Source: Kay Stewart, RVT, RLATG, CMAR; Valerie A. Schroeder, RVT, RLATG. University of Notre Dame, IN
Compound administration is often an integral component of an animal study. Many factors need to be evaluated to ensure that the compound is delivered correctly. The route of administration affects the mechanisms of absorption. The characteristics of the substance to be introduced (the pH, viscosity, and concentration) may dictate which route of administration is selected.1,2,3
The two main administration routes are enteral (via the digestive tract) and parenteral (administered outside of the digestive tract). The choice of the administration route is primarily determined by the requirements of the study. The main advantage to parenteral dosing is that the compound is not subjected to hepatic metabolism, resulting in higher bioavailability. However, there are many experimental protocols that require the compounds to be administered orally to better mimic the natural intake of a substance. The best route to utilize is determined by the properties of the test compound.
Oral dosing is the most commonly used method of drug administration in humans. This is done with tablets, capsules, or liquid forms of medication. Tablets and capsule administration is impractical in rodents, so the common oral dosing for mice and rats is accomplished by either dissolving the compound in drinking water, using specialized food that incorporates the test compound into the formula, or by placing the compound in treats. When dosing via drinking water, sucrose is often added to the water to enhance palatability. However, it should be finely balanced to encourage normal water intake and not excessive drinking because of the sugary taste.
Many studies have demonstrated that repeated oral gavage is stressful to both mice and rats.4,5 It is also labor intensive for the animal care staff. There is commercially available feed that has commonly dosed compounds-such as fenbendazole, which is used to eliminate parasites in rodents-incorporated into it. Other companies provide medicated treats that contain anti-inflammatories, analgesics, antiparasitics, or antibiotics. Feed companies that manufacture the formulated diets for mice and rats will create specialized test diets to meet an experimental requirement. Many laboratories have formulated their own treats with the use of honey, peanut butter, or fruit-flavored gelatin.4,5
Oral dosing lacks precision because the intake of a compound is dependent on the acceptance of the food or water, the stability of the compound, and the number of animals present in the cage. However, if exact dosing is not critical to the experiment, this noninvasive method of dosing is best for the wellbeing of the animals.
Oral gavage is a much more precise method of enteral dosing, as it allows for the deposition of the compound directly into the stomach of the animal at a specific time and at a specified volume. However, training is required to ensure success. Proper restraint of the animal is crucial to the success of this technique. Injury to the oral cavity, pharynx, larynx, trachea, esophagus, and stomach can result from improper positioning of the head and the body. The animal must be tightly gripped, such that the head is unable to twist, to prevent such injuries.
Dosing needles are available in reusable or disposable formats. Though costly, the use of disposable needles removes the threat of cross contamination when dosing multiple animals. There are several sizes of gavage needles, ranging from 20-22 gauge for mice and 18-20 gauge for rats. The choice of needle is determined by the size of the animal, the volume of liquid, and the viscosity of the solution to be administered.
Oral gavaging should be performed on a conscious animal to decrease the possibility of placing the needle into the trachea. An animal that is conscious will retain the swallowing reflex as well as the gag reflex, facilitating the proper placement into the esophagus. A study was conducted demonstrating that precoating the gavage needle with sucrose induced the swallowing reflex and decreased the amount of time needed to pass the needle into the esophagus.6
A topical agent that is placed directly on the surface of the skin is generally intended to affect only the area to which it is applied. These can be creams, lotions, ointments, foams, or gels. As animals will tend to groom the area where the agent is applied, the toxicity of oral consumption must be considered.
Compounds administered topically are formulated to be absorbed through the dermis layers and the subcutaneous space via the hair follicles and the accessory glands. Factors that affect the absorption of the compounds include the condition of the skin, the surface area utilized, the concentration of the substance, the lipid solubility of the compound, and the length of time it is in contact with the skin surface. Well-hydrated and thin skin layers will decrease the absorption time.
When applying a substance topically, there is the chance for systemic absorption due to the animal grooming the area. Preventative measures should be taken to avoid accident ingestion or early removal of the substance. Small collars or shields are available to prevent the animal from reaching affected body parts. Individual housing may also be required to prevent allogrooming from cage mates.
1. Topical application
- Before applying any ointments and creams, remove all fur from the area through the use of a depilatory cream or by shaving.
- Thoroughly clean and dry the skin.
- Apply the substance directly to the skin with a cotton-tipped applicator or via a directly-placed drop to avoid contact by the technician.
