Laboratory Animal Science

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 You can: - Give anesthetic overdose - Cervical dislocation - Decapitation - Gas - Shooting - Etc 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

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).

1) It is based on harm/benefit assessment, as before you can have a license, you 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. Therefore, it is important that the experiment could not have been performed using in vitro studies instead which leads to the next point. 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 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

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

Inbreeding E.g. BALB/c mouse and C57Bl/6 Different lines are brother sister mated in generation and some are closed down due to genetic lethal or recessively harmful genes. Due to many generations of sister x brother mating for at least 20 generations. They have the following properties: · Isogeneity: genetically identical - only one mouse should be genotyped to know the genotype for the whole strain. · Homozygosity: after mating in 20 generations - the change of two alleles are identical in more than 98%, no recessive genes we don't know anything about · Phenotype uniformity : fewer inbred animals will be needed to achieve a given level of statistic precision than outbred animals · Long-term stability: often no new mutations, the strain is relatively stable · Individuality: each strain have a unique combination of alleles and can be used for different things; strain AKR gets leukemia, SJL gets reticulum cell sarcoma, C3H get mammary tumors and C57BL/6 have low levels of cancer. · International distribution: a daughter strain can be set up other places relatively easy with the use of only one breeding pair. · Sensitivity: more sensitive to environmental influences than - extra care is needed to ensure that they have the optimum environment. Inbreeding pyramid : When starting an inbreeding strain 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. Hybrids Generate high heterozygosity you can mate a inbred mother from one strain with and inbred farther from another and get F1 hybrids Outbreeding Mating by random mating - a certain gene pole in the colony. 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 will be breeding in rotation and males are exchanged and from all groups the offspring can be used for studies. Estrous cycle relevant for breeding The female will pnly be able to be pregninated during estrous which is only 12 h and therefor you have to let the male be with the female for the whole estrous cycly of 72 h. You can do continuous mating, where they are together all the time or cylce mating where you remove male when the female is pregnant and have to give birth and be with the pups. Vaginal smear: · Very few cells in diestrus 52 h · In proestrus there are more cells · Estrous 10-15 h all the epithelial cells are curitinized · Post estrous there are also granual cells Time mating make a smear and know exactly where the mouse is in its cycle for mating. Animals suffer due to their genetic constitution Due to genetic malfunctions of inbred strains that can lead to death. These will be excluded when breeding the strain. Strains such as C57BL/6 mice and Lewis rats do not have genetically coded diseases. strain AKR and F344 gets leukemia, SJL gets reticulum cell sarcoma, C3H get mammary tumors and C57BL/6 have low levels of cancer. This is important to know and monitor when working with an inbred strain Besides this C57BL/6 mice is extremely aggressive NOD (non-obese diabetic) mouse will develop type 1 diabetes (after 12-30 weeks) - if they are kept alive beyond the outbreak of the disease it should be licensed, since it is stated that animals that are sick should be treated that way and euthanized. Immunosuppressant mice such as the nude mouse or SCIT mouse should be protected against microbes and other outside diseases.

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

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.

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

Accepted methods to carry out activities or operations in non-clinical laboratories (labs that carry out experimental research, not on humans). GLP refers to good quality system of management controls for laboratories where research is taking place. This is by law, and under quality assurance. Aims to ensure, consistency, quality, accountability and so on.

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.

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. 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.

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 humans. Good results cannot justify evil means. Contractarianism: approvers, no obligations, no rights, Man have no duties towards animals. 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, which means that the suffering from animals it justified, as long as it is beneficial to the society, it could also be to for the good of the animals.

