Experimental procedures

Administration of substances

In the case of procedures involving the administration of drugs or other substances, the following basic aspects must be considered.

Nature of the drug or substance administered.

• Before performing the procedure, its possible toxic or harmful effects must be considered. If such effects can be expected according to the information available, at systemic or local level, the form for that procedure must contain specific supervisory measures to detect these effects as they occur, and corrective measures to be applied to the animals affected.

Route of administration

• Enteral (mainly oral).
  • Intake through food or drinking water. This is the simplest method but account must be taken of the capacity given by the deign established to derermine the quantity of substance ingested and whether or not that could affect the results (for example, in substances administered in drinking water for animals stabled in groups). This type of administration is not possible for substances that irritate the gastric wall.
  • Animals could be made more willing to ingest the substance if it is made more palatable by, for example, dissolving it in gelatine or adding a sweetener.
  • In some cases it is advisable to use gastric tubes (0.8mm in outer diameter for mice and 1 to 2 mm for rats) or a curved needle for dosing in small animals.
     
• Parenteral.  For an administration of this type, the following basic aspects must be taken into account: the needles, which must be clean, sharp and sterile, and of the right size and gauge for the animal concerned, the route, the administration site and the viscosity of the fluid. Never exceed the maximum recommended volumes or the bolus injection speed. To inject large volumes, these must be spread over several administrations in different sites. When the pH is too high (>8) or too low (<4.5) it must be diluted in saline solution to avoid irritant effects. The solution to be administered must be at room or body temperature to avoid pain from injecting cold substances. The skin must be cleaned with a disinfectant solution (povidone iodine or chlorhexidine) to prevent infections, but not excessively, so as to cause no irritation or elimination of bacterial flora. The recommendations for intracerebral administrations are in the section on stereotaxy.
• The principal routes are as follows.
  • Intradermic (ID).
  • Subcutaneous (SC). This is the simplest and best tolerated route and it allows relatively large volumes to be administered, but the substances are absorbed quite slowly.
  • Intramuscular (IM). Necessary to aspirate before injecting to make sure there is no blood flowing through the needle (if there is, change the injection site). Does not allow very large volumes, so most cases require injections in different sites or using an alternative route.
  • Intraperitoneal (IP). Very common in rodents in which IV or IM access is difficult or inadequate; Care must be taken to avoid injuries from the injection (peritonitis, hepatic injury, etc.). For repeated administrations, the injection site should be on a different side of the body each time.
  • Intravenous (IV). Drugs have rapid, powerful effects and allow large volumes and small doses to be injected, and allow the administration of substances with high non-physiological pH or osmolarity values. For repeated, frequent administrations a catheter must be fitted and supervised periodically to keep it in optimum condition.

 

• Topical and inhalable
  • In topical administration (normally used for a local effect), the surface where the substance is to be applied (a solution or a cream) must be correctly shaved. In some cases depilatory creams are advisable. Depending on the type of animal, suitable physical restraint measures should be considered.
  • Signs of skin or mucous irritation should be checked for.
  • The inhalation route requires masks or vaporisers to match each animal species in order to ensure correct dosage.

Recommended volumes and sites depending on the administration route and the animal species

 

Blood extraction

The following are recommendations for procedures that involve extraction of blood, and a table with the blood volumes and usual entry sites for each species.

Volumes up to 0.1 (2-4 drops)

• Correctly immobilise the animal.
• Do not use injectable anaesthesia. Inhalable anaesthesia may affect the biochemical blood parameters. Consider applying anaesthetic cream.
• Shave and disinfect the entry site.
• It is advisable to use a sterile lancet rather than a scalpel.
• After making the extraction, press on the entry site for about 30 seconds.
• Check for possible adverse effects during the following 30 minutes. If any effects are observed, alert the centre's veterinarian.

