Evaluation on the effects of ageing factor, sampling and preservation methods on Asiatic black bear (Ursus thibetanus) noninvasive DNA amplification

Noninvasive genetic sampling allows studying wildlife without having to catch, handle or even observe individuals. In this study, factors which may affect the quality of noninvasive samples of Asiatic black bear (Ursus thibetanus) in the subtropical areas were identified. We collected hair and faecal samples from captive Asiatic black bears and quantitatively evaluated the effects of hair age (from fresh to 60 days), faeces age (from fresh to 14 days), faeces sampling locations (i.e. sample collected from either the surface, inside or a mixture of both the surface and inside of faeces), and faeces preservation methods (frozen or kept at room temperature in 95% ethanol) on amplification success rates of mitochondrial DNA fragments of different sizes (450bp, 900bp, and 1600bp). The results showed that the amplification success rates decreased with sample age and amplicon size in both hair and faecal DNA. In subtropical environment, there was no significant difference between amplification success of DNA extracted from fresh and 7-day-old samples of either the hair or faeces. The amplification success rates were not influenced by sampling location of faeces. For faeces preserved in 95% ethanol, the amplification success appeared unaffected by frozen at -20 °C or kept at room temperature in shorter mtDNA fragments, but was significantly influenced when amplicon size was 1600bp. The results of this study will reinforce the optimization of noninvasive sampling approaches in Asiatic black bear research, especially in the subtropics