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, it can affect their immunesystem, cardiovascular system, and reproductive system etc. Translatability denotes the transfer of basic in-vitro and in vivo research into human applications. In other words - If a study have a high translatability, the results and conclusions drawn from the experiment using an animal model can be transferred and applied to humans. Reproducibility (sometimes called replicability) denotes that the results can be reproduced or replicated - that is if the experiment is repeated, you will get the same results. Whether Animal Welfare is important for moral reasons, depends on the fundamental ethical view and the predominant ethical position in any given society. A Utilitarian, an Animal Rights view-person and a contractarian will have different views on the importance of animal welfare. The Utilitarian will claim that Animal welfare matters since animal are sentient being capable of suffering and it is our moral duty to limit or eliminate suffering. The animal Rights person will claim that Animals have intrinsic value; they must never be used as tools and their welfare is just as important as yours and mine. The Contractarian may claim that we do not have any direct moral obligations towards Animals and Animal Welfare matters only if a moral agent such as him/herself cares for animals and are willing to include animals in an agreement. Hence the moral obligations towards animals and the following importance of Animal welfare will depend on the predominant ethical view in a given society. In the EU, the predominant ethical view on which laboratory animal legislation is based, is the utilitarian view. It is also clearly stated in the EU directive that animals have intrinsic value - the essential, inner nature of the animal matters in itself and the animals should always be treated as sentient beings. To ensure valid data, it is important that animal welfare is not compromised to the extent where the animal is experiencing fear, pain or prolonged stress.

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)

Animals should be acclimatized to the facilities in order so settle to the new environment. This will lead to a faster recovery and less variation of the experimental results; this leads to better research results. It is a good thing to fast before anasthesia, this can prevent the risk of suffocating, and the body function will not be busy with digestion. However, this is stressful for the animals. Fasting is different between animal species, rodents and rabbits do not fast; they cannot vomit. Pre-anesthetic medication can be given to reduce fear and stress. However, in additional, careful handling is also helpful to reduce stress. The most difficult consideration is the choice of anesthesia. The anesthesia is chosen, which is best for the experimental outcome, this might cause loss of animals (however this is not desired). There are some advantages by using mixed drugs, is that a lower dose of each drug which gives less side effects. However, this can interfere with the protocol. There is also the choice between inhalation and injection. Monitoring of the anesthesia is also an important factor.

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

By being bias means that there might be something that influences our experiment in a non-random way. This has nothing to do with honesty, but rather hopes of that the experiment will have good results and fears that it goes wrong. Therefore, the researchers are blinded. Not only the researcher can be biased, differences among the animals can also be biased. For example, when doing research on pigs weight, and all the fat ones end up, randomized though, in one group and the smaller ones in another, this can lead to biased results. To prevent this, you can for example randomly sort out the bigger ones first and then the smaller ones. Or take two pigs who has the closest weight in pair and assign them randomly. Latin square design (more than 2) or full cross over (2).

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. The three R's are also included in the course, as there are 2 students per rat/mouse, and the suffering is kept to a minimum as they are anesthetized. We are allowed to practice on a toy rat as well, which replaced real life animals.

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)

Case-control study: fx tumors treated with drug and tumors not treated. We contrast experimental cases with controls. A more complex study is the cross-over design, here the animals are their own controls. Here the same group does the control and the experimental treatment, divided by a wash out period of time in between. 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) 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. Multifactor design, latin square.

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.

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

Ear tag: risk of infection, pain irritation, can be torn out Easy and cheap Ear punch: risk of infection, pain The sample can be used for genotyping, cheap Toe clipping: risk of infection, Limited amounts of variation Fast and easy Tattooing: risk of infection More human, since lidocaine can be used which causes less pain, permanent, minimal invasive 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 Microchipping: risk of infection, more expensive Data recording, and possible to use on more animals

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

External validity of animal models refers to how well a model translates to the species its modelling, which typically are humans. This is divided into 3 criteria: Fidelity and discrimination: high general similarity and high specific similarity Induced model: we gave the rat a tumor Spontaneous model: the rat developed cancer - Construct validity o Says something about to which extent both the animal model and the human phenomenon can be explainer, for example the origin, function, and mechanism. The cause of the disease is the same - Face validity o Similarity in appearance, for example the FIV in cast have a high face validity to HIV in humans - Predictive validity o Does the drug have the same effect in the human and the model? - - Induced animal model, for example the removal of the pancreas (model for TOD) • Low construct validity (the same underlying phenomenon): humans don't develop type 1 diabetes because their pancreas was removed • Low face validity (similarity in appearance): the rats of course also will develop symptoms due to the lack of more than just the beta cells. • High-low predictive value (similar performance in tests): o you want to test whether insulin treatment will alleviate diabetes, it will have similar effect in rats and humans à a high predictive validity o if you want to test interventions such as diet that targets the reason for developing type 1 diabetes à the predictive validity is of course low Spontaneous model: try with cancer

