Crossing and Rearing Protocols

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These protocols explain the techniques we use to cross and raise stickleback at the University of Oregon. We have pared down the technique to improve efficiency. We have also incorporated the use of sterile plates, media, and incubators to rear large numbers of embryos in minimal space with little mortality. Hundreds of families can be stacked on trays in the incubator. The precision of the incubator (+/-0.2C) allows the embryos to develop as predictably as can be expected for out bred organisms. In comparison to other techniques that use aeration in aquaria, we have few problems with infections by water mold or bacteria.



Contents

Fish Room SOP's (Standard Operating Procedures)

FishRoom_SOP's



Stickleback Husbandry Forum

Stickleback Husbandry Blog


Reagents and Equipment

Fry and Mom rack Huestis Hall
Fry and Mom rack Huestis Hall

Crossing

  • Sterile Ginzburg’s Fish Ringers solution containing antibiotic and antimycotics for testes
    • 6.6g NaCl
    • 0.25g KCl
    • 0.3g CaCl2
    • Add ddH2O to almost 1 liter
    • 0.2g NaHCO3
    • Finish bringing to 1 liter with ddH2O
    • Autoclave then add antibiotic/mycotic Gentamycin to a concentration of 50ug/ml, as well as antibiotic-antimycotic combination from Gibco-BRL (15240-096, 100X concentration) that contains penicillin, streptomycin and amphotericin B to a final concentration of 50ug/ml.
  • 25, 45 and 90mm disposable sterile petri dishes (standard)
  • MS222 Tricaine Methanesulfonate
  • 100% ethanol
  • Fine scissors and forceps
  • Wide-blade entomology forceps
  • Sterile Embryo medium (6ml of Instant Ocean into 1L npH2O) - autoclave
  • Squeeze wash bottles
  • Stress coat (standard from pet store)
  • Sterile flat razor blades
  • Sterile disposable large bore transfer pipettes (VWR cat# 691)
  • Dissecting microscope

Equipment for embryos

  • Refrigerating incubator (Fisher Model 146E cat # 97-990E) – expensive but necessary
  • 90mm disposable sterile petri dishes (standard)

Fish foods for fry, juvenile and adults

  • Fry - newly hatched baby brine shrimp, salt water rotifers (not essential)
  • Juvenile - newly hatched brine shrimp, juvenile dry food mix (Tropical flake food finely crumbled to 250-500 microns, Zeigler Larval Diet (Larva "Z" Plus 250-450 Microns), and Silver Cup trout chow starter, 440-590 microns)
  • Adult - adult dry food mix (Silver Cup trout chow no. 1 or no. 2, 590 microns - 1.38mm, and crumbled tropical fish flake), freeze dried blood worms, and freeze dried brine shrimp. (Worms and shrimp are feed to the fish once per week.)

