Laboratory animal science ABD

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)

It is important to evaluate the animal health before a surgery or procedure. They should be clinically healthy and should be examined by a veterinarian. The animals should not carry hidden infections and they should be acclimatised prior to surgery. In some cases you could also apply conditioning where you change the animal behaviour by a stimulus. So you could make the animal more comfortable around you by conditioning and therefore cause less stress in the animal. If you start your procedure with a health animal in good condition you would reduce the mortality, induce faster recovery, and reduce the variation in the experimental results and therefore get better research quality. In the textbook you can find more comprehensive text references regarding the important principles of surgery e.g. the British Laboratory Veterinary Association 1992. Before performing a procedure it must be carefully planned to make sure that everybody involved knows what is going to happen. You should therefore prepare a checklist of requirements that should be done prior to the procedure. This often includes: 1. Choice and availability of animals 2. Preoperative evaluation of animal health possible assisted by a veterinarian 3. Provision of facilities 4. Surgical instruments and equipment 5. Number of assistants required 6. Preparation of animal surgery 7. Management of the animal 8. Protocol of surgical and anaesthetic procedures 9. Adequate practice in both handling instruments and tissue 10. Record keeping All personnel involved in the procedure should have the adequate practice. You have to make sure that if they are performing any procedure on the animal, they should have a certificate to do so. They should also be able to euthanise the animals if necessary. Prior to the procedure it is also a very good idea to make sure that you can perform the procedure without causing unnecessary pain and suffering in the animals. So it is important to practice the procedure maybe in some test animals before you conduct the "real" experiment. In regards to the care of the animal before, during, and after a procedure you should always handle the animal gently to cause minimum stress. Before the procedure you should make sure that the animal is not stressed out. If the animal needs to be fasted you have to make sure that the have access to water. If they are fasted for a prolonged period of time you should also make sure that the water contains glucose or electrolytes. Under the procedure we should make sure that the oxygen saturation is acceptable and that the animal is not experiencing hypothermia. They should still be handle gentle and with compassion. After the procedure we should make sure that the animal is not in pain and administer analgesia if they are. We should make sure that they get adequate amounts of water which can be done by fluid therapy. We should make sure that they can maintain a proper body temperature and if not provide them with heating blankets, incubators, bedding, and such. We should also observe the wound healing and provide antibiotics if necessary. In general we should do whatever to optimise the health and well-being of the animal and we should make sure that they are in a good condition and that they do not experience unnecessary suffering, pain, or distress.

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

Mice and rats are homeotherm which means that they are able to maintain a stable internal body temperature regardless of external influences. Mice especially will regulate their temperature both via internal homeostasis regulation and e.g. by nest building. Both female and male mice prefer warmer temperatures but they may not prefer the same temperature for everything. They may prefer one temperature for active behaviour, one temperature for inactive behaviour, and one temperature for maintenance behaviour. This can be investigated by setting up three different temperatures in three different cages with no nesting material. The behaviour of the mice may be studies as well (active, inactive, maintenance). The cage where the mice stay the most may be the preferred temperature for that activity. It may therefore not be possible to select one single preferred temperature for all mice and it is important to provide mice with the opportunity to thermoregulate by nesting.

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

Reuse is the using of the same animal to yet another experiment after the animal has been used in the first experiment. You cannot however just reuse all animals. It is only allowed to reuse animals if the severity of the first experiment has been mild or moderate and the new experiment also is just mild or moderate. If it is not then it has to be non-recovery. You should also ensure that the health and wellbeing of the animals is fully restored in the first experiment and the veterinarian must advice that the reuse is acceptable. In exceptional cases after a veterinary examination, the inspectorate may allow reuse of animal used only once with severe pain, distress, or equivalent suffering.

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)

The animal facility can be located in a separate building or among other functional areas inside a shared building, but will often be located in isolated areas. The animal facility typically consists of one or more units depending on the size of the facility. Each unit can contain one or more rooms e.g. housing rooms for animals, rooms for experimental procedures, storage rooms, etc. The animals should be kept in enclosures inside the rooms and outside the room, there will be space for shared equipment. Everything must be constructed and managed to keep the animals healthy, happy, and clean. Animal facilities typically have a dedicated staff responsible for the care of animals, including feeding, housing, and monitoring their health. The staff also follows strict protocols to maintain a clean and safe environment, including regular disinfection of cages and equipment and the use of personal protective equipment (PPE) when handling the animals. To maintain an appropriate health status for the animals they can be housed in different ways. The animals may have barrier housing which is designed and managed to protect animals inside the barrier from unwanted microbes. It can also be used where experiments with pathogens are carried out. The air pressure is also regulated either to make sure that pathogens does not come in to the facility or allergens does not leave the facility. There will also be barriers for personnel before entering the facility to maintain high health status for the animals. Furthermore, the animal facility staff must monitor the animals for any signs of illness, disease, or abnormal behaviour. Acclimatization, habituation, and training are important processes that can have biological consequences for the animals. Acclimatization refers to the process of gradually adapting animals to a new environment, It allows the animal to maintain performance across a range of environmental conditions. The changes in condition is associated with effects of physiological systems. Before conducting any experiments, you should therefore make sure that homeostasis is restored and that all physiological systems are back to normal. A period of one to two weeks is regarded to be sufficient even though some physiological functions may require much longer to return to normal. This process can be stressful for the animals, and if not done correctly, it can compromise their immune system and increase their susceptibility to disease. Habituation refers to the process of animals becoming accustomed to handling or specific procedures, such as injections or blood sampling. This process can reduce the stress and anxiety associated with these procedures, making it easier and less stressful for both the animals and the staff. When animals are trained and habituated there is a risk that they might loose some of their natural behaviour. When animals are reinforced to do certain things, they might start doing these things as their natural behaviour instead of their actual natural behaviour. To sum up, acclimatization, habituation, and training are important processes that can have both positive and negative biological consequences for animals.

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)

When designing housing and husbandry it is important to remember the aims of the housing and husbandry as well as the regulatory demands. You should also consider the comfort and well-being of the animals and the animal welfare is also important. Different environmental factors must be controlled to ensure standardised conditions for scientific experiments. It is important that the holding rooms for animals should be constructed and ventilated in such a way that animals of different species can be separated physically and with no sight, sound, or smell of other species. Laboratory animals have various needs that must be considered, and these includes: A space to be in, a surface to stand on, lay down on, and walk on, a place to rest, perhaps a place to hide, food and drink should be provided, social animals need the company of other animals, the temperature should be appropriate, there should be fresh air to breath, there should be a cycle of day and night, the animals should be able to keep themselves clean and dry, and they should be able to perform activities. All in all the animals must feel safe and should be able to perform natural behaviour. Good hygiene is imperative for high quality animal husbandry, and it is essential to minimise the introduction and spread of infectious agents and to avoid stress in the animals. Fx the behaviour of rats can be affected when changing the cages and when provided with clean bedding, It is therefore recommended that a small amount of the dirty bedding stays in the clean cage in order not to totally disrupt the olfactory environment. All the husbandry routines must be adhered to. Changes from daily husbandry routines upsets the animal, and consistently and habit are important to allow the animal to feel confident and therefore thrive and reproduce. Cages, pens, and other enclosures used for housing laboratory animals should allow sufficient space for them to express behavioural, and the environment should provide reasonable complexity, rendering species-important behaviour possible. It would also be reasonable to provide sufficient space on the basis of what an animal needs to use, as opposed to a simple relationship with size. This is however not always the case and cage size is almost exclusively based on body weight rather than developmental stage. Laboratory animals should be housed in pair or groups with members of their own species. Only scientific or veterinary reasons should justify the practice of housing animals singly. Cages and pens should always be provided with appropriate bedding materials and shelters in which the animals can hide or escape. It allows the animal to remain dry and clean and to create and control its own microenvironment with regard to temperature and light if the bedding is present in quantities sufficient to allow burrowing. The provision of shelters reduces the ability to observe the animals and monitor their well-being from the outside. This can be solved by using transparent shelters (or red shelters). Acclimatisation of animals to a new room or cage takes some days, and it is important that this period of acclimatisation be permitted since it may affect the scientific data.

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

You can find information about laboratory animal science and laboratory animal care on different homepages. You can find information on the European Commission's homepage, as well as in various articles posted online in different journals. You can find information about the 3R's on the Danish 3R-center as well as on the national centre of the 3R's. It is important to continuously stay updated on how to optimise animal welfare during housing, handling, and procedures because we continuously learn and discover new things. What I mean is that for a very long period of time we have handled mice by the tail. It has been the recommendations for a long period of time. At some point somebody discovered that it was much more appropriate to handle mice with a tunnel. This would cause less stress to the animals.

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

A culture of care has come to indicate a corporate as well as personal commitment to improve animal welfare. The culture of care aims to improve welfare for both humans and animals and the 3R's will be an integrated part of a Laboratory Animal Facility's culture of care. A part of the culture of care is to implement the 3R's to the highest possible degree and thereby reducing animals used by replacement and reduction. It will also reduce animal stress, pain, and suffering by refinement. A high level of 3R compliance will reduce the emotional strain on animal care staff. The discomfort and suffering in the animals are reduced to a minimum and the staff know that all possible measures to reduce harm to the animals have been taken. This will therefore support job satisfaction.

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

A humane endpoint is the earliest point at which an experiment can or should be terminated with the purpose to eliminate unnecessary suffering and pain. If the animals reach a certain degree at being at risk of suffering, then we must stop the experiment before we have reached the experimental endpoints. In order to make these humane endpoints we should identify and use early behavioural and clinical signs in combination with model-specific signs. We need to know the signs of suffering in order to set humane endpoints. These may differ depending on the severity of the experiment. Body temperature, body weight, behavioural changes, pathological changes, and blood oxygen saturation are examples of criteria that can be used to implement humane endpoints. When the humane endpoints are reached, the animal must be withdrawn from the experiment. Depending on the suffering of the animal, it should either be treated or euthanised. You could contact the veterinarian for consultation about the actions that should be taken. You could also modify the experimental design to avoid pain and distress by identification of non-clinical humane endpoint criteria. These may occur prior to any observable suffering or clinical manifestation of a condition. An example of humane endpoints: A kid's competition where you have to eat 10 hotdogs in 5 min. This is our experimental endpoint. The humane endpoint could be that a kid has to be withdrawn from the competition if the kid pukes. Puking is causing pain and distress in the kids so in order to minimise the suffering of the kids we refine the method used. We can set another humane endpoint saying that if a kid turns pale or greenish, they must be withdrawn from the competition even though they have not puked or finish the 10 hotdogs. We don't want the kids to experience any suffering. When assessing and recording the welfare of animals a welfare protocol could be used to detect and define humane endpoints. You design a score sheet with relevant parameters that can help you identify if anything goes wrong with the animal. It could contain information about appearance, behaviours, and clinical signs.

