Quantification of lesions in nuclear and mitochondrial genes of Sparus aurata cryopreserved sperm

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 10 kb 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. © 2013 Elsevier B.V.This work was supported by MICINNAGL2009-06994, the Ramón Areces Foundation and the Ramón y Cajal program (RYC-2008-02339 and RYC-2007-01650, Spain). The authors would like to thank JCyL (E-24-2009-0036681), Fondo Social Europeo, Dr. Susana Martínez Guerra, David García Valcarce and Cintia Miranda Rodríguez.Peer Reviewe