- Transcriptome gene expression studies of sperm RNA have been utilized in a variety of different species to investigate causes of male infertility. Previous research investigators have optimized cell separation and RNA isolation techniques for each species of interest. To date, no study has been completed for dogs. The objective of this thesis research was to investigate the efficacy of various cell separation techniques in separating sperm cells from somatic cells in the ejaculate to yield a sample of total pure sperm RNA that could be used for a future downstream application. Comparisons were made between the conventional swim-up method and two commercial density gradient centrifugation (DGC) solutions (Bovipure[supercript]TM and Equipure[superscript]TM, Nidacon International, Mölndal, Sweden) Prior to and just following cell separation, total motility, normal morphology, and sperm count were determined for each method. Following cell separation, total RNA was isolated from each sample and RNA quantity and quality was determined via spectrophotometry and reverse transcriptase polymerase chain reaction. The presence of somatic cell RNA was used to determine purity of the cell separation method. The DGC methods were superior in separating sperm with higher percent normal morphology and higher total motility than the swim-up method. Additionally, the DGC methods were superior at producing a more pure RNA sample than samples not treated with any
separation technique (control). This research shows that the DGC methods should be used to separate canine sperm cells prior to RNA isolation for sperm-specific transcriptome applications.