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Dosing of experimental animals and the removal of blood are two of the most frequent procedures performed. Despite the apparent simple nature of these procedures they can, if not correctly carried out, have significant effect on the welfare of the animals and the scientific value of the results. Before a laboratory uses a new procedure it is essential that advice on the most refined method is sought, and the Home Office Inspector and the NC3Rs are two such sources. Advice for refinement of removal of blood can also be found in the NC3Rs Blood Sampling Microsite. LASA has a process, together with funding, which would enable an investigator to visit a laboratory that is performing current best practice. Note that training animals with positive reinforcement is one means of reducing the stress associated with some traditional methods of conducting procedures.
When administering a substance to an animal by any route the aim should be to achieve best practice, since mistakes at any stage can cause avoidable suffering. This will involve careful consideration of the experimental aims, substance, route, individual animals, technique and staff involved.
A general principle when administering substances is to keep the volume as low as possible. However, when using substances with low solubility it may be necessary to give larger volumes to satisfy both scientific and regulatory requirements. This has led to the introduction of numerous publications and guidelines, each one purporting to reflect best practice.
Three key publications are given in the Resources panel on the right-hand side of this page. The first (Morton et al. 2001) is a comprehensive report produced by the BVAAWF/FRAME/RSPCA/UFAW Joint Working Group on Refinement. It reviews the whole procedure from planning and preparation to refinement for individual routes, including advice on needle sizes. The second publication (Diehl et al. 2001), was produced by a consortium of European pharmaceutical companies. The maximum dose volumes given in this publication should not be used without exceptional justification. Both reports have been adopted by a number of regulatory authorities.
The third publication (Healing & Smith 2000) is devoted to the use of continuous intravenous infusion, a method of administration that is becoming increasingly popular. In addition to information on the surgical procedures, aseptic techniques, analgesia, choice of catheter and husbandry procedures for each species, this book has a chapter on the contribution of vehicles, rates of administration and volumes to infusion studies.
The principle when removing blood is to keep the volume as low as possible and to use a method which keeps to a minimum any pain, distress or discomfort. The calculation of limit volumes for blood sampling relies on accurate data on circulating blood volumes and the literature indicates considerable variation depending on the methodology used. The publication by Diehl et al. (2001) provides limit volumes and recovery periods based on the most up to date scientific data. It recommends up to 15% circulatory blood volume with a recovery period of 1 month for toxicity studies and up to 20% for toxico- or pharmacokinetic purposes where multiple, small samples are usually required. Single sampling beyond 15% is not recommended since hypovolaemic shock may ensue if it is not done very slowly (multiple, small samples are unlikely to produce such effects).
The Diehl et al. publication also reviews and makes recommendations on sampling sites, as does the earlier report produced by the BVAAWF/FRAME/RSPCA/UFAW working group (Morton et al. 1993). Recommended routes for bleeding are the lateral tail vein, the sublingual vein and the lateral tarsal (saphenous) vein for all rodents, and the marginal ear vein, central ear artery and the jugular vein for rabbits. Retrobulbar sampling with recovery should only be used in exceptional circumstances. Sampling by cardiac routes should only be carried out as a terminal procedure under general anaesthesia. The NORINA database demonstrates saphenous vein sampling in mice. This procedure can dramatically reduce the numbers of animals used for pharmacokinetic profiling of substances and other studies where repeated sampling is needed.
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