FELASA Flashcards
- Explain and discuss different ethical views on use of animals
Public perception of live animal use in experimental research and toxicology can be split into three positions:
Animal rights view (disapprovers): all sentient animals have rights which must not be denied.
o Represents the ethical position named Animal Rights View, which states that:
* A sentient being must never be used as a tool, and good results cannot justify evil means, which refers to the using or abusing of other living, sentient beings.
* The Animal Rights View is based on the same principle as our society in general: all humans have equal worth and an absolute right to be treated with respect. According to the animal rights view, not a single morally valid exclusion criteria of sentient animals from this principle exist, and thus it must be concluded, that all sentient animals, including humans, have an absolute right to be treated with respect.
Contractarianism (approvers): human interests are more important than the interests of animals, no matter the cost to the animals
o Contractarianism believes that morality is based on agreement, so as long as society allows for it, it is not a problem to use animals for experimental purposes.
Utilitarianism (approvers with reservations): Harms and benefits must be balanced, and animal suffering is not to be ignored.
o Utilitarianism builds on the principle of creating as much happiness as possible no matter the actions needed for doing so. However, the suffering caused by performing an experiment must be compensated by the resulting benefits, so that the overall consequence of the experiment is more welfare than before (“the needs of the many outweigh the need of the few, or the one”). The utilitarian focus thus lies on the consequences of actions, especially in terms of suffering, which must be diminished to the highest possible extent.
o In summary: Alternative actions must be considered, and the action chosen must be the one which creates most wellbeing and least suffering, which means to choose the action that will give the greatest welfare benefits for animals and humans and result in as little pain, discomfort, and suffering as possible.
- Present and discuss the demands for education and training of staff and license holders in Europe
According to the EU directive, any staff working with laboratory animals must be adequately educated and trained, that is, staff needs to be competent to do animal experimentation. In the EU directive, only a short list of subjects to be taught for staff involved in animal experimentation is provided. Therefore, the Danish legislation when it comes to staff competencies is based on guidelines issued by the EU Commission and is described both in the Animal Experimentation Act and in more detail in the Animal Experimentation Order.
According to legislation, staff must be adequately educated and trained before performing any tasks related to the following FELASA license functions:
A = required for staff carrying out procedures of experiments on animals
B = required for staff designing procedures and projects
C = required for staff taking care of animals at facilities
D = required for killing of animals
The Animal Experimentation Board of the competent authority in Denmark, the Animal Experimentation Inspectorate (= Dyreforsøgstilsynet), is responsible for issuing of licenses for animal experimentation. An animal experiment can be defined as:
“a procedure, which, for the purpose of research, teaching, or production of blood products, causes pain, suffering, distress, or lasting harm in a vertebrate including mammal fetuses in their last third or a cephalopod equivalent to, or higher than, that caused by the introduction of a needle in accordance with good veterinary practice (the injection criteria)”.
To get a license, the Animal Experimentation Board of the Animal Experimentation Inspectorate must evaluate whether:
the proposed experiments are beneficial to society (not just to an individual or their company)
the use of animals is necessary (if other appropriate non-animal methods are equally applicable, a license won’t be issued)
the experiments are causing strong pain, intensive fear or intensive suffering (upper threshold), as this is not allowed in Denmark.
Furthermore, the competent authority is responsible for ensuring that animal experiments are conducted in accordance with the obligations from the directive, and thus the implementation of the rules stated in the EU directive.
- Give examples of housing facilities and types of housing for rodents. Discuss different environmental factors and possible influence on animal welfare
Animal facilities
Animal facilities can be located as either a separate building or among other functional areas inside a shared building. Staff includes at least a facility manager, animal caretakers, and a veterinarian. At larger facilities supportive staff might include staff of administration, quality assurance workers, and laboratory technicians. An animal facility is usually composed of several units (e.g., units for breeding of animals, units for experiments, units for quarantine, units for large animals, etc.). Each unit has multiple rooms, and animals are kept in enclosures within these rooms such as cages, aquariums, or pens. Facilities are usually isolated and closed due to public relations, security, and for animal health reasons.
Different types of facilities exist:
– Barrier animal housing
A research barrier facility depends upon the existence of a microbiologically impervious perimeter enclosing a room or group of rooms used for housing animals. These infection barriers include physical barriers such as walls, and management barriers such as personal behaviour and disinfection. Barriers are used at animal facilities to protect animals from unwanted microbes, and can be present at cage level, room level, unit level, or at the entire facility.
Various barrier facility building designs have been developed to decrease the potential for cross-contamination between animal rooms:
o Single corridor (contamination can occur at the exit point from the washing machine OR due to dirty and clean materials being transported in the same corridor).
o Dual corridor with clean/dirty corridors.
Various approaches exist to avoid contamination, including separating work procedures in time or by covering clean and dirty cages by a sheet of plastic.
Air pressure is used as a tool at barrier facilities and in quarantine rooms:
o Hyper pressure is used at barrier units, as positive pressure rooms maintain a flow of air out of the room, thus protecting animals inside from possible contaminants and pathogens which might otherwise enter through exclusion of unwanted microorganisms.
o Hypo-pressure is used in quarantine rooms, as negative pressure rooms maintain a flow of air into the room, thus keeping contaminants and pathogens from reaching surrounding areas. Negative pressure can also be used to protect the outside from allergens present inside the animal facility.
