Noninhaled. **Sedation followed by IV barbiturate is preferred method. 1. Barbiturates sedation may be needed for IV, can give IP

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1 Acceptable Acceptable with Conditions Companion Animals (Usually Dog/Cat) Lab & Wild Caught Rodents Lab Farm Animals, Dogs, Cats, Ferrets, NHPs 1. Barbiturates IV preferred but can give IP if IV distressful/dangerous 2. Nonbarbiturate anesthetic overdose Ketamine/xylazine -IV, IP, IM Propofol- IV 3. T Tributame 1. Barbiturates IO, IC, intrahepatic, intrasplenic, intrarenal if IV access not achievable and /anesthetized (exception shelter cats, awake intrahepatic) 1. anesthetics via chamber if <7kg, ensure death b/c can recover 2. CO if equipment is designed and maintained and trained personnel (not recommended) 3. CO2 if equipment is designed and maintained and trained personnel; minimize distress, gradual fill, supplied regulated and purified form, pressure reducing regulator and flow meter must be used (not recommended) 4. Gunshot, recommend to sedate cats, trained personnel, safe for public, legal in area (not recommended) 5. Penetrating Captive Bolt rabbits and dogs in lab setting 1. Barbiturates IV preferred but IP more practical, 3x anesthetic dose 2. Barbiturate combo (local anesthetic & anticonvulsant) 3. Dissociative combo (ketamine w/ xylazine or diazepam) 1. anesthetics ± N2O 2. CO2 w/ or w/ out pre-med w/ inhaled anesthetic 10-30% displacement rate; gradual fill; supplied regulated and purified form, pressure reducing regulator and flow meter must be used 3. CO 1. Tribromoethanol when prepared, stored (dark/cool room, use w/in 2 wks) 2. 70% ethanol IP for mice if other agents aren t available or physical methods not desired 1. Cervical Dislocation personnel trained; mice & rats <200g 2. Decapitation mice and rats; trained personnel 3. Focused Beam Microwave Irradiation mice & rats for immediate fixation of brain metabolites **Sedation followed by IV barbiturate is preferred method. Lab Rabbits 1. Barbiturates sedation may be needed for IV, can give IP 1. anesthetic overdose if already under anesthesia 2. CO2 premed with sedative Physical 1. Cervical Dislocation if <1kg; personnel trained 2. Penetrating Captive Bolt good working order, positioned correctly and operated by trained people Lab Finfish, Aquatic Invertebrates, Amphibians, Reptiles Zebrafish 1. Rapid chilling (2-4 C) until loss of orientation and opercular movements; adults 10 min and fry 4-7dpf 20min; for < 3 dpf follow with another method like Na or Ca hypochlorite Small Bodied Tropical and Subtropical Stenothermic Species 1. Rapid Chilling Amphibians X. laevis and Rana spp best euthanized by a physical method while fully anesthetized 1

2 Adjunctive Methods 1. Potassium Chloride IV or IC after 2. N2 or Ar if 3. Electrocution dogs Unacceptable 1. SC, IM (except some anesthetics), and intrapulmonary, intrathecal administration of injectable euthanasia agents 2. Household chemicals, disinfectants, cleaning agents, pesticides 3. Hypothermia 4. Drowning Agents 1. Nitrogen and Argon Agents 2. Potassium Chloride 3. Neuromuscular blocking agents 4. Barbiturate with neuromuscular agent in same syringe 5. Opioids 6. Urethane 7. α Chloralose Special Cases - If fully anesthetized, bilateral thoracotomy, exsanguination, perfusion, IV or IC potassium chloride are acceptable Special Cases If fully anesthetized, adjunctive methods such as potassium chloride, exsanguination, or bilateral thoracotomy are acceptable Fetuses/Neonates 1. Mid or late term pregnancy barbiturate and leave fetuses in uterus for min; avoid IP inj late term pregnancy 2. IP pentobarbital in preweanlings Acceptable 1. Injectable barbiturates, in combination w/ local anesthetics & anticonvulsants 2. Dissociative agents with α2 adrenergic receptor agonist or benzodiazepine Acceptable w/ Conditions 1. anesthetics neonatal mice may take up to 50 min in CO2, need adequate exposure or adjunctive method 2. Hypothermia gradual cooling up to 7d of age; or altricial <5 d may be immersed in liquid N2 3. Decapitation of altricial <7d using scissors or sharp blades 4. Cervical Dislocation for mice/mats **Unnecessary to remove fetuses after dam is euthanized. 2

