Rapid and reliable DNA extraction method for the quantification of X- and Y- chromosome-bearing spermatozoa in ram semen

Accurate determination of X and Y chromosome-bearing spermatozoa ratios is a useful tool for reproductive management and livestock breeding efficiency. However, the highly condensed chromatin structure of spermatozoa presents a significant challenge for genomic DNA extraction, and current standard protocols are time-consuming. This study aimed to optimise ovine semen DNA extraction for the application of quantifying X and Y chromosome-bearing spermatozoa. A frozen ovine semen sample was divided into four groups (suspension of 30 ×10 6 spermatozoa cells). Three cell lysis methods; 1) 1 h + 10 µL DTT (dithiothreitol) (n = 8), 2) 1 h + 20 µL DTT (n = 7), and 3) 3 h + 20 µL DTT (n = 7), were designed to evaluate the genomic DNA (gDNA) compared to the 24 h + 10 µL of DTT (n = 8) (standard DNA extraction method). Quantitative Polymerase Chain Reaction (qPCR) SYBR Green assay was used to measure gDNA copy number from the extraction group using the housekeeping gene 18S rRNA . The ratio of X and Y chromosome-bearing spermatozoa was assessed using a qPCR TaqMan assay of Y-specific ( SRY ) and X-specific ( PLP ) genes. A gBlock was developed for each 18S rRNA , Y-specific ( SRY ) and X-specific ( PLP ) gene to generate the standard curve of both SYBR Green and TaqMan assays. The reference method of 24 h + 10 µL of DTT yielded the highest copies of extracted gDNA (3,040,919.6 copies ± 2,325,479.6), which was significantly greater than all other methods ( P < 0.05). Meanwhile, Y:X ratio differed significantly among methods ( P < 0.05) by the 24 h + 10 µL DTT and the 1 h + 10 µL DTT treatments yielded comparable Y:X ratios (1.32 ± 0.32 and 1.30 ± 0.21, respectively), indicating no significant difference between these methods. In contrast, 1 h + 20 µL DTT and 3 h + 20 µL DTT resulted in lower Y:X ratios (0.97 ± 0.22 and 0.97 ± 0.17). These findings indicated that the interaction between the DTT concentration and incubation period is critical for releasing spermatozoal DNA. Moreover, the amount of DTT is likely to affect the decondensation of X- and Y-chromosome-bearing spermatozoa differently. In conclusion, optimising the semen DNA extraction method, the 1 h + 10 µL DTT method is less time-consuming and reliable, making it suitable for routine laboratory verification of sperm sex ratio.