Identifying the minimum concentrations of cell-free fetal DNA in maternal blood required for bovine fetal sexing using PCR.

We evaluated the feasibility of cffDNA extraction from the maternal blood samples regarding the threshold concentrations required for fetal sexing in pregnant cattle by PCR. In four trials, we 1) compared the extraction efficiency of seven methods using freshly harvested plasma/blood of cows carrying male fetii (150-240 d gestation) bovine amelogenin (bAML) and Y-specific gene sequences, 2) identified the minimum amounts of spiked cffDNA needed for a PCR for fetal sexing, 3) determined the most optimal protocol among three commercial kits for cffDNA extraction from neat and spiked plasma samples (181-240 d gestation) for PCR detection of Y-specific sequence and 4) tested Y-specific sequence PCR on pregnant cows at different stages of gestation (60-150 versus 151-240 d pregnant). In these experiments, blood samples from unbred dairy heifers (Canadian Holstein, n = 10), pregnant dairy cows (Canadian Holstein, 60-240 d gestation, n = 25 with male fetii), and aborting beef cows (Angus cross, n = 5, 100-150 d pregnant) were used for DNA extraction, spiking, and PCR. Extracted DNA from the blood samples of unbred heifers (n = 5) and bull calves (n = 5) served as controls in all trials. In the first trial, DNeasy Blood and Tissue, Qiagen DSP Virus, and NucleoMag cfDNA isolation kits were relatively successful among seven methods to isolate cffDNA from freshly harvested maternal plasma/blood of pregnant cows. In trial 2, using serial dilutions of cffDNA from male fetii spiked in cow plasma samples, a positive and unambiguous detection by PCR targeting Y-specific sequence and bAML gene was observed when spiked cffDNA concentration in plasma was >31.3 pg/ml and >2 ng/ml, respectively. In the third trial, the DNeasy Blood and Tissue kit was most successful in extracting cffDNA spiked at the minimum concentrations in maternal plasma and subsequent PCR for Y-specific sequence. In our fourth trial, more cows in the second half (9/10) of gestation showed a positive Y-specific PCR outcome than those in the first half (3/9, Fischer's exact test; P < 0.05, 90%; CI: 55.5-99.75 vs 33%; CI: 7.5-70.1). In conclusion, we observed variability between different DNA extraction methodologies and stages of gestation results in the PCR for prenatal sexing. Thus, the current PCR methodologies are unreliable for detecting cffDNA in pregnant cows. Additionally, ≥10 (≥31.3 pg/ml of cffDNA) and ≥648 (≥2 ng/ml of cffDNA) copies of the whole fetal genome in bovine maternal plasma are needed for Y-specific PCR and bAML PCR, respectively.