LAS ABD

I.4 Describe who is responsible for compliance in relation to 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 organisation of animal work (LO 1.7, 1.9, 11.9)

- License holder - Legal person - Experienced scientist As the Animal Welfare Body (can not authorize nor prohibit any experiments) deals with husbandry (new shelters, cages, food, etc.) amongst other main tasks (project oversight, problems, new licenses, practices), they are partly responsible for how animals are housed in the facility. It is expected that the scientist knows about the organization of the animal facility, including housing and husbandry, but also how to organize procedures and operations in the animal facility.

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)

- Minimal threshold = when a procedure causes pain, suffering, distress or lasting harm. This is compared with the injection criterion, which is the lower limit to the amount of pain, etc., that can be elicited to an animal (introduction of a needle with good veterinary practice). - Animal Experiment: "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." - Lower threshold (injection criterion) - Upper threshold - intensive fear, harm or suffering - Purpose - teaching, research, blood sampling - Once an animal reaches ⅔ of its embryonic development, it is considered an animal that can suffer, receive pain (etc.) and therefore experiments with such animals are regulated as an animal experiment. - Invertebrates (except cephalopods) are not covered by the EU directive. - as a consequence in DK invertebrates are covered by the animal welfare act (can still be overruled by the animal experimentation act)

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 Animal Experimentation Authority needs to carry out inspections covering 1/3 of all animal facilities within the nation each year. - On a 3-year basis, they need to cover 100% of all the animals facilities - Facilities get both announced and unannounced inspections (approx. 1:1) - pros and cons for both. For each establishment (e.g. at KU it consist of 10 people, head, 2x vets, 2x techs, 2x facility managers, 2x scientist) there must be a committee called the animal welfare body: - Animal welfare officer - Scientist - Designated veterinarian (not needed) The National Committee are to advice the staff (animal welfare, the 3Rs (refinement, replacement, reduction), technical and scientific developments, rehoming) - SOPs - Follow the development/outcome of projects - "Culture of care" - going beyond the rules to strive for better animal wealth

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)

A = how to carry out the practical part of the procedure - requirement for receiving this function: to be a lab tech or to obtain another relevant technical or academic degree and pass an exam, "small course = 30h of work". - BUT you have to be trained in any given procedure you might want to perform (e.g. one can not perform surgery without being trained in surgery) B = how to set up the experiments and hold the license - requirements: relevant academic degree and larger course (>80 hour) in experimental animal science (this A/B/D course) C = how to take care of animals - requirements: laboratory animal caretakers, animal farmers and veterinary nurses. These individuals have 4+ years of education in animal caretaking. Equally, they need the presupposed knowledge within EU Function A/B/D that is general to everyone in lab animal science. D = how to kill animals The role of the manager (EU article 24): according to EU article 24, the manager needs to have specific competencies as a lab animal vet → Eventually, the manager is able to take care of animals (function C). The role of the designated vet (EU article 25): requires specific vet learning outcomes, presupposed knowledge and specific vet competencies for lab animals to become a designated vet.

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

A high level of culture of care and of 3R compliance will also reduce the emotional strain on animal care staff as discomfort and suffering in the animals is reduced to a minimum and the staff knows that all possible measures to reduce the harm to the animals have been taken. Such a culture will therefore support job satisfaction.

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).

AEA legislation is based mostly on a utilitarian framework of animal welfare. 1) Harm-benefit assessment (HBA). Based on utilitarian point of view. It includes both the impact on the animal and the benefit of the study. The harms (the costs) The benefits HBA · Animal welfare o Five freedoms · Animal rights o Intrinsic value of the animal o Respect for nature · Quality of the research · Compromising the animal welfare · Human harms by caretakers · Study outcome (scientific) o Human health o Veterinarian medicine o Nature · Educational · Economics · Social · Harms are certain o 3R compliance · Benefits are not guaranteed o Often difficult to predict · Should guaranteed harms during the study weight more than non-guaranteed benefits, which may lie many years in the future? · Check 3Vs to secure benefits The 3Vs: a way to secure benefits - construct validity: your model makes sense. The cause of diseases is the same in the model and real life. - face validity - predictive validity Additional: internal (to what extent does the study establish a trustworthy cause-and-effect relationship between intervention and the outcome specifically in our experimental context. This adheres more to the experimental design, randomization, blinding, blocking, statistics, PREPARE guidelines, sufficient reporting, GLP) and external validities (how well does the animal model resemble the disease we are trying to model in various aspects? This concerns construct, face, predictive validities) 2) The 3Rs and culture of care Replacement: Substitution of animal models. Reduction: less animals = same amount and quality of data or same number of animals = better quality of data. Refinement: apply noninvasive techniques, change scientific and management procedures. Culture of care: aiming at improving the caring for the welfare of the animals and the staff. Implementing the 3Rs. The animal welfare body. The national committee. No one is left behind feeling bad and sad about treating, harming etc. animals 3) good animal welfare assessment clinical examination (health and biological functioning), data evaluation (health and natural living), behavior/emotions/preferences, lower threshold: the injection criterion, upper threshold: strong pain, intensive fear, intensive suffering. Purpose: research, teaching, blood sampling. Animal severity classification. Based on consequences (utilitarian, approvers) and animal rights view (disapprovers).

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)

An example: multifactor case-control designs. Designs like this are good in that we can obtain a lot of information from a single animal. Testing the effect of the drug first, then the effect of sex on this effect, and then the effect of age, would require more resources and more animals. The drawback is that these designs are complicated. We may need to consult with a statistician when we are planning such an experiment. Take, for instance, the question of sample size. For some of the more complicated designs, there are no easily accessible tools that allows us to estimate an appropriate sample size. We need to take think about statistical power, but for all the potential factors... The elements of your study: Today, we can feed what we know of our experiment into a computer program that can then generate several thousand, if not more, potential outcomes. We can then gauge how well our planned analyses would fare in these theoretical scenarios, adjusting our sample sizes up and down, until we find an appropriate number. This is called a simulation-based approach. These methods often require some programming, and are consequently best left to statisticians or data scientists. But in order to be able to work with, for example, statisticians we need to understand the information that is needed for these simulations. This is still, as it is with planning a simple case-control study, variation, effect size, statistical significance and statistical power

VIII.9 Describe and discuss good working practices with regard to use, storage and disposal of anaesthetic and analgesic agents. (LO 20.13, 21.23)

Anesthetic gases: ensure adequate ventilation to scavenge waste gases, handle equipment correctly (put tubeings correctly together so that gas flow is correct) Gas cylinders: risk of explosion in case of fire, heavy so keep properly attached Needles: careful handling, disposal in designated waste containers Drugs: store in locked cupboards, keep record of purchase and usage, destroy non-used drugs according to legislation.

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)

Animal experiments are classified according to severity To do so you must consider some issues related to the procedure - The Type of manipulation and handling you do with the animals - The Nature of the pain, suffering, distress or lasting harm, and - Its intensity, duration, frequency and multiplicity - And also how high is the cumulative suffering, i.e. when you add up the effect of different procedures or multiple use of the same procedure Also you must consider some issues related to the animal - Which species and genotype do you use - How mature and old is it, age and which sex do you use - Do you prevent the animal from expressing natural behavior - Has the animal been trained for the procedure - And If reused, what was the severity of previous procedures Classification (only mildly or moderately treated animals can be re-used): - Nonrecovery: complete anesthesia, from which the animal shall not recover consciousness - Mild experiments (e.g. anesthesia) - Likely to experience: short-term mild pain, suffering, or distress - Moderate experiments (e.g. surgery under general anesthesia) - Severe experiments (e.g. severe restriction of movement or food over a prolonged period) Reporting: - Before, during and after completion. - In case of unexpected death of the animals - Diagnose - If not diagnosable → severe experiment

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)

Animals are used for education (LAS, veterinarians, surgeons/health care professionals) in Denmark and this is considered beneficial but needs to be subjected to an HBA. For example, some procedures are only recorded on videos (e.g. isoflurane anesthesia) where only one animal (reduction) is used for the means of showing this procedure. One could argue, however, that the attendants would benefit more from performing the specific procedure themselves, but it has been subjected to an HBA, the costs/harms outweighed the benefits from the this specific procedures. Dummies are very efficient for replacement in training and teaching purposes in LAS courses. Optimization of dummies to secure practice of procedures such as injections, blood sampling etc. Another example: in this course, one could say that it creates more harm than benefit if the persons attending the course are not going to use the knowledge acquired as the animals used during the practical exercises are then used without any beneficial purpose (probably just the purpose that these animals suffered to make sure that a specific person made their mind to not continue with animal experimentation)

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)

Applicants apply through the competent authority, the animal experimentation authority/animal experimentation inspectorate ("dyreforsøgstilsynet" that is within Fødevareministeriet) to review whether the application is fulfilling or not. If it is fulfilling, they hand over the application to the board for animal experimentation (the decision-making body of the animal experimentation inspectorate). They are the final authority to give out licenses. The Animal Experimentation Authority guides individuals to access or acquire the right information. The application (14-stages that are in Danish on AIRD system): - Non-technical project summary (scientist typically fail on this as they make it too complicated) - For ordinary people - Purpose - Utilization of 3Rs - Background - Effect - Technical project summary - For experts. - Type of experiment - Necessity - Procedures - Preconditions for licensing: - Cost-benefit analysis - 3Rs are implemented - Qualification (e.g. LAS course) To apply: - AIRD System of Dyreforsøgstilsynet - NEM-ID is required Licenses are granted only to specific experiments and numbers of animals: no more, no less. According to AEA, licenses are granted to "named individual" (a competent scientists which has the license as a personal allowance for him or her to carry out experiments) or "legal persons" (a company has a license and can appoint competent persons to be responsible and give notice to the competent authority about this)

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)

Avoiding bias → blinding (look at exam question 3). Classical parallel design is the simplest → cross-over (partial and full) → latin cross-over (most complex). Example of a more complex design: In a classical cross over study mice serve as their own control, where it undergoes both control treatment and test treatment separated by a washout period. This reduces bias in group-allocation (and even for randomization, it might be that one confounding parameter by chance is overrepresented in one group which we don't want and thus we do either blocking or pair-matching, however, we still might want each animal to serve as its own control and the experiment might not allow the set-up with both control and test treatment at the same time) but still a question about which treatment goes first arises which is important due to carry over effects. Thus, one might want to do either a full cross-over design (half of our animals start with one condition and the half with the control condition) or even latin square design (where we create a pattern where every animal undergoes each treatment, and each treatment is equally represented in each time period) - Or multifactor case control study: we need our subjects to be roughly equally distributed across the different testing conditions. If we balance our experiments right, we may even be able to look at an additional effect to our drug and the sex of our subjects. Maybe we also want to know if the effect changes with age. Designs like this are good in that we can obtain a lot of information from a single animal. Testing the effect of the drug first, then the effect of sex on this effect, and then the effect of age, would require more resources and more animals. The drawback is that these designs are complicated. Why we might one to use it: less bias, no need for a dedicated control group (fewer animals), animals acting as own control reduces biological variation to a minimum (fewer animals).

