Microsatellite analysis of cryopreserved stallion semen stored on FTA paper

The aim of this study was to establish and validate a method to permit microsatellite analysis of DNA profiles obtained from frozen-thawed stallion sperm cells. This would provide reliable and accurate verification of the identification of a semen donor. Ejaculates from 5 pony stallions were collected, processed and frozen in 0.5 ml plastic straws. Aliquots of 100 microl of the frozen-thawed semen thus obtained were either placed directly, or diluted (1:10; 1:100; and 1:1000) and placed on slides of FTA paper. Similarly, blood samples obtained from each of the stallions were placed onto slides of FTA paper. A punch was removed from each sample after drying Each sample was mixed with FTA purification reagent, Dithiothreitol and Proteinase K before incubation and processing. All samples were processed with a set of 13 microsatellite markers. Further analysis permitted a comparison of the DNA profiles of the frozen-thawed semen and the blood samples. A full profile of markers was obtained from the 1:10 and 1:100 dilutions of the frozen-thawed semen samples as well as from the blood samples. The DNA profiles from the frozen-thawed semen and blood samples obtained from the stallions matched in all cases.