Molecular Sex Determination in Caenophidian Snakes Using qPCR Amplification of Sex-Linked Genes: Validation and Interspecific Comparison

Accurate sex identification in reptiles with genotypic sex determination is essential for breeding management, veterinary care and evolutionary research, yet commonly used methods are often invasive, stressful or unreliable. This study aimed to evaluate a dosage-based quantitative PCR approach for molecular sex determination in caenophidian snakes, using naturally shed epidermal skin as a non-invasive DNA source. Genomic DNA extracted from shed skin was analysed by qPCR targeting conserved Z-linked genes (ADARB2, ARMC4 and TANC2), together with autosomal and reference genes, to assess sex-specific differences in gene copy number. Sixteen caenophidian snake species were examined, including taxa for which molecular sexing data are currently scarce or unavailable. The autosomal control gene showed dosage ratios close to parity between sexes, supporting DNA quality and reference gene reliability; meanwhile, Z-linked markers generally exhibited reduced dosage in females relative to males, consistent with a ZZ/ZW sex determination system. These results demonstrate that dosage-based qPCR applied to shed epidermal skin provides a promising and non-invasive framework for molecular sex determination in caenophidian snakes, without compromising animal welfare.