Identification of Individual Zebrafish (Danio rerio): A Refined Protocol for VIE Tagging Whilst Considering Animal Welfare and the Principles of the 3Rs

Abstract

In aquatic ecology, studies have commonly employed a tagging technique known as visible implant elastomer (VIE). This method has not been widely adopted by the zebrafish research community and also lacks refinement with regard to animal welfare. The current paper introduces a new VIE tagging protocol, with the aim of improving existing tagging techniques by placing particular emphasis on the Three Rs. To improve animal welfare and fish survival, we added the use of an analgesic compound (lidocaine) through the marking procedure, followed by after-treatment with antiseptics (melaleuca, aloe vera, and PVP-I as active ingredients) to improve tissue regeneration and healing. The newly improved protocol has been quantitatively evaluated on different populations and age groups of zebrafish. This study will be useful to the scientific zebrafish community and to the wider field including biologist and aquarists, especially in consideration of animal welfare, where tagging techniques are considered as a potential noxious stimulus for fish.

Migration and Histologic Effects of Visible Implant Elastomer (VIE) and Passive Integrated Transponder (PIT) Tags in the Marine Toad (Rhinella marina)

“…Operator error, product malfunction, or low ambient temperatures may have contributed to inadequate curing, allowing for migration of VIE. However, a study in larval Pacific lamprey ( Lampetra tridentata ) found that uncured VIE had equally long retention rates as cured VIE and a recent study in zebrafish ( Danio rerio ) found that uncured tags had a higher retention rate, suggesting that curing may not be required for tag retention [ 15 , 20 ].…”

“…Within the literature across taxa, tag retention appears to be variably affected by species, individual size, injection location, volume injected, experience of the injector, and observer variability [ 3 , 6 , 7 , 9 , 10 , 11 , 12 ]. Tag migration, breakage, granuloma formation, and complete loss have all been reported [ 2 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 ]. Microscopic evaluation of VIE tagging sites has only been reported in shrimp ( Penaeus vannamei ) and stertlet ( Acipense ruthenus ) in which mild, chronic inflammation and fibrosis were found at the injection sites [ 14 , 16 ].…”

Zebrafish (Danio rerio) meets bioethics: the 10Rs ethical principles in research

“…Reduction is an important principle to be taken into consideration when animals' replacement during scientific research is not possible (33) . Reduction means decreasing the number of animals used in protocols and experimental designs (33) ; therefore, the minimum number of zebrafish must be used to achieve scientific or educational aims when no alternative methods such as mathematical models, computer simulation, biological systems in vitro, or other non-animal (adjuvant) approaches are available to replace the use of living animals (52) . Researchers must initially choose an adequate model system for the analyses carried out in their experiments in order to avoid the unnecessary use of animals.…”

Simulated trapping and trawling exert similar selection on fish morphology

“…Fish ( n = 510) were housed in four identical aquaria, and each group (trap or trawl) was held simultaneously under the same husbandry conditions described above. A month before the start of the experiment, fish were tagged using visual implant elastomer (VIE) (Northwest Marine Technology, WA, USA) in four dorsal tag locations with an individual code identifier (Rácz et al, 2021 ). Wet mass and standard length were measured for each individual (Table S1 ), and each fish was also sexed based on a combination of color and morphology.…”