Storing and shipping zebrafish: Q & A with Zoltan Varga

Ken Poss, IZFS Board member and zebrafish researcher, Duke University

Zoltan Varga, Director, Zebrafish International Resource Center, University of Oregon

 

KP: What is ZIRC? Can you share a little about its history, its roles, and how it is supported financially?

ZV: The Zebrafish International Resource Center (ZIRC) is a central repository for mutant, transgenic, and wild-type zebrafish lines and other biological materials. ZIRC also provides diagnostic zebrafish health services and shares its more than 20 years of husbandry and fish health experience with researchers and fish facility staff. We are supported by an NIH Office of Research Infrastructure Programs (ORIP) P40 Grant that requires competitive renewal every 5 years.

The need for a centralized zebrafish resource center was recognized at the first international zebrafish meeting at Cold Spring Harbor in 1994. Large- and small-scale mutant screens were ongoing at the time in several laboratories, resulting in a rapid increase in the number of genetic lines. Individual laboratories were taxed with the burden of maintaining and distributing their lines. Although cryopreservation was used by some, most researchers could not afford the space or cost of maintaining lines they were no longer actively studying. The research community agreed that a centralized resource center was a top priority to ensure the highest possible levels of quality, uniformity, and long-term security. The NIH National Center for Research Resources (NCRR), which later became ORIP, agreed to consider an application for funding the ongoing costs of running a center. The initial funding for ZIRC at the University of Oregon was awarded to Monte Westerfield in 1999. The bulk of the funds for construction were provided by bonds issued by the State of Oregon. An NIH NCRR infrastructure construction grant provided additional money. Construction started in 2000 and the official opening was 2001.

 

KP: A lot of researchers are interested in freezing zebrafish sperm. What is the success rate for sperm freezing? Is it easy and can you refer readers to the latest protocol?

ZV: The post-thaw fertilization rate is around 60-70% in our hands, using the protocol we published (Matthews et al., 2018). We also provide the most recent modification of this protocol on our website. The method is not particularly difficult but requires upfront time investment to train personnel and to develop routine and proficiency. Success varies with the condition of the males, and we also provide recommendations for male conditioning before collecting milt, and for optimized in vitro fertilization after thawing.

 

KP: How long can sperm remain in liquid nitrogen and retain effectiveness?

ZV: We have successfully reactivated 16 and 17-year-old samples without any detectable decrease of fertilization rates or embryo survival. Cryoscientists suggest that correctly frozen and stored samples can be maintained for many decades, even centuries, without impact on quality or fertilization capability.

 

KP: Should all labs be freezing sperm rather than maintaining low-use stocks as live fish?

ZV: We strongly support the idea that all laboratories that develop or work with many fish lines also develop cryobanking capability. Millions of research dollars have been invested in generating more than 28,000 zebrafish research lines, and only about 12,600 of these are captured by ZIRC! Cryopreservation is the best alternative for long-term storage compared with live fish maintenance. There is tremendous space and cost-savings with cryopreservation. In addition, if a lab generates many lines but cannot characterize and study them all at once, cryopreservation helps with planning, and enables the laboratory to back-up presently unneeded stocks until they are used. To make the process easier, we are working with Terry Tiersch (Aquatic Germplasm and Genetic Resources Center at Louisiana State University) to develop a Cryo-kit together, which will come with all necessary tools, solutions, supplies, and straightforward instructions. It will enable laboratories to freeze in a standardized manner, even if cryopreservation is not part of their research focus or laboratory routine.

 

KP: Is it possible to ship frozen sperm?

ZV: Yes, there are specialized Dewars that hold liquid nitrogen vapor. Typically, these containers can hold cryogenic temperatures between 7 to 10 days and can be shipped by FedEx or other couriers. However, cryogenic transport requires specialized labeling and paperwork, and optionally also temperature and monitors indicating whether the Dewar was rocked or tipped in transit. Cost of shipping is higher than regular FedEx shipments, so it saves money to ship larger numbers of fish lines (dozens or more) at a time.

 

KP: Should labs send live fish to ZIRC to maintain and distribute? To avoid delays is it better to send sperm?

ZV: We prefer to receive live fish and freeze them ourselves from the quarantine room to ensure safe and efficient recovery. However, if everyone could produce samples of a predictable and reliable quality, we would consider cryogenic imports. To this end, we also offer to teach laboratories by visiting ZIRC and learning the technique. Please see the submission guidelines on our website.

 

KP: Why is it so expensive to receive fish from ZIRC?

ZV: I really appreciate the opportunity to speak about this. This is a complex issue, and I can see that the community wants us to be transparent about our cost recovery. We do our utmost to keep costs as low as possible for everyone. ORIP requires cost recovery as part of our budget, so we are obligated to charge at least a fraction of our costs.  ZIRC has not increased fish fees in over a decade and because of the COVID pandemic, increasing personnel costs, and stagnating cost recovery, we had to reduce our staff by 3 colleagues during the past 3 years. What drives cost for users, unfortunately, are changes in animal welfare laws, tighter regulations, and the associated shipping costs. 