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

According to the Animal Experimentation Act, you must have a licence in order to act as a user, breeder, or supplier of laboratory animals. You should therefore both have a licence in order to use laboratory animals, to breed laboratory animals, and supply laboratory animals. The holder of the licence may only allow others to perform the experiments, if they have the necessary training and work under management and supervisions of the holder of the licence. The licence will be issued by the Animal Experimentation Board which is a part of the Animal Experimentation Inspectorate (dyreforsøgstilsynet). The licences will be issued after careful evaluation of the applications and they will only be licenced to exactly what is implied in the applications - no more, no less. The experiment should have an acceptable cost-benefit balance and have 3R's implementation. This means that you should use the most refined procedures, use alternatives if possible, make sure that the severity is okay, and that you minimise the severity. The licence to do experimentation on animals is issued to a named individual or legal person. All experimentation should be performed by qualified staff in proper settings in destination bred animals. When acting as a user of laboratory animals you must have adequately education depending on your function. In the EU directive there is 4 function predefined. A. Carrying out the practical part of the procedures of an experiment B. Setting up the experiment and holding the licence C. Taking care of the animals in the facilities D. Killing the animals If you in some way are going to participate in the experimentation (function A) you must have gotten training in function D because there always should be one present that can euthanise the animals. Beside issuing licences, the Animal Experimentation Inspectorate inspects experiments and facilities, and accredit facilities for use in animal research. They are a part of the Ministry of Food and Environment and the inspectorate is led by an 11-member committee (Animal Experimentation Board). The board members can be experts or lay persons and they are appointed by the minister of Food and Environment after proposals from different specified organisations. · 4 members are proposed by the animal protection societies · 1 member is proposed by the animal ethics council · 1 member is proposed by the medical research council · 1 member is proposed by the technology production research council · 1 member is proposed by the patient NGO · 1 member is proposed by the industry · 1 member is proposed by the governmental health agency All members are led by a judge called the chairman which makes the 11 members of the board.

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).

According to the Danish legislation animal experimentation must be of essential benefit and this benefit must counterbalance the harm inflicted on the animal. This is therefore an utilitarian position because you consider both the human benefits and the cost of the animals. On the other side the legislation also states that animals must not experience strong pain, other intense suffering or intense fear, and must be killed if the condition must be assumed to exist at the discontinuation of anaesthesia or alleviating treatment. So here it is seen that the animals have been given the right not to be subjected to strong pain, intense fear, and intense suffering which thus can be argued to reflect the animal rights view. If we look at the European directive it is based upon replacement, reduction, and refinement. Member states shall ensure that whenever possible replacement options should be considered instead of using live animals. Member states should also ensure that the number of animals used in projects is reduced to a minimum without compromising the objectives of the study. Lastly, member states should ensure refinement of breeding, accommodation and care, and of methods used in procedures. This should be done to eliminate or reduce to a minimum any possible pain, suffering, distress, or lasting harm to the animals. In these 3R's it is also seen that animal suffering should be reduced to a minimum without compromising the study which could translate to that the risk or the cost should be minimised but without also minimising the benefits. So, we should have the lowest cost giving the highest benefits. In the European directive it is described how the essential, inner nature of the animal matters in itself. It is also described how we as human beings have moral obligations to ensure that the animals will experience as high a level of welfare as possible. The ethical view in the European directive is therefore predominantly the utilitarian view.

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

All European countries have to follow the European directive 2010/63/EU. The aim of the directive is to strengthen the legislation and improve the welfare of those animals still needed to be used. The aim is also to apply the principles of the 3R's Replace, - to Replace, Reduce and Refine the use of animals. The EU directive is unique in the world since its ultimate goal is full replacement of use of animals for scientific purposes. The 2010/63/EU directive is far more detailed and strict than the directive and convention from 1986. The directive is a harmonising directive which means that the member states can only have stricter rules if they were already applied according to the 1986 directive. The Danish legislation is therefore based upon the European directive. We do however have some stricter rules based on the directive from 1986. This is e.g. that there is an upper limit saying that you cannot under no circumstances induce strong pain, intensive suffering, or intensive fear in laboratory animals. This is no matter the purpose! In Denmark, the common rules for protection of animals can be found in the Animal Welfare Act. We have however implemented the common European legislation for animal experimentation into the Animal Experimentation Act and the Animal Experimentation Order. According to the Animal Welfare Act no one is allowed to cause pain, suffering, distress, or lasting harm in any species of animals. However, according to the Animal Experimentation Act pain, suffering, distress, or lasting harm can be allowed if the purpose is research, teaching, or the production of blood products and if there is a license in each specific case. The Animal Experimentation Act therefore overrules the Animal Welfare Act. The Animal Experimentation Act contains some general principles about animal experimentation. It also states that the experimentation should be beneficial, the use of animals must be necessary, and that no matter what the animals cannot experience strong pain, intensive fear, or intensive suffering. The Animal Experimentation Order contain details regarding how to make animal experimentation, how to set up the facilities, and how to take care of the animals. You can find information about the European legislation on European Commission's homepage with links to EUR-lex where the directive can be found in different languages. In Denmark you can find information about the legislation on retsinformation which is the official Danish online legal portal covering legislation, orders, circulars etc. from government departments.

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

All animals recovering from surgery and anaesthesia require monitoring and specialised care. The focus of the postoperative care should be on returning the animal to, and then maintaining, normal physiological parameters. The animals should also have dedicated rooms or areas to recover which should be warm and quite. Larger animals should get supportive care since it will provide a better and easier recovery. Rodents should not be given supportive care since it could cause unnecessary stress. There could arise different problems postoperatively and there are different solutions to these problems. The problems and solutions are · Postoperatively pain <> provide analgesia · Dehydration <> Fluid therapy o This could be body warm saline IV or SC, or the provision of easy access to drinking water · Hypothermia <> Heating blankets, infrared heating lamps, incubators o The body temperature should be maintained until the animal is fully recovered but without overheating the animal · Infections <> provision of antibiotics o The aseptically working could also help to avoid asepsis · Postanaesthetic restlessness and self-injury <> sedatives or analgesia o This can be caused by pain or severe distress and you should always consult a veterinarian if this happens

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)

An animal experiment is defined by a lower threshold, an upper threshold, and a purpose. The lower threshold is also called the injection criteria and the upper threshold is strong pain, intensive fear, or intensive suffering. The injection criterion means that a procedure must be licensed if it causes pain, suffering, distress, or lasting harm equivalent to or higher than that caused by the introduction of a needle in accordance with good veterinary practice. This also applies even if no injections are involved and could be in cases where you e.g. feed the animals a diet deficient of essential nutrients to induce specific deficiency symptoms. So mild procedures that does not violate the animal welfare act is not subject to a licence. In Denmark our legislation states that you under no circumstances can induce strong pain, intensive suffering, or intensive fear in laboratory animals no matter the purpose.

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

Case-control study: fx tumors treated with drug and tumors not treated. We contrast experimental cases with controls. A more complex study is the cross-over design, here the animals are their own controls. Here the same group does the control and the experimental treatment, divided by a wash out period of time in between. There are however drawbacks of cross-over designs, this is time consuming and is impossible to use if the effects remains after treatment + washout period (irreversible drugs). The tumour might not grow back to its original size. A more complex design is e.g. the Latin square design which is a more elaborate version of cross-over study. In these types of study designs we can test more than two conditions. We create a pattern where every animal undergoes each treatment and each treatment is equally represented in each time period. The guiding principle is that each set of subjects is exposed to every treatment and every treatment is equally represented in each time period to avoid bias. Like this, fewer animals are used and a lower biological variation because each animal serve as its own control (further reduced use of animals).

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

Animal welfare is important for scientific reasons relating to the quality of the data obtained by the use of living animals and the resulting translatability and reproducibility. To ensure valid data it is therefore important that animal welfare is not compromised to the extent where the animal is experiencing fear, pain, or prolonged stress. If animal welfare is not ensured it could have an adverse effect on physiology, immunology, neurology, and behaviour. The challenges arise from procedures that evoke fear and/or pain where stress will be the common denominator, fear and pain will affect animal behaviour, and the stressful conditions will affect the immune system, the cardiovascular system, and the reproduction. The challenges could also arise from insufficient housing environments. In relation to the ethical reasons to have good animal welfare this will very much depend on the ethical viewpoint. From a utilitarian viewpoint the animal welfare will matter since animals are sentient being and they are capable of suffering. It is therefore our moral duty to reduce or eliminate the suffering. From an animal rights view the animal welfare would be just as important as the human welfare. They will claim that animals have intrinsic value and that they must never be used as tools. Lastly, from a contractarian view the animal welfare will only matter if a moral agent such as him/herself cares for animals and are willing to include animals in an agreement. They will claim that we don't have any direct moral obligations towards animals.

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. This has nothing to do with honesty, but rather hopes of that the experiment will have good results and fears that it goes wrong. The largest source of bias is us as researchers. Not only the researcher can be biased, differences among the animals can also be biased. Bias could also be due to differences in experiment execution, e.g. the ordering of tests, the time of the day for tests, and the different experiment. There are also ambient parameters that cause bias, including light, sounds, smells, and temperature. The best approach to reduce the risk of bias is to randomise our experiment. The variation would then be random. We could randomise our experimental animals based on weight, age, sex, etc. Animals should be randomly allocated to treatment groups to reduce selection bias. We can also use blocking. An example of this is when doing research on pigs where weight has a strong influence in what we want to measure, and all the fat ones randomly end up together, in one group and the smaller ones in another, this can lead to biased results. To prevent this, you can for example randomly sort out the bigger ones first and then the smaller ones. Or take two pigs who has the closest weight in pair and assign them random. Another method to reduce the risk of bias is blinding: Investigators should be blinded to the treatment groups to reduce observer bias. One of the most common methods of blinding is the use of what seems like identical medications; one 'active' pill and one 'placebo' pill. As they are physically identical, it is impossible for patients and researchers to discern which pill is the active one based on appearance alone. When using blinding, there is a reduced chance that we as researchers will affect the result and cause bias. We could also let each laboratory animal be its own control and by this method obtain perfect matching. Fx transplant a tumour to each side of a mouse and only treat the one on the right side. We could also use different types of experimental designs, including a cross-over design, a full cross-over design, or the Latin square design.

