DNA sampling – To justify our current field sexing techniques to a 95% accuracy or create more accurate field sexing methods, blood samples were taken as a non-lethal method to determine the sex of the rails. The goal was to collect 100 DNA samples total, 25 from each age-sex group (AHY = after hatch year and HY = hatch year birds, based on fall plumage) (25 AHY-M, 25 AHY-F, 25 HY-M, and 25 HY-F). A Head shot picture was taken of all blood sampled birds to see if plumage characteristics had a recognizable pattern to distinguish sex.

To extract DNA samples from the rails the less invasive method of nail clipping was used. One nail from the middle toe of the banded (right) leg of an individual bird was disinfected using a Q-tip soaked in rubbing alcohol to remove any foreign DNA. The nail was then clipped just enough to nick the vein (usually 2/3 of the distance from the root of the nail) using sanitized nail clippers. A few drops of blood were then rubbed onto the DNA Sample Card provided by the DDC Veterinary Company. The card was then placed in Tupperware to allow the sample to dry for 60 to 90 minutes and remain protected from bacterial or fungal growth before placing into a properly labeled re-sealable plastic bag. All researchers participating in the DNA sampling were wearing sanitary gloves that were changed after each bird to prevent cross-contamination. After the blood sample is taken Wonder Dust, mainly used on horse hooves, is applied to the clipped nail. The Wonder Dust quickly clots the bleeding and prevents infection. DNA samples were then packaged and sent to DDC Veterinary in Ohio.

DDC Veterinary tests DNA using methods based on Griffith’s method published in 19986. Unlike humans where the sex chromosomes are XX for female and XY for male, bird sex chromosomes are WZ for females and ZZ for males. Using a Polymerase Chain Reaction, DDC Veterinary can detect the presence of a single CHD-Z band in male DNA samples and a second, distinctive CHD-W band in female DNA samples.