ALAT Chapter 18 Mice PDF

ALAT Chapter 18 Mice Mice General USDA regulated? Gestation Weaning Sex determination Maturity Uterus details Dental formula Housing needs Unique physio Common clinical concerns Mice Clinical Concerns Pathogens Rx Options Zoonotic concerns Sample collection sites Medication admins...

ALAT Chapter 18 Mice Mice General USDA regulated? Gestation Weaning Sex determination Maturity Uterus details Dental formula Housing needs Unique physio Common clinical concerns Mice Clinical Concerns Pathogens Rx Options Zoonotic concerns Sample collection sites Medication admins sites Meds to avoid Why mice? • Mice are the animal species most commonly worked with in biomedical research. International databases providing integrated genetic, genomic, and biological data for the mouse are readily available to facilitate the study of human health and disease. • Mice are efficient breeders and produce many generations of offspring in a short time period. Many mouse stocks and strains are available, with many different genetic backgrounds, as well as humanized mice. • Genetically modified mice are valuable in many types of research projects, including studies in genetics, cancer, and infectious diseases. Taxonomy and Nomenclature • The most widely used species of mouse in research is the common house mouse, Mus musculus. Field (Microtus spp.) and deer (Peromyscus spp.) mice, are also occasionally used in the laboratory. The information presented in this chapter refers to Mus musculus. • Outbred stocks are created by the mating of unrelated animals in a closed colony. This produces a population with a “stabilized” genetic composition. An outbred stock is defined as a closed population (for at least four generations) of genetically variable animals that is bred to maintain aximum heterozygosity.1 In an outbred stock, not every mouse shares the same genes as every other mouse, but the overall genetic makeup of groups of mice remains stable from experiment to experiment. Examples of outbred mouse stocks that are dependent on vendor source include:  Swiss Webster  CD-1  ICR  CFW Whether an inbred strain or an outbred stock is used in a study depends on the type of research being performed. Inbred strains are developed by breeding related individuals for many generations, until the animals produced all have nearly the same genetic composition. Generally, this is achieved by producing at least 20 consecutive generations of sister x brother matings. In some instances, an inbred strain is determined by a traceable ancestral pair in the 20th or subsequent generation. The genetic similarity of these animals is like that of identical twins. Some of the most common inbred mouse strains include:  BALB/c, an albino mouse  C3H, an agouti mouse (agouti means the hairs of the animals coat have alternating dark and light bands)  C57BL/6, a black mouse  DBA, the oldest inbred mouse strain, which is brown in color  FVB, an albino mouse  129, a light-bellied agouti mouse Nomenclature • Hybrids: Where animals from two different strains are bred together to produce an animal with a mixture of genes from each strain (like the doodle dogs). • Hybrids are designated by combining some of the designations of their parents. • For example, breeding a female C57BL/6 with a male C3H produces B6C3F1 offspring. • In the hybrid name, note that the C57BL/6 parent is abbreviated to B6, and the C3H parent is abbreviated to C3. F1 means that these animals are the first generation of this mating scheme. • Nomenclature of mouse stocks, strains, hybrids, and transgenic lines follow specific rules that are discussed in more detail in the Laboratory Animal Technician Training Manual and Laboratory Animal Technologist Training Manual. Other mouse models • Transgenic technology has enabled the creation of many strains of genetically modified mice. Transgenic animals are a valuable tool in research because they can be used to study human diseases that would not otherwise occur in unmodified animals. • For example, normal mice cannot be infected with the virus that causes HIV, but scientists have created transgenic mice that can be infected with this virus. • Knockout mice have been used to study cancer and other diseases. • Nude mice, a naturally occurring genetic defect in mice resulting in an animal with no hair, have a deficiency in their immune systems in addition to being hairless. These (and other )immunodeficient mice have become very valuable in the study of immune disease and cancer. Dental Formula • Dental formula: •1 0 0 3 • I C P M • 1 0 0 3 • Hypsodontic incisors http://www.vivo.colostate.edu/hbooks/pathphys/digestion/pregastric/rodentpage.html Which teeth in mice are hypsodontic? The front teeth (incisors) WORLD’S WORST VIDEO – totally memorable! Anatomy & Physiology • Teeth: Mice have only one pair of upper and lower incisors They have three upper and lower molars on each side of the mouth. They do not have canine teeth or premolars. They have no deciduous (baby) teeth, only adult teeth. Their incisors continue to grow throughout their life. The incisors are aligned to normally wear down through gnawing of hard structures, such as seed hulls and