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4 Acceptable Cattle Sheep and Goats Swine 1. Barbiturates IV 1. Barbiturates IV 1. Barbiturates IV Acceptable with Conditions Adjunctive Methods 1. Handguns close range, caliber, solid point lead bullets,.22 not recommended for adults 2. Rifles preferred firearm for distance,.22,.223,.243,.270, Shotguns - preferred gauge 20, 16, or 12; #6 or larger birdshot or shot-gun slugs 4. Penetrating Captive Bolt adjunctive method recommended 1. Potassium Chloride and Magnesium Sulfate 2. Second Shot 3. Exsanguination 4. Pithing Unacceptable 1. Manual blunt force trauma to head 2. Injection of chemical agent if conscious 3. α2 agonist followed by IV potassium chloride or magnesium sulfate 4. Drowning 5. Air embolism 6. Electrocution with 120V cord 7. Exsanguination if conscious Fetuses/Neonates 1. Barbiturate overdose fetus must remain in uterus for min. 2. Gunshot follow w/ adjunctive method 3. Penetrating or purpose built nonpenetrating captive bolt (Acceptable with Conditions) follow w/ adjunctive method 1..22LR rifle,.38 special,.357 magnum, 9mm handguns, shotguns, solid point bullets and larger caliber for large horned adult rams 2. Penetrating captive bolt must be followed by an adjunctive method 3. Non-penetrating captive bolt followed by exsanguination 1. Potassium Chloride and Magnesium Sulfate 2. Second Shot 3. Exsanguination 4. Pithing 1. Manual blunt force trauma to head 2. Injection of chemical agent if conscious 3. α2 agonist followed by IV potassium chloride or magnesium sulfate 4. Drowning 5. Air embolism 6. Electrocution with 120V cord 7. Exsanguination if conscious 1. Barbiturate overdose fetus must remain in uterus for min. 2. Gunshot follow w/ adjunctive method 3. Penetrating or purpose built nonpenetrating captive bolt (Acceptable with Conditions) follow w/ adjunctive method Agents 1. N2 w/ CO2 2. Ar w/ or w/o CO2 3. CO 1. Gunshot 2. Penetrating Captive Bolt follow with adjunctive method as necessary 3. Electrocution- 2 step or single-step; current must pass through brain and cardiac fibrillation must not occur before 1. Exsanguination 2. Pithing Nursery Pigs ( 70 lbs.) Acceptable 1. Barbiturate IV Acceptable w/ Conditions 1. CO2 alone or with N2 or Ar 2. Nonpenetrating captive bolt 3. Electrocution - if > 10 lbs. Suckling Pigs 1. CO2 2. Ar, N2 and CO2 mixtures 3. CO 4. anesthetics 5. Nonpenetrating captive bolt 4

5 6. Electrocution if > 10 lbs. 7. Anesthetic overdose 8. Blunt force trauma 9. Barbiturates 5

6 Acceptable Acceptable with Conditions Poultry Equids Avians Finfish in Research Facility Aquatic Invertebrates (2 step recommended) 1. Barbiturates IV 1. Overdose injectable anesthetic, including barbiturates 1. CO2 (most common) 2. CO concentration at least 6% 3. N2 or Ar mixed or used alone with approx. 30% CO2 residual O2 kept at minimum (2-3%) Physical 1. Cervical dislocation must result in luxation of cervical vertebrae without crushing 2. Decapitation commercial guillotine for species if available or sharp knife 3. Manual Blunt Force Trauma turkeys and broiler breeders too large for cervical dislocation; 1. Penetrating Captive Bolt 2. Gunshot 1. Barbiturates IV (awake), IC, IO (avoid humerus & femur), intracoelomic (avoid air sacs) if or anesthetized 1. anesthetics alone or before other methods 2. CO2 need high concentrations (> 40%) 3. CO 4. N2 or Ar only acceptable for poultry, not companion birds Physical 1. Cervical dislocation has been performed on birds as large as 2.3 kg 2. Decapitation birds <200g 3. Gunshot not recommended for captive birds you can restrain min. immersion in: buffered MS222 (tricaine methanesulfonate), buffered benzocaine, quinaldine sulfate, and 2- phenoxyethanol 2. Rapid chilling for zebrafish (2-4C water) & Aust. River gizzard shad (N erebri) adult min 10 min and fry 4-7dpf at least 20min following loss of opercular movements 1. Immersion in: CO2 saturated water (observers aware may exhibit hyperactivity), eugenol, isoeugenol, clove oil 2. Rapid chilling for small bodied tropical and stenothermic finfish for which lower lethal temp. is > 4C 3. Maceration when death is instantaneous; equipment designed for finfish 4. Decapitation followed by pithing 5. Manual blunt force trauma to head followed by pithing 1. 1 st step of 2 step method, immersion agents: magnesium salts (ineffective for crustaceans), clove oil/eugenol or ethanol 2. 2 nd step of 2 step method: immersion in 70% alcohol and neutral buffered 10% formalin or physical method pithing, freezing, boiling 6

