The use of cryopreserved boar semen for artificial insemination is limited due to significantly lower fertility rates as compared to their fresh counterparts. Cryopreservation procedures induce sub-lethal damages that dramatically decrease viability, motility, and fertility potentials of spermatozoa; while the existence of good and poor freezer boars suggests a possible molecular predisposition to cryopreservation resilience. The underlying molecular mechanisms remain unknown, and the identification and characterization of latent sperm cryosensitive molecules may contribute to the improvement of freezing-thawing media compositions and protocol development. Here, we conducted an RNA sequencing of boar spermatozoa to assess such RNA transcripts.
Three independent semen ejaculates were obtained from known good (GF: n=4) and poor (PF: n=4) freezer boars. Each ejaculate was divided for fresh dilution in commercial extender and cryopreservation in 5-ml plastic straws. Fresh samples (F) were immediately purified through a Percoll gradient and resulting motile spermatozoa were washed twice in a cold-PBS. Pelleted spermatozoa of three ejaculates (per boar) were mixed and stored at -80 oC. Three months later, the same procedure was applied for frozen-thawed samples (C). Total sperm RNA was extracted with rRNA depletion, subjected to in-column DNase digestion, and checked for purity and integrity. Samples were subjected to Illumina RNA-Seq technology. All reads were aligned to the pig genome (Hisat2). With ~ 48-62% alignment rates and features counted at the exon level (FeatureCounts), differentially expressed genes were discovered (EdgeR). Data were analyzed and compared as Group 1 (8F vs. 8C), Group 2 (4 Fresh GF vs 4 Fresh PF), Group 3 (4 Cryopreserved GF vs. 4 Cryopreserved PF), Group 4 (4PF fresh vs. 4PF cryopreserved), Group 5 (4GF Fresh vs. 4 GP Cryopreserved).
A total of 77919 transcripts were found, irrespective of groups. Approximately 2836, 2042, 1715, 6871 and 6552 transcripts were detected with confidence (P<0.05). Totals of 51, 14, 6, 65, and 18 were differentially detected within each respective Group 1, Group 2, Group 3, Group 4, and Group 5 (P and FDR<0.05). Using a 1% FDR threshold, a further analysis resulted in the differential expression of 19, 13, 6, 3, and 12 transcripts within Groups 1, 2, 3, 4, and 5, respectively. Numerous detected transcripts such as ryanodine receptors, protamines, and tubulins are already reported in the literature.
In summary, these preliminary data indicate that mature boar spermatozoa contain large pools of messenger RNAs that are affected by freezing-thawing procedures. These data suggest eventual predisposition of boars to semen freezability variations. We are investigating all cryosensitive transcripts for further comprehension of sperm cryobiology, as these transcripts may become or allow the development of reliable biomarkers for boar semen freezability status. This achievement will assist in better decision-making for sperm storage in studs. Supported by USDA-ARS Biophotonics #58-6402-3-018.