SUMMARY_LAS.pdf

## Introduction to Laboratory Animal Science Laboratory Animal Science is a multidisciplinary field that encompasses all aspects related to the use of animals in research and education, with a strong focus on animal welfare and care. This guide covers various facets of this field, from ethical considerations and experimental design to species-specific anatomy and disease control. ## Animal Welfare and Ethical Considerations The use of animals in research is strictly regulated to ensure animal welfare and to balance potential benefits against animal suffering. ### The 3Rs Principle A core concept in animal science, the 3Rs principle guides ethical research practices: * **Replacement:** Using non-animal methods whenever possible. * **Reduction:** Using the minimum number of animals necessary to obtain statistically valid results. * **Refinement:** Minimizing pain, distress, and suffering for animals used in research through improved housing, handling, and experimental procedures. ### Ethical Committees Ethical committees (ECs) are responsible for evaluating research proposals to ensure scientific merit, adherence to the 3Rs, and the safety of personnel. They assess the balance between potential benefits and animal suffering, classify pain levels, and set ethical criteria for animal use. ### Welfare Philosophies and Measures Different welfare philosophies exist, focusing on an animal's awareness of unpleasantness, its overall integrity, or the presence of positive feelings. Welfare is indirectly measured through resource-based indicators (e.g., food availability) and animal-based indicators (e.g., behavior, lesions). * **Species-specific behavior:** Understanding normal behavior is crucial for identifying abnormal behaviors indicative of stress or poor welfare. * **Abnormal behaviors:** These can include redirected behavior, decreased thresholds for behavior, apathy, automutilation, and stereotypies. * **Stress:** Defined as a disruption of homeostasis, stress can be classified as eustress (positive) or distress (negative). Control and predictability of unpleasant stimuli are key in managing stress. * **Learned helplessness:** A state where an animal believes it cannot avoid negative stimuli, leading to reduced stress response. * **Coping mechanisms:** Animals exhibit passive (defensive) or active (aggressive) coping strategies. ## Housing and Environmental Enrichment Proper housing is essential for animal welfare and experimental reproducibility. ### General Housing Requirements * **Ethology:** Animals should be housed to allow for natural behaviors and a wide behavioral repertoire. * **Environment:** Key factors include temperature, humidity, ventilation, lighting, noise, and hygiene, all of which must be species-specific. * **Ergonomics:** Building layout and working conditions should be optimized. * **Economy:** The cost-effectiveness of housing and the necessity of the experiment must be considered. * **Ecology:** Proper waste disposal and emission control are important. ### Environmental Enrichment (EE) EE aims to improve animal welfare and enhance experimental outcomes by providing environmental manipulations or additions that allow animals to express their species-specific needs. * **Types of Enrichment:** * **Social:** Contact with conspecifics (same species) or contraspecifics (other species, including humans). * **Nutritional:** Food balls or using food as a reward. * **Sensory:** Auditory (masking noise), olfactory (transferring nesting material), or visual enrichment. * **Occupational:** Providing materials for interaction, gnawing, or play. * **Physical:** Structuring the cage with shelters, nesting boxes, or tubes. * **Evaluation of EE:** Assessed by checking species-specific needs, reduction of abnormal behavior, stress levels (via behavior, growth, or physiology), and preference tests. * **Standardization:** While EE can improve welfare, it can also introduce variability. Standardizing EE protocols is crucial for reproducibility. ## Handling and Restraint Proper handling techniques are vital for minimizing stress and ensuring operator safety. This includes understanding species-specific behaviors and using appropriate methods for restraint, injection, and blood sampling. * **Mice:** Often picked up by the tail base (though tubes or cupping are preferred), scruffed for injections. * **Rats:** Handled over the shoulder, with restraint for oral gavage; injections can be subcutaneous, intraperitoneal, or intramuscular. * **Guinea Pigs:** Handled over the shoulder with hind leg support, can be cupped; prone to liver rupture if not supported. * **Hamsters:** Can be aggressive; cupped, ample scruff for injections. * **Rabbits:** Supported well, especially hind legs to prevent self-injury; covering eyes can reduce stress. * **Gerbils:** Never picked up by the tail due to skin release mechanism; handled over the shoulder or cupped. * **Fish:** Handle with gloves to protect the mucus layer; cover eyes if exposing to light. ## Transportation Animal transportation is governed by strict legislation to ensure animal welfare. Key considerations include: * **Transport Means:** Design and construction must provide adequate space, air quality, temperature control, and prevent injury. * **Transport Planning:** Involves understanding destination legislation, minimizing transit time, providing contact information, and ensuring fitness of animals. * **Legislation:** Regulations exist at national and international levels (e.g., Council of Europe, EU directives). Recognition by authorities is often required for transporters and facilities, especially for food-producing animals or long-distance transport. ## Genetics and Strain Selection Understanding genetics is crucial for choosing appropriate animal models and ensuring reproducibility. ### Genetic Standardization * **Goal:** To create groups of genetically similar animals that can be recreated. * **Methods:** Selection and breeding (e.g., monozygotes, inbred strains, F1 hybrids) or biotechnology (e.g., cloning). * **Inbred Strains:** Developed through at least 20 generations of sibling matings, leading to genetically identical individuals (homozygous). Can suffer from inbreeding depression. * **Outbred Strains:** Maintain genetic diversity through large breeding populations and controlled genetic management (e.g., Internal Genetic Standardization programs). * **Coisogenic/Congenic Strains:** Inbred strains that differ at specific loci or regions, often created through specific mutations or genetic transfers. * **Genetic Monitoring:** Essential to prevent contamination and drift, using observation or DNA markers. ### Transgenesis and Gene Targeting * **Transgenesis:** Introduction of foreign genes into the germline. * **Gene Targeting:** Precise modification of genes in embryonic stem cells to create knockout (KO) or knockin (KI) models. * **Conditional/Inducible Systems:** Technologies like Cre-loxP or Tet systems allow for tissue-specific and time-specific gene regulation. * **Genome Editing (CRISPR/Cas9):** A powerful tool for precise gene modification, although off-target effects are a concern. ### Choosing a Strain The choice of strain depends on the experiment: * **Diversity:** Outbred strains or F1 hybrids are used for testing drug efficacy, vaccines, or product safety where a diverse population response is needed. * **Similarity:** Inbred strains are used for disease models and hypothesis testing to reduce variability and increase reproducibility. ## Reproduction Understanding animal reproduction