wood in nature or rodent feed pellets in the laboratory. • Malocclusion: When the incisors are not in proper alignment, the teeth do not wear down and become overly long. This condition is known as malocclusion. Depending on the shape of the overgrown incisors, they can damage or even pierce the gums when the mouse chews. In some mice with malocclusion, the teeth protrude from the mouth. Typically, the only obvious sign of malocclusion is weight loss (relative to cage mates) because the mouse is unable to eat. In addition, the mouse may be dehydrated depending on the severity of the malocclusion. It is important for an animal technician to recognize the signs of this common problem. Clipping the overgrown teeth to the proper length corrects the problem; the animal will quickly begin to eat again. If the gums are injured, moistened/softened chow can be given until the mouth heals. Clipping does not permanently repair the malocclusion because of the continued growth of the incisors. Periodic clipping will be needed for the life of the animal.4 • Mice have firm, rice-sized, dark brown feces. • Urine is clear and yellow, with a strong odor. • Both respiration and heart rates are too rapid to be measured by observation alone, but can be measured by electronic recording devices. • Can’t vomit: Mice, and other rodents, do not vomit. This is because they lack the neural pathways to vomit and because the connection of the esophagus at the stomach prevents the regurgitation of feed. Therefore, feed and water are normally not withheld prior to anesthesia. Because of their small size, withholding feed and water prior to surgery makes mice prone to dehydration and hypoglycemia (low blood sugar). Preventing dehydration and hypoglycemia is important for the mouse’s recovery from surgery. Reproductive Data Sexing • Male • Scrotal sacs often evident (not a prolapse or tumor) • Greater anogenital distance • Female • Smaller anogenital distance • 3 orifices • Nipples Sexing How do you sex mice? What do you use as a reference? Female: Short AG distance Male: Greater AG distance Sexing neonates In a comparison of day-old pups, males may be recognized by the visibility of the testes through the abdominal wall and by their larger genital papilla. Nipple buds Biological Data Weight @ maturity: 2040gms Life span 1-3 years Body temperature (strain dependent) 97.7 – 100.4F Heart rate: 300800bpm Respiratory rate: 80-230 per minute Biological Data Water Consumption 6-7 mls/day Food Consumption ~5 gms/day Urine output ~2.5 ml/day Highly concentrated HIGHLY allergenic Reproductive Data • Puberty: Males 5-7 weeks • Females 4-5 weeks • Gestation: 19-21 days • Litter size: 4-12 • Strain dependent • Weaning: 21-28 days • Estrus: 4-5 days • Post-partum estrus • Non-seasonal polyestrus • Mating = copulatory sperm plug left in vagina • Seen for up to 24 hours What’s the gestation length of mice? How old can they be at weaning? Offspring • Weaning animals must be removed from the cage promptly to avoid overcrowding. If kept in the same cage, older juveniles can trample and kill neonates in the new litter. Therefore, weanlings are typically transferred to new cages before the new litter is born. • Newborn mice are altricial, meaning they are born hairless with eyes and ear canals closed. Shortly after birth, they develop a distinct white spot that is visible through the transparent skin on the left side of their abdomen. This spot, called the milk spot, is actually the stomach filled with milk; it indicates that the mice are healthy and nursing normally and that the mother is producing milk. • By 10 to 14 days, neonates grow fur and their eyes open. If possible, newborn litters and their parents should not be disturbed for several days. If females with newborn litters are disturbed by husbandry procedures, they may cannibalize their young. Some inbred mouse strains are less vigorous breeders than outbred mouse stocks, and may exhibit a decline in reproductive performance at an earlier age. This low productivity in breeding is also common in genetically modified strains. • Breeding Decline Issues: A decrease in reproduction in a breeding colony may be caused by environmental factors. • • For example, failure of the timer that turns the facility’s lights on and off may cause the lights to remain on constantly. Female mice exposed to constant light may stop having an estrous cycle. They will return to a normal reproductive pattern once the normal light cycle is restored. Loud noises and vibrations, such as from construction work, can also decrease reproductive performance in a mouse colony, even when the noise or vibrations are generated outside the laboratory animal facility. These noises and vibrations may be at such a high frequency that they are inaudible to humans, but within the hearing range of the mice. Special monitoring equipment may be needed to detect them. Behavior • Nocturnal: Mice are nocturnal animals, but they have alternating periods of activity and rest during both the day and night. Most of their eating and other activities take place when it is dark. • When sleeping, a mouse may curl its head under its body. • Mice have a high metabolic