Guide to Rigor and Reproducibility for the Zebrafish Aquaculture Core
- Consult with the core staff in the planning stage.
- In order to maximize robustness and reduce bias, the following standards apply:
- A minimum of 5 biological replicates should be performed for all quantitative experiments.
- Take sex into consideration when designing experiments with adult fish.
- Be sure consistent results are observed before publishing data.
- The methods and results sections should be completely transparent to allow for reproducible data.
- Student’s T-test and one-way ANOVA should be used (when appropriate) to assess significance with p<0.05.
- The NIH requires reagent validation and other relevant materials in the Resource Authentication Plan: https://grants.nih.gov/reproducibility/faqs.htm#V
Antibody registry: http://antibodyregistry.org/
CRISPR Plasmids: http://www.addgene.org/crispr/
TALEN Technologies: http://www.addgene.org/talen/guide/
Zinc Finger Consortium Reagents: http://www.addgene.org/zfc/
4. In addition to standard operating procedures (SOPs) found in the facility, these videos illustrate standardized procedures used in zebrafish.
Microinjection of zebrafish embryos: https://www.jove.com/video/1115/microinjection-of-zebrafish-embryos-to-analyze-gene-function
CRSPR-Cas9-generated gene KO: https://www.jove.com/video/56969/efficient-production-identification-crisprcas9-generated-gene
Retro-orbital injection: https://www.jove.com/video/1645/retro-orbital-injection-in-adult-zebrafish
- Everyone added to an Animal Use Protocol must first undergo online training for:
Animal Research: https://acap.research.unc.edu/iacuc_reg/
Then, each researcher undergoes complimentary hands-on training, which includes the use of anesthesia, tail fin clip procedure for genotyping, and euthanasia. Other training is available upon request, such as, in vitro fertilization, microinjection, retro-orbital injection, IP injection, and heart resection.
Once training is complete, researchers can be added to an existing protocol or new PIs may generate a protocol in ACAP.
6. All instruments are properly maintained and QC’d on a regular basis as follows:
Probes for temperature, pH, and conductivity are calibrated quarterly.
Physical and Chemical Parameters: (Testing modality, frequency of sampling) Temperature (Inline probes, continuous, value recorded daily). pH (inline probes, continuous, value recorded daily). Conductivity (inline probes, continuous, value recorded daily). Total Ammonia Nitrogen (TAN) (API freshwater test kit, once weekly). Nitrite (API freshwater test kit, once weekly). Nitrate (API freshwater test kit, once weekly). Hardness (Lamotte kit#4482, once monthly), Alkalinity (Lamotte kit#4491, once monthly). UV Dose (CLS, continuously). Dissolved Oxygen (NR). Carbon Dioxide (NR). Phosphate (NR). Light Intensity (Light Meter App, Trajkovski Labs, Nexus 6P, once annually).
Filters are changed at least monthly.
All logs are QC’d at least once per week.
Microscopes and injectors are maintained as needed (i.e. bulbs or gaskets replaced)
7. All husbandry data is recorded daily onto paper logs. Paper logs are periodically transcribed to Excel spreadsheets. Each Tecniplast system also has continuous data available for download. This data is used to populate the annual Environmental Summary for each system published on www.protocols.io.
8. Please acknowledge the Cancer Center Support Grant (P30 CA016086) or any other grant-supported equipment in publications.
The methods section of each publication involving the ZAC should include (please choose the appropriate protocols.io reference or contact the core for recent data):
“All experimental procedures performed in this study were reviewed and approved by the University of North Carolina Chapel Hill Animal Care and Use Committee. Animals were housed in an AAALAC-accredited facility in compliance with the Guide for the Care and Use of Laboratory Animals Guide as detailed on protocols.io (DOI:xxx).”
Published protocols include:
Acknowledgements should include:
“We would like to thank Michelle Altemara and all of the staff at the Zebrafish Aquaculture Core.”