All animal procedures described here must be conducted in accordance with institutional animal ethics guidelines and approved by IACUC. All procedures must follow the principles of the 3Rs—Replacement, Reduction, and Refinement—and must be performed by trained personnel.
1. Handling immunocompetent (conventional) mice
It has been demonstrated that immune disorders of mice are very similar to those in humans. As these animals have limited immunological capabilities, procedures in the handling and care of these animals have been designed to minimize the risk of infectious disease through the use of engineering controls, personal protective equipment for the animal care staff, and standard operating procedures; these mitigate contamination of caging and other equipment with which the animals are in contact.
2. Handling immunocompromised mice
Key Terms and Definitions
Housing/Caging
Mice and rats are primarily housed in shoebox-type caging with a solid bottom that contains bedding material. The typical shoebox mouse cage provides 75 square inches of floor space, which is adequate for a maximum of five adult mice. However, the experimental design may call for groups of four mice. In this case, the mice should be housed upon receiving in groups of four, to avoid the disruption of social groups when the experiment begins. In general, rat caging allows for a maximum of two fully-grown adult animals. If the rats are to be housed long-term, they should be established in pair housing. The cages can be static or individually ventilated (IVC). Static cages are either covered with a wire bar lid or are settled onto a rack under a shelf that is then the top of the cage. Filter material is added over the cage top either as a bonnet over the wire bar lid, or as a sheet of filter paper over the rack shelves. IVCs are set up as a cage, wire bar lid, and bonnet that are placed on a rack that provides air flow into the cage. The use of IVCs eliminates the need to change the cage as often as static cages are changed. Standard cage systems provide a source of water and an area for food distribution.
Water can be provided via a water bottle placed on the top of the cage or via a sipper tube that is a built in component of the animal cage. When the cage is engaged onto a rack, water is transported into the sipper tube for an automatic watering system that is designed to meet the needs of the animal throughout the animal facility. Caging must be properly prepared to provide proper care for the animals. 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. The Guide for the Care and Use of Laboratory Animals (the "Guide") 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. In general, the IVCs are scheduled for changing every two weeks unless there are four or more males or a female with a litter; those enclosures may require weekly changing. As cage changing is an opportunity for the mice to be exposed to pathogens, changing of the caging for immune deficient mice should be done as infrequently as possible.
Diet
There are many compositions of diets available with variations in protein, carbohydrate, and fat levels. Typically, feeds should be comprised of 15-19% protein, 44% carbohydrates, and 4-9% fat. Food with an increased fat content is often used for breeding colonies to enhance reproduction. As the food can be a source of contamination for immunocompromised mice, it must be either steam sterilized or irradiated. Food that is subjected to the autoclaving process must be specially formulated to withstand the process. However, the irradiation process does not alter the food contents. Water is another potential source of contamination for the immune deficient animals. Water should be processed either through an autoclave or filtration that includes the water passing through a UV filter. To steam sterilize water in an autoclave, individual bottles of water are covered with aluminum foil. Bottle lids with sipper tubes are sterilized separately in an autoclave bag. Automatic watering systems can be used with a series of filtration throughout the system that includes particulate filters and an ultraviolet light filter.
Environment
Environmental conditions within an animal care facility must be tightly controlled. Federal regulations and guidelines have established the standards for the care and handling of commonly used animals. Lighting, ambient temperature, humidity, and air flow are provided to meet the physiological needs of the animals. With the exception of the need for a slightly increased room temperature, the parameters for the immunocompromised animals are the same as those for conventional animals. The athymic nude animals also require additional bedding and shredded paper-type nesting materials in their cages to assist with thermoregulation.
Animal rooms should be maintained at a temperature between 68-72°F with a humidity level of 40-60%. Humidity levels below 40% can result in ringtail of mice and rats, a condition that causes the skin of the tail to dry to the point of it, constricting around the tail, which can lead to the tail sloughing. Lighting cycles should be set to avoid stressors to the animals caused by inappropriate photoperiods or light intensity. Normally, photoperiods are 12 hours on and 12 hours off. The lighting intensity should vary in accordance with the activity in the room. When technicians are working in the animal room, higher lighting levels can be used. However, the lighting should be lowered to approximately 325 lux once all activity is concluded. The lights should be on timers and the cycles verified through monitoring systems. As directed by the Guide, animal rooms must have a minimum of 10-15 air changes of 100% fresh air per hour. Air changes decrease microbes and odors in the animal room. In densely populated animal rooms, the number of air changes per hour may need to be increased.