is vital for colony management and experimental planning. ### Key Reproductive Concepts * **Estrus Cycle:** The cyclical period of sexual receptivity in females, distinct from human ovulation. * **Hormonal Control:** Reproductive processes are regulated by hormones like GnRH, FSH, LH, estrogen, and progesterone. * **External Influences:** Photoperiod, pheromones, and social factors can influence reproductive cycles (e.g., Lee-Boot, Whitten, Vandenbergh, Bruce effects). * **Spermatogenesis/Oogenesis:** Continuous sperm production in males and follicular development in females. Animals do not typically experience menopause. ### Species-Specific Reproduction * **Mice:** Nocturnal, fertile postpartum estrus, anogenital distance used for sexing, vaginal smears for estrus. * **Rats:** Similar to mice but lack Bruce and Whitten effects. * **Golden Hamsters:** Short gestation, flank organs for pheromones, postovulatory discharge indicates non-pregnancy. * **Guinea Pigs:** Long gestation, pups are precocial (developed at birth), vaginal membrane closure. * **Rabbits:** Induced ovulation, sensitive to environmental factors affecting fertility, require careful handling. * **Assisted Reproductive Technologies (ART):** Includes artificial insemination, embryo transfer, in vitro fertilization (IVF), cryopreservation, and cloning. ## Nutrition Animal nutrition must meet species-specific requirements, considering factors like genetic background, physiological state, environment, and experimental treatments. ### Diet Formulation * **Natural Ingredient Diets:** Formulated from natural ingredients; can be closed (manufacturer controls exact composition) or open (specific formulation known). * **Semi-synthetic/Purified Diets:** Use purified ingredients for greater control, often used for allergies or specific research needs. * **Synthetic Diets:** Chemically defined, offering precise control but potentially less palatable. * **Physical Form:** Meal, pellets, or gels, chosen based on ease of use and suitability for adding experimental substances. ### Feeding Strategies * **Ad libitum:** Animals have continuous access to food, often leading to obesity. * **Meal Feeding:** Limited feeding time per day. * **Restricted Feeding:** Amount of food is limited to nutritional requirements. * **Pair Feeding:** Control group is fed the same amount as the treatment group to isolate the effects of the treatment. ### Feed Manufacturing and Storage * **Particle Size, Homogeneity:** Affect passage rate and digestibility. * **Contamination:** Synthetic diets are susceptible to different contaminants than natural diets. * **Sterilization:** Heat sterilization can degrade thermolabile compounds. * **Storage:** Vitamins degrade; antioxidants are needed for high-fat diets. Segregation effects can occur in stored feed. * **Quality Assurance:** Routine analysis, contaminant checks, and proper sampling are crucial. ## Pharmacodynamics and Pharmacokinetics * **Pharmacokinetics (PK):** What the body does to the drug (absorption, distribution, metabolism, excretion). * **Pharmacodynamics (PD):** What the drug does to the body (effects and mechanism of action). ### Drug Targets and Mechanisms Drugs exert effects by binding to: * **Receptors:** Agonists mimic natural ligands; antagonists block them. * **Ion Channels:** Drugs can block or modulate ion channel activity. * **Enzymes:** Drugs can inhibit or activate enzymes. * **Carriers:** Drugs can block or modulate membrane transport proteins. ### Transduction Mechanisms * **Ligand-gated ion channels:** Rapid opening/closing of channels upon ligand binding. * **G-protein coupled receptors (GPCRs):** Involve intermediate signaling pathways. * **Kinase-linked receptors:** Ligand binding triggers phosphorylation cascades. * **Nuclear receptors:** Intracellular receptors that regulate gene transcription. ### Quantitative Pharmacology * **Receptor Theory:** Drug-receptor binding follows a key-lock mechanism, with affinity determining binding strength. * **Efficacy:** The maximum effect a drug can produce. * **Potency:** The dose required to produce a given effect (e.g., EC50). * **Antagonism:** Reversible (competitive/non-competitive), irreversible, functional, or pharmacokinetic antagonism. * **Tolerance:** Decreased drug responsiveness due to desensitization, receptor upregulation/downregulation, mediator exhaustion, increased biotransformation, or physiological adaptation. ## Disease and Disease Control Preventing and controlling diseases is paramount for animal welfare and data integrity. ### Impact of Diseases Diseases can significantly impact experimental results, animal welfare, and pose zoonotic risks. Subclinical carriers and facultative pathogens are particularly dangerous as they can spread unnoticed. ### Disease Types and Agents * **Infectious:** Caused by viruses, bacteria, or parasites. * **Non-infectious:** Metabolic, trauma, tumors, or hereditary conditions. * **Causal Agents:** Facultative pathogens cause disease only under predisposing factors; obligatory pathogens always cause disease. ### Recognizing and Diagnosing Diseases Symptoms can be general (e.g., fever, altered behavior) or specific to organ systems (skin, digestive tract, respiratory, etc.). Diagnosis involves clinical signs, necropsy, cytology, histology, and microbiology. ### Disease Prevention and Control * **Introduction of Non-contaminated Animals:** Sourcing from reputable suppliers with health reports, quarantine, and barrier systems. Rederivation and cryopreservation are advanced methods. * **Eradication Methods:** Stopping breeding, stamping out infected animals, or using antibiotics/vaccination (with caution regarding experimental impact). * **Hygienic Measures:** Facility design (compartmentalization, one-way traffic), personnel hygiene, disinfection of equipment and materials, and health monitoring are crucial. * **Containment Housing:** Isolators, cubicles, filter-top cages, and individually ventilated cages (IVCs) minimize pathogen and allergen spread. ## Biosafety Measures Biosafety levels (BSL) classify laboratories and procedures based on the risk posed by microorganisms. * **Risk Groups:** Microorganisms are categorized into four groups based on pathogenicity, transmission routes, and availability of preventive/therapeutic measures. * **Biosafety Levels (BSL):** Correspond to containment measures required, increasing with risk group. BSL-2 requires biohazard signs, restricted access, and inward airflow. BSL-3 and BSL-4 involve more stringent measures like showers, HEPA filtration, and specialized housing (isolators for BSL-4). ## Good Laboratory Practice (GLP) GLP is a quality system ensuring the reliability and integrity of non-clinical safety data for regulatory submission. * **Scope:** Applies to pharmaceuticals, pesticides, food additives, cosmetics, and industrial chemicals. * **Inspection:** Facilities undergo regular inspections to ensure compliance. * **Areas of Expertise:** Includes toxicity, mutagenicity, ecotoxicity, pharmacokinetic, and residue studies. * **Phase I (Environmental Exposure):** Predicts environmental concentration (PEC) and assesses degradation. * **Phase II (Effects on Organisms):** Evaluates toxicity to fauna and flora, considering persistence and dose-response relationships (LC50, EC, NOEC). * **Safety and Residues:** Depletion studies determine withdrawal times for food-producing animals. * **GLP Principles:** Emphasize documentation, traceability, quality assurance, and standard operating procedures (SOPs). ## Comparative Morphology and Physiology