Choice of method of administration when conducting minor procedures without anaesthesia

Substances are usually administered by the oral, subcutaneous, intramuscular, intraperitoneal or intravenous routes.

The selection of a particular route of administration must balance a number of factors – for example, the volume and physicochemical properties of the substance, the required speed of onset, and other factors such as the degree of tissue irritation that could be caused. These topics are discussed in more detail in a number of different guidelines (Diehl et al, 2001¹, Morton et al, 2001², Turner et al, 2011a³ and b⁴)

Oral administration

Material can be administered orally using a number of different techniques, although gavage with a stomach tube is the most widely used. To minimise the risk of adverse effects associated with this procedure, it is important that the operator is skilled both in the technique and the restraint method needed.

Although gavage can be undertaken using rigid dosing cannulae, flexible catheters or tubes are preferred, as these are less likely to cause oesophageal trauma. Inadvertent dosing into the lung can occur, which usually results in the animal showing immediate signs of respiratory distress. If such signs are observed, then the animal should be immediately humanely killed.

As an alternative to gavage, some materials may be consumed voluntarily in palatable mixtures (e.g. flavoured syrups, Leach et al, 2010⁵, Corbett et al, 2012⁶, Zhang, 2011⁷, Küster, et al, 2012⁸, Gonzales et al, 2014⁹). Rats and other species can also be trained to drink voluntarily from a syringe (see video) and this approach has been shown to be both effective and to result in accurate dosing (Atcha et al, 2010¹⁰). Material can also be dosed using a small flexible catheter introduced only into the animal’s mouth. When possible, these approaches are to be preferred to dosing using a stomach tube, since they are not associated with complications, such as tracheal dosing or oesophageal rupture.

Subcutaneous administration

Subcutaneous administration of material often causes minimal pain or discomfort, provided the material is non-irritant, has a near-neutral pH, and is not excessively hypertonic or hypotonic.

The usual site for injection in most species is over the shoulders, into the loose skin over the neck (the “scruff”), but other sites with loose folds of skin can also be used, for example over the flank. When repeated doses of material are needed, varying the site of injection can help reduce the likelihood of local skin reactions.

It is not usually necessary to try to sterilise the skin with antiseptics – their use is almost always ineffective and they simply prolong the duration of restraint needed and may cause additional disturbance to the animal.

As with all injection sites, using a new needle for each animal, and injecting fluid that is at body temperature will reduce any discomfort caused by the procedure.

As with other routes, if repeated injections of material are needed, consider alternatives such as the use of mini-pumps.

Intramuscular Injection

In the rat and other small rodents, the very small muscle mass makes intramuscular administration both technically difficult and painful for the animal because of the distension of the muscle.

If intramuscular injections are necessary, they can be made into the front or back of the thigh in all small rodents. In the rat and guinea pig, the muscle mass is usually sufficient for accurate administration of small volumes of material, ideally 0.05ml or less. Note that this volume, although small, would be equivalent to 13-15ml injected into an adult person.

It is not usually necessary to try to sterilise the skin with antiseptics – their use is almost always ineffective and they prolong the duration of restraint needed and may cause additional disturbance to the animal.

As with all injection sites, using a new needle for each animal, and injecting fluid that is at body temperature will reduce any discomfort caused by the procedure.

Material that is irritant or with a high or low pH can cause pain both during and following injection.

Intraperitoneal injection

Although widely used as a means of administering substances, particularly injectable anaesthetics, intraperitoneal injection is an inherently unreliable technique, since inadvertent injection of some material into the gut, abdominal fat and subcutaneous tissues is a relatively frequent occurrence (Steward et al, 1968¹¹, Gaines Das and North, 2007¹². For this reason, it may be preferable to use other routes such as subcutaneous or oral administration.

It is not usually necessary to try to sterilise the skin with antiseptics – their use is almost always ineffective and they simply prolong the duration of restraint needed and may cause additional disturbance to the animal.

As with all injection sites, using a new needle for each animal, and injecting fluid that is at body temperature will reduce any discomfort caused by the procedure.

Material that is irritant or with a high or low pH can cause pain both during and following injection.

As with other routes, if repeated injections of material are needed, consider alternatives such as use the of mini-pumps.

Intravenous injection

Intravenous administration of material can be technically difficult in small rodents, and use of a restraining device is often required. These should be selected carefully to be an appropriate size for the animal to be injected. Too small a device can result in injuries to the animal and can interfere with respiratory movements. Too large a restrainer can also result in injury, caused by movements during restraint. After use, restraining devices should be cleaned thoroughly, to avoid stress caused by residual odours and pheromones, or cross-infection with potential pathogens.

