Using frozen X. laevis sperm to improve animal welfare and reduce the resources required for research.

SUPPLEMENTARY MATERIAL Background: Most laboratories that use Xenopus obtain their embryos by in vitro fertilisation using fresh sperm. In vitro fertilisation provides an opportunity to use frozen sperm routinely, with the potential both to improve the welfare of male Xenopus and make research more straightforward and cost effective. Sending frozen sperm for dominant transgenic lines from resource centres also prevents the need for these lines to be held by the investigator’s laboratory, further reducing cost and harm. Methods: We recently tested and optimised existing sperm freezing protocols to make a robust, widely usable one. Here we test how this can be developed into a widespread method for the research community that produces Xenopus laevis by in vitro fertilisation. Results: We discover that 32-64 effective aliquots of frozen sperm can be made from a single male and that long-term survival and reproductive success of offspring made using frozen-thawed sperm are the same as those made with fresh. We find the biosecurity implications of using frozen sperm to be similar to exchanging fresh testes and less than moving animals. The technique can be transferred between laboratories but it requires training. Conclusion: Overall, the data support the use of frozen sperm for propagating both wild type and genetically altered (GA) Xenopus, providing significant advantages for researchers and their funding bodies.