Semen Assessments
The alkaline comet assay, also known as single-cell gel electrophoresis, is used to measure both single and double stranded DNA breaks. Spermatozoa are firstly embedded in agarose on a microscope slide. The cells are lysed to remove cell membranes, cytoplasm and nucleoplasm, and to solubilise nuclear packaging proteins, leaving behind the nucleoid containing tightly coiled DNA (‘comet head’). Under alkaline pH conditions, the supercoiled DNA loops are unwound, and subsequent electrophoresis causes damaged DNA strands to migrate towards the anode, forming the ‘comet tail’. Comets are imaged under fluorescent microscopy, and objectively analysed using the Comet IV software. The intensity of the comet tail relative to the head reflects the extent of DNA strand breaks. The larger the comet, the greater the extent of DNA strand breaks. DNA fragmentation assessed using the alkaline comet assay is highly predictive of sperm quality, male fertility and embryo survival [1-5].
1. Ribas-Maynou, J., Abad, C., García-Segura, S., Oliver-Bonet, M., Prada, E., Amengual, M.J., Navarro, J., and Benet, J. (2020) Sperm chromatin condensation and single- and double-stranded DNA damage as important parameters to define male factor related recurrent miscarriage. Mol Reprod Dev. 87(11): p. 1126-1132.
2. Ribas-Maynou, J., García-Peiró, A., Abad, C., Amengual, M.J., Navarro, J., and Benet, J. (2012) Alkaline and neutral Comet assay profiles of sperm DNA damage in clinical groups. Hum Reprod. 27(3): p. 652-8.
3. Ribas-Maynou, J., García-Peiró, A., Fernández-Encinas, A., Abad, C., Amengual, M.J., Prada, E., Navarro, J., and Benet, J. (2013) Comprehensive analysis of sperm DNA fragmentation by five different assays: TUNEL assay, SCSA, SCD test and alkaline and neutral Comet assay. Andrology. 1(5): p. 715-722.
4. Hamilton, T.R., Mendes, C.M., de Castro, L.S., de Assis, P.M., Siqueira, A.F., Delgado Jde, C., Goissis, M.D., Muiño-Blanco, T., Cebrián-Pérez, J., et al. (2016) Evaluation of Lasting Effects of Heat Stress on Sperm Profile and Oxidative Status of Ram Semen and Epididymal Sperm. Oxid Med Cell Longev. 2016: p. 1687657.
5. Hamilton, T., Siqueira, A.F.P., de Castro, L.S., Mendes, C.M., Delgado, J.C., de Assis, P.M., Mesquita, L.P., Maiorka, P.C., Nichi, M., et al. (2018) Effect of Heat Stress on Sperm DNA: Protamine Assessment in Ram Spermatozoa and Testicle. Oxid Med Cell Longev. 2018: p. 5413056.