Choosing the right animal model requires understanding its anatomy and physiology in comparison to humans. ### General Principles * **Model Selection:** Depends on the research question, cost-effectiveness, phylogenetic distance, and the 3Rs. * **Vertebrate Groups:** Fish, amphibians, birds, and mammals are commonly used. * **Comparative Morphology:** Examines similarities and differences in structures like integument, skeleton, bucal cavity, stomach, liver, lungs, and excretory systems to understand functional and evolutionary relationships. ### Species Comparisons (Rodents vs. Lagomorphs) Detailed comparisons are made for: * **Integument:** Skin, hair, mammary glands, sweat glands. * **Osteology:** Skull, limb bones, vertebral formula, digits. * **Myology:** Muscle groups for injections. * **Digestive System:** Teeth (hypsodont vs. brachydont, incisor overgrowth), stomach structure, cecum size, cecotrophy/coprophagy. * **Respiratory System:** Lung lobes, nasal passages. * **Urinary System:** Kidney structure (unilobular vs. multilobular). * **Genital System:** Uterine structure (duplex vs. bicornuate), sexual dimorphism, reproductive tract openings. * **Circulatory System:** Blood drawing sites, blood volume. * **Nervous System:** Brain complexity, olfactory bulbs, third eyelid. * **Eyes:** Vision capabilities, color vision (bichromatic vs. trichromatic). ## Immunology and Antibody Production Immunology in laboratory animals is crucial for vaccine development and understanding immune responses. ### Immune Cells and Antigens * **Antigen-Presenting Cells (APCs):** Dendritic cells, B-cells, macrophages. * **T-lymphocytes:** Helper (CD4+) and cytotoxic (CD8+). * **B-lymphocytes:** Produce antibodies. * **Antigens:** Foreign substances that elicit specific immune responses; immunogenicity is influenced by molecular weight, complexity, form, digestibility, route of administration, dose, genetics, age, and adjuvants. ### Antibody Production * **Polyclonal Antibodies:** Produced in vivo by immunizing animals, cheaper, but variable quality. * **Monoclonal Antibodies:** Produced in vitro using hybridoma technology, highly specific and sensitive, but more expensive. * **Immunoglobulin Isotypes:** IgG, IgM, IgA, IgE, etc., with different structures and functions. * **Antibody Response:** Characterized by a primary response (IgM first, then IgG) and a secondary response (faster, stronger, with memory). Affinity maturation increases antibody quality. * **Immunization:** Choice of animal, antigen dose, injection site, and adjuvants influence the immune response. ## Anesthesia and Analgesia Anesthesia and analgesia are critical for minimizing pain and distress during procedures. ### Anesthesia * **Phases:** Preparation, premedication, induction, maintenance, recovery, and postoperative care. * **Premedication:** Sedatives (e.g., acepromazine, $\alpha$-2 agonists) and opioids calm animals and reduce stress, requiring lower anesthetic doses. * **Induction:** Drugs like barbiturates, dissociative anesthetics (e.g., ketamine), and muscle relaxants are used. * **Maintenance:** Inhalation anesthetics (e.g., isoflurane) or continuous injection are preferred over repeated boluses. Balanced anesthesia (combination of agents) is often used. * **Recovery:** Fast recovery is crucial to minimize complications like hypothermia and respiratory depression. Inhalation anesthetics generally allow for quicker recovery. ### Analgesia * **Pain Components:** Nociception (signal transmission), perception, and response. * **Pain Evaluation:** Difficult, relies on objective parameters (scales), behavioral assessment, and experimental tools. * **Types of Pain:** Acute (often surgical) and chronic (more subtle). * **Nervous System and Pain:** Afferent fibers (A$\delta$, C for pain; A$\beta$ for touch), hyperalgesia (increased sensitivity), and allodynia (non-painful stimulus becomes painful). * **Pain Modulation:** Involves endogenous systems and can lead to phenomena like "wind-up." Pre-emptive analgesia (before pain occurs) is vital. * **Treatment:** Multimodal pain therapy combining non-pharmacological (immobilization, physical therapy) and pharmacological methods (NSAIDs, opioids, $\alpha$-2 agonists, local anesthetics, dissociative anesthetics) is most effective. ## Medical Imaging Various imaging techniques are used for diagnosis and research. ### Imaging Modalities * **Structural Imaging:** Radiology, Ultrasound, CT, MRI visualize anatomy. * **Functional Imaging:** Scintigraphy assesses physiological activity. ### Techniques and Applications * **Radiography (X-ray):** Transmission imaging, good for bone, limited soft tissue detail. Requires fast work and radioprotection. * **Ultrasound:** Reflection imaging, no radiation, good for soft tissues and fluids, relatively inexpensive. * **CT (Computed Tomography):** Transmission imaging using X-rays from multiple angles, excellent for bone, moderate for soft tissues, avoids superimposition. * **MRI (Magnetic Resonance Imaging):** Emission imaging using magnetic fields, superior for soft tissues (brain, cartilage), no radiation. * **Scintigraphy:** Emission imaging using radiopharmaceuticals to visualize functional processes. * **Image Fusion:** Combining images from different modalities to gain comprehensive information. ## Experimental Techniques Basic techniques for handling and administering substances to laboratory animals. * **Injections:** Subcutaneous (SC), intraperitoneal (IP), intramuscular (IM), and intravenous (IV) routes are used, with site and volume depending on species and substance. Always aspirate before injecting to check needle placement. * **Oral Administration:** Direct dosing to the esophagus or stomach using syringes or silicone tubes. * **Blood Sampling:** Methods vary by species and volume required. Tail veins, saphenous veins, ear veins, or cardiac puncture (terminal) are common sites. Minimizing trauma and ensuring proper volume withdrawal is crucial. ## Radiobiology The study of radiation's effects on biological systems. ### Ionizing Radiation * **Types:** Alpha ($\alpha$), Beta ($\beta$), Gamma ($\gamma$), and X-rays differ in ionization energy, charge, mass, and penetration. * **LET (Linear Energy Transfer):** A measure of radiation quality; high LET radiation (e.g., $\alpha$-rays) causes more damage per unit track length. * **Risk Quantification:** Effective dose (Sievert, Sv) accounts for radiation type, dose, and tissue sensitivity. * **Radiation Protection:** Based on distance, timing, and shielding (lead for photons, plastic for electrons). Personal dosimeters are essential. * **Biological Effects:** Deterministic effects (cell death) have a threshold dose; stochastic effects (DNA mutations, cancer) lack a threshold. Embryos and gonads are particularly radiosensitive. ## Euthanasia Euthanasia must be performed humanely, minimizing pain, fear, and distress. ### Principles and Methods * **Criteria:** Quick effect, minimal restraint, minimal pain/fear, suitability for species/condition, reliability, irreversibility, operator safety. * **Methods:** * **Physical:** Cervical dislocation (birds, rodents, rabbits), decapitation (birds, rodents), shooting, concussion, electrocution. Require skill and restraint. * **Chemical:** Inhalation (CO2 for rodents/birds, volatile anesthetics), absorption (anesthetic overdose for fish/amphibians), injectable anesthetics (barbiturates, T61). CO2 can cause distress if not administered gradually. * **Unacceptable Methods (unless anesthetized):** Hypothermia, drowning, neck