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)

For ethical reasons, it is important to balance the harm and benefits, but the benefits must always outweigh the harm. It is done to improve both human and animal health. The means justifies the deed. You should always have the three R's in mind: The choice of model should always be chosen based on a model that would predict/give the best results and give the least risk of experiencing pain, fear, and distress to the live animals. Origins: Animal species should be considered to obtain a good power of the study with the least number of animals used. Power analysis is when you do not the variance of the variables you are going to measure. Best models the phenomenon you are researching Life stages: Banana flies are considered inconsistent animals and you do not need a license. For ethical reasons, it is important to balance the harm benefit, but the benefits should always outweigh the harm. In general a we can justify our decisions, based on approvers with reservation views, because we do it as a means to an end, to improve animal and human health. The choice of model should always be chosen based on a model that would predict/give the best results and give the least risk of experiencing pain, fear, and distress to the live animals. Origins: Animal species should be considered to obtain a good power of the study with the least number of animals used. In general, as long as we use the 3 Rs, and do our best to optimize our experiments, we can justify our actions.

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: scalpel handle, forceps, scissors, needle holders, suture materials (such as syringes), stainless steel bowls, instrument tray. In addition: cotton wool, gauze, and drapes. Needles: aseptic, can only be poked once, and comes in different lengths and gauges.

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

ILAR codes for the big breeders · Tac: Taconic · Crl: Charles river · Rj: Janivier · Hsd: evigo Inbred = strain, outbred = stock · Strain names: o First strain name / breeder code § E.g. Cr57BL/6NTac · Stock name: o Breeder code : stock name § E.g. Crl:SD · F1 hybrids: o MotherFartherF1/Breeder code § E.g. a C57BL/6 (also called B6) mother with a DBA/2 (also called D2) farther : § Gives B6D2F1/Crl if they where from charles river · Transgenic animals: need to see the alterations in the name o First part is the background strain and then comes "-" and the gene alteration (gene is in kursiv) o Tg if it is a human transgene that has been put into o N if it is non-homologous way and H for homologous if that is the case (often left out since most are N) o Write a no. of the founding line o Then at last the founders lab coade e.g Rub o Then /and breeder e.g. /J (for jackson laboratories) § E.g C57BL/6-TgN(APOA1)1 Rub/J · Knockout mice o Background strain then a "." o Name of donor of stem cell o Then "-" and the name of the gene the animal normally would carry (e.g. ApoA1) o To show that the gene is no longer working the rest is written in this kind of writing tm (targeted mutation then number of founding line and lab code) o Then slash and the lab that breeded the mice § E.g. B6.129P2-ApoA1tm1Unc/J

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. In order to gain trust, it is important to be open with the handling of animals and the facilities.

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

It is possible to train animals to cooperate with certain procedures to reduce fear and stress. One way of improving the level of refinement, is to train the animals to some procedures this will reduce stress and fear -> better results. It can also be a good idea to train the staff to handle the animals the best possible way.

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)

It is the establishments welfare bodies own manager that is responsible for the specific experimentation, however, the competent authority carries out inspections to make sure that experiments are carried out in compliance with the animal experimentation act. They are also responsible for implementation of Culture of Care in the facility. Whenever you do an experiment, you should be familiar with the local organization. On KU it is the department of experimental medicine.

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)

Make a anesthetic protocol with timepoints and dose needed for maintenance of anesthesia. If done by injection. Using inhaled the anesthetic is given continuous. Monitoring can be done visual by checking reflexes (fx withdrawal reflex), but also to monitor the pulse rate, respiration, body temperature and so on. These are done on short term, low-risk procedures. The oxygen saturation should not be below 90%. At long term and high-risk procedures, more sophisticated monitoring is needed. This includes circulatory monitoring (heart rate by ECG, blood pressure and so on) and respiratory monitoring. Problems that can arise; cyanosis (less than 50% oxygen saturation) - always have oxygen nearby. Where breathing problems can occur, endotracheal intubation is used, for larger animals this is standard, is not that common in small rodents but can be used. Should always be used for procedures of longer duration. If the animal is moving, more anesthesia is used.

