User Guide Updated April 2019

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The Commandments of the Fish Facility 1) Always wear gloves and in-house shoes (Crocs/clogs) or shoe covers while working in the Main Facility. Sneakers are also acceptable as long as they have never been worn outside the facility. 2) No fish, embryos, tank components, or water taken from either the Main or Quarantine facilities may be returned to original location. Once something leaves, it never returns. Additionally, no animals or equipment can be transported between the Main and Quarantine Facilities. The only exception would be crossing adults in the Main facility to adults in the Quarantine facility. Adults and transport containers may not return to Main facility after crossing. 3) Any fish that accidentally falls to the floor must be euthanized by an approved method followed by proper disposal. 4) Use a new net each time you start working with a different tank/stock of fish while removing sick and dead fish from the tank. 5) Keep tanks covered with lids to prevent mixing of fish. 6) Please clean up after yourself. Place soiled equipment in designated locations to be cleaned and maintained by staff. Ethanol spray forceps, egg spatulas, and spoons after each use and place them in appropriate containers. Additionally, please put feed labels back on the appropriate pages in 3-ring binders. 7) Be sure to use the right type of water for the right situation. Any container that the fish will be living in should contain ‘system’ water obtained directly from the recirculating aquaculture system through the spigots or pump hose. Reverse Osmosis or ‘RO’ water is only used to rinse soiled equipment. This water does not contain proper levels of salts and will harm fish living directly in this water. Municipal tap water also contains high levels of chlorine and chloramines which are toxic to fish. Please only use tap water to rinse hands before leaving the facility.

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Quarantine System: Quarantine lights come on at 8:00 am and go off at 10:00 pm. The Quarantine must NOT be entered during Lights Off.

• All fish care (feedings, tank exchange, and tank checks) is the responsibility

of the Fish Facility Staff. • If you perform work in the Quarantine System, do not enter the Main

Facility until the next day and/or after showering/changing clothes.

Receiving Embryos or Fish from Other Labs/Facilities: • ONLY Fish Facility Staff are allowed to transfer adults/larvae into the

Quarantine Facility and administer disinfection (iodine) procedure to embryos and fluke tabs to adults.

• To obtain adults, a ‘clean bill of health’ must be obtained by the facility supervisor (Zak) from the veterinarian staff of the exporting institution prior to shipping.

• Adult Fish received start off-flow in Quarantine. They are kept off-flow for a week to undergo fluke tab treatment and are observed by Facility Staff. If given a ‘clean bill of health’, fish are placed on-flow on the Quarantine system. They are bred and their offspring iodinated by Fish Facility Staff to be transported into the Main Facility.

• If no ‘clean bill of health’ is provided or if QC reports positive hits for pathogens, the facility will only accept embryos.

• All embryos must be shipped on the day they are fertilized so they are received at 1dpf for the facility staff to administer their own disinfection procedure with iodine prior to going on the system. There are only two exceptions to this rule: If the shipment is coming from overseas and impossible to arrive at 1dpf, the exporter must guarantee that they will provide some form of disinfection prior to shipping. The other is if the embryos are being received from ZIRC who can only ship at 2dpf (IACUC). ZIRC will provide disinfection prior to shipping.

• Please notify and Cc Facility Staff (Zak/John) immediately when inquiring about receiving lines from outside institutions.

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Embryo Care: • Embryos need to be screened at the end of the day they are harvested on to

remove unfertilized. Distribute at maximum 50 embryos per Petri dish for normal development and survival.

• DO NOT write directly on Petri dishes. Lab tape must be labeled with: 1. Genotype 2. Date of Birth 3. Density 4. Lab/Owner initials.

• Add Methylene Blue to egg water to prevent fungal growth if desired. • Embryos should be kept within the 28.5 C incubators for normal

development. • Petri dishes need to be cleaned daily (exchange egg water and remove

dead/unfertilized embryos and chorions) • Embryos on day 0 can be discarded in “Retired Embryo” containers and

rinsed down the sink. Transgenic embryos must be retired in appropriately labeled containers for proper disposal. Embryos 1 dpf and older must be euthanized using an approved protocol (see Euthanasia section of this User Guide).

