Guidance for Ectothermic Vertebrates

Standard Operating Procedures

For researcher-maintained animals, Lead Researchers must include Standard Operating Procedures (SOPs) for the daily husbandry and care of animals, and the cleaning and maintenance of all housing areas and equipment as a part of their protocol submission for IACUC review and approval.

The SOPs must describe in detail all aspects of the care provided to the animals by the laboratory staff, including:

1. Feeding (feed type and vendor, frequency of feeding, method of feeding)
2. Regular monitoring of both the animals and their environment, including:
   • Daily checks for animal health, including weekends and holidays (required by Federal regulations)
   • Daily recording of critical environmental parameters, depending on the species
     • For aquatic species: examples of critical parameters include water temperature and pH
     • For terrestrial species: temperature and humidity of the primary and/or secondary enclosures
   • Regular recording of certain non-critical environmental parameters, depending on the species (e.g. concentration of nitrates, nitrites, ammonia, salinity, hardness and dissolved oxygen in the water of aquatic housing systems.)
     • The frequency of testing will vary depending on the species, life stage, system and specific parameter.
     • Newly-established aquatic systems generally require more frequent testing.
3. Standards for acceptable ranges of environmental parameters for the species concerned e.g. room temperature and humidity, water conditions, and response plans in the event that conditions are outside the acceptable ranges.
4. Description of the housing system (e.g. static, flow-through or recirculating aquatic system, biological filtration system, size of primary enclosures).
5. Description of the source and treatment of water for aquatic or semi-aquatic species (e.g. municipal water, ocean water, RO water, distilled water, filtration type, any water conditioners or additives such as salts or dechlorinating agents).
6. Description of handling procedures required for routine husbandry (e.g. capture of animals to transfer them to different primary enclosures).
7. Cleaning of primary enclosures and secondary enclosures rooms (cleaning methods and frequency). In general, each room should have its own stock of supplies, including nets and other animal handling devices and cleaning supplies. For aquatic species, this should include a description of the frequency and amount of water replaced.
8. Maintenance, cleaning, and replacement guidelines for all equipment used to house animals, including primary enclosures, filters, pumps, heaters, UV lights, nets, and water-quality probes.
9. Any regularly used disease prevention and/or treatment protocols.
10. Procedures used to acquire and introduce new animals.
11. Procedures used to monitor the health status of the existing population, including any procedures for monitoring animals and/or their environments for the presence of disease-causing agents or conditions.
12. An effective response plan for a pandemic animal disease outbreak, including procedures for large-scale culling of sick animals and for cleaning and disinfection of tanks and water systems.
13. An appropriate response plan in the event of equipment malfunction or failure, both on a small scale and large scale. This must include contact information for appropriate personnel, and plans for possible relocation or euthanasia of animals.

NOTE:  SOPs must be submitted to the IACUC for review and approval as part of the animal use protocol. Once approved, a copy of the SOPs should be kept in all areas where animals are housed. See sample SOP template.

The municipal potable water in Irvine contains chemicals making it unsuitable for use in aquatics tanks without treatment. Aquatics rooms should have a water supply incorporating appropriate treatment, such as conditioning, filtration, oxygenation, temperature control and ultraviolet sterilization. Ideally, the facility should include a water storage tank that can hold a supply of water that could be used to replace water in the primary enclosures in the event of an emergency. In the absence of such a storage facility, it is imperative that researchers develop a written emergency plan for situations where the regular water supply may be compromised.In addition, aquatics housing facilities should have:

1. An adequate electrical supply for filter pumps, water pumps, air supply, and/or heating/cooling systems, as well as an emergency power source to assure the continuance of fresh water and aeration to tanks.
2. Ground Fault Interrupt (GFI) electrical outlets.
3. A mechanism for appropriate temperature regulation of the entire room and/or individual tanks, when appropriate for the species.
4. An appropriate light source and light-dark cycle in all rooms or enclosures. Gradual changes in room light intensity are recommended, as sudden changes may elicit a startle response in some species (e.g.fish). Some aquatic and semi-aquatic species may need full-spectrum lighting and/or heat lamps to provide supplemental heating to facilitate adequate physiological function.
5. Sinks with hot and cold running water and functional floor drains are desirable in fish-housing areas.
6. Large, heavy equipment (e.g. racks, tanks, large filter canisters) must be secured to walls or constructed to prevent them from falling in the event of an earthquake.
7. Standards for acceptable water conditions appropriate to the types of animals housed.
8. Personnel managing aquatic systems must be trained on relevant aspects of water chemistry, how to monitor water quality, and how water quality can impact animal health.

Creation of new transgenic animals (including cross breeding of transgenic strains) requires approval of the UCI Institutional Biosafety Committee

Transgenic aquatic species require special procedures to comply with California Fish and Game Regulations and the Federal NIH Guidelines for Research Involving Recombinant DNA Molecules, as follows:

1. All transgenic aquatic animals shall be held, raised, and transported in a closed-water system or in a system which treats effluent discharge from the facility with a disinfection system adequate to ensure against the inadvertent release of live animals into the building drain.
2. Release of transgenic aquatic animals or their progeny into waters of the state is prohibited.
3. Access to facilities containing transgenic aquatic animals must be restricted through means determined to be adequate by the Department to assure against unauthorized removal of animals.
4. Movement of live transgenic aquatic animals from facilities is prohibited unless specifically permitted by the Department.
5. If transgenic aquatic animals are held with nontransgenic animals of the same species, all such animals that commingle with transgenic animals shall be treated as transgenic for the purposes of regulation and may not be introduced into waters of the state. Nontransgenic individuals that can be individually identified as nontransgenic may be exempt from this provision with prior department approval.

