Studies On Cryoinjury Mechanism In Red Seabream (Pagrus Major) Embryo Cryopreservation

Successful fish embryo cryopreservation has not been achieved due to its large volume, large content of water, complex multi-compartmental system, etc. During the process of cooling and thawing, embryos are subjected to a series of cryoinjuries such as cold shock, ice formation, cryoprotectant toxicity and osmotic effect. Cryoinjury is one of the most important obstacles for cryopreservation. Understanding the mechanism of cryoinjury is the basis for establishing fish embryo cryopreservation method. This study investigated the red seabream embryo tolerance for osmotic pressure, penetrability to cryoprotectant, and the extra-and intra-cellular ice formation temperatures at different cooling rates. The detailed results are as follows:1. We tested the hatching rates of embryos immersing in artificial sea water (ASW) with different concentrations (0%, 25%, 50%, 75%, 1×, 2×, 3×, 4×. osmotic pressure: 0³740 mOsm/kg). The optimal osmotic pressure ranges for embryos in different stages, and for heart-beaten embryos in different exposure time were obtained, respectively. The result revealed that 1) For 2-4 cells embryo, gastrulae, 10-14 somite embryo, heart-beaten stage embryo and embryo before hatching, when the osmotic pressure of artificial sea water 919¹391 mOsm/kg, 919¹391 mOsm/kg, 462 ¹391 mOsm/kg, 232¹878 mOsm/kg and 692¹391 mOsm/kg, respectively, the hatching rate were high than 50%. And heart-beaten stage embryo showed the best tolerance for osmotic pressure. 2) For heart-beaten stage embryos, after immersing in ASW with different concentrations for 10 min, 30 min, 1 h, 5 h and 10 h, the osmotic pressure range , 0²804 mOsm/kg, 0¹878 mOsm/kg, 232¹391 mOsm/kg, 232¹391 mOsm/kg and 919¹391 mOsm/kg, respectively, had no significant influence on embryo hatching rate. The result reveals that the heart-beaten embryo's tolerance of osmotic pressure decreased with the increase of exposure time.2. We tested the concentration of DMSO inside the embryo using capillary electrophoresis, and analyzed the relationship between inner-cellular DMSO concentration and outer cryoprotectent concentration. The results indicated that the hatching rate (y1) decreased with the increase of outer cryoprotectent concentration (x). When the exposure time were 10 min, 30 min and 60 min, the regression equation were y₁ = -2832.7x³ + 575.01x² - 37.011x + 99.641 (R² = 0.9722), y₁ = 30288x³ - 16322x² + 2077.3x + 27.603 (R² = 0.9876), y₁ = 16052x³ - 5985.2x² - 32.696x + 119.6 (R² = 0.9124), respectively. The inner-cellular DMSO concentration (y2) increased with the increase of outer cryoprotectent concentration (x). When the exposure time were 10 min, 30 min and 60 min, the regression equation were, y₂ = 0.2584e^6.7294x (R² = 0.9876); y₂ = 0.2521e^10.964x (R² = 0.9644); y₂ = 0.4054e^10.95x (R² = 0.8954), respectively.3. We examined the effect of cooling rate (20, 40, 60, 80, 100 and 120 oC/min) on the temperature of extra-and inner- cellular ice formation (TEIF and TIIF) under cryomicroscope. TEIF decreased with the increase of cooling rate, and TEIF decreased slightly when the cooling rate was more than 80℃/min. TIIF decreased with the increase of cooling rate (<80 oC/min), and increased with the increase of cooling rate (>80 oC/min). While TEIF - TIIF increased with the increase of cooling rate (<80 oC/min), and decreased with the increase of cooling rate (>80 oC/min).4. We observed the ice formation of four embryos cryopreservation methods under cryomicroscope. The results showed thatâ‘ In the cooling process, both TEIFs and TIIFs in programmed cooling groups were significantly higher than those in vitrification groups. And in vitrification groups, TIIF values were higher than those of TEIF, indicating intra-cellular ice formed earlier than extra-cellular ice, which was opposite to programmed cooling groups. In 40%PG vitrification group, the solutions outside the embryos vitrified, while other groups didn't.â‘¡In the cooling process, recrystallization had also occurred within the embryos in all four groups. After thawing, the percentages of embryo with intact morphology in vitrification groups (62.82%) were significantly higher than that in programmed cooling groups (9.21%).