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 animals that are bred specifically for use in experimental procedures. The EU directive states that member states shall ensure that animals belonging to the species listed in Annex I may only be used in procedures where those animals have been bred for use in procedures. The following animals have to be destination bred (purpose bred) · Mouse · Rat · Guinea pig · Syrian hamster · Chinese hamster · Mongolian gerbil · Rabbit · Dog · Cat · All species of non-humane primates · Frog, Rana · Zebra fish Farm animals can be used in experiments and they can be legally purchased from ordinary farms and they are therefore not a part of the list.

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

It is necessary to have uniform, precise, informative, systematic, and short ways of presenting the animals and therefore a international guideline has been made. Abbreviations are often used instead of full names but the full name shows the origin of breeding. · Inbred animals o Write the strain name, write a slash, write the breeders lab code § E.g. C57BL/6N/Tac (B6 mice from Taconic) o If their already is a slash in the name, we could just delete the second slash § E.g. C57BL/6NTac · Outbred animals o Write the breeder code, write a colon, write the stock name § E.g. Crl:SD (Sprague Dawley rats from Charles River) · F1 hybrids o Write the name of the mother strain, write the name of the father strain, write F1, write a slash for inbred, write the breeders lab code § E.g. B6D2F1/Crl (C57BL/6 mother, DBA/2 father, Charles River is the breeder) There is also ways to show in the nomenclature that the animal is a transgenic animal, a knockout animal, or a mutant.

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 is the humane killing of animals and it must be done without causing fear or stress in the animal. The method chosen should be easily performed by the person performing it and it should also be safe for the person. In the EU directive it is described how different laboratory animals should or could be euthanised. This depend very much on the species and also in some cases on the weight of the animal. You are allowed to euthanise animals by an anaesthetic overdose, a captive bolt, with carbon dioxide, by cervical dislocation, a percussive blow to the head, by decapitation (cutting the head of the animal), electrical stunning, inert gases, or by shooting with a free bullet. The cervical dislocation is one of those where the weight of the animal will determine whether the animal needs to be sedated prior to euthanasia. If you are using a chemical method to euthanise you animal you should always confirm it with an effective physical method, e.g. cervical dislocation. We cannot always predict what is going to happen to our animals. If you are in the middle of an experiment all by yourself and one animal all of a sudden shows' signs of suffering or pain, you should be able to euthanise that animal in order to minimise the suffering and pain. It could also be that your co-worker who normally euthanises the animal is sick one day and an immediate episode occur where the animal has to be euthanised. Then you must be able to do it to minimise the suffering and pain. Therefore, you cannot be a function A (doing the experiment) without also being a function D (euthanising).

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

Gene modification is a way to alter genes in animals either by inserting or deleting them. This can be done in different ways including with pro-nucleus microinjections, sperm precursor injection, targeted mutation by embryonic stem cells transfection, cloning and nuclear transfer as well as CRISP-R. · Simple pro-nucleus microinjection o The DNA is microinjected into one of the pro-nucleus of the fertilised eggs o This can only be used for inserting genes and not knock-outing genes and the placement of the genome will be random o It can be done in all species · Embryonic stem cell transfection o Some very specific embryonic stem cells can be harvested from the blastocysts and grown in culture where the gene modification technique can be applied o The stem cells can then be injected into another blastocyst and further injected into a pseudo-pregnant female o The genome placement will be targeted and it can be done in mice, rats, and humans · Cloning and nuclear transfer o Instead of a blastocysts, the cell is taking from the intact animal and is cultivated o The gene modification is then done on this cell culture and the modified cell nucleus is placed in fertilised eggs instead of the normal nucleus o This genome placement will also be targeted and it can be done in all species · CRISPR-cas9 o This technique uses DNA sequences which has been isolated from bacteria o The bacteria DNA sequence is linked to the nuclease cas9 which can induce a double break in the DNA o This break can stop the functioning of a gene (knockout) but it can also insert DNA constructs in the break to be an integrated part of the cell o It can be used for targeted insertion of genes and it can be done in relation to a microinjection, it can be done on embryonic stem cells, and it works in all species You can use these genetically altered animals in scientific research to either study the genetic ethology via forward genetics or to study what will happen to a mouse e.g. via backwards genetics. You can e.g. block the development of a specific organ and afterwards inject the mouse with human stem cells to develop the missing organ but it will off course be the human organ. You could make the animal or specific cells fluorescent to identify tumours, etc. Transgenic animals are animals that has genes from another species inserted into the genome. It will typically be from humans. The most simply way to do this is mating a female with a male. The day after the female gets euthanised and her fertile eggs are collected. They are then injected with a DNA construct with the desired gene. Another female is then mated with a sterile male and afterwards the female is anaesthetised to get the manipulated eggs inside her. At term she will then give birth to the gene manipulated pups. Under transgenic animals is also knockout animals and knock-in animals. In knockout animals the functioning gene is made non-functional and will therefore no longer encode a protein. In knock-in animals the opposite happens and you insert a gene version from a foreign species in exactly the site of the same gene in the homologue version of the animal. This is done if you e.g. want the mouse to produce the human version of the hormone and not the mouse version. Knockout and knock-in will only work in the homozygotic version whereas the simple gene insertion will work in both the hetero- and homozygotic version.

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)

Generally I think I would use the natural ingredient diet. This is because it is cheaper, the animal likes it, and it is nutritious. I would be aware of the risk that there could be variation between batches from the same supplier in which I would make sure that my animals would always if possible get from the same batch. If I were to study diabetes I could order a purified diet instead. Here I could choose that I wanted 10-70% of the metabolizable energy to come from fat. It is however a bit more expensive and the animal does not like it as much as the natural ingredient diet. In regard to ordering the diet I could order it online from an appropriate website. Upon arriving to the facility the diet should be stored in a place which is cool, dry, and rodent safe. I would make sure that I have receive the correct diet before doing anything.

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

Generally, there is three different positions on animals use in experimental research. These can be described as the disapprovers, the approvers, and the approvers with reservations. The disapprovers states that all sentient animals have rights that must not be denied and their view is the animal rights view. Their point of view is that sentient beings should not be used as tools and as a mean to obtain a goal and that good results cannot justify evil means. Our society builds on the principle that all humans have equal worth and an absolute right to be treated with respect. It is not possible to point out a single criteria for moral exclusion of sentient animals and inclusion of all humans. E.g. if the ability to communicate with spoken language is chosen as the moral criteria both animals and some humans will be excluded. Animals cannot communicate with spoken language but infants, severely demented people, or people with severe brain injury cannot either. So with this criteria it is therefore not possible to morally exclude animals and at the same time include all humans. Intelligence could also be chosen as the criteria and the story would be the same. The approvers are at the opposite end and they believe that human interests are more important than the interest of animals, no matter the cost to the animals. They have the contractarianism view and as long as the society allows for it, it is not a problem to use animals for experimentational purposes. The morality is based on agreements where the agreement is established between rational persons capable of negotiating such an agreement. So if you have no obligations, you have no rights. Humans therefore have no direct duties towards animals only indirectly if the animals in question matter to other people with rights. If an individual does not have the means or capability to enter the agreement, this individual does not have neither obligations, nor rights. Hence, animals and e.g. infants do not have ethical rights unless a rational person negotiates rights for these individuals. The approvers with reservations believe that harms and benefits must be balanced and animal suffering is not to be ignored. They have the utilitarianism view. They are often concerned with the suffering of the animals and the suffering must be counterbalanced by the benefits of the experiment. The needs of the many outweigh the needs of the few or the one. This statement says that it is perfectly logical and morally acceptable to sacrifice one or more individuals if this action will save a greater number of individuals. Their focus is on the consequences of their actions and you must create all the happiness you are able to create and remove all the misery you are able to remove.

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

Genetic monitoring is the current monitoring of the genetic status of strains and stocks. We want to show that mice from a colony of B6 mice are really all B6 mice. So there is three steps in how to monitor the genetics. First a sample from a mouse should be obtained. Then the DNA should be purified and lastly the analytically procedure is chosen. Here you can choose PCR, southern blot, SNP, or STR. · PCR is typically used for identifying single genes typically a transgene o It is easy and cheap · Southern blot can be used to identify single genes typically a transgene or knockouts o It is easy and cheap o It can also be used for the characterisation of strains or stocks but this is less common · SNP is used to characterise strains or stocks o It is a quite costly method but it can be used to e.g. identify differences between B6 mice produced from different vendors · STR can be used to show the inter-individual variation between inbred mice of the same strain o It is a quite costly technique

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)

Halstead's principle is a fundamental principle that can be used to minimise the trauma we inflict on the animals when handling tissue. The principle includes asepsis, gentle handling, haemostasis, closure of dead space, careful approximation of tissue, avoidance of tension, and minimisation of foreign materials. If the tissue is not handled correctly it could result in poor recovery, pain, suffering, and distress to the animal. Good hygiene and asepsis are two very important factors in regards to the surgical procedure. It could otherwise result in poor animal welfare and confounding factors, infected surgical wounds, poor healing, pain, sickness, distress, euthanasia, higher variability in our experimental outcomes, and the need for more animals. This is done by keeping a good hygiene and by working aseptically. The instruments should always be sterile and stored in a clean and dry environment. After they have been used they should be properly cleaned and sterilised again. All gross contamination must be removed manually by brushing the instruments. If the instruments are difficult to clean you could use ultrasonic cleaning equipment. It is also important to use an appropriate disinfectant since ethanol won't be enough to remove all fungi. You should however also be careful when cleaning the instruments, so that they don't break. The animals should also be prepared for surgery and the way to do it pretty much depend on the size of the animal. Large animals should be fasted 8-12 hours before surgery to prevent vomiting. They should be shaved in the region of interest and the area should be scrubbed with an antiseptic soap solution. The soap should then be removed with alcohol or iodine. The rest of the animal should be covered with sterile drapes. Smaller animals should be subjected to at least one week of acclimatation before they are used. There is no need to fast them since they cannot vomit. Otherwise the same preparation is used in regards to shaving the hair, etc. The person performing the surgery must also be prepared before the surgery. He or she should wear a scrub suit, sterile shoes or shoe covers, a surgical cap, a face mask, and sterile gloves. Long hair must be up and away, nails must be clipped short, there should be no jewellery on hands and arms, and you should wear comfortable and practical clothes. The hat and the mask should be put on before scrubbing. You should wash your hands and arms. If you are using a sterile gowning you should put this one on after scrubbing but before you put on your gloves. If you are using a non-sterile gowning, you should put this one on before scrubbing.