– Conventional animal housing
Conventional animal housing refers to non-barrier housing where no special precautions are required for access. This type of housing is not ideal, as there is a risk of carrying infections from the outside to the animals inside the facility.
Animal housing
Housing is related to the surroundings and environment of experimental animals, which must meet regulatory demands to ensure animal welfare and allow control of environmental factors (genetic, microbial, chemical, and physical factors) to ensure standardized conditions for scientific experiments.
Different types of rodent housing exist:
– Open cages are cages in which only a steel grid keeps the animal inside, which are usually used for small rodents such as mice or rats. The steel grid confers little microbial protection of the experimental animals, and no protection of their environment from allergens, dust, or smell. This can be avoided using a scantainer, a type of cabinet, in which all supply and exhaust air is filtered. This minimizes allergens, dust, and smell and experimental animals are protected against infections and environmental influences such as noise.
– Individually ventilated cages (IVCs) are airtight cages equipped with an air supply and exhaust system, which minimizes allergens and dust and leaves animal housing rooms odour free. IVCs can be kept in ventilation units which allow high air change rates, which can reduce the required cage change frequency.
– Isolators are used for animals in open cages and provide complete isolation of animals. Air pressure determines whether the animals are protected from the environment (positive air pressure) or the environment is protected from the animals (negative air pressure). This type of housing has been largely replaced by IVCs.
Husbandry
Husbandry relates to the caring for the animals and the daily work taking place at an animal housing facility (e.g., receiving, marking, cage changing, cleaning, feeding, observing, killing, etc.) which is typically carried out by animal caretakers. Acclimatisation describes the process during which an individual organism adjusts to a change in its environment. Acclimatisation is thus needed for reestablishment for homeostasis and is a legal requirement (1-2 weeks is regarded sufficient, but some physiological functions might take longer to return to normal).
Environmental factors in animal housing which could affect the animals and/or the experiments
– Temperature – must be kept at a stable level
– Humidity – must be kept at a stable level (too high can lead to undesired growth of microorganisms, too low might dry out the skin of the animals).
– Light/dark rhythm (often 12/12-hour light/dark cycle)
– Olfactory cues
– Noise
– Enrichment
– Density of animals (hierarchical populations)
- Explain and discuss the purpose, use and basic principles of Health Monitoring. Present an overview of the FELASA guidelines
The purpose of health monitoring, which refers to the assessment of microbiological status of animals, is to protect laboratory animals against infections. Animal facilities perform health monitoring based on recommendations from the Federation of European Laboratory Animal Science Associations (FELASA), stating which infectious agents to monitor, which methods to use, the number of animals which must be tested, as well as a recommended monitoring frequency and reporting method, for different species of experimental animals (e.g., rodents, rabbits, primates and ruminants). Health monitoring reports can be accessed from breeders upon request, and sometimes they will be given along with the animals, or can be found on the internet.
Facilities submit animals or animal samples to laboratories which perform routine health monitoring and issues a certificate based on their findings. Certificates are issued with some delay, and thus might not reflect the actual health status of the animals in the colony, if the animals develop an infection in the period between samples.
The laboratories base their health monitoring on certain assays performed on various samples from the animals:
Pelt and skin are assessed microscopically for ectoparasites
Serology on serum for antibodies for viruses and other agents
Bacterial swaps from genitals, ileum, caecum, trachea, nose
Faecal parasitic status
Assessment of pathological changes on organs
Experimental units perform health monitoring as well, but the programs differ slightly:
For small animals, sentinels are used for health monitoring. Sentinels are animals which are placed in an experimental unit for the sole purpose of health monitoring, often using the dirty bedding technique; when animals of the experimental unit are given clean bedding, the sentinels are in addition provided with dirty bedding from the other animals. Sentinels are routinely sampled and submitted to a laboratory as it would be done from a breeding colony. Sentinels are used to avoid killing of experimental animals.
Large animals can provide enough sample material without euthanasia, and health monitoring of large experimental animals is thus based on clinical examination and sampling. Samples are then sent to a laboratory and analysed.
Calculating how many animals to sample for health monitoring
It is important to know how many animals to sample, as if an inadequate sample size is used, no safety for the absence of an infection is provided, and the samples are therefore wasted.
The required number of sampled animals depends on an acceptable risk of false negatives, the sensitivity of the method, and the estimated prevalence of an infection.
Prevalence (P) describes the fraction of infected animals compared to non-infected animals. Prevalence depends on the method of entry of the infectious particle; air transmission results in a high prevalence, whereas transplacental transmission is associated with a low prevalence.
Sensitivity (N) describes the fraction of infected animals becoming positive in a diagnostic test, i.e., the ability of the detection method to detect true positives.
The confidence limit (C) is the acceptable risk of detecting a false negative.
The risk of getting a false negative can be calculated as 1-P*N.
