Laboratory Animal Science ABD

VIII.4 Describe the principles of humane killing (euthanasia) and give examples of different methods by which animals are allowed to be killed. In addition, explain why someone competent to kill animals should be available at all times. (LO 6.1.1, 6.1.3)

- Must be done without causing fear, pain or stress - Death as quick as possible - The method must be easy and safe to perform by the person performing it and the person should be competent in this. You can: - Killing can be done by overdose of anesthetic compound in all species. - The captive bolt can be used in amphibians, reptiles and large mammals. - Cervical dislocation can be used for birds and rodents. - CO2 or nitrogen can be used for rodents and birds (be vary to increase concentration slowly to not cause fear/discomfort in animal). -Shooting There should be someone competent at all times to ensure that if an humane endpoint is met or the animals welfare has been compromised that the animal can be euthanised instantaneously. There are a few methods to confirm death: - Confirmation of permanent cessation of the circulation - Destruction of the brain - Dislocation of the neck - Exsanguination (blood loss) or - Confirmation of the onset of rigor mortis

VII.1 Describe suitable housing and husbandry routines for laboratory animals, how conditions are monitored and identify the consequences for the animal resulting from inappropriate environmental conditions. (LO 4.1, 4.2, 4.3)

A house needs a bedding, nesting material, house, gnawing/wood sticks - they need a place to hide and some activity. The cages needs to be regulary cleaned with good circulation Husbandry: • Receiving • Marking • Cage changing • Cleaning • Feeding • Watering • Breeding • Weaning • Observing • Killing For laboratory animals, housing would normally be conventional housing where the staff can come and go. This does have a risk of carrying infections in because it does not keep out microbes. It should have positive pressure to keep microbes out. The cage has to have: Bedding, nesting material, a house, and gnawing/wood sticks. They generally like to be able to control their environment and they prefer cardboard things, instead of plastic. The cages should be regularly cleaned (circulation). This can be done in many ways, either single og dual corridors. If it is single corridors, it can be either bi-directional og undirectional. And here you have to either make sure clean and dirty cages aren't carried out at the same time (time slots), or that they are covered. Depending on the level of barriers, animals can either only enter via rederivation, or any animal can come in, or something in-between. The minimum requirements for controlled things in animal rooms are: ventilation, temperature, humidity, lighting, noise, alarm systems. The health is also very important, plus the caging should have food and water supply, bedding material, cleaning, dimensions, environmental enrichments and there should be good handling. Husbandry routines include: • Receiving • Marking • Cage changing • Cleaning • Feeding • Watering • Breeding • Weaning • Observing • Killing If these conditions aren't upheld, the animals can get sick, the caretakers or lab workers can get sick. It can also cause allergies in the caretakers, or stress in the animals. It can also make the animal frustrated. Amalie: Suitable housing should be done in according with the 5 freedoms, and can be divided into different enrichment: Behavioral enrichment: Suitable space, correct space, allows the animal to imitate wild behavior Social enrichment: Allow for social partners of the same species, and make sure potential predatory species are not able to be detected by the animal (Rats are considered predators for mice) Artificial enrichment: Artificially creating ways to engage the animal (Tunnels, running wheels) Nutrition enrichment: Measuring and determining the quality and quantity of food. Ad lib. Feeding leads to overweight mice, 75% of ad lib. Intake results in healthy mice. Husbandry routines should be consistent to avoid stressing the animals -> Better scientific results. Some animals might have specific needs for cleaning routines, e.g. olfactory (Pheromone) environment should be maintained, but resusing some of the old bedding Conditions can be monitored both through physiological parameters (BT, BW, Biomarkers) or through behavioral score sheets. When animals are housed in groups, more trained staff is required to monitor the behavior in the group to keep violence to a minimum. Failure to implement and monitor environmental conditions can lead to altered metabolism of the animals, as well as introducing unknown factors into the experiment, leading to higher variance in results, and more compromised scientific data.

I.7 Indicate the circumstances in which animals under the scope of the Directive should be humanely killed or removed from the study to receive veterinary treatment, and describe the legislative controls over the killing of animals bred or used for scientific procedures (LO 1.11)

Animals that has met humane endpoints are met or whose welfare has been compromised should be killed or remove from study to treat condition causing harm. An animals shall be killed when it is likely to remain in moderate or severe pain, suffering, distress or lasting harm. If the animal is likely to recover, it should receive veterinary treatment. If an animal used for experimentation is killed, it must be reported in the evaluation after the experiment.

IV.5 Explain international standards for nomenclature of laboratory animals (LO Additional KU)

Each of the breeders of lab animals has their own specific breeder code called ILAR (for instance CRL for charles river). Outbreed animals= stock. Nomenclature is, breeder code colon (:) and the stock name. Inbreed animals= strain. Nomenclature is, first strain name then slash (/) and the breeder code. There is some commen abbreviations acceptable for the most widely used strains, for instance B6 for the strain C57BL/6N. Genetically modified animals= end is added to the normal nomenclature with a horizontal slash (-) followed by gene name. TG is added if it's a transgene. F1 hybrids: MotherFartherF1/Breeder code

IV.2 Describe appropriate breeding programs for laboratory animals (LO 4.10)

Inbreeding strain programs uses inbreeding pyramids. In these you take a brother and a sister which will be the stem pair. These will have kids which will be the nucleus. And the brother and sisters from here will have offspring that will produce the production stock 1. These will be mated randomly and can be used for research or go on to production stock 2. The offspring from production stock 3 can only be used for research and not further breeding. All the animals from stock 3 must be used for research. Continuation of the strain is by taking another pair from the nucleus. THis way inbreed strains can be held genetically very similar. Outbreed stocks is breed by random mating - whereas they have more genetic varaiation. Some genetic selection will always be done since some animals live longer and breed better. Therefor an idea to not get selectivity is to make rotational breeding where the colony is divided into parts and Hybrids generate high heterozygosity you can mate a inbred mother from one strain with and inbred farther from another and get F1 hybrids. Rats and mice are fertile from around 7 weeks of age. One male can be mated with several females. Females are only fertile in estrous, u can determine how long the female is in the estrol cycle by a vaginal smear and a microscope-> if cells are keratinized the female is in estrous. Morning after mating there will be a vaginal plug.

VIII.8 Describe and discuss methods of optimising post anaesthetic recovery to ensure a smooth and rapid recovery from anesthesia. (LO 20.12, 21.16, 21.17, 21.18, 21.19)

To avoid pain, analgesia should be given. To avoid dehydration, fluid therapy with saline solution is essential. Hypothermia can be avoided with heating blankets or lamps, but be aware to not overheat the animal. Make sure tha animal has easy access to water and food source, as it may be harder for the animal to move now. Remember, pain and stress can lead to impaired recovery, as it results in superficial breathing - this causes poor oxygenation of the blood. Make sure that the animal is handled with care and is well acclimatized to the facilities before surgery. Infection is also a risk after operation, would healing should be observed, and usage of antibiotics if necessary. Postanesthetic restlessness and self-injury: the animal starts biting themselves due to pain or stress. A veterinarian should be contacted or provide anesthesia. Before: Pre-anesthetic medication, analgesia, when handling tissue, be careful, work aseptic. Acclimatization gives a faster recovery

VIII.9 Describe and discuss good working practices regarding use, storage and disposal of anesthetic and analgesic agents. (LO 20.13, 21.23)

When working with gases, make sure that there is ventilation! Needles should be disposed in designated waste containers. Gas cylinders have a risk of explosion in case of fire, make sure gas containers are stored with holding to ensure they dont get knocked over an cause injury. Drugs should be locked in cupboards, records of purchase and usage maintained. Non-used drugs are destroyed according to the legislation.

I.10 Describe the regulations regarding re-use of animals (LO 2.10)

You can only reuse animals if they have been used in the severity class mild or moderate and if the next experiment is mild, moderate, or non-recovery. The animal's health and well-being should be fully restored after first experiment. In exceptional circumstances the inspectorate can allow the reuse of an animal that has been caused severe pain, distress, or equivalent suffering.

II.11: Give examples on homepages where information on laboratory Animal Science, Laboratory Animal Care and 3R can be found. Discuss why you think it is important to continuously stay updated on how to optimise animal welfare during housing, handling, and procedures (EU LO 2.13; 2.14)

https://www.nc3rs.org.uk/ https://en.3rcenter.dk/ https://www.ccac.ca/ https://dyreforsoegstilsynet.dk/vejledninger/ There is no clear answer for good animal care, new methods and legislation is implemented all the time, so therefore it is always important to stay updated. Animals who are not feeling well in the facilities will not give good results.