- For best results, apply several thin layers of ointment or cream opposed to a onetime application of a thick layer.
- Apply liquid by parting the hair and applying a drop on the exposed skin.
- When applying a substance topically, there is a chance of systemic absorption due to the animal grooming the area. Preventative measures should be taken to avoid accident ingestion or early removal of the substance. Small collars or shields are available to prevent the animal from reaching affected body parts. Individual housing may also be required to prevent allogrooming from cage mates.
Figure 1. Topical application in mice.
2. Oral dosing
- When providing compounds to animals via feed or water, a minimum daily dose based on body weight must be calculated. The average weight of the mouse or rat strain being used in the experiment can be used to make the calculations, unless it is specified that each animal be weighed and the dose figured for each individual.
- For protocols that require that food and/or water intake be precisely measured, animals need to be housed individually.
- Specialized feed or treats can be manufactured to meet experimental needs.
- Replace the standard diet with the formulated one, which allows the animals to ingest the compound throughout the day.
- The food may need to be replaced more frequently than standard food due to compound instability.
- Substances may also be administered by dissolving them in the drinking water, allowing for the animals to receive a compound throughout the day
- A small amount of sugar is added to the water to make it palatable. The exact amount to add is determined by the acceptance of the water by the mice. Use the smallest increment possible.
- The frequency of changing the water is dependent on the stability of the compound. Some substances may be light-sensitive or precipitate over time.
3. Gavage procedure
20-25 gauge x 1.5 in straight or curved dosing needles are used for mice. For rats, 18-20 gauge x 2-3 in straight or curved dosing needles are used. These are available as reusable stainless steel needles or as a disposable needles. The disposable needles can have a metal or flexible plastic shaft with a silicone tip.
- Attach the dosing needle to a syringe selected based on the volume and viscosity of the test solution. For dosing thick liquids, a Luer lock syringe is preferred. These are generally available beginning with a 3 cc size that has 100 µl graduations. This may not be suitable for smaller volumes or when there is a need for exact-dose volumes at smaller increments.
- Affix the syringe to the needle such that the graduations on the barrel can be read without turning the needle. Once the needle has been placed into the esophagus, any rotation can rupture the esophagus, resulting in death of the animal.
- Fill a syringe with the appropriate volume of article to be administered (optimal volume = 5 ml/kg and maximum volume = 20 ml/kg)4
- The proper restraint is essential for this procedure.7
- Manually restrain mice by grasping the skin at the base of the head and scruffing them. It is important that the body be held such that it is suspended in a straight line from the head to the tail. Any twisting of the body will impede the placement of the gavage needle into the esophagus.
- Grasp the scruff tightly so that the head has minimal side-to-side movement. The hindquarters must be stabilized to prevent body rotation.
- For gavaging a rat, grasp it over the shoulders using the index and middle fingers on each side of the neck, and the palm on the animal's back. The thumb, third, and fourth fingers should encircle the chest to prevent the animal for being able to move forward or backward. The position of the fingers on each side of the neck prevents the rat from turning its head.
- It is important that the body be in a straight line and not twisted to assist in the placement of the gavage needle without damage to the mouth, pharynx, esophagus, or stomach.
- Make sure the head does not have excess side-to-side movement.
- The hindquarters may be stabilized to prevent body rotation. This is accomplished by allowing the back feet to rest on the counter or cage top.
- Administration of the substance
- Position the needle with the curve facing downward and inline with the natural curve of the neck. Introduce the needle into the mouth from the right or left side, between the incisors and molars at an angle toward the back of the pharynx.
- Once the needle is in the oral cavity, tilt the head back using the gavage needle until the nose is pointed up. This is especially important when using straight needles. The needle should slide easily into the esophagus. Do not force the needle down. If any resistance is felt, back the needle out and try again.
- Once the needle is in position in the stomach, gently deposit the compound.
- Remove the needle, making sure that no rotation of a curved needle takes place.
- If the animal begins to struggle, remove the dosing needle and restrain the animal more securely.
- While performing the procedure, if the animal shows signs of cyanosis or respiratory distress, or if there is solution flowing from its mouth, the dosing needle should be immediately removed and the animal released into its cage.
- In general, when gavaging rats and mice, particularly with a caustic substance, it is a recommended practice to intially deliver a small amount – to test for coughing – before the entire volume is given. That way, if the needle is incorrectly positioned, the volume delivered to the lungs is small and the animal has a better chance at recovery.