The minimum required sample size to sample in accordance with the confidence limit can be calculated using the following formula:
S >((Log C))/((Log(1-(P*N))) )
Example assuming a prevalence of 0.5 (50%), a sensitivity of 0.875 (87.5%), and a confidence level of 0.05 (5%):
S >((Log 0.05))/((Log(1-(0.5*0.875))) )=5.2
Thus, a sample size of at least 6 animals must be used in this example.
- Explain the basic rules for nomenclature of inbred, outbred and genetically modified laboratory mice and rats.
- Naming of inbred strains and substrains
o Strain name slash breeder code - Example: C57BL/6N (second slash omitted)
o Substrains are designated writing all the breeder codes with the historically oldest breeder first and the present breeder last, after the strain name.
Naming of outbred stocks
o Breeder code colon stock name
* Example: Crl:SD
o Substrains are designated writing all the breeder codes with the historically oldest breeder first and the present breeder last, before the stock name.
Naming of F1 hybrids (off-spring from 2 different inbred strains)
o Name of mother strain –> name of father strain –> F1 (indicating F1 hybrid) –> slash (indicating inbred) –> breeder’s lab code.
o To avoid long names, the official strain abbreviations are often used.
Naming of transgenic animals
o Name of background strain –> horizontal slash (-) –> gene name –> number of founding line –> founders lab code –> slash –> lab code of current breeder
* Gene names are usually written in italics
* Human genes are always written with all letters as capital letters, while the mouse of rat version is usually written with the first letter as capital letter, and most of the remaining part of the name as lowercase letters.
* Transgenes are not written in italics but are instead put in brackets.
* Insertion of a human transgene is designated by inserting Tg just after the horizontal slash, which is followed by a N if the transgene is inserted in a non-homologous manner. H is used for homologous insertion (i.e., for knockins only).
* Example: C57BL/6-TgN(APOA1)1Rub/J
Naming of knockout animals
o Name of background strain –> dot –> name of stem cell donor (if one exists) –> horizontal slash (-) –> animal version of gene –> written as superscript: tm (short for targeted mutation using embryonic stem cells) OR em (for endonuclease mediated mutation), number of founding line, founders lab code –> slash –> lab code of current breeder.
o Example: B6.129P2-ApoA^(1tm1Unc)/J
- Explain different ways of genetically modifying an animal and give examples of genetically modified animal models and their use in biomedical research.
Spontaneous mutants
Spontaneous mutants are animals characterized by a mutation which occurred spontaneously and has proven to serve a beneficial scientific purpose.
Examples:
Nu mutation in Foxn1 gene (Foxn1nu mice): nude, thymus lacking mice, which lack T-cells and thus are unable to reject transplants.
SCID mutation in the Prkdc gene of Severe Combined Immune Deficiency (SCID) mice leads to the generation of mice lacking both T and B cells.
Mice with a spontaneously occurred leptin deficiency are prone to the development of obesity and diabetes.
Selective inbreeding
Selective inbreeding is a kind of artificial selection and refers to the process of humans selecting which animals to mate to produce offspring with scientifically desirable/relevant characteristics.
Examples:
Spontaneously hypertensive rats (SHR)
Non-obese diabetic (NOD) mice
Genetically modified animals
Transgenic animals
Transgenic animals are animals the genome of which has been manipulated by insertion of foreign genomic material. Introduction of novel genes works through both heterozygous and homozygous expression.
Knockouts
Knockout animals are animals in which a gene has been removed or made non-functional. Knockout only works if the expression of both copies of a gene is impaired, that is homozygotic removal/impairment of function is required.
Knockins Knockin animals are animals in which a gene from a foreign species is inserted in the same site as the same gene in the homologue version (i.e., a one-for-one substitution of genomic information). Homozygous expression is required for knockin of genes to work. Examples of genetically modified animals: Rag1 mutants are analogous to spontaneous SCID mice, which lack both T and B cells. Cystic fibrosis models can be generated via creation of knockin mice, where the murine Cftr gene is swapped with one of the human mutations (e.g., ∆F508). Creation of fluorescent animals, tissues, or cell-populations within live animals. R6/1 mice = mice spliced with the human base pair repeats causing Huntington’s chorea. Methods of genetic engineering Pronucleus microinjection One pronucleus of a fertilized egg, which contains 2 pronuclei (one from each parent), from a mated female is injected with a DNA construct containing a desired gene. Often, DNA is injected into the male pronucleus. Genetically manipulated eggs are transplanted into a pseduopregnant female, which gives rise to heterozygous off-spring, as only one pro-nucleus is injected. Breeding for a couple generations is thus required to achieve homozygosity. Insertion of the genetic construct into the genome of the pronucleus is random, and this method is thus applicable only for the random insertion of a functional transgene, not the knockin nor the knockout of specific genes. This method is applicable to all species. Embryonic stem cell transfection Embryonic stem cells (ECs) are obtainable from blastocysts of mice, rats, and in principle humans, and their genomic contents can be manipulated using all available gene modification techniques. Upon genetic manipulation and selection, ECs are reintroduced into another blastocyst, which is then injected into an anaesthetised, pseduopregnant female, from which the injected blastocyst originated. The resulting offspring is chimeric, meaning that they consist of both wild type cells and gene modified cells. From this initial generation of chimeras, homozygotic gene modified mice can be obtained through breeding of multiple generations: chimeric mouse is mated with a wildtype mouse, which hopefully gives rise to off-spring of both sexes, both containing gene modified germ cells. These mice are then mated to generate homozygotic mice. Hopefully, both sexes are represented, so it is possible to continue breeding of homozygotic mice.