Environmental factors at the cage level include the caging type, the substrate used within the cage, the water delivery system, and the diet. Social housing is mandatory unless scientifically justified, or an animal is incompatible with all others. The provisions of environmental enrichment, such as paper huts and nesting materials, allow the animals to exhibit species-specific behaviors, which in turn decreases the stress level and increases the overall wellbeing 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. The nesting area is moved to the clean cage upon cage changing to help prevent aggression in the males as they are introduced into a new environment.
Questions that this video will help you answer
The proper care and handling of animals used in biomedical research is crucial to the outcome of the experiments. Regulations and established guidelines dictate many of the practices used. However, additional measures are required for mice and rats that have immune deficiencies to mitigate accidental exposure to opportunistic pathogens. When practices adhere to established procedures, the health and wellbeing of the experimental animals is enhanced, and better experimental results are attained.
Source: Kay Stewart, RVT, RLATG, CMAR; Valerie A. Schroeder, RVT, RLATG. University of Notre Dame, IN
Mice and rats account for a large proportion of the animals used in biomedical research. Ensuring their welfare is both an ethical responsibility and essential for scientific validity. There are general procedures that apply to the majority of these mice and rats, but some of the animals, such as the immunocompromised ones, require additional steps to be taken to sustain them for experimentation.
Commonly used immunocompromised mice include those that have naturally occurred in inbred mice and those that have been created through genetic engineering. The first immunocompromised mice used in research were "nude" mice. The BALB/c Nude (nu) mouse was discovered in 1966, within a BALB/c colony that was producing mice lacking both hair and a thymus. These athymic mice have an inhibited immune system that is devoid of T cells. The value of this animal was soon discovered for the use in studies of microbial infections, immune deficiencies, and autoimmunity. Although not as commonly used as the nude mouse, there is also a nude rat. The nude rat is T cell deficient and shows depleted cell populations in thymus-dependent areas of peripheral lymphoid organs. Another naturally occurring immune deficient mouse is the severe combined immunodeficiency mouse (SCID). These mice have a defect that impairs the production of functional B and T cells. With the inability to mount an adequate immune response, these mice can serve as host recipients for transplants of human cells. Several genetically-modified mouse strains with immune deficiencies are commercially available. The severity of the deficiency varies with the genetic modification; however, all of the immunocompromised mice and rats are handled similarly.
This manuscript will describe the care of both immunocompetent and immunocompromised animals. The housing, diet, environmental requirements, and handling will be discussed for both groups of animals
All animal procedures described here must be conducted in accordance with institutional animal ethics guidelines and approved by IACUC. All procedures must follow the principles of the 3Rs—Replacement, Reduction, and Refinement—and must be performed by trained personnel.
1. Handling immunocompetent (conventional) mice
It has been demonstrated that immune disorders of mice are very similar to those in humans. As these animals have limited immunological capabilities, procedures in the handling and care of these animals have been designed to minimize the risk of infectious disease through the use of engineering controls, personal protective equipment for the animal care staff, and standard operating procedures; these mitigate contamination of caging and other equipment with which the animals are in contact.
2. Handling immunocompromised mice
Key Terms and Definitions
Housing/Caging
Mice and rats are primarily housed in shoebox-type caging with a solid bottom that contains bedding material. The typical shoebox mouse cage provides 75 square inches of floor space, which is adequate for a maximum of five adult mice. However, the experimental design may call for groups of four mice. In this case, the mice should be housed upon receiving in groups of four, to avoid the disruption of social groups when the experiment begins. In general, rat caging allows for a maximum of two fully-grown adult animals. If the rats are to be housed long-term, they should be established in pair housing. The cages can be static or individually ventilated (IVC). Static cages are either covered with a wire bar lid or are settled onto a rack under a shelf that is then the top of the cage. Filter material is added over the cage top either as a bonnet over the wire bar lid, or as a sheet of filter paper over the rack shelves. IVCs are set up as a cage, wire bar lid, and bonnet that are placed on a rack that provides air flow into the cage. The use of IVCs eliminates the need to change the cage as often as static cages are changed. Standard cage systems provide a source of water and an area for food distribution.