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

Can be difficult to define the harms, define the benefits and balance those. Harms = "the costs" - Animal welfare: compromising the five freedoms - Animal rights: the intrinsic value of the animal (as well as an intrinsic moral to respect animals / Kantian ethics / categorical imperative), respect for nature, animal ethics dilemma on-line, - Quality of Research: a study with lack of relevant control or low power → the animals are used without any benefit - Human harms: animal caretakers/technicians (bites, allergens, psychology). Benefits = improvements in human health, diversity of the animal population on the planet - Usually not guaranteed. Good results must be rendered probable (thus, a good model has a high predictive validity). It is thus crucial that the competent authority has all the needed information. What makes HBA more complex and challenging is that harms occur during the experiments, while benefits are likely to occur in the far future. Other challenges when performing HBA: it is difficult to quantify the value of human vs. animal life and suffering.

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

Coisogenic = relationship between donor mouse and its background strain. Cogenic = relationship between a recipient mouse and its parental mouse strain Backcrossing = when one wants to transfer a genotype from a coisogenic mouse to a cogenic mouse to have the genotype in a specific background. Traditional backcrossing vs speed congenics: both methods are to select the correct background and genotype, however, speed congenics can achieve both in 4-5 generations (because one selects for the most background-true heterozygotes by e.g. SNP-assays.), whereas traditional backcrossing takes 10-1 generations Forward genetics = phenotype → genotype Reverse genetics = genotype → phenotype ILAR (Institute of Laboratory Animal Research) codes: - Provider code based on the company name: Tac (Taconic), Crl (Charles River Laboratories), Rj (Janvier), Hsd (Envigo) - When Janvier and Envigo have names that don't really fit logically to their company names because they have had other company names previously. - ILAR issue a short code for anyone who intends to breed rats and mice. Names for strains (isogenes, isogenic animals) and stocks (outbred): - inbred = strain (strain name/breeder code), outbred = stock - When giving names to inbred strains, we first write the strain name, then a slash and last the breeders code à C57BL/6NTac (C57BL/6N from Taconic, but we have here removed the second slash as there is already one in the name) - Substrains are designated by writing all the codes of all the relevant breeders with historically oldest breeder first and the present breeder last. That means at Taconic you can get both a C57BL/6NTac, which Taconic has received from NIH with the breeder code N, and a C57BL/6JBomTac, which Taconic has got when they purchased the former Danish breeding centre BomMice with the breeder code Bom, who had received it from Jackson Laboratories with the breeder code J. - When giving names to outbred animals we first with the breeder code, then a colon and last the stock name à Crl:SD is Sprague Dawley (SD) rats from Charles River Laboratories. - Again, if the outbreeding has happened at different origins this is put into the name. For example, HanTac:WH which is an outbred Wistar Hannover rat, abbreviated WH, which they have received from the former Central Institute for Laboratory Animal Breeding in Hannover, Germany with the breeder code Han F1 hybrids: When you produce F1 hybrids you will give these hybrids first the name of the mother strain, then the name of the father strain, then F1 for F1 hybrids, then a slash for inbred and then the breeders lab code. In this example you mate a C5BL/6 mother with a DBA/2 father. To avoid making the names too long you use the official abbreviations B6 and D2, which gives you the name B6D2F1/Crl, if these hybrids were bred at Charles River. Nomenclature of transgenic animals: C57BL/6-TgN(APOA1)1Rub/J - Background strain + horizontal line + designate that it is inserted as a transgene (Tg) + method of introduction of transgene (N for nonhomologous way such as pronucleus microinjection or H for homologous such as CRISPR/Cas9 Knock in) + gene name (human gene always in capital and surrounded by brackets) + number of the founding line (1 in this example as it is the first one) + founders lab code (Rub for Lawrence Berkely Laboratory in California) + slash + breeder actually breeding the mouse (J for Jackson Laboratories). Nomenclature of knockout: B6.129P2-ApoA1tm1Unc/J - Background strain is C57BL/6, donor of stem cells is 129P2, as ApoA1 is written with targeted mutation (tm) in superscript it means that the gene function has been deleted and 1 is the founding line number, Unc is the founders lab code in this case University of Northern California. Jackson Labs is breeding the mouse - If it was B6.129P2-ApoA1em1Unc/J it meant that the KO was not obtained by classical KO with embryonic stem cell method (transfection of ESCs with nonsense sequence instead of the gene into the blastocyst), but with endonuclease mutation (em) with CRISPR/Cas9 e.g.

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)

Common post-surgical care and monitoring: Common post-surgical complications and their causes: - if one has not performed a large enough incision, it can become difficult to see everything inside the animal and this might lead to damage inside the animal. - Organ damage because abdominal organs are friable, so avoid air and abrasive materials, wet tissues frequently, use proper instruments or fingers.

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 refers to a category of animals in which a license is needed to carry out breeding for animal experiments (* needs to be individually marked and followed by an individual protocol, so that they can be traced from birth to death). The rest cannot be bred. Wild animals can under certain circumstances be used for animal experiments. There're exempts with teaching institutions. According to the annexes 1 of both the directive and the animal experimentation order, you need a license to breed and deliver the following species - Mouse - Rat - Guinea Pig - Syrian hamster - Chinese Hamster - Mongolian gerbil - Rabbit - Dog* - Cat* - All Species of non-human primates* - Frog - Zebra fish Non-human primates under certain conditions and thereby these animals are above other animals. Non-human primates can only be used if the purpose is Diagnosis, treatment and prevention of disease, poor health or other abnormality and its effects in humans, animals and plants, including the manufacture of drugs, substances and products, as well as testing their quality, efficiency and safety. Basic research and research for species conservation

III.1: Describe the estrous cycle/ovarian cycle of rodents and describe how pheromones can affect the ovarian cycle and pregnancy (EU LO 3.1.1).

Estrous cycle/ovarian cycle: It is quite like the human. The main difference is the duration: 4.5 day cycle in rodents compared to 28d in humans. They have spontaneous ovulation. Mating results in a vaginal plug (this is practical to determine if a mouse has been mated overnight). Post-partum estrus normally happens 12-24h after female rodents have giving birth (goes into normal cyclicity after birth giving) - however, if a female rodent becomes pregnant with post-partum estrus, it might lead to lactation-induced delayed implantation (meaning that the blastocyst only implant into the uterus when the lactation period of the previous birth has past). Pseudo-pregnancy: sexual activity from male on female leads to reproductive hormones in pregnancy à returns to cyclicity Pregnancy is 19-20d. in mice, pregnancy can be determined around day 12 by maternal body weight increase from day 1. On day 8-10 it can be determined by pregnancy-specific proteins. Lactation is around 21d and normally one weans mice from the mother after 3 weeks (with transgenics it is recommended to wait 4weeks). Ovulation can be detected by a vaginal swap (presence of keratinized epithelium in the swap). How pheromones can affect the ovarian cycle and pregnancy: when you group-house females without presence of males, they will synchronize their ovarian cycles and suppress their estrus and therefore be often in a state of anestrus. This is called the Lee-boot effect. Mice can detect pheromones with the Grueneberg ganglion (mice have very sensitive olfactory sets), including fear pheromones, which have a huge impact on their reproductive physiology. Whitten effect: when the presence of males induces synchronization and estrus after 72h (interestingly, the females mated on the 3rd night ovulate more eggs and produce larger litters compared to 1st and 2nd night matings after whitten effect, which is important when comparing different groups in experimental designs). Bruce effect: foreign male smell induces fetal resorption in fertilized females. The Vandenberg effect: young female mice appear to become sexually mature more quickly when a male mouse is nearby.

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)

European Council: issues conventions serving as guidance for directives issued by the EU (voluntary). NGO's and industries partake in this discussion; however, they have no voting rights. EU Directive from 2010: Issues directives that all member states have to follow. Each member state must have a competent authority to carry out the obligations arising from the directive (licensing, inspections and accreditation of facilities). National directives: Can only be more strict than the EU directives. E.g. in Denmark under no circumstance it is allowed to induce harm, intensive suffering, and or fear. Therefore, the National Directive overrules the EU Directive. Where to find the relevant legislation: EU-directive, EU homepage on Laboratory Animal Legislation Activities of those carrying.out scientific procedures: one needs to have a license (in each specific case as stated in the animal experimentation act, the license can be given if it involves teaching, research, or collecting of blood samples) to perform animal research where the animal suffers (not allowed by the animal welfare act) = The animal experimentation act overrules the animal welfare act. Animal experiments must be the following: - Licensed - Beneficial to the society (well-reasoned) - Necessity of animal usage - Not be causing strong pain, intensive and long-lasting fear or suffering - Performed by qualified staff in the proper setting - Animals must be bred within the destination The Minister of Environment and Food have the right to change the animal experimentation act: - How to get the animals - Care and housing of the animals - Experimental design - Staff qualifications - Teaching experiments - Institutions or companies doing animal experiments - Releasing and re-housing of animals - Advisory committee on animal experimentation The Animal Experimentation Order: All the details of the Animal Experimentation Act are within the order. The Animal Experimentation Board: - Hands out all licenses - Led by a judge. - Meets 11 times a year - Interests are broadly represented to get a nuanced agreement/disagreement - All members (except by the judge) are appointed by the Minister of Food and Environment. However, they are all suggested by different organizations. The National Committee For Animal Experimentation and Alternatives: - Advice and to implement the 3Rs to create a culture of care for the animals. - Voice of the EU directive and networks with other authorities to inspire and to create a culture of care that is uniform throughout the member states. - 7 members (experts appointed by the minister of food and environment) from each of the 3Rs and the minister from the food and environment. - 2 from each R and the chairman

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)

Euthanasia means "good death", induction of death humanely without causing fear or stress in the animals. It should cause death as quick as possible, and the method chosen should be easily performed by the person performing it - do not use methods that you do not master. The method should be safe for the person performing it. Laboratory animals need to be killed at a defined time point, either as an experimental endpoint, or as a humane endpoint (legal requirement, no one can be function A without being function D). Examples of methods: confirmation of permanent cessation of circulation, destruction of the brain, dislocation of the brain, dislocation of the neck (comes with size dependence), exsanguination, confirmation of the onset of rigor mortis. The choice of methods depend on the species, but anesthetic overdose is allowed for all animals (shall, where appropriate, be used with prior sedation), CO2 can be used for birds and rodents (but for rodents, we do a gradual fill, but not allowed for fetuses and neonatal rodents as they are very resistant to CO2). Some methods e.g. decapitation is only allowed when other methods are not possible. Euthanisa of fetuses: nociceptiv system is not activated until after birth, so if fetus is not used we can leave it in the uterus. If the fetus is to be removed from the uterus while being alive, decapitation by a pair of scissors is performed. How do we know if the animal is dead: HR stops, respiration stops, reflexes are absent, pale skin, cold body surface, rigor mortis. We cannot be sure, so often chemical method is accompanied by a physical method such as cervical dislocation and experts should be present because assessing death can be difficult.