Cloning and nuclear transfer
A cell (e.g., a fibroblast) is isolated from an animal and cultivated, and its genomic material is manipulated with the desired changes. Then, the nucleus of a sow’s egg is replaced by a gene modified nucleus. After further development to a blastocyst, the gene modified embryo is transferred to the uterus of a pseduopregnant sow, which will result in the birth of a gene modified piglet.
This approach is applicable to all species, and can be used for both insertion of functional transgene, and as it offers spatial control, it can also be used for creation of knockin and knockout animals.
Nuclease based genome manipulation
Multiple nuclease-based genome manipulation tools exist:
Zinc-finger nucleases
TALENs
CRISPR/Cas9
Conditional gene modification Conditional gene modifications are gene modifications which are either done in specific tissues or for limited time periods only. Conditional gene modification is obtainable using the cre-lox system, which is enacted through a recombinase, Cre, which can cut the DNA string at specific LoxP cutting sites, and either flip or excise floxed sequences, depending on the orientation and number of LoxP sites. Both LoxP sites and Cre recombinase must be present in the same cells at the same time for the system to work. For example, knockout of a gene in a specific tissue can be achieved by introducing LoxP sites floxing the gene of interest in one animal, for example through embryonic stem cell transfection or nuclease-mediated genome manipulation techniques. In another animal, a construct encoding the Cre recombinase and a tissue-specific promotor is inserted into the genome by one of the techniques allowing targeted insertion. If these genetically modified mice the Floxed GOI mouse and the tissue specific Cre mouse, are now mated, mice which are knockouted for the GOI in a tissue specific manner are generated as a result. More complicated variants of the Cre-Lox system expand its applicability.
- Define and discuss the concept of a humane end-point and give some examples on humane endpoints. Discuss different ways of assessing suffering in animals.
Two distinct types of endpoints exist:
Experimental endpoints = predefined endpoint of the experiment, where all necessary data have been obtained.
Humane endpoints = the earliest point at which an experiment can or should be terminated, with the purpose of eliminating unnecessary suffering.
Humane endpoints are identified by assessment of early behavioral and clinical signs in combination with model-specific signs, to introduce as early endpoints as possible. Endpoints therefore differ depending on the severity of the experiment, of which 4 classes exist:
Non-recovery = procedures which are performed entirely under general anesthesia from which the animal shall not recover
Mild = procedures from which the animals are likely to experience short-term mild pain, suffering or distress, as well as procedures with no significant impairment of the well-being or general condition of the animals.
o Example: administration of anesthesia
Moderate = procedures from which the animals are likely to experience short-term moderate pain, suffering or distress, or long-lasting mild pain, suffering, or distress as well as procedures likely to cause moderate impairment of the well-being or general condition of the animals.
o Example: surgery under general anesthesia
Severe = procedures from which the animals are likely to experience severe pain, suffering, or distress, or long-lasting moderate pain, suffering, or distress as well as procedures likely to cause severe impairment of the well-being or general conditions of the animal.
o Example: severe restriction of movement over a prolonged period.
Thus, humane endpoints are present to minimize potential suffering and should be chosen in relation to the severity of the experiment. Humane endpoints are often defined in welfare protocols to ensure easy detection of their occurrence. Parameters defining humane endpoints are like those used in pain assessment and should be obtainable through cage side monitoring. Death is NOT ideal as humane endpoint (too extreme)!! An example of a more extreme humane endpoint is 20% body weight loss.
Pain and stress are very closely linked, as noxious stimuli will initiate a stress response. To achieve control of pain and stress, the following is required:
– Recognition
– Assessment
– Alleviation
It is important to recognize signs of pain and distress early on (you don’t want to end up in a situation where pain recognition is easy!), as a suffering animal in severe distress must be euthanized immediately.
Different parameters are used for pain and stress assessment
Physiological parameters (difficult to measure in smaller animals)
o Heart rate
o Blood pressure
o Body temperature
Biomarkers
o Corticosteroids (increases during stress)
o IgA (decreases during chronic stress)
o Noradrenaline or adrenaline in blood
o C-fos activation
o Oxidative stress markers
o “emotional” stress markers (8-OH-DPAT)
Behavioural and clinical signs
o Can be assessed either subjectively (not as accurate as objective assessment) or objectively (quantifiable). Generally, objective scoring of pain related behaviour is preferable over subjective scoring (greater precission).
o Behavioural readouts can be affected by drugs and time of day of the assessment (due to hormone level fluctuations). These parameters must thus be considered during pain assessment.
o General signs to look for (differs greatly among species – normal behaviour must be known in detail for assessment to be meaningful):
* Is the animal alert?
* Normal movement?
* Does it eat or drink as usually?
* Is it protecting a certain part of the body?
* Does it sound when being handled?
* Does it bite or lick itself more than normal?
* Does the animal exhibit abnormal posture?
* In rats: chromodacryorrea (= red coloured tears)
o Pain related behaviours (rats)
* stereotypic behaviour, which is used as a coping-mechanism by the animals.
