Welcome to the seminar „Humane killing of laboratory mice“ Two (main) reasons for killing animals in research:

1. taking organs for scientific purposes 2. end of an experiment (approved animal protocol)

Legal justification, approval and requirements are different. Skills and knowledge have to be the same and they have to be verifiable.

Important: this seminar will give knowledge for the humane killing of mice, nothing else!

For working with the living animals (applications, taking samples, surgeries etc.) the modules 1 – 3 offered by the animal welfare board have to be attended!!! To get the permission for killing laboratory mice for scientific purposes there are 3 prerequisites:

1. attendance of the seminar 2. expert talk 3. practical demonstration of the method in the actual project (no extra animals needed)

2 + 3 will be with the animal welfare officer or the substitute

Important: the permission is accepted by our local authority. No demand that other authorities will accept it, too. It is only issued for the particular species and the respective method to be applied. www.uni-saarland.de/tierschutzbeauftragte www.uni-saarland.de/tierschutzbeauftragte www.uni-saarland.de/tierschutzbeauftragte Development of the number of animals used for research in Germany

Versuchstierzahlen in Mio. animal numbers in mio. (vertebrates only)

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0 ‘91 ‘95 ‘00 ‘05 ‘10 ‘14

Nobel-prize for medicine 2007: How to knock out genes

Oliver Smithies (USA), Mario Capecchi (USA) and Martin Evans (GB) for developing the „Gene Targeting“ method, to modify single genes

... one of the most important biotechnological methods. With this people can create the so-called Knockout- mice; in their genome certain genes are turned off. These animals help to observe and analyse the function of a single gene and so contribute to understand the normal development of mammals as well as different diseases. Due to the committee the work has been so pioneering that modern research is hard to imagine without the technology of gene targeting. Transgenic mice in Germany

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0 2000 2005 2010 Species used in animal research (2014)

rabbits: 105.784 birds: 55.697 other rodents: 50.103 pigs: 14.374 rats dogs: 4.636 362.530 cats: 997 mice primates: 2.694 1.901.985 cattle: 4.408 Mice General remarks

• progenies of the „house mouse“ mus musculus

• diversity through english traders, who brought animals with unusual coat colors from China and Japan

• since the end of the 19th century systematic breeding for experimental purposes General remarks • wild mice are prey-animals • prefer dry areas with possibilities to hide • omnivorous animals with a preference for grain • mainly nocturnal • run, jump, climb; good swimmers, but not voluntarily General remarks

• order: rodentia • rodents: dental formular 1 0 0 3 1 0 0 3 human beings: 2 1 2 3 2 1 2 3

• incisor teeth grow lifelong General remarks

• coprophagy

• 5 pairs of mammary glands (rat 6) Physiology weight (adult): f: 40g, m: > 40g head-body-length: 5 – 10 cm tail-length: 8 - 10 cm life expectance: 1,5 – 2 (max. 3) years (influenced by genome and environment) Physiology food intake: 4 – 8 g / day water intake: 5 – 8 ml / day urine production: 1 – 3 ml / day heartbeats (/min): 300 – 800 breathing (/min): 100 – 200 (mean 160) body temperature: 36,5 – 38°C Handling Breeding and developing data • polyoestrus species • oestrus cycle: 4 – 5 days • oestrus duration: 12 h • vaginal plug: 12 – 16 h • gestation period: 18 – 22 days (19 – 20) • birth: occurs mostly at night or in the late afternoon • immature at birth • litter size: varies greatly (extremes from 2 to 20) • post partum oestrus: 12 – 24 h after parturition, causing simultaneous gestation and lactation in permanent breeding groups Post–natal development • birth weight: 1 – 1.5 g • weaning: at 3 weeks • weight at weaning: 10 g • sexual maturity at 4 – 6 weeks • breeding maturity: at 8 – 10 weeks

Newborns: blind ears closed no hair, except for vibrissae no teeth skin colour: red 1. day pink, rosy 2. day 24 h; 1,5g Post–natal development 3d, 3g

• pigmentation of the skin starts at day 2 • opening of the ears at day 3 hearing starts at day 10 7d, 4,5g • fluffy coat after day 3; • fur well developed after 2 weeks • from day 9 on 5 pairs of teats are clearly visible in the female mice • eyes open between day 11 and 16.; 14 d, 8g final visual acuity is reached around day 30

Animal facilities and health status „conventionel animals“

• in former times • microbiological status unknown • free from obvious diseases and parasites • animal room without barriers • today still the case with larger (farm) animals „gnotobiotic animals“

• the complete opposite • animals germfree or „purposely“ infected • microbiological status is positively defined • housing in isolators only • newborns developed via cesarean section or hysterectomy • immediate tansfer to gnotobiotic nurse • from the scientific point of view ideal (?) • very expensive and very labour intensive Isolators

• completely isolated from the environment • sterile inside • working only via gloves from the outside • ventilation via HEPA filters • anything brought inside has to be sterile • lock-systems (PES) • constant check of sterility (sentinels) „SPF-animals“ (specified pathogen-free)

