Procedure di assistenza di base

Since mice and rats account for over ninety percent of the animals used for biomedical research, taking proper care of these organisms is critical to the outcome of the experiments. These care procedures begin right at the start with unpacking the animals from shipping containers, to cage changing, to maintaining proper housing conditions, providing appropriate diet, and ensuring suitable environment. Although, there are general procedures that apply to the majority of mice and rats in the laboratory, some animals, such as those that are immunocompromised, require additional attentiveness in order to sustain them for experimental purposes.

This video will describe the standards and essential care procedures for both immunocompetent and immunocompromised animals. And lastly, we will illustrate some example experiments showing how scientists use this knowledge for different research purposes.

Receiving animals from other institutions poses a risk to the animals already housed within the facility, as during transit, the transport boxes can come in contact with pathogenic agents. Therefore, a proper procedure should be employed when unpacking the shipping containers.

Before handling any animals, it is important to wear the appropriate Personal Protective Equipment, or PPE. The minimum PPE to wear is a lab coat, gloves, safety glasses, and a surgical type mask that will reduce the exposure to dust and allergens. The next step is to select appropriate cages for incoming animals. Then, furnish the cage with appropriate bedding and nesting materials. And take the prepared cages to the temperature-controlled receiving room, so that they are ready for transfer prior to the arrival of the animals.

Upon shipment arrival, place the transport boxes in a secure place in the receiving room. Examine the container for any damage and disinfect the outer surfaces to destroy any harmful pathogens. Make sure that the disinfectant is in contact with the box for at least 15-30 minutes, as per manufacturer’s recommendations. Then, place the container in the Animal Transfer Station, or ATS. This station offers High Efficiency Particulate Air, or HEPA, filtered unidirectional airflow within the hood. HEPA protects the user from allergens and shields the animals from the surrounding room air, minimizing the chance of contamination. Next, open the shipping container and evaluate the overall health of the rodents. Make a note of any animal that appears to be injured or ill. After that, place them into the already assembled cage in the proper group sizes, cover the cage with the wire bar lid and secure the divider to create two areas. Select the appropriate food and place it in one side and leave the second side open to allow for better air circulation. Lastly, if the animals are not being placed on a rack with automatic watering, add a water bottle to ensure adequate water supply. Remember, the ATS has to be disinfected at the end of each session, and the blower must be off when disinfecting.

For cage changing, place the two cages-a fresh cage with the appropriate bedding material and the used cage with the animals-in the ATS. One can transfer the animals from the old cage to the new cage by hand or with the use of a padded forceps. Both these methods have been discussed in another video of this collection. Remember only one cage of animals can be opened at a time. For male mice or females with a litter, it has been demonstrated that transferring the nesting area of the mice into the clean cage assists the animals with the transition. Once the cage top is placed on the new cage, transfer food from the old cage top to the new top and add more food if necessary. Next, place the barrier top, and return the cage to the rack. It is a good practice to change gloves between cages or between groups.

The unpacking procedure for immunocompromised animals is a bit different, as things need to be sterile. It is recommended that the blower inside the hood be on at least 24 hours before use. Like for immunocompenent animals, upon shipment arrival spray the container with a disinfectant solution in the receiving room and leave it unopened for 15-30 minutes. At the ATS designated for immunocompromised mice, spray the laminar flow hood with 70% ethanol 30 minutes prior to manipulating animals. It is ideal to have two people participate in this unpacking activity — one person would be the sterile technician wearing the steam sterilized gown and double pair of sterile gloves to handle the animals using aseptic techniques and the other would be the non-sterile technician wearing a clean isolation gown and a pair of gloves to handle contaminated items. Both should be wearing a, mask, hair bonnet, and safety glasses.

Before opening the shipping container inside the hood, place all the supplies and caging in these and spray their exterior of with alcohol. And upon shipment arrival, place the received animal box in the hood and sprays it with 70% ethanol as well. There are two options for sterile caging, either standard caging that has been wrapped and steam sterilized, or disposable units that has been sterilized using irradiation by the vendor.

Start the unpacking procedure by opening the sterile food, followed by opening of the transport box. At this point, the handler should discard the first pair of sterile gloves. Next, the non-sterile person unwraps the sterile cage and aseptically passes it to the animal handler who then removes the bonnet and the wire bar lid, and places them upside down in the hood. Subsequently, the sipper tube on the cage is filled with water at the station located in the hood. Lastly, the animal handler places the animal into the cage, secures it with the wire bar lid, adds food and sets the bonnet in place. Note that the hood blower should remain on at all times to maintain the sterile environment. At the end, the non-sterile person removes the populated cage from the hood and places it on an appropriate rack. To avoid possible cross contamination between groups, the animal handler should change gloves after each shipping container.

For cage changing of the immunocompromised animals, the preparation of the hood and cages and PPE are the same as for the unpacking of these animals. And the transfer technique is same as the immunocompetent animals, but under sterile conditions.