Crossing Protocols

  1. Squeeze Female
    Cover gloved fingers with stress coat and gently squeeze gravid, ripe female. Ripe females have extended abdomens and their eggs have “dropped”(Picture can be added easily). If eggs do not emerge with very slight pressure female is not quite ready. Squeeze eggs with motion from pectoral girdle posterior to the cloaca into a 25mm sterile petri dish. The small size of petri dish allows the sperm to be concentrated on the eggs.
Collecting eggs
Collecting eggs
  1. Dissect Testes From Male
    Euthanize male by placing him into a finger bowl of embryo medium containing a lethal volume of MS222. Clean a large, 30cm by 60cm or similar size, glass sheet, fine scissors, and forceps with 100% ethanol. When male is motionless, remove and rinse fish with clean water and sever spinal cord using a razor behind skull to be sure of euthanization. Use scissors to make incision just posterior to the cloaca and cut from there anteriorly to the pelvic girdle making an incision along the mid line of the fish. Make another cut to each side of the fish from the cloaca dorsally approximately 15mm so that the body cavity is easily accessed. Incisions should be made just deep enough to cut the skin and body wall muscle while being wary to not cut into the stomach and intestines that are located just below the skin surface because this will release large amounts of unwanted bacteria into body cavity. Locate paired testes and vas deferens (testes are variable in shape and coloration, but are usually long and pigmented, usually having the same pigmentation as the skin surface, and sit in the dorsal part of the coelom near the kidneys. The vas deferens are threadlike and are usually as long as the testis to which it connects. Use fine forceps to grab vas deferens, sever near the cloaca, and remove one (or often) both testes. Doing so keeps the testes intact, allowing them to contain viable sperm for up to 2 months at 4°C in Ginzburgs Ringers.
  2. Store Testes
    If only a single cross is to be made with a male, proceed to step 4. Otherwise, place testes into 45mm petri dish filled with cold Ginzbergs with antibiotics and antimycotic medium. Store testes at 4°C. To store testes for extended periods, change out medium once a week.
  3. Prepare Testis Prep
    If multiple crosses are to be performed from a single pair of male’s testes, remove one testis to the inside lid of the 25mm petri dish into which a females eggs have been squeezed. Using a sterile razor, slice off small piece of testis (we’ve found a large testis can be used to fertilize approximately 5 clutches). Make cut as perpendicular as possible to the major axis of the testis and perform multiple cuts from the same end. This allows as much of the sperm to remain packaged and inactivated at the center of the testis. Use blade to macerate testis. With fiber optic light on a dissecting scope swimming sperm should cause a ‘sparkling’ refraction. Add a small amount, approximately 500 ul of sterile embryo medium to testis prep with disposable pipette, mix, and then add to the eggs on drop at a time, using same disposable pipette, dispersing drops between petri dishes so that all eggs are covered with sperm mixture.
  4. Fertilization (time = 0)
    Fertilization of most eggs will occur almost instantaneously. However, I leave the sperm on the eggs for about 10-15 minutes without stirring. At this point cover with sterile embryo medium. Mark fertilization time on the surface of the petri dish. Place in incubator at 20°C.
Fertilized stickleback embryo.
Fertilized stickleback embryo.
  1. Separation of Embryos and Second Cleaning (t = 2 hr)
    The animal pole of the embryo is established at the site of sperm entry, and at 20°C the first cell syncitium is usually visible at this point within 45 minutes. The first cell division usually occurs approximately 25 minutes later, and the two cell stage is fully visible approximately 1.5hr after fertilization. During this time, the chorion thickens and attaches to the petri dish as well as to other embryos. At or after 2 hours post fertilization, use the wide blade entomological forceps to detach the embryos from one another, as well as to dislodge embryos from the bottom of the petri dish. Remove all of the unfertilized eggs to limit mold contamination, and record the total number of eggs and embryos. Rinse embryos 3 to 4 more times then distribute embryos to 90mm petri dishes (30-50 embryos per dish) with fresh embryo medium. Enter cross information into database, print labels for dishes, and place embryos into incubator at 20°C. This should be done within 3 to 4 hours post fertilization.
  2. Raise Embryos (check daily)
    Embryos will develop at approximately 2.5 times zebrafish time [see http://zfin.org/zf_info/zfbook/stages/index.html], and hatch at approximately 7 days. 48hr stickleback embryos have completed most major morphogenetic processes, and the melanic pigment cells are just starting to migrate. A beating heart can be seen after ~72hr. Check petri dishes daily and remove deads and those with arrested development. A change of embryo medium may help at some point during the first 7 days as water quality may decline.

Raising Protocol

  1. Hatching, Begin Feeding Brine Shrimp, and Move to System (t = 7-8 days post fertilization)
    After hatching at 7-8 days post fertilization, remove chorions and allow young to absorb yolk (2-3 days). At approximately day 2 post hatching take petri dish containing fry out of incubator and place dishes on a table. To each petri dish add approximately the same volume of system water, as there is embryo medium; this will double the volume of liquid in the dish. This acclimates the fry to the system water. Allow the fry to acclimate for 6-10 hours, the longer the better. Then transfer fry to small aquaria on the system. Feed newly hatched brine shrimp once daily.
  2. Switch Food, Move to Main System
    Gradually incorporate juvenile food when fish reach approximately 4-5 mm in length, After 2-3 weeks, fry should have grown to about 7-10mm in length. At this point, move fry to 10 gallon aquaria. As fish grow and reach an approximate size of 25-30 mm, gradually mix in the adult food that will eventually be the primary food source. Feed fish twice daily with enough food that it is all eaten within 5-10 minutes and so that none remains on the bottom after 0.5 hour.
  3. Move Fish
    When fish are approximately 4-5 cm in length, and depending on the size of the group of fish, move the fish into a 20 or 30 gallon aquarium. We try to maintain a ratio of fish length to tank volume of 1 cm of fish per liter of tank volume.
  4. General Maintenance
    Once per week 10 gallon tanks will need cleaning. Scrub sides and bottom with rough pad and allow debris to settle. After detritus has settled, siphon the tank using siphon tube or water siphoning system sold through mail order or off the Internet. 20 and 30 gallon tanks will need to be cleaned less on average and can be done once every two weeks. Keep salinity of system water at 6 ppt, this is checked once per week using a salinity refractometer with salt, Instant Ocean, being added when needed. General water hardness is kept at 30 ppm and pH is kept at 7.0-8.0. These parameters are checked once per week using standard test kits obtained through mail order or off Internet. Temperature is kept at 20°C by chilling of room air at night. Anabolic waste of the fish will tend to lower the pH and when the ph is to low it can be brought up using aragonite, placed in mesh bags, in the sump of the water system.
  5. Adult Fish
    When fish are 1 year or older and more then 5 cm in length, they are placed into the breeding room with a light cycle as described above. The males start to show mating colors and females start to become gravid in 2- 3 weeks after being placed into the breeding room.

