Early fetal sexing in the rhinoceros by detection of male‐specific genes in maternal serum

Genetic sexing of animals with long gestation time benefits the management of captive populations. Here, X and Y chromosome-specific primers, based on equine gene sequencing data, were developed and tested on captive rhinoceroses (10 males, 20 females) representing four species (Diceros bicornis, Certaotherium simum simum, Rhinoceros unicornis, and Dicerorhinus sumatrensis). The Y chromosome-specific primer set targeted SRY (Sex-determining region Y), and amplified a 177-bp product following PCR of DNA extracted from males, but not females, of all species. A primer set based on the equine AMEL (Amelogenin) gene resulted in a 232-bp product following PCR of all rhinoceros species. These gene-specific primer sets were then evaluated for their ability to determine gender in cell-free DNA from rhinoceros serum. Modifications to the original extraction and PCR protocols were required to obtain sufficient DNA quantities from serum, and both DNA yield and PCR amplification were substantially reduced or absent following multiple freeze-thaw cycles of serum. When fresh serum from 14 pregnant rhinoceroses (ultimately bearing seven male and seven female calves), representing four species at different stages of gestation (Days 61-490), were probed in a PCR-based assay, an accuracy of 71% was achieved for male-specific gene detection of SRY, which improved to 100% by including a reamplification step into the protocol. Such early sex determination should be a valuable tool for current management practices as well as future assisted reproduction of rhinoceroses.