Assessment of cryotolerance of IVF bovine blastocysts after vitrification and one step-warming using an economical device

MARURI, A.; TELLO, MF.; LORENZO, MS.; LOMBARDO, DM.

Bologna

Congreso; 19th International Congress on Animal Reproduction; 2020

ICAR Committee

This study aimed to assess the cryotolerance of IVF bovine blastocysts after vitrification and warming in one step using a novel and economical device (eD) and to compare it with the well-known method of Cryotop® and warming in sequential steps. Cumulus-oocyte complexes (COCs) were obtained by follicular aspiration from slaughterhouse ovaries and washed three times with PBS supplemented with antibiotics. Only grade 1 oocyte were in vitro matured for 22 h (39°C, 5% CO2) and incubated for 5 h with 10x106 spermatozoa/mL in 100 µL-drops of modified 199 medium (M199), under mineral oil in the same condition of IVM. Previously, a straw of bull was warmed (35°C, 5 s), and sperm washed twice in M199 with caffeine and heparin. After IVF, presumptive embryos were pipetted to eliminate cumulus cells and sperm, and cultured in 50 µL-drops of SOF medium under mineral oil and incubated (39 °C, 5% CO2 and 5% O2) from day 2 to day 7-8. Only good-quality expanded blastocysts were selected and placed in a culture dish with PBS plus fetal bovine serum until vitrification. For the vitrification device, a 0.25 mL-straw was cut in half, and then each part again at both ends, creating a slightly concave surface where the embryos were placed. A total of 42 blastocysts was used. For vitrification, each blastocyst was placed in the equilibration solution for 5 min and then in the vitrification solution for 1 min. Afterward, the embryo was placed on the vitrification support (Cryotop® or eD), immersed in liquid nitrogen, and stored for at least one week in a tank. For warming, the eD was placed vertically in a 0.5 mL-straw loaded with 0.25 M sucrose solution at 40 °C. After 3 minutes, the straw was discharged on a culture dish with a washing medium, and the embryos were washed twice in SOF medium before in vitro culture in drops. The Cryotop® was placed directly in one of the wells of a four-well plate containing 0.25 M sucrose solution, followed by gradual dilutions of sucrose (0.125 M and 0.06 M) every 5 min and then proceeded in the same way than before. An additional group was added as control, using non-vitrified blastocysts that returned to the culture drops. Rates of re-expanded, hatching, and hatched blastocyst were determined at 24, 72, and 72 h after warming, respectively. There were no differences in these rates between Cryotop-vitrified and eD-vitrified embryos (94.4-93.3%, 61.1-53.3%, and 61.1-53.3%, respectively). In non-vitrified blastocysts, the rate of expanded embryos was 100%, and the hatching and hatched rates were significantly higher (P < 0.05) than in vitrified blastocysts (88.9 and 100%, respectively). In conclusion, the proposed device allows the vitrification of bovine blastocysts like the Cryotop® method. Besides, one-step warming facilitates ET under field conditions.