Gender determination by PCR assay for the sex-determining region Y (SRY) gene amplification in Linnaeus’s two-toed sloth (Choloepus didactylus)

In Linnaeus’s two-toed sloths (Choloepus didactylus), there is no distinct sexual dimorphism. It is an obstacle for gender determination from the external genitalia, especially in newborns or young sloths. Hence, easy, rapid, and reliable genetics-based methods for gender identification of the sloths are needed to continue captive breeding more successfully. In this study, a PCR-based technique that allows gender determination of two-toed sloths by using a sex-determining region Y (SRY) gene marker was described. The hair samples from young (suspect gender) and adult sloths (known gender) were used in genetic analysis. Initially, genomic DNA was isolated from hair root samples using Roche high pure PCR template preparation kit. The SRY primers were specifically designed based on the NCBI and Ensembl databases, and they were verified with the BLAST program concerning the two-toed sloth genome. PCR amplification with the SRY-specific primers was carried out by a programmable thermal cycler device using FastStart High Fidelity PCR System, Roche dNTPack. The samples were then electrophoresed on 2% agarose gels and were visualized by a gel documentation and analysis system. A specific band in the electrophoresis pattern is diagnostic for a male individual with a partial SRY region. Hence, the analysis demonstrated that the samples belonged to a male two-toed sloth. Two-toed sloth species are commonly preferred animals in zoos. Gender determination is inevitable for these animals in captivity to be raised successfully and healthily. Molecular genetic techniques allow high efficiency in taxonomic evaluations and gender identification in species that do not display sexual dimorphism. The PCR assay described here may be helpful for a rapid genetic analysis that can be widely used in gender determination for two-toed sloths.