Item talk:Q233995
From geokb
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"name": "Flow cytometric method for measuring chromatin fragmentation in fixed sperm from yellow perch (Perca flavescens)",
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"abstract": "Declining harvests of yellow perch,\u00a0Perca flavescens, in urbanized watersheds of Chesapeake Bay have prompted investigations of their reproductive fitness. The purpose of this study was to establish a flow cytometric technique for DNA analysis of fixed samples sent from the field to provide reliable gamete quality measurements. Similar to the sperm chromatin structure assay, measures were made on the susceptibility of nuclear DNA to acid-induced denaturation, but used fixed rather than live or thawed cells. Nuclei were best exposed to the acid treatment for 1\u00a0minute at 37\u00a0\u00b0C followed by the addition of cold (4\u00a0\u00b0C) propidium iodide staining solution before flow cytometry. The rationale for protocol development is presented graphically through cytograms. Field results collected in 2008 and 2009 revealed DNA fragmentation up to 14.5%. In 2008, DNA fragmentation from the more urbanized watersheds was significantly greater than from reference sites (P\u00a0=\u00a00.026) and in 2009, higher percentages of haploid testicular cells were noted from the less urbanized watersheds (P\u00a0=\u00a00.032) indicating better reproductive condition at sites with less urbanization. For both years, total and progressive live sperm motilities by computer-assisted sperm motion analysis ranged from 19.1% to 76.5%, being significantly higher at the less urbanized sites (P\u00a0<\u00a00.05). This flow cytometric method takes advantage of the propensity of fragmented DNA to be denatured under standard conditions, or 1\u00a0minute at 37\u00a0\u00b0C with 10% buffered formalin\u2013fixed cells. The study of fixed sperm makes possible the restrospective investigation of germplasm fragmentation, spermatogenic ploidy patterns, and chromatin compaction levels from samples translocated over distance and time. The protocol provides an approach that can be modified for other species across taxa.",
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