Specific requirements for amphibians vary by species. As with other ectothermic vertebrate species, expert advice should be sought when designing appropriate housing and husbandry SOPs.

1. Amphibians generally require cool, moist environments, although tropical species may prefer warmer temperatures. Primary housing enclosures should not be airtight, but should be covered if evaporation is a problem.
2. As with other ectothermic species, consideration should be given to species-appropriate temperature and lighting and maintenance of a power source. Light wavelength requirements for amphibians are largely unknown. Many amphibians can be maintained for long periods of time with standard fluorescent room lights, and no supplemental lighting at the tank level.
3. Many species of terrestrial salamanders do well on a substrate of moist paper towels, replaced at intervals sufficient that accumulated feces do not grow mold. Most species also do well on an earth/twig/leaf substrate. In either case, a small pool at one end of the enclosure and pieces of clay pots for shelter are appropriate additions.
4. Axolotls should be maintained in containers of at least one liter of water per adult animal, at a temperature of 16-24°C. An aeration system is usually required for long term care of axolotls and other aquatic salamanders, especially for stream-dwelling species.
5. Anurans (frogs and toads) should be housed in containers of sufficient depth that they do not injure themselves by leaping against the top. Rough wire screen or other abrasives should be avoided in places that the animals will contact. Generally, a few inches of water in the bottom of the container, with rocks protruding above the surface, represent optimal conditions for semi-aquatic anurans. Totally aquatic anurans, such as African clawed frogs (e.g.Xenopus laevis), may be housed in approximately two liters of water per adult animal, but a wide variety of housing systems are currently utilized in research settings. Researchers are encouraged to monitor on-going research and evaluate space needs as necessary, documenting their findings in the individual SOP and/or protocol as appropriate.
6. Some reptilian species (e.g., alligators) may require specific light wavelengths in order to thrive. As with space requirements noted above, researchers with particular expertise in this area are encouraged to develop SOPs and guidelines appropriate to their chosen species and area of research, documenting all requirements in the protocol and/or SOP with citations and references from scientific literature.

Temperature, proper lighting, sufficient space, and appropriate food are the key aspects of reptile care. Cages must be escape-proof and, for most species, should not have rough wire screen in places that animals can reach with their noses.

1. Temperature: As most reptiles actively thermoregulate during the photophase and exhibit voluntary hypothermia during the scotophase, heat in enclosures should form a gradient, with high temperatures not exceeding 40°C. Thigmothermal (contact) heat sources (e.g. heated rocks) are preferable. The use of infrared heat lamps should be avoided for non-desert species because of their intense drying effect. If lamps are used as a heat source, their intensity should be appropriate to the enclosure and species.
2. Lighting: A photocycle is preferable to continual light or continual dark, unless the photoperiod is an integral part of the experiment. Quality of the light is also important. A variety of fluorescent lamps mimic the color spectrum, while others produce the ultraviolet spectrum in the range that converts vitamin D2 to D3. Without exposure to these wavelengths, animals in long-term maintenance will eventually develop severe disorders in calcium metabolism.
3. Food: Reptiles can generally be divided into insectivores, carnivores, and herbivores. For long-term maintenance, insectivores, the small lizards, and some snakes, should be fed a diet of mixed insects, rather than one consisting largely or exclusively of mealworm larvae or crickets. Both of these food sources will eventually cause severe calcium deficiency. Insects should be dusted with a calcium carbonate/vitamin powder at least once each week. Carnivorous reptiles that feed on live prey should, if possible, be trained to eat killed prey items, in order to prevent accidental injury. Herbivores should be fed a complete diet consisting of a variety of vegetable matter, never lettuce or other greens alone. Supplemental protein sources, such as commercial dog/cat food, are preferable to mixed vegetable diets for some species.
4. Habitat: Aquatic turtles and crocodilians require clean water and a dry place to emerge. Lizards and snakes generally require mostly dry conditions with adequate water available for drinking. Species from moist areas often require higher humidity and sometimes regular misting.

The California Department of Fish and Game currently requires the following prior to issuance of a permit to maintain live venomous reptiles and other animals in the state:

1. All venomous species and any progeny shall be confined in escape-proof enclosures. Entrances providing immediate access to the animals shall be kept locked to prevent entry of unauthorized persons. Access to the animals shall be restricted to the permittee and his/her authorized representatives or associates. All individual cages or enclosures shall be of such design and construction as to preclude the escape of any animal from any human or natural cause, including earthquake.
2. All windows or other openings, including vents, in any room where venomous animals or their progeny are maintained, shall be screened and barred to prevent the escape of animals into the wild or the entry of unauthorized persons into the room.
3. Any room where venomous animals are confined shall be equipped with adequate temperature control to ensure that the animals are humanely maintained.
4. An up-to-date list of all subject anti-venom sources in California shall be posted in the immediate vicinity of the room where venomous animals are maintained.
5. Inspecting officers will not be limited by these requirements; they may impose additional restrictions which they feel are necessary in any particular case.

In addition to these requirements, it is required that all personnel dealing with these animals on campus be approved by the appropriate University personnel. Any cage containing a venomous animal must be labeled with the common name, family, genus, and species of the animal in such a way that the label can be easily removed and taken with a victim to the hospital. A first aid kit and typed instructions for treatment of snake bite (both first aid and initial hospital care) must be present and prominently displayed in each room containing live venomous animals. Venomous animals must be transported on campus only by approved personnel, and the animals must be in secure, properly marked containers. Whenever possible, venomous animals should be manipulated by indirect means (e.g., hooks or shift cages), rather than handled directly.