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)

In a harm/benefit analysis you weigh the harm to the experimental animals against the benefit of the study. A harm/benefit analysis of a study is done to assess whether the harm to the animals in terms of suffering, pain, and distress is justified by the expected outcome considering ethical considerations, and may ultimately benefit human beings, animals, or the environment. The harms should be considered all negative impact on the animals used. The harms can be related to animal welfare aspects, animal rights, the quality of research, and human harms. So, the harms can be anything that compromises the welfare of the animal depending on the definition of animal welfare, as well as the welfare of the caretakers, since subjecting animals to harmful procedures may lead to stress and frustration. Low quality research could also be a harm since the animals would have suffered for no reason and the use of animals would be irresponsible and with lack of respect. It may also produce misleading results that could potentially be harmful. The benefits mainly relate to the expected outcome of the studies in form of improvement in the field of human health. The outcome could also benefit the wildlife, the diversity of life on the planet, etc. Economic interests could also be counted as a benefit.

6. Discuss the preanesthetic 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)

In order to get a successful anaesthetic procedure, it is important to consider different things prior to the procedure. These include an evaluation of the animal where the animal should be clinically healthy and should not carry any hidden infections. They should also be acclimatised to the experimental facility and the duration may vary from species to species. This will allow the animal to adjust to a change in its environment since these changes can have an effect on physiological systems. These should therefore be allowed to return to normal before any procedures if performed on the animal. It is also important to consider whether or not the animal should be fasting, and whether or not an assistance is needed. The choice of anaesthesia is also important since it should comply with the objective of the experiment and still fulfil the anaesthetic triad. You could choose either one anaesthetic drug or a regimen. You should also consider pre-anaesthetic medication to reduce fear and stress and provide a smoother induction and recovery. The advantages by using mixed drugs, is that a lower dose of each drug which gives less side effects. However, this can interfere with the protocol. We have to make sure that the drugs we choose will result in the best experimental outcome. During the procedure we should monitor the anaesthesia to make sure that the anaesthetic triad is still fulfilled. We should consider if we want to administer analgesia to the animal either pre-emptive, intra-, or postoperative and we should make sure to observe, monitor, and treat the animals postoperative. All of these things should be listed in our anaesthetic protocol so everybody knows what is going on and how things are going to be performed. When these things are properly considered and chosen upon it will reduce the mortality, it will give the animal faster recovery, it will give less variation in the experimental results and thereby better research quality.

1. Give examples of behavioural 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)

In order to understand what behaviour is signs of discomfort, pain, suffering, or distress, it is important to know the animal's normal or basic behaviour. Pain and stress assessment can be seen in three different ways: · Physiological parameters o As describes: heart rate, blood pressure, body temperature e.g. due to sympaticus these will increase when the animal is in pain or stress · Biomarkers o Not really used unless it is a part of the study, but is e.g. hormones as adrenalin and Corticosteroids · Behavioral and clinical signs ehich is the tool we are mostly using, when studying animals. We can look for general signs, e.g. if the animal is more alert, if the movement is impaired, if the animal drinks or eats more than usually, does the animal protect certain body parts, does the animal make sound when being handled, does the animal bite of lick itself more than usually, etc. In rats you could also see red coloured tears which is a clear sign of stress. It can also be a decrease in the normal behaviour e.g. nesting and hiding, social behaviour, exploration, grooming, etc. It could also be an increase in abnormal behaviour or stereotypical behaviour. Stereotypical behaviour is repetitive, homologues, and apparently no-function behavioural patterns. This could e.g. be the animal running around in circles which is done to calm itself and cope with the changes. It does however have negative consequences for the animal involved. You could also assess clinical signs of pain and stress which manifests as body weight loss, decrease in food and water consumption, urination and defecation may increase, and the fur quality may also decrease. Pain-specific: twitching, back-arching, falling etc. Also, facial expression in mice show distress. Pain can generally be alleviated with anaesthesia (without sensation) or analgesia (without pain) but preferably we want to avoid pain from arise. One way could also be to administer analgesia beforehand and the pain treatment should continue after an intervention. You should also choose the appropriate type of analgesia depending on the object of our experiment, how invasive the procedure is, and the duration of our procedure. It will also depend on the pain intensity reaching from low, moderate to high. Sometimes it is also recommended that the animal receives multimodal analgesia especially if the pain intensity is high and, in some cases, where the pain intensity is moderate. Suffering and distress can also be managed by avoiding other sources of discomfort e.g. how the analgesia is administered, how we handle the animals (tunnel-handling instead of tail-handling), and making sure that the animal can perform natural behaviour.

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

In the general surgery pack, you find different tools. These include haemostats both curved and straight, scalpel handle, forceps, scissors, needle holders, needles and suture material, retractors, stainless steel bowls, instrument tray, cotton wool buds, gauze swabs, and drapes. When doing sutures, it is important to eliminate dead space. Sutures in the inner layers of the muscle and fascia should be tied tightly with resorbable materials. Sutures in the skin should be less tightly since we want to avoid pain and inflammation due to ischemia after swelling of the tissue. We should not use natural materials as catgut, linen, and silk but instead we should use proline, vicryl, etc. and we should use stainless steel needles. There are different types of sutures you could apply. The simple interrupted sutures are very secure since the loss of one suture does not open the entire wound but it is a bit more time consuming. The continuous sutures are easy and quick, they seal the wound better but they are generally less secure than the simple interrupted sutures. Your surgical knot must be based on the square knot and not the granny knot. You want the crossing to be the same on both sides.

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)

It is important that we monitor the animal after anaesthesia and that we take action if something is wrong. We don't want the animal to suffer. There is a different problem that may occur post-operatively. · Pain o The solution is to administer analgesia which can be done pre-operatively, intra-, or postoperatively · Dehydration o The solution is fluid therapy where body warm saline is administered either IV or SC. This can be done both during the procedure but also after wards o Make sure that the animal has easy access to drinking water · Hypothermia o The solution to this is keeping the animal warm with heating blankets, infrared heating lamps, and incubators depending on the size of the animal o Make sure that the body temperature is maintained until the animal is fully recovered and make sure that you don't overheat the animal · Infections o The solution to this is to work aseptically to avoid asepsis. You should observe wound healing and provide antibiotics if necessary · Postanaesthetic restlessness and self-injury due to pain or severe distress o The solution to this is to use sedatives or analgesic if indicated. It is always a good idea to consult the veterinarian There should also be recovery rooms that are warm and quit to minimise the stress on the animals. For larger animals it can be beneficial to have supportive care. For smaller animals like rodents, they should have bedding that is soft and comfortable and you should avoid unnecessary human contact and handling since they don't benefit from supportive care. The use of sedatives pre-operatively could also make the induction and recovery from anaesthesia smoother.

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)

It is never allowed that the animal experience strong pain, intensive fear, or intensive suffering, no matter the purpose. If the animal experience one of these, it should eventually after discontinuation of anaesthesia or other alleviating treatment, be humanely killed (euthanised). Sick animals must also be treated or euthanised in accordance with good veterinary practice. If the animal's welfare is at stake it should be humanely killed. Everybody cannot just kill an animal used or bred for scientific purposes. The member states should ensure that the animals are killed in the establishment of a breeder, supplier, or user and by a competent person. It should also be executed by the methods described in the directive as well. However, in emergency circumstances for animal welfare, public health, public security, animal health, or for environmental reasons, it does not have to be a competent person and it does not have to be by the ways described in the directive.

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)

It is the licence holder who is responsible for the compliance in relation to how the experiment is performed. The licence holder can have others performing the experiments but the licence holder must ensure that they have the right qualifications and that all legislation is being obeyed. The manager is responsible for the design and operation of stables and test rooms, for the competencies of the staff, and for the care and welfare of the animals. The national committee should protect the animal used for scientific purposes.

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).

It is vital that scientific communities and the laboratory animal facilities make their ethical principles for handling and use of animals accessible for the public to facilitate communication and trust. In Denmark this is done with seminars. These seminars are implemented to address various aspects of the use of experimental animals. These seminars are open to the public and they are presented by the Animal Experimentation Inspectorate and some of the Animal protection societies. Communication and transparency is key to maintain the trust and acceptance from the public. It should always be given a high priority. If the public feels like a pharmaceutical company is hiding something and that they are not transparent then the public would be much more resistant towards animal experimentations. If you are not open and honest then the public would believe that you are hiding something. Pharmaceutical companies and academia could publish information on their webpages to inform the public on different matters that happens within their facility. In this way the information will be available for the public to read which will give this transparency.

7. Discuss relevant maintenance of anaesthesia, 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)

Make an anaesthetic protocol with timepoints and dose needed for maintenance of anaesthesia. If done by injection. Using inhaled the anaesthetic is given continuous. It is important to make sure that the anaesthetic triad is fulfilled during the whole procedure. This means that you have to administer additional anaesthesia if the animal starts to show signs of awakening. We should always monitor the animals when they are sedated since we should make sure that the physiology is as close to normal as possible and we should also make sure that the animal can recover well. In general, we should monitor the absence of reflexes, we should monitor the circulatory and respiratory functions and well as body temperature. In short and low-risk procedures the animals can be non-electronically monitored by visually assessing the animal. You should monitor the pulse rate, the respiration, body temperature which can be done by a rectal thermometer, and the colour of the skin. This will give an indication of whether or not the normal physiological functions are working properly. You could also use a pulse oximetry to assess the circulatory and respiratory functions. In long-term and high-risk procedures, you need to apply more sophisticated monitoring. You should monitor the circulatory functions by either an electronic monitoring of the heart rate and rhythm or by direct arterial blood pressure. The respiratory functions should be monitored by the tidal volume, percentage of end-tidal CO2 and O2, you should measure the concentration of volatile agents and blood gases. During anaesthesia you could run into potential problems. You could have an animal that responds to noxious stimulus or an animal that is actually moving. This would be very bad and it would be caused by insufficient anaesthesia. You can solve the problem by administering more anaesthesia or administer analgesics or local anaesthesia. You could also have an animal with poor respiration, poor circulation or poor oxygenation. This could be due to too much or too long-lasting anaesthesia or it could be due to low body temperature. In these cases, you could solve the problem by reducing the amount of anaesthesia, you should provide oxygen, heat, and fluid. If action is not taken within reasonable time, the animal may suffer or die.

