Basic Care Procedures

Lab Animal Research

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Overview

Source: Kay Stewart, RVT, RLATG, CMAR; Valerie A. Schroeder, RVT, RLATG. University of Notre Dame, IN

Mice and rats account for over 90% of the animals used for biomedical research. The proper care of these research animals is critical to the outcome of experiments. 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.1 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.

Cite this Video

JoVE Science Education Database. Lab Animal Research. Basic Care Procedures. JoVE, Cambridge, MA, (2017).

Principles

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 Animals2 (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. Acidification of the water to a pH of 2.5-3 helps control the growth bacterial organisms in the water bottles. Water is acidified by adding two drops of 12 mole (M) hydrochloric acid (HCl) to eight liters of reversed osmolality (RO) water. 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%.2 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.2 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.2 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.3

Procedure

1. Handling immunocompetent (conventional) mice

It has been demonstrated that immune disorders of mice are very similar to those in humans.2 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.

  1. Unpacking from shipping containers: Receiving animals from other institutions or animal vendors poses a risk to the animals already housed within the animal facility. During transit, the animal transport boxes can come in contact with pathogenic agents. Therefore, proper procedure should be performed when unpacking the shipping containers.
    1. Minimal Personal Protective Equipment (PPE) requirements are gloves, clean lab coat or scrubs, a particulate mask, and safety glasses when handling the animals.
    2. Select the appropriate cage, including the wire bar lid and the barrier top. The cage should be furnished with appropriate bedding and nesting materials. All caging should be prepared and placed in the receiving area prior to the arrival of the animals.
    3. Upon arrival, the transport boxes are placed in a secure and temperature controlled room. Then, examine the container for damage.
    4. Disinfect the outer surfaces of the shipping container with a choline dioxide product to destroy any harmful pathogens. Make sure that the disinfectant is in contact with the box in accordance with the manufacturers recommendations, usually 15-30 minutes.
    5. Place the container in an Animal Transfer Station (ATS). The ATS provides HEPA-filtered airflow within the hood that provides allergen protection by creating a vacuum air barrier between the user and work zone. This ensures minimal exposure to animal hair/dander and cage bedding material. The unidirectional air also protects the animals from the surrounding room air, minimizing the chance of contamination.
    6. Once the shipping container is opened, assess the overall health of the animals. Make a note of any injured or ill animals.
    7. Place the animals into the assembled cages in the proper group sizes. The grouping should be in accordance to the space regulations, and the experimental protocol to which they will be assigned.
    8. Cover the cage with the wire bar lid. Secure the divider on the wire bar lid to create two areas on the lid.
    9. Select the appropriate food and place the food in one side of the wire bar lid. Leave the second side open to allow for better circulation of air.
    10. Water will be provided via water bottles or an automatic watering system. Water bottles are used for newly weaned mice to ensure that they are able to maintain their hydration levels. For IVC caging with automatic watering, charge the watering tube prior to placing it on the rack.
    11. Place the cage on a rack. If using IVCs, check that the cage is fully engaged onto the rack, and that the sipper tube is secured to the watering system.
  2. Cage changing conventional mice
    1. Animal cages are changed in an ATS. The ATS is disinfected at the end of each session.
    2. Only one cage can be opened at a time.
    3. Caging prepared with the appropriate bedding and nesting materials is brought into the animal room. A small number of cages are placed in the ATS.
    4. IVC cages with internal sipper tubes must be charged to force water into the sipper to remove the dead space of air in the sipper tube. If not properly charged, the mice may not be able to provide enough pressure on the sipper tube to remove the air from it.
    5. Minimal Personal Protective Equipment (PPE) requirements are gloves, clean lab coat or scrubs, a particulate mask, and safety glasses when handling the animals.
    6. A cage of animals to be changed is placed into a new cage, and then in the ATS.
    7. Animals are either transferred by hand or with the use of a padded forceps. Gloves are either changed between cages or between groups of mice. Gloves can also be disinfected with a nontoxic chemical between cages.
    8. It has been demonstrated that transferring the nesting area of the mice into a clean cage assists with the transition into the clean cage, especially for group-housed males. Other enrichment items are also transferred to the new cage.
    9. Once the cage top or feeder is placed in the new cage, food is added.
    10. The barrier top is placed on the new cage and the cage is returned to the rack.
    11. IVC cages must be properly docked into the rack to ensure that the air and water valves are engaged.