Water can be provided via a water bottle placed on the top of the cage or via a sipper tube that is a built in component of the animal cage. When the cage is engaged onto a rack, water is transported into the sipper tube for an automatic watering system that is designed to meet the needs of the animal throughout the animal facility. Caging must be properly prepared to provide proper care for the animals. 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. The Guide for the Care and Use of Laboratory Animals (the "Guide") 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. In general, the IVCs are scheduled for changing every two weeks unless there are four or more males or a female with a litter; those enclosures may require weekly changing. As cage changing is an opportunity for the mice to be exposed to pathogens, changing of the caging for immune deficient mice should be done as infrequently as possible.
Diet
There are many compositions of diets available with variations in protein, carbohydrate, and fat levels. Typically, feeds should be comprised of 15-19% protein, 44% carbohydrates, and 4-9% fat. Food with an increased fat content is often used for breeding colonies to enhance reproduction. As the food can be a source of contamination for immunocompromised mice, it must be either steam sterilized or irradiated. Food that is subjected to the autoclaving process must be specially formulated to withstand the process. However, the irradiation process does not alter the food contents. Water is another potential source of contamination for the immune deficient animals. Water should be processed either through an autoclave or filtration that includes the water passing through a UV filter. To steam sterilize water in an autoclave, individual bottles of water are covered with aluminum foil. Bottle lids with sipper tubes are sterilized separately in an autoclave bag. Automatic watering systems can be used with a series of filtration throughout the system that includes particulate filters and an ultraviolet light filter.
Environment
Environmental conditions within an animal care facility must be tightly controlled. Federal regulations and guidelines have established the standards for the care and handling of commonly used animals. Lighting, ambient temperature, humidity, and air flow are provided to meet the physiological needs of the animals. With the exception of the need for a slightly increased room temperature, the parameters for the immunocompromised animals are the same as those for conventional animals. The athymic nude animals also require additional bedding and shredded paper-type nesting materials in their cages to assist with thermoregulation.
Animal rooms should be maintained at a temperature between 68-72°F with a humidity level of 40-60%. Humidity levels below 40% can result in ringtail of mice and rats, a condition that causes the skin of the tail to dry to the point of it, constricting around the tail, which can lead to the tail sloughing. Lighting cycles should be set to avoid stressors to the animals caused by inappropriate photoperiods or light intensity. Normally, photoperiods are 12 hours on and 12 hours off. The lighting intensity should vary in accordance with the activity in the room. When technicians are working in the animal room, higher lighting levels can be used. However, the lighting should be lowered to approximately 325 lux once all activity is concluded. The lights should be on timers and the cycles verified through monitoring systems. As directed by the Guide, animal rooms must have a minimum of 10-15 air changes of 100% fresh air per hour. Air changes decrease microbes and odors in the animal room. In densely populated animal rooms, the number of air changes per hour may need to be increased.
Environmental factors at the cage level include the caging type, the substrate used within the cage, the water delivery system, and the diet. Social housing is mandatory unless scientifically justified, or an animal is incompatible with all others. The provisions of environmental enrichment, such as paper huts and nesting materials, allow the animals to exhibit species-specific behaviors, which in turn decreases the stress level and increases the overall wellbeing 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. The nesting area is moved to the clean cage upon cage changing to help prevent aggression in the males as they are introduced into a new environment.
Questions that this video will help you answer
The proper care and handling of animals used in biomedical research is crucial to the outcome of the experiments. Regulations and established guidelines dictate many of the practices used. However, additional measures are required for mice and rats that have immune deficiencies to mitigate accidental exposure to opportunistic pathogens. When practices adhere to established procedures, the health and wellbeing of the experimental animals is enhanced, and better experimental results are attained.
Since mice and rats account for a large proportion of animals used in biomedical research, ensuring their welfare is both an ethical responsibility and essential for scientific validity.
Animal care procedures begin with receiving animals from shipping containers and continue through cage changing, appropriate housing, providing proper nutrition, and environmental management.
These practices should be performed in accordance with institutional guidelines and the principles of the 3Rs, particularly refinement, to minimize stress and promote well-being.
This video describes the standards and essential care procedures for laboratory animals.
Receiving animals from other institutions poses a risk of introducing pathogens; therefore, proper procedures should be followed when unpacking shipping containers.
Before handling any animals, it is important to wear the appropriate Personal Protective Equipment, or PPE.
Minimum PPE includes a lab coat, gloves, safety glasses, and a surgical type mask, which helps reduce exposure to dust and allergens and minimizes the risk of contamination to the animals.
