Abstract
Cryopreservation is a common procedure for long-term storage of sperm in animal reproduction and is also employed in the aquaculture industry. The freezing–thawing process originates reactive oxygen species (ROS) which cause serious damage at chromatin level. Analyzing and characterizing this type of damage, especially in specific DNA regions, could be particularly interesting for germplasm banking purposes. This type of study could provide information about damage in some regions of interest or specific genes with important roles in fertilization and embryo development. This is the first report on quantification of DNA lesions in specific genes after cryopreservation in any species. We performed a specific DNA assay based on the quantitative PCR (qPCR) approach to quantify lesions generated after sperm cryopreservation in two Sparus aurata nuclear genes with important roles in embryo development (Igf1 and Gh) and two mitochondrial genes (Cytb and CoI). We tested different cryopreservation protocols using DMSO as permeable cryoprotectant, with or without BSA supplementation. The maximum number of lesions per 10kb observed after cryopreservation was 2.28 (for Igf1) and 0.8 (Gh), significantly lower than that detected for mitochondrial genes, 8.43 (Cytb) and 5.34 (CoI). A complementary analysis of DNA fragmentation (Comet assay) and telomere length measurement (qPCR) was done. The results demonstrated that the protocol used did not induce telomere shortening and that DNA integrity was very well preserved (%DNAt lower than 2.47±0.19%). The results suggest that this protocol offers a high degree of DNA protection; however the qPCR assay demonstrated different vulnerability of the DNA regions to undergoing damage during freezing/thawing processes. This study demonstrates the usefulness of using qPCR approaches to study gene-specific damage after cryopreservation, which can be an excellent complement for traditional techniques.
Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