There are many activities behind the scenes for which ZIRC cannot, and is not, recovering costs directly. These activities are particularly focused on quality assessment and quality control. More than 44,000 alleles are ready to ship within a week or two. Once a line is established at ZIRC and we have become its custodians, there is a strong expectation from the community that we are also the primary source for line-specific information, helping them with trouble shooting to identify their lines, or to provide husbandry information on how best to propagate a line. Because we also continuously import new lines, we routinely monitor fish health in our quarantine and main fish facility to keep our colony healthy and further minimize the risk of distributing pathogens to the community. ZIRC also puts significant effort into collecting and curating the currently available husbandry and sequence information to characterize, identify, and advertise newly imported fish lines. There is a lot of trouble shooting, including re-importing lines several times. By the way, for most submissions, the data and fish are not as high quality as those from the Poss lab! 

ZIRC is asking that all shipping costs are carried by the recipient, and ZIRC does not mark up any shipping costs. Shipping prices are determined by the shipper’s Volume-Weight cost, and for FedEx, these currently start around $175. Internationally, ZIRC uses FedEx where possible; however, most countries, especially in the European Union, require specialized animal couriers. ZIRC is looking to find reliable courier services that are more cost effective. Unfortunately, however, the COVID pandemic has made all such services more expensive recently. We are painfully aware of this trend. To keep shipping costs low, our best advice is to collect requests from as many labs as possible from the same institution. By sharing shipping costs proportionally for large orders and avoiding one-off requests, each lab can save money.

For shipping, ZIRC staff manages all shipping paperwork, customs papers, and health documents, and we ensure that fish are shipped safely and arrive alive. With quality control (health and line identification) and shipping routines, ZIRC saves laboratories who do not have this level of routine and efficiency significant personnel cost and effort that can be better directed to research goals instead.

 

KP: What is the most effective way to send zebrafish to international colleagues? And as adults, embryos, or frozen sperm?

ZV: Deposit lines at ZIRC! Have ZIRC ship the line for you. You spend more on personnel costs (and nerves) to research the destination’s import regulations, fish breeding, preparing, packaging, paperwork, and shipping than you think. In our hands, adults and embryos survive equally well.

 

KP: I understand, but how do I best get the lines to my colleagues quickly for a collaboration or in those first few months after describing the line in a preprint or publication?

ZV: When lines need to be exchanged between collaborators or to meet deadlines such as addressing reviewer requests, I recommend shipping embryos, because if anything happens, they are more easily replaced in another shipment than adults. But if it is necessary to send adults, I would recommend trying a test shipment with few fish first.

For live animal shipments in general, one can start with the APHIS IRegs for the country of destination. This is also a good starting point to determine whether one needs a licensed animal courier service in the destination country or a postal courier. For example, the only countries in Europe where animals can still be sent with postal couriers are the Benelux states and France, all others to our knowledge require a licensed animal courier. At the IRegs country page, scroll down to Research / Laboratory Animals section, or aquatics section. 

The paperwork is straightforward, and ZIRC can provide samples of these:

  1. Customs Statement,
  2. Customs Invoice (in triplicate),
  3. Import Permit (of the receiving country), if required and
  4. A Declaration of Health 

For the DoH, start with the signature of the Attending Veterinarian at your Institution. However, sometimes the importing country requires a United States Department of Agriculture (USDA)-endorsed Health Certification. If so, the reciprocal import permit from that country is required and you must obtain the appropriate certificate form from the USDA website. Step (4) is not necessary in this case. For some countries (e.g., Japan) the signature of a USDA-certified veterinarian is sufficient (not of a USDA-accredited veterinarian). If you need the signature of an accredited veterinarian, it can be a matter of luck how your USDA district is structured and whether the signature has a quick enough turn-around time and is still valid when the shipment enters the destination country. If electronic signatures are acceptable, it is usually not a problem. However, if destination officials expect an ink signature and you have to FedEx health certificates back and forth between USDA and your institution, the timeline can get extremely short. Britain, for example, does not acknowledge the electronic signature since Brexit. Again, these are starting points and only examples. ZIRC is always happy to assist with the latest information we have, so researchers can call or email ZIRC and we will advise as best we can. Also – the community could develop a “shipping” blog on the ZFIN wiki pages. This would aid to exchange recent shipping experiences.

 

KP: Still, more effort, time, and cost than one hopes for. And what are the current challenges at ZIRC?

ZV: One of our biggest concerns is funding for ZFIN, our sibling organization. As I mentioned, we curate a lot of practical data for fish identification and husbandry. ZFIN information, nomenclature curation, and the widespread support we receive from our colleagues at ZFIN is simply invaluable. We are extremely concerned about funding for all MODs, but of course particularly for our friends at ZFIN. I would very much like to grab the opportunity and mention that the NIH has published an RFI on User Experience with Scientific Data Sources and Tools. I would really appreciate it if the zebrafish community would signal their dedicated support for MODs by responding to this RFI and the survey link. ZIRC also took a financial hit during the COVID pandemic, and we are very slowly on the way to recovering that business. Our biggest challenge remains to improve our import pipeline and make it more streamlined and efficient, without losing essential information.

We also have great news to share. ZIRC is currently expanding its facility with the support of an ORIP C06 construction grant. We are modernizing our water filtration systems, and we are creating space for better workflow, equipment cleaning, quarantine, husbandry research, and cryogenic storage. An upcoming challenge of the expansion is that we must move all our fish stocks several times around the facility, while the floors in the main fish room are sealed and the new water filtration systems are installed. During this time, it will be all hands on deck and the community may experience longer turn-around times. However, we are well on track to conclude the construction by May 2022, and we will then have space to improve current services, improve colony health even more, work more safely, and offer new services, for example backup of sperm samples for laboratories.

 

KP: Thanks so much, Zoltan, for sharing all this information in this forum.

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