The next step is to select appropriate cages for incoming animals. Then, furnish the cage with appropriate bedding and nesting materials, and place the prepared cages in the temperature-controlled receiving room prior to animal arrival.
Upon shipment arrival, place the transport boxes in a secure area in the receiving room. Examine the container for any damage and disinfect the outer surfaces to reduce potential contamination.
Ensure that the disinfectant remains in contact with the box, as per manufacturer's recommendations. Then, place the container in the Animal Transfer Station, or ATS.
This station provides HEPA-filtered unidirectional airflow, which reduces airborne contaminants, protecting both personnel and animals.
Next, open the shipping container and evaluate the overall health of the rodents. Any animals that appear injured or ill should be reported and managed according to institutional veterinary guidelines.
After that, place them into the already assembled cage in the appropriate group sizes, based on species, sex, and compatibility, cover the cage with the wire bar lid, and secure the divider if required.
Select the appropriate food and place it in the designated area of the cage. Lastly, 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 remains on during disinfection.
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. Animals should be transferred using appropriate handling techniques, such as cupping or tunnel handling, to minimize stress.
Only one cage of animals should 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 clean 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.
For the unpacking of immunocompromised animals, aseptic conditions must be maintained. It is recommended to turn on the blower inside the hood before use to allow proper airflow to be established.
Like for immunocompetent animals, upon shipment arrival spray the container with an appropriate disinfectant and leave it unopened as per manufacturer's recommendations.
At the ATS designated for immunocompromised mice, disinfect the laminar flow hood using an appropriate disinfectant prior to manipulating animals.
Ideally two personnel may be involved in this unpacking activity-a sterile technician handling animals aseptically, and a non-sterile technician handling contaminated items. Both should wear appropriate PPE.
Before opening the shipping container inside the hood, place all the supplies and caging in the hood and disinfect their exterior using an appropriate disinfectant.
Sterilized caging can include either reusable autoclaved cages or sterile, ready-to-use disposable cages.
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 in the hood without contaminating the inner surfaces.
The animal handler should change outer gloves as needed to maintain aseptic technique.
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 aseptic conditions.
At the end, the non-sterile person removes the populated cage from the hood and places it on an appropriate rack.
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 can accommodate up to 4-5 adult mice, depending on strain, sex, and compatibility.
On the other hand, a rat cage has an area of 142 square inches and can accommodate up to 2 adult rats, depending on strain and compatibility. Housing should also allow rats to exhibit normal postures, including the ability to rear.
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 can reduce the frequency of cage changes compared to 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 the drinking valve option, when the cage is engaged onto a rack, water is delivered through an automated valve system connected to a central supply that is designed to meet the needs of the animal throughout the animal facility.
The Guide for the Care and Use of Laboratory Animals recommends that caging implements be sanitized regularly, typically about 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.
Proper food and water supply are essential for animal care. There are many commercially available feeds with variable protein, carbohydrate, and fat compositions.
Feed composition may vary depending on the species, strain, and study requirements. For immunocompromised animals, feed is typically provided in a sterilized form according to institutional guidelines.
Water should be provided in a form that is safe and appropriate for the animals, with treatment or sterilization applied as needed according to institutional guidelines.
Standard environmental conditions are also essential for maintaining animal welfare.
At the cage level, animals should be socially housed unless scientifically justified for single housing 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 such as 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 familiar sleeping area with scent cues that can assist with housing multiple male mice in the same cage.
As mentioned previously, moving the nesting area to the clean cage during cage changing helps reduce aggression, especially in males, as the animals are introduced into a new environment.
At the room level, the temperature for mice and rats is typically between 68 and 79°F, according to institutional guidelines. 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 light-dark cycles.
Normally, the animal room is set to a 12-hour light/12-hour dark cycle. Lighting intensity should be adjusted according to the activity in the room and maintained at levels appropriate for the species and strain when the room is unoccupied.
Lastly, airflow within the room should also be carefully controlled. Ventilation should provide an appropriate number of air changes per hour, according to institutional standards, to help reduce odors and microbial load 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 is associated with reduced stress and can serve as an indicator of animal well-being. This behavior can be influenced by environmental conditions such as ambient temperature, highlighting the importance of maintaining appropriate housing conditions to support animal welfare.
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.
Chapters in this video
0:00
Overview
1:06
Unpacking and Cage Changing Procedures
6:50
Housing
9:18
Diet
10:03
Environment
12:10
Applications
12:55
Summary
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