The zebrafish were bred and housed at the University of Queensland (UQ) Biological Resources zebrafish facility. This study was approved by the UQ Molecular Biosciences Animal Ethics Committee (2018/AE000384). The study was conducted in compliance with Animal Research: Reporting of In Vivo Experiments (ARRIVE) 2.0 guidelines.
Zebrafish were allocated to two groups of 10 adult Tüebingen (TU) (five female, five male) and placed in individual, single-use, static immersion baths containing 10 mg L–1
of either preservative free alfaxalone (group 1) or alfaxalone with preservative (group 2). Water was collected from the main recirculating system and the drug added immediately prior to immersion. Water quality was measured prior to the addition of any drugs. Treatment order was assigned using an online randomization tool (randomization.com
, accessed 18 December 2018) with each fish assigned a number between 1 and 20 and subsequently anaesthetized in numerical order. One of two veterinarians, both blinded to treatment, used a composite score to assess each fish.
The composite scoring scale (CSS) used in this study (Table 1
) was a modified version of the scale described by
Bauquier et al., 2013
- Bauquier S.H.
- Greenwood J.
- Whittem T.
Evaluation of the sedative and anaesthetic effects of five different concentrations of alfaxalone in goldfish, Carassius auratus.
used to evaluate the effects of alfaxalone in goldfish. Equilibrium, opercular movement, activity and tactile response baseline scores were recorded for each fish prior to anaesthesia immersion and then every 30 seconds until induction of anaesthesia criteria was met. Tactile response was assessed by gently swiping along the lateral surface of fish with a cotton tip as soft stimulus. Hard stimulus was assessed by application of a 2 g force with a von Frey filament (San Diego Instruments Inc., CA, USA) to the tail fin.
Table 1Composite scoring system used to assess immersion anaesthesia in 20 adult zebrafish, Danio rerio (Tuebingen strain) using a preservative-free formulation of alfaxalone (10 mg mL–1) and a formulation of alfaxalone (10 mg mL–1) with a preservative containing chlorocresol 1 mg mL–1, benzethonium 0.2 mg mL–1 and ethanol 150 mg mL–1
Fish were then removed from the anaesthetic bath, weighed and immediately placed in a clean, static, water recovery bath and scored every 60 seconds until criteria for recovery from anaesthesia were met. The time between removing a fish from the anaesthetic bath and placing it into a recovery bath was < 20 seconds.
Fish were observed for adverse effects such as excitatory behaviour, increased ventilation, thrashing and jumping during the induction period.
The following anaesthetic time variables (measured in minutes) were recorded and defined as follows:
Anaesthetic induction time (AIT) = time from when fish were first placed in anaesthetic bath (time zero) until loss of equilibrium (equilibrium = 3) and loss of response to soft stimulus (tactile score ≥ 2)
Total procedure time (TPT) = time from when fish were first placed in anaesthetic bath (time zero) until fish met anaesthesia recovery criteria (CSS of ≤ 1) when observed in recovery bath.
Anaesthetic recovery time (ART) = TPT – AIT.
Fish were assessed for evidence of gross external pathology from the effects of each drug solution during the procedure. Once recovered, the first two and last two male and female fish from each group were euthanized by rapid cooling. This occurred by submersion in an ice water slurry (0–4 °C) until cessation of opercular movements and visible cardiac movements. Fish were then immediately fixed in 10% neutral buffered formalin (Point of Care Diagnostics, NSW, Australia). Following fixation, the ventral gill arch was trimmed by cross-section at the caudal aspect of the head, and a parasagittal section medial to the operculum was made to view the lateral gill arch. A board-certified pathologist blinded to the allocated group inspected tissues for any morphologic tissue or cellular changes with a particular focus on gill pathology. The gill tissue was assessed for the presence of diapedesis of erythrocytes, overt haemorrhage, the presence of surface debris on the gills, and the presence of lamellar fusion and immune cell infiltrates. The severity of lesions was scored on a 5-point semi-quantitative scale: 0, normal; 1, minimal change; 2, mild change; 3, moderate change; 4, marked changes; and 5, severe change.
Data were analysed using descriptive statistics and summarized using tables and graphs. Initially, the distributions of the anaesthetic time variables were assessed using box plots for presence of outliers that may have affected the normality of the data. Shapiro Wilk test was performed to test the normality of the data of the two groups. Results were reported as mean ± standard deviation. Independent t
tests were used to compare the difference in the mean anaesthetic time variables between the two groups. Data management and analysis were performed using the statistical package R Version 3.3.3 (
; R Foundation for Statistical Computing, Austria). Values of p
< 0.05 were considered significant.