Abstract: Przewalski's horse is the world's endangered species. There are less than 2 000 in the world,and the vast majority of them are captive or semi-wild individuals. It is of great significance to study the breeding and development of Przewalski's horse for the rejuvenation and returning to wild state of this species. Methanol has particular effect on detecting fecal steroids. To determine optimal dilution ratio when using ELISA method to detect fecal steroids of Przewalski's horse,we used PBS buffer to dilute the extracted samples which stored in methanol at a series ratios before detecting. The results showed when detecting progesterone (P),sample was diluted 15 times; detecting testosterone (T),sample was diluted 15 times; detecting estradiol (E2),sample was diluted 30-50 times,the enzyme-labelled antigen binding rate B/B0(%) closed to 50%. It means that 50% coating antibody was bound by the enzyme-labelled antigen,and at that time detecting results were more sensitive. The result provides support in the way for researching breeding and development of non-invasive species.

Key words: Przewalski's horse, fecal steroids, methanol, ELISA, dilution ratio