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 rats are homeotherm, which means that they are not affected by surroundings. Mice and rats are homeotherm and able to maintain a stable internal body temperature regardless of external influence. Mice especially 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 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 Temp preference: they do not have a specific temp they prefer, so you have to give them materials for nesting. Tt may not be possible to select a single preferred temperature for all mice. Therefore, it is important to provide mice with the opportunity to thermoregulate by nesting. However, fun fact: fasting is dangerous, as it can induce torpor (reduced body temp and metabolism)

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.

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

Monitoring -> to see if e.g. B6 is actually B6. A tip of the tail is cut of and used in PCR (could also be blood etc.) . Southern blot, SNP, and STRs can also be used. PCR: used to identify single genes Previously southern blot has been used to find transgenes (gel electrophoresis) - Can also be used for characterization of strains and stocks Single nucleotide polymorphism (SNP) chip used to characterize strains and stocks test on all chromosomes but is quite costly Short tandem repeats (STR) chip is multiple repeats of a double nucleotide - often in non-coding regions and can be used to show inter-individual variations between inbred strains - also quite costly When we do genetic monitoring, we want to show that for instance mice from a colony of B6 mice they are really B6 mice. You cut of tip of a tail, but it can also be other tissues such as fur, a piece of the ear or some blood. And then we purify the DNA. Polymerase chain reaction or PCR is typically used for identifying single genes. It is an easy and cheap method. Another way is: southern blot Cheap and easy way to identify a single gene. Here we move the restriction enzymes so that the genes are different length, and we will be able to see a difference in a gel was also used to identify single genes, also typically transgenes or knockouts. It is also a cheap and easy method, and it is also possible to find protocols. With southern blot, we move the restriction enzymes, so that the genes are different lengths, so we can see the difference in a gel. Southern blot can also be used for the identification of strains or stocks. It is less common today. Another method is: Single Nucleotide Polymorphisms or SNPs ("SNIPS") are used to characterize strains or stocks. They test thousands of polymorphisms on all the chromosomes. They are stable over generations and therefor they can be used to characterize an inbred mouse. It is a quite costly method. 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.

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: An example given in the directive is the administration of anesthesia Moderate: An example given in the directive is surgery under general anaesthesia Severe: 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 use of the same procedure

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: An example given in the directive is the administration of anesthesia. Another example is short term restriction of movement. Moderate: An example given in the directive is surgery under general anaesthesia Severe: 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 use of the same procedure. This can increase the severity of the harm that has been caused to the animals.

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

Procedures to ensure welfare during transport: -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).

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.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, give more analgesia if the animal still is in pain. Do literature search in order to see possible effects of the used anesthetics, and possible interference with other factors

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.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. EU Directive issues mandatory directives. EU has directives which has to be followed by all the member states. EU made a directive for all the member states in 2010, which all the countries must follow. There can also be stricter rules in each individual member state, which is still valid in the countries. - Directive says that each member state needs a competent authority o Rådet for dyreforsög - Each member state must have a national committee to give advice on experimentation and alternatives for the implementation of the three R's and to ensure a culture of care of the animals Implementation in Denmark The animal experimentation act overrules the welfare one if a license is given to a specific case. These two acts have been discussed in folketinget. The animal experimentation act contains some general principles about animal experimentation in accordance with the EU Directive. They must be: - Licensed - Beneficial to the society - Animals must be necessary o Preformed by qualified staff in proper settings on destination bred animals - - Not be causing o Pain o Intensive fear o Intensive suffering The experimentation act is way too detailed for folketinget, therefore one minister has been given the assignment to make rules on the appendices, fx how to get the animals, care and housing for them, staff qualifications and so on. Additional to the act, we have the order, which is a larger much more detailed document. This has some of the same annexes as the directive/experimentation act. Fx the care and housing, legal methods for killing. According to the directive, each member state should have a competent authority to carry out the obligations of the EU directive, which includes giving licenses, inspecting facilities and accretes facilities. In Denmark, this competent authority is called the animal experimentation inspectorate (dyreforsøgstilsynet), which go under the Ministry of food and environment. They are lead by a 11 member committee, The animal experimentation board (rådet for dyreforsøg). They issue all the licenses. Rådet for dyreforsøg consists of: - 4 animal protection societies - 1 animal ethics council - 1 Medical research council - 1 Technology production research council - 1 Patient NGO - 1 Industry - 1 Health agency This committee is lead by a judge This committee has no rules to whether it conists of experts or laypersons. According to the EU directive, each nations must have a committee for animal experimentation and alternatives. The committee consists by 7 experts, who gives advice on animal care and the three R-s. This committee is also an authority in itself under other smaller animal welfare bodies.

