title: Is there a relationship between the chromatin status and DNA fragmentation of boar spermatozoa following freezing-thawing?
abstract: In this study a radioisotope method, which is based on the quantitative measurements of tritiated-labeled actinomycin D ((3)H-AMD) incorporation into the sperm nuclei ((3)H-AMD incorporation assay), was used to assess the chromatin status of frozen-thawed boar spermatozoa. This study also tested the hypothesis that frozen-thawed spermatozoa with altered chromatin were susceptible to DNA fragmentation measured with the neutral comet assay (NCA). Boar semen was diluted in lactose-hen egg yolk-glycerol extender (L-HEY) or lactose ostrich egg yolk lipoprotein fractions-glycerol extender (L-LPFo), packaged into aluminum tubes or plastic straws and frozen in a controlled programmable freezer. In Experiment 1, the chromatin status and DNA fragmentation were measured in fresh and frozen-thawed spermatozoa from the same ejaculates. There was a significant increase in sperm chromatin destabilization and DNA fragmentation in frozen-thawed semen as compared with fresh semen. The proportions of spermatozoa labeled with (3)H-AMD were concurrent with elevated levels of sperm DNA fragmentation in K-3 extender, without cryoprotective substances, compared with L-HEY or L-LPFo extender. Regression analysis revealed that the results of the (3)H-AMD incorporation assay and NCA for frozen-thawed spermatozoa were correlated. Boars differed significantly in terms of post-thaw sperm DNA damage. In Experiment 2, the susceptibility of sperm chromatin to decondensation was assessed using a low concentration of heparin. Treatment of frozen-thawed spermatozoa with heparin revealed enhanced (3)H-AMD binding, suggesting nuclear chromatin decondensation. The deterioration in post-thaw sperm viability, such as motility, mitochondrial function and plasma membrane integrity, was concurrent with increased chromatin instability and DNA fragmentation. This is the first report to show that freezing-thawing procedure facilitated destabilization in the chromatin structure of boar spermatozoa, resulting in an unstable DNA that was highly susceptible to fragmentation.
authors.affiliation: Department of Animal Biochemistry and Biotechnology, University of Warmia and Mazury in Olsztyn, Olsztyn, Poland. fraser@uwm.edu.pl
authors.forename: L
authors.initials: L
authors.lastname: Fraser
authors.forename: J
authors.initials: J
authors.lastname: Strzezek
mesh_terms: D000818:Animals; D002843:Chromatin / Q000187:drug effects / Q000648:ultrastructure*; D015925:Cryopreservation; D053938:DNA Fragmentation; D006493:Heparin / Q000494:pharmacology; D008297:Male; D013094:Spermatozoa / Q000187:drug effects; D013552:Swine / Q000235:genetics*
chemical_list: D002843:Chromatin; D006493:Heparin