Now let’s discuss appropriate housing conditions for the animals, which is another important aspect of providing essential care. Mice and rats are primarily housed in shoebox type caging with a solid bottom containing bedding material. The typical mouse cage provides 75 square inches of floor space, which is adequate for a maximum of 5 adult mice. On the other hand, a rat cage has the area of 142 square inches and is adequate for 2 adult rats. Static cages are usually covered with a wire bar lid and filter material is added over the cage top as a bonnet. Individually ventilated cages, or IVCs, also have a wire bar lid and bonnet, but they are placed on a rack that provides airflow into the cage. This eliminates the need to change the cage as often as static cages, which may be beneficial for immunocompromised animals.

All caging systems have a provision for food and water supply. There are either feeders placed within the animal cage or a section of the wire bar lid is used for food storage. Food must be placed such that it is not soiled by the animals. Water is usually supplied via a water bottle placed on the top of the cage, or via an automatic watering drinking valve that is a built in component of the animal cage. For drinking valve option, when the cage is engaged onto a rack, water is transported into the tube for an automatic watering system that is designed to meet the needs of the animal throughout the animal facility.

The Guide for the Care and Use of Laboratory Animals states that all caging implements must be sanitized at least every 2 weeks. Some cages and accessories may need to be changed more often as dictated by the level of odor and ammonia in the enclosure.

Now let’s talk about proper food and water supply for immunocompetent and immunocompromised animals. There are many commercially available feeds with variable protein, carbohydrate, and fat compositions. Ideally, feeds should comprise 15-19% protein, 44% carbohydrates, and 4-9% fat. For immunocompromised animals, make sure that the feed has been sterilized.

Tap water, or reverse osmosis that is RO water, can be used for immunocompetent animals, but for immunodeficient animals, water can be a source of contamination, and therefore needs to be sterilized. Sterility in the water is maintained by adding 2 drops of 12 molar hydrochloric acid to 8 liters of RO water, which will acidify the water to a pH of 2.5-3 and limit bacterial growth. Then pour the water in individual bottles and cover them with aluminum foil. Place the bottle lids with sipper tubes separately in an autoclave bag, and put everything in an autoclave for steam sterilization.

Now let’s discuss the standard environmental conditions essential for animal care.

At the cage level, social housing is mandatory unless scientifically justified or if an animal is incompatible with all others. Enriching the environment with paper huts and nesting materials allows the animals to exhibit species-specific behaviors like nest building and burrowing, which in turn decreases the stress level and increases the overall well-being of the animals. Nesting materials also provide a sleeping area that is saturated in calming pheromones that assists with the housing of multiple male mice in the same cage. As mentioned previously, moving the nesting area to the clean cage upon cage changing helps prevent aggression, especially in males, as the animals are introduced into a new environment. For athymic nude animals, one should consider providing additional bedding and shredded paper type nesting materials in their cages to assist with thermoregulation.

At the room level, the temperature for mice and rats should be between 68 and 79° F. The room humidity level should be 30-70%, as lower levels can result in a condition known as ringtail. The third room parameter is lighting. This includes light intensity and cycles. Normally, the animal room is set to 12-hour light/12-hour dark cycle. The lighting intensity should vary in accordance with the activity in the room. When technicians are working, higher lighting levels can be used. However, it should be lowered to approximately 325 lux once all activity is concluded. Lastly, the airflow within the room should also be carefully controlled. A minimum of 10-15 air changes of 100% fresh air should occur every hour. This decreases microbes and odors in the animal room.

Lastly, let’s see how researchers are using this knowledge about lab animals housing, diet and environment preferences in their experiments.

As discussed earlier, lab animals generally exhibit nest-building behavior, which reduces stress, and researchers have used this as an indicator of animal welfare. In this particular experiment, scientists developed an unbiased system to score the nest built and then used it study the effect of ambient temperature on the nest-building behavior. As you can see, an increase in temperature caused a significant drop in the score, highlighting the role of surrounding temperature in animal welfare.

Given that mice and rats love carbohydrates, researchers have used feed of different shapes to test animal’s motor function. This includes analyzing dexterity during different types of food handling tasks, and examining reaching behavior following surgical or pharmacological interventions.

Lastly, it is known that mice and rats are nocturnal and they don’t prefer lit-up areas. Scientists use this knowledge to build environment to study anxiety in these species. For example, here the researches introduced animals into simple open field and studied the time spent by the wild type and knockout mice in the inner and outer filed zones, to analyze the effect gene knockdown on anxiety-like behavior.

You’ve just watched JoVE’s introduction to essential care procedures for mice and rats. You should now have a better understanding of the measures one should take to ensure that the environment and handling of these animals does not cause any harm to animal’s health and you should also know how researchers can modify these parameters to achieve different scientific goals. As always, thanks for watching!