IX.8 How can pilot studies help us when planning our experiments? Can we use a pilot study to estimate an effect size? (LO 11.6)

Pilot studies can help us identify experimental and humane endpoints. Thus we can identify the best ways to measure on the animals and improve data extraction, while also helping us abide the 3 R's more easily, as we may identify better humane endpoints. Pilot studies can be used to give a rough estimate of the effect size. By testing on a few test subjects we can gain a rough estimate of how big effect for instance a compound has on a measured outcome, while also allowing for a rough estimate of how small an effect we can observe using current methods. If we don't have a pilot study, a big study is done which maybe has useless results as the compound could prove to be ineffective.

VIII.12 Describe Halstead's principles and discuss the importance of hygeine and asepsis, correct handling of tissues, preparation of instruments, personnel and animals for a successul surgery and avoidance of post-surgical complications (LO 22.3, 22.3, 22.4, 22.5,-22.6)

Poor hygiene and work without asepsis can result in infected wounds, poor healing, pain (thus also stress) and distress in animal, more difference in outcomes leads to higher variability and thus lower reproducibility. Higher mortality leads to the need for using more animals. Preparation: -aseptic techniques are used to lessen likelihood of exposure to microorganisms. To work aseptically sterile objects must not be in contact with unsterile objects. They should be stored in a clean dry environment. The facilities, environment, surgical site equipment and staff should be suiatable for aseptic work, for instance with the proper ventilation and trained staff. For sterilization autoclavation and irradiation can be used. -Larger animals should be fasted (not rodents as they cant vomit). Acclimatize animals (1-2 weeks for rodents). You shave area where surgery is to be performed, and clean area with anti-septic soap solution and remove with alcohol or iodine. Whole animal is covered in sterile drape up to procedure. Halsted's principles: -work under asepsis -use gentle handling -ensure hemostasis -Closure of dead space and suturing tightly (suture=row of stitches) -Carefull approximation of tissues -Avoidance of tension -Minimization of foreign material

VIII. 14 Discuss the principles of post-surgical care and monitoring, and describe common post-surgical complications and their causes (LO 22.11, 22.12)

To avoid pain, analgesia should be given. To avoid dehydration, fluid therapy is essential. Hypothermia can be avoided with heating blankets but be aware to not overheat the animals. Infection is also a risk after operation, would healing should be observed, and usage of antibiotics if necessary. Postanesthetic restlessness and self-injury: the animal starts to bite themselves due to pain or stress. A veterinarian should be contacted.

IX.1 Describe the concepts of fidelity and discrimination, and discuss validation criteria for an induced and a spontaneous animal model. (LO 10.1 & LO Additional KU)

- A model has high fidelity if it has a high overall similarity to what is being modeled (in many ways is comparable to the phenomenon it tries to imitate.) - A model has high discrimination if it has a specific similarity for one particular property of what is being modeled (in a very specific way is comparable to the phenomenon it tries to imitate, while in other aspects it is not similar.) -In spontaneous models the animals develops the phenomenon we want to investigate by itself -> for instance in inbreed mice strains where tumors develop more often -In induced models we induce the phenomenon we want to investigate-> for instance via injection of a compound that gives rise to diabetes.

III.4: Give examples of stressors (i.e. any adverse stimulus; physical, mental, or emotional, internal or external, that tends to disturb the homeostasis of an organism) and discuss the physiological consequences and manifestations of stress in rodents (EU LO 3.1.2).

-Isolation of social animals will result in stress and increased stress hormones in animals. As stress is related to depression, the animals may become depressed. Stress and depression is related to pain and thus if animals are recovering from surgery or have an impaiment they may experience increased pain! Rough handling can of course also stress mice. As rodents can hear ultrasound they may experience audible stressors which humans cant hear. Disturbing the circadian rythm may also stress animals. Different oudours and pheromones could stress animals and proximity to other stressed animals could thus increase stress through pheromones. Fasting can induce torpor: this means that the body temperature is reduced and metabolism is slowed down. A too low temperature, 19-21 degrees, can challenge the homeostasis of a mouse and can be a stressor. -In standard laboratory environments mice are housed at 20-24 °C. However, their thermoneutral zone ranges between 26 °C and 34 °C and the lower critical temperature for a single-housed mouse with no nesting material is 34 °C Such a challenge to homeostasis is by definition stressful, and could therefore affect many aspects of physiology and behavior.

VII. 4 List the correct procedures for ensuring health, welfare and care of animals during their transport. (LO 4.12)

-Persons transporting animals must ensure that the animals are transported in a way that is not likely to cause injury or unnecessary suffering and under conditions suitable for the animal. -Everything has been done to minimize the length of the journey -The animals are fit for the journey -the means of transport is designed to avoid injury and ensure safety -The personnel handling animals are trained or competent in the transport of animals -Sufficient floor area and height is provided for the animal -the animal is watered and fed Transportation is a very stressful process for animals. Proper caging required, as well as procedures are for each specific animals (e.g. providing food and water for rodents are associated with better acclimatation).

II.10: Explain what is meant by "A Culture of Care" and discuss why it is important to promote a Culture of Care (EU LO 2.12)

A culture of caring for both the animals and the animal staff to decrease the stress on both parties. How to develop a good culture of care: - Senior staff should lead by good example - Hire staff with focus on CoC - Award programs and high expectations - Encourage communication - Sharing of good practices Key factors of A Culture of Care - Management is important o Know of animal care and use o Signal commitment and supply the funds o Expect high standards o Promote a shared responsibility o Support a positive attitude o Empower the care staff and veterinarians o Encourage communication o Allow for voices of concern "A culture of care" broadly indicate a corporate and personal commitment to improve animal welfare and thus scientific quality (translatability and reproducibility is better), while supporting a good working environment for the staff. People who work in this field often cares greatly for the animals and thus it may be distressing to observe or take part in procedures where animals are suffering or are euthanized. Part of a culture of care ofc includes implementing the 3Rs, supply enough funds for facility, hire staff with focus on good culture of care and senior staff leading by example, while supporting a positive attitude. According to EU regulation all states should have a animal-welfare body, in Denmark this is called dyrevelfærdsorganet. It does: · Communicates on the 3Rs · Drives a culture of care · Collaborates with senior management · Encourage collaboration between scientists and animal staff Other things that can be implemented: Award programs and high expectations - Encourage communication - Sharing of good practices o Promote a shared responsibility o Empower the care staff and veterinarians o Encourage communication o Allow for voices of concern

VIII.2 Describe what a humane endpoint is. Identify criteria to be used to set humane endpoints. Define action to be taken when a humane endpoint is reached and consider possible options for refining methods to finish at an earlier endpoint. Discuss also factors to be considered and methods available for assessing and recording the welfare of animals e.g. score sheets. (LO 5.3, 5.4)

A humane endpoint is the earliest point, at which an experiment should be terminated, with the purpose of eliminating unnecessary suffering. When the humane endpoint is reached the harmfull condition is stopped, either via aleviation (could be analgesia) or through euthanasia. Criteria to be used to set humane endpoints: To set humane endpoints you need to identify clinical signs (like heartrate, weight, apperance, temperature and stress hormones), in combination with modelspecific signs (signs that are relevant in the specific animal and model you are using, for instance tumor weight as percentage of body weight). This is done to set as early endpoints as possible. Possible options for refining methods to finish at an earlier endpoint: -Methods to finish at an earlier endpoint is to design a score sheet with relevant parameters for the experiment, the species and the strain. -Better and more precise measurement methods for clinical signs. -Use pilot studies to find the first clinical signs - done on a few animals - identify critical periods in the experiment. -Have skilled staff that know the signs of distress in the animals Factors to be considered and Methods available for assessing and recording of the welfare of the animals: -The use of welfare protocols can help with assesing and recording humane endpoint. In these a score sheet based on things like appearance, behavior and other clinical/behavioral signs are used to keep track of the animals welfare. If a predetermined score is reached in a category the humane endpoint has been reached. -A factor to consider could for instance be torpor in mice if temperature is used as humane endpoint.

