Inside the IACUC

Zebrafish continue to be a very popular laboratory animal model in biomedical research, with more investigators studying immediate post-fertilization changes in embryos. Caring for these precious embryos has become a subspecialty in laboratory animal medicine, in and of itself. Incubator systems for fish, typically housed outside of traditional animal facilities, tend to be the preferred storage site.

This issue of Laboratory Animal Science Professional gave us the opportunity to highlight the experience of Stephen Frederickson, Wendy Pridgen, Irene Ginty, and Dr. Tannia Clark with IACUC oversight of incubator management for zebrafish. The article touches upon some important topics including incubator setup, care, husbandry, maintenance, and disaster planning. Requirements for IACUC oversight of incubators, and how the IACUC should accomplish its oversight, are also discussed.

The authors hoped to provide a detailed discussion of how to properly use and maintain incubators and explain how the IACUC can best support their use, and we think we did just that! The article contains valuable information that can assist any program with their zebrafish oversight and certainly adds to the body of literature of care for this important aquatic species.

We hope you enjoy this “deep dive” into zebrafish care!

Stacy Pritt, DVM, MS, MBA, CPIA, CHRC, DACAW is the Associate Vice President of Research Support and Regulatory Management and Assistant Professor in Psychiatry at the University of Texas Southwestern Medical Center in Dallas, TX.

F. Claire Hankenson, DVM, MS, DACLAM, is the Associate Vice Provost for Research and Attending Veterinarian and Executive Director, University Laboratory Animal Resources, at the University of Pennsylvania in Philadelphia, PA.

By Stephen C. Frederickson, B.S; Wendy Pridgen, B.S.; Irene Ginty, B.S. & Tannia Clark, DVM, MS, DACLAM

Incubators are a great way to maintain a consistent micro-environment for laboratory animals, embryos, and larvae, such as zebrafish, medaka, Xenopus tadpoles, or even chicken eggs. In addition, incubators can be located outside animal facilities and closer to benchtop work, can be under the exclusive care of research staff, and the same model of incubator used for bacterial plates, cell cultures can alternatively house thousands of fish larvae! This great asset for the investigators presents a unique challenge to an Institutional Animal Care and Use Committee (IACUC), as incubators housing fish larvae, such as zebrafish may meet the PHS definition of satellite facility (SF).1 This standard piece of laboratory equipment easily blends into the research setting, and benign oversight of investigators could lead to unexpected surprises during facility inspections. Sharing methods of SF documentation, oversight, and inspection can only benefit the animal research community as we work towards a shared goal of maintaining animal welfare. This article will focus particularly on the use of incubators housing fish used for biomedical research.

The PHS Policy on Humane Care and Use of Laboratory Animals (PHS policy) applies to “Any live, vertebrate animal used or intended for use in research, research training, experimentation, or biological testing or for related purposes,” therefore the PHS policy applies to fish. It also defines SF, as any containment outside of an animal facility in which animals are housed for more than 24 hours.4 The Office of Laboratory Animal Welfare (OLAW) further clarify that egg-laying vertebrates, like zebrafish, offspring are considered live animals upon hatching.2 In zebrafish reared under standard husbandry practices, hatching occurs approximately 3 days post fertilization (dpf).5 Therefore, under the PHS policy, any incubators outside of a core facility that house zebrafish at 3+ dpf for more than 24 hours should be considered a satellite facility. These satellite facilities should be approved by the IACUC and include documentation of animal care.

Since laboratory incubators may have multiple uses for purposes other than animal housing

Figure 1. Zebrafish being socially housed and interacting within their environment. Photo credit: Stephen C. Frederickson.

(i.e., bacteria growth, cell culture, etc.), there should be clear signage delineating which incubator(s) have been designated for animal use. We recommend using only dedicated satellite facility incubators for animals.

Larval fish development is often temperature-dependent1 so a method for monitoring the temperature within the incubator is recommended, as extreme heat (or cold) could be harmful to the animals. Most incubators currently on the market utilize a digital display of the internal temperature sensor value. Alternately, an analog display or a small, independent thermometer could be placed in the incubator to indicate the status. Additionally, we recommend there should be a nearby sign or resource that notes the acceptable temperature range as well as emergency contacts, such as: the lead investigator, veterinarian and/or facility manager for the incubator.

Incubators can be a good solution for off-system housing of zebrafish used for chemical screens and other research that would not be possible on a system with shared, recirculating water. There are special considerations that need to be given when animals are being exposed to drugs, chemicals or other agents while also being housed in an incubator. The appropriate Animal Biosafety Level and precautions for the specific hazard(s) should be determined by consultation with an institution’s safety department/official and cautions should be clearly posted on the incubator. Designated supplies (trays, embryo disposal cups, etc.) should be used for chemical exposure and not shared for any other purpose or area.

Maintaining documentation of equipment and animal care within the incubators are required and should be standardized via standard operating procedures (SOPs) across an institution. The frequency of the health check, husbandry care and the resulting documentation of such will vary with species, age, research study endpoints, the animal care and use program, as well as veterinary care requirements.

