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

Abstract

In Linnaeus’s two-toed sloths (Choloepus didactylus), there is no distinct sexual dimorphism. It is an obstacle for gender determination fromthe external genitalia, especially in newborns or young sloths. Hence, easy, rapid, and reliable genetics-based methods for gender identificationof the sloths are needed to continue captive breeding more successfully. In this study, a PCR-based technique that allows gender determinationof two-toed sloths by using a sex-determining region Y (SRY) gene marker was described. The hair samples from young (suspectgender) 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, andthey were verified with the BLAST program concerning the two-toed sloth genome. PCR amplification with the SRY-specific primers wascarried out by a programmable thermal cycler device using FastStart High Fidelity PCR System, Roche dNTPack. The samples were then electrophoresedon 2% agarose gels and were visualized by a gel documentation and analysis system. A specific band in the electrophoresis patternis diagnostic for a male individual with a partial SRY region. Hence, the analysis demonstrated that the samples belonged to a male two-toedsloth. Two-toed sloth species are commonly preferred animals in zoos. Gender determination is inevitable for these animals in captivity tobe raised successfully and healthily. Molecular genetic techniques allow high efficiency in taxonomic evaluations and gender identification inspecies that do not display sexual dimorphism. The PCR assay described here may be helpful for a rapid genetic analysis that can be widelyused in gender determination for two-toed sloths.