2. Handling immunocompromised mice

  1. Unpacking from shipping containers
    1. Immunocompromised animals must be unpacked under sterile conditions. All individuals handling immune deficient mice must wear a steam sterilized gown, mask, hair bonnet, and sterile gloves. Aseptic technique should be used at all times when handing these animals.
    2. All equipment and caging should have been sterilized by steam, UV radiation, or chemically sterilized with a soak in 70% ethanol for at least 30 minutes There are two options for sterile caging: standard caging that has been wrapped and steam sterilized, or caging that is sterilized-usually through irradiation-by the vendor. These cages are one use, disposable units.
    3. Prior to manipulating mice under the laminar flow hood, the hood is sprayed with 70% ethanol with the blower operating. The hood blower should remain on at all times to maintain the sterile environment. After each use, the hood is sprayed with a disinfectant and wiped of all debris and again sprayed with 70% ethanol.
    4. Optimally, two people are required for the proper receiving of the immunocompromised mice: one to handle the animals using aseptic techniques (the sterile technician), and one that will handle contaminated items (the nonsterile technician). The nonsterile person should still wear a clean, long-sleeved gown; lab coat or smock; mask; bonnet; and sterile gloves to prevent contamination of the sterile interior should they need to reach into the hood.
    5. Prepare a laminar flow hood as previously described. It is preferable to have a hood dedicated for immunocompromised animals. It is recommended that hood be equipped with ultraviolet (UV) sterilization lights that can be used to sterilize the interior of the hood between uses. A timer is utilized to ensure that the surface of the hood has adequate exposure to the UV rays.
    6. The exterior of all supplies that are placed into the hood are sprayed with 70% alcohol, including the outer packaging of food, water, and caging.
    7. Shipping containers are sprayed with a chlorine dioxide solution before they are placed in the hood. Once placed in the hood, they are to remain unopened for 15-30 minutes.
    8. The designated animal handler opens the sterile food and water under the hood.
    9. The nonsterile technician opens the shipping box under the hood. Care must be taken by the nonsterile technician to avoid contact with the inner surfaces of the hood and the inside of the shipping box.
    10. The animal handler opens the sterile cage, and prepares the cage by placing food and water onto the cage top. The mice are then removed from the shipping box and placed into the sterile cage.
    11. Animal condition is noted by the sterile animal handler. Any animals that appear ill or have obvious injuries will be segregated. All cages marked with animals requiring veterinary attention will be placed for handling, cage changing, or experimental manipulation last, to reduce potential contamination of other animals.
    12. The nonsterile person will remove the populated cage from the hood. Cages can be placed on a standard rack as static cages, or on a ventilated rack as IVCs. In the IVCs, the mice should be provided with additional nesting materials to assist with thermal regulation.
    13. The animal handler should change gloves after each shipping container to avoid possible cross contamination of groups of animals. The shipping container number is recorded on the cage card to track the origin of the animals in the event of illness.
    14. Unused supplies can be saved for future use provided the original container can be resealed. Unused food is removed from the room.
    15. Mice should undergo a minimum one week acclimation period prior to experimental use.
  2. Cage changing for immunocompromised mice
    1. The mouse cages are changed at least once weekly, or more often if deemed necessary.
    2. Steps for hood preparation, cage preparation, and personnel PPE are the same as for the unpacking of the animals.
    3. Animals are either transferred by hand or with the use of padded forceps. The designated animal handler should reglove after every 5-10 cages, if the gloves are torn or contaminated, or if an animal appears ill.
    4. Any cages with animals appearing sick will be placed at the end of the cage changing order, to avoid possible exposure of healthy animals to pathogens or contaminants.
    5. If food and/or water must be added to the cage before the next scheduled cage change, it must be done aseptically under the prepared hood.

