156 Somatic cell lysis influences protamine 1 mRNA abundance in stallion frozen-thawed sperm

The study of sperm RNAs requires species-specific extraction protocols and is particularly challenging because of its reduced content and fragmented nature. The aim of this study was to optimize RNA isolation techniques in frozen-thawed (FT) stallion sperm, with particular focus on the importance of somatic cell lysis (SCL), and to identify differences in gene expression ascribable to the treatment. The FT semen from different stallions (n = 10) was included. After thawing, semen was assessed for sperm kinetics (CASA), morphology (Hancock), and chromatin condensation (Aniline blue). The remaining sample underwent one centrifugation step with Equipure followed by two centrifugation steps in PBS. At this point, each sample was divided into two parts: one was treated for SCL (group SCL), and the other was not (group NSCL). From each aliquot, bead-based homogenization (3×) in TRIzol was performed and RNA was isolated using Direct-zol columns. The RNA contamination by solvents (260/230 ratio) and by proteins (260/280 ratio) was evaluated, together with RNA integrity (DV200 Index) and RNA yield (TapeStation). Differences in the abundance of genes previously proven to be correlated with stallion fertility (PRM1, PRM2, PRM3, CRISP3, PLCz1, ACR, ZPBP, PRDX5, NOX5, SOD) between groups were determined byRT-qPCR (reference genes: ACTB and RPL32; somatic cells markers: PTPRC and CDH1). Statistical analysis was performed with univariate analysis and chi-squared test (IBM-SPSS statistics 29.0.1.0). The inclusion of SCL did not influence (P > 0.05) RNA contamination (260/230 and 260/280), RNA integrity, or RNA yield. Among somatic cell markers, PTPRC was detected in 3 of 10 samples of the NSCL group but not in any SCL sample (P > 0.05). CDH1 was detected in one SCL and five NSCL samples (P < 0.05), respectively. With regard to RNA abundance, only PRM1 was overexpressed (P < 0.05) in SCL. In conclusion, SCL did not influence RNA quality, but inclusion of SCL prior to RNA extraction might be beneficial because somatic cell markers are almost absent, while PRM1 (sperm-specific) shows higher abundance. This result suggests that the extraction protocol affects RNA abundance in FT stallion semen. Further studies are needed to identify the best and most reliable method to isolate RNA from sperm.