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)

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.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)

The animals, whose welfare would be compromised should be killed. An animal shall be killed when it is likely to remain in moderate or severe pain, suffering, distress or lasting harm. If it is likely to recover, it should receive veterinary treatment. If an animal used for a specific procedures is killed, it must be told in the evaluation after the experiment.

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 diet to maintain health and vigour. - Freedom from discomfort, by providing an appropriate environment - Freedom from pain, injury, and disease, by providing an appropriate environment - 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

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).

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

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.

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.

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)

There are three definitions. - Health and biological functioning - Natural living - Emotions and preferences How to measure and evaluate the welfare is dependent on how we define welfare. Behavioral measurements go through them. Health and biological functioning: The ability of the animal to have normal biological functions e.g. stress hormones, mortality rate fertility, normal growth etc. measured: · Clinical examinations: o measuring physicological parameters (HR, temp, respiration rate) o Appearance of animal (Fur, skin etc) · Clinical chemistry o Blood, urine, fecal samples o Metabolic, immunobolic, hormonal parameters o Animal products (milk, meat, eggs) Natural living: Species-specific purpose in life - not being but doing well. Express behaviors that define the species. Rodents by nesting and grooming - more of these expressions the higher the welfare; Ethogram (list of species specific behaviours) Emotions and preferences: The presence of positive mental state and absence of negative mental state. Depends on the acceptance that animals feel emotions. Also have your own preferences fulfilled Measure: Joy: o Ultrasonic vocalization (mimics laughter) o Judgement / emotional bias: see how animal perceive unknown stimuli Pain, fear: o Pain reactions o Fair in plus maze Preferences: o Choice test between resources Scientific impact: an animal with more natural behavior resembles true biology of said animal -> more valid results. The metabolic state is influenced by fear and pain, so pain free resembles the true metabolic state. More accurate results.

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)

They are A, B, C, and D. Function A: to carry out the experimental parts of the experiment Function B: To set up the experiment, and to hold the licenses. Function C: To take care of the animals in the facilities Function D: Killing the animals Article 24 states that there must be one or several persons who are in charge of the: - Welfare of the animals - Facility design - Training of staff Article 25 states that there must be a designated veterinarian who gives veterinary advice.

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.

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)

Triad of anesthesia: loss of consciousness, muscle relaxation and no sense of pain (analgesia). Sedation is a method to calm down the animal, general anesthesia is the loss of consciousness, muscle relaxation and no sense of pain. General anesthesia is no sense of pain. Balanced anesthesia is to apply two or more medications to help patients to achieve an anesthetic state. The can be done through a anesthetic protocol where the animal is evaluated.

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

Underpowered studies and usefulness 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.

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: It has been shown that different mice from different breeders gave different results. In order to control variation, it would be an idea to use the same number of mice from different breeders in order to obtain results. This has the influence that more animals need to be used, as some might respond good to a new drug candidate, while some will not. Less variation - the more uniform tumors. Therefore, a smaller need for animals, however, if we remove too much variation, we cannot say anything about the real world. Technical (instruments, tools, planning) and biological (genetics, gut-flora and so on) variability. Level of significance: It is a limit we set before the study starts, which determines how willing we are to accept false positives. (A false positive is a result can be due to variation, we incorrectly think that the drug works), the most common is 5%. If our p-value is lower than the level of significance, we can say that our result is significant - we can say that the drug works, but there is a chance that it doesn't. Effect size: the quantitative measure of the experimental effect. The smallest effect of when the drug is seen to be useful. Let animals grow larger tumors, or use of more of the drug would be easier to measure. For example, when measuring tumors, if they are bigger, they are easier to measure and see, and to let them grow bigger, a reduced number of animals would have to be used. More of the drug could also lead to more side effects. However, remember, that this might cause more pain in the individual mouse.

