Author(s): Date: Kara E. Powder, Principle Investigator. AARL Facility

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1 Page 1 of 6 Title: Daily Care of Zebrafish (Danio rerio) Facility: Aquatic Animal Research Laboratory (AARL) Room 3 Author(s): Date: Kara E. Powder, Principle Investigator AARL Facility Manager: Attending Veterinarian: John A. Smink Dr. John Parrish Date: Date: 1.0 OBJECTIVE This SOP describes daily care of zebrafish (Danio rerio). This SOP is based on standard aquarium trade procedures and care protocols from The Zebrafish Book: A Guide for the Laboratory Use of Zebrafish (Danio rerio), by Monte Westerfield. 2.0 HEALTH AND SAFETY All personnel will be enrolled in the Clemson University Medical Surveillance Program. Attire appropriate to the task must be worn at all times. 3.0 PERSONNEL/TRAINING/RESPONSIBILITIES All personnel must have completed the required Animal Care and Use Committee on-line training. Thereafter, any Clemson employee familiar with the techniques and trained in this and referenced SOPs may perform this procedure. Prior to being assigned full responsibility for performing this procedure, personnel must have demonstrated proficiency in the use of the technique in a closely supervised environment. Documentation of training should be kept for each person performing the procedure. 4.0 REQUIRED AND RECOMMENDED MATERIALS 4.1 Required Materials Aquaneering zebrafish system, GM300 adult food, larval food (Ziegler brand <100-micron), zebrafish tanks and baffles, tape for labeling, permanent markers, transfer pipette, API brand (or comparable) water test strips, API brand (or comparable) ammonium test kit, handheld monitor for conductivity, ph and temperature 5.0 GUIDELINES The following procedures were developed to meet or exceed the National Institute of Health (NIH) Guide for the Care and Use of Laboratory Animals. Westerfield, M, 2007, The Zebrafish Book, 5 th edition. Eugene: University of Oregon Press PROCEDURE 6.1 Zebrafish System

2 Page 2 of 6 Zebrafish are maintained on a purpose-built recirculating system purchased from a leading manufacturer, Aquaneering. This system includes all necessary mechanical and biological filtration, UV sterilization, RO water preparation, automated timed water exchange (10% per day), automated chemical dosing, and automated monitoring of ph, temperature, conductivity, and water level. Necessary components are on a backup generator in case of power failure. Per standard zebrafish protocols described in Westerfield 2007, all zebrafish will be maintained on a 14 hour light/10 hour dark cycle, managed by built in timer systems. 6.2 Animal acquisition and identification Zebrafish will be obtained from established commercial facilities like the Zebrafish International Resource Center (ZIRC), donated from other academic institutions, or produced via breeding within AARL Room #3. All additions to the system will be noted in the Zebrafish Inventory and Mortality Log. 6.3 General Housing Animals are kepts in tanks between 1.8 and 6L with a density of 10 adults per liter of system water. Different transgenic strains are housed in separate tanks on the same water system. All tanks should be labeled using tape and permanent marker with the strain name and birth date. 6.4 General Care of Fish The below points will be addressed daily, with information recorded on the Daily Care Log and Checklist. If any of the listed parameters are outside the acceptable range, the care taker should take immediate action to bring the value to an acceptable value (or tell the PI or facility manager if you don t know what to do) and record the action taken in the comments section of the Daily Care or Water Quality Logs. A monitoring system provides text and alerts to relevant lab members, the PI, and John Smink, Aquatics Facility manager, if there are significant deviations for water temperature, water level, ph, and conductivity. In the event of an alert, the alert and action taken will be recorded in the comments section of the Daily Care or Water Quality Logs. A. Room maintenance As needed, clean floors, put away equipment, and clean zebrafish tanks from accumulated food or algae through siphoning. As needed, tanks will be manually scrubbed and put on the rack to dry. Tanks will be disinfected periodically using a bleach solution (1/4 cup or 60mL sodium hypochlorite in 10 gallons of water). Tanks should be rinse VERY well after bleaching and allowed to dry completely before use. B. Water temperature Optimal temperature is 28.5 C. Zebrafish can be can be grown +/- 5 C (23-33 C) without any side effects as would be expected from animals that live in rivers in the wild that are subject to changes in the weather and temperature fluctuations. However, acceptable range for the system will be considered +/- 2.5 C, or C. Water temperature readings from the automated system will be recorded daily in the log. Room temperature should be between F (21-27 C). As needed, room temperature will be adjusted using a space heater. Room and water

3 Page 3 of 6 temperature will be periodically verified independent of the system using a thermometer or handheld device and recorded in Water Quality Log. C. Water conditions The system is automated to adjust ph and salt levels. Conductivity should be between microsiemens (µs) ( µS optimal) and ph should be Readings from the automated system will be recorded daily in the log. Other deviations (e.g. an increase in water cloudiness) should be recorded in the Water Quality Log and reported immediately to the PI. Water conditions will be verified independent of the system by the PI through occasional checking by test strips and kits (e.g. API 5 in 1 aquarium water test strip and API Ammonium test kit) to measure nitrate, nitrite, alkalinity, general hardness, and ammonium levels. Conductivity and ph will be verified independent of the system with a handheld device or test strips. Results of all independent verifications will be recorded in the Water Quality Log. All water quality checks and verifications independent of the automated system will be conducted monthly, or as needed. Acceptable water quality parameters are in below table and on Daily Care and Water Quality Logs. Values are based on industry standards, as reported in Westerfield s The Zebrafish Book 5 th edition (2007) and Lawrence s The Laboratory Zebrafish: A Volume in The Laboratory Animal Pocket Reference Series (2012). Parameter Optimal Acceptable Measured by* Range Water temperature 28.5 C C Daily readings from automated system, periodically verified independently by second thermometer or handheld device ph Daily readings from automated system with meter calibrated every 2weeks-1 month, periodically verified independently by handheld device or test strips Conductivity µS µs Daily readings from automated system with meter calibrated every 2weeks-1 month, periodically verified independently by handheld device or test strips Nitrate <200 ppm Test strips Nitrite <0.5 ppm Test strips Alkalinity (Carbonate ppm Test strips or titration hardness) General hardness ppm Test strips or titration Ammonium <0.5 ppm Test kit *All measurements not obtained daily from the automated system will be collected monthly, or

