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

Abstract

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.

Perspectives on the assessment of human sperm chromatin integrity

“…The SCD assay also involves acid denaturation that detects only single-stranded DNA. The similar findings of the TU-NEL and SCD assays is a result of the fact that they are scored on individual spermatozoa; in fact, they can be applied simultaneously on the same cell (66,70). Overall, these microscopy assays are very useful for the DFI assessment of cryptozoospermic, virtual azoospermic men, and for surgically retrieved specimens in which only a few cells are available.…”

Effect of Cryopreservation on the Sperm DNA Fragmentation Dynamics of the Bottlenose Dolphin (Tursiops truncatus)

“…We have demonstrated in a range of species (López‐Fernández et al. , ; Gosálvez et al. ; García‐Peiró et al.…”

“…Our group has also progressively introduced the concept of dynamic assessment of SDF (López‐Fernández et al. , , ; Gosálvez et al. ; Imrat 2012a,b), where the rate of DNA fragmentation is evaluated over time from the point of ejaculation (fresh semen) or after thawing (cryopreserved semen).…”

Sperm quality assessments for endangered razorback suckers Xyrauchen texanus

“…3). The NOMP method provides an additional means by which spermatozoal haplotypic DNA damage can be used in broodstock selection or for cryopreservation, and in examining betweenmale variation, as with a sperm chromatin dispersion assay used with tench Tinca tinca (López-Fernández et al 2009). In fact, sperm chromatin has been considered an independent measure of sperm quality apart from standard sperm parameters (Agarwal & Said 2003).…”