A FELASA Working Group Survey on Fish Species Used for Research, Methods of Euthanasia, Health Monitoring, and Biosecurity in Europe, North America, and Oceania.

An international survey was conducted regarding species used for research, methods of euthanasia, health monitoring, and biosecurity in fish laboratories. A total of 145 facilities from 23 countries contributed. Collectively, over 80 different species (or groups of species) were reported to be used for research, of which zebrafish (Danio rerio) was the most common by far. About half of the participating laboratories used multiple species. Anesthetic overdose was the preferred method for euthanasia for adult, fry (capable of independent feeding), and larval (not capable of independent feeding) fish. For all developmental stages, the most popular anesthetic compound was tricaine (MS-222), a substance associated with distress and aversion in several species. Moreover, around half of the respondents did not perform a completion method to ensure death. One-quarter of the responding facilities did not have a health monitoring system in place. While most respondents had some form of quarantine process for imported fish, only a small fraction reported quarantine routines that ensure reliable biological barriers. Furthermore, less than one in five screened fish for pathogens while in quarantine. In sum, there was little consensus amongst facilities in how to perform biosecurity measures. Regarding euthanasia, health monitoring, and biosecurity processes, there is a need for updated and universal guidelines and for many laboratories to adjust their practices.

FELASA-AALAS Recommendations for Biosecurity in an Aquatic Facility, Including Prevention of Zoonosis, Introduction of New Fish Colonies, and Quarantine.

FELASA and AALAS established a joint working group to advise on good practices for the exchange of fish for research. In a first manuscript, the working group made recommendations for health monitoring and reporting of monitoring results. The focus of this second related manuscript is biosecurity in fish facilities. First, we define the risk of contamination of personnel by zoonotic pathogens from fish or from system water, including human mycobacteriosis. Preventive measures are recommended, such as wearing task-specific personal protective equipment. Then we discuss biosecurity, highlighting the establishment of biosecurity barriers to preserve the health status of a facility. A functional biosecurity program relies on integration of the entire animal facility organization, including the flow of staff and animals, water treatments, and equipment sanitation. Finally, we propose 4 steps for introducing new fish colonies: consideration of international trade and national restrictions; assessing risk according to fish source and developmental stage; establishing quarantine barriers; and the triage, screening, and treatment of newly imported fish. We then provide 3 realistic sample scenarios to illustrate practical biosecurity risk assessments and mitigation measures based on considerations of health status and quarantine conditions.

FELASA-AALAS Recommendations for Monitoring and Reporting of Laboratory Fish Diseases and Health Status, with an Emphasis on Zebrafish (Danio Rerio).

The exchange of fish for research may expose an aquatic laboratory to pathogen contamination as incoming fish can introduce bacteria, fungi, parasites, and viruses capable of affecting both experimental results and fish and personnel health and welfare. To develop risk mitigation strategies, FELASA and AALAS established a joint working group to recommend good practices for health monitoring of laboratory fish. The recommendations address all fish species used for research, with a particular focus on zebrafish (Danio rerio). First, the background of the working group and key definitions are provided. Next, fish diseases of high impact are described. Third, recommendations are made for health monitoring of laboratory fishes. The recommendations emphasize the importance of daily observation of the fish and strategies to determine fish colony health status. Finally, report templates are proposed for historical screening data and aquatic facility description to facilitate biohazard risk assessment when exchanging fish.

A Multi-Site Assessment of Anesthetic Overdose, Hypothermic Shock, and Electrical Stunning as Methods of Euthanasia for Zebrafish (Danio rerio) Embryos and Larvae.

Euthanasia in zebrafish (Danio rerio) younger than 5 days post fertilization (dpf) is poorly described in the literature, and standardized protocols are lacking, most likely because larvae not capable of independent feeding are often not protected under national legislations. We assessed the euthanasia efficacy in laboratories in different countries of a one hour anesthetic overdose immersion with buffered lidocaine hydrochloride (1 g/L, with or without 50 mL/L of ethanol), buffered tricaine (1 g/L), clove oil (0.1%), benzocaine (1 g/L), or 2-phenoxyethanol (3 mL/L), as well as the efficacy of hypothermic shock (one hour immersion) and electrical stunning (for one minute), on zebrafish at <12 h post fertilization (hpf), 24 hpf, and 4 dpf. Based on the survival/recovery rates 24 h after treatment, the most effective methods were clove oil, lidocaine with ethanol, and electrical stunning. For 4 dpf larvae, signs of aversion during treatment demonstrated that all anesthetics, except lidocaine, induced aversive behavior. Therefore, the most suited euthanasic treatment was lidocaine hydrochloride 1 g/L, buffered with 2 g/L of sodium bicarbonate and mixed with 50 mL/L of ethanol, which euthanized both embryos and larvae in an efficient and stress-free manner. Electrical stunning also euthanized embryos and larvae efficiently and without signs of aversion; this method needs further assessment in other laboratories to draw firm conclusions.

Screening of Anaesthetics in Adult Zebrafish (Danio rerio) for the Induction of Euthanasia by Overdose.