4 Page 4 of 6 as needed. D. Feeding Adult and larval zebrafish food will be stored in the dedicated fridge in AARL Room #17, labeled with PI name, purchase date, and date opened. Zebrafish must be fed once a day per the following ratios: 15 adults receive roughly 1/3 of a drop scoop of GM300 feed, using the provided measuring spoons. This is equivalent to roughly 5% of their body mass. Larval food is prepared by mixing 1/3 of a drop scoop of larval (<100micron) food per 50mL system water. 50 larvae receive 2 fills of a transfer pipette (~5mL) of this prepared solution. E. Monitoring of fish and water flow Each day, every tank containing animals should be observed. Check that water is actively flowing into the tank; use the valve to adjust as needed. Fish are monitored daily for health through observation of normal swimming and feeding behaviour, correct orientation, normal interaction within the school, smooth skin, and normal coloration. See section 6.4.A for further instructions of sick or dead animals. F. Cleaning screen The filtration screen should be rinsed daily and replaced. G. Inventory Any changes to fish number will be noted daily on the Inventory and Mortality Log. H. Sanitation Tanks which can be pulled off line will be disinfected periodically using a bleach solution (1/4 cup or 60mL sodium hypochlorite in 10 gallons of water). Tanks should be rinse VERY well after bleaching and allowed to dry completely before being returned to the system for use. Dirty nets will be placed into the appropriately marked container. Nets are sanitized by soaking in a 5% bleach (sodium hypochlorite) solution for 24 hours in the dirty net container. Nets should be rinsed VERY well after bleaching and allowed to dry completely before being placed with other clean nets in the appropriately marked area. Petri dishes will be manually scrubbed and sprayed with 70% ethanol prior to reuse. 6.5 Specialized Animal Care A. Sick animals Animals that look sick should be transferred to a separate tank to be assessed by the PI. The tank should contain system water, but not be on the system to prevent spread of sickness. The AARL Facility manager and the Attending Veterinarian will be notified immediately. Sick and injured animals may be treated with antibiotics if this is deemed necessary. If animals are too distressed or deemed by the PI or the AV to be unable to recover, they will be euthanized.

5 Page 5 of 6 B. Euthanasia Animals are sacrificed as described in Powder SOP EU and Powder SOP EU C. Dead animals If any dead animals are noticed during daily inspection, it will be noted in the Zebrafish Inventory log. Carcasses are disposed of as described in Powder SOP EU Specialized System Care The following system maintenance will be done by the PI or a senior member of the lab. If there are any alarms or monitors going off for these, notify the PI or senior lab members immediately. All system maintenance will be recorded under the comments column on the daily log or in the Water Quality Log. If you have any doubts about any of this, ask the PI before touching anything! A. Preparation of stock solutions As needed, the stock solution of bicarbonate and salt will be added to the automated dosing system reservoirs and noted on the comments column. THIS SHOULD HAPPEN EVERY FEW WEEKS AT MOST IF THIS IS HAPPENING MORE FREQUENTLY, SAY SOMETHING (the probe may be broken or in need of calibration). The solution should be prepared at roughly ¼ lb (113g) sodium bicarbonate per 3 gallons, and ½ lb (226 g) Instant Ocean salt per 3 gallons of system water. Before adding, double check that you are adding the salt solution to the salt container and the bicarbonate solution to the bicarbonate container. B. UV bulb A UV bulb is used to kill control biological contaminants such as bacteria. This will be replaced by the PI roughly every year, or as necessary. Crystal sleeve will be cleaned roughly yearly, and replaced roughly every 2 years, or as necessary. C. Carbon filtration Carbon filtration sleeves and activated carbon will be replaced roughly every 3 months or as necessary, per manufacturer s instructions. Per manufacturer s instructions, replacement of carbon will be alternated between the two filters (i.e. change A after 3 months, then B after another 3 months, then A again after another 3 months). D. RO system filters The carbon and sediment filtration units on the RO water maker will be replaced roughly every 6-12 months or as necessary, per manufacturer s instructions. E. Cleaning and calibration of probes The ph and conductivity probes will be cleaned and calibrated roughly every 2wks-1 month or as necessary, per manufacturer s instructions.

6 Page 6 of 6 F. Bead biofilter As necessary, glass beads will be added to the biofilter. 7.0 QUALITY CONTROL CHECKS AND ACCEPTANCE CRITERIA All procedures are subject to review by the Attending Veterinarian(s) and the Institutional Animal Care and Use Committee. 8.0 LITERATURE CITED 1. Westerfield, M, 2007, The Zebrafish Book, 5 th edition. Eugene: University of Oregon Press Aquaneering system manual. 3. Lawrence, C. (2007) The husbandry of zebrafish (Danio rerio): A review Aquaculture 269, p Harper, C and C. Lawrence, 2012, The Laboratory Zebrafish: A Volume in The Laboratory Animal Pocket Reference Series. CRC Press. 9.0 APPENDICES Zebrafish Daily Care Log and Checklist Zebrafish (Danio rerio) Water Quality Log Zebrafish (Danio rerio) Inventory and Mortality Log