COMPARISON OF THE SEMEN SWIM-UP AND SOMATIC CELL LYSIS PROCEDURES FOR RAM SPERM RNA EXTRACTION

Male infertility is an important aspect of animal reproduction, which has a high economic impact on the livestock industry. From rapidly increasing list of different sperm fertility biomarkers, the sperm RNA could serve as a promising diagnostic tool to assess male fertility or could have prognostic value for fertilization and embryo development. The aim of this preliminary study was to compare swim-up and somatic cell lysis buffer (SCLB) procedures for extraction of high-quality ram sperm RNA suitable for downstream molecular biology applications. A modified TRI REAGENT RT procedure with glycogen and lysis step at 65 °C was carried out in order to extract total RNA. Spectrophotometric measurement of quality and quantity of extracted RNA showed A260/280 ratio 1.8 – 1.9, indicating the absence of contaminants and the amount of RNA 24 ± 3.9 µg (unpurified sperm), 0.9 ± 0.11 µg (swim-up) and 1.5 ± 0.2 µg (SCLB). Sperm RNA quality was further validated by RT-qPCR using primers for WBP2NL and MKRN1 genes. The CD18 and CDH1 markers for leucocytes and endothelial cells, respectively, have been used to check a successfull removal of somatic cells from ram sperm by both procedures. Unlike comparable relative amount of WBP2NL and MKRN1 transcripts among unpurified and swim-up or SCLB purified sperm RNA samples, relative amounts of CD18 and CDH1 transcripts were significantly lower in purified sperm RNA samples (p < 0.001), confirming an effective removal of leucocytes and endothelial cells from sperm by both purification techniques. Further investigations could reveal a potential of sperm RNA as a novel biomarker and promising diagnostic tool to assess ram fertility.