* Twitching (often seen with a hop either preceding or following the twitch)
* Back-arching
* Falling
* Abdominal press
* Writhing
* Staggering (= vaklende gang)
* Facial expressions (grimace scale)
o Clinical signs
* Body weight loss
* Food and water consumption (decreases –> anhedonia)
* Urination and defecation (increases –> diarrhoea)
* Fur quality (decreases)
- Explain and discuss the 3 R’s.
Russel and Burch proposed the 3Rs in 1959:
o Replacement = methods which avoid or replace the use of animals.
* Replacement options should be the first step when planning and designing a research study.
Substitution = the replacement of conscious, living, sentient beings with insentient material (plants, microorganisms, primitive endoparasites).
Absolute replacement = techniques do not involve animals at any point. Obtainable using computer modelling (in silico approaches) or human volunteers.
Relative replacement = the use of established animal cell lines instead of live experimental animals.
o Reduction = methods which minimize/reduce the number of animals used in each experiment. Scientific output in terms of amount and quality of data must not be compromised, as this will lead to the wasting of animals. Both using less animals to obtain the usual amount and quality of data OR using the same number of animals to obtain more data or data of improved quality is regarded as reduction.
Reduction approaches involve:
* Improved experimental design and statistical analysis
* Improved techniques/methods (e.g., imaging)
* Establishment of biobanks for sharing of animal tissues and other resources
* Reduction of variation in animal models
o Refinement = methods which minimize suffering and improve animal welfare in situations where the use of animals is unavoidable. Relates to breeding, accommodation, care, and procedures, and applies to the entire lifetime experience of an animal.
Methods of refinement include:
* Using non-invasive techniques if possible
* Continuously improving techniques and methods used and always choosing the most gentle technique available
* Using appropriate anesthetic and analgesic regimes for pain relief
* Optimizing methods for pain recognition
* Ensuring that housing and accommodation facilities meet the basic biological and ethological needs of the animals
* Provide sufficient environmental and cognitive enrichment
* Training animals to cooperate with certain procedures to reduce fear and stress
The principles of the 3Rs clearly overlap; replacement of a mouse with a cell culture (insentient material) can also be considered reduction and refinement, as the number of animals needed for the experiment is reduced and only 1 mouse is painlessly killed (refinement).
The principles may however also be mutually exclusive and are thus not always compatible. For example, using fewer animals may risk increasing the strain on each individual animal, indicating how reduction is not always compatible with refinement.
The implementation of the 3Rs is important for establishment of a culture of care, which can be defined as a commitment to improve animal welfare, scientific quality, care of the staff, and transparency for stakeholders. The animal welfare body of laboratory animal facilities is an ideal place to put responsibility for the establishment and maintenance of an appropriate culture of care. The National Committee (in Denmark called “Udvalget for Forsøgsdyr og Alternativer”) can support the role of the animal welfare bodies in the implementation of culture of care as well by facilitating a national forum for sharing of good practice.
- Explain and discuss basic procedures and principles for experimental surgery including e.g. principles and methods of aseptic technique, suturing techniques, tissue handling and strategies for minimizing inflammation, infection and post-operative pain.
Aseptic techniques
o Aseptic techniques can be defined as methods and practices that prevent cross contamination during surgery, which involves proper preparation of the facilities and environment, surgical site, surgeon, and the surgical equipment.
o The purpose of aseptic techniques is to limit the animal’s exposure to both endogenous and exogenous microorganisms and no pathogen has yet developed resistance to aseptic techniques.
o The concept of working aseptically involve sterile things only touching other sterile items and unsterile things only touching other unsterile items, thus keeping sterile and unsterile items separated.
Tissue handling should follow Halsted’s principles:
o Asepsis
o Gentle handling
o Hemostasis (stopping bleeding)
o Closure of dead space
o Careful approximation of tissues
o Avoidance of tension
o Minimization of foreign materials
Working in accordance with Halsted’s principles requires proper training and selection of appropriate instruments, as instruments are designed to prevent undue trauma and aid in gentle handling.
Wound closure and suturing
o Elimination of dead spaces is important
o Sutures in inner layers (muscles, fascia) must be tied tightly
o Sutures in skin must be less tight to avoid induction of inflammation and pain.
o The correct suture material must be chosen, as different materials elicit distinct tissue reactions (use synthetic material is possible!). Tissue absorbability of the chosen materials must also be considered.
o Different suture techniques exist:
* Simple interrupted sutures
Time consuming
Secure; loss of one suture does not open entire wound
* Continuous sutures
Easy and quick
Better seal of wound edges
Less secure
Examples of experimental surgical techniques include:
o Laparotomy = abdominal surgery
* Various approaches (midline, flank, paramedial, transverse) exist depending on organs of interest. Medial incision along linea alba is least traumatic.
* Necessary incisions must be made large enough to improve accessibility and prevent/limit undue trauma.
o Thoracotomy = thoracic surgery
* Obtainable through intercostal space or via median sternotomy
* Requires ventilator
o Catheterization = the insertion of a catheter into a body cavity (e.g., venous catheter, urine catheter etc.).
o Craniotomy = the removal of parts of the skull to access the brain
- Lab animal feeding and nutrition: Describe various types of diets and feeding schedules and discuss the possible effects in animal studies.
