TDK conference 2020

Fetal-sex determination of unipara mammals with long gestation can benefit the management of economically important as well as captive wild populations, but feasibility of currently available methods is limited to very few species. Based on the hypothesized presence of cell free fetal DNA (cffDNA) in maternal circulation of all unipara species, the primary objective of this study was to develop a single, universal cffDNA-based assay for fetal-sex determination.

Highly conservative regions of Y-specific genes (SRY, TSPY, Amelogenin) and an autosomal gene (GAPDH, used as a reaction control) were identified by in silico analysis, and universal primers were designed for the most appropriate gene segments. Selected primer pairs were tested on control samples (hair, feces, muscle, blood) from individuals of known sex collected from a wide range of relevant species, representing primates (e.g. gorilla), even and odd toed ungulates (e.g. cattle, mouflon, takin, equine, Grévy’s zebra, rhinoceros), proboscideans (e.g. african elephant) and carnivores (e.g. sea lion).

After primer testing with simplex (single marker) PCR programs, the base sequence of specific products was confirmed by sequencing. Subsequently, we optimized a duplex (two-marker) reaction, using touch-down PCR technique. The amplified fragments were detected by agarose gel and capillary electrophoresis.

After the optimization process, as expected, both Y-chromosomal and autosomal segments were detected in high-quality male control samples, while only the GAPDH gene segment was detected in female samples.

Following preliminary testing, to evaluate the ability of the assay to determine fetal sex from maternal blood, plasma samples of pregnant zebra, equine and cattle with a known gender of the offspring were tested up to the current stage of the study. Testing of samples from additional species is necessary and in progress to accurately determine reliability, specificity, and sensitivity of the test.

By successful development of such a universally applicable method our research could rapidly promote both economic-, but most notably conservation-related breeding activities of a wide range of species, and beyond it could be the first to provide information about the presence of cffDNA in maternal blood of various, not yet studied mammalian species.