Laboratory animal science

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

There will be different rooms for breeding, housing, and experimental rooms where procedures can be performed. The facilities are often in a closed area to keep the animals free from infection and to reduce the risk of people outside the facility to develop an allergy. We have something called a barrier facility which is designed to keep unwanted microorganisms away from the animals inside the barrier, or where experiments with pathogens are carried out and keep the experimental pathogens inside the barrier. All though there is also something calles conventional housing a "non barrier" housing where the animals are in open cages and people are allowed to go in and out without any rules for entry og exit. Animal with normal behavior are better representative of true biology and thus provides more valid research results. Environment and enrichment: Behavioral enrichment: mimicing the habitat world fx if you provide the mice with nest material it's gonna build a nest. Social enrichment: gives the animal the opportunity for social interaction and performance of species-specific social behavior. Artificial enrichment: use a variety of artificial objects to achieve enrichment, they must be object that the animal is interested in. Food enrichment: makes the animal exhibit species specific behavior. Making a game out of getting food can help poorly eating animals more interested in nutritious food. Lack of control: If the animal feel lack of control it behaves in a manner that indicates frustration you can then give the animal some choices so itll feel in control ex different food choices.

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.

According to the EU legislation the killing of animals shall be completed one of the following methods, which depends on the species: confirmation of permanent cessation of the circulation destruction of the brain dislocation of the neck exsanguination (death caused by the loss of blood) confirmation of the onset of rigor mortis (muskelstivhed efter døden er trådt in). There is two different techniques used for euthanizing animals Euthanasia by pharmacological - chemical methods Euthanasia by mechanical - physical methods Chemical methods: lethal drugs are injected such as carbon dioxide (cheap and easy). physical methos: disloaction og the neck. Remember ALWAYS to confirm death! There is some Immediate signs which includes: • Heartbeat stops • Respiration stops • reflexes are absent • pale skin Some later sign which includes: • Cold body surface •Rigor mortis (stiffness)

Define sedation, local and general anaesthesia, including the triad of anaesthesia, balanced anaesthesia, preanaesthetic considerations selection of agents and their use, storage and disposal

Anaestesia can either be local where you block some certain nerves to get the anaestesia in a local area, or it can be general, where the subject is put into unconsciousness. There are some basic elements of general anaesthesia, first of all it has to be reversable and controllable. General anaestheisa cosists of three components called the anaesthetic triad: - Loss of consciousness - No feeling of pain -Muscle relaxation Considerations regarding the animal: - assistent needed? fasting needed? pre-anastehtic needed? (Pre-anaesthetic medication often consists of a cocktail of different drugs, and we mainly use it in larger animals.) which drug to use the estimated time of surgery. Monitor the animal and have a plan in case something goes wrong. - make sure the animal is healthy (no infections ect) fasting needed? - we dont want the body busy with digestion and prevent vomitting. Give the animal time to settle in its new enviromment (1-2 weeks for mice and rats) One anasthehic drug or a combination? - With a combination you have less side effect as a smaller dose is used, but each drug can effect various systems. More than often a combination is needed to fulfill the anastethic triad. Make sure the drugs doesn't effect the outcome of the experiment. Surgery in patients: you chose the anesthesia to make sure the patient vill survive in good condition. Experimental surgery: the anesthesia is chosen to collect scientific data as accurate as possible and loss of animal is accepted.

Explain and discuss different ethical views on use of animals for experimental research and how this is relevant for communication in public and for the choice of animal or non-animal models

Animal Ethics is a discipline describing different views on how we should treat animals in our care and to what extent we should be working with animals. There are three main positions and ethical views on animal use in experimental research and toxicology. - Disapprovers - Approvers -Approvers with reservations Disapprovers (animal rights view): They think that animals have rights that must not be denied and they only consider animals which are considered being sentient. Approvers (contractarianism): They accept the use of animals and assume that human interest are more important than the interest of animals, no matter the cost to the animals as long as society allows for it - morality based on agreement. Approvers with reservations (utilitarianism): the suffering in the experiment must be compensated by the resulting benefits, so that the overall consequences of the experiment are more welfare than before. They are far more concerned with the suffering of the animals and argues that animals that are capable of suffering do deserve moral concern. This view is based on Utilitarianism, which includes the principle of creating as much well-being as possible no matter the actions - though all sufferings must be deminished to the highest possible extent.

