125 Investigation of the effects of different cryopreservation parameters on the genome of zebrafish (Danio rerio) embryos

In recent years, a large number of studies implicated cryopreservation to increased occurrence of various forms of mutations, increased DNA fragmentation and the event of apoptosis. However, the evidence emerged from such studies is somewhat inconclusive; and the current study argued that analysing the DNA damage response (DDR) from the cryopreserved cells would help characterise the nature of the putative DNA damage resulting from cryopreservation. To that end, the current study set out to investigate the effects of different cryopreservation parameters on the genome in the context of the event of double strand breaks (DSBs), single strand breaks (SSBs), and various forms of sequence alteration using 51/4 h post fertilisation (hpf) zebrafish (Danio rerio) embryos as the model organism. The cryopreservation parameters under which the investigation was carried out were chilling of the embryos at 0 °C for up to 120 min, treatment of the embryos with two cryoprotective additives (CPA), MeOH and Me2SO, for 30 min, and cooling of the embryos to −35 °C and subsequent warming. Assays for DSB-activated DDR proteins and SSB-activated DDR proteins in 51/4 hpf zebrafish (Danio rerio) were developed under the current study. The assays were then utilised to investigate the event of DSBs and SSBs in the genome of the embryos treated with different cryopreservation parameters. The study also analysed the expression profiles of a set of genes unique to the base excision repair (BER), nucleotide excision repair (NER) and mismatch repair (MMR) as indicators of the occurrence of various forms of sequence alteration in the genome of the embryos treated with the cryopreservation parameters. The study found that chilling and CPA treatment did not induce DSBs or SSBs but up-regulated the MMR and BER mechanisms, respectively. CPA treatment also down-regulated NER and MMR mechanisms. Cooling and warming, on the contrary, did not induce DSBs but induced SSBs in the genome, which were repaired when the embryos were provided with a recovery time. Cooling and warming also up-regulated the NER and BER mechanisms in the embryos. The findings of the study provided important insights into how eukaryotic cells respond to different cryopreservation parameters, which will significantly enhance the knowledge regarding the effects of cryopreservation on the genome of biological objects.