I.5 Describe minimum and maximum threshold of pain, suffering, distress or lasting harm, i.e. when a procedure becomes regulated as an animal experiment, and when it cannot be allowed no matter the purpose (LO 1.8, 9.3)

According to the animal welfare act, no one is allowed to cause: - Pain - Suffering - Distress - Lasting harm This act is overruled by the animal experimentation act. This act can be applied by either: - Research - Teaching - Production of blood products Thereby, the animal welfare act can be allowed to cross if licensed in each specific case. The injection criteria. This means that a license is required for an experiment when the pain is higher than the injection of a needle. They may not be causing: - Strong pain - Intensive fear - Intensive suffering If they do, the animal should be humanely killed (euthanized). Some experiments, such as dog training, are mild enough to not require a license. Invertebrates are not covered by the EU directive, meaning that all experiments done with these species should be carried out with respect for the animal welfare act. Cephalopods (octopuses) and fetuses are covered by the animal experimentation legislation act. -The lower treshold (the injection criterium)→ "A procedure must be licensed if it causes: Pain, suffering, distress or lasting harm equivalent or higher than that caused by the introduction of a needle in accordance with good veterinary practice." -The upper threshold → Strong pain, intensive fear, intensive suffering and a purpose (research, teaching, or production of blood products). Definition of an experminet → "An animal experiment is a procedure for the purpose of research, teaching or production of blood products, which causes pain, suffering, distress, or lasting harm in a vertebrate including mammal fetuses in their last third or a cephalopod equivalent to, or higher than, that caused by the introduction of a needle in accordance with good veterinary practice"

VII. 5 List potential human health hazards associated with contact with laboratory animals and how these can be prevented. (LO 4.13)

Allergy: -Keep levels of allergens in facility as low as possible (for instance by: use isolation cages like scantainers) -use protective equipment and don't carry allergens with you out from facility -monitor allergen levels -proper ventilation (negative vs positive pressure) -educate people. Physical injuries: Bites and scratches. Kicks from larger animals. Needles. Prevention-> proper handling of animals. Treatment-> wash with soap and keep in warm water, elevate bit limp, get medical assistance if swelling or persisting pain. Harmful substances: Contact with biologicals, radioactive isotopes, chemicals and adjuvants (immune stimulants) in experiments. Also remember that they can be present in larger amounts in urine and feces from animals. Precautions: Warning labels, gloves, eyewear, mask, good needle practice proper restrain of animals disinfection/autoclavation of waste use of biosafety cabinet, focus and hazardous properties via databases like kemibrug. Zoonoses: Infections that can infect both humans and animals. Can be latent in carry animal. Most servear is from non human primates. Precautions: Work with animals where microbiology is defined (health certificates from vendors), use barrier units instead of conventional units in facilities, good working routines-> don't eat in work space, good needle practice and so on. Protective equipment, proper actions in case of accident.

IX.4 When planning an experiment, what do we mean by "experimental unit?" Give two examples of experimental units (of different "size"). (LO 10.4)

An experimental unit is the smallest element from what we can draw any conclusions. Sometimes this is animals, but sometimes it is something else. For example, if you want to test kidney function and need to determine how much a mouse is drinking, it is hard to do on only one mouse. Mice are social animals and in one cage you need at least three as to not have stress/anxiety result have an impact on the results (and also to allign with the 3 Rs). However, you cannot know how much 1 specific mouse drank, and therefore the experimental unit becomes the cage, where it is 1 bottle and 3 mice. Another example of experimental unit size would be if we ant to measure how social isolation impact weight gain (or something else) in mice. Here we would need to isolate the mice so 1 mouse in one cage is now our experimental unit.

II.2: Describe how the public perception of Animal experimentation overall relate to the ethical theories of Utilitarianism, Contractarianism and Animal Rigths View (EU LO 2.1)

Animal rights view: animals have the same rights as humans and should not suffer because of human desires, thus experimentation on animals is morally wrong. Good results can not justify mean. One argument is it is also hard to make a law that allows for experimentation on animals without also allowing for the experimentation on some humans within the scope of this law, as distinguish via intelligence level, speach and cognitive skills could still allow for some humans. Contractarianism: are approvers of animal experimentation. Man have no obligations towards animals and animals have no inherent rights. As long as the society allows for it, it is not a problem to use animals for animal experimentation. Utilitarianism: the means justifies the deeds. Benefit vs harm. Animals are sentient beings with instrinsic value so unesecary harm is morally wrong, but experimentation is allowed if benefit (could also be for the good of the animals or enviroment) outweighs harm.

II.7: Present and discuss different viewpoints on/definitions on the nature of Animal welfare and discuss methods to measure/evaluate animal welfare(EU OL 2.7)

Animal welfare can be defined and measured as: Health and biological functioning: measured via · Clinical examination-> Heart rate, body temperature, respiration rate, appearance of animal. · Clinical chemistry-> Blood and urine samples to measure immunologic, metabolic and hormonal biomarkers for wellbeing or disease. Appearance of animal products (milk, eggs..) Natural living/behavior: measured via occurenec of natural behavior->-Natural behavior thus needs to be defined, which can be done from: Semi natural environments (big cages with lots of different stimuli and enciroment, releasing animals into the wild and observing their behavior, observing behavior of wild ancestors) · Data evaluation on individual or group behavior: Mortality, morbidity, injuries, reproduction rate, yield of products (milk, egg..), quality of meat. · Measure of abnormal behaviors in animals such as aggression, apathy and anorexia. · Use of ethogram to classify specific behaviors (enabling the choice to track these): Ethogram is a list of species specific behaviors, for instance stereotopy (an atypical behavior which can further be subdivided into other behaviors like "bar mouthing") Emotions and preferences: Measured via -Behaviors indicating positive mental states like joy, carrying, playfulness, can be measured by: -Ultrasonic vocalization (some wavelengths are equal to laughter in rodents). -Emotional bias/judgement (measure of how optimistic animal is)-> can be measured via behavioral/trace experiments, like the food-bowl-placement experiment:

II.9: Describe the importance of good animal welfare including its effect on scientific outcomes as well as for ethical reasons

Animal welfare is important for scientific reasons relating to the quality of the data obtained by use of live animals and the resulting translatability and reproducibility. If the animals are in fear, stress or mishandled it can affect their immunesystem, cardiovascular or reproductive system and behavior, which can affect the measured outcomes in the experiment. Thus the results become less valid and the varriance in the study may increase, making it less reproducible for others. Furthermore legislation requires good animal welfare, so there is also a legal perspective. As most legislation is build on the utilitarian principle in the EU and Denmark, animal welfare is a key aspect. In the utilitarian principle we try to maximice benefit and animal welfare at the same time, that is maximise benefit while reducing harm. In the utilitarian principle it is acknowledged that animals are sentein beings with intrinsic value and thus performing unnesecary harm on these are morally wrong.

VIII.6 Discuss the preanaesthetic considerations necessary for a successful anaesthetic procedure, including acclimatization and evaluation of the animal, as well as selection of agents. (LO 20.4, 20.5, 20.6, 20.7, 20.8, 21.1, 21.2, 21.3, 21.4, 21.5)

Before procedure make an anesthetic protocol. Make sure animals are healthy via clinical examination (look at fur, food and water intake, body weight, indicators of good health..), make sure animal don't carry hidden infections (rely on good health monitoring system). Make sure they are acclimatized (1-2 weeks for mice and rats) to avoid stress as stress also impacts healing and pain. Sometimes animals should fast to prevent risk of suffocating/vomiting (in rodents no fasting as they cant vomit). Pre anasthetic medication can also be used to reduce fear in animal.-> these considerations will lead to reduced mortality, faster recovery and better reproducibility.Choice of anesthesia-> Anesthesia should be chosen on a platform best for the experimental data (it should not influence experiment), this is higher prioritized than giving the anesthesia best for the animal, as in the end the results are the reason for the experiment and without these the sacrifice will have been useless. Combination of several anesthesia results in less dose needed for each drug resulting in minimization of side effects and easier fufillment of the anesthetic triade. However it also gives rise to different side effects, which could effect experimental results. Anasthesia can be given by inhalation or injection, so also a choice to make here.

II.4: Discuss the use of live animals for training and teaching purposes in Laboratory Animal Science in courses such as the one, you are taking right now (EU LO 2.3)

By the use of animal experiments in education, it can be seen through utilitarianism eyes, that it is for a good cause, as it is done in the name of science which in the future can improve both human and animal health. Thus we benefit greatly from the animals used in our practicals, which outweigh the harm they where caused doing the exercise. The three R's are also included in the course, as there are 2 students per rat/mouse (reduction), and the suffering is kept to a minimum as they are anesthetized (refinement). We are allowed to practice on a toy rat as well, which replaced real life animals (replacement).

I.6 What is meant by destinational breeding in relation animal experimentation legislation, and give examples of some species covered and some species not covered (LO 1.10)

Destinational breeding is the breeding of certainanimals for which you require a license from the competent authority to breed. The animals are to be trackable (for instance via chip) and in good health, without disease. Most animals that are not farm animals are destinational breed, like: • Mice (Mus musculus) • Rats (Rattus norvegicus) • Guinea pigs (Cavia porcellus) • Hamsters (Mesocricetus auratus) • Mongolian gerbil (Meriones unguiculatus) • Rabbits (Oryctolagus cuniculus) • Dogs (Canis familiaris) • Cats (Felis catus) • All species of non-human primates • Frog (Xenopus (laevis, tropicalis), and Zebra fish (Danio rerio) Farm animals like pigs, sheep and cows do not have to be licensed to breed. Non-destinational bred animals can be allowed for experimental use by the animal experimentation board, as well as wild animals. This has to be properly supported by scientific arguments. If you are allowed to use wild animals, you will need a license from the Nature Agency aswell. Cloning and gene modification has an act on its own. The cloned or gene modified animals must be housed properly as well as licensed.

