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Development of a novel flow cytometric approach to evaluate fish sperm chromatin using fixed samples

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Abstract

The integrity of the paternal DNA is essential for the accurate transmission of genetic information, yet fertilization is not inhibited by chromatin breakage. Some methods are available for the sensitive detection of DNA damage and can be applied in studies of environmental toxicology, carcinogenesis, aging, and assisted reproduction techniques in both clinical and experimental settings. Because semen samples obtained from remote locations undergo chromatin damage prior to laboratory assessment, the present study was undertaken to evaluate treatments for effective chromatin staining in the development of a DNA fragmentation assay using fixed milt from yellow perch (Perca flavescens). Similar to the sperm chromatin structure assay (SCSA), susceptibility of nuclear DNA to acid-induced denaturation was measured by flow cytometry (FCM). Use of 10% buffered formalin for milt fixation allowed easier peak discrimination than 4% paraformaldehyde. The effects of time and temperature of incubation in 0.08 N HCl were evaluated in order to determine the ideal conditions for promoting DNA decondensation and making strand breaks more available for staining and detection by FCM. The best results were obtained with incubation at 37°C for 1 minute, followed by cold propidium iodide staining for 30 minutes.

Additional Publication Details

Publication type:
Book
Publication Subtype:
Conference publication
Title:
Development of a novel flow cytometric approach to evaluate fish sperm chromatin using fixed samples
Year Published:
2013
Language:
English
Publisher:
Applied Animal Andrology
Publisher location:
Wallingford, Oxfordshire, UK
Contributing office(s):
National Wetlands Research Center
Conference Title:
8th Association for Applied Animal Andrology Biennial Conference
Conference Location:
Vancouver, British Columbia
Conference Date:
2012-04-28T00:00:00