crushing, strangulation, ether, chloroform. * **Confirmation of Death:** Checking body temperature, reflexes, heartbeat/breathing, rigor mortis, and potentially brain destruction. * **Human End-points:** Predetermined criteria to terminate or alleviate animal suffering during experiments. ## Viral and Bacterial Zoonoses Understanding zoonotic diseases is critical for occupational health. ### Viral Zoonoses from Rodents * **Hantaviruses:** Transmitted via rodent excreta aerosols; cause flu-like illness or Hantavirus Pulmonary Syndrome (HPS). * **Arenaviruses:** RNA viruses, can cause hemorrhagic fever. * **Lymphocytic Choriomeningitis Virus (LCMV):** Found in mice; can cause neurological disorders or affect fetuses. * **Lassa Virus:** Endemic in West Africa, transmitted by rodents; can cause hemorrhagic fever. * **Encephalomyocarditis Virus (EMCV):** Can infect pigs and humans; RNA virus, causes abortion. * **Orthopoxvirus:** e.g., Cowpox, Mousepox; cause skin lesions. ### Bacterial Zoonoses * **Prevention:** Training, hygiene, appropriate handling, wearing protective clothing, and reporting exposures are crucial. * **Bite-Related Infections:** * **Pasteurella multocida:** Common in dog/cat oral flora; can cause local infection or bacteremia. * **Capnocytophaga canimorsus:** Particularly dangerous for immunocompromised individuals; can lead to sepsis. * **Rat Bite Fever (Streptobacillus moniliformis):** Asymptomatic in rats but causes flu-like symptoms and potentially fatal illness in humans if untreated. * **Cat Scratch Disease (Bartonella henselae):** Transmitted via cat scratches; typically causes local lesions and lymph node swelling. * **Other Bacterial Zoonoses:** * **Tularemia ("rabbit fever"):** Hosted by rabbits and mice; dangerous, potential biological warfare agent. * **Leptospirosis:** Spiral bacteria inhabiting kidneys of rodents (rats); shed in urine, transmitted through skin/mucous membranes; can cause liver/kidney failure or meningitis. * **Dermatomycosis (Fungal Infection):** Common zoonosis causing skin lesions in animals and humans. Fungal spores are environmentally resistant. ## Statistics in Animal Research Statistical analysis is essential for interpreting experimental data and drawing valid conclusions. ### Basic Concepts * **Random Variable:** An outcome that cannot be predicted with certainty. * **Distributions:** Binomial (two outcomes) and Normal (continuous data). * **Standardization:** Converting data to a standard normal distribution (Z-score). * **Hypothesis Testing:** Formal procedure to test a null hypothesis (H0) against an alternative hypothesis (Ha). * **Type I Error ($\alpha$):** Rejecting H0 when it's true (false positive). * **Type II Error ($\beta$):** Failing to reject H0 when it's false (false negative). * **P-value:** Probability of observing results if H0 is true. P < $\alpha$ suggests rejecting H0. * **Confidence Interval (CI):** A range likely to contain the true population parameter. If the CI does not include the H0 value, H0 is rejected. * **Power (1 - $\beta$):** The probability of correctly rejecting a false H0. ### Statistical Analysis Planning * **Hypothesis Formulation:** Specific, testable hypotheses are required. * **Experimental Design:** Influences the number of animals needed and the statistical analysis used (e.g., completely randomized, randomized block, crossover, factorial designs). * **Sample Size Calculation:** Determines the number of animals required to achieve adequate statistical power, considering expected differences, variance, and desired significance level. * **Pilot Studies:** Used to estimate variance and relevant differences when prior data is unavailable. ### Data Interpretation * **Statistical Significance vs. Relevance:** A statistically significant result does not always mean a practically relevant difference. * **Reporting:** P-values and confidence intervals are crucial. Avoid data snooping; formal experiments are preferred over explanatory ones for definitive conclusions. * **Multiple Comparisons:** Adjust significance levels (e.g., Bonferroni correction) to control Type I error inflation. ## Postoperative Care Care after surgery is critical for recovery and preventing complications. ### Key Aspects * **Monitoring:** Body temperature, blood loss, wound status, and animal activity. * **Recovery:** Fast recovery is desirable to minimize complications. Anesthetics are metabolized or exhaled to facilitate recovery. * **Pain Management:** Analgesia is crucial, especially in the first 24-48 hours. * **Hydration and Nutrition:** Maintaining fluid balance and encouraging feed intake are vital, as small animals can quickly become hypoglycemic. * **Wound Care:** Preventing self-trauma, infection, and ensuring suture integrity. * **Recordkeeping:** Detailed notes on observations and treatments are essential. ## Immunology and Antibody Production Immunology in laboratory animals is crucial for vaccine development and understanding immune responses. ### Immune Cells and Antigens * **Antigen-Presenting Cells (APCs):** Dendritic cells, B-cells, macrophages. * **T-lymphocytes:** Helper (CD4+) and cytotoxic (CD8+). * **B-lymphocytes:** Produce antibodies. * **Antigens:** Foreign substances that elicit specific immune responses; immunogenicity is influenced by molecular weight, complexity, form, digestibility, route of administration, dose, genetics, age, and adjuvants. ### Antibody Production * **Polyclonal Antibodies:** Produced in vivo by immunizing animals, cheaper, but variable quality. * **Monoclonal Antibodies:** Produced in vitro using hybridoma technology, highly specific and sensitive, but more expensive. * **Immunoglobulin Isotypes:** IgG, IgM, IgA, IgE, etc., with different structures and functions. * **Antibody Response:** Characterized by a primary response (IgM first, then IgG) and a secondary response (faster, stronger, with memory). Affinity maturation increases antibody quality. * **Immunization:** Choice of animal, antigen dose, injection site, and adjuvants influence the immune response. ## Viral Zoonosis Infectious diseases transmissible between animals and humans, with specific viral examples. ### Transmission Routes * **Interspecies Transmission:** Generally rare due to species-specific receptors, but mutations can facilitate transmission. * **Zoonosis Classifications:** By organ system, animal origin, mode of transmission, or time of appearance (old, recent, established, emerging). * **Parazoonoses:** Involve reassortment of genetic material between different viruses. ### Viral Zoonoses from Rodents * **Hantaviruses:** Transmitted via rodent excreta aerosols; cause flu-like illness or Hantavirus Pulmonary Syndrome (HPS). * **Arenaviruses:** RNA viruses, can cause hemorrhagic fever. * **Lymphocytic Choriomeningitis Virus (LCMV):** Found in mice; can cause neurological disorders or affect fetuses. * **Lassa Virus:** Endemic in West Africa, transmitted by rodents; can cause hemorrhagic fever. * **Encephalomyocarditis Virus (EMCV):** Can infect pigs and humans; RNA virus, causes abortion. * **Orthopoxvirus:** e.g., Cowpox, Mousepox; cause skin lesions. ## Bacterial Zoonoses Bacterial zoonoses are a significant occupational health concern. ### Prevention and Transmission * **Prevention:** Training, hygiene, appropriate handling, protective clothing, and reporting exposures are crucial. * **Transmission Routes:** Inhalation, ingestion, skin contact, bites, scratches, and handling contaminated materials. * **Bite-Related Infections:** * **Pasteurella multocida:** Common in dog/cat oral flora; can