Describe national and EU laws and guidance regulating the scientific use of animals and the activities of those carrying out scientific procedures involving them, and how you get an authorisation before acting as user, breeder or supplier of laboratory animals

Animal welfare act: no one can cause any species of animals any pain, suffering, distress or lasting harm. Animal Expermentation act: Overulls the animal welfare act hereby allowing inflicting animals pain, suffering, distress or lasting harm IF the purpose is research, teaching or the production og blood products and a specific license is needed. The Animal experiments must be: Licensed, beneficial, animals must be necessary. You shouldn't be causing strong pain, intensive fear or intensive suffering. And it should be performed by qualified staff in proper settings and on destination bred animals. The members of the Europe Union: If the EU issues directives which is agreed, it's then mandatory for the member states to implement these rules. The member states can in their national legislation have stricter rules but not weaker ones.

Present and discuss different viewpoints on/definitions on the nature of Animal welfare and discuss methods to measure/evaluate animal welfare and the impact of good welfare on scientific outcome

Animal welfare can be defined as the animal's state of health and biological functioning. It can be defined as its natural living where the animal can express its natural behaviour and lastly the definition can be chosen to focus on animal emotions. Welfare is good health If animal welfare is defined as good health with good biological functions, then clinical examination and measurements of physiological parameters (for example stress hormone) can be established to tell if the animal has good welfare. Statistical parameters such as the mortality rate and the assessment of abnormal behaviours can also be used to indicate whether the animal is healthy. Welfare is natural living If animal welfare is defined as natural living then welfare of the animal depends on whether it can realize its potential and perform its natural behaviours. Welfare is positive emotions In this definition, animal welfare is based on experienced feeling or mental states, where presence of positive feelings add to welfare, meanwhile presence of negative mental states reduces welfare. Furthermore it includes whether the animal gets its preferences fulfilled, does the animal get what it wants and does it avoid what it does not want. The five freedoms - Freemdom from hunger and thirst - easy access to water etc. - Freedom from discomfort - provide good environment.

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.

Experimental endpoint: Predefined endpoint of the experiment When all necessary data have been achieved Humane endpoint: The earliest point at which an experiment can or should be stopped/terminated With the purpose of eliminating unnecessary suffering The human endpoint can be reached before the experimental endpoint. Non- recovery experiment: procedures are performed under general anasthesia. Mild experiment: procedures will result in a short-term mild pain/suffering. Moderate experiments: procedures will result in shorm.term moderate pain/suffering or long lasting mild pain. Severe experiments: procedures will result in severe pain/suffering or long term moderate pain. Example on a clinal humane endpoint: Frightened and unusually aggressive when handling the animal Example on a pathophysical humane endpoint: When the animal has a weight loss over 5 % compared to pre-procedure. Example on a biochemical humane endpoint: When the level of a specific biomarker is over a certain percentage. In order to detect and define the humane endpoints it is a good idea to create a welfare protocol - where you design a scoresheet with relevant parameters that can help you identify if something is wrong with the animals.

Give examples of stressors and consequences and manifestations of stress in rodents, including both basic biological factors relating to the animals and factors relating to the environment and physical handling

Freezing in open field. Bruce effect. Red light has an effect on rats' circadian rhythm Handling of the rats - if you don't know how to handle the rat you'd probably pick it up in an incorrect way thereby the rat will feel uncomfortable and react to it which won't make you succeed right away the rat will end up being stressed

Describe how genetically altered animals can be generated, how they can be used for scientific research, and how they can be genetically monitored

Gene modification of animals allows for insertion or deletions of genes. Gene modification can produce transgenic animals, knockout or knockin animals. Knockout: deleting a gene Knockin: inserting a gene The most simple way to make transgenic animals: Knockin: Mate a female animal with a male. The female is euthanized the naxt day and her fertilized eggs are flushes from her salpinx and injected with a DNA construct with the desired gene - an incidental break will occurre and the gene is randomly inserted without influence of the operating person. Another female animal is mated with a sterilized male animal, she is then anesthetized and the manipulated eggs are surgically plaed in her salpinx. In larger animals sperm precursor cells are frequently sampled from the seminiferous tubules for microinjection. The desired DNA contruct is injected into these precursor cells, which can subsequently be propagated in culture. The propragated manipulated cells are placed back into the tubules of another male. At mating this male will now ejaculated both some normal sperms and some gene modified sperms. From the blastocytes of some very specific inbred strains of rats and mice embryonic stem cells can be harvested and grown in culture. For mice it is the agouti coated 129 mouse which is normally used. When in culture the whole set of gene modification techniques can be applied, which enables that after gene modification techniques specific cultivations can select only those cells in which a targeted mutation has occurred. These can be injected into another blastocyst, which is then injected into an anesthetized pseudopregnant female mouase made by mating with a sterilized male. She will give birth to chimaeras, that means mice containing both wild type cells and gene modified cells. Newer techniques such as CRISPR-Cas9 will probably replace most of the techniques as it's easier and allows knockin/knockout on all animal species. Genetically altered mice can be used for scentific research - they can be used to discover treatments and causes to different diseases

