Evaluating the Suitability of Cryoprotectants and Cryopreservation Solutions for Olive Barb, Puntius Sarana Embryos

The purpose of the present study was to examine the suitability of cryopreservation solutions for the embryos of olive barb Puntius sarana, a small-sized, freshwater, endangered fish. The specific objective was to examine the toxicity of various cryoprotectants (CPA) and to test the effectiveness of solutions of cryoprotectant mixtures (CS) to different developmental stages of olive barb embryos. Embryos at three developmental stages (gastrula, somites, and tail elongation stages) were exposed to 10, 15, 20, 25, and 30 % solutions of five cryoprotectants, propylene glycol (PG), methanol (MeOH), dimethyl sulfoxide (DMSO), dimethylformamide (DFA), and ethylene glycol (EG). Further, thirteen cryoprotectant mixture solutions comprising these five cryoprotectants were also used at different concentrations to test the toxicity to the embryos. Hatching rates decreased with increasing cryoprotectant concentrations and toxicity to olive barb embryos varied in the order of PG < EG < MeOH < DMSO < DFA. Embryos tolerated the addition of CPA mixtures in five-steps (5 × 3 min) better than in four-steps (4 × 3.45 min). The tail elongation stage was more tolerant to single CPA and CPA mixtures than earlier stages. In terms of embryo viability after equilibration, CPA mixture solutions CS3 (PG-30 %, MeOH-10 %, EG-10 %), CS6 (PG-25 %, MeOH-10 %, EG-10 %), CS8 (PG-20 %, MeOH-15 %, EG-15 %), CS9 (PG-20 %, MeOH-10 %, EG-10 %), and CS13 (PG-15 %, 10 % each of MeOH, EG and DMSO, DFA-5 %) appeared to be suitable for cryopreservation of the olive barb embryos.