cause local infection or bacteremia. * **Capnocytophaga canimorsus:** Particularly dangerous for immunocompromised individuals; can lead to sepsis. * **Rat Bite Fever (Streptobacillus moniliformis):** Asymptomatic in rats but causes flu-like symptoms and potentially fatal illness in humans if untreated. * **Cat Scratch Disease (Bartonella henselae):** Transmitted via cat scratches; typically causes local lesions and lymph node swelling. * **Other Bacterial Zoonoses:** * **Tularemia ("rabbit fever"):** Hosted by rabbits and mice; dangerous, potential biological warfare agent. * **Leptospirosis:** Spiral bacteria inhabiting kidneys of rodents (rats); shed in urine, transmitted through skin/mucous membranes; can cause liver/kidney failure or meningitis. * **Dermatomycosis (Fungal Infection):** Common zoonosis causing skin lesions in animals and humans. Fungal spores are environmentally resistant. ## Safety and Hazards Occupational Health and Safety (OHS) programs aim to prevent injuries and illnesses in the workplace. ### Hazard Identification * **Physical:** Animal bites/scratches, sharps, flammable materials, ergonomic strains, noise, UV radiation, lasers, ionizing radiation. * **Chemical:** Disinfectants, anesthetic gases, tissue preservatives (e.g., formaldehyde), bedding materials, pesticides. * **Protocol-Related:** Hazards from chemicals or infectious agents used in experiments, including viral vectors and transgenic animals. * **Allergens:** A significant concern, often causing respiratory symptoms and skin reactions, with sensitization requiring prevention strategies. ### Allergy Prevention * **Goals:** Reduce sensitization and manage symptoms. * **Programs:** Screening (identifying at-risk individuals), facility design (ventilation, filtration, separate areas), work practices (hygiene, job rotation, wetting down dust), and personal protective equipment (respirators, gloves, lab coats). ## Viral Zoonosis Infectious diseases transmissible between animals and humans, with specific viral examples. ### Transmission Routes * **Interspecies Transmission:** Generally rare due to species-specific receptors, but mutations can facilitate transmission. * **Zoonosis Classifications:** By organ system, animal origin, mode of transmission, or time of appearance (old, recent, established, emerging). * **Parazoonoses:** Involve reassortment of genetic material between different viruses. ### Viral Zoonoses from Rodents * **Hantaviruses:** Transmitted via rodent excreta aerosols; cause flu-like illness or Hantavirus Pulmonary Syndrome (HPS). * **Arenaviruses:** RNA viruses, can cause hemorrhagic fever. * **Lymphocytic Choriomeningitis Virus (LCMV):** Found in mice; can cause neurological disorders or affect fetuses. * **Lassa Virus:** Endemic in West Africa, transmitted by rodents; can cause hemorrhagic fever. * **Encephalomyocarditis Virus (EMCV):** Can infect pigs and humans; RNA virus, causes abortion. * **Orthopoxvirus:** e.g., Cowpox, Mousepox; cause skin lesions. ## Genetics and Strain Selection Understanding genetics is crucial for choosing appropriate animal models and ensuring reproducibility. ### Genetic Standardization * **Goal:** To create groups of genetically similar animals that can be recreated. * **Methods:** Selection and breeding (e.g., monozygotes, inbred strains, F1 hybrids) or biotechnology (e.g., cloning). * **Inbred Strains:** Developed through at least 20 generations of sibling matings, leading to genetically identical individuals (homozygous). Can suffer from inbreeding depression. * **Outbred Strains:** Maintain genetic diversity through large breeding populations and controlled genetic management (e.g., Internal Genetic Standardization programs). * **Coisogenic/Congenic Strains:** Inbred strains that differ at specific loci or regions, often created through specific mutations or genetic transfers. * **Genetic Monitoring:** Essential to prevent contamination and drift, using observation or DNA markers. ### Transgenesis and Gene Targeting * **Transgenesis:** Introduction of foreign genes into the germline. * **Gene Targeting:** Precise modification of genes in embryonic stem cells to create knockout (KO) or knockin (KI) models. * **Conditional/Inducible Systems:** Technologies like Cre-loxP or Tet systems allow for tissue-specific and time-specific gene regulation. * **Genome Editing (CRISPR/Cas9):** A powerful tool for precise gene modification, although off-target effects are a concern. ### Choosing a Strain The choice of strain depends on the experiment: * **Diversity:** Outbred strains or F1 hybrids are used for testing drug efficacy, vaccines, or product safety where a diverse population response is needed. * **Similarity:** Inbred strains are used for disease models and hypothesis testing to reduce variability and increase reproducibility. ## Reproduction Understanding animal reproduction is vital for colony management and experimental planning. ### Key Reproductive Concepts * **Estrus Cycle:** The cyclical period of sexual receptivity in females, distinct from human ovulation. * **Hormonal Control:** Reproductive processes are regulated by hormones like GnRH, FSH, LH, estrogen, and progesterone. * **External Influences:** Photoperiod, pheromones, and social factors can influence reproductive cycles (e.g., Lee-Boot, Whitten, Vandenbergh, Bruce effects). * **Spermatogenesis/Oogenesis:** Continuous sperm production in males and follicular development in females. Animals do not typically experience menopause. ### Species-Specific Reproduction * **Mice:** Nocturnal, fertile postpartum estrus, anogenital distance used for sexing, vaginal smears for estrus. * **Rats:** Similar to mice but lack Bruce and Whitten effects. * **Golden Hamsters:** Short gestation, flank organs for pheromones, postovulatory discharge indicates non-pregnancy. * **Guinea Pigs:** Long gestation, pups are precocial (developed at birth), vaginal membrane closure. * **Rabbits:** Induced ovulation, sensitive to environmental factors affecting fertility, require careful handling. * **Assisted Reproductive Technologies (ART):** Includes artificial insemination, embryo transfer, in vitro fertilization (IVF), cryopreservation, and cloning. ## Nutrition Animal nutrition must meet species-specific requirements, considering factors like genetic background, physiological state, environment, and experimental treatments. ### Diet Formulation * **Natural Ingredient Diets:** Formulated from natural ingredients; can be closed (manufacturer controls exact composition) or open (specific formulation known). * **Semi-synthetic/Purified Diets:** Use purified ingredients for greater control, often used for allergies or specific research needs. * **Synthetic Diets:** Chemically defined, offering precise control but potentially less palatable. * **Physical Form:** Meal, pellets, or gels, chosen based on ease of use and suitability for adding experimental substances. ### Feeding Strategies * **Ad libitum:** Animals have continuous access to food, often leading to obesity. * **Meal Feeding:** Limited feeding time per day. * **Restricted Feeding:** Amount of food is limited to nutritional requirements. * **Pair Feeding:** Control group is fed the same amount as the treatment group to isolate the effects of the treatment. ### Feed Manufacturing and Storage * **Particle Size, Homogeneity:** Affect passage rate and digestibility. * **Contamination:** Synthetic diets are susceptible to different contaminants than natural diets. * **Sterilization:** Heat sterilization can degrade thermolabile