• Between 3-5 dpf, embryos to be grown up should be cleaned and new stocks of fish are to be recorded into the Facility Stock List and assigned a stock ID number (S#). All applicable information regarding the stocks’ pedigree and starting quantity of larvae should be included. The Facility Stock List is located on the Nursery computer.

• Embryos/larvae should be added to the Rotifer rack on 4 or 5 dpf. Individual users can do this or embryos should be placed within the Walker Nursery incubator for Fish Facility Staff to put on the system.

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Nursery (Walker) Guidelines: Nursery lights come on at 7:00 am and go off at 11:00 pm. The Nursery must NOT be entered during Lights Off.

• Tank labels are to be printed from the above-mentioned stocks list and

affixed to the front of the tank in a manner that does not obstruct the view of the fish or the flow of water or the water level of the tank.

• Each Lab is assigned a color. Please attach the appropriate color flag to your tank labels. This is required for facility organization and accurate billing.

• Larval fish are to be placed into appropriate tanks, at appropriate densities: Ø 1-10 fish: 1.1L tank Ø 11-40 fish: 3.5L tank Ø 41-80 fish: 8L tank (Petri dishes are combined to reach 80

embryos, do not house 80 embryos in a single Petri dish.) • An 800-micron BABY BAFFLE must be used for all tanks going onto the

Nursery system. Be sure not to use an Adult Baffle on embryos, as they will flow through and be lost.

• Fill tank with about 2 inches of system water and place on Rotifer Rack. • Once tanks are added to the Nursery system, they become the Fish Facility

Staff’s responsibility. No additional care is required from the user until the tank leaves the Nursery, with the exception of checking on your fish’s health and status.

• In general, fish will reach sexual maturity by 6-8 weeks. Once the majority of fish in a tank have reached sexual maturity, they will be moved to a clean tank and placed within the user’s adult section in Streisinger or Chien systems.

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Common Nursery Requests: Below are common Nursery requests that can be performed by Facility Staff. If you would like any of these done, please CLEARLY label your tank with said request.

ü Grow Fast: we can push the growth of fish to reach sexual maturity by 6

weeks. However, survivorship can be affected and sex will likely be skewed towards females. Also, please note that the quicker fish reach sexual maturity, the faster their fertility declines as Adults.

ü Grow Big: Tank is housed in the Nursery until all individuals have reached sexual maturity. High productivity should be expected immediately out of the Nursery.

ü Grow Small: Tank is removed a week or two before sexual maturation. People do this for stocks that are not immediately needed or in hopes of extending productivity.

ü Want Males: Sex can be skewed toward males through reduced feeding. It takes longer for these fish to reach sexual maturity due to reduced feedings.

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Adult Rooms (Streisinger and Chien) Guidelines: Adult room lights come on at 8:00 am and go off at 10:00 pm. The Adult Rooms must NOT be entered during Lights Off.

• Fish within the Adult rooms are the responsibility of Tank Owner and Fish

Facility Staff. • An exchange of all tanks is required every 3 months; Fish Facility Staff can

be request to perform this for a fee. • For optimal fish health, we suggest users spend 5 minutes per day with their

fish. During this time the following should be checked: 1. Sick and dead fish should be removed (one net per tank),

recorded in “Sick and Dead” fish log (located on book shelf in each room) and disposed appropriately.

2. Clogged and overly soiled lids should be exchanged. 3. Excessively soiled and over-flowing tanks should be

exchanged. 4. Fish health and feeding labels should be checked, for desired

results, and exchanged if needed. Food label books are located on the bookshelf within each room. Users should consult with Fish Facility Supervisor about appropriate feeding labels.

5. Check tank water flow.

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Euthanasia: Euthanasia may be accomplished using one of the following approved methods:

1. Rapid cooling using an ice bath is the preferred method because it results in more rapid and humane death, lower cost, and less chemical disposal into the environment.

a. Prepare an ice bath using a container of appropriate size to hold all of the fish to be euthanized.

b. Fill container with crushed ice (machine located in the Cleaning Room).

c. Add system water to create a slurry and mix to ensure even cooling of the water. Wait 5 minutes to ensure water temperature is evenly distributed in ice bath.

d. Place fish in ice bath without adding excess additional water (e.g. from a net).

e. Fish may be properly disposed of in a sink with a mechanical disposal 10 minutes after complete cessation of opercular movement.