Figure 2. Oral gavage in mice (left) and rats (right).
Many experiments require that a compound be administered into an animal via an oral or topical route, so that the method better mimics the natural intake of the experimental substance.
For oral dosing, the compound can be dissolved into the drinking water or incorporated into the food that the animals consume. Another, more precise method of oral dosing is oral gavage in which a large needle is used to place the compound directly into the stomach. On the other hand, a topical agent can be placed directly on the skin surface, and is generally intended to affect only the area to which it is applied. Each of these strategies has advantages and limitations, which we will discuss in this video. Following that we will demonstrate how to perform these compound administration techniques in lab animals, and some of their applications.
Before delving into protocols, let's review the considerations, benefits, and shortcomings of these techniques. Oral dosing with tablets, capsules, or liquid is the most commonly used method of drug administration in humans. However, tablets and capsule are impractical for rodents, so the common oral dosing is accomplished by dissolving the compound in the drinking water or food.
When dosing via drinking water, sucrose is often added to the water to enhance palatability. However, it should be finely balanced to encourage normal water intake and not excessive drinking because of the sugary taste. A good rule is to start with a 10% sucrose solution, and then adjust depending on the intake. Another way to orally dose the animal is through specialized food. There is commercially available feed that has commonly dosed compounds, such as fenbendazole, incorporated into it. The rodent feed companies can also create specialized test diets to meet an experimental requirement.
The advantages with this form of oral dosing are that it is non-invasive, administers the compound continuously, and mimics human oral drug administration. However, it lacks precision because compound intake is dependent on the acceptance of the food or water, the stability of the compound, and the number of animals present in the cage. If exact dosing is not critical to the experiment, this non-invasive method is best for the well being of the animals.
For more precise oral administration, one can use oral gavage, which deposits the compound directly into the stomach at a specific time and at a specified volume. However, proper training is required to perform this technique because serious damage to the animal can occur if they are not properly restrained.
Coming to topical agents, these include creams, lotions, ointments, sprays, and gels. Typically, topical agents are intended to affect only the area to which they are applied. However, they can also be absorbed into the bloodstream, which may be intentional or unintentional. This absorption depends on: the condition of the skin, the surface area utilized, the concentration of the substance, the duration of contact, and the lipid solubility of the agent.
Now that we have discussed the background of oral and topical administration methods, let's learn the protocols starting with oral administration via feed or water.
For dosing calculations, first you need to know the intended dose to be administered. Second, you have to determine the body weight. Rather than weighing individual animals, you can use the average weight of the mouse or rat strain for your calculations, unless otherwise specified. Third, you should be aware of the number of animals per cage, and fourth, you need to know the average daily water or food consumption per cage.
Remember, the formulated food or the drinking water with the dissolved compound may need to be replaced more frequently than standard food or water due to compound instability. Some compounds may be light sensitive or may precipitate over time.
Now, let's learn how to orally administer precise doses by performing oral gavage in mice and rats.
First step is to select an appropriate needle for the procedure. These are either reusable made up of stainless steel or disposable with a flexible plastic shaft and silicone tip. For mice, 20-25 gauge straight or curved dosing needles that are 1.5 inches in length are appropriate. For rats, select 18-20 gauge straight or curved dosing needles that are 2-3 inches long. The correct needle gauge and syringe selection also depends on volume and viscosity. Review the Compound Administration I video to understand the impact of these factors. For dosing thick liquids a Luer lock syringe is preferred.
After making the selection, affix the syringe to the needle such that the graduations on the barrel can be read without turning the needle. This is important as once the needle has been placed into the esophagus any rotation can rupture the esophageal wall resulting in death of the animal. Next, fill the syringe with the correct volume of the solution to be administered.
This method should always be performed on conscious animals with intact swallowing reflexes to prevent accidental placement into the trachea. Using proper restraint technique is critical for this procedure. For mouse, grasp the skin at the base of the head and hold the scruff tightly. This ensures that the head has minimal opportunity for side-to-side movement. Also, make sure that the body is suspended in a straight line from the head to the tail; any twisting will impede the placement of the gavage needle into the esophagus. The hindquarters must be also stabilized to prevent body rotation.
To restrain a rat, grasp it over the shoulders using the index and middle fingers on each side of the neck. This will prevent the rat from turning its head from side to side. The thumb, third, and fourth fingers encircle the chest to prevent the animal from moving forward or backward.
