Design and Husbandry Considerations for a Containment Level 2 Aquatic Facility.

The greatly increased use of aquatic species to study disease over the past 20 years necessitates understanding their husbandry and housing requirements to optimize research and welfare and to ensure compliance with regulations. To achieve these goals, aquatic systems have expanded from pet shop and home aquaria to research-grade systems incorporating designs and features to increase their robustness, practicality, and flexibility. Moreover, these last decades have seen the increasing use of aquatic animals for infectious disease research using containment level 2 (CL2)/biosafety level 2 pathogens. In this study, we discuss the facility design requirements and modifications, which must be considered for the planning, construction, and use of an aquatic facility for zebrafish infected with CL2 pathogens. These include decontamination of water and equipment, racking and filtration design, personal protective equipment, and husbandry procedures. This guidance is based on our experience in the design and ongoing management of such facilities.

Delay of Initial Feeding of Zebrafish Larvae Until 8 Days Postfertilization Has No Impact on Survival or Growth Through the Juvenile Stage.

The use of early-stage zebrafish for biomedical research spans early organogenesis to free-swimming larva. A key benefit of this model organism is that repeated assessments spanning several days can be performed of individual larvae within a single experiment, often in conjunction with administered drugs. However, the initiation of feeding, typically at 5 days postfertilization (dpf), can make serial assessments challenging. Therefore, delayed feeding would increase the utility of the model. To ask whether feeding could be delayed without adversely affecting larval growth and development up to 39 dpf, we systematically raised zebrafish and introduced feeding at 5 dpf or delayed initial feeding up to 9 dpf. We assessed survival into the juvenile stage (39 dpf) and anterior-posterior length at this age as proxies for growth and development. Delaying feeding initiation up to 8 dpf did not decrease baseline survival of greater than 90%; survival decreased to 66% only when delayed to 9 dpf. Larval length was no different under any of these conditions. Our findings define 9 dpf as the critical age before which larval zebrafish must be fed when raising to 39 dpf. The option to delay feeding to 8 dpf will broaden experimental applications for the zebrafish larval model.

Standardized Welfare Terms for the Zebrafish Community.

Managing the welfare of laboratory animals is critical to animal health, vital in the understanding of phenotypes created by treatment or genetic alteration and ensures compliance of regulations. Part of an animal welfare assessment is the requirement to record observations, ensuring all those responsible for the animals are aware of their health status and can act accordingly. Although the use of zebrafish in research continues to increase, guidelines for conducting welfare assessments and the reporting of observations are considered unclear compared to mammalian species. To support the movement of zebrafish between facilities, significant improvement would be achieved through the use of standardized terms to ensure clarity and consistency between facilities. Improving the clarity of terminology around welfare not only addresses our ethical obligation but also supports the research goals and provides a searchable description of the phenotypes. A Collaboration between the Wellcome Trust Sanger Institute and Cambridge University (Department of Medicine-Laboratory of Molecular Biology) has led to the creation of the zebrafish welfare terms from which standardization of terminology can be achieved.

Evaluating and Optimizing Fish Health and Welfare During Experimental Procedures.

Many facilities house fish in separate static containers post-procedure, for example, while awaiting genotyping results. This ensures fish can be easily identified, but it does not allow for provision of continuous filtered water or diet. At the Wellcome Trust Sanger Institute, concern over the housing conditions led to the development of an individual housing system (GeneS) enabling feeding and water filtration. Trials to compare the water quality measures between the various systems found that fish housed in static containers experienced rapid deterioration in water quality. By day 1, measures of ammonia were outside the Institute's prescribed values and continued to rise until it was 25-fold higher than recommended levels. Nitrite levels were also outside recommended levels for all fish by day 9 and were twofold higher by the end of the trial. The water quality measures for tanks held on the recirculating system were stable even though food was provided. These results indicate that for housing zebrafish, running water or appropriately timed water changes are a critical component to ensure that the ethical obligations are met.

Intensive, time-series measurement of upper limb recovery in the subacute phase following stroke.

OBJECTIVES: To discover if intensive monitoring of wrist extension would produce consistent recovery curves during the subacute period, and whether any impact of additional physiotherapy could be detected. We also investigated improved approaches to statistical analysis in single-case experiments. DESIGN: A randomized multiple-baseline experiment with very frequent assessment. SETTING: Stroke rehabilitation unit. SUBJECTS: Four patients with some active wrist movement less than seven weeks after stroke. INTERVENTIONS: Wrist extension was measured twice daily with an electrogoniometer for 3-4 weeks. Additional upper limb physiotherapy 115 minutes, twice per day) commenced after a randomly determined period. MAIN OUTCOME MEASURES: Speed and range of wrist movement. RESULTS: A logarithmic function was fitted to the data to produce recovery curves. In all cases, active range and maximum velocity of wrist extension rose gradually over time. Mean variability in range was <5%, but with occasional outliers. Range of passive movement decreased in two cases in association with pain and increased tone. There were no large improvements coinciding with additional physiotherapy but autoregression analysis indicated statistically significant changes in three cases. A randomization test confirmed an increase in active range associated with additional physiotherapy. CONCLUSIONS: Intensive electrogoniometry provided a detailed recovery pattern for each of these patients. The data were surprisingly consistent over time, showing that it is feasible to use a time-series approach to investigate subacute recovery. Changes associated with additional physiotherapy were observed on some measures, demonstrating the potential of this approach for exploratory evaluation of interventions.