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

Movement and transportation are considered stressful to the animals and should therefore be kept to a minimum. When animals are transported by vehicle it can be very stressful and, in some cases, you must allow a 10-12-day period where the animal can return to normal again. When animals are transported over longer distances, a careful choice of transport caging, transport means, and route should be made in order to make the journey as short as possible with minimum stress to the animals. -Persons transporting animals must ensure that the animals are transported in a way that is not likely to cause injury or unnecessary suffering and under conditions suitable for the animal. -The animals are fit for the journey -the means of transport is designed to avoid injury and ensure safety -The personnel handling animals are trained or competent in the transport of animals -Sufficient floor area and height is provided for the animal -the animal is watered and fed During transportation you may see an increase in heart rate, body temperature, and animal activity. I guess it would be a good idea also to notice the behaviour of the animal to assess whether the animal is stressed or not or if the well-being of the animal is compromised.

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)

One could argue that there is no purpose in just killing animals for training and teaching. This will not directly give any benefits to the humans but it will give a huge amount of cost to the animal. The indirect benefits of the training and teaching would however be a lot higher. As Daniel said in the laboratory that even though we euthanise animals giving no immediate benefits, one of us could might as well in the future find the cure for cancer or any other devastating disease. The benefits would therefore outweigh the cost many times. So here we see the long term benefits instead of the immediate benefits and we hope that the lives taken will be beneficial in the future. When performing procedures on animals you should also be sure that you can perform them quit well. If you are unexperienced and have not tried a procedure before you could mess up the procedure and potentially ruin the results. Then you would have wasted many more animal lives compared to the once used for training and practice. So in order to get beneficial results in the future there must be a cost of the animals in the present.

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 animals are animals maintained by random mating which will result in unintended selection. Some animals grow more, breed better, survive better, etc. Inbred animals on the other hand is close relatives that are mated. They are originally based upon outbred animals which have then been inbred. So you take the brother and sister or father and daughter and mate these for at least 20 generations. When genetically screened there will be a huge difference between animals from different colonies in outbred animals. Inbred mice will on the other hand only have very few percent heterozygosity left in their genome making them quite alike. This will give lower variation in inbred animals which will increase the power of the study. In outbred animals the variation will be higher. In inbred animals some lines will be closed down due to the appearance of recessive lethal or defect genes. These will be sorted out during the initial inbreeding. This also means that there will be no unexpected diseases in inbred animals. Either the strain has some genetic diseases or it does not. E.g. the Fisher 344 rat will when aging get a high incidence of large granular lymphocyte leukaemia. It is therefore important to know and monitor these diseases especially when working with a specific inbred strain of rats or mice where the disease is known. Some outbred colonies of mice of rats may have a high incidence of specific diseases. E.g. the Sprague-Dawley rat may have a high incidence of mammary gland tumours.

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

Perioperative is from the time the animal arrives until the end of the procedure. The overall strategy is to prevent pain from occurring at all. This is not always inevitable but we strive to do it. Before the procedure it could be beneficial to administer analgesia. This would make the pain less apparent when the animal wakes from the procedure and the animal does not have to wait minutes before the analgesic effect kicks in. Under the procedure we should make sure that we avoid the nociceptive signalling so we avoid the risk of stress response, impaired recovery, and chronic pain in our animals. We should as the operator be technical skilful so we cause less trauma and therefore less pain. We should also work aseptically and maintain a good hygiene. We should also minimise other sources of discomfort. This includes gentle handling to avoid stress. This applies also to before and after the procedure. You should also administer supplementary analgesia and in a way that does not stress the animal. After the procedure the animal should be monitored and it should be assessed whether the animal is in pain. If the animal is in pain, you should take action and in worst cases the animal should be euthanised to avoid the pain and suffering. The treatment with analgesia should also be continued after the procedure in an adequate duration and at relevant timepoints. This depends of the drug used and the wanted effect. When an animal experiences pain it can be quite difficult determine the intensity of the pain and therefore the choice of analgesia. Should we just administer a mild analgesia or should we use two different types of analgesia? We should also consider whether the analgesia chosen could affect our outcome data. We should also consider the duration of our procedure since it will also affect the choice of analgesia.

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 is the state on temporary unconsciousness (semiconscious). You could also administer sedatives to make the animal relax before putting it under general anaesthesia. This will make the induction and recover more smooth. It will help the animal relax. Local anaesthesia is where you block certain nerves to get anaesthesia in a local area whereas general anaesthesia is where you put somebody into unconsciousness and you can do procedures without the patient or the animal being aware of it. Triad of anaesthesia: loss of consciousness (anaesthesia), muscle relaxation and no sense of pain (analgesia). You can make sure that the animal does not experience pain by administering analgesia as well. This is in fact called balanced anaesthesia which is an anaesthetic regimen where you combine different drugs. So instead of using a high dose of just one drug, you could use lower doses of each drug which will minimise the side effects while still fulfilling the anaesthetic triad. This can be done by e.g. mixing ketamine with xylazine or fentanyl with isoflurane.

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).

Stressors are defined as factors that trigger stress and in turn stress is the body's response to stressors. Stressors can be many different factors including environmental, physical, and biological factors. Examples of these are temperature, housing, light, noise, ventilation, fasting, physical handling, movement, human contact, different life experiences, genetics, gut flora, behaviour, and microbiota. Exposing animals to stressors may have a major impact on physiological and endocrine functions. It may result in altered physiology, impaired recovery after surgery, and the risk of developing chronic pain. We may see altered metabolism, immune suppression and antiinflammation, reduction of reproductive functions, and stress induced analgesia. We will also see an immediate increase in heart rate and blood pressure as well as an increase in glucose levels and lipolysis. This will in turn affect our experimental results giving an increase in the variation between animals since they may respond completely different to what a normal unstressed animal would respond. This will force us to use more animals and there will be a risk that the stress turns chronic which will make the animal end up in distress and suffering. Stress is not avoidable and it is sometimes good. This is when the stress is acute and it can be classified as positive stress. This is our survival mechanism and it aids the animal to adapt and cope with challenges. Acuta stress is related to an increased activity of the HPA-axis and the sympathetic nervous system resulting in the consequences as just mentioned. When the animal is out of the stressed situation there will be a rapid return to the basal levels and the body will not be damaged from this situation. However if the stress persists and we have prolonged elevated levels of stress hormones the animal will develop chronic stress also called negative stress. This is very problematic to the animal since we have gone beyond stress and really start to having risks of suffering. In humans we have seen a negative circle where stress, anxiety, and fear leads to depression, leading to fatigue, pain, tense muscles, anger and frustration, and in the end this will increase the stress, anxiety, and fear as well. This could also apply to rodents and therefore we really want to avoid long term stress.

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

Tail-handling in mice is a widely used method to handle mice. However, to minimise stress reactions, mice should be removed from their cage without grasping and lifting the mouse by the tail. If they are tail-handled they will be unwilling to interact voluntarily with handlers and they will even show high anxiety. This will affect the animal welfare negatively and the anxiety will be visible in their behaviour e.g. when studying the behaviour in the elevated maze test. Tunnel-handling or cupping will on the other hand be much less stressful for the mouse. They will show lower levels of anxiety and they will readily approach the handler. This will off course affect their welfare since you don't stress the animals and causes them unnecessary suffering.

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

The 3R's focus on the animal's wellbeing and the ethical issues regarding pain applying and the amount of stress the animals are under. It is therefore some common recommendations in order to reduce the number of experimental animals and to reduce the suffering of the animals. The 3R's comprises of replacement, reduction, and refinement. Replacement imply methods which avoid or replace the use of animals. This could be the substitution of conscious, living, sentient beings with insentient material (e.g. plant, microorganisms) or the absolute replacement of living animals with e.g. a computer model or human volunteers. It could also be the relative replacement where living animals are replaced with established animal cell lines. This means that no sentient animals are used per se, but at some point, in time, an animal was used to establish the cell culture. Reduction imply methods which minimise the number of animals used per experiment. Here we should however be aware that the less number of animals should still give the same amount and quality of data. The scientific output and the quality of the data must not be compromised since the data then would be useless and the animal lives would have been wasted. The reduction could also be where you use the same number of animals but you get more or better-quality data. Lastly, refinement imply methods which minimise suffering and improve animal welfare. It aims to minimise actual or potential pain, suffering, distress, or lasting harm in situations where the use of animals is unavoidable. Refinement should be implemented both in scientific procedures, husbandry, and management procedures. It applies to the entire lifetime experience of the animal which means that it should be implemented in breeding facilities, during transport, in all housing facilities as well as doing procedures and interventions. The reduction of animals is not always compatible with refinement since using fewer animals may risk increasing the strain on each individual animal. This could e.g. be if you were to reduce the number of dogs from 12 to 6, you could increase the amount of pain on each dog. This could go from one fracture on each of the 12 dogs to 2 fractures on each of the 6 dogs. Dogs that are subjected to 2 fractures will give more pain to the dogs that dogs subjected to only one fracture.

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

The 3R's is the concept of replacement, reduction, and refinement. It is a way to minimise the harm in animal experimentation by using methods that avoid or replace the use of animals, methods that minimise the number of animals used per experiment, and methods that minimise the suffering and improve animal welfare. We want to make sure that our animals suffer less and that the welfare of the animal is always as good as it can be. Since the scientific field is continuously developing and improving, new findings are also being discovered. This means that we should always improve the techniques and methods that we use. We should always use the most gentle technique available and since science is always improving we as staff and researchers should too. At one time point a specific technique or method could be the most gentle but after a period of time other scientists may have found another technique or method that is more gentle to the animals. It is therefore important that we always improve the level of refinement in animal experimentation in order to make sure that the animals actually suffer less and that the welfare of the animals is actually good.

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 EU directive states that each member state should have a National Committee. Their role is to advise on Animal Experimentations and Alternatives, to implement the 3R's, and to enhance a culture of care for animals. It is the overall authority for all the smaller committees known as the animal welfare bodies which is found in each facility. In Denmark this committee consists of 1 chairman and 6 members with high professional knowledge and experience in at least one of the 3R's in relation to animal experiments (2 members in each R). The members are administered by the Ministry of Food and Environment. They should work to promote the application of the principles of the 3R's (replacement, reduction, and refinement). They shall advice the competent authorities and animal welfare bodies in matters relating to acquisition, breeding, housing, care and use, and they should ensure the exchange of best practice. Their job is to exchange information on the activities of animal welfare bodies and project evaluation in connection with animal testing and exchange best practice with the union.

