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Rapid rates of sperm DNA damage after activation in tench (Tinca tinca: Teleostei, Cyprinidae) measured using a sperm chromatin dispersion test

Unidad de Genética, Departamento de Biología, Edificio de Biología, Universidad Autónoma de Madrid, C/Darwin no 2, 28049 Madrid, Spain¹ School of Biological Sciences, University of East Anglia, Norwich NR47TJ, UK² Instituto Tecnológico Agrario de Castilla y León, Ctra. Burgos Km 118, Finca Zamadueñas, 47071 Valladolid, Spain³ Complejo Hospitalario Universitario Juan Canalejo, As Xubias 84, 15006 A Coruña, Spain

Correspondence should be addressed to J Gosálvez; Email: jaime.gosalvez{at}uam.es

Spermatozoal haplotypic DNA is prone to damage, leading to male fertility problems. So far, the assessment of sperm DNA breakage has been challenging because protamines render the nuclear chromatin highly compacted. Here, we report the application of a new test to quantify DNA fragmentation in spermatozoa of an externally fertilizing teleost fish. The sperm chromatin dispersion (SCD) test uses a species-specific lysing solution to generate controlled protein depletion that, followed by DNA-specific fluorescent labelling, allows an easy morphological discrimination between nuclei affected by DNA damage. Using tench (Tinca tinca) as our model, we first trialled the test against established, but more technically demanding, assays employing in situ nick translation (ISNT) and the comet assay. The SCD test showed high concordance with ISNT, comet assay measures and a chromatin-swelling test, confirming the application of this straightforward SCD technique to various aspects of reproductive biology. Second, we examined between-male variation in DNA damage, and measured changes through time following spermatozoal activation. Between-male variation in the basal levels of average DNA damage ranged from 0 to 20% of sperm showing damage, and all showed increases in DNA fragmentation through time (0–60 min). The rates of DNA damage increase are the fastest so far recorded in sperm for a living organism, and may relate to the external fertilization mode. Our findings have relevance for broodstock selection and optimizing IVF protocols routinely used in modern aquaculture.