Ejaculated bovine spermatozoa retain a pool of RNAs that may have a function in early embryogenesis and be used as predictors of male fertility. The bovine spermatozoal transcript profile remains incomplete because previous studies have relied on hybridization-based techniques, which evaluate a limited pool of transcripts and cannot identify full-length transcripts. The goal of this study was to sequence the complete cryopreserved bovine spermatozoal transcript profile using Illumina RNA-Sequencing (RNA-Seq). Spermatozoal RNA was pooled from nine bulls with conception rate scores ranging from -2.9 to 3.5 and confirmed to exclude genomic DNA and somatic cell mRNA. After selective amplification of poly(A)(+) RNA and high-throughput sequencing, 6166 transcripts were identified via alignment to the bovine genome (UMD 3.1/bosTau6). RNA-Seq transcript levels (n = 9) were highly correlated with quantitative PCR copy number (r(2) = 0.9747). The bovine spermatozoal transcript profile is a heterogeneous population of degraded and full-length predominantly nuclear-encoded mRNAs. Highly abundant spermatozoal transcripts included PRM1, HMGB4, and mitochondrial-encoded transcripts. Full-length transcripts comprised 66% of the top 368 transcripts (fragments per kilobase of exon per million fragments mapped [FPKM] > 100) and amplification of the full-length transcript or 5' and 3' ends was confirmed for selected transcripts. In addition to the identification of transcripts not previously reported in spermatozoa, several known spermatozoal transcripts from various species were also found. Gene ontology analysis of the FPKM > 100 spermatozoal transcripts revealed that translation was the most predominant biological process represented. This is the first report of the spermatozoal transcript profile in any species using high-throughput sequencing, supporting the presence of mRNA in spermatozoa for further functional and fertility studies.

Spermatozoal messenger RNA (mRNA) has the potential as a molecular marker for sire fertility because this population can reflect gene expression that occurred during spermatogenesis and may have a functional role in early embryonic development. The goal of this study was to compare the oligo-dT selected spermatozoal transcript profiles of higher fertility (Conception Rate (CR) 1.8-3.5) and lower fertility (CR -2.9 to -0.4) sires using Ribonucleic Acid Sequencing (RNA-Seq). A total of 3227 transcripts and 5366 transcripts were identified in the higher and lower fertility populations, respectively. While common transcripts between the two populations were identified (2422 transcripts), several transcripts were also unique to the fertility populations including 805 transcripts that were unique to the higher fertility population and 2944 transcripts that were unique to the lower fertility population. From gene ontological analysis, the transcripts unique to each fertility population differed in Biological Processes (BP), including enrichment of regulatory transcripts for growth and protein kinase activity in the higher fertility bulls. Biological variation in transcript presence among individual sires was also found. Of the candidate fertility spermatozoal transcripts chosen from the RNA-Seq population analysis reported here and previous publications, COX7C was negatively correlated with sire fertility. Using high-throughput sequencing, candidate spermatozoal transcripts were identified for further study as potential markers for sire fertility.

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Ejaculated bovine spermatozoa retain a pool of RNAs that may have a function in early embryogenesis and be used as predictors of male fertility. The bovine spermatozoal transcript profile remains incomplete because previous studies have relied on hybridization-based techniques, which evaluate a limited pool of transcripts and cannot identify full-length transcripts. The goal of this study was to sequence the complete cryopreserved bovine spermatozoal transcript profile using Illumina RNA-Seq. Spermatozoal RNA was pooled from nine bulls with conception rate (CR) scores ranging from-2.9 to 3.5 and confirmed to exclude genomic DNA and somatic cell mRNA. After selective amplification of polyA + RNA and high-throughput sequencing, 6,166 transcripts were identified via alignment to the bovine genome (UMD 3.1/bosTau6). RNA-Seq transcript levels (n=9) were highly correlated with qPCR copy number (r 2 =0.9747). The bovine spermatozoal transcript profile is a heterogeneous population of degraded and full-length predominantly nuclear-encoded mRNAs. Highly abundant spermatozoal transcripts included PRM1, HMGB4 and mitochondrial-encoded transcripts. Full-length transcripts comprised 66% of the top 368 transcripts (FPKM>100) and amplification of the full-length transcript or 5' and 3' ends was confirmed for selected transcripts. In addition to the identification of transcripts not previously reported in spermatozoa, several known spermatozoal transcripts from various species were also found. Gene ontology analysis of the FPKM>100 spermatozoal transcripts revealed that translation was the most predominant biological process represented. This is the first report of the spermatozoal transcript profile in any species using high-throughput sequencing, supporting the presence of mRNA in spermatozoa for further studies.

No abstract