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

The Five Freedoms concept is a guide on how to avoid suffering in animals and it is a combination of the different animal welfare definitions. The Five Freedoms are: 1. Freedom from hunger and thirst a. This is done by allowing the animals ready access to water and a diet to maintain health and vigour 2. Freedom from discomfort a. This is done by providing an appropriate environment 3. Freedom from pain, injury, and disease a. This is done by prevention or rapid diagnosis and treatment b. This is highly important in laboratory animals since we intentionally apply pain, injuries, and disease in different studies. We therefore need to do our best to reduce these effects whenever possible and we should never accept more pain, injury, or disease than absolutely needed for the purpose of the study. 4. Freedom to express normal behaviour a. This is done by providing sufficient space, proper facilities, and appropriate company of the animal's own kind b. In association appropriate environmental enrichment is mandatory 5. Freedom from fear and distress a. This is done by ensuring conditions and treatments which avoid mental suffering since it is our legal and moral obligation to reduce fear and distress as far as we can b. This is very relevant for laboratory animals since they often are subjected to procedures evoking fear and distress

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

The challenges when performing a harm/benefit analyses of a study using live animals are that harms are certain but benefits are not guaranteed. The harms should off course be reduced to a minimum according to the 3R's but they are sometimes unavoidable. On the other hand, there is no way to ensure that the results of a study will contribute to human health improvement or save lives. It requires high quality research and good results must be rendered probable. It is sure that the harms are done during the study and that they are certain but the benefits may lie many years in the future and these expected benefits may never become a reality. It is therefore a very delicate and difficult discussion where there is no definite right or wrong.

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)

The classification system contain four categories of pain. They are non-recovery, mild, moderate, and severe. · The non-recovery category contains procedures which are performed entirely under general anaesthesia from which the animal shall not recover consciousness o e.g. our teaching in the laboratory · The mild category contains procedures on animals as a result of which the animals are likely to experience short-term mild pain, suffering, or distress. It is procedures with no significant impairment of well-being or general condition of the animals o e.g. administration of anaesthesia · The moderate category contains procedures on animals as a result of which the animals are likely to experience short-term moderate pain, suffering, or distress. It is procedures that are likely to cause moderate impairment of the well-being or general condition of the animal o e.g. surgery under general anaesthesia · The severe category contains procedures on animals as a result of which the animals are likely to experience severe pain, suffering, or distress. It is procedures that are likely to cause severe impairment of the well-being or general condition of the animal o e.g. severe restriction of movement over a prolonged period Cumulative severity is when you add up the effect of different procedures or multiple uses of the same procedure. So you can say that it is the cumulative suffering the animal in total will experience when all things are put together. I guess that if you do the same mild procedure many times on the same animal the cumulative suffering would maybe be moderate or even severe. So it could potentially change the scoring from mild to moderate or even more severe.

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)

The classification system contain four categories of pain. They are non-recovery, mild, moderate, and severe. · The non-recovery category contains procedures which are performed entirely under general anaesthesia from which the animal shall not recover consciousness o e.g. our teaching in the laboratory · The mild category contains procedures on animals as a result of which the animals are likely to experience short-term mild pain, suffering, or distress. It is procedures with no significant impairment of well-being or general condition of the animals o e.g. administration of anaesthesia · The moderate category contains procedures on animals as a result of which the animals are likely to experience short-term moderate pain, suffering, or distress. It is procedures that are likely to cause moderate impairment of the well-being or general condition of the animal o e.g. surgery under general anaesthesia · The severe category contains procedures on animals as a result of which the animals are likely to experience severe pain, suffering, or distress. It is procedures that are likely to cause severe impairment of the well-being or general condition of the animal o e.g. severe restriction of movement over a prolonged period Cumulative severity is when you add up the effect of different procedures or multiple uses of the same procedure. So, you can say that it is the cumulative suffering the animal in total will experience when all things are put together. I guess that if you do the same mild procedure many times on the same animal the cumulative suffering would maybe be moderate or even severe. So, it could potentially change the scoring from mild to moderate or even more severe.

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)

The concept of a model is that is accurately resembles something else. In this regard you have to consider whether the model is of high fidelity or high discrimination. Fidelity, also known as accuracy or precision, refers to the degree to which a measurement or observation represents the true value or state of a phenomenon. Fidelity is the extent of resemblance to the target which means that what we have looks in many ways like it is supposed to look. It could e.g. be the look-a-like of a seagull which will very much look like a real seagull. So, the lookalike is a model of high fidelity, that means it looks in many ways like the seagull it is supposed to model. Discrimination on the other hand is how much what we have looks like something else in one relevant phenomenon while there are no similarities in other aspects. Fx a stick with some red spots on it would resemble the real seagull in just that place where the red dot is, but everything else would be very different. the stick is a model of high discrimination, that means in one relevant phenomenon it looks exactly like the seagull, while there are no similarities in other aspects. Validity is a phenomenon used to describe how well a model translates to the species it is modelling meaning how applicable are our findings to the real world? The external validity can be divided into three criteria - construct validity, face validity, and predictive validity. · Construct validity describes the extent to which both the animal model and the human phenomena can be explained by the same theory o E.g. the cause of the disease is the same · Face validity describes the similarity in appearance. What we see in the animal model also resembles what we observe in the modelled phenomena o E.g. the symptoms of FIV in cats are of high face validity to HIV in humans · Predictive validity describes how the performance in the test predicts the performance in the condition being modelled o E.g. how much a drug has the same effect in human and models tested An induced model is a model where the phenomenon is induced chemically or surgically e.g. the surgical removal of the pancreas in a rat diabetes model. This will cease the production of insulin in the rat. In this model we will have low construct validity, low face validity, and high-low predictive validity. · Low construct validity: Humans do not develop type 1 diabetes because their pancreas was removed. In humans the development of diabetes is rather a failure of the immune system to prevent an autoimmune attack of the pancreatic insulin producing beta cells · Low face validity: The rats will develop symptoms due to the lack of more than just beta cells · High-low predictive validity: o High: If you want to test whether an insulin treatment will alleviate diabetes, it will have similar effects in rats and in humans o Low: If you want to test the interventions such as diet that targets the reason for developing type 1 diabetes, it will not be similar in rats and in humans A spontaneous model is a model where the phenomenon will occur spontaneously e.g. the NOD mouse in diabetes models. The insulitis in NOD mice is initiated much earlier compared to humans and is in many ways different from human insulitis. The clinical symptoms are quite the same but mice have larger resistance to ketoacidosis development compared to humans. If their diabetes is not treated they can remain alive about 2-4 weeks after establishment, but eventually they will die from dehydration rather than ketoacidosis. · Medium construct validity: In NOD mice the disease begins with the lymphocytes surrounding the islet perimeter and continues with an infiltration of the whole islet by an unusually large number of leukocytes. After a period of subclinical β-cell destruction overt diabetes will be presented when more than 90% of the pancreatic β-cells are destroyed. · Medium face validity: The typical clinical symptoms are present in humans and in the NOD mice. The mice do however have larger resistance to ketoacidosis development than humans. · High predictive validity: Nasal and orally administered insulin will reduce the diabetes in NOD mice as well as in humans

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)

The definition of animal welfare can be divided into 3 viewpoints. These are health and biological functioning, natural living, and emotions and preferences. So, if we think of animal welfare as having a life in good health and with good biological functioning we can perform clinical examinations, measure physiological parameters, and evaluate statistic parameters. The physiological parameters could e.g. be stress hormones to establish whether the animal is in fact healthy and able to function within normal, biological parameters. The statistical parameters could e.g. be mortality rate and assess the occurrence of abnormal behaviours or changes in behaviours. Changes in behaviour can also indicate whether the animal is healthy and able to cope with the environment. A clinical examination will give an idea on whether the animal is overall healthy e.g. by monitoring heart rate, body temperature, and respiration rate. It could also be different appearances of the animal e.g. the mucosal membranes, the fur, the skin, the condition of the feathers, etc. The welfare of the animal could also be assessed with laboratory tests e.g. with blood-, urine-, or faecal samples, metabolic, immunological, and hormone parameters as well as the appearance of animal products such as eggs, milk, meat, etc. If we think of natural living as animal welfare you have to realise essential species-specific potentials in order to have a high a level of welfare. You should realise your potential by fulfilling one's purpose in life. The animal has to perform natural behaviour which means that they not necessarily have to be well but by doing well considering the genetics and culture of the being. This could translate to a pig, that has to be allowed to be a good pig and therefore do what pigs do best. The animals should be able to express their nature and perform those behaviours that define them as a species, e.g. nest-building, etc. The more piggy-ness the higher welfare. Animals should be allowed to perform their natural living with natural behaviours. So, the occurrence of natural behaviours could be one way of assessing the welfare of the animals. If we think of welfare as emotions and preferences then animal welfare is based on experienced felling and mental states. This means that there should be a presence of positive mental states and an absence of negative mental states. The presence of positive mental states would increase welfare whereas the presence of negative mental states would reduce welfare. This viewpoint demands the acceptance of animals having the ability to experience emotional states such as fear, caring, joy, positive anticipation, loneliness, and anger. Within this definition lays the preference theory which states that the more you experience to get what you want, do what you want to do, and avoid what you do not want, the better the welfare you have. So, when you seek to having your preferences fulfilled it will bring positive anticipation as the goal I approached. It is also a good feeling to get what you have been working for. In this case behavioural tests could also be applied to measure the welfare of the animals. This could for example be studying behaviours that indicate positive mental states and behaviours that demonstrates preferences. The behaviour indicating negative mental states should also be studied.

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)

The effect size is the magnitude of the effect we are looking for. This could for example be how much a cancer drug could shrink a tumour. The bigger the effect we are looking for is, the easier it is to find. So the bigger the effect, the smaller our group size has to be. When designing an experiment we want to estimate the smallest effect size that we are interested in since bigger effects can potentially induce greater suffering in the individual animal. Fx if we let animals grow larger tumours, or use of more drug would be easier to measure. For example, when measuring tumours, if they are bigger they are easier to measure and see, and to let them grow bigger, a reduced number of animals would have to be used. More drug could also lead to more side effects. However, remember, that this might cause more pain in the individual mouse. So the bigger effect, the smaller our sample size needs to be, but we need to have reduction and refinement in mind and therefore we should determine the smallest effect size of interest. This could influence the amount of animal needed and we should consider a bigger sample size in order to observe the effect we are interested in. Variation: The less variation we have in our measures - the more uniform our tumors are in our example experiment - the fewer animals we need to demonstrate an effect. Reducing variation in our measurements is one of the most effective ways of reducing the number of animals we need. It has been shown that different mice from different breeders gave different results. In order to control variation, it would be an idea to use the same number of mice from different breeders in order to obtain results. This has the influence that more animals need to be used, as some might respond good to a new drug candidate, while some will not. Less variation - the more uniform tumors. Therefore, a smaller need for animals, however, if we remove too much variation, we cannot say anything about the real world. For example in some new promising drug candidates for Alzheimer's and cancer have been found to only show an effect under very narrow circumstances.