Laboratory animal diets can be broken into nutrient fractions
o Macronutrients (burnable)
* Carbohydrates
Digestible forms
Indigestible forms = fibers
* Protein
* Fat
o Minerals (un-burnable)
o Vitamins
* Fat soluble: A, D, E, and K
* Water soluble: B vitamins, C vitamin (only essential in guinea pigs and primates, as other species can produce it themselves.
Only a fraction of the energy intake is available as metabolizable energy. Indigestible contents pass through the gut and leave the animal as faecal energy, while some digestible energy is excreted through urine.
Metabolizable energy must be distributed between catabolic (break-down of larger molecules into smaller ones), energy releasing processes, and anabolic (building larger molecules from smaller constituents), energy consuming processes.
Diets must be regulated based on the current state of the animal, e.g., growing, pregnant, and lactating animals need more energy. As animals differ in how much of their GI-tract is constituted by small or large intestine, respectively, diet composition must reflect these differences (protein vs. fiber contents).
Diets for laboratory animals are produced by commercial laboratory animal diet producers, and complete diet fulfilling all nutrient requirement for each species in different life stages are available. Some are based on natural ingredients, referred to as natural ingredient diets or chow, while diets based on synthetic compounds are called purified diets.
Natural ingredient diets
o Natural ingredients include whole grains (corn, barley, and wheat), mill by-products (bran), high protein meals (soybean, fish, casein) and mineral sources (bone meal). Mineral and vitamin mixtures are added as well.
o Both breeding and maintenance natural ingredient diets, which vary in their nutrient composition, exist.
o Natural ingredient diets exist as powders, pellets, extrudates and hybrid pellets.
* Pellets are not cooked during manufacturing and the ingredients are thus raw when fed to animals, which causes a low digestibility associated with increased intake amounts. The optimal/preferred pellet size differs across species.
* Extrudates are cooked during manufacturing, which increases the digestibility, and animals thus must eat less to fulfil nutrient requirements. Extrudates are usually more expensive than pellets.
o Natural ingredient diets can be made using either fixed or variable formulas:
* In fixed formula natural ingredient diets, a fixed amount of each ingredient is mixed into the diet, which means that the level of nutrients will vary, as nutrient levels vary in natural ingredients.
* In variable formula natural ingredient diets, a variable amount of ingredients is mixed into the diet, with the aim of standardizing/fixing nutrient levels in the diet.
Advantages and disadvantages of natural ingredient diets
Advantages
o Cheap
o Palatable (liked by animals)
o Nutritious
Disadvantages
o Variation between batches
o Single components are non-removable
o Contains uncontrolled biologically active compounds (* This can also be an advantage, as these compounds are needed by certain models.)
o Contamination risk, as most components originate from fields which might be inhabited by other animals.
Purified diets
o Different molecules constitute the macronutrients of purified diets:
* Protein
Casein, simple amino acids
* Carbohydrates
Sucrose and starch
Fibers: cellulose
* Fat
Corn oil
Mineral and vitamin mixtures are added as well.
o Purified diets are mostly used for specific research purposes. It is important to match control diet and experimental diet when making diets deficient of one single component.
o Purified diets are often artificially coloured to ensure correct feeding. However, this might be a potential source of bias, as blinding is impaired if it is known what group is fed what diet.
Advantages and disadvantages of natural ingredient diets
Advantages
o Standardized (low variation between batches)
o Single components removable
Disadvantages
o Aversive
o Expensive
o Lack biologically active compounds of importance for specific models
Feeding schedules:
o Ad libitum feeding, meaning that food is always available, is the most common way to feed rodents
o Restricted feeding means that animals are given exactly the amount of food needed to secure intake in accordance with daily nutrient requirements every day.
o Gavage (= oral dosing with a gastric tube) removes oral digestion, and data thus might be less translational compared to humans. However, it is more precise and individual compared to dosing through the diet or drinking water.
o Pair feeding is used to obtain two groups eating the exact same amount: the food intake in one group is registered, and the same amount of food is supplied to the other group.
The impact of different feeding schedules and diets:
o Ad libitum fed animals have a higher growth rate than restrictedly fed animals. However, the expected life span of ad libitum fed animals is lower than that of restrictedly fed animals, as ad libitum fed animals have a higher disease rate than restrictedly fed animals.
o Fasting of mice can have significant impact on experimental results, as fasting induces a state of topor in rodents, a sort of pseudohibernation during which rodents reduce their metabolism and body temperature due to lack of food, which appears in bouts. This is observed as changes in body temperature, corticosterone levels, most blood parameters, and body weight.
o Diet affects the microbiota. Therefore, animals of a study should be fed from the same batch of the same diet throughout a study.
- Present and discuss different ways of eradicating infections in laboratory animal facilities including rederivation, cessation of breeding or stamping out, antibiosis and vaccination.