VI.1 Describe different types of diets for laboratory animals and how they are constructed to meet the dietary requirements for the animals (LO 3.1.5, 4.6)

Diets include macronutrients in various concentrations depending on animal: · Fats-> including essential fatty acids · Proteins-> including the important essential amino acids (as the animals dont produce these) · Carbohydrates-> divided into fibres and digestible carbohydrates · Minerals-> calcium, phosphate, iron and more · Vitamins-> A, D, E, K and Bs Pellets are used a lot and divided into: A natural diet (chow): whole grains (carbohydrates), soybean (protein), bone meal (minerals)-> Cheap and nutritious, but there is variation between batches from producers. Synthetic pellets/powder: Amino acids, Starch, cellulose, corn oil and different minerals-> Low variation between badges, but is less liked by animals and more expensive than natural diet. Synthetic pellets are easier to order special made from vendors without certain compound like gluten.

II.1: Explain and discuss different ethical views on use of animals for experimental research (EU LO 2.1)

Disapprovers - All animals are equal and have rights that must not be denied Approvers - Human interests are more important than the interest of animals, no matter the cost to the animals Approvers with reservations - Harms and benefits must be balanced and animal suffering is not to be ignored

VIII.11 Explain the relevance and need for pre-operative assessment and, where appropriate, conditioning, and give examples of sources of reference for good surgical practice. Describe the planning of surgical procedures and discuss the competencies required of all personnel involved and describe particular aspects of care appropriate for animals before, during and after surgical or any other potentially painful intervention. (LO 22.1, 22.2, 22.13, 22.15)

Do literature search in order to see possible effects of the used anesthetics, and possible interference with other factors. It is important to have a checklist before performing a surgery for all the requirements. This includes preparing of the facilities and equipment. This differs between larger animals and rodents. Before/During/after: Make sure they are relaxed, comfortable. You can give pre-anesthetic medication, to make a smoother induction or recovery of anesthesia. During you must keep them sedated, test their reflexed, and make sure you give them eyedrops. After you must give them a comfortable space to recover, that is heated and has nice bedding. If it's rodents, minimize physical contact and make the space body temp. Competence of all the staff, they must be qualified. The educational demands for staff needs to be fulfilled. Assistants, care takers, ABCD person. To follow Halsteads principles. Rikkes: Surgery and anesthesia always pose a risk for the animals which is why they must undergo a pre-operative assessment to account for the suitability of anesthesia and surgery. • It will minimize the risk of complications both during and after surgery • Will help detect possible underlying problems • Divided into three parts o Physical examination o Blood testing Comprises a complete blood count (CBC) will indicate oxygenation of the animal (anemia) o Extended/ specific testing Good surgical practice: • Surgical procedures should be planned down to the smallest detail to prevent mistakes and accidents from happening. • Everything from the choice of animal, acclimatization of the chosen animal, preoperative evaluation of the animals' health, preparation of the animal for surgery, postoperative management of the animal to the provision of facilities, choice of instruments, number of assistants, and anesthetic protocols should be carefully considered. Preoperative assessment, conditioning, and care before surgery: • Larger animals need to have fasted before surgery. Food is restricted to prevent vomiting under the procedure. • Water is not restricted until a few hours before surgery to prevent dehydration. • Rodents must be acclimated in their home cage before surgery. • Rodents are incapable of vomiting, so unless the gastrointestinal tract needs to be empty for surgery, there is no need to restrict food. • The skin must be prepared to minimize the risk of infection. o First, the hair is removed in the area of the surgical incision and antiseptic soap is used to scrub the area. o The area can be swapped with an antiseptic (alcoholic chlorhexidine or povidone-iodine). The animal is covered with a sterile drape during surgery. If there is potential for contamination an antibiotic should be injected prior to surgery. The presurgical administration of antibiotics will allow the drug to achieve maximum effect during surgery when contamination is most likely. Competencies of personnel • For major procedures, the surgical team includes a surgeon, an assistant surgeon, an instrument nurse, and an anesthetist. • The surgeon should be fully trained in the procedure that is to be performed. • The roles of the surgeon and the assistant(s) should be known. • All personnel should be trained to work aseptically to minimize the risk of sepsis. • A veterinarian should be attending to consult in case of risk of contamination. • The surgeon should use surgical techniques and understand animal physiology and anatomy. • A person trained in performing euthanasia on the selected animal should be present in case of adverse events and accidents requiring euthanasia of the animal to prevent suffering. Care for animals during surgery include monitoring of vitals such as pulse, respiration, oxygenation, and temperature. Placing the animal on a heating pad will prevent hypothermia. It is important to maintain and monitor the anesthesia during the entire procedure. • A way of doing this is by checking the withdrawal reflexes. It is also important to keep mucous membranes moist, fx. By moistening the cornea with eye ointment or eye drops. After surgery, the animal will need to recover and requires monitoring and specialized care. The animal should be allowed to rest in a dedicated recovery room/area. It should have supplementary fluids and oxygen available as well as analgesics. NSAIDs and opioids are preferred for postoperative treatment of pain.

I.4 Describe who is responsible for compliance in relation how the experiment is performed, and how the animals are housed in the facility. Describe what is expected from the scientist in relation to being aware of local organizations of animal work (LO 1.7, 1.9, 11.9)

Each facility has a designated manager that is responsible for the specific experimentation, and animal welfare/husbandry. However, the competent authority (animal experimentation expectorate in denmark) carries out inspections to make sure that experiments and housing are carried out in compliance with the animal experimentation act. They carry also a part of the responsiblity for implementation of a Culture of Care in the facility through awarity local organization. Scientist should cooporate with all staff and organizations including the veterenarians, manager and husbandry staff, to foster a culture of care and deepen the bond and understanding across fields. On KU it is the department of experimental medicine.

IX.7 Sometimes when planning an experiment we may need outside help with our statistics. Give an example of when this might happen and of the elements of your study that you need to be able to present to, for example, a statistician. (LO 10.8 & LO 11.4)

For multifactorial designs, help is needed to calculate the needed sample size, as this is complicated calculations where in silico simulations are used. (contraty simple case control setups could be calculated without staticians) • The elements that you need to be able to present to a statistician is statistical power (how willing are we to have false negatives), effect size (what is the smallest effect we are interested in), significanse (how willing are we to have false positives) and variation (both biological and technical.)

IX.9 Explain the principles of GLP in experimental animal research. (LO 11.8)

GLP means quality assurance is in place, that is ensuring good quality and validity of data produced. GLP insures consistency, reliability, tracability, uniformity and quality, while ensuring reproducibility. Means for GLP could be: Good training of staff to wear correct equipment, record procedures...

VIII.13 Give examples of commonly used surgical instruments, suture materials and needles and how they are applied. (LO 22.7, 22.8, 22.9)

General surgery pack includes: -scalpels for cutting in tissue -forceps for graping organic material -scissors to cut -Hemostatic clamp to control bleeding. -Needle holders to hold needles while suturing -Things like the metal tray, steel bowls and such -Suture materials like prolene or PDS Needles: aseptic, can only be poked once, and comes in different lengths and gauges.

IV.3 Describe how genetically altered animals can be generated and how they can be used for scientific research (LO 4.11)

Genetically altered animals can be made by * maybe not toally correct, not sure about CRISPR, rewatch lecture if time. 1. Mating a female and male, female is euthanized and fertilized eggs are extracted and altered genetically using the CRIPSR Cas9 technique. Eggs are know inserted into another female which has just mated, eventually she will birth the genetically modified pups. Alternatively for larger animals sperm is extracted from male and genetically modified and placed in the tubulus of another male, which then mates with the female and ejaculates the genetically modified sperm. Pups are the breeded for several generations to ensure similar genecity. 2. Using embryonic stem cells from a female and genetically modifying using the CRISPR Cas9 technique, whereafter they are placed in a new female who will eventually give birth to pups. Genetically modified animals can be used for: Researching how altered gene functions affect the animals, make new traits/disease models to be investigated (like diabetes or high blood pressure, no thyroid (no T-cells), proneness for obesity and how new treatments affect these), or making animals transgenic carrying human/other animals genetics (or immune system using stem cells), making results more translatable.

