Researchers at the Federal University of Pelotas, Brazil, recently completed a study identifying circulating cell-free fetal DNA (ccffDNA) to determine fetal gender in pregnant mares. While ccffDNA has already been explored in humans, this is the first study to successfully demonstrate its presence ccffDNA–and thus aid in sex determination–in horses.

Generally, veterinarians determine fetal sex-typing via transrectal or transabdominal ultrasound of the mare. In the current study, researchers isolated ccffDNA in the blood plasma of 20 Thoroughbred mares in their final three months of pregnancy.

The current study was conducted in two steps:

• Step 1 involved a polymerase chain reaction (PCR) assay to detect ccffDNA in the plasma of pregnant mares. PCR determines sex at the molecular level by detecting the presence or absence of the Y chromosome. The expressed sex-determining SRY gene suggests a male, while the absence of SRY indicates female gender.
• Step 2 sought to validate results of the first PCR product through reamplification, producing 2nd-PCR and quantitative real-time PCR (qPCR) results.

The first stage PCR/SRY analysis produced an overall accuracy of 85%, with the second stage of analysis after reamplification achieving 95% accuracy. Researchers then confirmed the test results after foaling