Basic of Animal Research Methodology Mehran Hosseini Birjand

Basic of Animal Research Methodology Mehran Hosseini Birjand University of Medical Sciences Birjand, Iran

Why animal research methodology and study design are important? - Ethical - Economical For ethical and economic reasons, it is important to design animal experiments well, to analyze the data correctly, and to use the minimum number of animals necessary to achieve the scientific objectives—but not so few as to miss biologically important effects or require unnecessary repetition of experiments.

Ethical considerations: The use of animals in scientific experiments likely to cause pain, distress, or lasting harm generates important ethical issues. Animals should be used only if the scientific objectives are valid, there is no other alternative, and the cost to the animals is not excessive “Validity" - high probability of meeting the objectives, - objectives have a reasonable chance of contributing to human or animal welfare, Possibly in the long term

Russell and Burch (1959) 3 Rs - Replace Animals should be replaced by less sentient alternatives such as invertebrates or in vitro methods whenever possible. - Refine Experimental protocols should be refined to: minimize any adverse effects for each individual animal( e. g. appropriate anesthesia and analgesia should be used for any surgical intervention), Staff should be well trained, Animals should be protected from pathogens - Reduce The number of animals should be reduced to the minimum consistent with achieving the scientific objectives of the study

General Principles All research should be described in such a way that it could be repeated elsewhere. Authors should clearly state the following: 1 - The objectives of the research and/or the hypotheses to be tested 2 - The reason for choosing their particular animal model 3 - The species, strain, source, and type of animal used 4 - The details of each separate experiment being reported, including the study design and the number of animals used 5 - The statistical methods used for analysis.

Experiments and Surveys An experiment is a procedure for collecting scientific data on the response to an intervention in a systematic way to maximize the chance of answering a question correctly(confirmatory research) or to provide material for the generation of new hypotheses (exploratory research). A survey, in contrast, is an observational study used to find associations between variables that the scientist cannot usually control. Any association may or may not be due to a causal relation. These guidelines are concerned only with experiments.

Confirmatory and Exploratory Experiments: Main difference: Confirmatory research normally involves formal testing of one or more prespecified hypotheses. By contrast, exploratory research normally involves looking for patterns in the data with less emphasis on formal testing of hypotheses.

Need to Control Variation Models should be sensitive to the experimental treatments by responding well, with minimal variation among subjects treated alike. Uncontrolled variation, whether caused by infection, genetics, or environmental or age heterogeneity, reduces the power of an experiment to detect treatment effects. The animals should be adequately described in the materials and methods or other relevant section of the paper or report.

Experimental Design Virtually all animal experiments should be done using one of the formal designs described briefly below: - Experimental Unit Randomization Blinding Pilot Studies

Formal Experimental Designs: Scientific papers should give sufficient detail so that the experiment(s) can be repeated 1 - The objectives of the research and/or the hypotheses to be tested 2 - The reason for choosing the particular animal model 3 - The species, strain, sex, age/weight, source and type of animal used and brief details of environment, diet and husbandry 4 - Each experiment should be numbered giving details of Purpose of the experiment 5 - The experimental design, Completely randomised, randomised block, factorial design etc. 6 - The “experimental unit” 7 - Method of randomisation and whether blinding was used (where appropriate) 8 - Method of sample size determination and number of animals used 9 - The statistical methods used for analysis 10 - Conclusions

Experimental design: Initial steps 1 - Literature search 2 - Scientific method(s) 3 - Problem Statement, Objectives, and Hypotheses 4 - Identification of Animal Model 5 - Identification of Potential Collaborators

1 - Literature search The search should include current and past journal articles and textbooks, as well as information available via the internet. Journal searches can be performed in any number of appropriate journal databases or indexes (e. g. , MEDLINE, TOXLINE, PUBMED, NCBI, AGRICOLA). The goals of the literature search are to learn of pertinent studies and methods, identify appropriate animal models, and eliminate unnecessary duplication of research.

2 - Scientific Method The core aspect of experimental design is the scientific method The scientific method consists of four basic steps: (1) observation and description of a scientific phenomena, (2) formulation of the problem statement and hypothesis, (3) use of the hypothesis to predict the results of new observations, (4) the performance of methods or procedures to test the hypothesis.

3 - Problem Statement, Objectives, and Hypotheses It is critical to define the problem statement, objectives, and hypotheses clearly. problem statement should include the issue that will be addressed experimentally and its significance (e. g. , potential application to human or animal health, improved understanding of biological processes). Objectives should be stated in a general description of the overall goals for the proposed experiments and the specific questions being addressed. Hypotheses should include two distinct and clearly defined outcomes for each proposed experiment (e. g. , a null and an alternate hypothesis)

Problem statement: Which diet causes more weight gain in rats: diet A or diet B? Null hypothesis: Groups are expected to show the same results (e. g. , rats on diet A will gain the same amount of weight as rats on diet B). Alternate hypothesis: Experimental groups are expected to show different results (e. g. , rats will gain more weight on diet A than diet B, or vice versa).

4 - Identification of Animal Model (1) Use the lowest animal on the phylogenic scale (2) Use animals that have the species- and/or strain-specific characteristics desirable or required for the specific study proposed. (3) Consider the costs associated with acquiring and maintaining the animal model during the period of experimentation. (4) Perform a thorough literature search, network with colleagues within the selected field of study, and/or contact commercial vendors or governmentsupported repositories of animal models to identify a potential source of the animal model. (5) Consult with laboratory animal veterinarians before final determination of the animal model.