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 experimental unit is a physical entity that is the primary unit of interest in a specific research objective. It is the smallest element that we can assign independently to a group in a study. It is the smallest element that we conclude anything about. The experimental unit could be the individual animal or it could be the cage where more animals are gathered. When mice are housed in groups you cannot measure the water intake per mouse. The experimental unit therefore becomes the cage. The measurement becomes a bit less exact, but if control mice are housed with control mice and experimental mice are housed with experimental mice, it would be possible to conclude if water intake is changed when comparing controls with experimental mice. It is only when the results we obtain from an individual animal is independent of one other animals, that we can considered the individual animal our experimental unit of the study. For example, if you want to test kidney function and need to determine how much a mouse is drinking, it is hard to do on only one mouse. Mice are social animals and in one cage you need at least three. However, you cannot know how much 1 specific mouse drank, and therefore the experimental unit becomes the cage, where it is 1 bottle and 3 mice.

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

The four functions in relation to educational demands for staff involved in animal experimentation is function A, B, C, and D. · Function A has to do with carrying out the practical parts of the procedures of an experiment o To do function A you must either be a professional laboratory animal technician or have another relevant technical or academic education. You should also have necessary training, meaning that you cannot perform surgery without having surgical training · Function B has to do with setting up the experiment and holding the licence o To do function B you must have a relevant academic degree within health or natural science, e.g. a medical doctor, a veterinarian, a biochemist, a pharmacist, a biologist, or similar · Function C has to do with taking care of the animals in the facilities o To do function C you should be a laboratory animal caretaker, agricultural animal assistant, or a veterinary nurse. This means that you should have completed 4 years of technical education with both theory and practice within taking care of animals · Function D has to do with the killing of animals The manager is also called the animal welfare officer and it is one or several persons who is/are responsible for the welfare, access to species specific information, and for staff training. They are responsible for the design and operation of stables and test rooms, for the competencies of the staff, and for the care of the animals and the welfare of the animals. The designated veterinarian is also called the inspecting veterinarian. He or she should have expertise in laboratory animal medicine and should be able to give veterinary advice. If the scientist is a veterinarian there is nothing against that they can be their own designated veterinarian. A more suitable expert could also be appointed as the designated veterinarian, e.g. in fish or insect facilities.

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

The ovarian cycle in rodents is a bit different from that in humans in regard to the length of the cycle where the rodent cycle is a 4.5 day cycle. The oestrus cycle has four phases namely proestrus, oestrus, metestrus, and dioestrus and rodents will have spontaneous ovulation. · Proestrus is equal to the human follicular stage o It is associated with a rise in circulating oestradiol concentrations and little surge in prolactin which leads to a rise in LH and FSH · Oestrus is equal to the human ovulation o The peak in FSH concentration leads to a rapid decline in oestradiol levels · Metestrus is equal to the early secretory (luteal) stage o High levels of progesterone shows here · Dioestrus is equal to the late secretory (luteal) stage o High levels of progesterone shows here Mouse pheromones have a huge impact and regulation of mouse reproduction. As with women, group housed female mice (without the presence of male mice) will synchronise their cycles. You will have suppression of oestrus which is called the Lee-Boot effect. When the female mice detect male pheromones it will induce oestrus which will normally happen after 3 days. Females that are mated on the 3rd night will ovulate more eggs and produce larger litters. This is actually regulated by FSH. Also, foreign male pheromones impacts the mouse reproduction. If you have a mated female and she is subjected to a male that is genetically more foreign to the male she was first mated with, it will induce foetal resorption (Bruce effect).

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

The principles of GLP shall help ensure safety and it also has a positive influence on the quality if the results. It is a part of the quality assurance that ensures that organisations consistently produce and control goods to a high quality standard. It aims to ensure the consistency, reliability, uniformity, and quality of chemical non-clinical safety tests. There are different elements of good laboratory practice, including: · Certification of analysis · Reagent and material certification · Accountability · Statistical procedures · Instrumentation validation · Documentation and records · Quality assurance · Specimen and sample tracking · Certification of laboratory facilities GLP is not a set of guidelines but it has the force of law. It include sections regarding animal care facilities, animal supply facilities, and animal care. It includes requirements for GLP research facility design to ensure separation of test systems, adequate space, and specialised rooms for conducting non-clinical laboratory studies. It includes requirements for animal housing, feeding, handling, and care with an emphasis on identification of animals and elimination of contaminants of variables that might affect the conduct of the study.

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)

The role of a pilot study is to test the practical feasibility of an experiment. The design of these pilot studies can be a bit more relaxed and it can be changed underway. We use a small number of animals (or no animals if possible) to test the execution of methods and to obtain a small dataset that allows for the design of a "proper" study. You will in a pilot study obtain information regarding the source of variability in our experiments and estimate their magnitude. One of the most important functions of a pilot study is that they allow us to discover pitfalls in the experimental design that are not immediately obvious when siting in front of a computer making the plans. A pilot study cannot be used to estimate the effect size since. It gives information about how variable the results might be.

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)

The sense of hearing in rodents is highly developed and the hearing range is well into the ultrasonic frequencies. Humans are not able to hear ultrasound which means that humans are not able to hear a large part of rodent communication since some of their communication is within human audible ranges and some is outside the human audible ranges. This means that humans may not be able to hear many of the sounds that may stress mice and rats and we may not hear their communication to each other. So when we think that the mice and rats may be quite, they may actually communicate to each other. Rodent visual acuity is most likely low which means that the surrounding may very well be quite blurry to them. Their colour vision is also very different from humans since humans are tri-chromatic and rodents are di-chromatic. Humans have three photoreceptors and we are able to perceive blueish, greenish, and red colours. Rodents only have two photoreceptors and are therefore only able to perceive greenish and ultraviolet light. We don't really know what they use their ultraviolet vision for but we know that their urine is fluorescent and therefore we could speculate that they communicate via urine markings. It is also quite important to remember that regular lighting in animal facilities should include the UV-wavelengths because without this range we provide unnatural lighting for the rodents and it might change their perception and we don't know how it actually affects them. Since rodents cannot perceive red light you can build houses and hiding spots for rodents in red see-through plastic. In this way the rodents will perceive the e.g. tunnel as dark but we as human beings can still see through it. The olfaction sense in rodents is very important in regards to communication. The social communication is mediated by pheromones released by one rodent to signal something to another rodent. The pheromones can be found in the urine, faeces, and in the saliva. This chemical communication can be used to identify other mice and decide how to approach and interact with them. They use their olfaction sense to establish a hierarchy and also for reproduction. It is very important to be aware of the three senses in rodents. They might hear things that we humans cannot hear and these sounds may stress the animals. They may smell pheromones from other rodents stressing them or increasing their aggression and anxiety due to foreign smells. They may also perceive things differently than us humans which can affect them. So if we don't think about these senses then we may end up with animals that are stressed, anxious, aggressive, etc. which in the end can interfere with our experimental results in a negative way. Therefore it is very important to be aware of these senses.

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)

The term culture of care has come to indicate a corporate as well as personal commitment to improve animal welfare and thereby scientific quality. The culture of care aims to improve welfare for both humans and animals, to give good scientific quality, to care for the staff, and to give transparency for the stakeholder. There is however no clear definition of culture of care and every facility has its own culture of care. The way the staff communicate and mutually respect each other will vary more of less between any two facilities. The 3R's will be an integrated part of the culture of care. It is really important to promote a culture of care in order to obtain good animal welfare as well as human welfare. It is important that there is an appropriate behaviour and attitude towards animal research and there should be great cooperation. A good culture of care will also reduce animals used by replacement and reduction as well as animal stress, pain, and suffering by refinement. The animals should be treated and handled with compassion and respect. A good culture of care will also reduce the emotional strain an animal care staff which supports job satisfaction.

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

There are different methods on how to identify your laboratory animals. The method chosen depends on the species, duration of the study, age and skin pigment of the animals as well as the availability of technical expertise. The ideal identification technique should provide efficient individual identification, be easy to apply and read without inducing pain and/or distress. The methods are: 1. Ear tags: Ear tags are plastic or metal tags that are clipped onto the animal's ear. They are inexpensive, easy to apply, and can be customized with different numbers or symbols. However, ear tags can become lost or damaged, and they can cause discomfort or injury if not applied correctly. 2. Tattoos: Tattoos are applied to the animal's skin using ink or a branding iron. They are permanent and easy to read. However, tattoos can be difficult to read if they become faded or obscured, and they may cause discomfort or stress to the animal during application. 3. Microchips: Microchips are small electronic chips that are implanted under the animal's skin. They are permanent and can be read using a scanner. However, microchips can be expensive, and the animal must be scanned to read the chip, which may not be possible if the scanner is not available. 4. Collars: Collars are worn around the animal's neck and can be customized with different colors or markings. They are easy to apply and can be used to track the animal's movements. However, collars can become lost or damaged, and they can cause discomfort or injury if not applied correctly. 5. Toe-clipping: Toe-clipping involves removing a portion of one or more toes from the animal. It is a permanent method of identification and does not require any additional equipment. However, toe-clipping can cause pain or injury to the animal, and it may be considered inhumane or unethical. 6. Dye marking: Dye marking involves applying a non-toxic dye to the animal's fur or skin. It is temporary and easy to apply. However, dye marking can be difficult to read if the animal's fur grows or if the dye fades, and it may not be suitable for all species or colors of animals. Each of these methods has its own advantages and disadvantages, and the choice of method will depend on the species, age, and purpose of the animal being marked. The method chosen should prioritize the animal's welfare and minimize any discomfort or stress caused by the marking process.

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

There are different safety and precautions that you need to be aware of when working with anaesthetics and analgesics. With anaesthetic gases you have to ensure that there is adequate ventilation to scavenge waste gases since you don't want to inhale them yourself. You should also handle the equipment correctly and make sure that the tubes are assembled correctly. Drugs should always be stored in locked cupboards and destroyed according to legislation in designated waste containers. You should always be careful when using these agents since they could potentially be harmful to you and you don't want to anaesthetise yourself during a procedure. You should also be careful if you are handling drugs for euthanasia since you do not either want to hit yourself with this or put them anywhere on yourself. Needles that has contained "dangerous" drugs should also carefully be disposed in designated waste containers. Gas cylinders have a risk of explosion in case of fire.

