Feasibility study on bovine fetal sexing utilizing circulatory cell-free fetal DNA in maternal peripheral blood

Abstract

The studies conferred in this thesis were performed to establish an appropriate PCR methodology to target sex-chromosome specific genes for prenatal fetal sex determination in pregnant cows. The first study determined the effect of frozen and fresh plasma on cell-free fetal DNA (cffDNA) extraction for PCR-based fetal sexing and compared different DNA extraction methods on downstream PCR for fetal sexing in cows by detecting sex-chromosome-specific gene products. We found that none of the DNA extracts obtained from frozen plasma samples of cows (120-150 days pregnant) yielded any male fetus-specific PCR products, although all cows were carrying male fetii. In contrast, male fetus-specific amplicons were successfully amplified in 5/6 DNA extracts obtained by the phenol-chloroform method when fresh plasma samples were used. Although the smaller sample size was a limitation, we further found that DNA extraction methodologies compared in the present study (DNeasy Blood & Tissue Kit, QIAamp DSP Virus Kit, QIAamp DNA Blood Midi Kit, NucleoMag cfDNA Isolation Kit, MagMAX cfDNA Isolation Kit, KAPA Express Extract Kit, Salting-out protocol, and Phenol-chloroform method) appeared to show variability in their ability to isolate fetal DNA from freshly harvested maternal plasma/blood of pregnant cows. The second study determined the minimum concentrations of cffDNA that must be present per milliliter of maternal plasma for successful extraction and PCR. In addition, we also compared Y-specific sequence PCR-based fetal sexing results in the cows divided into two gestational stages. We concluded that the spiked serum and cellular DNA from aborted male fetii could be successfully re-extracted (Blood & Tissue Kit) from maternal plasma at all dilution rates (0.5-100%). Furthermore, the lowest concentration of spiked fetal DNA for successful extraction (from maternal plasma) followed by Y-specific PCR and bAML PCR was found to be >31.25 pg/ml and >2ng/ml, respectively. Besides, we also found that the proportion of cows with a positive Y-specific PCR outcome were higher (P<0.05) in 151-240 days pregnant cows (90%; CI: 55.5-99.75) than 60-150 days pregnant cows (33%; CI: 7.5-70.1). Lastly, we found that the lower limit (35 pg/ml) of spiked male fetal DNA could be successfully recovered from maternal plasma of advanced pregnant cows (using Blood & Tissue Kit, DSP Virus Kit, and NucleoMag cfDNA Isolation Kit) for Y-specific PCR. However, there was variability in the Y-specific PCR results when neat plasma samples from the same pregnant cows were processed for DNA extraction using these kits. In conclusion, fresh but not frozen plasma could be used for PCR-based prenatal fetal sexing. Moreover, due to variability between different DNA extraction methodologies, the PCR assay for fetal sex determination was unreliable for fetal sexing in pregnant cows. Besides, cffDNA could be consistently extracted from maternal plasma for Y-specific PCR when present at amounts ≥31.25 pg/ml. Although a significantly higher proportion of samples from advanced pregnant cows yielded Y-specific fetal DNA amplicons, the study needs to be replicated on a large dataset to confirm our observations.