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

Monitoring: The absence of reflexes - Circulatory functions - Respiratory functions -Body temperature Keep oxygen at hand as the oxygen levels could drop under 90% which could do damage to the body. Oxygen levels under 70% is critical/crucial for the animal - hypoxia --> cyanosis Reflexes: Righting reflex: an four legged animal will try to turn back on its feet if it falls over. Withdrawel reflex: respondence to noxious stimulus - If you pinch the animal fx between the toes, do you see any movement/reflex? If the animal shows reflex during surgery - provide more anesthesia or local anesthesia If the animal has low oxygen levels - provide oxygen, heat, fluid or try to reduce the anesthetics if possible.

Explain international standards for nomenclature of laboratory animals

Necessary: Uniform, informative and systemaic way of presenting the animals. The line for inbred animals is called strain, whereas it is called stock for outbred animals. When we name inbred animals, we first write the strain name, then a slash and last the breeders lab code. When we name the outbred animals we first write the breeder code, then a colon and last the stock name. For a gen modified rodent is it necessary to show the gene modification in the name of the rodent. Naming transgenic animals, we first write the name of the background strain and thereafter the gene name. The background strain and the gene name are separated by a horizontal slash. This is all done to help the research by allowing the researcher to follow each step/history).

Describe appropriate breeding programs, including inbreeding and outbreeding for laboratory animals, how the estrous cycle is relevant for breeding, and how animals can suffer due to their genetic constitution

Outbreeding: usually maintained as a closed colony without the introduction of animals from another stock. Outbreeding is when animals are mated randomly, but unintended selection will always occur under random mating, as some animals grow more, breed and survive better etc. Researchers have created a system to randomize the breeding. It is called Rotational breeding. We have four subcolonies, A, B, C and D. The females that are produced in A can be used for breeding in B, where the females produces in B can be used for breeding in C and so on. The males are exchanged between the four subcolonies, A, B, C and D. This helps prevent inbreeding and protects against natural selection and breeder selection. Inbreeding: inbred animals are close relatives - a father and daugter or a pair of siblings for at least 20 generations. Researchers have also created a system to inbreed rotens. It is called the Pyramidal breeding Traffic light system. In production stock 1 there is green light for producing animals for further breeding, in production stock 2 the light is yellow and in production stock 3, the light is red and the animals produced can only be used for research and not further breeding.

Describe different types of diets for laboratory animals, how they are constructed to meet the dietary requirements for the animals, how you would use specific diets for specific studies, how you would order them, and how you would store them

Pellets - consisting of carbohydrates, proteins, fat (containing essentiel biochemical acids such as arachidonic acid), essential minerals and some vitamins depending on the animal. Diets: The natural ingredient diet - natural ingredients such as whole grains, high protein melas such as fish and bone meal. Its cheap and liked by the animals but batches of the same diet can vary and there is a risk of the batches being contaminated by other animals from the field. The purified diet: based on synthetic compounds such as amino acids, sucrose, vitamins and minerals in amounts to fulfill the entire need of the animals. This diet is mostly used for specific research purposes and there is low variation between batches also one single component is removable. But the animals does not like this diet, it's expensive and may lack biologically active compounds of importance for the models. Feeding schedule: Ad libitum Restricted feeding - fed with exactly the food they need to make sure that the intake corresponds with their daily nutrient requirements. Pair feeding - You can get two groups to eat exactly the same amount by registering the food intake in the group that eats the most, where the other group can be supplied with exactly the same amount of food. Fasting - necessary for some experiments such as a glucose tolerance test. The correct way to fast rodents is to remove the diet when the lights turn on where they are active. Furthermore, fasting can cause torpor of the rodents, which means that their metabolism and body temperature become reduced due to the lack of food. Gavage - this is a precise and individual dosing through the gastric tube. This also means that there will not be any oral digestion and therefore data may be less translational compared to humans.

