Development of a quantitative assay for the non-invasive determination of fetal sex in Sable and Roan antelope

Abstract

The research field of cell free DNA (cfDNA) and cell free fetal DNA (cffDNA) has expanded in the past decade, with the inclusion of animal based cfDNA studies. This has resulted in an increase in research making use of cffDNA in prenatal sex determination studies on animals. This study attempted to be the first study to confirm the presence of cfDNA as well as cffDNA in the maternal plasma of pregnant female sable and roan antelope species. The aim of this study was to develop a non-invasive cffDNA-based quantitative assay for foetal sex determination of the sable and roan antelope prenatally. cfDNA was isolated from pregnant female sable antelope plasma samples to determine the foetal sex of the animal using a non-invasive technique. Due to lockdown regulation imposed by the COVID-19 pandemic, pregnant female roan antelopes blood samples could not be obtained. The SRY gene, which is only present in the male species, was used as the target gene. The sex determining region y (SRY) gene of the sable and roan antelope was sequenced to determine the level of conservation of the SRY gene between and within species as well as to design primers for the quantification assay methods. Mitochondrial DNA sequences were used to identify antelope sub-species. There were no differences found between the SRY gene sequence of the sub-species of sable antelope as well as between the SRY gene sequence for the roan antelope sub-species. Two different qPCR assays, SYBR Green qPCR and probe-based qPCR assay, as well as the MassARRAY® system was developed to identify the optimal method to be used for the foetal sex determination using cffDNA. However, due to low cfDNA yields from the blood samples, only the SYBR Green qPCR and the MassARRAY® methods could be evaluated to determine the sex of the calves. The results obtained were compared to the sex of the calves at birth. For the SYBR Green qPCR assay, 60% of the samples gave positive results, while 40% were inconclusive. For the MassARRAY® system only one out of five single nucleotides polymorphisms (SNPs) gave a result for 6 samples and only 20% of these results corresponded to the sex at birth. Based on these results, the isolation of cfDNA from plasma samples needs to be optimised to improve yield. Further testing of these two techniques should be done for future studies to determine which techniques would be more reliable to determine the sex of the calf prenatally using cffDNA.