• possess a physiological microbiological gut flora • microbiological status is negatively defined • problem: individual specification • unmentioned pathogens / micororganisms • air HEPA-filtered • cages, bedding autoclaved • food autoclaved or irradiated (sometimes) • BUT: personnel can enter with PPE SPF- animals

• animals of a reliable and well-defined SPF- quality are today‘s „first choice working basis“ • specification und timepoint of analysis are extremely important (FELASA recommendations) • technical und hygienic barriers shall prevent infections • harmless microorganisms are tolerated animals of a reliable and well-defined SPF- quality have a higher life expectance and a better breeding outcome than „conventional animals“ IVCs – isolated ventilated cages Methods of killing laboratory mice humanely in agreement with the animal welfare legislation Development of the number of animals used for research in Germany

animal numbers in mio. 3,5

3

2,5

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1,5

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‘07 ‘08 ‘09 ‘10 ‘11 ‘12 ‘13 ‘14

„killing for taking organs“ approved animal protocols

§ 4 Animal Welfare Law

Killing of animals

• vertebrates only under anesthesia or under avoidance of pain • only by people with the appropriate knowledge and practical skills § 2 Decree on laboratory animal welfare (TierSchVersV)

Requirements for the killing of vertebrates and cephalopodes

(1) … only by a person, who fulfills the requirements of Appendix 1 section 2 under anesthesia or under avoidance of pain and suffering

(2) … killing only in accordance with Appendix 2; the chosen method has to be the least stressful one compatible with the purpose of the experiment Appendix 1 section 2 Decree on laboratory animal welfare (TierSchVersV) Requirements for people killing animals Appendix 2, Decree on laboratory animal welfare

Appendix 2 Decree on laboratory animal welfare Euthanasia

derived from the Greek terms eu meaning good thanatos meaning death

„A good death“

A „good death“ would be one that occurs with minimal pain, fear and distress Primary criteria for euthanasia in terms of animal welfare:

• painless method • rapid achievement of unconsciousness and death • minimum of restraint required • minimum of fear and excitement • method appropriate for the age, species and health status • method reliable, reproducible, irreversible • simple to administer • (safe and acceptable for the operator) In laboratories and breeding facilities animals are killed for various reasons:

• to provide blood and /or other organs for scientific purposes • when levels of pain, distress and suffering are likely to exceed the designated level (humane endpoints!)

• when they are no longer suitable for breeding

• unwanted stock or those with unsuitable character- istics (sex, genotype etc.)

vertebrates are only allowed to be killed by people with the appropriate knowledge Euthanasia mechanical / physical methods: correct application causes immediate loss of consciousness with subsequent death through physical trauma to the brain application requires personal engagement and skills, it takes strong efforts to overcome the natural aversion to actively killing an animal Physical methods a) cervical dislocation recommended for mice, in rats < 150 g only b) decapitation guillotine in mice, rats and hamsters after sedation in newborns with scissors without sedation c) shooting (free bullet or captive bolt) for large mammals; with the bolt exsanguination is required Euthanasia

Injectable agents:

• Barbiturates are the first choice (Pentobarbital) • whenever possible: application i.v. (i.p. in small rodents). • suitable for the euthanasia of pregnant animals, since it easily crosses the placenta and the blood-brain- barrier Inhalational agents

• are either vaporized or delivered as a gas into chambers or anaesthetic circuits

common agent: isoflurane Inhalational agents common agent: isoflurane

also possible as Open–Drop–Exposure: • cotton pad • bell jar or small container with a secure lid • wire mesh • hood! Euthanasia

Inhalational methods:

Lead to unconsciousness first, then to death Isoflurane is to prefer but still very common is the

killing with CO2 Points to consider when killing mice with CO2

1. for adult mice only (NOT suitable for newborns!!!) 2. not within the animal holding room 3. homecage preferred 4. otherwise chambers of sufficient size 5. clean chamber between animals 6. influx of CO2 without noise 7. no prefilling of the chamber 8. not too slow 9. not too fast 10. optimum: exchange 20 % of the volume of the chamber per minute (CO2 against air) 11. only from a central gas supply (no dry ice!) Guideline concerning adequate, species-specific and humane killing of (laboratory) animals

AVMA 2013

Euthanasia

The following methods are never to be applied:

 oral oder rectal application of anesthetics (too slow!!)  death by freezing / liquid nitrogen ( too slow!!)  electric shock, (only allowed in fish); might cause cramps and pain  all methods involving suffocation (drowning, inhalation of nitrogen or helium, application of curare, strychnine, hydrocyanoic acid) would cause maximum fear and distress important in any case, no matter what kind of method for euthanasia is used:

removal of the carcasses only after confirmation of death:

cessation of heartbeat cessation of respiration absence of reflexes lowering of the body temperature below 25°C (small rodents) rigor mortis