2. An overdose of Tricaine methane sulfonate (a.k.a. Mesab, MS-222, Tricaine, Finquel) solution of 4 g/L buffered to pH 7.4 by Trizma Base.

a. Place Tricaine solution in finger bowl or breeder tank bottom or similar container, and fully immerse fish in solution.

b. Fish may be properly disposed of in a sink with a mechanical disposal 10 minutes after complete cessation of opercular movement.

Dispose of wild type and mutant euthanized fish in the sinks that have a garbage disposal. Transgenic lines should be placed in a biohazard bags located in appropriately labeled cabinets and then permanently stored in biohazard freezer. Facility staff will dispose of frozen carcasses in Mouse House. Facility age limit of fish is 2 years; all stocks must be euthanized by this age to maintain colony health.

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Breeding: • Fish health and appropriate nutrition is vital in achieving high crossing

success. The image below shows the ideal size and health for actively breeding female (top) and male (below) zebrafish:

• Fish can be too skinny, as well as obese. Both negatively affect productivity and egg quality.

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Breeding Continued: • Fish are most productive shortly after attaining sexual maturity. Productivity

starts to tail off after 9 months – 1 year or possibly sooner if fish were grown fast. However, fish can be productive up to 2 years, if appropriately managed.

• Keeping mixed sex groups in housing tanks is ideal when possible. Male productivity in general, is not affected by isolation. Isolated females have to be regularly set-up/managed to avoid becoming egg bound.

• Breeding of fish in the Main Facility can be accomplished by either allowing the fish to spawn naturally or by in-vitro fertilization (a.k.a. IVF, squeezing).

• Fish that are regularly bred and set-up (trained) perform better. Infrequent or irregular crossing reduces productivity.

• Mating crosses are set up on the “wet” mating racks in Streisinger or Chien in the afternoon or evening usually with one male and one female, or 2-3 males and one female. Dividers can be used to separate males and females, if one wishes to control the time of breeding the next day (see below for details). Staff will pull dividers at 8am the following morning if the user signs up for the request on the sign up sheet.

• The mating cages should be set up with a trickle or slow flow of water into the tanks.

• A reduction of the water level the morning of crossing increases breeding success.

• Maturation of eggs and the initiation of spawning behavior in zebrafish is thought to be triggered at sunrise (the onset of the photoperiod at 8:00 am in Streisinger and Chien). The closer to 8:00 am you pull your dividers, the better breeding success you will have.

• Fish MUST be returned to their tanks on the day of breeding (i.e. they cannot spend more than 24 hours on the mating rack).

• Fill out breeding success on appropriate clip boards. • When finished, all soiled equipment associated with breeding fish is the

responsibility of the individual researcher to put in the designated racks and containers in the Cleaning Room to ensure that the equipment is cleaned and returned for use in a timely manner.

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In Vitro Fertilization: This procedure requires training by an experienced technician or researcher and some practice to ensure success without unnecessary loss of fish life. Training is a service provided by Facility Staff for a fee.

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Feedings: The amount and frequency of feedings is up to the Tank Owner, provided the desired feeding regimen is covered by animal use protocols. The facility has a default feeding schedule and recommendations posted within each room. (See page 14 of this User Guide). Facility Users are encouraged to discuss breeding and feeding strategies with Facility Staff. Together we can decide what plan best fits your research needs and ensures the optimal well-being of fish. Tritones (Feeding Robots):

• Tritone feeding times are posted within each room and attached (Page 13). • The Tritone scans each tank at the start of each feed time, and then feeds the

appropriate amount. • The indicator lights at the top end of each aisle show the Tritone’s current

status: 1. No Light = not in operation 2. Green Light = in operation 3. Yellow Light = in hold mode 4. Red Light = Error

• If a tank is moved in between the scan and feed out phase, the Tritone will still feed that location creating a mess.

• If the number of fish are reduced or increased within the tank, the corresponding feeding label must be changed. Most overfed/underfed tanks occur when the number of fish in a tank is changed without updating the tank’s feeding label.