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!

Summary

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 resulted are attained.

References

  1. Belizário J.E. 2009. Immunodeficient Mouse Models: An Overview. The Open Immunology Journal. 2. 79-85.
  2. Institute for the Laboratory Animal Research. 2011. Guide for the care and use of laboratory animals, 8th ed. Washington (DC): National Academies Press.
  3. Van Loo, P. 2001. Modulation of aggression in male mice: influence of cage cleaning regime and scent marks. In Male Management: Coping with Aggression Problems in Male Laboratory Mice. Surrey: UK.

1. Handling immunocompetent (conventional) mice

It has been demonstrated that immune disorders of mice are very similar to those in humans.2 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.

  1. Unpacking from shipping containers: Receiving animals from other institutions or animal vendors poses a risk to the animals already housed within the animal facility. During transit, the animal transport boxes can come in contact with pathogenic agents. Therefore, proper procedure should be performed when unpacking the shipping containers.
    1. Minimal Personal Protective Equipment (PPE) requirements are gloves, clean lab coat or scrubs, a particulate mask, and safety glasses when handling the animals.
    2. Select the appropriate cage, including the wire bar lid and the barrier top. The cage should be furnished with appropriate bedding and nesting materials. All caging should be prepared and placed in the receiving area prior to the arrival of the animals.
    3. Upon arrival, the transport boxes are placed in a secure and temperature controlled room. Then, examine the container for damage.
    4. Disinfect the outer surfaces of the shipping container with a choline dioxide product to destroy any harmful pathogens. Make sure that the disinfectant is in contact with the box in accordance with the manufacturers recommendations, usually 15-30 minutes.
    5. Place the container in an Animal Transfer Station (ATS). The ATS provides HEPA-filtered airflow within the hood that provides allergen protection by creating a vacuum air barrier between the user and work zone. This ensures minimal exposure to animal hair/dander and cage bedding material. The unidirectional air also protects the animals from the surrounding room air, minimizing the chance of contamination.
    6. Once the shipping container is opened, assess the overall health of the animals. Make a note of any injured or ill animals.
    7. Place the animals into the assembled cages in the proper group sizes. The grouping should be in accordance to the space regulations, and the experimental protocol to which they will be assigned.
    8. Cover the cage with the wire bar lid. Secure the divider on the wire bar lid to create two areas on the lid.
    9. Select the appropriate food and place the food in one side of the wire bar lid. Leave the second side open to allow for better circulation of air.
    10. Water will be provided via water bottles or an automatic watering system. Water bottles are used for newly weaned mice to ensure that they are able to maintain their hydration levels. For IVC caging with automatic watering, charge the watering tube prior to placing it on the rack.
    11. Place the cage on a rack. If using IVCs, check that the cage is fully engaged onto the rack, and that the sipper tube is secured to the watering system.
  2. Cage changing conventional mice
    1. Animal cages are changed in an ATS. The ATS is disinfected at the end of each session.
    2. Only one cage can be opened at a time.
    3. Caging prepared with the appropriate bedding and nesting materials is brought into the animal room. A small number of cages are placed in the ATS.
    4. IVC cages with internal sipper tubes must be charged to force water into the sipper to remove the dead space of air in the sipper tube. If not properly charged, the mice may not be able to provide enough pressure on the sipper tube to remove the air from it.
    5. Minimal Personal Protective Equipment (PPE) requirements are gloves, clean lab coat or scrubs, a particulate mask, and safety glasses when handling the animals.
    6. A cage of animals to be changed is placed into a new cage, and then in the ATS.
    7. Animals are either transferred by hand or with the use of a padded forceps. Gloves are either changed between cages or between groups of mice. Gloves can also be disinfected with a nontoxic chemical between cages.
    8. It has been demonstrated that transferring the nesting area of the mice into a clean cage assists with the transition into the clean cage, especially for group-housed males. Other enrichment items are also transferred to the new cage.
    9. Once the cage top or feeder is placed in the new cage, food is added.
    10. The barrier top is placed on the new cage and the cage is returned to the rack.
    11. IVC cages must be properly docked into the rack to ensure that the air and water valves are engaged.