Zebrafish are often euthanized by overdose of anaesthesia. However, fish may have aversion towards some anaesthetics, and protocol efficacy varies between species. Using wild type adult Danio rerio, we assessed time to loss of opercular beat, righting, and startle reflexes during induction of anaesthetic overdose by either tricaine (0.5 g/L or 1 g/L), benzocaine (1 g/L), 2-phenoxyethanol (3 mL/L), clove oil (0.1%), isoeugenol (540 mg/L), lidocaine hydrochloride (1 g/L), or etomidate (50 mg/L). Initial screening demonstrated that benzocaine and buffered lidocaine hydrochloride achieved the fastest loss of reflexes. The rapid induction times were confirmed when retesting using larger batches of fish. The fastest induction was obtained with 1 g/L lidocaine hydrochloride buffered with 2 g/L NaHCO3, in which all adult zebrafish lost reflexes in less than 2 min. Next, we monitored signs of distress during benzocaine or buffered lidocaine hydrochloride overdose induction. The results indicated that buffered lidocaine hydrochloride caused significantly less aversive behaviors than benzocaine. Finally, we tested several buffers to refine the lidocaine hydrochloride immersion. The most efficient buffer for euthanasia induction using 1g/L lidocaine hydrochloride was 2 g/L NaHCO3 with 50 mL/L 96% ethanol, inducing immobility in less than 10 s and with only 2% of adult zebrafish displaying aversive behaviors during treatment.

Assessment of Microbial Reduction by Cage Washing and Thermal Disinfection using Quantitative Biologic Indicators for Spores, Viruses and Vegetative Bacteria.

Cage washing is a key process of the biosecurity program in rodent facilities. For the current study, we developed systems (i. e., magnet attachments, quantitative biologic indicators (Q-BI), and measurement of thermal disinfection at equipment level) to assess the microbial decontamination achieved by a rodent equipment washer with and without thermal disinfection. 99% of the magnets remained in position to hold Q-BI and temperature probes inside cages, water bottles or at equipment level across a cabinet washer chamber with loads dedicated to either housing or drinking devices. Various types of Q-BI for Bacillus atrophaeus, Enterococcus hirae and minute virus of mice were tested. To simulate potential interference from biologic material and animal waste during cage processing, Q-BI contained test soil: bovine serum albumin with or without feces. As a quantitative indicator of microbial decontamination, the reduction factor was calculated by comparing microbial load of processed Q-BI with unprocessed controls. We detected variation between Q-BI types and assessed the washer's ability to reduce microbial load on equipment. Reduction factor results were consistent with the Q-BI type and showed that the washing and thermal disinfection cycle could reduce loads of vegetative bacteria, virus and spore by 5 log10 CFU/TCID50 and beyond. Thermal disinfection was monitored with temperature probes linked to data loggers recording live. We measured the period of exposure to temperatures above 82.2 °C, to calculate A0, the theoretical indicator for microbial lethality by thermal disinfection, and to assess whether the cabinet washer could pass the minimum quality standard of A0 = 600. Temperature curves showed an A0 > 1000 consistently across all processed equipment during thermal disinfection. These data suggest that, when sterilization is not required, a cabinet washer with thermal disinfection could replace an autoclave and reduce environmental and financial waste.

Anaesthesia of laboratory, aquaculture and ornamental fish: Proceedings of the first LASA-FVS Symposium.

Following on from the Annual Fish Veterinary Society Conference, this symposium was organised with the Laboratory Animal Science Association and brought together experts from ornamental (pond and aquarium) fish practice, aquaculture and aquatic-research facilities to discuss good practice of anaesthesia. This proceedings paper gives an overview of relevant experiences involving a range of immersion drugs including tricaine, benzocaine and isoeugenol, as well as a summary of the main topics of discussion. While fish anaesthesia is commonplace, administration methods, drugs and monitoring procedures may often be regarded as antiquated when compared with mammalian practice. These limitations notwithstanding, individual fish will benefit from good anaesthetic monitoring. Although the most common anaesthetic drugs may be perceived as equally efficacious and therefore interchangeable for different settings, challenges are different for the anaesthesia of grouped fish, when determining species-dependent anaesthetic dosing in a multi-species tank, or adapting to farming requirements, nationally licensed products, costs and withdrawal periods. The fish anaesthetic arsenal fails to address premedication, analgesia and issues of averseness. The two latter factors should be part of the evaluation of anaesthetic protocols; therefore, instructions for the analgesic provision of lidocaine to fin clipped zebrafish are proposed. Euthanasia practices could sometimes be refined too. Alternative physical methods such as electrical stunning are options to be considered.

Refining the unilateral ureteral obstruction mouse model: No sham, no shame.

Fibrosis, as a common final pathway in many renal diseases, contributes significantly to the decline of organ function and to progression to end-stage renal disease. To establish therapeutic interventions that target fibrosis, animal models are essential. The most widely used model of renal fibrosis is the unilateral ureteral obstruction (UUO) model. Typically, the control for this model is a sham-operated animal. Sham surgery causes pain and distress to these control animals, and here we aim to show that there is no difference in the main read-outs of this model between sham-operated animals and non-operated C57BL/6J mice. In five experiments, quantification of Picro Sirius Red stained collagen in the renal cortex did not show any difference between 15 sham and 25 non-operated individuals. A comparison of the regulation of genes involved with fibrosis did not show any difference between sham and non-operated groups at 21 days post surgery either. We conclude that there are no significant differences between non-operated controls and sham animals with respect to collagen deposition and fibrosis pathways in the UUO mouse model.