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)

Examples of behavioral changes and other signs of discomfort, pain, suffering, or distress in rodents: behavioral and clinical signs (activity, posture, moving patterns, writhing, twitching, back arching, facial expression assessed by a scoring-system ethogram usually + alertness, does it eat or drink normally, is it protecting a certain part of the body, does it sound when being handled, does it bite or lick it self more than normal, in rats: chromodacryorrea (porphyrin - red coloured tears). Clinical signs are body weight loss, food and water consumptions, urination and defecation and fur quality (piloerection). Physiological parameters (HR, BP, body temp), biomarkers (acute pain/stress: corticosteroids, adrenaline, NE, cFos activation. Sub-acute or chronic stress: corticosteroids, IgA, cFos activation. Oxidative stress: 8-oxo-dG, lipid peroxidation -> isoprostanes. Distress/depression: 8-OH-DPAT challenge to see how temperature is normalized). Abnormal behavior is also repetitive, homologous, and apparently no-function behavioral patterns with negative consequences for the animal involved. Stress-induced pain: ACTH-transcript includes beta-endorphins (ACTH expression leads to pain) Distress: defined as something always negative for the animal (whereas stress can be positive as a physiological response to adapt to a change in environment) à reads of corticosteroids don't always show distress Signs of positive well-being: they are prey animals, so they may hide. Basic behaviors: nesting and hiding, social behavior, exploration, foraging, grooming, locomotor activity and movement. Principles of how pain, suffering, distress can be monitored: - implantation of telemetric device, blood sampling (NE e.g.), gene expression or IHC of euthanized animals or bioluminescence of live animals (cFos activation), visual analog scale (behavior but subjective), scoring-system ethogram (data that can be quantified or graded and put into score sheet, more objective than the VAS), analgesiometry (measure latency to avoid the unpleasant stimulus such as tail flick, hot plate, paw pressure, writhing test, formalin test, von frey filament) - increased activity of HPA-axis and SNS à increases in HR, BP, hyperglycemia, lipolysis, altered metabolism, immunosuppression, anti-inflammation, reduced reproduction, stress-induced analgesia, prolonged levels of stress hormones Principles of how pain, suffering, distress can be managed: - analgesia, anesthetics, environmental enrichment (does it suffer or show distress due something specific then provide specific enrichment such as socialization, food, temperature) intensive suffering or pain à euthanasia

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)

Fidelity: whether or not a certain stimulus/model resembles the real model/animal/world. (general/overall similarity to what is being modeled) Discrimination: specific similarity for one particular property that is being modeled. Induced model: the animal model can be induced chemically (HFD) or by surgery (surgical removal of the pancreas). It can even be genetically (even though the model spontaneously develops a condition, it is genetically induced). Spontaneous model: Spontaneous animal models are those for which a particular disease appears naturally in the animal studied (e.g. NOD mice) Primary vs. secondary readout: The primary readout would be the most important for your phenomenon and the statistics related to that, whereas secondary readouts can give more information on the variety in your models. Construct (the extent to which both the animal model and the human phenomenon can be explained by the same theory, e.g. in terms of mechanism, origin), face (what is observed in the animal models resembles what is observed in the modeled phenomenon, same symptoms → high face validity, same phenotype → high face validity), and predictive validity (performance in the test predicts performance in the condition being modeled) for induced and spontaneous animal models: depends on the context - example with the pancreatic surgery for diabetic modeling. Negative vs. orphan models: The negative is a model for which the phenomenon that you're investigating does not exist. Orphan models refer to diseases for which there is no human analog and occur exclusively in the species studied.

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)

Four different classes of severities: nonrecovery, mild, moderate, severe. Cumulative severity:

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 surgical instruments: hemostats (curved and straight), scalpel handle, forceps, scissors, needle holders, needles and suture material, retractors, stainless steel bowls, instrument tray, cotton wool buds, gauze swabs, sterile drapes Microsurgical instruments are very fragile. One must be extra careful when cleaning those instruments. Wound closure and suturing: important to eliminate dead spaces, sutures in inner layers of muscles and fascia should be tied tightly, sutures in the skin less tight - or this might induce pain and inflammation, choose correct suture material (inside the body, the suture material must be reabsorbable). Material can be silk, polyamide, prolene etc. and all have different tissue reactions and other features. There exist both simple interrupted sutures (secure, loss of one suture does not open entire wound) and continuous sutures (easy and quick, better seal of wound edges, less secure)

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

Good laboratory practices (GLP) are accepted methods to carry out activities or operations in non-clinical laboratories. Regulatory agencies and laboratory organization say that these practices help ensure safety - in the context of the animal research, both the humans and animals. GLP has a positive influence on the quality of the result. GLP refers to a quality system of management controls for laboratories where research is taking place. The labs are not treating humans. GLP does not refer to a set of guidelines. It has the force of law. GLP is a part of a quality assurance that ensures that organizations consistently produce and control good to a high quality standard. It is not only concerned with production, but also quality control. GLP aims to ensure the consistency, reliability, uniformity and quality of chemical non-clinical safety tests. It also aims to ensure their consistency and reproducibility.

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

Halsted's principles (describes how to handle tissue): asepsis, gentle handling, hemostasis, closure of dead space, careful approximation of tissues, avoidance of tension, minimization of foreign materials. - Instruments are designed to prevent undue trauma and aid in gentle handling, appropriate instrument must be selected for a particular procedure, surgeon must familiarize with the use of instruments Consequences of poor hygiene: poor animal welfare and confounding factors, infected surgical wounds, poor healing, pain, sickness, distress, euthanasia, higher variability, more animals needed. Aseptic techniques and practices are defined as those that prevent cross contamination during surgery and involves proper preparation of the facilities and environment, surgical site, surgeon, surgical equipment. It limits the animal's exposure to microorganisms (might be exogenic from air, surgeon, instruments or might be endogenic from the animal). Aseptic techniques have the advantage over antibiotics that no pathogen has yet developed resistance to aseptic techniques. All invasive animal experimentation on awake animals or where animals are supposed to wake up, should be performed aseptic (survival surgery, injections or blood sampling). Even long-term terminal surgery should be performed aseptic (infections affects physiology and impairs the animal model). - How to work aseptically: sterile things must only touch sterile items, unsterile things must only touch unsterile items, sterile and unsterile items must not touch each other. Preparation of instruments: instruments should be sterilized and stored in a clean, dry environment when not used. Thoroughly cleaned after use to remove blood and tissue, then sterilized. Sterilization techniques include autoclaving (steam or heat), irradiation, ethylene oxide gas). Required instruments depends on the procedure - Cleaning of instruments: surgical instruments must be cleaned carefully so that delicate instrumnes will not be damaged. After operation, all gross contamination must be removed manually (open the instrument jaws, soak them in water with washing-up liquid or cleaning agent containing enzymes, use brush and pay particular attention to box locks, serration and hinges). Ultrasonic cleaning equipment. Use appropriate disinfectants. Preparation of animals: large animals must be fasted 8-12h prior to surgery to prevent vomiting (water until a few hours before surgery), shave or clip to remove hair at region of interest, scrub area with anti-septic soap solution, remove soap with alcohol or iodine, cover whole animal with sterile drape - Rodents: one week acclimations before use, no need to fast (cant vomit), slip or shave to remove hair, swap if dirty - disinfect with alcohol or iodine, cover animal with sterile drape (use transparent drape if available; respiration might be difficult to observed otherwise) Preparation of surgeon: scrub suit, sterile shoes or shoe covers, surgical cap, face mask, sterile gloves. - Before scrubbing: long hair must be taken up, nails must be clipped short, no jewelry on hands and arms, wear comfortable and practical clothes. - Scrubbing: scrub the hand and forearm with soap or surgical scrubs, the sink should be activated by other means than the hands, the soap dispenser should also work without use of the hands, dry then with paper and disinfect. Gloving: If using sterile gowning, put this on after scrubbing and before gloving. The assistant opens the outer package. The surgeon opens the inner sterile package.

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 optimize animal welfare during housing, handling and procedures (EU LO 2.13; 2.14)

Homepage: The Danish 3R center, National center of the 3Rs, Canadian Council for Animal Welfare. Homepages for EU directive and Danish legislation. Textbook: The Principal of humane experimental techniques (first to implement the 3Rs). Why it is important to continuously stay updated: as improvement in new technology often allows housing, handing and procedures with less harm and more benefit for science and animals, and as this development is often progressing faster than the legislation, it is important to continuously stay updated on how to optimize. Additionally, scientific knowledge can present new features of animal behavior and preferences that suggest e.g. that a given animal experiences stress under current housing, handling, procedures which is also another reason why one should stay updated.

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)

How the animal facility is organized to maintain an appropriate health status for the animals and the scientific procedures: - animal facility can be placed in a separate building or among other functional areas inside a shared building. Staff include manager, animal care takers, a veterinarian. Typically consists of one or more units (it depends on the size of the facility, sometimes units for breeding, experimental procedures, quarantine etc. are separate, whereas in small they may be all placed in one unit). One unit contains several rooms, one for storage, shared equipment, one for procedures etc. - In large facilities, administration, quality assurance and laboratory technicians can be present as well. - The animal facility is isolated and closed in order to keep public relations out, ensure security, animal health and so that allergens don't spread. - A barrier animal housing facility: designed and managed to keep out microbes for animal health, to keep in pathogens for human health. This can be done at cage level, room level, or level of an area within a facility, or the entire facility. Infection barriers include physical barriers such as walls and management barriers such as personal barrier and disinfection (autoclaving of cages and material). Irradiation of food and bedding. Control of the ways in which supplies and personnel enter the facility. - Conventional housing: any type of laboratory animal housing facility. You can come and go from this area without special precautions. - Movement of air in the animal facility unit: hyper-pressure for barrier units (positive pressure room to maintain a flow of air out of the room, thus protecting the animals inside from possible contaminants and pathogens which might otherwise enter) and hypo-pressure for quarantine rooms (negative pressure room to maintain a flow of air into the room, thus keeping contaminants and pathogens from reaching surrounding areas). - Circulation: the movement of clean and dirty material within a unit. Can be either single corridor or dual corridor with clean/dirty corridors. This allows for the decrease in the potential for cross-contamination between animal rooms. - Entering a barrier facility: the most important restrictions relate to the entry of animals. At the highest level of restriction, animals can only enter via rederivation (only aseptically obtained eggs and fetuses can enter). At lowest level, all animals can enter. At the personnel level, people might only enter after showering and after clothing change. Supplies will be autoclaved. Sterile supplies will go through a chemical lock. Sometimes people are also quarantined if they have e.g., entered another animal facility which is considered dirtier. - Containment facility: constructed to keep experimentally or naturally occurring hazards safely inside to protect workers, animals and the environment. Below is a very high level containment facility where: air and waste is filtered, people wear a suit, gets disinfected and under low pressure. Biological consequences of acclimatization, habituation, and training: acclimatization (in general 1-2 weeks) allows an animal to maintain performance across a range of different environmental conditions. After transportation an animal is often placed in a new social environment where it has to find its place in the hierarchy (these changes manifest as perturbations in homeostasis which is why acclimatization is both desired and a legal requirement). Habituation (e.g. sham injections, where there is a decrease in response to a stimulus after repeated presentations) leads to the animals being less stressed at the day of the experiment (thus less variation, thus reduction of animals). Training of staff and/or animals usually also leads to reduced number of animals due to less biological and technical variation when performing the experiments.

