Reducing the use and refining the distribution of male Xenopus

To understand the normal biological processes in humans and animals, together with the diseases that occur when these go wrong, we need to study "model organisms". In addition to being ethically preferable, there is a long history of research using the models, so we have both a great deal of information about them and a wide range of experimental tools to work with. Two of the four main vertebrate model organisms are aquatic Xenopus frogs that come from Africa. Much of our knowledge about basic animal biology comes from research using Xenopus, including: cell division control, intercellular signaling, cell reprogramming, regeneration and healing, gene control networks and DNA repair. More recently they have been used to study human genetic diseases, more than 90% of human genes are found in Xenopus and the structure of over two-thirds of their genomes is the same.

To support the use of model organisms, resource centres make, collect, quality assure and distribute all of the molecular and animal resources needed. For Xenopus the main resource centre is the EXRC, which collaborates closely with centres in the US and Japan to ensure that researchers' needs are met worldwide. Part of the EXRC's mission is to improve the welfare of frogs used for research and this project contributes to that aim.
Currently male frogs are sent to research laboratories where they are kept in tanks and then, when embryos for experiments are needed, they are killed humanely and their testes removed to provide the sperm for in vitro fertilization. This has two disadvantages we aim to overcome: first the frogs are stressed by travel and second, because so many embryos are created by one fertilization (often more than can be analysed in an experiment) the removed testes often become inactive before all of the sperm are used, leading to wastage.

Together with our collaborators and the US stock centre we have developed robust methods for freezing sperm and recently performed three successful trials of sending male gametes as frozen sperm to researchers based abroad. We now need to develop this process to the point where it can be widely used. To do so we will first test whether we can divide the testes from each male into more than the 8 sections that we currently use, without affecting fertilization rates and the developmental fate of the embryos produced. We will then assess the variability of embryo production from each piece of testis, allowing us to understand how best to quality assure the sperm we send to our users. The next stage of the programme is to develop the distribution process, first by testing different courier's services using dummy packages to a group of 7 labs around the world and optimizing the packaging and paperwork to speed passage through customs. Once we are sure of the delivery process, we will send frozen sperm and discuss the use of these in the test laboratories by Skype. We will use these discussions to improve the written and video instructions that accompany the sperm as more batches are sent out. Once we are consistently distributing frozen sperm to the test laboratories we will then extend the service to all of our users who want it and encourage the other stock centres to do so too. We are confident that users will take up this service due to the substantial cost savings it represents over sending live animals.

This programme will deliver a more cost effective way of sending male Xenopus gametes whilst improving animal welfare and decreasing the number of male frogs used.

Xenopus frogs are arguably the most versatile vertebrate model organism, especially now that highly efficient gene editing has added genetic approaches to their established strengths of gain of function screens, explant culture, transplants, ease of transgenesis, extracts for biochemistry, regeneration, single embryo transcriptomics and proteomics. Three major resource centres and two small, specialist ones currently provide resources for researchers using Xenopus. These distribute inbred lines, transgenic and mutant animals to users, but sending all of these as live animals incurs stress to the animals during transport and significant costs for users and funding bodies.

To address these challenges, we have worked with the US resource centre (NXR) to develop existing methods for Xenopus sperm freezing to the point where they are robust. We have already successfully sent a few transgenic lines as frozen sperm and now aim to develop this process so it can be used to distribute GA and wild-type gametes worldwide. We will first optimize the number of aliquots testes can be divided into without compromising fertilization and development of the embryos produced; this will minimize the number of male Xenopus used for research. Experience sending other resources shows that the key to success in this programme is how the sperm are sent, the paperwork that accompanies them to facilitate passage through customs and the instructions with them. We will develop these by an iterative process, first sending dummy packages and then cryopreserved sperm to six test labs around the world, then getting feedback about the timing of arrival, success of embryo production and accuracy of the instructions. We will modify our methods and test again. Once the sperm sending process is robust we will roll it out to other labs progressively, this will allow us to make any necessary changes to the infrastructure and organization at EXRC to support this programme.

The main impact of this project is centrally within the 3Rs and focused on male Xenopus frogs; they will suffer less from stress associated with transport and fewer of them will be used. Additionally, the reduced cost of transport and use of fewer males for experiments will improve the cost effectiveness of research grants from funding agencies. The mechanism by which we will reach as much of the research community as possible is outlined in the "communications" section.

There will be training benefit for the graduate RA on the programme. They will be working in a large team that is expert at Xenopus husbandry and sperm cryopreservation, together with managing the logistics of transporting biological samples. They will work closely with that team and gain skills in those areas. Added to this the RA will have excellent opportunities to hone their networking and communication skills whilst they work with the test laboratories and in the initial stages of rolling out the service. Finally, they will develop skills that they are unlikely to have had before, in video design and production.

In terms of bringing this work to the wider public, the EXRC staff have an excellent record of outreach. We welcome School visits to the EXRC (including ones from overseas, e.g. Sweden and Russia) and all University of Portsmouth second year biology and biomedical science undergraduate students are introduced to animal research and basic ethics via the EXRC. We have recently started going to local schools to talk about the animal research and the senior post-doc in the EXRC, Dr Ania Noble, has been trained to do so by the organization, Understanding Animal Research. Finally, we represent the University at the annual Hampshire science fair which reaches over 2000 primary school children. We will use the example of this project as good 3Rs practice in all of these activities.