title: An optimized method for cryogenic storage of Xenopus sperm to maximise the effectiveness of research using genetically altered frogs.
abstract: 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.
authors.affiliation: National Xenopus Resource, 7 MBL Street, Woods Hole, MA, 02543, USA.
authors.forename: Esther
authors.initials: E
authors.lastname: Pearl
authors.affiliation: European Xenopus Resource Centre, Institute of Biomedical and Biomolecular Sciences, University of Portsmouth, Portsmouth, PO1 2DT, UK.
authors.forename: Sean
authors.initials: S
authors.lastname: Morrow
authors.affiliation: European Xenopus Resource Centre, Institute of Biomedical and Biomolecular Sciences, University of Portsmouth, Portsmouth, PO1 2DT, UK.
authors.forename: Anna
authors.initials: A
authors.lastname: Noble
authors.affiliation: School of Biological Sciences, University of Portsmouth, Portsmouth, PO1 2DY, UK.
authors.forename: Adelaide
authors.initials: A
authors.lastname: Lerebours
authors.affiliation: National Xenopus Resource, 7 MBL Street, Woods Hole, MA, 02543, USA.
authors.forename: Marko
authors.initials: M
authors.lastname: Horb
authors.affiliation: European Xenopus Resource Centre, Institute of Biomedical and Biomolecular Sciences, University of Portsmouth, Portsmouth, PO1 2DT, UK; School of Biological Sciences, University of Portsmouth, Portsmouth, PO1 2DY, UK. Electronic address: matthew.guille@port.ac.uk.
authors.forename: Matthew
authors.initials: M
authors.lastname: Guille
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D015925:Cryopreservation / Q000662:veterinary*; D005307:Fertilization in Vitro / Q000662:veterinary; D008297:Male; D009865:Oocytes; D012662:Semen Preservation / Q000379:methods / Q000662:veterinary*; D013084:Sperm-Ovum Interactions / Q000502:physiology; D014981:Xenopus / Q000196:embryology / Q000235:genetics / Q000502:physiology*
keywords: 3Rs; Cryopreservation; Genetically altered lines; Sperm; Stock centres; Xenopus