In zoo husbandry and wildlife conservation, accurate sex identification is critical for American mammals with indistinct sexual dimorphism, yet relevant molecular studies are limited. In this study, the Y-chromosomal SRY gene, a core male sex-determining factor in mammals, was investigated. Primers were designed for its conserved regions, and PCR amplification, sequencing, and bioinformatic analysis were conducted on seven Neotropical mammals (e.g., two-toed sloth, jaguar). Their SRY nucleotide sequence similarity reached 83.2-95.0%. Over 60 American mammals from Carnivora, Xenarthra, Primates, Artiodactyla and Rodentia were further analyzed via GenBank data: a phylogenetic tree was built, primer binding sites were predicted, and the target fragment was found to have purifying selection (dN/dS = 0.352) and span three HMG-box motifs. This study provides a potential cross-species sex identification method for American mammals, offering molecular support for precise zoo management and wild conservation, with important practical and scientific value.
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