rate. They are constantly active during waking hours. They labor meticulously to keep their quarters organized. • Mice groom themselves almost constantly, which helps them maintain a smooth, glossy hair coat. • Fighting: Although they are social animals, mice can be aggressive to cagemates. • Aggression may be due to many factors, including gender, strain, territory, fear-induced defensiveness, and social dominance. • Male mice in particular tend to be more aggressive, as are female mice defending a litter of pups. • Male mice tend to establish social dominance hierarchies in a group, but they will continuously compete for dominance. This often results in fighting and subsequent injuries. • Fighting may be increased by changes in group composition, the presence of female mice in the room (pheromone stimulation), or manipulation of the mice (e.g., cage changing, temporary removal for experimental procedures). • If multiple mice are in the cage, removal of the dominant mouse will not necessarily stop the injuries, as the remaining mice will fight to reestablish a social order. Behavior – Mouse management • Combine small stable groups at 3 to 4 weeks of age, so they are familiar with one another prior to puberty. • Avoid providing mice, males in particular, with items or enrichments that can be monopolized and guarded by dominant animals, such as rigid shelters with only one entrance. • Barbering: Behaviorally dominant mouse may overgroom (barber) the fur of more subordinate cagemates. • This should not be confused with fur loss caused by skin disorders or parasites. • This is not considered a clinical issue and should not lead to injury. • If the dominant mouse is moved to another cage, it will often start barbering the hair of its new cagemates. Barbering is usually harmless. • To decrease this behavior, mice should be offered various forms of environmental enrichment. Handling • Tail restraint: Mice may be picked up by the base or middle of the tail; do not pick them up by the tip of the tail. Picking up mice by the tip of the tail can cause the skin to tear off the tail – an injury known as “degloving.” When holding mice by the tail, they will typically grasp at the cage lid or another nearby surface with their front feet (Figure 18.7A). This behavior can enable you to change your grasp of the mouse. • Scruff of neck: Mice may be further restrained by grasping the scruff of the neck while securing their tail, as pictured (Figure 18.7B). This restraint grasp takes some practice but is easily mastered. • Forceps: Rubber-tipped forceps may also be used to grasp adult mice by the scruff of the neck when changing cages (Figure 18.7C). • Hands: Very young mice (pups less than 10 days old) should be picked up by cupping the hands around the whole body, by grasping the skin across the shoulder blades, or by picking up a group of pups together with a small amount of bedding material. • Tunnels or cups: Mice may also be handled using the techniques called tunnel handling and cup handling. Tunnel handling uses the mouse’s natural desire to be in an enclosed tube, which is then used to transport the animal between cages. The cup method involves keeping the animal in a cupped hand, without restraint. These techniques work best with mice that are calm and used to being handled, as neither technique uses any physical restraint. As such, these methods are best used for transferring animals between cages rather than to restrain animals for procedures, such as blood draws or substance administration. • Plastic restraint devices: Can be used to hold mice in a confined space for certain research procedures. Holes in the restraint device allow the animal to breathe freely and provide access to the animal. Use of restraint devices and the duration of restraint need to be approved in the animal study protocol. Restraint - Injection • When working with an assistant to restrain a mouse for an injection, use one hand to grasp the base of the tail and the other hand to grasp the scruff of the neck. • Solo: When giving an injection by yourself, grasp the scruff of the neck while wrapping the tail between your little finger and ring finger of the same hand. Your other hand will be free to hold the syringe. • It is important to grasp the skin close enough to the head to prevent the animal from being able to turn its head and bite. Hold the head firmly but gently, making sure that the animal can breathe normally. Identification Methods • Various methods are available for identifying mice. • Cage card: Most common. Lists the strain or stock, the responsible investigator, the protocol number, dates of birth or arrival, and other information. • Ear notching: To mark individual mice • Ear tags • Tattoo: tail or toe tattoo • Microchips: SQ implants read with special scanners. • Nontoxic color marker: For temporary identification of individual animals by marking the fur. • Toe clipping: as a method of identification of neonate mice requires IACUC approval. Institutions will have their own policies on use of toe clipping as a method of identification and specific restrictions. The Guide states that toe clipping “should be used only when no other individual