40 million short cDNA reads were produced using Illumina RNA-Seq technology (Illumina Inc., San Diego, CA, USA). All reads were aligned to the pig reference genome and the produced genome-scale transcription maps consisted of both the transcript structure and the expression level of each gene mapped. Analysis of FD sperm RNA revealed a total of 18 357 sequence tags that were successfully mapped to all pig chromosomes and the mitochondrial genome. Frozen‐thawed spermatozoa showed only 16 864 sequence tags. In both FD and FT samples, chromosomes 1, 2, 6, 7, and 13 contained, in total, the highest density of mapped transcripts (>42%). Chromosome Y and mitochondrial RNAs had the lowest sequence tags mapped (<0.08%). A comparative analysis of FD and FT datasets revealed a net decrease in the total number of sequence tags (1493) with each chromosome being affected, except mitochondria. Chromosomes of FT samples showed a strong (>10%; 17, 7, 4, Y, and X) to moderate (10 to 5%) or weak (≤5%) reduction in RNA numbers. Structural annotation revealed a diverse population of sperm transcripts comprising both coding (mRNA) and noncoding (rRNA, snRNA, and mtRNA) RNAs. In both FD and FT samples, noncoding RNAs were among the most abundant sequence tags. Approximately 12 355 of sequence tags in FD v. 10 948 in FT spermatozoa were annotated with ENSEMBL and the selected genes are under investigation for comparative analyses using RT-PCR. In conclusion, mature boar spermatozoa contain a large pool of coding and non-coding RNAs that can be affected by the freezing-thawing procedure. Inflicted damage affects RNAs of all chromosomes with a great effect being seen on chromosome X. Generated datasets have the potential to lead to further study of the cryo-damage associated with reduced fertility of cryopreserved spermatozoa. Study was supported by USDA-ARS Biophotonics initiative grant # 58-6402-3-0120 and MAFES-SRI grants.