April 1, 2017;
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.
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Fig. 1. Cryogenic preservation of X. laevis sperm is less effective and consistent than for X. tropicalis sperm. Testes from individual X. tropicalis (A) and X . laevis (B) were either used fresh or frozen and thawed using the Harland method to fertilise eggs from a single female of known good quality. A-I on the X-axis refers to sperm samples, each from a different male. The experiment was repeated using three female frogs, to eliminate this as a possible source of variability, data from a single female are shown. Eggs that divided were counted as fertilised, and morphologically normal embryos were counted at NF32. C) The mean values from the experiments above are shown for X. laevis and X. tropicalis ± SEM. There is a significant effect of the species on the percentage fertilisation (**: p = 0.002) and on the percentage of normal embryos at NF32 (***: p = 0.0003), whether fresh or frozen sperm was used.
Fig. 2. The Mansour and Harland methods are the most effective for long-term cryopreservation of X. laevis sperm. Sperm from 3 separate male frogs was either tested fresh on eggs from two females of known good quality or was tested following cryopreservation by the methods shown. Sperm were recovered after 2 days, 3 months or 13 months and fertilisation rates were recorded. Means ± SEM are shown. There is no significant difference between the Mansour and Harland methods (p = 0.82). There is a significant difference between the Harland and Sargent methods (*: p = 0.04) and between the Mansour and Sargent methods (*: p = 0.03).
Fig. 3. Embryos from frozen sperm develop normally. A) Eggs were fertilised using sperm frozen by the methods shown and stored on LN2 for 3 months, the number of normal and abnormal embryos was scored at NF32, normal embryo percentages are expressed as a % of the surviving embryos at NF32. Means ± SEM are shown. B) Example of normal, left, and abnormal, right, embryos fertilised with cryopreserved sperm. C) Embryos produced using fresh or frozen sperm (Harland method) were fixed at NF32 and 12 of each analysed for the expression of the markers shown by in situ hybridisation. D) Once the embryos had developed to metamorphosis they were weighed. Means ± SEM are shown.
Fig. 4. There is no difference in sperm activity between cryopreserved sperm stored in liquid nitrogen and cryopreserved sperm stored at -80C. Eggs were fertilised using sperm frozen by the Harland method and stored on LN2 or at −80C for 2 days, 12 weeks or 24 weeks. Means ± SEM are shown. The storage method does not have an effect on the fertility (p = 0.08), and neither does the time stored (p = 0.45).