Clarification of early end-points for refinement of animal experiments, with specific reference to fish

Abstract

Appropriate end-points are integral to the refinement of laboratory animal experiments. Our recent experience has highlighted that ambiguity around end-points is hampering their adoption in experiments that cause severe suffering to fish. In toxicology, the term endpoint (single word) refers to the response variable to the treatment that is measured and analysed. This differs to usage within laboratory animal experimentation, where end-point (hyphenated) refers to the time-point when exposure of the animal(s) to the treatment (and suffering) ends. Within laboratory animal experimentation, standardised terminology is needed for different types of early end-point which are based on the condition of the animal(s) or progress of the experiment. We propose that those involved in regulating and conducting animal experiments consider seven distinct types of early end-point (aim, technical error, biological error, mortality, moribundity, prognostic humane, non-prognostic humane) in addition to the planned experimental end-point (i.e. maximum duration). Moribundity (not morbidity) refers to an animal in a severely debilitated state close to death. Moribundity in fish is not yet defined, so we propose identification via a lack of response to external stimuli, loss of equilibrium (i.e. loss of righting reflex), and a slow opercular ventilation rate. As these clinical signs equate to those of deep/surgical anaesthesia, this moribundity end-point cannot be considered a humane end-point as the fish is likely to be unconscious and have passed the point of maximum suffering. We believe that identification of earlier humane end-points based on clinical signs and wider recognition of other types of early end-point can reduce suffering in experiments.

Antarctic teleosts with and without hemoglobin behaviorally mitigate deleterious effects of acute environmental warming

“…The latter was defined as a thermal point at which locomotion of the animal becomes disorganized, leading to the loss of ability to escape from harmful conditions and prompt death. The choice of variables that identify physiologically and ecologically relevant end-points during the state of moribundity (a severely debilitated state close to death of the animal) including that in the CT max method has been a subject of debate [ 26 – 29 ]. Of those, the onset of Loss of Equilibrium (LOE) represents a reasonably humane end-point, because it does not constitute irreversible injury or death of fishes [ 30 ].…”

Pain management in zebrafish

“…Identification of earlier humane end-points before mortality have been proposed. 9 For zebrafish used in toxicology studies, this is aided by methodical measurements of behavioural and physiological welfare parameters. Use of analgesia may be advantageous if chemicals are acidic (pH <6).…”

“…Since zebrafish are adopted in a wide variety of experimental contexts they are subject to a wide variety of procedures which potentially cause tissue damage (summarised in Table 1 8 , 9 ). Invasive procedures typically involve the use of anaesthesia to render the fish unconscious to allow the procedure to be conducted safely, efficiently and to safeguard the welfare of the fish.…”