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 and a culture of care to minimise the harm, maximise benefits and 3) promote good animal welfare practices, and explain minimum and maximum threshold of pain, suffering, distress or lasting harm

So in order to get permission to do an experiment, the animal experimentation act has to decide whether the benefits outweigh the harms. And the more beneficial you experiments are, the more you're allowed to "harm" the animals, the more animal distress they will allow. The animal experimentation act also says that you can only house the animals and do the experiments in premises that are suitable and they have to be designed to fulfill the 3R's. This is going to help minimize the harm and maximize the benefits. The staff also has to be advised amongst other things, animal welfare. So when we talk about an animal experiment we have a lower and an upper threshold. The lower threshold is the injection criterion, and the upper threshold is known as strong pain, intensive fear and intensive suffering. We also have a purpose, when talking about animal experiment, which is either research, teaching or production of blood products. And according to the animal welfare act, we are not allowed to cause pain, suffering, distress or lasting harm in any species of animals, but the animal experimentation act overrules the animal welfare act. It says, that if the purpose Is research, teaching or the production of blood products, pain, suffering, distress or lasting harm can be allowed if it is licensed.

Describe who is responsible for compliance in relation to how the experiment is performed, and how the animals are housed in the facility, including scientist awareness of local organization, such as the animal welfare bodies

The animal Experiment: If you apply for a license to do the animal experiment, you'll get a license through the license holder, and basically you have the responsibility in relation to this. So even if you have a PhD student, that does something that is not according to the license you're the responsible one. The animal care and housing: This is something the EU has asked Denmark to change, because it's a central responsibility. At present in Denmark, for instance the University or a company like Novo can ask the animal experimentation inspectorate to place alle the responsibilities with a specific person at the University or the company. According to the EU directive this is always a central responsibility, and it will also be like that in the future. So for instance if you have a license and you work at the University, you have a license to do the experiment and everything that happens in the relation to the experiment is your responsibility, and then you will place your animals in the central laboratory facility, and the head of that department will have the responsibility for animal care and housing. That means that it isn't your responsibility that that someone comes in the weekend and takes care of the animals, that's their responsibility.

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

The disease can be: fluminant - there are clinical symptoms and the disease is shown. Latent - you cannot see the disease/infection etc. and there is therefore no clinical expression. So, infections may cause disease, but but they dont always do. However, infections that do not cause disease can interfere with animal research in other ways. Spontaneous infections: This is caused by viruses, parasites or bacteria, that can all infect the animal. Spontaneous infections are infections that the animals (or humans) catch by chance i.e. coronavirus in mice or humans. In laboratory animals it is in contrast to 'experimental infections', which the scientist gives to the animals by will. Importance for animal experimentation: Reduces the health of animals fx latent infection (direct/indirect) Direct interfering; Immunomudlation: infection may change immune system Physiological modulation: infection may change the physiology Ocological modulation: change sensitivity to cancer Fertility modulation of animal; fertility of the animal Microbiological competition: the infection can compete with experimental infections that you want to infect the animal with Indirect: The infection can indirectly contaminate some of the products you sample from animals. Furthermore, when you stress the animals, by experiments, some of the above direct effects may be activated. Fx mouse hepatitis is cause by the coronavirus. For protecting animals against infections we use a three-step rocket. Rederivation (''producing'' germ-free animals) Protection (protection of the germ-free animals) Health monitoring (ensterm-9uring that the animals are not infected with hazardous infectious agents) Protection of animals: barrier housing - In the barrier housing the the animals are kept free of certain species the air is ventilated and the staff will enter through a three-room shower. Furthermore there are quarantine rules if the staff has had contact with other animals isolators - which means fully closed systems as the animals are only being handled through gloves integrated into and tightly sealed to the isolator wall. Health monitoring: The Federation of European Laboratory Animal Science Associations has issued recommendations for health monitoring of various species. They list which agents they consider relevant to test in those animal species and they also list how frequently this should be done. So, the facilities will submit animals or samples from animals to laboratories, who will do routine health monitoring and after that they will issue a certificate. Get rid of infection: Cessation of breeding - have a break in the breeding and no new animals are added as the colony has developed immunity. Stamping out - remove the infected animals from the colony. Antibiotic treatment - Antibiotics have been used for total eradication of specific pathogenic bacteria in immune-competent animals. Can't be used for virus infections as these do not have a cellmembrane which the antibiotics attack. Vaccintation - he use of vaccinated animals may still result in undesirable microbial effects on research, such as the immunosuppressive effect of Sendai virus. So, if the animal colony can be kept free of an infection using other techniques, without resorting to vaccination, then these techniques should be implemented.