We recommend researchers conduct both incubator (equipment) checks and animal health checks (Figure 2). When incubators house zebrafish, daily checks are common. However, the IACUC may approve an alternative schedule with reduced checks. An alternative schedule should be based on performance standards that take into account housing aspects such as animal density, housing tank/container, age of animals, and feeding requirements. We recommend establishing an alternative schedule, using performance standards, as they can apply to a disaster scenario. Incubator Animal Welfare N/A Alive, no illness

Labelling/ Signage

Physical Environment Emergency contact(s), temperature range Age of embryos/ animals, owners of containers

Powered, properly functioning (no odd sounds) Housing containers are intact

Documentation Temperature, semi-annual temperature verification and light cycle verification (if applicable) Frequency of health checks, feeding/water changes (if applicable)

Figure 2: Recommended checks and documentation for an incubator Satellite Facility

Incubators require minimal to no maintenance; however, periodic (annual/semi-annual) temperature verification of each incubator is important as temperature is directly linked to development1 and temperatures can vary during times when not observed by staff. We recommend a temperature logging probe, such as the Hobo MX100, to trend temperature over time, and if a light cycle is used, we recommend a logger with a light sensor, such as the Hobo UX100003. Temperature verification for 24+ hours should catch the typical temperature swings and variation that comes from normal use, as well as the variance in the environmental conditions created by the unit working to maintain a stable temperature (Figure 3).

We recommend that incubators housing animals are included within an institution’s disaster planning. While a typical incubator is not overly complex, there are a few circumstances that could arise and require a response that should be addressed in your disaster plan. When available, the incubator should be placed on the emergency power circuit to prevent issues from potential power outages. In the event that an incubator loses power (or fails) and function cannot be restored in a timely manner, do not open the door. The insulation built into the unit will allow maintenance of a suitable temperature for the longest time possible. A slightly lower or higher temperature may change the rate of development but is not an immediate animal welfare concern. If function cannot be restored and relocation to other incubators is not feasible, alternate methods of heating or cooling may be considered. Supplemental temperature assistance, like hand warmers or similar heating pads (often used in the shipment of fish) or cold packs, may be used to help maintain temperatures within an acceptable range. However, it is important to note that all disaster planning must include euthanasia and researchers should know how to appropriately euthanize based on species and development.

The Guide for Care and Use of Laboratory Animals provides requirements, guidelines and language regarding the environment, housing, and management of fish in a laboratory setting.3 However incubator satellite facility management is not outlined and any particular husbandry areas may be altered based on research needs:

Health & Equipment Checks: Using performance standards, the IACUC with veterinary input, should ensure the frequency of the health check, husbandry care and the resulting documentation of animals cared for within incubators (Figure 2) are appropriate based on the species, age, and research study endpoints. Our IACUC has outlined that zebrafish satellite incubators (with animals present) receive both equipment and health checks a minimum of every three days, with daily checks being optimal.

Feeding: Incubators and typical larval containers (e.g., petri dishes) are not designed to allow for easy access with feed devices, though feeding and care can be provided during temporary removal. Research and veterinarian staff should establish within an SOP when first feedings and frequency are expected for animals housed within an incubator. If feeding is withheld for research purposes, then it should be approved of by IACUC review or veterinary consultation.

Semi-Annual Facility Inspection: IACUCs are required to inspect satellite facilities. During semi-annual walk-through, members should verify that their established requirements are being met, such as: documentation and signage is correct, that any animals in containers

Figure 3. Recorded temperature and relative humidity inside an incubator over several days. Each sudden temperature drop (and corresponding humidity increase) is associated with the incubator door being opened. Graph created using HOBOware.

appear healthy, and that the temperature is in the desired range.

Animal Numbers: The PHS Policy requires that investigators approximate number of animals to be used and as such institutions must have mechanism to track animals, including zebrafish in incubators, that are acquired and use including animals euthanized when not needed.2 In many institutions much of the research is done on fish, embryos and larvae that are housed in an incubator. For example, while those 3+ dpf zebrafish may not ultimately be placed on system, they should be accounted within the animal numbers.

Reporting Animal Welfare Concerns: Institutions should always have a method for reporting any animal welfare concerns. As incubators are considered satellite facilities, we recommend posting the same animal welfare requirements established for other animal holding areas and regularly reviewing or training to ensure that the policy is known to the staff so that they would feel comfortable using it, if needed.

Incubators are a great research tool that provide proper environmental conditions for animals. It allows investigators to bring the subjects of the research closer to the lab space where most of the work is done. An IACUC can easily embrace this solution while still ensuring animal welfare and regulatory compliance are met. Stephen Frederickson, BS, LATG, Contractor- Charles River, is the NHGRI Zebrafish Research Specialist at the National Institutes of Health in Bethesda, MD. Wendy Pridgen, BS, LATG, ILAM, is the NHGRI Animal Resources Program Coordinator at the National Institutes of Health in Bethesda, MD.

Irene Ginty BS, RLATg, CMAR, Contractor- Priority One Services, Inc., NHGRI Animal Operations Specialist at the National Institutes of Health in Bethesda, MD. Tannia Clark, DVM, MS, DACLAM, is the NHGRI Animal Program Director at the National Institutes of Health in Bethesda, MD. REFERENCES: 1. Bartlett DH, Silk SB. Office of Laboratory Animal Welfare Comments. Zebrafish 2016;13:563-564.

Care and Use of Laboratory Animals. [Cited 29 November 2022]. Available at: https://olaw.nih.gov/faqs#/guidance/faqs?anchor=50289 3. Guide for the care and use of laboratory animals. 8 ed, 2011 4. PHS Policy on Humane Care and Use of Laboratory Animals. 2015.

Temperatures. Zebrafish 2021;18:316-325.