Once you've properly restrained the animal, position the needle with the curve facing downward and in line with the natural curve of the neck. Then, introduce the needle into the mouth from the right or left side, through the diastemata and between the incisors and molars at an angle toward the back of the pharynx. Now that the needle is placed in the oral cavity, use it to tilt the head back until the nose is pointed up. At this point, the needle should slide easily into the esophagus. Once the needle is positioned in the stomach, gently deposit the compound. Then, remove the needle from the animal, making sure not to rotate it. It is important NOT to force the needle down. If you feel any resistance, remove it and try again. While performing the procedure, if the animal shows signs of cyanosis or respiratory distress or if there is solution flowing from its mouth, the dosing needle should be immediately removed and the animal should be released into its cage.
Lastly, let's review the procedures for topical administration in lab animals. Start by anesthetizing the animal. This is commonly done using a fast acting inhalant, which allows quick animal recovery. For more details on anesthesia administration, please see another video in this collection.
Next, using a hair clipper remove all fur from the area of application. To avoid any cuts, place the clipper's flat surface against the skin and shave in the direction opposite to that of the hair growth. Then, clean the skin with water and dry it with a gauze pad. Now, using a cotton-tipped applicator, apply the substance directly to the skin. For best results, apply several thin layers of ointment or cream, as opposed to a one-time application of a thick layer. To prevent accidental ingestion of topically applied compound due grooming, place a small collar or a shield to prevent the animal from reaching the affected body part. Furthermore, to prevent allogrooming from cage mates, the animals should be housed individually.
Now that you have an understanding of these alternate routes of administration, let's see how they are being used in biomedical research today.
One of the important applications of oral gavage is precise administration of substances to study their direct effect on the gastrointestinal tract. Here, the researchers used this method to deliver a transformed probiotic yeast directly into the animal's stomach. And then they dissected the Peyer's patches, which are organized lymph nodules from small intestine, to study the adherence of the delivered microorganism.
The non-invasive oral route can be used to mimic the natural modus of food-borne infection. In this experiment, the scientist developed a model for oral transmission of Listeria monocytogenes in mice via ingestion of contaminated food. Following infection, the researchers harvested various organs like small intestine, colon, spleen, liver and gall bladder, to analyze spread of infection to these tissues.
Lastly, some researchers are interested in studying the mechanism by which UV radiation may lead to sunburns. Here, the scientists used topical route to apply a pharmacologic substance that induces production of epidermal melanin-a UV protective agent. Following that, they used a protocol that is similar to human studies to examine the protective effect of the topically applied agent against erythema induced by UV radiation.
You've just watched JoVE's second installment of compound administration methods dealing with oral and topical dosing. You should now understand how one can orally dose the animal using feed or water, and how one can perform the more precise, oral gavage administration. Lastly, you should know the considerations and method for topical administration, and applications of these various techniques. As always, thanks for watching!
Applications and Summary
The safest and most humane method of compound administration is through oral dosing if it is possible to incorporate it into the feed or water. Oral gavage is a precise dosing method but is also very stressful to the animal, and it requires technical proficiency. Topical administration is usually accomplished with little or no restraint of the animal, once the area is properly prepared. Careful consideration of the route of administration should factor in the potential stress to the animals.
- Turner, P.V., Pekow, C., Vasbinder, M. A., and Brabb, T. 2011. Administration of substances to laboratory animals: equipment and considerations, vehicle selection, and solution preparation. JAALAS. 50:5. 614-627.
- Shimizu, S. 2004. Routes of Administration in The Laboratory Mouse. Elsevier.
- Machholz, E., Mulder, G., Ruiz, C., Corning, B. F., Pritchett-Corning, K. R. 2012. Manual Restraint and Common Compound Administration Routes in Mice and Rats. J. Vis. Exp. (67), e2771, doi:10.3791/2771.
- Turner, P. V., Brabb, T., Pekow, C., Vasbinder, M. A. 2011. Administration of substances to laboratory animals: routes of administration and factors to consider. JAALAS. 50, 600-613.
- Hoggart, A.F., Hoggart, J., Honerlaw, M., and Pelus, L.M. 2010. A spoonful of sugar helps the medicine go down: a novel technique to improve oral gavage in mice. JAALAS. 49:3. 329-334.
- Gonzales, C., Zaleska, M.M., Riddell, D.R., Atchison, K.P., Robshaw, A., Zhou, H., and Rizzo, S.J. 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. 126:28-35.
- 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. JAALAS. 51:2. 219-223.