138 CELL INTEGRITY OF HOLSTEIN AND JERSEY EMBRYOS: COMPARISON OF TWO FREEZING METHODS

Abstract

The aim of this study was to compare cell integrity of Holstein and Jersey embryos using the standard method of freezing and vitrification. Embryos were harvested and processed following standard procedures approved by IETS (International Embryo Transfer Society), and then underwent two methods of freezing. (1) Standard method: embryos were put into 1.5 m ethylene glycol (EG) for 5 min at room temperature, and then at –6.5°C for 10 min for stabilization. After seeding, the temperature was lowered by 0.5°C until it reached –32°C, and then straws containing embryos were plunged into liquid nitrogen. (2) Vitrification: embryos were put for 3 min into vitrification solutions V1: 5 m EG in EmCare (ICPbio, Ltd., Auckland, New Zealand) and V2: 7 m EG, 0.5 m galactose (Sigma Chemical Co., St. Louis, MO, USA), and 18% Ficoll70 (Sigma) in EmCare for 45 s. Embryos were loaded into straws and then plunged into liquid nitrogen. De-freezing was done as follows: 5 s at room temperature, and then 30 s in water at 25°C. Vitrified embryos were de-frozen for 10 s at room temperature followed by 30 s in water at 25°C. All embryos were subjected to a combined staining, Hoechst 33342 and propidium iodide (Sigma-Aldrich Canada, Toronto, Ontario, Canada). The combined staining with these dyes makes analysis of cell death possible. Propidium iodide is specific to dead cells, whereas Hoechst stains all of the cells. Embryos were put in a PBS solution containing 20 µg mL^–1 of Hoechst 33342 and propidium iodide for 15 min at 37°C and placed between a slide and coverslip. Fluorescence microscopy was then used to assess blue nuclei (live cells) and red nuclei (dead cells) of the cow embryos. For this experiment, a total of 51 grade 1 embryos (IETS classification) at early blastocyst or blastocyst stages were used (25 Holstein embryos from 4 donor cows and 26 Jersey embryos from 3 donor cows). The standard method of freezing was performed on 26 embryos (13 from Holstein cows and 13 from Jersey cows) and vitrification was performed on 25 embryos (12 from Holstein cows and 13 from Jersey cows). Embryos from a given cow (Holstein or Jersey) were evenly allocated to the standard and vitrification methods. The GLM procedure of SAS (SAS statistical software version 8; SAS Institute, Inc., Cary, NC, USA) was used to compare numbers of dead and live cells between vitrification and the standard method of freezing in Holstein and Jersey embryos. The mean numbers (±SE) of live cells of Holstein embryos were, respectively, 87.92 ± 8.48 and 85.5 ± 6.46 for standard method and vitrification. For Jersey embryos, means (±SE) of live cells were 94.42 ± 2.6 and 73.93 ± 7.39, respectively, for the standard method and vitrification. Means (±SE) of dead cells of Holstein embryos were, respectively, 5.58 ± 1.37 and 13.67 ± 2.91 for the standard method and vitrification. For Jersey embryos, means (±SE) of dead cells were 8.08 ± 1.01 and 27.6 ± 7.06, respectively, for the standard method and vitrification. In conclusion, vitrification significantly increased (P ≤ 0.05) the number of dead cells of embryos over the standard method of freezing. This effect was more evident (P ≤ 0.05) in Jersey than in Holstein embryos.

The authors thank Dr. Vincent Girard for his help with statistics.