Development of molecular sex identification method for Asian palm civet (Paradoxurus hermaphroditus) and Small Indian civet (Viverricula indica): a step towards forensic identification

Civet coffee and perfume are premium products in demand worldwide. Consequently, the civet population is threatened by several factors (e.g., the rapidly growing trend of civets being removed from the wild and held in captivity), raising concerns about civet welfare and conservation. In light of this, our aim was to develop a molecular sex identification method for the Asian palm civet ( Paradoxurus Hermaphroditus ) and the Small Indian civet ( Viverricula Indica ). We collected five Asian palm civet tissue samples (two male, one female, and two of unknown sex) and one female Small Indian civet from the repository, Advanced Institute for Wildlife Conservation (AIWC). DNA was extracted from all tissue samples using the DNeasy Blood and Tissue Kit. For sex identification, we optimized the PCR conditions to amplify partial fragments of sex-determining region Y ( Sry ) and the zinc finger X ( Zfx ) genes using DNA from two male Asian palm civet samples. The PCR amplicons were examined on a 2% agarose gel, and two male civets showed one distinct band at 250 bp (for Sry ) and another at 120 bp (for Zfx ). To confirm this finding, the PCR amplicons ( Sry and Zfx genes) from one male sample were sequenced and % similarity and nucleotide pairwise analysis were calculated. The Sry and Zfx sequences showed 96.99% (for Sry ) and 97.78% ( Zfx ) similarity to the masked palm civet, the only species in the families Paradoxurinae or Viverridae for which sequence data was publicly available. These results confirmed that the optimized PCR conditions are suitable for amplifying the Sry and Zfx genes in the Asian palm civet, which will be helpful for sex identification. Subsequently, we used this optimized method for the sex identification of three Asian palm civets and one Small Indian civet sample obtained from the repository, AIWC. Additionally, we used the method for identification of sex in a seized civet tissue sample received for forensic analysis. This report is the first to focus on developing a method for identifying the sex of Asian palm civets and Small Indian civets. This method will be helpful for researchers working on the ecology and conservation of civet population.