• There are 3-ring binders of Tritone feeding labels within Chien and Streisinger on the bookshelf. If you cannot find the label you need, please notify Facility Staff to print more.

• If the Tritone is in operation and you need to access the aisle, please press the orange hold button at the start of the aisle before you walk in to pause the Tritone. When you are finished, press the orange hold button once more to take the Tritone back into operational mode.

• Tritones have light curtain barriers programmed to stop when encountering a physical obstacle, e.g. a researcher. On occasion, these light curtains break down and would not stop upon encountering a person. In such cases, a sign will be posted. Please, be very careful around Tritones during this time.

Please try to avoid your aisle during the Rotifer Feeding (12:00-2:00pm)

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Facility Services and Resources: The Facility offers a wide variety of services and training. Services available include:

• Tissue collection for genotyping • DNA isolation from tissue samples • Genotyping of fish – PCR based assays or scoring larvae for

transgenics/phenotypes • Cryopreservation of sperm • Micro-injection • Generating fish and embryos from frozen sperm by in vitro

fertilization • Tank exchange • Fish line maintenance

As the number of users grows, space and equipment may have limited availability. To avoid conflict and ensure that the needed equipment is available on the day of your experiment, we ask that you sign up for equipment use on the ZFISH Google calendar. Username: wustlzfish@gmail.com Password: zebrafish2018 Equipment Available:

• Fluorescence microscopes • Injection rigs • I-Spawn (for mass embryo production) • Presentation microscope

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Facility Contact List: Zak Kupchinsky Zebrafish Facility Supervisor Email: zacharyk@wustl.edu Office Phone: (314) 362-4006 Facility Phone: (314) 362-1431 Cell Phone: (570) 225-5111 Contact For: facility training, orientation, access, quarantine Anna Hindes Lab Supervisor Email: ahindes@wustl.edu Contact For: cryopreservation, IVF, facility ordering, fish/embryo shipping Isa Roszko Staff Scientist Email: roszkoi@wustl.edu Contact For: microinjection, genotyping, sequencing John Engelhard Senior Technician Email: jengelha@wustl.edu Contact For: quarantine, fish/embryo receiving, genotyping

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*Volumes indicate amount per feeding* Feeding Schedule:

Time J-Junior S-Senior A-Adult F-Feed a lot 7:30am Dry Dry Dry Dry 9:30am Dry Dry 12:00pm Rotifers Rotifers Rotifers Rotifers 2:00pm Rotifers Dry 5:00pm Dry Dry 8:00pm Dry Dry Dry #Dry-Feedings 1 2 4 5

Feeding Recommendations: J-labels— Intended for low density <5, individuals, and split 1.1L tanks. S-labels—Intended for infrequent breeding, storage of stocks, or slow growth of 3.5L and 8L stocks that will not be needed in the near future. Recommend for high density (>5) and frequent breeding within 1.1L tanks. A-labels—Intended for frequently bred fish with less hassle of dirty tanks. You still can achieve good breeding success rates with these labels. Not recommended for 1.1L tanks. F-labels—These are intended for fish that are removed from the nursery early and need additional growth, and for fish that are bred weekly (this is the feeding regiment that used for daily WTs). With this feeding regimen, more time may be needed to change dirty tanks or clogged baffles. Not recommended for 1.1L tanks.

What does each Label feed? J-Junior S-Senior A-Adult F-Feed a lot ml of Dry Fed x1 Dry Fed x2 Dry Fed x4 Dry Fed x5

Label #Fish Rotifers A 1-5 5 30 mg 30 mg 60 mg 60 mg B 5-10 6 30 mg 30 mg 60 mg 60 mg C 10-15 7 30 mg 30 mg 60 mg 60 mg D 16-25 8 60 mg 60 mg 60 mg 60 mg E 26-35 10 90 mg 90 mg 120 mg 120 mg F 36-45 11 150 mg 150mg 180 mg 180 mg G 46-55 12 180 mg 180 mg 180 mg 180 mg H 56-65 14 240 mg 240 mg 240 mg 240 mg I 66-75 16 270 mg 270 mg 300 mg 300 mg L 76-85 18 300 mg 300mg 360 mg 360mg