compounds. * **Storage:** Vitamins degrade; antioxidants are needed for high-fat diets. Segregation effects can occur in stored feed. * **Quality Assurance:** Routine analysis, contaminant checks, and proper sampling are crucial. ## Pharmacodynamics and Pharmacokinetics * **Pharmacokinetics (PK):** What the body does to the drug (absorption, distribution, metabolism, excretion). * **Pharmacodynamics (PD):** What the drug does to the body (effects and mechanism of action). ### Drug Targets and Mechanisms Drugs exert effects by binding to: * **Receptors:** Agonists mimic natural ligands; antagonists block them. * **Ion Channels:** Drugs can block or modulate ion channel activity. * **Enzymes:** Drugs can inhibit or activate enzymes. * **Carriers:** Drugs can block or modulate membrane transport proteins. ### Transduction Mechanisms * **Ligand-gated ion channels:** Rapid opening/closing of channels upon ligand binding. * **G-protein coupled receptors (GPCRs):** Involve intermediate signaling pathways. * **Kinase-linked receptors:** Ligand binding triggers phosphorylation cascades. * **Nuclear receptors:** Intracellular receptors that regulate gene transcription. ### Quantitative Pharmacology * **Receptor Theory:** Drug-receptor binding follows a key-lock mechanism, with affinity determining binding strength. * **Efficacy:** The maximum effect a drug can produce. * **Potency:** The dose required to produce a given effect (e.g., EC50). * **Antagonism:** Reversible (competitive/non-competitive), irreversible, functional, or pharmacokinetic antagonism. * **Tolerance:** Decreased drug responsiveness due to desensitization, receptor upregulation/downregulation, mediator exhaustion, increased biotransformation, or physiological adaptation. ## Disease and Disease Control Preventing and controlling diseases is paramount for animal welfare and data integrity. ### Impact of Diseases Diseases can significantly impact experimental results, animal welfare, and pose zoonotic risks. Subclinical carriers and facultative pathogens are particularly dangerous as they can spread unnoticed. ### Disease Types and Agents * **Infectious:** Caused by viruses, bacteria, or parasites. * **Non-infectious:** Metabolic, trauma, tumors, or hereditary conditions. * **Causal Agents:** Facultative pathogens cause disease only under predisposing factors; obligatory pathogens always cause disease. ### Recognizing and Diagnosing Diseases Symptoms can be general (e.g., fever, altered behavior) or specific to organ systems (skin, digestive tract, respiratory, etc.). Diagnosis involves clinical signs, necropsy, cytology, histology, and microbiology. ### Disease Prevention and Control * **Introduction of Non-contaminated Animals:** Sourcing from reputable suppliers with health reports, quarantine, and barrier systems. Rederivation and cryopreservation are advanced methods. * **Eradication Methods:** Stopping breeding, stamping out infected animals, or using antibiotics/vaccination (with caution regarding experimental impact). * **Hygienic Measures:** Facility design (compartmentalization, one-way traffic), personnel hygiene, disinfection of equipment and materials, and health monitoring are crucial. * **Containment Housing:** Isolators, cubicles, filter-top cages, and individually ventilated cages (IVCs) minimize pathogen and allergen spread. ## Biosafety Measures Biosafety levels (BSL) classify laboratories and procedures based on the risk posed by microorganisms. ### Risk Groups Microorganisms are categorized into four groups based on pathogenicity, transmission routes, and availability of preventive/therapeutic measures. ### Biosafety Levels (BSL) Correspond to containment measures required, increasing with risk group. BSL-2 requires biohazard signs, restricted access, and inward airflow. BSL-3 and BSL-4 involve more stringent measures like showers, HEPA filtration, and specialized housing (isolators for BSL-4). ## Good Laboratory Practice (GLP) GLP is a quality system ensuring the reliability and integrity of non-clinical safety data for regulatory submission. ### Scope and Inspection * **Scope:** Applies to pharmaceuticals, pesticides, food additives, cosmetics, and industrial chemicals. * **Inspection:** Facilities undergo regular inspections to ensure compliance. * **Areas of Expertise:** Includes toxicity, mutagenicity, ecotoxicity, pharmacokinetic, and residue studies. * **Phase I (Environmental Exposure):** Predicts environmental concentration (PEC) and assesses degradation. * **Phase II (Effects on Organisms):** Evaluates toxicity to fauna and flora, considering persistence and dose-response relationships (LC50, EC, NOEC). * **Safety and Residues:** Depletion studies determine withdrawal times for food-producing animals. * **GLP Principles:** Emphasize documentation, traceability, quality assurance, and standard operating procedures (SOPs). ## Comparative Morphology and Physiology Choosing the right animal model requires understanding its anatomy and physiology in comparison to humans. ### General Principles * **Model Selection:** Depends on the research question, cost-effectiveness, phylogenetic distance, and the 3Rs. * **Vertebrate Groups:** Fish, amphibians, birds, and mammals are commonly used. * **Comparative Morphology:** Examines similarities and differences in structures like integument, skeleton, bucal cavity, stomach, liver, lungs, and excretory systems to understand functional and evolutionary relationships. ### Species Comparisons (Rodents vs. Lagomorphs) Detailed comparisons are made for: * **Integument:** Skin, hair, mammary glands, sweat glands. * **Osteology:** Skull, limb bones, vertebral formula, digits. * **Myology:** Muscle groups for injections. * **Digestive System:** Teeth (hypsodont vs. brachydont, incisor overgrowth), stomach structure, cecum size, cecotrophy/coprophagy. * **Respiratory System:** Lung lobes, nasal passages. * **Urinary System:** Kidney structure (unilobular vs. multilobular). * **Genital System:** Uterine structure (duplex vs. bicornuate), sexual dimorphism, reproductive tract openings. * **Circulatory System:** Blood drawing sites, blood volume. * **Nervous System:** Brain complexity, olfactory bulbs, third eyelid. * **Eyes:** Vision capabilities, color vision (bichromatic vs. trichromatic). ## Immunology and Antibody Production Immunology in laboratory animals is crucial for vaccine development and understanding immune responses. ### Immune Cells and Antigens * **Antigen-Presenting Cells (APCs):** Dendritic cells, B-cells, macrophages. * **T-lymphocytes:** Helper (CD4+) and cytotoxic (CD8+). * **B-lymphocytes:** Produce antibodies. * **Antigens:** Foreign substances that elicit specific immune responses; immunogenicity is influenced by molecular weight, complexity, form, digestibility, route of administration, dose, genetics, age, and adjuvants. ### Antibody Production * **Polyclonal Antibodies:** Produced in vivo by immunizing animals, cheaper, but variable quality. * **Monoclonal Antibodies:** Produced in vitro using hybridoma technology, highly specific and sensitive, but more expensive. * **Immunoglobulin Isotypes:** IgG, IgM, IgA, IgE, etc., with different structures and functions. * **Antibody Response:** Characterized by a primary response (IgM first, then IgG) and a secondary response (faster, stronger, with memory). Affinity maturation increases antibody quality. * **Immunization:** Choice of animal, antigen dose, injection site, and adjuvants influence the immune response. ## Anesthesia and Analgesia Anesthesia and analgesia are critical for minimizing pain and