title: Validation of the sperm chromatin dispersion (SCD) test in the amphibian Xenopus laevis using in situ nick translation and comet assay.
abstract: The integrity of sperm DNA is becoming increasingly recognised as an important parameter of semen quality, but there are no published reports of this procedure for any amphibian. The primary aim of this study was to apply a modified sperm chromatin dispersion (SCD) test (Halomax) to an amphibian sperm model (African clawed frog; Xenopus laevis) and to validate the assay against in situ nick translation (ISNT) and the double-comet assay procedure. Inactivated spermatozoa were collected from fresh testes (n=3). Sperm DNA fragmentation (SDF) for each sperm sample was conducted immediately following activation (T0) and again after 1h (T1) and 24h (T24) of incubation at room temperature in order to produce a range of spermatozoa with differing levels of DNA damage. The SCD procedure resulted in the production of three nuclear morphotypes; amphibian sperm morphotype 1 (ASM-1) and ASM-2 showed no evidence of DNA damage, whereas ASM-3 spermatozoa were highly fragmented with large halos of dispersed DNA fragments and a reduced nuclear core. ISNT confirmed that ASM-3 nuclei contained damaged DNA. There was a significant correlation (r=0.9613) between the levels of ASM-3 detected by the SCD test and SDF revealed by the double-comet assay.
authors.affiliation: School of Agriculture and Food Science, The University of Queensland, Gatton, Qld 4343, Australia.
authors.forename: K
authors.initials: K
authors.lastname: Pollock
authors.affiliation: Department of Biology, Genetics Unit, The Autonomous University of Madrid, Madrid, 20849, Spain.
authors.forename: J
authors.initials: J
authors.lastname: Gosálvez
authors.affiliation: Department of Biology, Genetics Unit, The Autonomous University of Madrid, Madrid, 20849, Spain.
authors.forename: F
authors.initials: F
authors.lastname: Arroyo
authors.affiliation: Department of Biology, Genetics Unit, The Autonomous University of Madrid, Madrid, 20849, Spain.
authors.forename: C
authors.initials: C
authors.lastname: López-Fernández
authors.affiliation: European Resource Centre, The University of Portsmouth, King Henry 1 Street, Portsmouth, PO1 2DY, UK.
authors.forename: M
authors.initials: M
authors.lastname: Guille
authors.affiliation: European Resource Centre, The University of Portsmouth, King Henry 1 Street, Portsmouth, PO1 2DY, UK.
authors.forename: A
authors.initials: A
authors.lastname: Noble
authors.affiliation: School of Agriculture and Food Science, The University of Queensland, Gatton, Qld 4343, Australia.
authors.forename: S D
authors.initials: SD
authors.lastname: Johnston
mesh_terms: D000818:Animals; D002843:Chromatin / Q000378:metabolism*; D020552:Comet Assay; D004249:DNA Damage; D053938:DNA Fragmentation; D020287:In Situ Nick-End Labeling; D008297:Male; D055101:Semen Analysis / Q000379:methods; D013094:Spermatozoa / Q000502:physiology*; D014982:Xenopus laevis
chemical_list: D002843:Chromatin