7 Adjunctive Methods alternatives encouraged 4. Electrocution must ensure death or apply adjunctive method afterwards 5. Gunshot for free-ranging poultry and ratites 6. Penetrating and Nonpenetrating Captive Bolt large poultry (turkeys, broiler breeders, ratites, waterfowl, etc); must ensure death or use 2 nd shot/adjunctive method 1. Potassium Chloride or Magnesium Sulfate IV or IC if 2. Exsanguination 1. Potassium Chloride IV or IC *Emergency situation euthanasia neuromuscular blocking agent followed by immediate euthanasia once animal controlled 1. Potassium Chloride IV or IC - if 2. Exsanguination if or anesthetized 3. Thoracic compression insentient only Unacceptable 1. Chloral Hydrate 1. Thoracic compression in unanesthetized Fetuses/Neonates Embryonated Eggs 1. Prolonged CO2 exposure (20min) 2. Cooling (4hrs at 40F) 3. Freezing 4. Egg addling 5. anesthetics via air cell in some cases 6. Decapitation if egg has been opened 7. Maceration Chicks/Poults Embryos >50% incubation 1. Anesthetic overdose, 2. Decapitation 3. Prolonged (>20min) CO2 Eggs <50% incubation 1. Prolonged (>20 min) CO2 2. Cooling (<4C for 4hrs) 3. Freezing 4. Addling Rapid chilling followed by dilute sodium or calcium hypochlorite immersion for zebrafish embryos and larvae (2 step method); acceptable w/conditions as a 2 step method for non-zebrafish embryos/larvae Removing from water resulting in hypoxia or desiccation, water without adequate aeration, caustic chemicals, traumatic injury without being 7

8 1. Maceration up to 72hrs of age Acceptable Acceptable with Conditions Captive Invertebrates 1. Pentobarbital ideally into hemolymph but can do intracoelomic 1. anesthetics when injectables are not available 2. CO2 Physical/Chemical 1. Boiling, pithing, chemical Captive Amphibians/Reptiles 1. Sodium pentobarbital IV, SQ lymph space, lymph sacs, intracoelomic, direct injection into the parietal eye for some lizzards 2. Dissociatives or combinations, propofol IV recommend adjunctive method 3. External or Topical: MS222 water bath or injected into lymph sacs (amphibians) or coelomic cavity (amphibians & reptiles) 4. Benzocaine HCl bath or 7.5%/20% gel applied to ventrum of amphibians 1. anesthetics many can hold their breath and survive anoxia 2. CO2 if alternate methods not practical; verify death or apply secondary method Captive Nonmarine Mammals 1. Pentobarbital- IV or IP, IC in 2. Nonbarbiturate anesthetic overdose IV or IM when animal size, restraint requirements or other circumstances indicate this is the best option 1. anesthetics when more practical 2. CO, CO2, inert gas 1. Penetrating captive Captive Marine Mammals 1. Barbiturates IV (preferred) or IP 2. Barbiturate given IC if anesthetized or Can give IM sedatives or anesthetics beforehand to immobilize. 1. anesthetics may be appropriate for small pinnipeds after administration of a sedative or anesthetic Free Ranging Wildlife 1. Injectable anesthetic overdose (incl. barbiturates) 2. T61 3. Other agents listed as acceptable for domestic animals/captive wildlife 1. anesthetics 2. CO2 3. CO 4. Other inert gases Physical 1. Gunshot targeted to brain Free Ranging Marine Mammals 1. Overdose injectable anesthetics multiple routes (e.g. IV, IP, blowhole) Physical 1. Gunshot for small marine mammals 2. Manual blunt force trauma to head for small juvenile marine mammals 3. Implosive 8

9 Adjunctive Methods Unacceptable methods (e.g. alcohol, formalin) adjunctively after anesthesia, nonresponsiveness, or death 2. Pithing 1. Penetrating captive bolt/firearm crocs and large reptiles 2. Manual blunt force trauma well trained, no other options, adjunctive method used 3. Rapid freezing - <4g in liquid nitrogen 1. Decapitation after anesthetized, part of 3 step method (injectable anesthetic, decapitation, pithing) 2. Pithing used as a 2 nd step method in animals; foramen magnum in frogs Hypothermia unless animals are sufficiently small (<4g) to permit immediate and irreversible death if placed in liquid nitrogen bolt/firearm knowledge of species target sites and safety, trained personnel 1. Potassium chloride IV or IC after anesthetized or 2. Exsanguination 2ndary or tertiary method 3. Cervical dislocation or decapitation birds <3kg, rodents <200g, rabbits <1kg 4. Thoracic compression while deeply anesthetized, or when animal s status is uncertain Same as for other domestic species unless deeply anesthetized. 1. Potassium chloride IV or IC after anesthetized or 2. Exsanguination 3. Cervical dislocation or decapitation birds <3kg, rodents <200G, rabbits <1kg 4. Thoracic compression decerebration large mysticietes and odontocetes 1. Potassium chloride or succinylcholine IV or IC 1. agents not practical under field conditions 2. Exsanguination as a sole 9

10 Fetuses/Neonates Freezing likely appropriate for newly oviposited eggs as would be methods of maceration Similar to those described in other sections. Same as for domestic species. method, only as an adjunct to ensure death of an animal 10