Rabbit morulae were exposed to a vitrification solution-modified PBS [PB1] medium containing 40% ethylene glycol + 18% Ficoll + 0.3 M sucrose (EFS) for 2, 5, or 10 min at 20 degrees C and were vitrified in liquid nitrogen. When morulae were rapidly warmed, 96% had an intact zona pellucida. When embryos were cultured after removal of the mucin coat, high proportions of them formed blastocoel (79-100%), but the percentage of embryos developed to fully expanded blastocysts decreased with increased exposure time 87%, 40%, and 17%). The survival rate of morulae vitrified after removal of the mucin coat was lower than that of mucin-intact embryos. To assess the development potential in vivo, 131 embryos were vitrified after 2 min of exposure to EFS solution; all the embryos were recovered and 120 were transferred to recipients without removal of the mucin coat, resulting in 78 (65%) full-term fetuses or young. This simple method, which yields high survival both in vitro and in vivo, will be of practical use for vitrifying rabbit embryos.
Mouse morulae were exposed in one step to a vitrification solution (EFS, a modified PBS containing 40% ethylene glycol, 18% Ficoll, and 0.3-M sucrose) at various temperatures, then cooled rapidly in liquid nitrogen, and then warmed rapidly. All of the embryos exposed to the EFS solution for 0.5 min at 25 degrees C before vitrification developed in culture. However, survival rates were lower if the duration of exposure was prolonged to 2, 5, or 10 min. At lower ambient temperatures (20, 10, and 5 degrees C), high survival rates were associated with longer exposure to the EFS solution. The toxicity of the EFS solution was also lower at lower temperatures. The toxic injury of morulae was manifested as decompaction of the blastomeres. Among the three additives in the EFS solution, ethylene glycol, which can cross cell membranes, was responsible for the toxicity. The results show that the optimum time for exposure of the embryos to the EFS solution before rapid cooling varies with the ambient temperature, i.e., 0.5 min at 25 degrees C, 0.5-5 min at 20 degrees C, 2-5 min at 10 degrees C, and 2-10 min at 5 degrees C. If they are exposed for an optimum period, almost all mouse morulae can survive vitrification (94-100%).
To examine the factors affecting the survival of refrigerated embryos, rabbit and mouse morulae were stored at 0 degrees C in modified phosphate-buffered saline (PB1) or in PB1 containing 0.75 M sucrose. Survival was defined as the ability to develop into an expanded blastocyst in culture. The data was analyzed with special reference to the presence of a mucin coat around the embryos. When rabbit morulae were stored in isotonic PB1 for 2, 4, 6, and 8 days, survival rates were 98%, 88%, 85%, and 50%, respectively. However, if the mucin coat had been removed before storage, the rates were lower (95%, 75%, 36%, and 3%, respectively). Sucrose impaired the survival of rabbit morulae irrespective of the presence of the mucin coat. Only 11% of mouse morulae survived 2 days of storage in PB1 medium, but if the medium contained sucrose, survival rates after storage for 2, 3, 4, and 5 days were higher (83%, 55%, 31%, and 7%, respectively). To provide them with a mucin coat around the zona pellucida, mouse embryos were incubated in a rabbit oviduct. Survival rates of these embryos after storage in the presence of sucrose did not decrease over 4 days of refrigeration (98-92%), and the rates after storage for 5, 6, and 7 days were 65%, 40%, and 30%, respectively; embryos that had been stored for 5 days were transferred to recipient mice, and live young were born. Agar embedding of mouse morulae did not have the same effect as the mucin coat.(ABSTRACT TRUNCATED AT 250 WORDS)
Abstract. Experiments were conducted to find optimal conditions for obtaining high survival of expanded mouse blastocysts after vitrification in glycerol-based solutions. The solutions were GFS20, GFS30, GFS40 and GFS50, which contained 20%, 30%, 40% and 50% glycerol, respectively, diluted in PB1 medium containing 30% Ficoll + 0.5 M sucrose. The toxicity tests of the solutions and glycerol showed that longer exposure to a higher concentration of glycerol is more injurious to the embryos. For vitrification, expanded blastocysts were exposed to the GFS solution at 20 or 25 C for various periods; they were then vitrified in liquid nitrogen, and were warmed rapidly. When the embryos were directly exposed to GFS40 for 2 min at 25 C, 68% of them re-expanded during 48 h of post-warming culture. The re-expansion rates decreased, when exposure time was shortened or extended, when the exposure temperature was lowered (20 C), or when embryos were vitrified in GFS20, GFS30, and GFS50. When embryos had been pretreated in a dilute (10-20%) ethylene glycol solution for 5 min, followed by short exposure (0.5 min) to GFS40 at 25 C, the post-vitrification survival rate increased to 82-87%; furthermore, the rate reached 92% when GFS40 was replaced by GFS50. Expanded blastocysts cryopreserved in GFS50 had the ability to develop into live young. The findings showed that glycerol-based solutions are as effective as ethylene glycol-based solutions, although the optimal solution needs a higher concentration of glycerol, probably because glycerol permeates the embryos more slowly than ethylene glycol.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.