Environmental conditions in the animal unit

Transcription

1 Environmental conditions in the animal unit Means Aims Regulations and ethics Contributing to the humane use of animals in biomedical research and to the collection of informative unbiased and reproducible data. Characteristics of the animals Influence of external factors Planning experiments and research design Managing the human factor Aurora Brønstad Laws, regulations, Anatomy, guidelines, 3R, physiology, genetics, ethics biochemistry, microbiology Environmental conditions, Interventions, Anesthesia, surgery etc.. Quality control systems, SOP, GLP, statistics, experimental design Communication Cooperation, Coordination and Contracts APPENDIX A OF THE EUROPEAN CONVENTION (ETS NO. 123) The great majority of animals used in procedures are kept in facilities ranging from outdoor corrals to cages for small animals in a laboratory animal house. This is a situation where there are often highly conflicting interests between the scientific requirements and the needs of the animal. In this conflict, the basic physiological and ethological needs of the animals (freedom of movement, social contact, meaningful activity, nutrition, water) should be restricted only for the minimum necessary period of time and degree. Subjects to address: How can environmental conditions in the animal unit influence our results? What environmental conditions are important? What standards apply for environmental conditions? Such restrictions should be reviewed by scientists, animal technicians and those competent persons charged with advisory duties in relation to the well-being of the animals before procedures are undertaken to ensure that the extent of the compromise to animal welfare is minimized to a level consistent with the scientific objectives of the study.

2 Human versus animals Body H um an Anim al Large, relatively hairless Small, furry Heat loss Sweat Cant sweat (rodent) Thermoreg. M ostly behavioral M ostly metabolic Activity Day-active Nocturnal Sense of smell Relatively poor Excelllent!! Homeostasis mechanisms Change in environment causes physiological responses to sustain optimal physiological conditions Immune response, cardiovascular response, metabolic response, behavioral response These responses may effect experimental results eyesight Hearing Excellent detail view High colour sensitivity Limited (15Hz K Hz) Poor detail view Limited colour view Ekstensiv (100 Hz til 80+ khz) Minimize environmental variation: minimize unwanted variation in research results (UFAW handboook) Temperature Ex. LD-50, Clorpromazine in mice: Neuroendocrine interactions 400 L D m g / k g sympathetic hypothalamus anterior pituitary thyroid thymus islets, gonads somatotrophin Thyroxin T-cells Thymic hormones Interleukin-1 Interleukin-4 sensory pathways sympathetic L ymphoid tissue Romtemperatur C 40 Berti & Cima (1955) adrenals insulin, sex hormones Cathecholamines, corticoids corticoids, catecholamines

3 The 3 Rs Replacement Reduction Refinement (Russell & Burch, 1959) Reproducibility Experiments should be repeated giving same results any place at any time Experimental interventions are the only source of difference Everything else is held constant Strict environmental control is necessary Standardized environment - less animals necessary Less animals necessary comply with the demand of reduction Standardization Regulation of Animal Experimentation 13. Planning and conducting an experiment Standardizing environment + Standardizing the animal = Increased reproducibility Experiments are to be thoroughly planned, carried out and quality controlled in order to ensure that the minimum number of animals is used.

4 Sources of environmental variation Temperature control Affect behavior and metabolism Environmental factors influence several systems in the body Variation in room temperature max: ± 1 C max: ± 1/2 C Temperature in animal room should be logged continuously, and saved (3 / 10 years) STRESS! Environmental factors - mammals Temperature Relative humidity (RH) Light Sound/Noise Air quality Smell Animal density Acclimatization Temperature control species Opt. temp. C Mice Rat Guinea pig Rabbit Cat Sheep Pig APPENDIX A OF THE EUROPEAN CONVENTION (ETS NO. 123)

5 Relative humidity For rodent Optimal RH = 55±5% (UFAW) 45-65% Appendix A Cold winter with blue sky Low humidity Added humidity necessary in Norway, especially during winter Air condition system has to compensate for all weather conditions to offer stabile climate in the animal room Rainy days - high humidity Relative humidity (RH) RH > 80% disturbances in metabolism poor hygiene increased bacteria growth

6 Relative humidity (RH) Temperature and RH < 35% RH: General dehydration Dry mucous membranes Irritated airways Increased problems with dust Increased static electricity Decreased reproduction rate eating of offspring ringtail in rat Important for reproducibility of many experiments, and should therefore be mentioned when publication of results. Relative humidity (RH) Common reason for unwanted experimental variation How can temperature and RH affect your experiments?? Air humidity should be logged continuously, and data saved

7 Light Rodents are usually housed 12 L+/12 M. Only artificial light (no windows) Reproduction problems when day- or night- gets changed Nocturnal Physical activity is strongly influenced by light Light as environmental factor Identical lighting in the whole room is often difficult Rotation devices Compromise between optimality for animals and people who work with them Light as environmental factor light in nature 80 Permanent lightning Strong light Lux shadow shadow 1 m 0 May Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec

8 Light as environmental factor Lux Suggested intensity, 1m above the floor (UF AW 300 handbook, 1987) 250 Light as environmental factor Wavelengths - Rat: Activity high medium low activity Lower level for work (Schlingmann & al. 1993c) 100Preferred maximum for rats (Bloom & al 1993) 60 Max to avoid retinal damage in albinos (Schlingmann & al Albino rats own preferrence, max (Schlingmann & al. 1993c) Adaptation to changed light regime: 2 8 weeks. A period of red light at frequencies undetectable to the rodents can be useful during the dark period so that staff can monitor the rodents in their active phase. * *APPENDIX A OF THE EUROPEAN CONVENTION (ETS NO. 123) Light as environmental factor Sound and noise Rodents are nocturnal or twilight animals Live in tunnels below the ground Outside; Lux (day) Moonlight 5-10 lux Recommendation: Lux 1 m above floor in middle of room. No windows in room Albino rats prefer less than 25 Lux Lux does not take wavelength into consideration

9 Sound and noise < 55 dba: Acceptable level of background noise in empty animal rooms Equal Level sound better than unexpected/unknown sounds. Ultrasonic noise produced by many common laboratory fittings, including dripping taps, trolley wheels and computer monitors, can cause abnormal behavior and breeding cycles. Noise can lead to: reduced fertility kid-eating non reproducible results cramps Sound/Noise The animals themselves (dog, pig, poultry) can be the source of damaging noise How do rats react to noise? H-M. Voipio 1997 Noise How can noise affect Reproductions/breeding of animals Behavior studies What sources of noise do you have in your animal facility? What sources of ultrasound do you have in your animal facility? Gas Ideal cage atmosphere = fresh air Carbon dioxide (CO2) irritates mucous tissue influences respiration Ammonia (NH3) Very irritating for eyes and airways indicator of poor hygiene and ventilation

10 Air change air changes/hour in the room* Distributed throughout the room Frequent air changes, but avoid drafts Nesting material protect from draft Air pressure influence animals activity - and thereby experimental results Smell Change clothes and be careful with perfumes when you Work in the lab animal house Predators (Dog cat) Your own, or the one next door Pheromones in perfumes increases NA level (Wang & Tsai 1991) Pavlovskian effects Specific smells, mobilization of NK cells in sensitive animals. (Solvason & al. 1991) *APPENDIX A OF THE EUROPEAN CONVENTION (ETS NO. 123) Individually ventilated cages Drug administration Air in - air out Traditionally open cage 70 air changes/hour Handling/stress +/- Administration method: i.v., s.c., i.p. etc., including error injections. Volume, concentration, osmotic pressure, viscosity, administration speed,. Solvents, adjuvant (Freud)

11 Bedding as environmental factor Absorb urine and droppings Clean and dry in the cage Environmental enrichment nesting material Bedding as environmental factor Wood chips, paper, corn-cobs Avoid toxic tree species such as teak or mahogany Induction of liver-enzymes: pine Pollution Dust: irritated mucous membranes Sharp or pointed parts, can harm the animals SPECIAL MADE BEDDING: EXPENSIVE!!! Bedding as an environmental factor Vermiculite, SA Corn cobs, US Sugar cane, CU Aspen, SF Shredded paper, AUS Bedding 3, NZ Bedding 2, NZ Pine, AUS Pine, Russia Bedding 1, NZ Pine, Greece Toxic index, hepatocyte toxicity test Pelkonen & Hänninen, 1994

12 Animal concentration Solitary confinement Depression / aggression Heat loss Lack of social contact Common reason for GREAT variations in experimental results. Crowded conditions Unstable environment, fighting, great variation in food intake Reduced quality (air and bedding) Variation in results Diet Small but considerable variations i the feed will seldom be noticed Large variations between the manufacturers Variation between batches Variation among batches (humidity damage, wrong storage, light, temperature) How can diet influence your project? Maybe allot - little not at all What should we believe???? Do a search in literature much is known Avoid change of feed during an experiment Report feed in the articles Diet/Feeding Well documented influence on many types of experiments Sources of pollution: Bacteria (spores) Fungi Aflatoxins Chemicals Insects Unintentional source active substances Fatty acid in fish Phytoestrogen in some plant Avoid unintended variation in diet Feed change as well as batch change Other environmental factors Cage material Social factors Handling Moon phases Magnetic fields Radiation Placement of cage in room Water quality and availability

13 Acclimatisation A period of acclimatization is needed to allow animals to recover from transport stress, to become accustomed to a new environment and to husbandry and care practices. Even when the animals are seen to be in good health, it is necessary for them to undergo a period of acclimatization before being used in a procedure. The time required depends on several factors, such as the stress to which the animals have been subjected which duration of the transportation, age of the animal, change of the social environment. International transport may necessitate an extended period of acclimatisation due to disturbance of the diurnal rhythm of the animals. Order your aniamals well ahead!! Quarantine, acclimatisation and isolation The objectives of quarantine and isolation periods are: a. to protect other animals in the establishment; b. to protect man against zoonotic infection; and c. together with an acclimatisation period, to foster good scientific practice. APPENDIX A OF THE EUROPEAN CONVENTION (ETS NO. 123) Acclimatization Acclimatization Changes in feed temperature RH airstreams light sound/noise Without proper Acclimatization it is hard to obtain reproducible results Practical consequence: Buy animals EARLY BEFORE THE EXPERIMENT! Species days. (minimum) Primates Dog, Cat Rabbit Rodents 5-15 Pig 7-10 European Convention

14 Avoid environmental variation Accreditation systems ISO 9001 GLP/GMP Association for Assessment and Accreditation of Laboratory Animal Care International Environment influences the body STRESS! Control with environmental factors are taken care of! Environmental factors: references The European convention for the protection of vertebrate animals - Appendix A ( Europakonvensjonen ) Arev.pdf

15 Do you have control with the environmental factors??