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

We cannot guarantee that there will be a good outcome, harm is guaranteed, but the benefits are not. It all depends on what view you have, where disapprovers will say that the harm is above everything, while approvers with reservations would say that the outcome justifies the means. There is also hard to justify, how many animals does it take to safe one life? And how big will the population be that will be saved? Since there are no clear answers, it is very hard to measure.

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)

What it is: The earliest point, at which an experiment can or should be terminated, with the purpose of eliminating unnecessary suffering. Criteria to be used to set humane endpoints: You need to identify early behavioral and clinical signs in the animals, in combination with model specific signs - signs that are relevant in the specific animal and model you are using. This is done to set as early endpoints as possible. The endpoints might differ in the different severity classifications. A welfare protocol is a good tool to identify endpoints, those contain relevant parameters. Clinical signs can be divided in quantitative data and quality data. Examples of quantitative date is body weight, temperature, respiration and so on. Qualitative is fx the quality of respiration, posture, appearance such as closed eyes, ruffled fur or feathers and so on. Action to be taken when a humane endpoint is reached: The animal should either be euthanized or a veterinary should be contacted 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. There is used 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: Welfare protocol: a score sheet which is : appearance, behavior (provoked, natural). Clinical signs can be divided in quantitative (kan blive målt) data and quality (kan ikke blive malt, have en reference) data. Examples of quantitative data is body weight, temperature, respiration and so on. Qualitative is fx the quality of respiration, posture, appearance such as closed eyes, ruffled fur or feathers and so on.

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 ventilations! Needles should be disposed in designated waste containers. Gas cylinders have a risk of explosion in case of fire. Drugs should be locked in cupboards, records of purchase and usage. Non-used drugs are destroyed according to the legislation.

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 a couple of days to adapt and get used to the new surroundings. 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. You can train animals to cooperate in some procedures.

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

You can only reuse animals if the pain has been 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, therefore it is always important to stay updated, as explained earlier, animals who are not feeling well in thein facilities will not give good results.

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)

• Mouse (Mus musculus) • Rat (Rattus norvegicus) • Guinea pig (Cavia porcellus) • Syrian (golden) hamster (Mesocricetus auratus) • Chinese hamster (Cricetulus griseus) • Mongolian gerbil (Meriones unguiculatus) • Rabbit (Oryctolagus cuniculus) • Dog (Canis familiaris) • Cat (Felis catus) • All species of non-human primates • Frog (Xenopus (laevis, tropicalis), Rana (temporaria, pipiens)) • Zebra fish (Danio rerio) Farm animals do not have to be licensed to breed Destinational bred animals is breeding when you need a license from the competent authority to breed and deliver animals, fx experimentation. Non-destinational bred animals can be allowed by the animal experimentation board as well as wild animals. This has to be properly supported by scientific arguments. You are not allowed to use animals from the wild according to the EU directive otherwise. 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.

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, as this is complicated calculations. • For multifactor design a statistician, may be necessary, as the design and statistical analysis are very complicated. For some of the more complicated designs, there are no easily accessible tools that allows us to estimate an appropriate sample size. Computer programs that can be used by statisticians to calculate our sample size, are based on a simulation-based approach • The elements that you need to be able to present to a statistician is variation, effect size, statistical significance, and statistical power.

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)

Hygiene is highly essential, as poor hygiene can cause a lot of negative consequences. Fx healing worsen and slower, animals will be in pain, infection, this will lead to distress. Infections can also lead to higher variability -> this can mean that we need more animals as some will have to be euthanized. Aseptic techniques means that methods and practices that prevent cross contamination during surgery. This involves not just the surgeon, but also the facilities and the environment, surgical site and the equipment. You want to limit the animals exposure to microorganisms. There are different sources from microorganism: exogenic - from air, surgeon, instruments. Endogenic from the animal. Handling of tissues: Harlsteds principles - Asepsis - Gentle handling - Hemostasis - Closure of dead space - Careful approximation of tissues - Avoidance of tension - Minimisation of foreign materials This will minimize the trauma inflicted on the animal. Suturing inside of the body needs to be able to absorb.

