Cryopreservation of early-stage male Black Sea trout (Salmo trutta labrax) germ cells and comparison of the whole tissue and enzymatically isolated cells

Abstract

The high plasticity, or the ability to differentiate into various cell types, and capacity to become both gametes of early-stage germ cells (eGCs) allows them to be used for the long-term storage and recovery of genetic information. In this study, species-specific protocols (isolation period, enzymatic separation solution, incubation period, appropriate cryoprotectant, and cryopreservation protocol) were established for the isolation and long-term preservation of Black Sea trout eGCs. In addition, the difference between the application of cryopreservation to the whole gonad tissue and to the enzymatically separated cells has been shown in terms of viability and the number of cells obtained. According to the cell count made from the histological sections of the testicular tissue throughout the year, the period with the highest concentration of eGCs in the tissue is between May and June. To identify the optimal enzymatic dissociation solution, we subjected testicular tissues to digestion using various combinations of trypsin (T) and collagenase (C) (0.3-0.5% T and 0.1-0.3% C). Our findings revealed that the 0.3% C provided the highest yield of viable cells (90.9%). We cryopreserved the enzymatically dissociated cells (EDCs) using six different cryoprotectants (Dimethyl sulfoxide, Glycerol, and CryoSOfree™, in both L-15 and phosphate-buffered saline - PBS mediums). Additionally, whole tissue exclusively with CryoSOfree™. After storing the samples at -152 °C for two years, the group of EDCs cryopreserved in L15+Glycerol exhibited the highest viability at 93.3%. Meanwhile, the whole tissue group yielded the greatest number of cells per ml after thawing, with a count of 33 210 000 cells. When considering both the viability (91.11%) and the number of cells obtained (33 210 000 cells/ml), the whole tissue cryopreservation group outperformed all the other methods.

Keywords

• enzymatic dissociation
• long-term storage
• salmonidae
• spermatogonia