TDK conference 2019

From the literature reviewed concerning elephant population and management, fetal sex diagnoses and molecular sexing methods, we can resolve that there is a clear need to develop accurate methods of early fetal sex diagnosis of elephants, as the potentiality of ultrasound and hormonal measurements for fetal sex diagnosis are unfeasible or are presently limited. Concerning placentation types and period of gestation it is apparent that although these factors have potential to limit the successful detection of cell free fetal (cff) DNA. It has been shown also, that with appropriate methods of DNA extraction along with increased sensitivity of PCR techniques it is possible to investigate circulating cffDNA from animals with varied placentation and at early points of gestation.

Accurate molecular methods of sex diagnosis of mammals have been developed for mammals including elephants. The primary objective of this study was to investigate the presence of cell free fetal DNA in the maternal plasma of African elephants by developing and using a reliable PCR system combined with a highly sensitive capillary electrophoresis for detection of amplified PCR products.

Firstly, we designed three primers specific to Asian and African elephant species. The PlpX gene is present in the X-sex chromosome and the SRY and AmelY genes are specific for the Y-chromosome therefore can only be detected in males. Hair samples from both species and both sexes have been tested for accurate sex diagnosis in different dilutions of isolated DNA. The Y-chromosome marker was only detectable in male samples as expected, and the X-chromosome specific marker gave positivity in both sexes after PCR amplification by capillary electrophoresis detection.

In the next step, two samples originating from African elephant cows being in the last trimester of pregnancy have been tested for fetal sex. From one sample, both markers gave positive results and was diagnosed as male, and the other sample was positive only in the X-chromosome marker and signed as female. The calves’ sexes have agreed with the test results.

To summarize, this is the first study on confirming the presence of fetal cell-free DNA in pregnant elephant plasma, demonstrating a new opportunity for non-invasive assessment for fetal sex determination. We hope that our method will have great potential as a management tool for zoos and other captive elephant institutions that currently breed elephants. By prenatal sex diagnosis of elephants in captivity they can make further arrangements to the placement of such offspring or even the outcome of the pregnancies, in order to help maintain appropriate sex ratio and healthy elephant populations