IX.3 Describe relevant factors and possible sources of bias, when planning an animal experiment, and possible actions to prevent them. (LO 10.3)

Humans are a big source of bias. Due to our hopes, fears and expectations we can unconsciously skew results. For instance end points used may rely on visual scoring sheets for behavior, humans will unconsciously skew scores depending on their expectations. Different treatment of animals by humans or environment, like light sources, sounds, temperature etc. may also bias experiments, while bias could also be introduced when sampling the populations for case and control. To avoid bias, blinding is used in studies to avert human bias. Thus the humans cant differntiate between the case and control in case control experiments. We can also use animals as their own control! For instance placing two tumors in mice and only treating one of them. Now we have a treated and control for comparison. We could also use the full cross-over design, where half the animals start with control and the other half with the case, then we use a wash out period and switch around case and control.

IX.6 Sometimes when we plan experiments, we realize that there are benefits to using something other than a simple case-control study. Give an example of a more complex design, of when we might use it, and why. (LO 10.7 & LO 11.3)

If we want to exclude biases (both human and enviromental/animal specific) we might introduce other experimental desgin. One such design could be the multifactoral design. In this design we test and find out how different factors affect the experimental outcome, this could be things like sex and age, which in the end could make results more translatable. To make the multifactoral design we often need more subgroups of animals and the help of statician, as the most optimal sample sizes is often calculated using in silico simulations, where we take into account the effect size, significance, power and variation in the studies. Another study is the cross-over design. Here the same group does the control and the experimental treatment, divided by a wash out period of time in between. There are however drawbacks of cross-over designs, this is time consuming and is impossible to use if the effects remains after treatment + washout period (irreversible drugs). The tumor might not grow back to its original size. For more treatments: Latin square design: Different treatments are carried out on the same subjects, but on different times. Like this, fewer animals are used and a lower biological variation (further reduced use of animals)

II.5: Explain how the pharmaceutical industry and academia can inform the public on animal experimentation and discuss the benefits of such openness and transparency (EU LO 2.4).

In Denmark, the Animal experiments inspectorate and some of the animal protection societies are presenting aspects on experimental animal use in seminars each year, which are open to public. On some companies' web pages, such as Novo Nordisk, they also have a few videos where the animal housing facilities can be seen, the companies can also make award programs for employees and puplic to partake in covering things like innovative refinement methods. In order to gain trust, it is important to be open with the handling of animals and the facilities. The transparency allows for the puplic to be aware of the many things the industy and legislative facilites are doing to promote good animal welfare. Simultaneously the pupblic may gain access to knowledge about what aspects of the animal experimentation that could be better, which may translate into more political power or innovation/room for solutions.

IV.1 Describe the differences between outbred and inbred animals, and in which ways different strains can differ from one another, and how they can suffer due to their genetic constitution (LO 3.1.7)

Inbreeding refers to mating of related individuals. It results in a decline in survival and reproduction (reproductive fitness), known as inbreeding depression, in most species of plants and animals. Along the way lines will be closed down due to the appearance of recessive lethal or defect genes. Outbreeding refers to matings between individuals from different populations, subspecies, or species. Pyramid breeding is used here. Inbreed strains: Have very similar genetics (almost homozygote) due to inbreeding of brother & sister or father & daughter for at least 20 generations. Animals used for research can be first second or third cousins, as random breeding between offspring is allowed for 3 production stocks. As variance is very low in inbreed strains, different strains has different characteristics (slow learners vs quick learners, high vs low metabolism, proneness for obesity, tumor growth etc.) When breeding inbreed strains survival, reproduction and overall phsyiological functioning may be harmed due to the appearance of recessive lethal or defect genes. Thus certain lines will be cloased as the animals are not prone for breeding/survival. In other strains these properites are favorable, as they make for good disease models. As the mice from certain strains will inherently suffer, a licence is of coursed also needed for breeding and purchasing these strains. Outbreed stocks: have a genepool and animals are genetically different (heterozygote) and thus with higher variance. However some genetic selection are still present in the different stocks, as selection will always occur if random mating is performed, due to some animals growing more, breed better, survive better etc, and breeders have intentionally also used this (etc faster breeding rate preffered). Outbreed stocks are cheaper than inbreed, but as genetic material is different reproducibility is lower for experiment. Outbreed may be advantageous for some studies as variance in response to experiment may not be determined if only of inbreed strain is used.

II.14: Discuss the importance of being able to justify on ethical grounds, the decision to use living animals, including the choice of models, their origins, estimated numbers and life stages. Describe the ethical and welfare factors influencing the choice of an appropriate animal or non-animal model (EU LO 11.5)

It is important not only for puplic perception of animal experimentation, but also for limiting animal experimentation to what is ehtically and morally arguable as just, while raising awareness about key isues with animal experimenentation. As we take basis in the utilitarian principle and the 3 Rs, the model should if possible be in alternatives like in vitro and in silico studies (replacement). If animals are chosen, the model should be chosen on wthat would predict/give the best results and give the least risk of experiencing pain, fear, and distress to the live animals, while allowing for the use of few animals. Origins: Animal species should be considered to obtain a good power of the study with the least number of animals used. There may exist suitable disease models in for instance in inbreed mice strains, or you may need to develope your own model. Either way the choice of the most suitable animal to ensure good results is important, as if the results are not valid the animals sacrifice would have been in vain. Life stages: You have to take into account the life stages of the animals to plan for the duration and developement of the experiment and at what stages in life the experiment is most suiatble (biologically) to happen.

VIII.7 Discuss relevant maintenance of anesthesia, and how to monitor the anesthetic level and vital signs during both short-term and long-term anesthetic procedures. Describe what problems that might arise during anesthesia and discuss what actions to be taken to prevent or counteract these. (LO 20.9, 20.10, 20.11, 21.9, 21.10, 21.11, 21.12, 21.13, 21.14, 21.15)

Maintenance-> Make an anesthethic protocol. re-dose if needed or alter concentration if using inhalation, use eye ointment to prevent drying of eyes. Monitor for: -Absence of reflexes - Righting reflex-> put animal on back and it will want to turn over if conscious - Withdrawal reflex-> pinch animal between toes · Circulatory functions-> measure heart rate/rhythm with ECG. (for high risk/long term procedures) · Respiratory functions -> CO2 and Oxygen levels. Use of endotracheal intubation and ventilation. · Body temperature-> use heating pad or body warm saline to keep animal warm. Problems: - animal responds to stimuli-> more anesthesia or analgesia -Poor respiration or circulation of oxygen-> provide oxygen, provide heat, reduce anesthetic

III.3: Discuss what temperature is optimal for mice, how the preference for a specific temperature can be established and how mice can thermoregulate (EU LO 3.1.1).

Mice and ratsy will regulate their temperature both via internal homeostasis regulation and by for example nest building. In standard laboratory environments mice are housed at 20-24 °C. However, their thermoneutral zone ranges between 26 °C and 34 °C it may not be possible to select a single preferred temperature for all mice as this also cahnges based on activity (active vs inactive), therefore, it is important to provide mice with the opportunity to thermoregulate by nesting. Mice torpor (reduced body temp. and metabolism) when in fasted state for even few hours and thus fasting mice for few hours may result in reduced body temp. via thermo regulation.

VII.3 Name different methods for marking individual animals and state an advantages and disadvantage for each method. (LO 4.8)

Microchipping: risk of infection, more expensive but allows for data recording, and possible to use on more animals. Ear tag: risk of infection, pain irritation, can be torn out but is easy and cheap Tattooing: risk of infection, but less invasive than other methods. Quite humane, since lidocaine can Freeze marking: Relatively stress-free and cause little or no damage, benefits over hot branding: no scar formation. Can't be done on mice with white fur Hot branding: Painful, more infectious, Cheap.

V.1. Discuss potential disease risks in the animal facility, including specific predisposing factors which may be relevant, and describe methods available for maintaining appropriate health status (LO 4.9).

