Validation of the sperm chromatin dispersion (SCD) test in the amphibian Xenopus laevis using in situ nick translation and comet assay
K. Pollock A D , J. Gosálvez B , F. Arroyo B , C. López-Fernández B , M. Guille C , A. Noble C and S. D. Johnston A
+ Author Affiliations
- Author Affiliations
A School of Agriculture and Food Science, The University of Queensland, Gatton, Qld 4343, Australia.
B Department of Biology, Genetics Unit, The Autonomous University of Madrid, Madrid, 20849, Spain.
C European Xenopus Resource Centre, The University of Portsmouth, King Henry 1 Street, Portsmouth, PO1 2DY, UK.
D Corresponding author. Email: kate.pollock@uqconnect.edu.au
Reproduction, Fertility and Development 27(8) 1168-1174 https://doi.org/10.1071/RD14070
Submitted: 22 February 2014 Accepted: 13 April 2014 Published: 3 June 2014
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 1 h (T1) and 24 h (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.
Additional keywords: African clawed frog, halomax, sperm DNA fragmentation.
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