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

Allergy, Physical injury, Harmful substances, Zoonoses Allergy: Skin contact but also inhalation and contact with urine etc. intermitted exposure. Keep levels of allergens in the environment as low as possible (air flow), using protective equipment and do not carry the allergens with you when leaving the facility. Protective equipment of the staff, education Physical injury: -Bites may be infectious and wounds should be washed with soapy water -Injury by handling of needles -kicking etc. of bigger animals Can be avoided by: -proper handling of animals -practical scalpel and needle handling Harmful substances: -Biologics, radioactive isotopes, adjuvants, chemicals - Isoflurane - watch out for it if you are pregnant - Streptozocin - induces diabetes Can be avoided by: -Handling substances with care - Proper protective equipment (PPE) - Know the hazardous properties of all compounds - Special education - Special facility - High pressure bottles Zoonoses -Infections from animals to humans Can be avoided by: -working with pathogen free animals ->microbiology defined -working in barrier units and at last resort isolators

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).

Fasting can induce torpor: this means that the body temperature is reduced and metabolism is slowed down. Rough handling can also stress mice. Ultrasound is a sound which is very stressful for rodents, as they can hear it. We humans can not A too low temperature, 19-21 degrees, can challenge the homeostasis of a mouse and can be a stressor. Mice and rats should be group housed, however, this will induce some stress for some individuals, depending on where they are in the hierarchy -Fasting Torpor: can give a reduced body temp -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. -Ultrasound/fluorescent lighting. Humans are not able to hear ultrasound and therefore we are not able to hear neither a large part of rodent communication nor many of the sounds that may stress rats and mice. -Rough handling -Group housing can cause a higher level of stress hormones When rats are stressed, they can release an alarm odour: rat welfare and experimental aims might be compromised if neighbouring conspecifics are distressed by illness, injury, or experimental procedures bias rats' decisions in choice tests increase 'baseline' stress in subsequently tested rats or supposed control ones alter behaviour in tests such as swim tests and open field or novelty tests the physiological responses may increase to levels where the effects potentially interfere with experimental results, e.g. leading to suppressed immune function

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. There is a low variation within the strain, however, between different strains, there could be a lot of variation. Some are for example good learners while some are not. Outbred animals: Unintended selection will always occur if random mating is performed, as some animals grow more, breed better, survive better etc, and of course during history some commercial breeders have intendedly selected those animals, which were best for the economy, that means those who grow fast, because when selling animals after weight fast growth means fast money. Rotational breeding is used here.

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).

Mice: 1000 hz- 100 khz Rats: 200-800 khz Humans: 20 hz - 20 khz They have a way better hearing than us, and can hear in ultrasound, it can therefore be hard for us humans to know which sounds are stressful to rodents. Florescence light is also stressing the rats, as they can hear them. Vision: rodents: bad vision, dichromatic (we only see three), cannot really see. They do not see red light, therefore there is a phenonomen with red cages, where only red light will come in. This will appear dark and safe. However, it will also alter the physiology. They cannot see very well, and have sensitive eyes, and comfortable light for us animals can cause eye diseases in rodents. Smell/olfactions: Mice and rats are nocturnal, and burrowing by nature. Their sense of smell is highly developed. Olfaction is important for communicating for example in mice in relation to reproduction and when to uphold a hierachy. These various effects can be explained by the effect of pheromones. Pheromones are chemical substances released by an animal to signal something to another animal. Pheromones can be found in for example urine (bound to major urinary proteins) , feces and saliva and many animals have scent glands in the skin as well. This chemical communication can be used to identify other mice (sex, age, status etc) and to decide on how to approach/interact

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.

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 diets are used for specific study purposes, fx if you need to study obesity you can use a diet which consists of a lot of fat. These diets might not fulfill the requirements for diets for animals, and therefore you need a license to feed them this. Ordering is done on the homepages of laboratory diet producers e.g.: envigo, ssniff, LabDiet etc. here you can buy both standardized diets and natural ingredient diet. Handling of the diet: You get a large amount for natural diets while you get a smaller amount for purified diets. Must be kept cool, dry and rodent safe. Have to be aware of the shelf life of the diet and whether the diet is able to be used within its life span. It is important that the correct mice get the corret diet and especially if natural - then from the same batch. If it is not possible to use the diet within its life span then it should be frozen down in portions. It is important to note batch number so that the right animal get the right diet. Autoclave or evaluation - two different ways to sterilize.