Monitoring - by taking random samples Disease risks: · Infections o Bacterial o Virus o Parasites · Microorganisms in laboratory facilities can be zoonotic meaning they can cause disease in animals and human and interfere with research · They can be o Fulminant : causes clinical symptoms that can be observed o Lantent: do not cause clinical symptoms o The latent disease may influence the experiment directly or indirectly § Direct: immunomodulation of animal, physiological modulation etc. § Indirect: contamination of sample - thereby activation of direct effect Predisposing factors: · Some strains of mice are more sensitive than others; B6 mice · Nude mice are immunodeficient meaning they do not have a thymus and cannot produce T-cells - therefor more predisposing to get hepatitis e.g. and other infectious diseases. Methods for maintaining appropriate health status (barriers, containment levels, sentinels) The three step rocket 1. Rederivation (if you have an infected strain you want to survive) a. Caesarian section ; mate a female and kill her - take out pubs and place pups with germ free mother b. Embryo transfer ; take mother from a strain you want to keep and make her over-ovulate with hormones- produces a lot of eggs mate with male of same strain - take out embryo and freeze them - put into germ free female and mate her with vasectomized male - she will give birth to not her own pubs. 2. Protection - of rederived animals a. Conventional house keeping: no ventilation, the staff has unrestricted entrance, the biological status is not secure b. Barrier protected: i.Chemical lock ii.Autoclave iii.3-room shower iv.Animal are kept free from species-specific agents v.Specific pathogen free (SPF) vi.Filtered ventilation c. Isolator 3. Health monitoring a. Range of test of the animals from a colony - if one animal is infected it is looked at as the entire colony is infected b. Federation of European Laboratory animal Science associations FELASA has issued guidelines for health monitoring of various species - here there are some agents to look for, which methods to use, how many animals to test and how often. i.Macroscopic pathology ii.Genitalia iii.Feces iv.Pelt and skin v.Serum vi.Nose vii.Etc.

I.9 Describe the severity classification system, and give examples of each category. Describe cumulative severity and the effect this may have on the severity classification (LO 2.8, 2.9)

Non-recovery: Procedures which are performed entirely under general anaesthesia from which the animal shall not recover consciousness shall be classified as 'nonrecovery' Mild: Short term of mild pain, suffering or distress. Also procedures with no significant impairment of well being or general condition. An example given in the directive is the administration of anesthesia. Another example is short term restriction of movement. Moderate: - Short term moderate pain, suffering or distress. OR long lasting mild pain, suffering or distress. Also procedures that cause moderate impairment of well being or general condition. An example given in the directive is surgery under general anaesthesia Severe: Severe pain, suffering or distress. OR long lasting moderate pain, suffering or distress. Also procedures that cause severe impairment of well being or general condition. An example given in the directive is severe restriction of movement over a prolonged period. Cumulative severity: When you add up the effect of different procedures or multiple times use of the same procedure on same animal. This can increase the severity of the harm that has been caused to the animals.

VIII.3 Describe the severity classifications included in the Directive and give examples of each category; explain cumulative severity and the effect this may have on the severity classification. (LO 5.5)

Non-recovery: Procedures which are performed entirely under general anaesthesia from which the animal shall not recover consciousness shall be classified as 'nonrecovery' Mild: Short term of mild pain, suffering or distress. Also procedures with no significant impairment of well being or general condition. An example given in the directive is the administration of anesthesia. Another example is short term restriction of movement. Moderate: - Short term moderate pain, suffering or distress. OR long lasting mild pain, suffering or distress. Also procedures that cause moderate impairment of well being or general condition. An example given in the directive is surgery under general anaesthesia Severe: Severe pain, suffering or distress. OR long lasting moderate pain, suffering or distress. Also procedures that cause severe impairment of well being or general condition. An example given in the directive is severe restriction of movement over a prolonged period. Cumulative severity: When you add up the effect of different procedures or multiple times use of the same procedure on same animal. This can increase the severity of the harm that has been caused to the animals.

VI.2 Describe how you would use specfic diets for specific studies, how you would order them, and how you would store them (LO Additional KU)

Purified syntethic diets are normally used for specific study purposes, as they are easier to change in composition than natural diets. Lets say we want an experiment where we test the effect of gluten on the digestion of mice. I would order a standardized syntethic diet suitable for the mice im using, from a professional laboratory diet producersr homepage (ex Sniff) and change the composition to not include gluten. I would of course need to replace this component with something else and have to be aware of what I use instead. I would be aware of batch size when ordering, as it is most optimal to use the same batch for the whole experiment. I would then keep the diet in a cool, dry and rodent safe place, while being aware of the shelf life and note batchnumber. If it is not possible to use the diet within its life span then it should be frozen down in portions. I would autoclave or irradiate to sterilize diet, if facility requires this.

IX.5 Why do we decide on the number of animals we will be using in an experiment before we start; and how can we do that in a sensible way? (LO 10.6)

Remember the three R's: one of them is reduction. This means that we want to have an as low number of animals as possible. However, to obtain reliable results, we need to have a larger variation which means a higher number of animals. Why do we decide on the number of animals we will be using in an experiment before we start • The size of the experiment must be as small as possible (reduction), while providing the adequate statistical results (refinement). • A tendency to reduce the number of animals, has led many studies to have insufficient statistical power, meaning the experiments become unethical in nature, as they harm animal without achieving anything. • They will be unethical because money, time and animal lives are wasted, and any pain or discomfort they might have suffered will have been needless. • Legal requirements implemented presently, force the applicants for a license required to demonstrate that they have calculated an appropriate animal number for the study. Piece of information needed: smallest effect size of interest, estimated variation, level of significance (false positives) and the desired statistical power (false negatives). (the last two says how much we're willing to accept false positives and false negatives - you have to have in mind, how big would the disaster be if there were too many false negatives?

VIII.5 Define sedation, local and general anaesthesia. Explain the triad of anaesthesia and the term balanced anaesthesia, and give examples of how to achieve this. (LO 20.1, 20.2, 20.3, 21.6, 21.7, 21.8)

Sedatives calmes the animal and often carry a muscle relaxing effect. They work through an depressing effect on the CNS, most comonly via activating the GABAnergic system. Local anaesthesia numbs a part of the body (through peripheral nerves) while the animal remain conscious. General anaesthesia makes the animal unconscious (through the CNS) and unable to feel pain. The triade of anesthesia is: -Loss of consciousness -Muscle relaxation -No sense of pain (analgesia). Balanced anesthesia is to apply two or more medications to help patients to achieve a proper anesthetic state (fufill the triade). This can be done through a anesthetic protocol keeping the specific animal and its state in mind.

II.12: Discuss the 3Rs and why it is important to continuously improve the level of Refinement in animal experimentation (EU LO 9.2)

See other questions for deff on the 3 Rs. By continuously improving the level of refinement the animals will suffer less harm fear and disstress, all things that correlate to physical responses like stress. By reducing these we will not only help the animals but also ensure more valid results with higher reproducebility as the variance will be lower. Methods for refinement: -Use of non invasive techniques and improving techniques used for procedures -Optimizing methods for pain recognition -Using appropriate anastatic and analgetic dosing regimens -Ensure good housing and transportation facilities for animals -Ensure environmental and cognitive enrichment -Reduce fear and stress in animals by training them for certain procedures

VIII.1 Give examples of behavioral changes and other signs of discomfort, pain, suffering, or distress in a rat or a mouse, as well as signs of positive well-being and principles of how pain, suffering and distress can be managed. (LO 5.1, 5.2. 5.6)

There is a difference between acute and chronic pain and thereby stress of the animal. Signs of stress is in acute circumstances related to an increase in the HPA-axis and sympaticus and is shown as higher heart rate, blood pressure, hyperglycemia, and lipolysis, it is fight or flight. Also, an altered metabolism and lowered immune response. Fear, stress and so on will increase pain. Pain and stress assessment can be seen in three different ways; · Physiological parameters o As described: heart rate, blood pressure, body temperature e.g., due to sympaticus - these will increase when the animal is in pain or stress · Biomarkers o Not really used unless it is a part of the study, but is e.g., hormones as adrenaline, c-fos and corticosteroids o IgA decrease is also a sign of distress (rarely with mice) · Behavioral and clinical signs · There are visual factors which we can observe (activity, posture, moving patterns, writhing etc) however this is hard to observe, as there is a range of variation of those observations among animals How do we know that the animal is experiencing pain? - Is it alert? - Is it moving normally? - Does it eat or drink more or less? - Is it protecting a certain part of the body? - Does it sound when being handled? - Does it bite or lick itself more than normal? - Is the animal's posture abnormal? - In rats: chromodacryorrea (porphyrin - red colored tears) o Abnormal behavior: stereotypical behavior as grooming too much or too little, a way for the animal to try and cope and calm down, repetitive homologues and no apparent function behavioral pattern o Calming/coping effect · Clinical: weight loss, stop drinking water, urination goes down etc. · Pain-specific: twitching, back-arching, falling etc. · Also facial expression in mice show distress. Positive well-being is basic behavior: Nesting and hiding, social behavior, exploration, grooming etc. It is important to know the normal behavior to see if something is wrong. Pain management in animals: Alleviation of the pain of animals is done by either analgetic (without pain) and anesthesia (without sensation). Analgesics are drugs as NSAID, opioids, local anesthetics. Besides this you need to keep the animal free of discomfort by a suitable environment Technical skillfulness - a good operator will cause less pain.