Eradication of infections at laboratory animal facilities can be achieved in multiple ways:
Rederivation = ensuring that mice are breed from infection-free ancestors.
o Caesarean section approach (used less frequently)
A female is mated and euthanized at term. The pups are removed through a C-section in a disinfecting bath inside an isolator. The pups are then placed with a germ-free foster mother inside an isolator.
o Embryo transfer
A female from a strain we want to preserve is superovulated using hormones, to make the female produce a lot of eggs. The superovulating female is then mated with a male of the same strain. The morning after, the female is killed, and embryos are sampled and frozen.
Rederivation is achieved by mating a germ-free female with a germ-free vasectomized mouse. The germ-free female is then anaesthetised, and thawed embryos are placed inside the uterus. The female will give rise to germ-free pups of the strain from which the transferred embryos originated.
Alternatives to rederivation:
Cessation of breeding: Cessation of breeding is stopping the breeding of a line for a while (waiting disease out)
Burn out: let disease have its course. virus strong enough to induce a immune response that is strong enough to eliminate the virus and protect against reinfection. virus may be eradicated by a break in breeding procedures, and thus no naïve animals are introduced (I think they mean bred) before all animals have been infected and developed immunity.
So burn out and cessation of breeding are complementary, but not the same.
stamping out: killing/removing all the animals in the strain that have been infected in hopes to stop the infection from spreading further.
Antibiotics: fungus penicillium will produce penicillin, which will hinder the growth of infectious bacteria.
Vaccinations: only protect against the specific intended infection. Vaccines prime the immune system to detect a particular virus or bacteria by showing it a harmless version of the pathogen, or part of it so that it can remember it and is able to mount a defence if the person ever becomes infected
- Discuss physical and chemical methods of humane killing of animals including both rodents and larger animals such as pigs.
Experimental animals must be killed at a predefined time point, either as an experimental endpoint or as a humane endpoint.
Euthanasia must be fast/efficient, safe, and painless/stress-free. Therefore, only methods known and managed well by the experimenter should be utilized.
How animals are euthanized depends on the species, and chemical methods must often be complemented with an effective physical method to ensure/confirm death.
Legislation:
Always confirm death
Immediate signs of death:
- Heart beat stops
- Respiration stops
- Reflexes are absent
- Pale skin
Later signs
- Cold body surface
- Rigor mortis (stiffness)
Two overall methods: pharmacological/chemical, or physical/mechanical methods.
Pharmacological: overdose of anesthetic. Injections can cause inflammations (bad for science). Consider metabolic/pharmacological effects.
Physical: good when histological or pharmacological analysis after death. Decapacitation or cervical dislocation fx in small rodents. Risk of physical methods is that they fail and cause suffering. Should be practiced on anesthetized to sedated to normal animals first
- Captive bolt for pigs (but with other method after)
Most commonly used methods:
Mice and rats
Physical methods of killing
* Decapitation
o Most often used in rats
o The head is instantly removed by the use of a guillotine
* Cervical dislocation
o Especially for mice, but also for rats
o The animal dies from rupture to the brain stem and the carotid arteries
* Both methods can be used for conscious animals, but extensive training is required
o Training stunned or anesthetized animals only, until sufficient skill.
Chemical method of killing
* Carbon dioxide
* Cheap, quick, and easy way of anesthesia
* Gradual fill is important to avoid stress in the animals.
* Prior sedation or anesthesia may be beneficial, but is still debated (ie. Is the stress from prior sedation worse than the co2?) – Klas thinks sedation is good
Pig
* Captive bolt (be aware in Danish legislation this is only considered a method of stunning! if it hits just a bit off target, the animal does not die, then you have to follow up with another method of killing) – make sure animals is dead by examination afterwards.
* Shot between the eyes, right above
* In remaining lecture they mention fx overdose of anesthesia – I think this is the safer option to mention.
Permitted methods for each species:
Fish: anasthetic overdose, concussion/blow to head, electrical stunning (book also says cervical dislocation)
Amphibians: anasthetic overdose, concussion/blow to head, electrical stunning
Reptiles: anasthetic overdose, concussion/blow to head, captive bolt, shooting with free bullet
Birds: anasthetic overdose, carbon dioxide, cervical dislocation, decapacitation, concussion/blow to head, electrical stunning, inert gasses
Rodents: anasthetic overdose, carbon dioxide, cervical dislocation, decapacitation, concussion/blow to head, inert gasses
Rabbits: anasthetic overdose, captive bolt, cervical dislocation, concussion/blow to head, electrical stunning
Dogs/cats/ferrets/foxes: anasthetic overdose, concussion/blow to head, electrical stunning, shooting with a free bullet. Book: Physical methods not recommended. Prior sedation for anxious animals. Isoflurane but not CO2 accepted.
Large mammals: anasthetic overdose, captive bolt, electrical stunning, inert gasses, shooting with free bullet
Non-human primates: anasthetic overdose. Minimize stress/anxiety prior to killing. Done in home surroundings. Sedation to facilitate handling.
Decapacitation / cervical dislocation only for smaller animals,
Captive bolt for larger animals
Gassing mostly for smaller animals (CO2 or inert gasses, but latter also for pigs)
- Discuss spontaneous infections in lab animals, their importance for animal experimentation and ways to prevent spontaneous infections
Diseases in laboratory animals which arise spontaneously are often infectious, meaning that they arise from infection by viruses, bacteria, and parasites. These diseases can affect experimental animals to different extents and examples include:
o Hepatitis in susceptible mice and rat strains caused by coronavirus infection.
o Tyzzer’s disease caused by Clostridium piliforme infection of both mice and rats.
o Glässer’s disease caused by Haemophilus parasuis infection in pigs.
