Application of restriction endonuclease enzymes for sex determination in selected mammalian species.

BACKGROUND: In livestock breeding and forensic cases, it may be necessary to determine the sex of the species, as well as the sex of animal-derived products intended for consumption. For this purpose, in addition to molecular-based techniques, several different approaches are employed. The aim of this study was to investigate the feasibility of determining the sex of various mammalian species by different restriction endonuclease enzymes (REs). METHODS AND RESULTS: In this study, DNA was isolated from blood samples previously stored in our laboratory, obtained from one male and one female individuals belonging to five mammalian species (goat, sheep, cattle, horse, and dog). The DNA samples were amplified via PCR using primers specific to the zinc finger genes (ZFX/Y). The PCR products were digested using 10 different REs (EcoRI, MspI, SmaI, AlwI, PstI, NdeI, HaeIII, TaqI, DdeI, and AluI), and were visualized via gel electrophoresis. PCR products were successfully obtained from all species, and six of the REs yielded species-specific band patterns that allowed for sex differentiation. The enzymes AlwI, PstI, and NdeI were found to differentiate sex in cattle; HaeIII in horses; and HaeIII, TaqI, and DdeI in dogs. CONCLUSIONS: This is the first study to employ a large panel of REs for sex determination in common domestic animal species. Furthermore, this study is the first to demonstrate that two enzymes (AlwI and NdeI) in cattle, one enzyme (HaeIII) in horses, and two enzymes (TaqI and DdeI) in dogs can be used for sex differentiation within these species.