Volumes above 0.1 ml

• As a rule, in healthy, well-nourished animals, the maximum volume to be extracted in a single extraction should not exceed 10% of the volume of circulating blood.
• In series of extractions it is important to take into account the volume of blood to be extracted in each sample, the number of samples and the intervals between each extraction. Depending on the frequency and volume of the required samples, researchers will need to carefully consider using a butterfly needle or a percutaneous cannula attached with adhesive tape.
• The animal should previously be placed in a thermostatic chamber (30ºC) to dilate the veins. If the extraction is from the tail vein the tail may be placed in a thermostatic bath (45ºC). Never use irritants that cause vein dilation due tot this effect.
• Correctly immobilise the animal.
• Do not use injectable anaesthesia. Inhalable anaesthesia may affect the biochemical blood parameters. Anaesthetic creams seem to be quite beneficial when applied 30-60 minutes before the extraction.
• Shave and disinfect the entry site to avoid infections and contamination of the sample and to make it easier to find the vein.
• Needle sizes vary from 15 to 50 mm in length and from 14 to 26G in diameter, depending on the diameter of the vein and the volume of blood required. It is advisable to administer a local anaesthetic (SC or topical) during the insertion of 14 to 18G needles.
• Choose a suitable entry site (see table) and draw the blood quickly enough to prevent the vein from collapsing.
  After making the extraction press on the entry site for 30-60 seconds.
• Observe for 30 seconds to detect haemorrhaging. Check for possible adverse effects during the following 30 minutes. If any effects are observed, alert the centre's veterinarian.
• Change the entry site for any subsequent extractions.

 

More information and videos of interest in this area can be found at https://www.nc3rs.org.uk/3rs-resources

In vitro procedures
In vitro procedures are those in which the animals are slaughtered humanely using euthanasia.

The following should be considered when using this procedure.

• The form submitted for each procedure should contain the total number of animals to be used, however approximate, based on scientific criteria, whether these are statistical, bibliographic or estimates calculated from the amount of biological material that can be obtained from an animal.
• The euthanasia method must be in line with the recommendations for euthanasia of experimental animals contained in FELASA, a document published in Laboratory Animals (1996) Vol.30(4):293-316 (see Agents and Methods of' Euthanasia).
• If the chosen euthanasia method is not one of the first options, such as decapitation, the scientific criteria for the choice must be explained in the form for the corresponding procedure.
• If decapitation is shown to be the best option for obtaining valid results, the following recommendations should be observed.
  • The personnel performing the procedure must be suitably trained in the decapitation technique.
  • All the material must be perfectly clean and in optimal condition.
  • Use a physical restraint method, such as a decapicone, and let the animals get used to being handled in this way during the procedure.
• The use of ether as a chemical agent in euthanasia is not allowed under any circumstances.

Tumours in rodents

All procedures in which experimental neoplasias are induced must define the end-point criteria: the set of conditions under which the animals affected will be euthanised.

Some of these general criteria are as follows.

• The weight of the tumour is 10% greater than the weight of the animal. Given that in some cases it is difficult to calculate the weight of the tumour, due to its shape for example, the form for the corresponding procedure must contain an explanation of how the weight of the tumour is to be monitored and what measures will be adopted if the above limits are exceeded.
• The tumour ulcerates before reaching this weight.
• There could be a significant weight loss due to the tumour itself or the experimental design (for example, procedures that involve antitumour therapy). In these cases an end-point criterion would be a 15-20% weight loss or a 15-20% reduction in weight gain in animals in the growth stage.
• Some tumours can interfere with the correct functioning of certain vital organs, especially those needed for correct nutrition (inability to move in order to obtain food or water, inability to swallow, etc.). The animal should be euthanised in such cases, regardless of the weight of the tumour or of the animal.

Animals with induced tumours should be supervised daily by the personnel involved in the procedure.


Euthanasia

Agents and methods of euthanasia

Decree 214/1997, Chapter 9, Article 28 c): it is a function of ethics committees on animal experimentation to ensure that humane euthanasia methods are used.

The basic criteria for euthanasia, in terms of welfare, are that the method should not be painful, should lead rapidly to unconsciousness and death, require minimum restraint, avoid agitation, be suitable for the age, species and state of health of the animal, minimise fear and stress for the animal, and be reliable, reproducible, irreversible, simple to administer (in low doses if possible) and safe for personnel. As far as possible it should be aesthetically acceptable for the personnel.

The following recommendations are based on the document published by FELASA in Laboratory Animals (1996) Vol. 30(4):293-316 Recommendations for Euthanasia of Experimental Animals.

 

Statistical analysis
Decree 214/1997, Chapter 9, Article 28a). It is a function of ethics committees on animal experimentation to inform on the possibility of reaching valid conclusions with the least possible number of animals.
Applied Statistics Service (SEA)

Humane end-points in animal experimentation