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

How they are generated: - Pronucleus microinjection: (30 eggs for an optimal success rate, applicable for all species), random insertion/random insertion of transgenes. Breeding for multiple generations is needed to obtain homozygosity. - Sperm precursor injection: only for knock-in - Targeted mutation by ESC transfection → breeding of chimeras for a couple of generations to obtain homozygotes. - Done by transferring ESCs into a blastocyst (around E3-4 before lineage commitment) that then serve to differentiate into a germ layers, and importantly giving rise to reproductive cells. - Cloning and nuclear transfer - Nuclease and restriction enzyme-based techniques (zinc finger, talens, CRISPR/cas9) - One can transfer microinjected two-cell embryos in the oviduct or transfer microinjected embryos with ES cells with targeted mutations in the uterine. How they can be used: mice lacking thymus → accept any cells, models developing a given disease/phenotype One can achieve tissue-specific/conditional and time-specific KOs (CRE-LoxP systems)

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).

How: Videos on how animals are housed in their facility are on their homepage, a resource for the general public. Local management (e.g. AWBs) can encourage companies and academic labs to lecture about CoC as well as write about it online. Dorthe also mentioned that they have a annually organized laboratory animal experimentation event which can lead to openness and transparency as well. Also, when receiving a license the layman description will be publicly available for everyone which also leads to openness and transparency to the general public. Benefits: Communication and transparency equal trust and acceptance in the public. For some people, this transparency would likely lead to some laymen taking on an animal rights view but probably for many, it would lead to a utilitarian view with some animal right view. It definitely would depend on the framing of how the information is presented to the public.

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)

Humane endpoint: the earliest point at which an experiment can or should be terminated with the purpose of eliminating unnecessary suffering. If the animal reaches a certain degree at risk of suffering, we most stop the animal in advance before we may have reached the experimental endpoint (when all necessary data have been achieved). E.g. in an induced or spontaneous rheumatoid arthritis, the humane endpoint is legally set by Danish legislation (10/24 points is maximum - where 1 point from each enlarged toe, 1 point from each involved metatarsal/metacarpal, 1 point from each involved tarsal/carpal). Or for cancer models, the AEI has said upper limits for the size of tumors in specific organs. This is also a humane endpoint (it could also be described as a percentage of bw). Criteria to be used to set humane endpoints: In order to detect and define the humane endpoint, it is desirable to design a welfare protocol, a score sheet with relevant parameters specific for characteristics related to the experiment/model, species and strain and may include appearance of the animal, behavior (spontaneous, provoked, in tests) as well as early general clinical (quantitative: bw, body temp, respiration, HR, behavior + qualitative: respiration, posture, appearance of closed eyes, disturbed fur as with ruffled fur or feathers, diarrhea, coughing) and model dependent clinical (e.g. if working with a tumor model: ulceration/infection of tumor site, invasion of surrounding tissues, size of tumor etc.) signs. Endpoints may differ depending on severity of the experiment. Define action to be taken when a humane endpoint is reached and consider possible options for refining methods to finish at an earlier endpoint: humanely killed (euthanized), veterinarian to be contacted, exclusion of study progression. Possible options for refining methods to finish at an earlier endpoint would be to take both general and model-specific clinical signs into consideration, and in the format of a score sheet and welfare protocol we might be able to finish at an earlier endpoint. Factors to be considered and methods available for assessing and recording the welfare of animals: welfare protocol with score sheets of the above-mentioned parameters. Parameters relevant for data collection can also be included.

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

If you after having done one experiment want to do yet another experiment on the same animals this is called Re -use. You can only re-use animals if the severity has been only mild or moderate in the first experiment and this will be the same in the new experiments; or eventually the new experiment will be non -recovery. Also the health and well -being should be fully restored and the veterinarian must advise that the re -use is acceptable. Reuse should not be considered as if you e.g. do one blood sampling and then another one after some hours, if this is stated in your license as one procedure. Thus, reuse is directly linked to the procedures that one has applied a license for.

II.9: Describe the importance of good animal welfare including its effect on scientific outcomes as well as for ethical reasons (EU LO 2.11)

In science, one wants to achieve high translatability from a studied phenomenon in a studied model to the actual reality as well as high reproducibility. One factor determining the level of these is animal welfare (e.g. procedures evoking fear and/or pain, insufficient housing environment). To ensure valid data, it is important that animal welfare is not compromised to the extent that the animal is experiencing fear, pain, or prolonged stress because this could interfere with the studied phenomenon and the present readout. Also, if some animals in one's cohort experience stress and others don't, this would introduce variation in one's data and therefore uncertainty as well as a requirement to use more animals (violating refinement criterion). Another thing to bear in mind is that often a specific model is evaluated as the extent to which it resembles human physiology (construct, face, predictive validities), but this is done with the premise of the animal being healthy and without experiencing stress, suffering, pain, harm, distress. Therefore, whenever animal welfare is compromised, we also in some sense compromise the translatability of the phenomenon studied and the data generated to that of human pathogenesis.

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

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. But also other views besides utilitarianism exist such as contractarianism and animal rights view (see below).

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

Inbred mice do not necessarily carry out disease-causing genes (because it may be deselected during breeding), but it may occur as some disease-causing genes are inherited. Therefore, it is important to monitor these types of strains. For example, certain mouse strains are known to develop specific benign tumors and need to be monitored for malignancy. Similarly, some are known to have blood in their urine. Some strains of mice are extremely aggressive. Large offspring syndrome: clonal disease in large animals → however, with Crispr/cas9 this is not a problem. If one wants to keep disease models alive (for experimental purposes) after the disease phenotype has occurred, this needs to be licensed according to the animal welfare act. Immunodeficient mice strains such as: RAG1 KO, SCID, NUDE (rat), are to be kept in highly restricted/monitored environments to protect them against diseases. These strains can be used to e.g. transplant human cancer cells underneath the skin of the animals. It is good practice to genetically monitor the mouse strains by SNP / STR / LTR screens to ensure the background.

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

It is a guide on how to avoid unnecessary animal suffering and promote health. 1) Freedom from hunger and thirst: by ready access to water and a diet to maintain health and vigor. (fundamental refinement) 2) Freedom from discomfort, by providing an appropriate environment. This freedom emphasizes the need for providing laboratory animals with an appropriate environment. (refinement) 3) Freedom from pain, injury, and disease, by prevention or rapid diagnosis and treatment (refinement) 4) Freedom to express normal behavior (similar to viewpoint 2 in animal welfare in definitions) 5) Freedom from fear and distress (refinement)

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

It is important to realize that any transportation of animals must be considered a stressful event and should thus be minimized as much as possible. In general, there exist inter and intra facility transport. Even for intra-facility transport between rooms, one must consider that every stressful event compromise both animal welfare and maybe scientific outcome. Animals should be transported in the least stressing way, and under optimal environmental conditions, e.g. ventilated, climate-regulated trucks are better than public transports such as trains and planes, and transport should be short.On this course, the acclimatization part is the most important, i.e. animals should be properly acclimatized before use in experiments, and the period may differ dependent on species and type of transport We therefore must ensure the following list: The regulation sets the following general requirements for the transport of animals. No person shall transport animals or cause animals to be transported in a way likely to cause injury or undue suffering to them. Transport arrangements must be made in advance to minimise the length of the journey and meet the animals' needs. The animals must be fit for transport. The means of transport and loading and unloading facilities must be designed, constructed, maintained and operated so as to avoid injury and suffering and ensure the animals' safety. Personnel handling animals must have received adequate training and be competent. Transportation to the destination must take place without delay and involve regular checks on the animals' welfare. Sufficient height and floor space must be available for the animals. Water, feed and rest must be provided when needed. Transporters must: have authorisation from the relevant national authority for all journeys over 65 km; provide documentation containing details such as the animals' origin and owner, their destination and expected journey time; ensure that an attendant accompanies the animals, unless they are transported in adequate containers with sufficient feed and water. Competent authorities must inspect and approve means of transport for animals by road and sea for long journeys before these may be used. Keepers of animals and operators of assembly centres (holdings, collection centres and markets) must ensure that the rules and welfare standards are followed at the various points of departure, transfer and destination. Competent authorities must certify that transporters: are based in a Member State; have sufficient and appropriate staff, equipment and operational procedures in place; have no record of serious breaches of EU or national animal protection rules during the previous 3 years. For long journeys between Member States and to destinations outside the EU: transporters must have the necessary authorisation, documentation, satellite navigation system and contingency plans for emergencies; competent authorities must carry out checks on a random basis during the journey and checks at the point of departure. In the event of an emergency or failure to apply the welfare rules, national authorities can insist that the transporter take the necessary actions to safeguard the welfare of the animals being transported, such as: changing the driver or attendant; making a temporary repair to the means of transport; transferring the consignment to another vehicle; returning the animals to their point of departure; unloading the animals and holding them in suitable temporary accommodation.