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 Key factors for a good culture of care: It all starts with the management who must start a good atmosphere.

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 is essential. Hypothermia can be avoided with heating blankets but be aware to not overheat the animals. 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

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)

To be able to have a small idea if it is effective, therefore it can be good to have some clinical designs which gives you an idea on the effect and the variation of the drug candidate. This is testes on a few test subjects. If we don't have a pilot study, a big study is done which maybe has useless results.

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

Transgenic animals: Pronucleus microinjection: A male fertilizes a female, and the fertilized eggs are taken out, those are injected with DNA constructed gene. Another female is mated with an infertile male, and the eggs are placed in her. She will then give birth to gene modulated pups. This will produce heterozygotes, as one gene is from the mom and one from the dad. Therefore, it takes some generations to reach homozygosity. This is a technique which can be used in all animals. In larger animals, sperm precursor injection, is used instead. Gene modified sperm and normal sperm will be ejaculated by the male. Targeted mutation by embryonic stem cells transfection: Conditional GM animals: Used to create humanized mice as disease models or just to have a human functional gene to look at how PK-PD values for drugs in this model. E.g. humanized mouse with human immune system. Look at genetic diseases as cystic fibrosis and make a knock-in where you insert the human gene for the genetic mutation responsible for this disease. Transgenic: gene inserted into non-coding region Knock-out: working gene is silenced (only works homozygotic) Knock-in: Humane gene set into mice own gene (also only works homozygotic) Generation of genetically altered mice: Simple pronucleus microinjection: most simple way to make transgenic mice. Mate two mice take fertilized eggs inject with DNA construct and put into another mated female. Injection is done into pronucleus (usually male sperm cell). DNA is incorporated into random DNA-breaks - therefore not for knockout or in. Offspring is heterozygotic and mating for generations ensures homozygote. Embryonic stem cell transfection: From blastocytes embryonic cells can be harvested and grown and here the different gene-modification techniques and be applied and only the cells that are gene-modified can be selected and put into another blastocyte a nd put into a female pseudo pregnant mouse. Give birth to "Chimeras" both GM and wild type cells. Only mice, rats, and humans Cloning and nuclear transfer: Take an intact cell from animal and cultivate it and do gene-modification in this culture. Nucleus from egg is removed and nucleus from gene-modified cell is inserted instead. The cell is developed into an embryo and inserted back into the animal and this animal will give birth to a gene-modified piglet CRISPR-Cas9: Nuclease is an enzyme with makes a break in the DNA-string and there are DNA-sequences coupled to this Cas9 nuclease enzyme, so you get a targeted break of the DNA. By clipping in a functional gene, a knockout has been made but also a wanted sequence can be put into this clipped region making a transgenic or knock in mouse. Usage: Used to make humanized mice as disease models or have humane functional Genes

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)

· The fat: o Here is is important that there are essentiel fatty acids as linoic acids · The protein: o Important with essential amino acids that the animal cannot produce itself as arginine and histidine · The carbohydrates: o Is divided into digestible carbohydrates and fibers · Minerals: o Macrominerals: Calcium, sodium, magnesium etc. > 100 mg/day o Trace minerals: Selenium, iron, cobalt < 100 mg/day · Vitamins: o Fat soluble: ADEK o Water soluble: B o Vitamin C is only essential for guinea pigs Pellets: Nutrition fractions: macronutrients Carbs comes in digestible carbs and fibers In the fats and proteins, there are the essential fatty acids and amino acids Different animal species have different needs Natural ingredient diets: Based on natural ingedients - Whole grains - Mill by-products - High protein meals (soybean, fish, casein) - Mineral sources (bone meal) This is cheap, nutritious and animals likes it, however, there is a lot of variations, single components are not removable. They also do not have any control of bioactive compounds. It might be contaminated. Comes as powder, pellets, extrudate and hybridpellets Purified diets - based upon synthetic compounds - Protein - Carbs - Fibers - Fats - Various mineral and vitamin mixtures Advantages is that they are standardizes and single components removable, however the animals might not like them, they are expensive and may lack biologically active compounds of importance for the modelf