Relationship between TUNEL assay and sperm chromatin structure assay for assessing DNA damage in equine spermatozoa

During this project two methods for assessing DNA damage in equine spermatozoa are compared to the ‘golden standard’, the sperm chromatin structure assay (SCSA). The two methods are variations of the TUNEL assay. During preliminary trials the protocols were standardized. During this development two protocols were optimized. The difference between them is the use of a different technique to create a binding place in the DNA for the dye in the TUNEL mixture. With the Triton-X-HCl technique the cells are challenged with an acid. Thereby the cells with poor quality are more damaged and take up more stain. With the DTT (Dithiothreitol) technique the disulfide (S-S) bonds in the protamine proteins that surround the sperm DNA are broken. Thereby the compact sperm DNA expands and becomes accessible for the dye. The more damaged the DNA is, the more nicks are present and the more stain is taken up by the cells. The SCSA, which uses an acridine orange staining, uses Triton-X-HCl to measure the resistance of the sperm DNA. The aim of the project is to compare the two techniques to determine if they are independent methods to assess DNA damage in equine sperm. The results show a significant difference between the TUNEL DTT technique and the SCSA, which suggests an independent method. There is no significant difference between the SCSA and the TUNEL Triton-X-HCl technique which suggests that the Triton-X-HCl has a bad effect on the DNA and creates more free 3’-OH-termini. In the future more research is needed to confirm these results. There is also research required to find out the clinical relevance