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

Method Advantage Disadvantage Ear tag Readily identified Skill (correct placement), may cause pain and irritation Ear punch Tissue used for genotyping Skills for correct placement and numbering system, can be hard to identify if not placed correctly, hard with many animals Toe clipping Permanent Hard to justify, invasive Tattooing Permanent (useful for neonates), minimally invasive, Local anesthetics / general sedation/general anesthesia. Skills to put the ink intradermally. Microchipping Very precise identification, can be integrated with data recording and programs. Need for surgical glue to seal the hole Need for equipment (expensive)

VIII.7 Discuss relevant maintenance of anesthesia, and how to monitor the anaesthetic level and vital signs during both short-term and long-term anaesthetic procedures. Describe what problems that might arise during anaesthesia 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)

Monitoring in short and low-risk procedures: - nonelectronic monitoring: absence of reflexes (righting reflex - responding to up-side-down, withdrawal reflex - responding to noxious stimuli), pulse rate, circulatory functions, respiratory functions (color of skin or mucous membranes to assess arterial oxygenation), body temperature (rectal thermometer) - pulse oximetry: a simple method to obtaining relevant information about circulatory and respiratory functions, provides information of arterial pulse frequency and arterial oxygenation (oxygen saturation < 90 % = too low) monitoring long-term and high risk procedures: - circulatory monitoring: electronic monitoring of HR and rhythm using ECG, direct arterial BP - respiratory monitoring: tidal volume, percentage of end-tidal CO2 (using capnograph) and O2, concentration of volatile agents (gases), blood gases - we can also do endotracheal intubation and ventilation: standard practice in larger animals, less commonly used in small rodents due to technical difficulties, should be applied for all anesthetic procedures of longer duration, hypoxia can be circumvented by providing oxygen - but CO2 levels cannot be controlled without ventilation à risk of hypercarbia and respiratory acidosis. Problems: - animal responds to noxious stimulus / animal is moving - cause is insufficient anesthesia à action is to provide more anesthesia, provide analgesic or local anesthesia. - Poor respiration / poor circulation or oxygenation (animal pale or cyanotic) - Cause is too much or too long lasting anesthesia / too low body temperature à action is reduced anesthetic, provide oxygen, provide heat, provide fluid. If the nociceptive system is not properly turned off during anesthesia (if proper analgesia is not provided pre- or intraoperatively) and acute pain persists, central mechanisms may be altered at spinal or supraspinal levels à chronic pain, where pain becomes pathological because hyperalgesia or allodynia can occur

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

One male can have one or several females. Rodents are fertile at around 7 weeks, female rodents have a 102h cycle, including 12h estrus (4,25d). One can ensure estrus by vaginal smear (most epithelial cells are keratinized when observed under the microscope). Continuous mating: male with female all the time. Cycle mating: remove males every time they have mated → female alone with offspring. Time mating (when one needs to know exactly when murine animals are mated due to an experimental setup): vaginal smear → microscope → identify proestrus → put male and female together during the night → assess success rate by plug formation or microscopic examination (e.g. presence of sperm tails). Gestation periods for murine animals are approximately 3 weeks. Weaning is also at 3 weeks ish. Inbreeding program: pyramidal breeding, propagating the use of inbred animals such as brother x sister mating. Outbreeding program: rotational breeding. OBS: for species such as hamsters, guinea pigs, and rabbits other programs are more appropriate according to the biology of the species. e.g. for gerbils, a male and female stay together life-long, and must be put into a cage before becoming fertile. Case 1: take into account maturity, mating, gestation, and weaning when calculating how long it takes to have a given amount of pups for genotyping. (and productive mating/success rate).

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).

Optimal temperature for mice: 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 How the preference can be established: Brianna Gaskill and co-workers (2009) tested mice housed in groups of three with bedding, but no nesting material. Each group was given free access to 20, 25 and 30 °C in a preference test set up consisting of three cages connected with a tunnel. The mouse cages were submerged into a waterbaths to keep the temperature inside the cages constant. Of course no water was spilling into the cages. The location of each mouse and the occurrence of three behavioral categories (Active, Inactive, and Maintenance) were recorded to assess the preferences of the mice. Active behaviour was general locomotion; rearing and sniffing, whereas inactive behaviours were sleeping and still, but alert. Maintenance behaviours was grooming; eating; drinking; and nest-building(that is making shallow nestssites in the bedding, as no nesting material was present). The results show that both sexes preferred warmer temperatures only for maintenance and inactive behavior and this effect was most pronounced in females . It can therefore be concluded that C57BL/6J mice at 20-24 °C are not housed at their preferred temperature for all behaviors or genders, and that it 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. How mice can thermoregulate: mice are homeotherm (maintaining body temperature at a constant level, usually above that of the environment by its metabolic activity). Mice especially will regulate their temperature both via internal homeostasis regulation and by for example nest building. Mice are extremely efficient nest builders, and it has been shown that mice - provided with an adequate and sufficient amount of nesting material such as cotton wool - are able to build a nest in which the temperature is more than 30 °C even though the temperature outside the nest is below 0 °C. Body temperature is a very common parameter to score welfare. Someone uses a drop in temperature as an indication that the animal needs to be humanely killed. However, it could also be that the animal is just in torpor. The body temperature for mice is 36.5-38C. Fasting might induce torpor (reduced body temp and metabolism) in mice. This might make them limit the need for calorie intake, simply because they don't need the same number of calories to keep body temperature.

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

Ordering and storage: - Ordering at webpages - Stored cool, dry and rodent safe - Check batch number - Sterilization of the diet maybe (heat autoclave or irradiation) There are different ways of feeding laboratory animals for specific studies: - Ad libitum feeding - Restricted feeding e.g. for rabbits - Fasting (causes changes in the mice) - Gavage (tvangsfodring) oral dose with a tube - Pair feeding: Registration of the amount of diet eaten by the highest eating group in a two-group study, and subsequently feeding the other group the same amount. - Purified diet without a specific chemical (e.g. B12, C-vitamin for guinea pigs, essential fatty acids, amino acids) - Usually pathogen free OBS: Torpor (reduce metabolism and body temperature and activity as a result of fasting)

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)

Outbred: maintained by random mating, theoretically alleles are distributed in the populations as Heidel-Weinberg equilibrium. Different breeding types (Swiss Websters, Wistar etc.). rotational breeding. Inbred (isogenes): Mating close relatives (brother x sister mating e.g.) for at least 20 generations. Inbred BALB/c or BALB/k mice (albino mice). Inbred C57BL/6 or C57BL/10 (black mice). 99% of genes are homozygous due to inbreeding. There will always be a small genetic drift from generation to generation, and this will accumulate if you just keep on doing random mating. E.g., when passing more than fur generations away from inbreeding you will get late rejctions if you do transplantation randomly within the colony. So, it is a general agreement that we can do only these 3 generations aways from brotherxsister mating and still claim that the mice are inbred. Differences: price (outbred are cheaper), reproducibility (as inbred mice are more similar, the reproducibility is higher), purpose (researcher may want to investigate heterozygosity, therefore, outbred animals are more purposeful), variation (lower for inbred). In which ways different strains can differ from one another: "There now exist colonies of mice derived from the original C57BL/6 colony that have been bred in isolation from one another for many hundreds of generations. Owing to genetic drift these colonies differ widely from one another (and, it goes without saying, from the original mice isolated at the Bussey Institute). Responsible scientists, including those at accredited repositories, are careful to point out this fact and take pains to distinguish sublines such as C57BL/6J (the established subline at The Jackson Laboratory) from C57BL/6N, etc. But even within these sublines, the potential for drift exists in colonies maintained by individual laboratories who do not have a systematic practice of reestablishing breeders from a centralized, vetted stock" Suffering from their genetic constitution: inbreeding in general à less fitness (recessive traits evolutionary advantage to be so get more manifestation, less variation in genome of offspring, there might be meiotic disadvantages of inbreeding) when working with laboratory animals with specific genetics they must be intensely monitored as they might have welfare issues, genetic diseases, immunodeficiencies etc. specifically for inbred mice: mice with certain traits e.g. high blood pressure through inbreeding or might be that certain inbred mice are prone to spontaneously develop T1D,

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 are small experiments, the main aim of which is to test the logistics and feasibility of some larger study and, in some cases, to obtain preliminary information, such as whether dose levels seem appropriate. This information can be used in planning the larger study. Generally, the results of such studies will not be published. Pilot studies do not fix bias, variability or stuff like that. They help us discover preventable issues before doing the proper study. You need to have some assumption on the factors to put into the group size calculation. Often, you need to do a pilot study to get an idea of the variation and if it is a totally new disease/treatment might even the smallest effect size of interest. In the case, where you have data from a previous study, it may not be that needed with a pilot study. It is inadvisable to use a pilot study to estimate an effect size as the pilot study is not dimensioned to give such information (it is not big enough). By definition, if the pilot study could give information about effect size, there would be no reason to carry out the larger study. The way to estimate the smallest effect size of interest is by asking "what is relevant for me, what is relevant for my study, what is relevant for this experimental design to secure enough benefit to outweigh the harms". We can not use a smaller experiment to obtain information about a larger study. Due to publication bias where more positive effects than negative effects tend to get published (we are likely to get an overestimate of effects à we a likely to create an underpowered study), it is inadvisable to only consult present literature when deciding on an effect size.

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

Postoperative care: - Postoperative pain à provide analgesia - Dehydration à fluid therapy (1) body warm saline IV or SC during anesthesia and/or post-operatively 2) provide easy access to drinking water, extra-long drinking nipples or jelly liquid on the floor for easy ingestion) - Hypothermia (heating blankets (larger animals), infrared heating lamps or incubators (rodents), body temperature must be maintained until animal is fully recovered, be aware not to overheat the animals) - Infection à work aseptically, observe wound healing, provide antibiotics if necessary - Postanesthetic restlessness and self-injury à can be caused by pain or severe distress, consult veterinarian, use sedatives or analgesic if indicated Special needs: - Large animals: dedicated recovery room, warm (20-25) and quiet, supportive care Rodents: separative recovery area, incubator/warmed cage to maintain body temp (25-30 for adults, 35-47 for neonates, bedding should be soft and comfortable (avoid sawdust or similar), avoid unnecessary human contact and handling)

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).

Potential disease risks in the animal facility: zoonoses (bacteria, viruses, fungi, parasites spread between species) + humans + contaminated experimental equipment. Predisposing factors that may be relevant: animal models with higher risk of getting diseases + immune deficient mice with no thymus or T cells (nude, SCID, RAG1 KO) are very predisposed. Methods available for maintaining appropriate health status: look for infections, showers, chemical lock, autoclaving of equipment, circulation systems, PCR, MFI (bacteria cloning), health monitoring due to risk of infections such as parasites, viruses etc (microscopy, parasites in feces, serology, bacteriology, flotation etc). Protection against infections during breeding (caesarian section). Health reports are available online according to the FELASA. Presence of some bacteria is actually preferable (e.g. segmentated filamentous bacteria). But one can by these animals SPF (specified pathogen-free). Animals are bought with a certificate. - Another method: placing a sentinel (guard) animal in the animal facility with the only purpose of monitoring its health status as a pilot (guard) for other animals coming in Methods: · According to the EU directive 2010/63, we need to protect laboratory animals from infections. This can be done by: 1. Rederivation 1. C-section (old way do this) → transfer of pubs by c-section, clean them, and host them with a foster mother 2. Embryo transfer → sampling of fertilized eggs → fertilized eggs are placed into a germ-free female mice that has been mated with vasectomized and germ-free male (pseudo-fertilization). 2. Protection 1. Conventional open facility housing - not really used 2. Barrier → filters in ventilation, materials are autoclaved, staff entering through a 3-room shower, quarantine after contact with other animals → specific pathogen free and microbiologically defined 3. Isolator 3. Health monitoring 1. Health reports are available online according to the FELASA. Presence of some bacteria is actually preferable (e.g. segmentated filamentous bacteria) 2. Facilities send animals to laboratories that do their routine health monitoring → they do various assays to inspect various pathogens. Methods available for maintaining appropriate health status: PCR, MFI (bacteria cloning): 3. A sentinel (a guard): Animals placed in an animal facility with the sole purpose of being sampled for health monitoring