identification method is feasible.” • If any physical method is used, the method must be scientifically justified and approved by the IACUC. Husbandry & Diet • Mice are normally fed and watered ad libitum, meaning that feed and water is always available. • Standardized, nutritionally complete rodent diets are readily available from multiple vendors. Most commonly, the diets come in pellets or chunks that are gnawed by the mice. Gnawing the feed helps to wear down their incisors, which helps prevent malocclusion. • The pellets are normally placed in feed hoppers, which are often integrated into the wire-bar lids of the cage. To avoid contamination with urine and feces, avoid placing feed pellets on the floor of the cage, unless approved in the animal study protocol or required for supportive care. • Sterilized feed is generally used for animals in barrier housing to protect them from exposure to environmental microorganisms and to preserve their microbiological status. Husbandry • Mice are usually group housed, generally 4 to 5 to a cage, depending on the size of the cage. The density is specifically indicated in the Guide for the Care and Use of Laboratory Animals. • Various types of caging systems are available, depending on the needs of the mice and the type of research being performed. Solid-bottom plastic cages with bedding are used in most facilities. To prevent exposure of the animals to airborne pathogens, microisolation lids are used with these cages. More advanced ventilated caging systems provide air circulation within each individual cage to reduce moisture buildup from feces and urine, which reduces the frequency of required cage changes. • Generally, mouse cages are changed in an animal work station for Animal Biosafety Level (ABSL) 1 to prevent exposure of mice to the environment and reduce human exposure to allergens (Figure 18.10). ABSL-2/3 requires the use of specialized biosafety cabinets. • Wire bottom cages or metabolic caging, as shown in chapter 6, are often used in toxicology research. These cages allow urine and feces to fall through the floor of the cage, minimizing the mouse’s exposure to any experimental drugs excreted in the urine or feces and/or allowing for collection of urine or feces. This type of cage is not used for standard housing and must be approved by the IACUC. Mouse species typical behaviors • Social interactions • Mice are a social species, and the most common form of environmental enrichment is group housing • Nesting • Burrowing • Investigative • Foraging Why do we provide enrichment? Enrichment • Purpose: • • • • Exhibit species typical behavior Allows for choices in environment Decrease stress Should be meaningful What type of enrichment would they find meaningful? What are typical species behaviors for mice? Additional enrichment options include providing nesting material, structures that can be used as hiding places (shelters or cardboard tubes), and foraging material. In addition, nutritional enrichment (e.g., peanuts or sunflower seeds) may be used to promote species like behavior and reduce stress around parturition. Clinical Signs of Illness Sick or injured mice often display similar signs regardless of the cause. You must be able to recognize these signs to identify sick mice within a colony so they can get veterinary attention. Each institution should have a mechanism to identify and report clinical health concerns. Health checks should be a component of your daily monitoring. Shining a red-filtered flashlight into the cage is a common method of rousing the mice to check their activity level. Enrichment items may inhibit your ability to see all the mice in the cage, so you may need to open the cage lid in an animal work station to complete your health check. 1. Hunched posture 2. Ruffled fur 3. Anorexia 4. Weight loss 5. Poor body condition* 6. Lethargy 7. Huddling 8. Facial grimace* 9. Lumps & bumps 10. Dehydration 11. Staining 12. Licking Mouse Grimace Scale https://nc3rs.org.uk/grimacescales • Reliable & rapid means of assessing pain • Looking at “facial action units” • Squinting eyes • Tight whiskers • Flattened, lateral ears • A non-invasive and rapid method to assess health • Scores are established by both visual evaluation and handson examination. Euthanasia At the end of a study, mice may have to be euthanized. • Acceptable: Historically, the most common euthanasia method for laboratory mice has been carbon dioxide (CO2) asphyxiation. • Acceptable with conditions: • An overdose of an anesthetic gas, such as isoflurane, is an “acceptable with conditions” method of euthanasia. • Inject an overdose of anesthetics, such as pentobarbital, into the peritoneal cavity (in the abdomen) • physical methods of euthanasia, such as cervical dislocation or decapitation (used when chemical exposure impacts the research) • Unacceptable: It is not appropriate to use dry ice to generate CO2 for euthanasia. • Whatever method is chosen, death must be verified. Your institution’s SOPs may require an additional procedure, such as cervical dislocation, exsanguination, or opening the thorax, in order to ensure death. Carcass disposal should be according to the facility’s guidelines.