Assessment of double ovulation to halve Xenopus laevis use for eggs

The African claw-toed frog, Xenopus laevis, is used in many laboratories because of its eggs. Unlike the eggs of many other species, frog eggs (spawn) are laid in large numbers and develop outside the other in ordinary pond water. This makes them ideal for observing embryonic development and doing experiments such as testing the consequences of a tissue graft or injecting small amounts of DNA (genetic material) to find out how genes work in a living organism. Xenopus frogs are particularly useful because they normally live in water and so can be kept easily in water-filled tanks all year round. To get them to lay eggs (ovulate) researchers inject the females with a hormone. If injected in the evening, the frogs lay eggs into the tank the following day. (This used to be used clinically as a pregnancy test since the hormone is elevated in a pregnant woman's urine.) Currently, labs using Xenopus ovulate frogs and then rest them for three months ("single ovulation"). However, although not routine, experiments have shown that a frog can be ovulated weekly over several weeks with only gradual decline in the number of eggs produced. This may reflect breeding in the wild where frogs have a limited breeding (rainy) season. We have conducted some pilot experiments to show that ovulating females two times, a week apart ("double ovulation") with three month's rest before this is repeated, yields similar numbers per ovulation that are not obviously less than single ovulation. In this project we will test this double ovulation regime to see if yield numbers are reliably maintained. Importantly, to ensure that the procedure does not cause additional stress to the animals, either immediately or over time, we will assess stress levels by tracking egg numbers and quality (which are both known to deteriorate for sick or otherwise stressed animals) and measure levels of a stress-hormone (corticosterone) that is released by the animals under stressful conditions. If these measures show that egg yields hold up with little or no increase in stress or harm to the animals, they will allow labs to use half as many animals as they do now. Worldwide, Xenopus users have a strong community and new methods are rapidly shared. There are no barriers to take-up of double ovulation, and we plan to disseminate our results using proven channels of the Xenbase website, national Stock Centres in the UK and USA and through training courses, as well as publication of our data and methods in journals and conferences.

The African clawed-toed frog Xenopus laevis is a widely-used research animal owing to its large egg and embryo size and reliable production of large numbers of eggs. The standard Xenopus ovulation protocol involves hormone injection to induce females to lay eggs, with reuse following three month's rest. Animals used this way flourish and lay useful egg numbers for many years. It has been reported that after an ovulation, a second ovulation just one week later yields an unchanged number of eggs and that successive weekly ovulations show only gradual decline in numbers. This is consistent with reports that Xenopus in the wild mate multiple times in a season and suggests that Xenopus have significant egg-laying capacity above what is currently used. We have piloted a Double-Ovulation protocol, re-ovulating frogs 7-10 days after a previous ovulation, followed by the standard 3-month rest. Preliminary observations show no signs of reduction in egg yield or health of the animals, despite doubling egg yield in a given calendar period. The proposed project will determine rigorously whether indeed doubling egg yield is possible while maintaining quality and without increasing stress to the animals, either acutely or over multiple cycles. We will directly compare single- and double-ovulation protocols, counting total eggs laid and measuring fertilisation rates. In addition to monitoring animal weight and general health, we will also, innovatively, determine stress levels in the animals by measuring secreted corticosterone levels using a newly established assay. We will assess both acute stress (during ovulation) and chronic stress (testing for build-up versus return-to-baseline between cycles). The results of this study will show whether it is possible to reduce the size of Xenopus colonies by half worldwide, potentially reducing the number of animals by half (up to 25,000 animals) while maintaining animal welfare and research productivity.

How will this project have impact?
The primary potential impact of this project will be to fulfil the Reduction aim of NC3Rs with respect to the most commonly used amphibian species, namely Xenopus. This work aims to establish whether double the number of eggs can be collected from each female Xenopus per ovulation cycle, thereby halving the number of females needed in any laboratory colony engaged in egg and/or embryo-based research. Xenopus has long been and remains one of the most powerful and pioneering vertebrate model systems in biomedical research (http://www.xenbase.org/entry/doNewsRead.do?id=220). Given that the current laboratory population of Xenopus used in the way that this project addresses is over 50,000 animals, the potential for reduction is strightforward, substantial, and realistic.

Who will this impact?:
In conjunction with Xenbase (www.xenbase.org), we have conducted a survey of the worldwide Xenopus research community to ascertain current ovulation protocols, estimate the number of research animals, and to predict the potential impact of this study. Our survey results show there are approximately 50,000 Xenopus in research laboratories around the world. Of surveyed laboratories, 90% ovulate their Xenopus females once in an ovulation cycle before recuperation, 0.5% rest their frogs for one month between ovulations, 2.5% use a Double-Ovulation protocol, while 7% do not ovulate their X. laevis at all. Therefore, should our Double-Ovulation protocol hypothesis be supported, we could potentially halve the size of Xenopus colonies in greater than 90% of laboratories that use Xenopus laevis.

More than 200 laboratories worldwide use Xenopus, including the large stock centres in the UK (the European Xenopus Resource Centre - EXRC) and the USA (the National Xenopus Resource - NXR). These two important centres, as well as a number of other current single-ovulation Xenopus laboratories have expressed interest in the double ovulation protocol pending the results of our study. This suggests that our impact would be far-reaching. The resource centres function as training centres and points of contact for the Xenopus community for advice regarding experimentation and animal husbandry. Letter of support from the directors of both centres demonstrate that the results of this project are of major interest in the heart of the Xenopus research community.

Should the results turn out as expected from pilot studies, namely showing minimal or no additional harm to the frogs from double-ovulation, impact could be rapidly achieved. Dissemination would be community websites (e.g. Xenbase), via research conference presentations (e.g. the biennial International Xenopus conference); and via the international Xenopus stock centres, the EXRC and NXR; as well as appropriate journal publications. We envisage the combined animal welfare and financial benefits caused by reduced Xenopus colony sizes will elicit rapid and widespread takeup of in the improved protocol.

The project will also provide additional validation of the corticosterone assay developed by a previous recipient of NC3Rs funding as an assay for wild-caught and other aquatic species in diverse contexts.