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)

Preanesthetic considerations: - evaluation of the animal - is it clinically healthy - look for discharge from eyes or nose, matting fur, soiling of perianal region with feces, if inexperienced ask for assistance from veterinarians and animal care takers, monitoring of food and water intake as well as bw change pre- and postoperatively - no hidden infections relying on good health monitoring system in our animal facility because animals do not manifest hidden infections - acclimatized: often 1-2weeks housed before procedures for mice and rats) - practical considerations (assistance needed, fasting) - the reason for fasting animals could be for specific experimental purposes (GI tract), prevent risk of suffocating (stomach content leaking into esophagus), body function not busy with digestion, prevent vomiting post-operatively - drawbacks for fasting: stressful, weakening, harmful for digestive function in species which eat continuously (most herbivores) - fasting in various species are different as the amount depends on the intestinal food passage rate as well as breed, age, sex, stress level etc. For example, rodents and rabbits we generally don't fast as they cannot vomit. However, we may want to for experimental purposes such as GI tract examination. - preanesthetic medications - reduces fear and stress, provides smoother induction of anesthesia, provides smoother recovery from anesthesia, reduces the amount of other anesthetic agents required (and be a part of a balanced general anesthesia), if usage of anticholinergics it can reduce the volume of salivary and bronchial secretions and block vasovagal reflex - preanesthetic medication often consists of a cocktail of different drugs - preanesthetic medication is often used in larger animals - in addition to the use of drugs, careful and expert handling of laboratory animals is an essential part of the humane preanesthetic management - choice of anesthetic drug/regimen (objective of experiment, surgical procedure, estimated time of surgery) - in experimental surgery, the anesthetic is chosen to make sure that the scientific data collection will be as accurate as possible. We don't want to choose some anesthesia that affect the particular system we are studying à we may need to accept loss of some animals, although not desired, because we want to have a as valid data collection as possible - advantage of using a regiment with combination of drugs (balanced anesthesia): lower dose of each drug can be given, minimization of the side effects from each drug while still fulfilling the anesthetic triad. - Disadvantage of balanced anesthesia: different side effects of each can interfere with the research protocol. - Inhalation vs. injection: - Advantages of inhalation include rapid induction and recovery, easy to control anesthetic level, can be used for prolonged surgery, while disadvanteges include need for anesthetic machinery and technical competence, could be harmful to the fetus in pregnant women - Advantages of injection include simplicity (no requirements of equipment), easy induction (allowing the animal to pass quietly into surgical anesthesia), while disadvantages include depth of anesthesia is less readily controlled, when a drug is injected it has to be metabolized or excreted by the kidney - it is therefore easy to inject an overdose, prolonged anesthesia calls for repeated injections or continuous infusion through IV access. - The effects of anesthetic drugs on model parameters: the anesthetic chosen affect several physiological, pharmacological and endocrine systems which is why the researcher should ask the designated vet and colleagues for guidance, consult the present literature, do a pilot experiment (so the researcher does not introduce bias by choosing a specific anesthetic), use checklists of criteria for selection of an anesthetic regimen for laboratory animals - monitoring of anesthesia and plan for action (if something goes wrong) - analgesia (pre-emptive, intra-, or postoperative = before, during and after the operation) - postoperative supervision and treatment (avoidance of any suffering after anesthesia) In all cases, make an anesthetic protocol, where all these preanesthetic considerations are listed so one has a clear view of how it is going to be performed and where one can also make notes (if something does not go as planned) Why do we do evaluations of animals: reduced mortality, faster recovery of the animals, less variation in exp. Results à better research quality

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

Public perception can be split into 3 overall positions related to animal research and toxicology: 1) Disapprovers (all sentient animals have rights that must not be denied) 2) Approvers (human interests are more important than the interest of animals, no matter the cost to the animals) 3) Approvers with reservations (harms and benefits must be balanced and animal suffering is not to be ignored). Disapprovers relate overall to the animal right view that states that a sentient animal can not be used as a tool or mean to reach a goal (opposing the view of the utilitarians). The animal rights view is based on the same principles that our society builds upon, namely that all humans have equal worth and an absolute right to be treated with respect. It is not possible to point out a single criterion for the moral exclusion of sentient animals which includes all humans (hence, all sentient animals, as well as humans, have an absolute to be treated with respect). Approvers relate overall to contractarianism, stating that as long as society allows for it, it is not a problem to use animals for experimental purposes. In other words, it is stated as "morality is based on an agreement". Narveson, 1983: "the contract view of morality is based on an agreement between individuals defined as rational, independent, self-interested persons, persons who have something to gain from entering into such an agreement". Approvers with reservations relate overall to utilitarianism, where a quality/value is assigned to an act and the total value of actions should be maximized no matter the means used. Here, people focus on the consequences of their actions. However, still, all suffering needs to be minimized. Danish Legislation: Utilitarian position with a hint of animal rights view.

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)

Relevance and need for preoperative assessment: Examples of sources of reference for GSP: The surgical procedure has to be planned by preparing a checklist of requirements including: 1) choice and availability of animals 2) preoperative evaluation of animal health 3) provision of facilities 4) surgical instruments and equipment (choice, provision, sterilization) 5) number of assistants required 6) preparation of animal for surgery 7) management of the animal (postoperatively and in an emergency) 8) protocol of surgical and anesthetic procedures 9) adequate practice (handling instruments and tissues) 10) record keeping. - Facilities and equipment for large animals: operating room, animal preparation room, surgeon preparation room, storage + instrument preparation + animal recovery, gas anesthesia, electrocautry, ECG monitoring, BP monitors, monitors for exhaled CO2. Operating and preparation rooms must be easily cleaned and have adequate ventilation. - for rodents: surgery should be carried out in a designated area - for survival surgery: a designated area separated from other activity, surgery and preparation of animal and surgeon could occur in the same room, but the sections should be separated from each other - for terminal surgery: a designated area on a lab bench is sufficient

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).

Rodents' senses of hearing: 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. Some rodent communication is audible to humans such as agonistic encounters in mice and distress/fear calls in mice and rats (for example during rough handling). However, other kinds of communication are outside out audible range and we are not able to hear it. This goes for all ultrasonic vocalizations (20-55 kHz) in rats including juvenile and adult rats in aversive situations (vocalize at 22 kHz), pups during social isolation (vocalize at 40 kHz) and rats engaging in rough-and-tumble play which vocalize at 50 kHz. Apart from communication, rats also use ultrasonic vocalization for navigating in the dark. Rats seems to emit very fast "clicks" in the same way bats do and use the reflections from the surrounding to navigate in total darkness. Whether mice do the same, remains to be shown. Rodents' senses of vision: Rodents most likely have a low acuity of vision, so the surroundings may very well be quite blurry to them. The challenge, when it comes to mice and rats, is that these species are dichromatic. They have only two classes of photoreceptors, namely ultraviolet and greenish and we have no idea what their worlds look like. Humans have four photoreceptors, and we can't see UV. Rodents' senses olfaction: as rodents are nocturnal animals, they have highly developed sense of smell. Olfaction is important for communicating for example in mice in relation to reproduction and when to uphold a hierarchy. 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. Also olfaction most likely also plays an important role in underground-navigation. Another point to make on senses: different rodent species have different senses à different handling is needed (example on albino rats having bad hearing etc)

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)

Sedation=compromission of consciousness (degree definition system) Anesthesia serves the purpose of bringing the animal in a state, where you can perform a surgical or other procedure, without the animal suffering. It contains some basic elements: reversibility and controllability (physiology after recovery should not be change ideally) Local anesthesia: block some certain nerves to get anesthesia in a local area. General anesthesia: all elements of the triad anesthesia are fulfilled. Before performing surgery, general anesthesia must be performed. Triad anesthesia = unconsciousness, muscle relaxation, analgesia (no feeling of pain) must be achieved in order to have general anesthesia. Balanced anesthesia = when applying two or more medications or techniques in order to help patients to ease pain, relax muscles and have autonomous reflection suppression (achieved by).

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 previous questions on their definitions, including examples of how to implement them - especially II.6. Why it is important to continuously improve the level of refinement: more refined procedures, models, handling à less harm (to the individual animal as well as leading to less variation and thus reduced number of animals), more benefit (better model quality). Additionally, as technology and science progress faster than legislation, it is important to always improve level of refinement if new technology allows or if new scientific data show that certain procedures, models, handling etc. can be refined.

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

Special hazards for scientists working with laboratory animals: allergy (LAA, laboratory animal allergy), physical injuries, harmful substances, zoonoses. And for animal caretakers, in addition: injury from heavy lifting, repetitive movements, noise etc. LAA: - Allergens can be exposed by skin context, inhalation, and injection accidents. Inhalation is must common exposure and manifest with rhino-conjunctivitis, asthma (wheezing, coughing, chest tightness, shortness of breath), skin reactions (urticaria, itching), anaphylactic shock. - How these can be prevented: keep the levels of allergens in the environment as low as possible, use personal protection equipment, do not take with you animal allergens when leaving facility. - Reduce level of allergens: use isolation caging, keep animal density down, use correct bedding material, effective and safe cleaning, safe transportation of animals, reduce number of transportations, monitoring allergen levels, ventilation related separation (positive/negative pressure), increased humidity - Reduce contact with allergens: protective clothing (face masks, gloves, particle-filtering respirators), restrictions of access to animal facilities, separate animal work from other work, clean/dirty areas, reduce the number of exposed persons - AND: monitor allergen levels, educate people, evaluation before employment (markers of atopy - sensitized people), health surveillance Physical injuries: - Bites and scratches from animals: are they infections or non-infectious? How to avoid?: learn how to handle animals, use proper restraint. - Treatment of animal bit (depending on size and depth): wash with soap, keep soaked in warm soapy water, elevate limp, if pain/swelling visit your doctor. - Sharp objects: from needles, scalpels, glass pipettes à details about how to handle and dispose these materials are found in the handbook of practical Harmful substances: - Biologicals: infectious agents used for experiments (viruses), cells that may contain viruses, toxins (PTX to induce EAE, diphtheria toxin used to activate genes in certain GMO mice) - How to prevent: warning on label on cage, only open cage in a biosafety cabinet or hood, gloves, eyewear, mask, coverall, good needle practice, screening of materials for human pathogens, disinfection/autoclaving of waste - Radioactive isotopes - Deterministic effects: cell deaths. Stochastic effects: cancer. Consider half-life, consider radiation type, - How to prevent: follow legal requirements for rooms and labs, education of personnel, have expertise available, keep and transport radioactive substances safely, prepare syringes before experiments, restrain animals properly e.g. anesthetize, inject in safety cabinet, use personal protection, keep animals enclosed, handle waste properly, clean up after work procedures. - Adjuvants - Used to make water-in-oil emulsions of immunogen. Often used to make antibodies. - How to prevent damage from adjuvants: Prepare any syringe in advance for each animal, work under safety cabinet, when injecting keep animals in safety cabinet, wear nitrile gloves, wear eyewear, ensure proper restrain and anesthesia can be helpful. Do not separate needle and syringe when discarding them. - Chemicals - Tamoxifen (to induce tissue-specific gene expression, but is a carcinogen), streptozotocin (to induce diabetes in animals by killing beta-cells, but is a carcinogen), inhalation anesthetics (isoflurane) - How to prevent: known the hazardous properties (kemibrug database at KU), take precautions during preparation (injection etc.), isolate animals, take precautions during waste handling, use proper PPE (personal protection equipment). Zoonoses (diseases that are naturally transmissible from vertebrate animals to humans): - Bacteria, viruses, fungi, parasites from animals, cells lines, tissue cultures, transplants, biological products by common routes such as ingestion, inhalation, inoculation, contamination of skin and mucous membranes. How to prevent: microbiologically defined animals, biological products from safe sources, barrier units (in the animal facility), good working routines (SOPs) - sanitary routines for staff, animals, biological substances, buildings, equipment etc. adequate PPE, actions in case of accident.