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

There are different types of breeding, including pyramidal breeding and rotational breeding. The pyramidal breeding is used for propagation of an inbred strain whereas rotational breeding is used to maintain a small colony of outbred stocks. With the pyramidal breeding it will happen as followed: · You will mate a brother and a sister called the stem pair 1. The stem pair will get a litter with pubs that are brothers and sister called the nucleus 2. These brothers and sisters from the nucleus will be mated and their offspring will be used as breeding animals a. These offspring may be cousins and they can either be used for research or breeding 3. If they are used for breeding they will make offspring which can either be used for further breeding or research 4. If they are used for breeding they will make offspring a. At this point (3 steps away from the nucleus) all of the offspring is used for research and no further breeding is allowed With the rotational breeding, one colony is subdivided into e.g. four sub-colonies (A, B, C, D). Females produced in sub-colony A can be used for breeding in sub-colony B. Females produced in sub-colony B can be used for breeding in sub-colony C. Females produced in sub-colony C can be used for breeding in sub-colony D. Females produced in sub-colony D can be used for breeding in sub-colony A. The males will be divided into two groups. One group of males will be exchanged between A and C, where the other group ill be exchanged between B and D. Off-spring from all four sub-colonies can be used in research. In this way we get systemisation of the randomness and it is very useful if the colony is small.

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

There are different types of human health hazards when working with laboratory animals. These include allergies, physical injuries, harmful substances, and zoonoses (an infectious disease that has jumped from a non-human animal to humans). For animal caretakers there could also be potential injuries from heavy lifting, repetitive movements, noise, slippery floors from cleaning, etc. In this connection there are also many things that can be done to prevent these hazards. In regards to the development of allergies you should make sure that you keep the levels of allergens in the environment as low as possible. You should use personal protective equipment and you should make sure that you do not carry animal allergens with you when you leave the animal facility. To avoid accidents due to harmful substances, different precautious actions must be taken. These include warning labels on cages and only opening of cages in a biosafety cabinet or hood. You as personnel should wear gloves, eyewear, mask, and coverall. You should be able to perform good needle practice. You should screen materials for human pathogens and use disinfection and/or autoclaving of waste. You should also follow legal requirements for rooms, laboratories, and education of personnel. You should restrain the animal properly and have experts available. You should keep and transport radioactive substances safely and only inject the animals in safety cabinets. The syringes should be prepared before the experiments and you should take precative action during the preparation, injection, etc. In regards to zoonoses this can be prevented by using animals that do not carry the zoonoses (using microbiologically defined animals). You should make sure that biological products and barrier products come from safe sources. You can establish a barrier unit in our animal facility and you should imply good working routines. You should also be able to use adequate personal protective equipment. If you experience injuries you should action immediately. You should wash the exposed body part with water and soap and make sure to disinfect it afterwards. In case of symptoms of infection, you must seek medical treatment and report what you have been working with to get the proper care.

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)

There are generally two different types of diets - the natural ingredient diet and the purified diet. The natural ingredient diet is based upon natural ingredients and it is produced to fulfil specific requirements of specific animal species depending on whether you are using animals for breeding or maintenance. If the animals are used for breeding then you can get a diet that stimulates growth, pregnancy and lactation. The natural ingredient diet contains whole grain, milk by-products, high protein meals, and mineral sources. The purified diet is on the other hand based upon synthetic compounds. It contains proteins, carbohydrates, fibres, fat, various mineral mixtures and various vitamin mixtures. These types of diets are most often used for scientific purposes where you e.g. make a diet deficient of one single component. The natural ingredient diet is made to fulfil the nutrient requirements of the animal whereas the purified diet is not.

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)

Utilitarianism states that morality is about maximising human and animal well-being and that animals deserve moral considerations just like humans. The welfare consequences for animals must be considered as well as the potential benefits for humans. This means that activities that may have an adverse impact on the well-being of animals may be justified if they lead to a net increase in welfare for humans or other animals. The public might believe that some animal research may be justified by its vital importance as it may enable us to find cures for alleviate painful diseases. They might also believe that it sometimes is better for stray cats to be euthanised as they would otherwise live very poor lives. The remaining stray cat population may benefit as well because there will be less competition for food. Contractarianism states that morality is based on agreement. The ethical obligations originate in mutual agreements or contract between people. Each of us has his or her own interest and we are perfectly entitled to pursue these. In most situations we can benefit from the help of others. The view is based on mutual cooperation and that this is in all of our interests and best for everyone. Animals neither create nor have moral duties because non-humane animals cannot make agreements. They lack the control and understanding needed to enter a contractual arrangement. But we as human beings may have indirect ethical obligations towards animals since they can matter to other human beings. The public might believe that to improve the quality of animal research, one should be concerned about animal welfare and that the animals should be treated well enough to suit a purpose but it is not worthwhile doing any more than that. Animal Rights View believe that good results cannot justify evil means and that fixed ethical rules place limits on our treatment of animals. This means that there are some things that we are not permitted to do to an animal whatever the circumstances. The animal rights view comes in more or less radical forms. With the most radically view animals have rights just like our human rights. This view includes the right not to be killed for human benefits except in self-defence. In the other end the less radically would believe that animals have the right to be treated with respect or humanely. This means that we must not do avoidable harm to animals. The public might believe that animals are not out slaves and that animals have inherent value which should be respected. They might believe that experiments on animals are unacceptable, regardless of the potential benefits involved.

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

When publishing experimental animal studies a good way is to use the ARRIVE guideline since it includes very specific details on what to include in the paper. It should include a title, abstract, introduction, material and method, results, discussion and a conclusion. It has the same outline as a regular scientific paper the material and method as well as the discussion has to be a bit different. In the material and method you should describe the ethical statement, study design, experimental procedures, sample size, allocation of animal to experimental groups, experimental outcomes, statistical methods, experimental animals as well as housing and husbandry. It should include information regarding the species used, the genetic status, age, sex, microbiological status, quarantine or acclimatisation period, etc. You should describe what measure you have taken to protect the microbiological status, what housing equipment used, the number of animals per cage or housing unit, the type and quality of the bedding, the environmental temperature, the relative humidity, the lighting schedule, the ventilation scheme, the measures done to refine the experimental techniques to benefit animal welfare, the diet, and the water. In the discussion there should be information regarding the limitations of the results including any potential sources of bias, any limitations of the animal model, and the imprecision associated with the results. You should describe any implications or you experimental methods or findings for the 3R and you should comment on whether and how the findings of this study are likely to translate to other species or systems, including any relevance to human biology. So to conclude you should include information regarding everything that could have an impact on the results in regard to the animal. So everything from how the animal is received and from where to how they were housed, fed, etc. All of this should be put in perspective when discussing the results.

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)

When we are conducting multifactorial designs we are testing multiple effects in a single large study. Fx when testing the effects of a drug, we might want to know if males and females react the same. This is not a problem; the important thing is just that 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. We can therefore obtain more information per mouse compared to carrying out many small studies since we test multiple effects concurrently. The drawback is that the design and statistical analysis of the future results become complicated. · For multifactor design a statistician, may be necessary, as the design and statistical analysis are very complicated. For some of the more complicated designs, there are no easily accessible tools that allows us to estimate an appropriate sample size. Computer programs that can be used by statisticians to calculate our sample size, are based on a simulation-based approach · The elements that you need to be able to provide information regarding to a statistician are statistical power, effect size, level of significance, and variation. I guess that you also have to be able to provide information regarding the study design and the sample size.

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)

When we are going to decide upon the number of animals we need in our study, we need to look at the variation, effect size, statistical significance, and the statistical power. We need to decide this before our experiment so that we don't use an excessive number of animals when there is no need. We should therefore carefully consider what aspects that will affect our study outcome and then decide upon this. It very much depends on what we are studying. The sample size can be determined or estimated with different tools. There is a way to do it in the book and you could also do it on the internet when it is just simple case-control studies. You could use different types of equation where all parameters are included, but I guess it is always important to keep in mind that we want to reduce the number of animals used. You should always consider if it makes sense to you and you experiment. You should balance the number of animals used with the things we are looking for. Because if you choose a sample size that is too small and you don't observe what you want, then the animal's lives would have been wasted. On the other hand, if you just use a low of animals and you see the effect quite fast or in all animals, then you maybe didn't need all of the animals and some animal lives could have been spared.

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)

When we decide to use live animals in experiments, it is important, that we can justify it. The 3R says that whenever possible we should replace live animals with insentient material. But sometimes it is inevitable and therefore is should be justified on ethical grounds. So when we plan an experiment using live animals we should strive to maximise the benefits and minimise the harm. This should also be applied to the choice of model but here we should also consider to use the model causing the least suffering. The animals should be in good health, and we should use as few animals as possible without compromising the quality of our outcome data. We should make sure that the animal is in good health and that their welfare is optimal. The animal should not experience fear, pain, or prolonged stress. If you know that your model may cause any of these, then you should consider to refine your model to minimise the harm to the animals. It is our prime goal!

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).

When we talk about diseases in laboratory animals, we normally talk about infectious diseases that may infect the animals. These are caused by viruses, bacteria, or parasites. The microorganisms in laboratory animals may be zoonotic (cause disease in humans), cause disease in the animals, or interfere with research. It is therefore important to protect the animals against these diseases and take precautionary action. Some species are more prone to different infections than others. Both the nude mouse and B6 mice are prone to get infected with mouse hepatitis which in both cases will cause symptoms. The nude mouse is immune deficient and the immune system may therefore not battle the virus. If hepatitis infects the mouse and causes impact on the liver and its function, the mouse will eventually die. In order to protect the animal against the infections, the three-step racket of laboratory animal hygiene is used. The three steps include: 1. Rederivation where the ancestor breeders have been produced either by caesarean or embryo transfer. In this way you can create infection free animals in flow cabinets or disinfected baths. Embryo transfer is the most widely used method for rederivation since the eggs of many strains can get frozen and kept in the freezer. The eggs are harvested from the mother the morning after superovulation. 2. Protection of the infection free animals could be behind a barrier, in a barrier room, or in isolated surroundings. If the animals were just housed in conventional rooms there is no guaranty that the animals will stay uninfected. Offspring bred by rederivation should be housed in one of these to make sure that they do not get infections. It requires stricter rules regarding the staff entrance, material entrance, and the ventilation of the room. 3. Health monitoring is done routinely to make sure that the animals are still infection free. FELASA has issued recommendations regarding the health monitoring of various species. They make recommendations on the frequency of different monitoring procedures. You can send either samples of tissue or a whole animal to be subjected to one of these health monitoring procedures. The laboratories performing these tests will make a certificate where the results can be seen and easily assessed.