2. Handling immunocompromised mice

  1. Unpacking from shipping containers
    1. Immunocompromised animals must be unpacked under sterile conditions. All individuals handling immune deficient mice must wear a steam sterilized gown, mask, hair bonnet, and sterile gloves. Aseptic technique should be used at all times when handing these animals.
    2. All equipment and caging should have been sterilized by steam, UV radiation, or chemically sterilized with a soak in 70% ethanol for at least 30 minutes There are two options for sterile caging: standard caging that has been wrapped and steam sterilized, or caging that is sterilized-usually through irradiation-by the vendor. These cages are one use, disposable units.
    3. Prior to manipulating mice under the laminar flow hood, the hood is sprayed with 70% ethanol with the blower operating. The hood blower should remain on at all times to maintain the sterile environment. After each use, the hood is sprayed with a disinfectant and wiped of all debris and again sprayed with 70% ethanol.
    4. Optimally, two people are required for the proper receiving of the immunocompromised mice: one to handle the animals using aseptic techniques (the sterile technician), and one that will handle contaminated items (the nonsterile technician). The nonsterile person should still wear a clean, long-sleeved gown; lab coat or smock; mask; bonnet; and sterile gloves to prevent contamination of the sterile interior should they need to reach into the hood.
    5. Prepare a laminar flow hood as previously described. It is preferable to have a hood dedicated for immunocompromised animals. It is recommended that hood be equipped with ultraviolet (UV) sterilization lights that can be used to sterilize the interior of the hood between uses. A timer is utilized to ensure that the surface of the hood has adequate exposure to the UV rays.
    6. The exterior of all supplies that are placed into the hood are sprayed with 70% alcohol, including the outer packaging of food, water, and caging.
    7. Shipping containers are sprayed with a chlorine dioxide solution before they are placed in the hood. Once placed in the hood, they are to remain unopened for 15-30 minutes.
    8. The designated animal handler opens the sterile food and water under the hood.
    9. The nonsterile technician opens the shipping box under the hood. Care must be taken by the nonsterile technician to avoid contact with the inner surfaces of the hood and the inside of the shipping box.
    10. The animal handler opens the sterile cage, and prepares the cage by placing food and water onto the cage top. The mice are then removed from the shipping box and placed into the sterile cage.
    11. Animal condition is noted by the sterile animal handler. Any animals that appear ill or have obvious injuries will be segregated. All cages marked with animals requiring veterinary attention will be placed for handling, cage changing, or experimental manipulation last, to reduce potential contamination of other animals.
    12. The nonsterile person will remove the populated cage from the hood. Cages can be placed on a standard rack as static cages, or on a ventilated rack as IVCs. In the IVCs, the mice should be provided with additional nesting materials to assist with thermal regulation.
    13. The animal handler should change gloves after each shipping container to avoid possible cross contamination of groups of animals. The shipping container number is recorded on the cage card to track the origin of the animals in the event of illness.
    14. Unused supplies can be saved for future use provided the original container can be resealed. Unused food is removed from the room.
    15. Mice should undergo a minimum one week acclimation period prior to experimental use.
  2. Cage changing for immunocompromised mice
    1. The mouse cages are changed at least once weekly, or more often if deemed necessary.
    2. Steps for hood preparation, cage preparation, and personnel PPE are the same as for the unpacking of the animals.
    3. Animals are either transferred by hand or with the use of padded forceps. The designated animal handler should reglove after every 5-10 cages, if the gloves are torn or contaminated, or if an animal appears ill.
    4. Any cages with animals appearing sick will be placed at the end of the cage changing order, to avoid possible exposure of healthy animals to pathogens or contaminants.
    5. If food and/or water must be added to the cage before the next scheduled cage change, it must be done aseptically under the prepared hood.

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!

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