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 perioperative pain management are similar to those described for pain management in VIII.1. - Analgesic drugs affect physiology and thereby the experimental data. Yet, it must be remembered that the stress related to the surgical procedure and possible intra- and post-operative pain may have at least as high as impact as the drug itself. - Examples: morphine stimulates the HPA axis and the SNS to suppress the immune system (thus this analgesic cant be used when studying immune function), NSAIDs affect immune system by targeting COXs as well - For pain models: we should, if the model allows, treat against avoidable and unnecessary pain such as post-operative pain and when relevant pain is not tested - according to our morality, the legislation, maybe scientific data say we should too - How to avoid bias by analgesics? Choose the most appropriate analgesic regimen, but how? Study the literature, consult experts and discuss with colleagues, investigate if and how the model actually is affected. A key difficulty that may arise is when pain relieve is not possible due to pain response being the model. Here we must use other forms of refinement: non-pharmacological refinement: - Introduce early experimental endpoints: the animals do not have to be very sick in order to obtain relevant data - Strictly defined humane endpoints with actions specified - If animals are clearly affected within approved severity, facilitate the situation by easy access to food and water, comfortable bedding, tender love and care etc.

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).

Stress at a physiology-or-organism wide level can be divided into to different parts: 1) acute stress (sometimes referred to as positive stress) which can be considered as a biological reaction to any stimulus both physical, mental, internal, external that tends to disturb the homeostasis. Here, there is a rapid return to basis. Stress hormone in mice is corticosterone. 2) chronic stress (sometimes referred to as negative stress), which is a state of distress. This is an aversive mental state where the organism no longer can adapt (no production of stress hormones). There is a huge risk of suffering. State of dystress: prolonged levels of stress hormones. A state inbetween acute and chronic stress. Examples of stressors that cause fear, anxiety, pain, stress etc. in rodents: captivity (unnatural living), noise, transport, handling, restraint, procedures (exposed to pain/stress during experiments), lack of food and water, isolation, extreme temperatures, changes in circadian rhythms. Physiological consequences and manifestations of stress in rodents: pain and stress are closely related, and the consequences can be measured by physiologically and biochemically assessment: - Physiological parameters: HR, BP, BT. - Biomarkers: corticosteroids, 8-OH-DPAT - Behavioral and clinical signs: comparison to normal/stereotypic behavior, BW loss, food and water consumption, urination and defecation, facial expression. All this affect experimental data and animal welfare a lot.

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

The EU directive is firmly established on the basis of the 3Rs. Replacement (substitution of a conscious, living, sentient being with insentient material such as plants or microorganisms or in other words: Achieving the stated purpose of the study without conducting any form of procedures on sentient animals): - Substitution → if possible, use non-sentient animals to achieve the same scientific goal. - Absolute replacement → techniques that do not involve animals, e.g. in silico modeling. Another example: instead of expressing and purifying a protein in an animal, one could use bacteria for this if it does not compromise scientific outcome. Human volunteers can also be used instead of animals. - Relative replacement → cell cultures, animals defined as less sentient animals or invertebrates. Reduction: - Use as few animals as possible without compromising scientific animals. - Same quality of data → less animals - Better quality/more of data → same number of animals - Improved experimental design and statistical analysis, making sure that only the amount of animals needed to get valid, robust statistics is used - Improved techniques/methods (e.g. imaging) reducing the number of animals needed to obtain the data needed Establishing biobanks for sharing of animals tissues and other resources - Reducing variation in animal models - Establish better models, less bias, less variation Refinement: - To minimize actual or potential pain, suffering, distress, lasting harm and/or improve animal welfare in situations where the use of animals is unavoidable (when one has replaced and reduced as much as possible) - Shall be implemented in all stages of the experimental setup (breeding, housing, husbandry, transport, procedures/interventions) - Methods: Non-invasive if possible, Better staff, Most gentle methods/intervention, using appropriate anesthetics and analgesics, Better recognition of pain (staff education), Environmental enrichment (housing and accommodation facilities meet the basic biological and ethological needs of the animal), Cognitive enrichment → training of animals to behave with staff before intervening. The 3Rs overlap: if the phenomenon can be studied in cell culture instead of a mouse (replacement), then it also means that instead of using 20 mice, 1 mouse can be used to establish multiple cell cultures (reduction). At the same time, the previous experiment e.g. needs 20 mice to have enough going through sampling procedures for the appropriate scientific readouts, whereas now only 1 mouse gets painlessly killed (refinement). (in)Compatibility: - The 3R principles may be mutually exclusive. Reduction and refinement being incompatible in the bone fractures in hind dog legs (12 single fractures vs 6 double-fractures).

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)

The definition: "an experimental unit is the smallest element that we can assign independently to a group in our study" For some measures, limitations in the experimental design (e.g. water intake) we cannot identify the individual animal as experimental units, but instead, their cage becomes the experimental unit. Additionally, we can have more experimental units in the same animal - if we e.g. do an injection of two tumors, one on each side of the animal.

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

The importance of having an ultraviolet light source and being able to utilize it is unknown in rodents. Rodent urines are fluorescent, so it could be speculated that mice and rats also use vision when communicating by urine makings. Many light-sources, adapted to humans, in the animal's facility - such as a standard fluorescent light tube - have close to no ultraviolet wavelengths. Hence the rodents are provided with an un-natural source of light that may change their perception. We cannot be sure how this may affect the behavior of rodents; but we know that birds such as chickens and turkeys prefer a light-source including the uvpart and shows signs of stress, if they are denied this environmental feature. It could be speculated if this could also be the case with the mice and rats. Tail handling vs. cupping vs. tunnel à different amount of stress à differently effective in terms of anesthesia etc. - Hurst and West, 2010: mice handled with tunnel and cupped contact more voluntarily compared to tail-handling and they do not urinate and defecate as much as tail-handled mice. Mice handled with tunnel or cupping also do less protected stretch attend (how much they look out to discover if there is something dangerous in the maze) and they spend more time in the open field compared to tail-handled mice. Anxiety and fear in mice during handling can be assessed by: - If fear of humans, à no voluntary interaction with handlers hand + urination and defecation during handling - Anxiety can be tested by elevated plus maze, open filed (the more anxious, the less the animal will go on the open arms of the maze), other mazes (e.g. latency to move, freezing, stretching, thigmotaxis (staying along the walls), defecation)

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 of the HBA in animal experimentation is that the researchers should weigh the harm to the animal and the potential societal, economic, and social benefits from the experiment. It should be carried out by license holders (company and their legal persons or a named person within a university setting). The animal experimentation inspectorate (Dyreforsøgstilsynet) has an animal experimentation board (decision making body of the AEI) which reviews the license application and can reject the application instead of granting it, if harms outweigh the benefits or if procedures, handling, housing etc. cause intensive fear, distress, suffering, harm to the animals used (AEB can also reject based on other parameters e.g. lack of 3R implementation, no other approach causing less harm is appropriate). The harms (the costs) The benefits HBA · Animal welfare o Five freedoms · Animal rights o Intrinsic value of the animal o Respect for nature · Quality of the research · Compromising the animal welfare · Human harms of caretakers · Study outcome (scientific) o Human health o Veterinarian medicine o Nature · Educational · Economics · Social · Harms are certain o 3R compliance · Benefits are not guaranteed o Often difficult to predict · Should guaranteed harms during the study weight more than non-guaranteed benefits, which may lie many years in the future? · Check 3Vs to secure benefits Difference between construct and face validity: example with pancreatic surgery (a mouse model of human T1DM induced by removal of the pancreas results in high face validity as there is absence of insulin in the blood stream, however low construct validity as it does not represent human etiology of T1DM)

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)

There's no clear definition of culture of care in the laboratory animal community. Culture of care is a commitment to improve both care of animals and staff. The Term "Culture of Care" is NOT explicitly described in the EU directive. Yet, the animal welfare body "needs to foster a climate of care". - The animal welfare body can initiate a good culture of care by: - Collaboration with senior management - Encourage contribution from staff - Collaborations between scientist and care staff - Communicating the 3Rs (conferences, articles, etc.) Why it is important: "happy animals make good science" (like II.9, for both ethical and scientific reasons, by promoting a culture of care, we reduce the harms while increasing the benefits). Loop effect: a culture of care among scientists/animal caretakers/lab techs/vets leads to better animal welfare which lead to better job satisfaction etc. etc.

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)

Think this question as securing animal welfare definitions, 5 freedoms etc. (those measurements are essentially monitoring housing and husbandry routines at the cage level, whereas establishment and holding room monitoring is more about ventilation, air, temperature, humidity etc.) Suitable housing and husbandry routines: - legislation on housing concerns the establishment (opstaldningssted), holding room (opstaldningslokale), animal enclosure (forsøgsdyrsanlæg, e.g. a cage) - aims of the housing and husbandry include: it needs to meet regulatory demands, ensure animal comfort and well-being, control the animal's environment for GSP. Many variables should be considered: genetic, microbial, chemical and physical. - Minimum requirements according to Danish and European legislation: - The environment in the animal rooms: ventilation and air, temperature, humidity, lighting and circadian cycle, noise, alarm systems - Health and care: an animal needs space to be in, surface to be on, a place to rest, hide, feel safe, and have company of other animals. - Caging: environmental enrichment, dimensions, food and water supply, bedding material and cleaning, handling - Cage types: open cages (can be either put or not put in scantainers which protect both immunodeficient and transgenic animals as wells personnel from allergens as all supply and exhaust air is filtered), individually ventilated cages (IVCs=airtight animal cage with air supply and exhaust system, animal room with very few allergens, animal room odor free), isolators - Husbandry is more about caring for the animals: tasks typically carried out by animal care takers such as receiving, marking, cage changing, cleaning, feeding, watering, breeding, weaning, observing, killing. - Enrichment: any initiative which increases the amount of species-specific behavior and reduces or eliminates abnormal behavior. - There are legal, ethical (animal welfare) and scientific (an animal with a natural behavior gives a more valid interpretation of nature) reasons - Five methods for enrichment: behavioral enrichment (mimicking the wild habitat, e.g. nest-building allowing behavior for mice), social enrichment (e.g. company of other animals), artificial enrichment (e.g. mice very much like running wheels), food enrichment (the animal must spend time searching for the food) How conditions are monitored: for example, the rodent cage needs to have bedding, housing, nesting material and something to chew on. Is this monitoring of the conditions? Consequences for the animal resulting from inappropriate environmental conditions: lack of control of the environment can cause frustration and stress (when the animal can't control feeding, lighting, whether it wants to be hidden or out in the open field etc.).