VIII.10 Discuss strategies for successful perioperative pain management, and difficulties that may arise in connection to this. (LO 21.20, 21.21, 21.22)

Strategies for successful perioperative pain management: Skilled worker causes less pain, work aseptic then no infections, minimize other sources of discomfort, monitor councioness of animal and clinical signs. Have a protocal for surgery and pain management ready. Do literature search in order to see possible effects of the used anesthetics, and possible interference with other factors Problems that could arise: - Animal responds to stimuli-> more anesthesia or analgesia - Poor respiration or circulation of oxygen-> provide oxygen, provide heat, reduce anesthetic (if gasous administrations is used).

III.5: Discuss how different handling methods of mice can affect animal welfare and animal behaviour (Additional KU LO).

Tail handling: induced stress, however when used quick it is ok, Tunnel handling: tunnel handling is ok and easier for beginners to use and you should always put the animal down with the butt first. Cupping handling: For more experienced users, cupping can be used. It makes a dark environment which is calming. The method of handling is all based on how experienced the handler, as it can induce a lot of stress. It is important to do no sudden movements and not too much noise.

I.3 Describe the roles and responsibilities of the local animal welfare bodies and the national committee for the protection of animals used for scientific purposes (LO 1.6)

The Danish animal welfare body and national committee for experimental animals and alternatives. The national committee is an authority of 7 members, they are all experts covering the three R's and give advice to the board (the competent authority in Denmark) and enhance culture of care. Each establishment who uses animal experimentation using animals needs a welfare body, who oversee the animal welfare. This is according to the EU directive. They should include a person in charge, a scientist, and a veterinarian - those are a must. Otherwise, you can construct the body as you want. The Danish animal welfare body consists of 10 members currently. Their main goal is to discuss the effect of how we keep animals, their husbandry and the practices performed. **** LOOK AT THIS

II.15: Describe and explain the challenges when performing a Harm/Benefit analysis of a study using live animals (EU LO 9.5)

The benefit harm analysis has both scientific and an ethical aspect to it and is very complex. For benefit we want to establish the benefit to humans, animals and enviroment, how big of an impact will this have and for how many people. Here we also have to take into account the likelyhood of the experiment resulting in positive results and also the likelyhood of these result being realised to something beneficial. This is not certain at all and we should also take into account the time horizon on such events taken place (this could take many years). As for the harm we want to evaluate if this outweighs the benefit. However how do we determine the overall harm performed in the study? Does one animal life correspond to one human life? If not how many animals correspons to a human? Is one mice equal to a pig in worth?

I.8 Describe how the animal experimentation act is based on an ethical framework which requires 1) weighing the harms and benefits of projects (the harm/benefit assessment)2) applying the Three Rs to minimise the harm, maximise benefits and 3) promote good animal welfare practices (LO 2.5, 9.4).

The animal experimentation act is based on the utilitarian principle. 1) It is based on harm/benefit assessment. We acknowledge the intrinsic value of animals and that they are sentient beings that should be respected on not dealt unesecary or avoidable harm. Thus we try to maximise the benefit from experiments, while minimising the harm done to animals. Before you can be granted a license, we need to evaluate if the experiment is beneficial to the society, and to ensure that the animal does not experience intensive pain, suffering or fear. It is important, that the beneficence to society of the experiments outweighs the harm caused to the animals. 2) The three r's are implemented to limit the animal suffering. You should always try to reduce, replace and refine animal experiments. 3) The act promotes and implements good animal welfare practice to reduce harm through: - appropriate structures in place to promote a good culture of care - Animals have intrinsic value which must be respected - Always anesthesize the animals to limit pain unless anesthesia is considered more stressful than the procedure itself or makes the experiment incompatible - Ensure staff is encouraged to come with ideas

I.2 Describe the authorisation that is needed before acting as user, breeder or supplier of laboratory animals and especially the authorisation required for projects and where applicable individuals. Describe who will grant you a license in Denmark, how this authority is organized, and which other functions this authority has (LO 1.3) **** NEED TO LOOK

The animal experimentation inspectorate issues the licenses for animal experimentation after careful evaluation of the application. You apply through the AIRD system. The animal experimentation board (rådet for dyreforsøg) is the authority who does this. The experiments must be: - Licensed - Beneficial - Animals must be necessary And not be causing: - Strong pain - Intensive fear - Intensive suffering The board consists of 11 members, who are led by a judge. They also inspect/accredit facilities and experiments. In the EU directive article 10, it is stated that you need a license to breed and deliver animals for research. Annex 1 specifies what species are acceptable For gene modified animals, a specific license for breeding and housing from both the Danish working environment authority and the nature agency. The competent authority carries out inspections to make sure that experimentations are carried out in compliance with the animal experimentation act.

I.1 Describe and present which national and EU laws and guidance which regulate the scientific use of animals and in particular the activities of those carrying out scientific procedures involving them, as well as other forms of relevant animal welfare legislation, and discuss how to find the relevant legislation (LO 1.1, 1.2, 1.4, 11.2)

The council of europe issues voluntary conventions, while the EU directives are mandatory for EU member states to follow. The EU directive from 2010 alligned legislation in EU regarding animal experimentation, therefore in general animal experimentation legislation is the same in the European union, with a few exceptions. For Denmark special addon is no strong pain or intensive fear or suffering can be induced in laboratory animals, independently of how strong the benefit is conceived to be. The 210 directive stated member states must have 1: A competent authority. In Denmark this is called the "animal experimentation expectorate". It is led by an 11 member commitee from various background like NGOs, the industry, ethical experts. 2: a comitte for advice on animal experimentation and alternatives. In Denmark this is called "The national committee for Animal Experimentation and Alternatives" and includes experts in the 3 R's and a culture of care. Danish legislation follows the EU directive. We have the "Animal Welfare Act" wich prohibits the harm of animals and the "Animal Experimentation Act" which overrules and allow for experimentation under certain circumstances in alignment with the EU directive. The circumstances are that the animal experimentation must be -Licensed -Beneficial -Use of animals must be necessary -Experimentation must not cause strong Pain, intensive fear, intensive suffering. -Experimentation must be performed by qualified staff, in proper settings and on destination bred animals.

II.8: Present the Five Freedoms and explain how these apply to laboratory species (EU OL 2.7)

The five freedoms focus on farms animlas, guide on how to avoid unnecessary suffering, physiological, emotional and behavioral factors. Freedom from hunger and thirst, discomfort, pain injury and disease, to express normal behavior, from fear and distress. - Freedom from hunger and thirst, by ready access to water and a nutritious diet to maintain health. - Freedom from discomfort, by providing an appropriate environment. - Freedom from pain, injury, and disease, by prevention or rapid treatment of disease. - Freedom to express normal behavior, by providing sufficient space, proper facilities and appropriate company of the animal's own kind. - Freedom from fear and distress, by ensuring conditions and treatment, which avoid mental suffering

I.11 Describe the four functions in relation to educational demands for staff involved in animal experimentation, as well as the role of the manager (EU article 24) and the designated vet (EU article 25) (LO 1.5, 11.1)

The four functions are called A, B, C, and D. Function A is to carry out the experimental parts of the experiment Function B is to set up the experiment, and to hold the licenses. Function C is to take care of the animals in the facilities Function D is euthanisia of the animals (killing) The manager is in charge of the: - Welfare of the animals - Facility design - Training of staff The designated veterinarian gives veterinary advice for the welfare and wellbeing of the animals

III.1: Describe the oestrous cycle/ovarian cycle of rodents and describe how pheromones can affect the ovarian cycle and pregnancy (EU LO 3.1.1). **** NOT COMPLETE, REMAKE IF TIME

The ovarian cycle of rodents is 4.5 days. High ranking female rodents are pregnant at almost all times and will reach post-partum estrus within 12-24 h pp, so there are a few hours where she will not be fertile. Pregnancy in mise and rats are 19-20 days, and the pregnancy can be determined around day 12 Lactation lasts ca 21 days, and can induce delayed implantation of the egg the timespan between individual litters will be much shorter when you have a continuous mating system you basically have a free floating egg in the uterus, that is fertilized, but it will not implant directly ones it has developed. It will implant ones the lactation is declining Fertile after 7 weeks. Will only alow mating during estrous Continues mating /group mating Pheromones: Group housed females have synchronize cycles where estrus is suppressed When males are introduced to females, the pheromones from the males will induce estrus, which happens 3 days after the animals are put together. This is called whitten effect. The females mated on the 3rd night ovulate more eggs and produce larger litters. When female mice meet male mice, the pheromones will induce estrous cycle. If a female mouse can not scent a male mouse, the oestrous cycle will slow down and eventually stop in anestrous. This is called the Lee-Boot effect. However, if a female mouse is put together with a male mouse again, or can scent the urine, the oestrean cycle will start again. (Whitten effect). This can be used to synchronize cycles If a female is mated with a male, and can scent the smell of another male, this can lead to abortion (the Bruce effect). Vanderbergh: young female mice can be sexually mature at a younger age. The fertilized eggs will not implement in the female after birth, they remain free floating in the uterus It will not implant directly once it is developed, only once the lactation is declining, this means that continues mating system is much faster than with an unfamiliar male. (They are always together - higher productions of babies) However - foreign male smell induction of fetal resorption in fertilised females

