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.

The paperwork for standard international distribution may vary due to country-specific requirements for customs and health declarations/certifications. In addition, many of these processes are still being developed between countries and information may be outdated or inaccurate. However, most international shipments must be accompanied by at least the following documents:

1. Customs Statement
2. Customs Invoice (in triplicate)
3. Import permit (of the receiving country), if required and
4. A Declaration of Health
5. If a the importing country requires a USDA endorsed Health Certification, then the reciprocal import permit from that country is required and the sender must obtain the appropriate certificate form from the USDA website (see below). 4) Is not necessary in this case.
   

ZIRC provides templates of documents 1. – 4. upon request and on its site: http://zebrafish.org/documents/protocols.php. These documents should be copied onto your institute’s official letterhead.
If a USDA endorsed Health Certificate is required, information can be obtained from the Animal & Plant Health Inspection Service (APHIS) at the United States Department of Agriculture (USDA): (http://www.aphis.usda.gov/regulations/vs/iregs/animals/). It is a good idea to get in touch with your USDA Area Office to ensure the appropriate steps are taken. Select the country of destination in the pull-down menu on the iRegs site. The guidelines detailed by USDA/APHIS and the receiving country must be met in order to obtain a USDA endorsed health certificate for export. Download the appropriate form for aquatic species and all supporting documents for the country of destination. If the country specific form is not available, the USDA’s APHIS Form 7001 must be used: http://www.aphis.usda.gov/library/forms/pdf/APHIS7001.pdf
If a country requires certification that the exporting facility is free of a specific OIE reportable pathogen you should contact the USDA Area Office. In this case a USDA Veterinary Medical Officer may need to schedule sampling and diagnostic testing of fish and possibly conduct a facility inspection.
Complete the health certificate for the shipment and add additional information as specified on the import permit. Note: this is a reciprocal process - the USDA will only issue a Health Certificate endorsement if a copy of the import permit from the recipient country is available, to ensure all country-specific information is provided. A USDA accredited veterinarian must sign and date the health certificate (or Form 7001), which then must be endorsed by the USDA/APHIS. To this end, mail the form to your local USDA Area Office with payment (currently $38).
Importantly, the health certificate is only valid for 30 days, and the importing country may limit shipment to a shorter time frame. The sender has to ensure that the shipment occurs before the earlier of the two dates expires. Hence it is important to schedule steps carefully and have a good working relationship with your local USDA office.

When receiving a shipment of zebrafish from abroad, a custom declaration (Form 3-177) must be filed with the US Fish and Wildlife Service (USFWS). Visit the http://www.fws.gov/le/declaration-form-3-177.html site, download the four pages, fill in the form and file it with the appropriate office (see instructions). Alternatively, you may with eDecs, an electronic declaration system offered by USFWS. In this case, create a new personal account on the eDecs site https://edecs.fws.gov/eDecsHome.cfm, create an Importer/Exporter Account and a new eDec file, and file it electronically. For academic institutions filing must occur, but a license is not needed and the fees are waived.

• VWR International, https://us.vwr.com/
• Small box dimensions 8“ L X 6” W X 9“ H; Catalog number 14100-412
• Medium box dimensions 12” L X 10“ W X 7” H; Catalog number 14100-402
• Extra-large box dimensions 22” L X 15” W X 15” H; Catalog number 15714-704, Superior Shipping, http://superiorshippingsupplies.com/category And http://www.thermosafe.com/.

• Ammonia binder: ClorAM-X, Reed Mariculture, http://reedmariculture.com/product_cloram_x.php
• Sterile Fish System Water

• do not use water dissolvable cellulose!

• Superior Shipping, http://superiorshippingsupplies.com/category
• For the UK: http://www.heatpacksuk.co.uk/heat-packs.php
• 40 hour pack for domestic (overnight) shipments
• 60-72 hour packs for international (2-4 day) shipments

• Customs and veterinary health certification documents for international shipments (Fig. B)
• address, courier labels and document pouch (Fig. I)

• 250 ml Tissue culture flasks, Fisher Scientific, catalog # 10-126-5; http://www.fishersci.com
• VWR International, catalog # 82050-854; https://us.vwr.com/
• 0.5X Embryo Medium (E2)
• Methylene Blue (optional)

• Polyethylene plastic bags: at least 0.003 mm or 1.2×10-4 inches thick).
• Gulf Coast Plastics for bulk quantity, http://www.gulfcoastplastics.com/BottomSeal.htm
• Superior Shipping, http://superiorshippingsupplies.com/category
• Check any pet store for small quantities
• Average size for holding 10-20 fish (10“ X 18” (.003 mil))
• Note: Square bottom bags can be mad from the above using a plastic heat/bag sealer. You can also tape the corners of the bag to create square bottom.
• Pure oxygen (not required, recommended)
• Rubber-bands, plastic ties. or metal clips

Packaging of embryos and adults for shipping. A) Thick-walled Styrofoam box inside cardboard box, labeled as indicated in panel B). C) Adults are prepared in labeled breeding cages, embryos in labeled 250 ml flasks. D) Adults and their label are transferred to two leak-proof polyethylene bags and closed with plenty of air or oxygen. The Styrofoam box is partially filled with Styrofoam packing peanuts before the bag or flask is added (E,F). Completely filled Styrofoam box. Packing peanuts prevent movement of bag or flask. For the cold season or for cold-climate destinations, a hole is punched into the Styrofoam lid with a pencil or screwdriver to provide oxygen for a heat pack (H) which is taped to the lid over the hole. I) Address labels and pouch with shipping documents (Invoice, Customs statement, Copy of import permit, Health Declaration/Certification) are added in triplicate copy to the pouch.

Varga, Z.M. (2016) Aquaculture, husbandry, and shipping at the Zebrafish International Resource Center. Methods in Cell Biology. 135:509-34.
http://zfin.org/ZDB-PUB-160725-11

and

4th Edition (2000) Chapter 1.7 (online): http://zfin.org/zf_info/zfbook/chapt1/1.7.html

5th Edition (2007) Pages 1.24 - 1.39 (print): http://zebrafish.org/orders/buyBookQ.php?item=Book&id=book&detail=The%20Zebrafish%20Book