Shipping Zebrafish Protocols

Zebrafish shipments require special packaging to provide a controlled environment for fish while in transit and careful documentation to ensure expedient delivery. Because customs, animal welfare and biosafety regulations, vary from country to country, international shipments may require modifications of the labeling and documentation provided below. Packaging, however, must always conform to the International Air Transport Association (IATA) Live animal Regulations (LAR, container requirement 51; https://www.iata.org/whatwedo/cargo/live-animals/pages/index.aspx). International shipments should be sent Mondays or Fridays to allow time for customs and veterinary inspection clearances (1-3 days) and avoid weekend delivery. When shipping embryos, these should be bleached before packaging. For a detailed bleaching protocol see the link: Customer_Egg_Bleaching.pdf

• Label tissue culture flasks with line specific information such as line name, allele designation, stock #, date of fertilization, and number of embryos.
  
• Add 200 ml of 0.5X Embryo Medium with Methylene Blue to the flask(s).
  
• Add 100 embryos per flask and close lid tightly, if in doubt, add Parafilm around the cap (Fig. C,D).
  
• Fill shipping box about 1/3 with Styrofoam packing peanuts, then place flask(s) with embryos in its center (Fig. F), and fill any remaining space with additional packing peanuts (Fig. G).
  

• Write information on the plastic bag (line name and allele, # of fish, date of birth) or attach a label with the information (Fig. D).
  
• Add the following to a square bottom plastic bag:
  
  1. Approximately 1/3 fish water
     
  2. ClorAM-X ammonia binder (1.5 ml ClorAM-X per liter of fish water)
     
  3. Fish. For US or priority over night shipments, place 20 fish/bag (1.5 liters of water); for international or 2-3 days shipments, place 10 fish/bag (1.5 liters water).
     
• Close the bag trying to trap as much air as possible: Open the top of the bag wide, quickly grasp the top closing it off and then twist keeping pressure in the bag. The bag should contain approximately 2/3 air and 1/3 water when closed. If more air is needed, reopen the bag and try closing it again. Do not blow into the bag to add more air (CO2!). If available, use pure oxygen, then the proportions can be inverted to 2/3 water and 1/3 Oxygen.
  
• Close bag by knotting or twisting the bag so the top doubles over. Rubber-band this portion. Push the knot down to maintain pressure in the bag. The bag should remain tight and be leak free when turned upside down. Metal (Fig. D) or plastic clips can also be used instead of rubber bands.
  
• Place the bag of fish inside another plastic bag! Line bags up (by their seams), so they are in the same orientation. Remove as much air as possible from the second bag, to provide no space for water if the inner bag spills. Close the outer bag as above. Ensure there are no leaks.
  
• Place bagged fish in the shipping box and fill the remaining space with Styrofoam peanuts.
  

• During winter months, a heat pack should be placed in the box to avoid or minimize temperature drops when the package is exposed to outside temperatures. Punch a small hole in the center of the Styrofoam lid (not cardboard!) to allow oxygen to reach the heat pack. Activate heat pack and tape it to the inner side of the lid, covering the hole (Fig. H).
• Place the Styrofoam lid on the box. Thick Styrofoam insulates the fish from outside temperature changes and also prevents water spills in case the plastic bags leak.
• Close the cardboard box and seal with sturdy packing tape.
• Place the following labels and documents on the outside of the box, including:
  1. Live fish label (Fig. B)
  2. Address label (on top of box)
  3. Pouch for customs and health certification documents for international shipments (usually provided by courier).
• ZIRC currently uses World Courier for the following countries: Austria, Australia, China, Germany, Ireland, Israel, Italy, New Zealand, Spain, Saudi Arabia, Singapore, all South American countries, South Korea, Sweden, and the United Kingdom. Typical World Courier shipment cost is approximately $1700.