In larger species, an assistant is usually required to restrain the animal.

Since small movements of the animal can dislodge a hypodermic needle from the vein, alternative delivery systems may be found useful, especially in the rat. “Over-the-needle” catheters allow a flexible catheter to be introduced into a vein through the skin. These can be anchored securely to provide a more stable route for injection or sampling. Small diameter catheters are available, suitable for use in rat (24g) or mouse (26g) tail veins.

Although rapid buffering by the blood and rapid dilution can allow a wider range of materials to be administered, injection into a peripheral vein can result in irritation, and in some circumstances flushing with normal saline is advisable.

It is common practice to stimulate dilation of the tail veins in rats and mice either by placing them in a warmer environment (e.g. at 28-30 ºC) for up to 30 minutes, or by placing the tail in warm (30-35 ºC) water. If a warming box or incubator is used, its temperature should be monitored carefully. It is good practice to place an electronic thermometer adjacent to the animal’s cage in the incubator, as the temperature registered by the device thermostat may not be accurate.

Selection of needle size

Generally, the smallest diameter needle should be used, as this causes less discomfort on insertion. However, this must be balanced against the speed of administration that can be achieved, since injecting high viscosity material, or large volumes of material, with a small diameter needle can be a slow process.

1. Diehl, K.H., Hull, R., Morton, D., Pfister, R., Rabemampianina, Y., Smith, D., Vidal, J.M. and Vorstenbosch, C.V.D., 2001. A good practice guide to the administration of substances and removal of blood, including routes and volumes. Journal of applied Toxicology, 21(1), pp.15-23.
2. Morton, D.B., Jennings, M., Buckwell, A., Ewbank, R., Godfrey, C., Holgate, B., Inglis, I., James, R., Page, C., Sharman, I. and Verschoyle, R., 2001. Refining procedures for the administration of substances. Laboratory animals, 35(1), pp.1-41.
3. Turner, P.V., Brabb, T., Pekow, C. and Vasbinder, M.A., 2011. Administration of substances to laboratory animals: routes of administration and factors to consider. Journal of the American Association for Laboratory Animal Science, 50(5), pp.600-613.
4. Turner, P.V., Pekow, C., Vasbinder, M.A. and Brabb, T., 2011. Administration of substances to laboratory animals: equipment considerations, vehicle selection, and solute preparation. Journal of the American Association for Laboratory Animal Science, 50(5), pp.614-627.
5. Leach, M.C., Forrester, A.R. and Flecknell, P.A., 2010. Influence of preferred foodstuffs on the antinociceptive effects of orally administered buprenorphine in laboratory rats. Laboratory animals, 44(1), pp.54-58.
6. Corbett A, McGowin A, Sieber S, Flannery T, Sibbitt B. A method for reliable voluntary oral administration of a fixed dosage (mg/kg) of chronic daily medication to rats. Laboratory animals. 2012 Oct;46(4):318-24.
7. Zhang, L., 2011. Voluntary oral administration of drugs in mice. Protocol Exchange, 10.
8. Küster, T., Zumkehr, B., Hermann, C., Theurillat, R., Thormann, W., Gottstein, B. and Hemphill, A., 2012. Voluntary ingestion of antiparasitic drugs emulsified in honey represents an alternative to gavage in mice. Journal of the American Association for Laboratory Animal Science, 51(2), pp.219-223.
9. Gonzales, C., Zaleska, M.M., Riddell, D.R., Atchison, K.P., Robshaw, A., Zhou, H. and Rizzo, S.S., 2014. Alternative method of oral administration by peanut butter pellet formulation results in target engagement of BACE1 and attenuation of gavage-induced stress responses in mice. Pharmacology Biochemistry and Behavior, 126, pp.28-35.
10. Atcha, Z., Rourke, C., Neo, A.H., Goh, C.W., Lim, J.S., Aw, C.C., Browne, E.R. and Pemberton, D.J., 2010. Alternative method of oral dosing for rats. Journal of the American Association for Laboratory Animal Science, 49(3), pp.335-343.
11. Steward, J.P., Ornellas, E.P., Beernink, K.D. and Northway, W.H., 1968. Errors in the technique of intraperitoneal injection of mice. Applied microbiology, 16(9), p.1418.
12. Gaines Das, R. and North, D., 2007. Implications of experimental technique for analysis and interpretation of data from animal experiments: outliers and increased variability resulting from failure of intraperitoneal injection procedures. Laboratory animals, 41(3), pp.312-320.