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

The ethical reason is since pain may be a source of suffering, we have a moral obligation to avoid it whenever possible. The scientific reasons: 1. Pain induces a stress response which will alter the normal physiology of the animal ànd this can change the way the animal will respond in the experiment. 2. We also have impaired recovery after surgery 3. And thirdly we have the risk of developing chronic pain. Signs the mouse/rat is uncormtable: Pain specific behavior: Twitiching, back-arching, falling, adominal press. Is the animal eating or drinking normal? does it bite or lick itself more than normal? Physiological parameters: Heart rate, bp, body temp can be assessed. As these parameteres increase when an animal is in pain. Cliniqal signs: body weight, food/water consumotion, urination, pooping and fur quality. Try to prevent the pain in the first place by handling the animal in a correct way and give it analgesic drugs before surgery and good hygiene.

Explain the purpose of a harm/benefit analysis in animal experimentation, and describe the severity classification system, including cumulative severity and the effect this may have on the severity classification

The purpose of the harm/benefit analysis is to try to decrease harm to both the animal and the staff as much as possible and try to get as much benefit as possible out of it. Harm relating the animal welfare aspect compromises the welfare of the animal - For instance it may be considered a harm to the animals, if the quality of the research is low. The animal is not per se harmed more due to a low-quality study, but the data are useless and the use of the animals irresponsible and with lack of respect.

List the correct procedures for ensuring health, welfare and care of animals during their transport, and describe the biological consequences of acclimatisation, habituation and training.

There is a term called acclimatization which is the process in which an individual organism adjusts to change in its environment. It allows an animal to maintain performance across a range of environmental conditions. Fx, if the animal is bred one place, put in a truck for hours and then placed in a new facility. We have to then give the animal time to adjust and receive homeostasis and "build up" its circadian rhythm again. Some period of acclimatization following transportation is generally suggested to restore homeostasis and is a legal requirement. In general, a period of 1-2 weeks is regarded to be sufficient. However some physiological functions may require much longer to return to normal.

List potential human health hazards associated with contact with laboratory animals and how these can be prevented.

There are som hazards that include: allergy, physical injury, harmful substances and zoonoses. Hazards for animal caretakers: Injury from heavy lifting, repetitive movements, noise etc. Allergens are proteins from urine, skin, hair, saliva. These can be airborne. Exposure through inhalation, skin contact. Some of the symptoms can be hay fever, asthma, skin reactions, anaphylactic shock. Management: Reduce levels of allergens - Isolation of animals - Ventilation in animals housing - Effective and safe cleaning of materials Reduce contact with allergens - Protective clothing - Clean/dirty areas - Restrictional access -Separate animal workers - Minimize nr of animal workers. Kicks which can be very serious and fatal can be caused by larger animals

Discuss strategies for successful perioperative pain management as well as post anaesthetic recovery and post-surgical care, and difficulties that may arise in connection to this

To prevent perioperative pain you first of all need to know how to handle the animal. Due to the anasthesia the animal won't form any memory of the surgery thereby reducing the risk of post-traumatic-stress. Difficulties connected with postoperative care could for example be post-operative pain, dehydration and hypothermia (low body temperature). These "side effects" could be treated or prevented by providing analgesia hereby relieving the pain. To treat/prevent dehydration the animal can be given fluid therapy - which includes giving body warm saline I.V or subcutaneously and provide easy drinking access. To make the drinking easier one could use extra long drinking nipples or jelly liquid for easy ingestion. Hyperthermia can be prevented/treated by using heating blankets on larger animals or heating pads/infrared heating lamps on smaller animals, maintaining the body temperature of the animal until it's fully recovered. Make sure not the overheat the animals. - Post operative pain o We can provide analgesia as a solution - Dehydration o Provide fluid therapy o Body warm saline IV o Provide easy access to drinking water - Hypothermia o Heating blankets, infrared heating lamps or incubators o Body temperature must be maintained until animal is fully recovered o Be aware not to overheat the animals - Infections o Work aseptically o Observe wound healing o Provide antibiotics if necessary - Postanaesthetic restlessness and self injury (where the animal fx starts biting themselves) o Can be caused by pain or severe distress o Consult a veterinarian oUse sedatives or analgesic if indicated Large animals recovery: in dedicated recovery room, quit, room temp and supportive care. Rodents recovery: separate area, higher temp., soft and comfortable bedding, avoid unnecessary human contact and handling