Water System

  1. 10,000-liter re-circulating system with fluidized sand bed and a polyethylene bead filter that self cleans and drained once a day. Main grow-out room is kept on a light cycle of 10 hr light and 14 hr dark. The breeding room is kept on a light cycle of 20 hr light and 4 hour dark. A 50-watt light is turned on 0.5 hour before the main light is turned on and is on 0.5 hour after the main light is turned off. This simulates dawn and dusk and seems to aid in getting the fish ready to breed. Approximately 5% of system water is changed daily through general maintenance and evaporation. Make-up water, that is building water that is ran through a particle filter and a carbon filter, is added automatically through a float valve into the sump. System is monitored using a scada system by Sensaphone, Inc.. Dissolved Oxygen, pump activity, dirty water flow, and water on floor are all monitored. This allows for persons on call to be paged if problems arise. Someone is on call at all times in case problems arise.

Online Resources

Fish Room Protocols and Procedures

Cleaning Tanks

(Source: Westerfield, M. (2000). The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio). 4th ed., Univ. of Oregon Press, Eugene.)

  1. Siphon (cleaning by "vacuuming") the bottoms of all tanks at least once per week. Use siphon tips with a length that corresponds to the size of the tank: tips for 5-gallon tanks are 12" long; the 10-gallon tips are 18" long; and the 30-gallon tips are 26" long. Each tip is made of 0.5" I.D. x 1/16" Wall Plexiglas tubing (Port Plastics, Portland, Oregon). Cover one end with a one inch tip of 3/8" I.D. x 3/32" Wall Amper Latex tubing at one end (VWR Scientific Co.). (See Embryo Collection, for a diagram.) The siphon tips, which are attached to the hoses, are the only parts of this apparatus that can be immersed in the tank. Use each tip in only one tank and then sterilize it by soaking in a bleach solution (solution is 20 parts water to 1 part bleach) followed by a rinse in a sodium thiosulfate.
  2. Scrubbing (removal of algae from sides, front, and back of tank) is done on an "as needed" basis. (If you can't see into the tank, it's past time to scrub.) Make scrubber handles from 0.5" x 1.5" Plexiglas Plastic (Port Plastics, Portland, Oregon). The heads are made by sewing Scotch Brite Pads (United Grocers, Eugene, Oregon) with 20 lb test monofilament fishing line so they slip tightly over the scrubber handles. Use each handle and head assembly in one tank only and then sterilize by bleaching as described above for the siphon. The scrubber.i.Scrubber; heads can be autoclaved. Use two lengths of scrubber handles. The size for 5 and 10 gallon tanks is 0.5" x 1.5" x 16" and for the 30 gallon tanks use 0.5" x 1.5" x 24" handles. Heads are the same for both size handles.
  3. Replace basket of bulkhead. Take the dirty basket off and sterilize in bleach as described above. Obtain clean basket and replace.
  4. Tanks that are emptied of fish need to be cleaned and sterilized before another batch of fish can be introduced. Drain the tank and remove it from the rack. All parts (lid, back, and bulkheads) are kept with that particular tank. Clean all parts with brushes and a scrub pad. Clean the tank thoroughly with a scrub pad, taking care not to damage the silicon water seals on the inside (algae should be left if very gentle rubbing will not remove it). Once the tank and parts are clean they need to be sterilized in bleach. Place all parts except the lid into the tank and cover them with about 3" of water. Add 1/4 cup (~65 ml) of bleach to a 10-gallon tank (125 ml to a 30-gallon tank; 30 ml to a 5-gallon tank). Wash the bleach water thoroughly around the inside of the tank by hand, using a pad, sponge, tank back, or other means to expose all inside portions of the tank to bleach. Immerse the lid in the bleach water. Rinse the tank thoroughly with tap water. Reassemble the tank and put it back on the rack. Fill with system water and allow water to recirculate for about 10 minutes before adding fish.

Decapsulating Brine Shrimp

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