5 - Identification of Potential Collaborators It is important to identify and consult with potential collaborators at the beginning of project development to determine who will be working on the project and in what capacity (e. g. , as coinvestigators, consultants, or technical support staff).

Design of the Animal Experiment a. Research Plan b. Experimental Unit c. N Factor: Experimental Group Size d. Controls including; Positive, Negative, Sham, Vehicle and comparative e. Randomization

a). Research plan: A description of the experimental manipulations required to address the problem statement, objectives, and hypotheses should be carefully devised and documented Practical issues that may need to be addressed include the lifespan of the animal model (for chronic studies), the anticipated progression of disease in that model (to determine appropriate time points for evaluation). If the animals are to receive chemical or biological treatments, an appropriate method for administration must be identified (e. g. , per os via the diet or in drinking water [soluble substances only], by osmotic pump, or by injection). All experimental procedures should be detailed through standard operating procedures, a requirement of good laboratory practice standards

b). Experimental Unit The entity under study is the experimental unit, which could be an individual animal or a group. For example, an individual rat is considered the experimental unit when a drug therapy or surgical procedure is being tested, but an entire litter of rats is the experimental unit when an environmental teratogen is being tested.

Summary: The steps listed below comprise a practical sequence for designing and conducting scientific studies. We recommend that investigators 1 - Conduct a complete literature review and consult experts who have experience with the techniques proposed in an effort to become thoroughly familiar with the topic before beginning the experimental design process. 2 - Ask a specific question and/or formulate an appropriate hypothesis. Then design the experiments to specifically address that problem/question. 3 -Consult a biostatistician during the design phase of the project, not after performing the experiments. 4 - Choose proper controls to ensure that only the variable of interest is evaluated. More than one control is frequently required. 5 - Start with a small pilot project to generate preliminary data and work out procedures and techniques. Then proceed to larger scale experiments to generate statistical significance. 6 - Modify original question and procedures, ask new questions, and begin again.

Laboratory Animal Allergens and Zoonotic Diseases

Working with animals can be dangerous business! § Physical and chemical hazards § Protocol related hazards § Allergens § Zoonotic diseases

Physical Hazards Ergonomic issues Strained postures Heavy lifts Repetitive motion Slips/Trips/Falls Noise/Heat in Cage Washing Rooms Bites

Risks of Work with Research Animals Heavy lifting and awkward postures are part of the daily work.

Zoonosis Infectious diseases capable of being transmitted from animals to humans

Routes of Transmission or Exposure • Injection (contaminated sharps) • Inhalation (airborne contaminants) • Ingestion (eat lunch with dirty hands) • Skin contact • Mucous membranes (rub eyes with dirty fingers)

Rodents Rat Bite Fever. Spirillum minor, Streptobaccilis moniliformis Leptospirosis Lymphocytic choriomeningitis

Laboratory Animal Allergy 10% of those without previous history will develop allergy to lab animals 70% of those with pre-existing allergies will develop a new allergy Overall risk is 30%

Common allergy sources Rats/ Mice--major allergens in urine/saliva Cats--sebaceous glands, hair, saliva Dogs--saliva, hair, skin Rabbits--fur, saliva, urine Birds--droppings

Prevention of Lab Animal Associated Allergies Employee selection Biosafety cabinets Filter top cages Ventilated cage racks Choice of bedding Animal density Proper humidity Personal protective equipment

Exposure Control Engineering controls: enclosure dilution ventilation Administrative controls reduce time with animals reduce density of animals housekeeping practices Personal protective equipment respirators and clothing Medical surveillance (screening and ongoing monitoring)

Personal Protective Equipment Hands • Gloves disposable – nitrile gloves offer puncture resistance and reduced risk of allergic reactions seen with latex • Change often Breathing • N 95 dust and mist respirators are filters will prevent workers from inhaling airborne allergens by excluding 95% of airborne particulates from the workers breathing zone. See illustration below. Body (cover or replace street clothes and prevent contamination) • Greens (normally worn by husbandry staff) • Lab coats or back fastening gowns according to facility policy • Pants/clothing that covers the legs Feet • Full shoes (no sandals, flip flops or crocs!)

Hygiene • Food/beverages not allowed in animal holding facilities • Wash your hands (soft soap best) • When entering and before leaving the animal holding room • Hand washing is the best contamination control method

GUIDELINES FOR ANESTHESIA AND ANALGESIA IN LABORATORY ANIMALS

Anesthesia characters: 1 - Analgesia 2 - Amnesia 3 - Immobilization Animal Anesthesia route: 1 - Parenteral Anesthesia (i. p, I, v, i. m) 2 - Inhalation Anesthesia (Open drop, Non-rebreathing system, Rebreathing system)_

Inhaler Anesthetic drugs Drug MAC% Response Toxicity Description Ether 2 -3. 2 Slow Liver Flammability Hard recovery Chloroform 0. 77 Slow Liver and carcinogen brain anoxia Methoxyflur ane 0. 2 Slow Renal if co. Tetra Expired Halothan 0. 7 -0. 9 Medium Renal and Liver in hy- Hypotermia Isofluran 1. 5 Fast No Heart. Respiratory Influsan 2 Fast NO -

Topical Anesthesia • • Procaine Bupivicaine Lidocaine Proparicaine