An optimized method for cryogenic storage of Xenopus sperm to maximise the effectiveness of research using genetically altered frogs

Cryogenic storage of sperm from genetically altered Xenopus improves cost effectiveness and animal
welfare associated with their use in research; currently it is routine for X. tropicalis but not reliable for
X. laevis. Here we compare directly the three published protocols for Xenopus sperm freeze-thaw and
determine whether sperm storage temperature, method of testes maceration and delays in the freezing
protocols affect successful fertilisation and embryo development in X. laevis. We conclude that the
protocol is robust and that the variability observed in fertilisation rates is due to differences between
individuals. We show that the embryos made from the frozen-thawed sperm are normal and that the
adults they develop into are reproductively indistinguishable from others in the colony. This opens the
way for using cryopreserved sperm to distribute dominant genetically altered (GA) lines, potentially
saving travel-induced stress to the male frogs, reducing their numbers used and making Xenopus experiments
more cost effective.