Usually, spontaneous infections are unwanted, as microorganisms in laboratory animals may be zoonotic, cause disease in the animals, or otherwise interfere with research. However, some microorganisms may be important for the induction of specific animal models, and presence of microorganisms might improve translatability.
Diseases can be either fulminant, i.e., causing observable clinical symptoms, or latent, which refers to diseases with no clinical expression/manifestation.
Infections can affect animal research both directly and indirectly:
o Direct influences
* Immunological modulation
* Physiological modulation
* Oncological modulation
* Fertility modulation
* Microbiological competition (compete with experimental infections)
o Indirect influences
* Contamination of biological products
* Activation of direct effects
Protection against infections in laboratory animals in accordance with EU guidelines is achieved using a three-step rocket:
o Rederivation
* C-section
* Embryo transfer
o Protection
* Barrier housing (instead of conventional housing)
* Isolator
o Continuous health monitoring
Furthermore, prevention of infection from contaminated biological materials used in research through proper screening and cleaning is important.
- Explain and discuss the purpose of Environmental enrichment and provide examples of ways of enriching the environment of laboratory rodents and pigs.
– Enrichment is defined as any initiative which increases the allowance and thus the amount of species-specific behaviour and reduces or eliminates abnormal behaviour.
– Legal requirements for the rodent cage
o According to legislations, the rodent cage must as a minimum contain:
bedding (only provides enrichment indirectly)
nesting material
a small house (e.g., cardboard tube, red shelters, as these are perceived as dark by rodents, because rodents can perceive red light)
gnawing sticks.
– Legal, ethical, and scientific reasons exist to provide enrichment to laboratory animals:
o Enrichment is required by law.
o Ethically, as enrichment is believed to allow more species-specific behaviour and reduce abnormal behaviour, enrichment is interpreted as improving/increasing animal welfare.
o Scientifically more natural behaviour is believed to be more representative of true biology and thus provide more valid research results.
– Different methods of enrichment exist:
o Behavioural enrichment
* Created by providing an environment which imitates the wild habitat of the experimental animal
o Social enrichment
* The act of giving the animal the opportunity of social interaction by providing social partners
* Most species used as experimental animals are social (except the hamster, which is solitary).
o Artificial enrichment
* Refers to enrichment through the use of a variety of artificial objects which must be of interest to the animals to which they are supplied to obtain the enrichment effect.
o Food enrichment
* Achieved by forcing the animal to spend time searching for food to promote species-specific behaviour.
o Environmental control enrichment
* Providing an ability of the animal to make choices, e.g., structuring the caging of an animal in a manner which allows the animal to decide whether the light should be on or off.
For pigs (although we likely won’t be tested on pigs according to Dorte Bratbo in Plenary session 3)
* Pigs are also social animals and need to be housed socially.
* I guess we would just want to include things that encourage species-specific behavior. Maybe the inclusion of some dirt/mud or including some straw bedding could be implemented
- Give an overview of the European legislation forming the basis for national member state legislation on animal experimentation.
Danish legislation is based upon common European legislation. The council of Europe includes nearly all European states and issues conventions which are voluntary for member states to follow, while the European Union (EU) includes only some European states as members, and issues directives which are mandatory.
The European Convention for the Protection of Vertebrate Animals used for Experimental and Other scientific purposes issued by the Council of Europe from 1986 and the Council Directive 86/609/EEC regarding the protection of animals used for experimental and other scientific purposes issued by the EU later that same year, which was revised in 2010, ensure that animal experimentation legislation is, with few exceptions, somewhat the same in all member countries of the European Union. Member states are, however, still somewhat allowed to subject animal research to higher standards/stricter rules.
In Denmark, the common rules for protection of animals can be found in the Animal Welfare Act, but Denmark has 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.
The animal experimentation act overrules the animal welfare act by stating that if the purpose is research, teaching, or the production of blood products, pain, suffering, distress, or lasting harm can be allowed if licensed in each specific case.
The animal experimentation act contains some general principles about animal experimentation in accordance with the EU Directive, and states that animal experiments must be:
o Licensed
o Beneficial
o The use of animals must be necessary
o Not be causing
* Strong pain
* Intensive fear
* Intensive suffering
o Performed
* By qualified staff
* In proper settings
* On destination bred animals
All the details on how to make animal experimentation, how to take care of the animals, and how to set up the facilities are put into the animal experimentation order. So, in addition to the animal experimentation act we have the animal experimentation order, which is a larger and much more detailed document.
In Denmark, the Animal Experimentation Inspectorate (= Dyreforsøgstilsynet), is the competent authority responsible for ensuring that animal experiments are conducted in accordance with the obligations from the directive, and thus the actual implementation of the rules stated in the directive. The Animal Experimentation Inspectorate is part of the Ministry of Food and Environment and is led by the Animal Experimentation Board, an 11-member committee, who issues all licenses.
