Reproduction

Rapid, Cost-Effective and Reliable semen DNA Extraction Method for quantification of X and Y chromosome-bearing spermatozoa

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Abstract

This study aimed to optimize ovine semen DNA extraction for the application of quantifying X and Y chromosome-bearing spermatozoa (X and Y-CBS). A pooled frozen ovine semen sample was divided into four groups (n=10/group, suspension of 30 ×106 spermatozoa cells). Three cell lysis methods: 1) 1h+10 uL DTT (Dithiothreitol), 2) 1h+20 uL DTT, and 3) 3h+20 uL DTT were designed to evaluate the genomic DNA (gDNA) compared to the 24h+10 uL of DTT (control) DNA extraction method). Quantitative Polymerase Chain Reaction (qPCR) SYBR Green assay was used to measure gDNA from the extraction group using 18S rRNA (a housekeeping gene). The ratio of Y and X - CBS was assessed using a qPCR TaqMan assay of Y-specific (SRY) and X-specific (PLP) genes. A gBlock Gene fragment was used to develop the standard curve of both SYBR Green and TaqMan assays. The result showed that 24 h + 10 µL of DTT method (control) yielded the highest amount of extracted gDNA, which was significantly greater than all other methods (p < 0.05). Meanwhile, the ratio of Y and X-CBS (Y:X ratio) differed significantly among methods (p < 0.05) by the 24 h + 10 µL DTT (Control) 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 significantly lower Y:X ratios (0.97 ± 0.22 and 0.97 ± 0.17). These findings indicated that the interaction between the Dithiothreitol (DTT) concentration and incubation period is critical for achieving the release of spermatozoal DNA. Moreover, the amount of DTT is likely to affect the decondensation of X- and Y-chromosome-bearing spermatozoa differently. In conclusion, optimizing the semen DNA extraction method, the 1 h + 10 µL DTT method is less time-consuming and reliable. These would be beneficial for inspecting the quality of imported or costly sex-sorted semen, making it suitable for routine portable laboratory setup or field use for quantifying X and Y-CBS before artificial insemination.

Keywords: 2026

How to Cite:

SAENGRUANG, N., (2026) “Rapid, Cost-Effective and Reliable semen DNA Extraction Method for quantification of X and Y chromosome-bearing spermatozoa”, World Congress on Genetics Applied to Livestock Production Digital Archive 2026(1): 2286127. doi: https://doi.org/10.31274/wcgalp.23856

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