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Bringing new fish or embryos into a facility can also bring in new pathogens. The use of strict quarantine practices (i.e., introducing only surface sanitized embryos) can eliminate a large number of potential pathogens from entering a facility and is strongly recommended for all laboratory zebrafish colonies regardless of the source. When adult fish are acquired, they should be held in quarantine until they can successfully breed and all embryos introduced into the main facility or system should be bleached. The ideal quarantine area is a room on a flow-through water system that is both spatially and procedurally separated from the main fish facility. Small facilities often utilize individual aquaria or small recirculating systems in a designated quarantine area. Fish should be observed and acclimated in quarantine for a minimum of two weeks before breeding. If fish show clinical disease within this period, they should be treated or euthanized, depending on the value of the particular fish. Quarantined fish are bred, and only bleached embryos leave the quarantine area. Once the line has been established in the main facility, the fish in quarantine are culled. Fish should be disposed of in a manner that will not contaminate the main facility.
Embryos received from other facilities within 36 hours post-fertilization can often be bleached upon arrival and introduced immediately into the facility, thus avoiding quarantine. If the embryos are too old to be bleached (embryos with a compromised chorion or already hatched) when they arrive, they should be reared and bred in quarantine similar to adult fish.
A procedure for bleaching embryos can be found in The Zebrafish Book (Westerfield, 2000), which is available on-line through ZFIN, and is distributed in print by ZIRC. The specific ZIRC bleaching procedure is also outlined in the protocols link from the ZIRC homepage. Organisms can be protected from bleach exposure when sequestered within detritus or nonviable embryos. Embryos should therefore be cleaned of all debris, fecal material, and unfertilized or dead eggs before the bleaching procedure.
The “sanitized egg only” type of quarantine is the preferred and recommended procedure. Other approaches allow greater possibility of pathogen introduction and spread. However, sanitized egg approach requires the ability to raise larval fish and is a limitation for some facilities. In these cases, it is recommended that new arrivals be quarantined for observation for a minimum of three to four weeks before being introduced into the main system. A sample of fish can be sacrificed for diagnostic workup along with any fish showing signs of disease. Treatment of the entire group can then be instituted based on the diagnostic evaluation.
For some frozen lines, embryos shipped to customers are generated by in vitro fertilization using eggs from AB females at ZIRC and cryopreserved sperm from males not raised at ZIRC. The health status of the males contributing sperm was not evaluated and neither the embryos nor paternal stocks have been on the ZIRC water system. The ZIRC recommends raising the first generation of an imported line on a quarantine system.