2) PHYSICAL METHODS:
a) Decapitation followed by pithing (2 step). Rapid severance of the head and brain from the spinal cord, followed by pithing of the brain, will cause rapid death and unconsciousness. Decapitation alone is not considered a humane approach to euthanasia, especially for species that may be particularly tolerant of low O2 concentrations. Pithing helps ensure rapid loss of brain function and death for those species.
b) Cervical transection using a knife or other sharp instrument inserted caudal to the skull to sever the spinal cord and cervical vertebrae, followed by pithing (2 step). The rationale for this approach is similar to that for decapitation (destruction of connections between brain and spinal cord) and pithing (destruction of brain tissue), except that the head is still physically
attached by musculature to the body
c) Manually applied blunt force trauma (cranial concussion) followed by pithing (2 step). Manually applied blunt force trauma (a rapid, accurately placed blow of sufficient energy to the cranium with an appropriate-sized club) can cause immediate unconsciousness and potentially death, but should be followed by pithing to ensure death. The finfish’s size, species, and anatomy and characteristics of the blow (including its accuracy, speed, and club mass) will determine the efficacy of manually applied blunt force trauma. This procedure requires training and monitoring for proficiency. Anatomic features, such as the location of the eyes, can help serve as a guide to the location of the brain
d) Captive bolt (most commonly nonpenetrating; 1 step). This is a method usually applied to large finfish species.
e) Maceration (1 step). When applied correctly, using a well-maintained macerator specifically designed for the size of finfish being euthanized, death is nearly instantaneous. The process is aesthetically unpleasant for some operators and observers
f) Rapid chilling (hypothermic shock; 1 step or 2 step). It is acceptable for zebrafish (D rerio) to be euthanized by rapid chilling (2° to 4°C) until loss of orientation and operculum movements316,461,462 and subsequent holding times in ice-chilled water, specific to finfish size and age. Zebrafish adults (approx 3.8 cm long) can be rapidly killed (10 to 20 seconds) by immersion in 2° to 4°C (36° to 39°F) water. Adult zebrafish should be exposed for a minimum of 10 minutes and fry 4° to 7 °F for at least 20 minutes following loss of operculum movement...
...Until further research is conducted, rapid chilling is acceptable with conditions for other small-bodied, similarly sized tropical and subtropical stenothermic species. Species-specific thermal tolerance and body size will determine the appropriateness and effectiveness of rapid chilling for euthanasia of finfish. Finfish size is important because the rate of heat loss via thermal conduction from a body is proportional to its surface area. Based on these 2 factors, it has been suggested that rapid chilling in water associated with an ice slurry is a suitable killing method for small tropical and subtropical finfish species 3.8 cm in length (tip of the snout to the posterior end of the last vertebra) or smaller, having lower lethal temperatures above 4°C. To ensure optimal hypothermal shock (ie, rapid killing), transfer of finfish into ice water must be completed as quickly as possible. This means rapid transitions from acclimatization temperature to 2° to 4°C must be achieved. This can be accomplished by using minimal water volume to transfer finfish (ie, using a net to place finfish in chilled water). In addition, finfish should not be in direct contact with the ice in the water; rather a depression should be formed in the ice slurry to expose the entire surface of the finfish to the chilled water. Full contact with cold water ensures optimal exposure and rapid chilling of the finfish temperature must not exceed 2° to 4°C. Well-insulated containers, such as coolers, will assist in maintaining the ice slurry and a probe thermometer can be used to confirm water temperature. This method of euthanasia is not appropriate for temperate, cool, or cold-water–tolerant finfish, such as carp, koi, goldfish, or other species that can survive at 4°C and below.