The first in a series on refinement of scientific procedures and laboratory aninial

This report is the first in a series on refinement of scientific procedures and laboratory aninial
husbandry. Its aim is to help those removing blood from animals to do so in the most humane
and efficient way so that any pain, distress or discomfort for the animals is kept to a minimum.
The report should also be useful in training courses, or as basic reading before applying for a
personal or project licence under the UK Animals (Scientific Procedures) Act 1986. The
emphasis throughout is on practical detail. It is hoped that publications on refinement of
experimental procedures such as these will lead to 'best practice becoming common practice' in
all laboratories.

Blood is removed from animals for a variety of scientific purposes. Scientists should be aware
that the process may well be unnecessarily stressful for an animal, simply because of the
handling, the type of anaesthetic, or the discomfort associated with a particular technique. The
physiological changes associated with increased stress may even invalidate results (Ajika et al.,
1972; O'Neill & Kaufmann, 1990; Sarlis, 1991). Comparison of 'normal' blood obtained
through chronic indwelling canulae in unrestrained animals with blood obtained by more
conventional methods has shown significant differences, for example in the levels of prolactin,
cortisol, corticosterone and glucose, as well as in counts for red and white cells and platelets,
and packed cell volunie. Since stress may cause physiological reactions which may affect the
research, the method of blood sampling used should be checked for any associated changes,
e.g. in blood corticosteroid levels. It would then be possible to see if an animal adapted to a
procedure and becomes less stressed as a result. It is obviously in the interests of good
science, as well as of animal welfare that stress should be kept to a minimum.
It is worth noting that when collecting blood for antibody production, it may be
desirable to collect blood into an anticoagulant and process the plasma. The yield of serum
from blood is relatively, poor and the yield of antibody can be 20-50% higher from plasma than
from serum.
The report is divided into sections which describe the removal of blood from veins
(Sections 3 & 4), arteries (Section 5), and by cardiac puncture (Section 6). Where the route for
blood removal may also be used for the administration of substances, or for measuring blood
pressure, further details are given. Each section is subdivided to describe possible techniques
and ttieir advantages and disadvantages. The potential adverse effects of each are discussed
together with recommendations on how these can be minimized. The report concludes with a
guide to the severity banding of blood sampling techniques for project licence applications,
together with recommendations for training of personal licensees.

The volume of blood removed from an animal will normally be determined by the scientific
protocol which, in turn, will depend on aspects such as the sensitivity of assays to be used
subsequently. Section 3.2 deals with removal of small volumes of blood of less than 0.1 ml.
Volumes over 0.1 ml are dealt with in Section 3.3. Methods of venepuncture of particular
concern are described in Section 3.4.
It is important that scientific techniques are continually refined so that only small
sample volumes are needed. However, there may still be occasions when the small size of the
animal is critical because of the volume or frequency of blood samples needed (e.g. in mice).
In such cases, the welfare of an individual animal should not be jeopardized and either more
animals should be used, or some form of compensatory blood transfusion or replacement
considered as appropriate. Frequent sampling increases the stress for the animal and, if this is
necessary the scientist should consider cannulation. Even in the short term, this technique is a
favourable alternative to repeated venepuncture. It is addressed as a separate topic in Section

A superficial vein can be punctured to obtain blood for haematological or chemical estimations
requiring only 50-200 l (approximately equivalent to 1-4 drops). Anaesthesia is normally not
necessary since the associated stress would probably be greater than the discomfort of a needle
prick or of a puncture with a small sharp sterile object.

A sterile needle or lancet should be used to puncture the skin and underlying blood vessel. A
scalpel blade is not recommended as its use is imprecise, and may lead to accidental mutilation
of the animal, or operator if the animal is not adequately restrained.

The recommended sites for venepuncture in a variety of species are given in Table 1.
To familiarize oneself with the location of a vein, it is strongly recommended that the
relevant anatomy first be studied on dead animals to avoid having to make repeated
unsuccessful entries when trying to locate a blood vessel.
A common site for venepuncture in a small animal is the coccygeal or tail vein. In
small rodents the tip of the tail may be removed and in mice - unlike rats - this seems not to
involve the removal of any coccygeal vertebrae. Tail cutting should be carried out only once or
a maximum of twice. (In naked mole rats, nicks can be made at the end of the tail which
regrows in 4-6 weeks and can then be used again.)
For small, tail-less animals, such as guineapigs and hamsters, the ear or jugular veins
may be used, but this requires considerable skill; in these mammals cardiac puncture under
general anaesthesia may be the preferred method. In large animals it is more likely that a small
sample will be taken directly from a superficial vein (see Section 3.3). In birds, puncture of the
wattles, combs or snoods can be used.
The use of the footpad for obtaining blood is unacceptable because of the sensitivity of
the area and the risk of infection, since laboratory animals are usually kept near, or on, bedding
contaminated with urine and faeces. Infection can result in lameness and unnecessary