IX.10 Explain key issues to be reported when publishing experimental animal studies. (LO 11.9)

The power of the studies and the methodology used, so people can reproduce with same biological and technical variation. Also usefulness/implications of the studies. There is an increasing concern of how useful animal model studies are, as we are becoming experts in treating mice and rats for various diseases, but most of it does not work in humans. Besides the criteria for external validation of animal models, it is important to consider internal validity of an experiment, in other words, the extent to which a study establishes a trustworthy cause-and-effect relationship between an intervention and the outcome, and thereby eliminate alternative explanations for a new result. Internal validity is dependent on procedures such as the experimental design, randomization, including proper controls, and the ability to reproduce the results. This applies to all scientific research but is highly relevant for research where animal models are used. The reproducibility crisis highlights the failure of replicating up to half of all studies, especially in the field of social and medical sciences. Cherry-picking data, publication bias when only positive results are published, insufficient reporting of experimental settings to enable replication are parts of the problem.

II.13: Explain the purpose of a Harm/benefit analysis in animal experimentation and give examples on parameters included in a Harm/benefit analysis (EU LO 9.5)

The purpose is to evaluate the project if it considers both the impact on the individual animal and the benefits of the study. In other words: what problem do the researchers expect to solve; what are the objective and hypothesis of the study and how much suffering, pain and distress will the animal be subjected to. Harm could be - Animal rights - Five freedoms - Physical harm It may be considered a harm to the animals if the quality of the research is low. Animal caretakers can also be harmed. Cost and pain is also some parameters. The Benefits mainly relate to the expected outcome of the studies in the form of improvements in the field of human health incl. safety testing and veterinary medicine.

II.6: Present the 3 Rs, the purpose of the 3R and give an example on how the principles of the 3 Rs are not always compatible (EU LO 2.6)

The three R's are: Replacement: When animal use can be avoided, it should Reduction: Reduce the animals to the lowest possible number Refinement: Use methods which minimize the suffering the most The purpose is to minimize the animal use in experiments and their suffering Reduction is not always compatible with refinement: Two scenarios are possible: Either the researcher use 12 dogs (6 treatment and 6 control) who all are subjected to a fracture of the left fibula, a bone in the hind leg of the dog. Or the researcher uses 6 dogs (3 treatment and 3 control) who are now all subjected to a fracture of both left and right fibula, bringing the total number of fractures up to 12 as in the first scenario. Clearly, dogs subjected to two fractures will risk more harm than dogs subjected to only one fracture; so reducing the number of dogs in this hypothetical scenario will compromise Refinement. Or the tumor example.

II.3: Describe how 3Rs and a Culture of Care combined can promote animal welfare and support staff job satisfaction EU LO 2.2)

The three R's: replacement, reduction, and refinement. This can promote welfare by restraining the emotional stress on animals, the caretakers, who are mostly very fond of animals, won't have to see that the animals are suffering A culture of care for both the animals and the animal staff will decrease the stress on both parties. Key factors for a good culture of care: - Senior staff should lead by good example - Hire staff with focus on CoC - Award programs and high expectations - Encourage communication - Sharing of good practices Key factors of A Culture of Care - Management is important o Know of animal care and use o Signal commitment and supply the funds o Expect high standards o Promote a shared responsibility o Support a positive attitude o Empower the care staff and veterinarians o Encourage communication o Allow for voices of concern

IV.4 Describe principles of genetic monitoring of laboratory animals (LO Additional KU)

To confirm the genetics of a mouse a sample from typically tail is taken (could also be fur or blood). Then DNA is purified PCR is used to amplify DNA, followed by electrophoresis (protein sorting by size in gel) of DNA and sample with reference gene is made, to verify the gene (often transgenes) is present. Southern blot can also be used as electrophoresis technique (normal sorting by size and then visualization via fluorescent compound binding to target gene/DNA). Normally the vendor who cells or produce the animals also supply a protocol for how to identify the target gene in the strain. Inbreed mice may vary depending on vendor (so same name may not mean 100% identical genetics, although very close), SNP (single nucleotide poly morphism) can be used to characterize the difference in the strains, however this method is expensive. Another method: Short tandem repeats or STRs are short repeats of a double nucleotide. They are often found in non-coding regions, and they can be used to show inter-individual variation between inbred mice of the same strain. It is also a costly technique.

IX.2 The size of our experiment is dictated by four elements: Variation, effect size, level of significance, and statistical power. Choose two of these elements, explain what they are, and how they influence how many animals we need to use. (LO 10.2 & LO 10.5)

Variation can be either biological, that is variation in animal population due to genetics, age, sex and things such as liver enzyme activity and personality. Strain variation is even found within inbreed strains, as animals that are from same strain still has variation depending on which vendor is used. Variation may also technical and arise from experimental measurement methods. By using animals with more biological variation we may have higher chance to find translatable results and paint a bigger picture (as different variations may react different in experiment). However use of more biological variation require more animals to be used for obtaining significant statistical results. Level of significance: If significance is lower than p-value we reject the null hypothesis and our results are not significant. If significance is higher than p-value we will accept the null hypothesis. The P-value is thus essential. It reports the willing risk that observed experimental results are false, that is observed positives are in reality false (thus false positives =type 1 error). Normally a p-value of 5% is used (in 5% of cases we conclude wrong).

III.2: Describe the rodents' senses of hearing, vision and olfaction and discuss the importance of being aware of how these senses differ between rodents and humans (EU LO 3.1.1).

Vision: Rodent have poor vision and thus things seem blurry. Rodents are dichromatic (meaning they have to fotoreceptors) as they have ultraviolet and green receptors, compared to humans which are trichromatic (red, blue, green). (Rodents have fluorescent urine so they can see this). As rats cant see red light their houses and hiding spots can be built with structures only transparent to red light-> humans can see in, but rodents cant see out (making the hiding spots preferable). However the house me still affect circadian rhythm in rodents as they are subjected to the red light. Olfaction: Rodent sense of smell (in mice/rats) are highly developed. Pheromones is used to social communication and is used to identify sex, age and status (for instance fear pheromones from other mice), which can be used to approach how to interact with each other. Male mice use pheromones to establish hierachi, which allows for less aggression. Thus when cleaning cages only nesting material must be transferred (no urine or feces) as to not disturb the senses and promote violence. Four things are important for olfaction and reproduction in mice: · Housing of only female mice without males will result in oestral cycle will slow down and stop. (lee-boot effect) · If female mice that has stopped oestral cycle (due to no male mice) are exposed to male pheromones from example urine thay will begin cycle at same time synchronized. If the female mice are put with males 3 days after exposure of male pheromone approximately 75% of these will become pregnant by the following night. (whitten effect) · If female mice are exposed to unfamiliar male pheromones (from example different mice strain) they will risk insufficient implantation or abortion if pregnant. (Bruce effect)-> thus use new gloves when changing in handling of different mice strains. · Young female mice becomes sexually mature quicker if a male mouse pheromones is nearby (Vandenberg effect) Hearing: Hearing is highly developed in mice and rats and also within the ultrasound frequencies. Thus humans may not be able to hear external sounds that stress mice/rats and their communication. Stress and fear calls from mice can be heard by humans, but other communication cant be heard like for example laughter when tickled. Mice and rats use ultrasonic vocalization to communicate but rats also use it to navigate in dark, as they use echolocation like bats!

VII.2 Describe how the animal facility is organized to maintain an appropriate health status for the animals and the scientific procedures. Do also describe the biological consequences of acclimatisation, habituation and training. (LO 4.4, 4.5)

You can decorate the facility in different ways in order to maintain appropriate health status, for example dual corridors. Air pressure: Hyper: Keep the pathogens outside Hypo: E.g., a pathogen is used in the trial; microbiomes may not be let outside. If the animals are social, you have to give them the opportunity to socialize. Acclimatization: If you transfer an animal to a new facility, you must give them time to adapt (1-2 weeks for rats/mice) and get used to the new surroundings, as to mimimise stress. Animals in groups need to find their place in the hierarchy. Habituation: you should take the animal out of the cage at least once per day and pet/play with it, in order to give the animal a habit and to get used to animals. Training: You can train animals to cooperate in some procedures, this will results in less stress when procedures are performed and mor valid results.