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Prolonging Egg Viability for IVF using High Salt Solution

Percent of dividing embryos from eggs squeezed into 0.2% BSA in 1x MBS at 0 hrs

This high salt solution can be used to extend the viability of tropicalis or laevis eggs for IVF by several hours. We have been able to obtain consistently successful fertilization up to 2 hours after eggs are laid into this solution. Fertilization can be achieved later than 2 hours, though the percentage of eggs fertilized tends to become variable. The table below shows percentage of tropicalis eggs fertilized after sitting in high salt solution for 2, 4 and 6 hours. There is some variability between individual females at each timepoint, and likely some variability due to the male, as a different male was used for IVF at each timepoint. We would suggest boosting 2 -3 females and allowing them to lay into the salt solution in order to have the best chance at success. You may have better success with laevis eggs in high salt, though we have not tested this extensively.

High Salt Recipe – 1X MBS + 0.2 % BSA

1L 2 L 4 L
10X MBS Salts 100 ml 200 ml 400 ml
0.1 M CaCl2 7 ml 14 ml 28 ml
H20 893 ml 1786 ml 3572 ml
Bovine Serum Albumin (BSA) 2 g 4 g 8 g
Dissolve BSA in solution, pH to 7.8, filter sterilize (0.22µm). Store at RT or 4 degree. Do not use if cloudy.
Allow boosted frogs to lay in High Salt solution. Remove eggs, remove the high salt solution from the dish by pipetting, and proceed with fertilization as usual. We do not leave the frogs in high salt solution longer than 24 hours.