distress during procedures. ### Anesthesia * **Phases:** Preparation, premedication, induction, maintenance, recovery, and postoperative care. * **Premedication:** Sedatives (e.g., acepromazine, $\alpha$-2 agonists) and opioids calm animals and reduce stress, requiring lower anesthetic doses. * **Induction:** Drugs like barbiturates, dissociative anesthetics (e.g., ketamine), and muscle relaxants are used. * **Maintenance:** Inhalation anesthetics (e.g., isoflurane) or continuous injection are preferred over repeated boluses. Balanced anesthesia (combination of agents) is often used. * **Recovery:** Fast recovery is crucial to minimize complications like hypothermia and respiratory depression. Inhalation anesthetics generally allow for quicker recovery. ### Analgesia * **Pain Components:** Nociception (signal transmission), perception, and response. * **Pain Evaluation:** Difficult, relies on objective parameters (scales), behavioral assessment, and experimental tools. * **Types of Pain:** Acute (often surgical) and chronic (more subtle). * **Nervous System and Pain:** Afferent fibers (A$\delta$, C for pain; A$\beta$ for touch), hyperalgesia (increased sensitivity), and allodynia (non-painful stimulus becomes painful). * **Pain Modulation:** Involves endogenous systems and can lead to phenomena like "wind-up." Pre-emptive analgesia (before pain occurs) is vital. * **Treatment:** Multimodal pain therapy combining non-pharmacological (immobilization, physical therapy) and pharmacological methods (NSAIDs, opioids, $\alpha$-2 agonists, local anesthetics, dissociative anesthetics) is most effective. ## Medical Imaging Various imaging techniques are used for diagnosis and research. ### Imaging Modalities * **Structural Imaging:** Radiology, Ultrasound, CT, MRI visualize anatomy. * **Functional Imaging:** Scintigraphy assesses physiological activity. ### Techniques and Applications * **Radiography (X-ray):** Transmission imaging, good for bone, limited soft tissue detail. Requires fast work and radioprotection. * **Ultrasound:** Reflection imaging, no radiation, good for soft tissues and fluids, relatively inexpensive. * **CT (Computed Tomography):** Transmission imaging using X-rays from multiple angles, excellent for bone, moderate for soft tissues, avoids superimposition. * **MRI (Magnetic Resonance Imaging):** Emission imaging using magnetic fields, superior for soft tissues (brain, cartilage), no radiation. * **Scintigraphy:** Emission imaging using radiopharmaceuticals to visualize functional processes. * **Image Fusion:** Combining images from different modalities to gain comprehensive information. ## Experimental Techniques Basic techniques for handling and administering substances to laboratory animals. * **Injections:** Subcutaneous (SC), intraperitoneal (IP), intramuscular (IM), and intravenous (IV) routes are used, with site and volume depending on species and substance. Always aspirate before injecting to check needle placement. * **Oral Administration:** Direct dosing to the esophagus or stomach using syringes or silicone tubes. * **Blood Sampling:** Methods vary by species and volume required. Tail veins, saphenous veins, ear veins, or cardiac puncture (terminal) are common sites. Minimizing trauma and ensuring proper volume withdrawal is crucial. ## Radiobiology The study of radiation's effects on biological systems. ### Ionizing Radiation * **Types:** Alpha ($\alpha$), Beta ($\beta$), Gamma ($\gamma$), and X-rays differ in ionization energy, charge, mass, and penetration. * **LET (Linear Energy Transfer):** A measure of radiation quality; high LET radiation (e.g., $\alpha$-rays) causes more damage per unit track length. * **Risk Quantification:** Effective dose (Sievert, Sv) accounts for radiation type, dose, and tissue sensitivity. * **Radiation Protection:** Based on distance, timing, and shielding (lead for photons, plastic for electrons). Personal dosimeters are essential. * **Biological Effects:** Deterministic effects (cell death) have a threshold dose; stochastic effects (DNA mutations, cancer) lack a threshold. Embryos and gonads are particularly radiosensitive. ## Euthanasia Euthanasia must be performed humanely, minimizing pain, fear, and distress. ### Principles and Methods * **Criteria:** Quick effect, minimal restraint, minimal pain/fear, suitability for species/condition, reliability, irreversibility, operator safety. * **Methods:** * **Physical:** Cervical dislocation (birds, rodents, rabbits), decapitation (birds, rodents), shooting, concussion, electrocution. Require skill and restraint. * **Chemical:** Inhalation (CO2 for rodents/birds, volatile anesthetics), absorption (anesthetic overdose for fish/amphibians), injectable anesthetics (barbiturates, T61). CO2 can cause distress if not administered gradually. * **Unacceptable Methods (unless anesthetized):** Hypothermia, drowning, neck crushing, strangulation, ether, chloroform. * **Confirmation of Death:** Checking body temperature, reflexes, heartbeat/breathing, rigor mortis, and potentially brain destruction. * **Human End-points:** Predetermined criteria to terminate or alleviate animal suffering during experiments. ## Viral and Bacterial Zoonoses Understanding zoonotic diseases is critical for occupational health. ### Viral Zoonoses from Rodents * **Hantaviruses:** Transmitted via rodent excreta aerosols; cause flu-like illness or Hantavirus Pulmonary Syndrome (HPS). * **Arenaviruses:** RNA viruses, can cause hemorrhagic fever. * **Lymphocytic Choriomeningitis Virus (LCMV):** Found in mice; can cause neurological disorders or affect fetuses. * **Lassa Virus:** Endemic in West Africa, transmitted by rodents; can cause hemorrhagic fever. * **Encephalomyocarditis Virus (EMCV):** Can infect pigs and humans; RNA virus, causes abortion. * **Orthopoxvirus:** e.g., Cowpox, Mousepox; cause skin lesions. ### Bacterial Zoonoses * **Prevention:** Training, hygiene, appropriate handling, wearing protective clothing, and reporting exposures are crucial. * **Transmission Routes:** Inhalation, ingestion, skin contact, bites, scratches, and handling contaminated materials. * **Bite-Related Infections:** * **Pasteurella multocida:** Common in dog/cat oral flora; can cause local infection or bacteremia. * **Capnocytophaga canimorsus:** Particularly dangerous for immunocompromised individuals; can lead to sepsis. * **Rat Bite Fever (Streptobacillus moniliformis):** Asymptomatic in rats but causes flu-like symptoms and potentially fatal illness in humans if untreated. * **Cat Scratch Disease (Bartonella henselae):** Transmitted via cat scratches; typically causes local lesions and lymph node swelling. * **Other Bacterial Zoonoses:** * **Tularemia ("rabbit fever"):** Hosted by rabbits and mice; dangerous, potential biological warfare agent. * **Leptospirosis:** Spiral bacteria inhabiting kidneys of rodents (rats); shed in urine, transmitted through skin/mucous membranes; can cause liver/kidney failure or meningitis. * **Dermatomycosis (Fungal Infection):** Common zoonosis causing skin lesions in animals and humans. Fungal spores are environmentally resistant. ## Safety and Hazards Occupational Health and Safety (OHS) programs aim to prevent injuries and illnesses in the workplace. ### Hazard Identification * **Physical:** Animal bites/scratches, sharps, flammable materials, ergonomic strains, noise, UV radiation, lasers, ionizing radiation. * **Chemical:** Disinfectants, anesthetic gases, tissue preservatives (e.g., formaldehyde), bedding materials, pesticides. * **Protocol-Related:** Hazards from