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)

Variance (technical/biological): the overall difference in primary readout values from individual samples (higher → more animals). More complexity in the animal model means a need for a larger sample size. Is key that you try to limit the variance by having the right tool for measurement. Some variance makes sense to limit, yet, we cannot standardize everything so that the experimental outcome no longer reflects the real-world phenomenon. Effect size: This is the difference between the mean of two groups (quantitative data) or the proportions of events in two groups (qualitative data). The effect size should reflect the limit on how small of an effect that we consider an effect. Therefore, we cannot use pilot studies or published data on similar studies to estimate the effect size. Instead, this should be reflected on the investigators expertise within the field, e.g. a drug that should lower blood pressure should lower it by xx mmhg to have an effect. Level of significance: how willing we are to accept false positives (the thing we are studying has not effect, but it looks like we have, type I errors), a limit we set before our study. For each added readout (2nd readout), one wants to increase the significance level according to the Bonferroni Correction Statistical power: Power deals with false negative results. It is our ability to avoid false negative results (the thing we are studying has an effect, but we are unable to see it, type II errors). "the smallest effect size of interest", typically research has a statistical power of 80 % - meaning 80 % chance to find the smallest effect size of interest.

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

Viewpoint 1 (Health and biological functioning, welfare = good health): physiological parameters, clinical examinations, and evaluating statistics - Measurement: studies of reproduction, clinical examination of heart rate, blood oxygenation, temperature, appearance (more qualitative). - It is important to consider how the measurements were obtained (could the procedure introduce a bias). - Combining multiple measurements can give better assessments - One can use an ethogram to assess health and biological functioning - Specie-specific behavioral scheme - Very extensive Viewpoint 2 (Natural living, welfare = natural living): realize your potential, perform natural behaviors, species-specific - Measurement: behavioral studies by assessing natural behavior - Semi-natural environments - Released into the wild - Ancestor behavior in the wild Viewpoint 3 (Emotions and preferences, welfare = positive emotions): presence of positive mental states, absence of negative mental states, having your preferences fulfilled - Measurement: also behavioral studies (joy, care, playfulness, safeness) - Vocalization is often a measure of mental state - Negative mental states can be measured by the elevated maze (does it stay in the safer zones or not). - Assessing the preferences of animals → what type of bedding would the pig prefer could indicate certain behavior. - Strength of preference can also be assessed → how willing is the animal to do a task that is more preferable than a less preferable but with higher performance Evaluation of animal welfare: defining animal welfare is important as we need to separate the nature of its existence (animal can feel well or not) and how to measure it (the animal feels well or not due to the existence of this or that behavior/parameter/measurement). This allows for the evaluation of animal welfare and thus adheres directly to assessing harms/benefit as well as trustworthy science (well and happy animals make good science). If a caretaker observes that an animal is misbehaving, the following actions to prevent this would depend on the viewpoint of animal welfare (anti-anxiety drugs vs. socializing with other animals in an anxiety situation - the former being viewpoint 3, the latter being 2). However, those animal welfare definitions overlap. Whether animal welfare is important for moral reasons depends on the fundamental ethical view and the predominant ethical position in any given society.

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)

We decide on the numbers of animals before we start to fulfill the 3Rs - underpowered studies are ethically problematic, as no usable knowledge is acquired. Also, if the reduction or refinement are not implemented prior to starting the study, we could actually have reduced the number of animals used due to the lower variation secured by refining the study in order to have the same power and significance. Legally is it required to do proper calculations on the amounts of animals used for the experiment (also when publishing, many journals require you to explain how group sizes were calculated). So basically, the sample size needs to be calculated prior to experiments so we do not waste animals unnecessarily (underpowered studies e.g.). The sample size can be calculated by equations that always use the following four inputs: smallest effect size of interest, estimate of the variation (standard deviation), desired levels of significance and desired level of statistical power. Usually, we need to carry out a pilot study, consult literature or similarly in order to know something about variation, effect size.

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)

When breeding animals, it is important to use high-protein diets. Dietary requirements Carbohydrates Fat Proteins Minerals Vitamins Digestible Fibers Essential fatty acids Essential amino acids Na+ Ca2+ Zn2+ Mg3+ Fat soluble Water soluble These nutrients factors can be in food pills (pellets using compression) or as extruded diet with highest digestibility (using 180°C hot steam "cooked diet"). The natural ingredient diet (CHOW) Used for Based upon Pros Cons · Breeding · Maintenance of ordinary adult animals · Natural ingredients · Whole grains (corn, barley, wheat) · Mill by-products (bran) · High protein (soybean, fish, casein) · Minerals (bone) · Cheap · Nutritious · Contamination risk · Variation · Single components non-removable High calorie diet is to make the mice obese The purified diet Used for Based upon Pros Cons · Specific studies · Synthetic diet · Protein (casein, amino acids) · Carbohydrates (sucrose, starch) · Fiber (cellulose) · Fat (corn oil) · Standardized · Single components removable · Aversive (animals don't like it) · Expensive · May lack biologically active compounds important for the model Purified diets are used in studies with specific dietary requirements e.g. if you want to investigate the function of caecum in the gut. Then you could use a diet with and without fibers. Purified (pure chemicals) diets are not necessary for animals as such. Natural diets would be perfectly fine. Purified diets are much more expensive; however, they might be useful if one wants to achieve e.g. a diet without an essential amino acid such as leucine, isoleucine, valine etc. Extruded diet has higher digestibility than the pelleted diet. The difference between the pelleted (ordinary, a bit like a cold meal, a "raw" diet) and extruded (a bit like a warm meal. a "cooked diet") diet is that the pelleted diet is made on a meat mincer mixed with water, whereas the extruded diet is made through a "shotgun" warming up the food.

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)

When one plans one's study, one has to write down the termination end points for animals (humane end points). How to properly kill animals is described as function D of the four staff functions (A,B,C,D) of the EU Directive. One can not allow an animal to stay in the experiment if it becomes sick (for example, if it gets bloody diarrhea). It either needs to be treated or removed from the study. According to AEA, animal experiments can only be performed if the animal is: 1) locally or generally anesthetized 2) pain releivers or other measures are used to limit pain, suffering, anxiety or lasting harm as far as possible UNLESS anesthesia is considered: - more stressful than the procedure itself - incompatible with experiment 3) not experiencing strong pain, other forms of intensive suffering, intensive fear IF SO at the termination of anesthesia/a relieving treatment, THE ANIMAL MUST BE HUMANELY EUTHANIZED.

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

Why are the ARRIVE (Animal Research: Reporting In vivo Experiments) guidelines important? It is all about reproducibility, there are certain criteria that many scientific journals see as requirements to publish data. ARRIVE GUIDELINES: - Title: correct description of the content - Abstract: accurate summary of the scientific findings - Introduction: - Why the animal species and model being used can address the scientific objectives - Study's relevance to human biology - Material and methods: - Ethical statement - Ethical review permissions - Relevant licenses - National and institutional guidelines for the care and use of animal - Study design - Number of groups - Steps to minimize bias - Randomization - Blocking - Blinding - The experimental unit - Species, genetic status (outbred, inbred, hybrid, mutant), source, age & weight, sex, microbiota status, QC, and acclimatization. - When describing the genetic status, use the ILAR nomenclature. - Experimental procedures - How/when/where/why - Sample size - total number of animals used and how this number was achieved - Independent replications - biological - technical - How was the animals allocated - Experimental outcomes - Primary and secondary readout - Statistical method description - Housing and husbandry - detailed description of everything from bedding material to status of the water, microbiome and diet. (ventilation, humidity) - Discussion: - Limitations to the animal model, bias, etc. - 3Rs implications - Relevance to humans

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)

Why it is important: it is in our legislation + it makes us consider if our models and their characteristics are most appropriate to secure best quality science (data about a modeled phenomenon as closed to "reality" as possible à data depicting "real world" à data can be used to most efficiently and correctly intervene with the modeled phenomenon, e.g. in the context of pharmaceutics) and to secure animal welfare. If we can't justify on ethical grounds (that is, based on HBAs/utilitarianism, animal rights view etc.) why we use living animals, then we shouldn't. Same goes for origin of models, estimated numbers, life stages etc. It makes us deeply consider our experimental design and the potential outcome. The benefits should always outweigh the harms and that is part of justifying it. When we choose the model, it is stated in the legislation that we should choose the model that best models our phenomenon. However, we should also consider choosing the model that is less prone to experience harm, suffering and distress. Also, when choosing a model, we need to consider that maybe one model can be trained for one procedure (dog) that others cant (mice need to be restrained for the same procedure). The estimated numbers should always be estimated based on power analyses (no waste of animals based on under- and overpowered studies). For life stages: if you can use a life stage where the sentience of the animal is less developed without compromising the scientific outcome, you should do so. Some life-stages are considered insentient. Life stage is also defined according to trimester (e.g. if you go into the last trimester of pregnancy in mammals and study fetuses, this needs a license).

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

bias is something that influences our experiment in a non-random way. Factors: the ordering of tests, time of day for tests, different experimenters, lights, sounds, smells, temperature Actions to prevent them: blinding, randomization (turning bias into random variation) e.g. of random assignment of case and control groups, blocking (creating blocks to ensure that one parameter is not present in only one group - sometimes we don't know which parameter to take into account) or even more blocks such as matched pairs (e.g. if we do blocking according to BW, we create pairs of animals closest to each other in terms of weight and then flip a coin where one goes into control group and the other into treatment group). As it might be difficult to choose the parameter from which the blocking is done, we can reframe experimental units (e.g. 2 tumors in each side of the animal being case and control) to create the "same" parameters for the experimental units (same mouse is its own control and then animals are perfectly matched with respect to every possible characteristic). Instead of having set-ups where one mouse exists as its own control at the same time (1 tumor in each leg at the same time), we can do cross-over designs (e.g. as full cross over design or as Latin square design, but one has to take the "carry-over" effect into account) where we have to treatment periods (control or treated) separated by a wash-out period. Simulation-based approach to sample size estimation is a modern way to calculate sample size.