chemicals or infectious agents used in experiments, including viral vectors and transgenic animals. * **Allergens:** A significant concern, often causing respiratory symptoms and skin reactions, with sensitization requiring prevention strategies. ### Allergy Prevention * **Goals:** Reduce sensitization and manage symptoms. * **Programs:** Screening (identifying at-risk individuals), facility design (ventilation, filtration, separate areas), work practices (hygiene, job rotation, wetting down dust), and personal protective equipment (respirators, gloves, lab coats). ## Legislation and Ethics * **Belgian Legislation:** Law of 14 August 1986 and Royal Decree 2013 regulate animal experimentation, user recognition, origin of animals, and ethical committees. * **Ethical Committees (EC):** Evaluate projects based on scientific quality, the 3Rs, safety, and cost-benefit analysis. Belgium utilizes local ECs. * **Ethical Perspectives:** Anthropocentrism, welfarism, egalitarian views, biocentrism, and ecocentrism offer different frameworks for animal ethics. * **Moral Status:** Discusses criteria like sentience, consciousness, language, rationality, and emotions in determining moral status for animals. * **Historical Views:** From Aristotle's hierarchy of souls to Descartes' dualism and Darwin's evolutionary theory, views on animals have evolved. * **Animal Ethics Debate:** Explores consequentialism (utilitarianism) and deontology, with key figures like Peter Singer and Tom Regan. ## Common Mistakes to Avoid * **Ignoring the 3Rs:** Failing to adequately replace, reduce, or refine animal use. * **Inadequate Training:** Handling animals improperly, leading to stress or injury. * **Poor Experimental Design:** Lack of proper randomization, blinding, or controls, compromising reproducibility and generalizability. * **Overlooking Zoonotic Risks:** Failing to implement appropriate hygiene and protective measures. * **Inadequate Postoperative Care:** Neglecting pain management, hydration, or monitoring, leading to animal suffering. * **Using Incorrect Strains:** Selecting animal models that do not appropriately mimic the human condition or experimental question. * **Failing to Adhere to GLP:** Compromising the integrity and regulatory acceptance of safety data. * **Neglecting Ethical Committee Approval:** Conducting research without proper authorization. * **Improper Euthanasia:** Using inhumane methods or failing to confirm death correctly. * **Ignoring Legislation:** Non-compliance with national and international regulations for animal use, transport, and facility standards. * **Over-reliance on Statistical Significance:** Not considering practical relevance or potential biases in the data. * **Misinterpreting Results:** Drawing conclusions without appropriate statistical analysis or understanding of animal models. * **Ignoring Potential Hazards:** Underestimating risks associated with chemicals, radiation, or biological agents. * **Inadequate Environmental Enrichment:** Failing to provide conditions that support species-specific behaviors and welfare. * **Poor Sample Size Calculation:** Leading to underpowered studies or the unnecessary use of animals.

## Glossary | Term | Definition | |---|---| | Laboratory Animal Science | A multidisciplinary field concerned with the welfare and care of animals used in research and education. | | Animal Experiment | An experiment that uses living animals, with specific restrictions and regulations applied to certain species. | | FELASA | The Federation of European Laboratory Animal Science Associations, which provides education and certification for professionals working with laboratory animals. | | Zoonosis | Microorganisms or diseases that can be transmitted from animals to humans. | | Necropsy | A post-mortem examination of an animal's body to determine the cause of death or to collect samples for further research. | | Amniogenesis | The formation of the amnion, a membrane that encloses the embryo in amniotic fluid. | | Implantation | The process by which the embryo attaches to the uterine wall and establishes a link with the mother, leading to placental formation. | | Placental Barrier | The layers of tissue separating maternal and fetal blood vessels, determining the exchange of nutrients and waste. | | Maternal Relation | The fate of the maternal endometrium during pregnancy, classified as deciduate (shedding) or adeciduate (retained) at birth. | | Epizootic | A disease that spreads rapidly and severely within a population during a specific period, exceeding expected rates of spread. | | Enzootic | A disease that is endemic to a population, with sporadic cases appearing over time without the need for external introduction. | | Pharmacokinetics | The study of how a drug enters the body, is distributed, metabolized, and eliminated. | | Pharmacodynamics | The study of the biochemical and physiological effects of drugs and their mechanisms of action on the body. | | Receptor | A target molecule (often a protein) in or on a cell that a drug binds to, initiating a biological response. | | Agonist | A drug or substance that binds to a receptor and produces a biological effect, mimicking the action of the natural ligand. | | Antagonist | A drug or substance that binds to a receptor but does not produce a biological effect, blocking the action of agonists. | | Efficacy | The maximum effect a drug can produce when all receptors are occupied. | | Affinity | The strength of binding between a drug and its receptor. | | Potency | The amount of drug required to produce a given effect, often expressed as EC50. | | Tolerance | A gradual decrease in responsiveness to a drug upon repeated administration, requiring higher doses to elicit the same effect. | | Homeostasis | The maintenance of a stable internal environment in an organism despite external changes. | | Ethology | The scientific study of animal behavior. | | Stereotypies | Repetitive, morphologically identical behaviors that are performed regularly and apparently without function, often indicative of welfare issues. | | Xenotransplantation | The transplantation of organs or tissues from one species to another. | | GLP (Good Laboratory Practice) | A quality system for research and development facilities to ensure the consistency and reliability of non-clinical safety data. | | Biosafety Level | A set of containment measures and precautions required when working with microorganisms, categorized into levels based on the risk they pose. | | Zoonosis | Infectious diseases naturally transmissible between vertebrate animals and humans. | | Euthanasia | The practice of intentionally ending a life to relieve pain and suffering. | | SPF (Specific Pathogen Free) | Animals that are free from a specific list of pathogens, crucial for reproducible research. | | Transgenesis | The process of introducing an exogenous gene into the germline genome of an organism, which is then heritable by offspring. | | Gene Targeting | A technique to modify specific genes within embryonic stem cells, allowing for the creation of knockout or knockin mice. | | CRISPR/Cas9 | A gene-editing technology that uses a guide RNA and Cas9 nuclease to precisely modify DNA sequences. | | ANOVA (Analysis of Variance) | A statistical method used to compare the means of three or more groups. | | P-value | The probability of obtaining the observed results (or more extreme results) if the null hypothesis were true. | | Confidence Interval | A range of values that is likely to contain the true population parameter with a certain level of confidence. |