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. Author manuscript; available in PMC: 2020 Dec 3.
Published in final edited form as: Cold Spring Harb Protoc. 2020 Dec 1;2020(12):pdb.prot106146. doi: 10.1101/pdb.prot106146

Animal maintenance systems: Xenopus tropicalis

Nikko-Ideen Shaidani 1, Sean McNamara 1,*, Marcin Wlizla 1, Marko E Horb 1
PMCID: PMC7666031  NIHMSID: NIHMS1616056  PMID: 32404312

Abstract

Modular recirculating systems incorporate biological, mechanical, activated carbon, and UV sterilization to create a nearly self-contained stable housing system. Nonetheless, minimal water exchange is necessary to mitigate accumulation of metabolic waste and regular weekly, monthly, and yearly maintenance to ensure t accurate and efficient operation. This protocol describes the methods for establishing a new recirculating system and the maintenance, and water quality parameters that are necessary for keeping Xenopus tropicalis.

MATERIALS

Reagents

  • Ammonia Fresh Water and Salt Water Test Kit (API, Chalfont, PA, USA).

  • Bleach (6.0% NaClO).

    Dilute with Type II water (ASTM International 2018) to make a 10% bleach solution (final concentration of NaClO is 0.6%)

  • Nitrite NO2 Fresh Water and Salt Water Test Kit (API, Chalfont, PA, USA).

  • Nitrate NO3 Fresh Water and Salt Water Test Kit (API, Chalfont, PA, USA).

  • Dechlorinator (Na2S2O3). ProLine brand (Pentair AES, Apopka, FL, USA).

    Contains both the anhydrous (CAS# 7772-98-7) and pentahydrate salt (CAS# 10102-17-7) molecules. 1.6–2.6 ppm of Na2S2O3 per 1 ppm of chlorine is typically sufficient to dechlorinate water.

  • Ethanol (190 proof).

    Dilute to 70% with Type II water.

  • Nitrifying bacteria (ProLine brand, Pentair AES, Apopka, FL, USA).

  • Reef Salt from Seachem Laboratories (Madison, GA, USA).

  • Sodium Bicarbonate (NaHCO3) (ProLine brand, Pentair AES, Apopka, FL, USA).

  • Virkon Aquatic (DuPont, Wilmington, DE, USA).

Method

Establishing a new recirculating system

  1. Rinse biomedia with Type II water and place in the biofilter.

  2. Keep the carbon filter empty and disable the water effluent exchange and UV sterilization.

    The filtration and sterilization functions of the system need to be initially disabled to allow for establishment of a stable biofilter.

  3. Allow flow to a single tank on the end of each rack and add 5–10 frogs to the system.

  4. After 24 hours, disable the water flow but keep the biomedia aerated.

    Biomedia can be aerated using an external air pump.

  5. Add nitrifying bacteria to the biofilter.

    Larger systems require more nitrifying bacteria. Follow the instructions outlined by ProLine.

  6. Wait 1–2 hours before starting the water flow again.

    During this brief period, the frogs present in the system should be kept in their tanks. The absence of water flow should not be detrimental to their health.

  7. Perform daily NH3/NH4+, NO2−, and NO3− level measurements.

    Wait for NH3/NH4+ levels to measure at least 1–2 ppm.

  8. Initiate UV sterilization, water exchange, and add carbon to the system.

    Flow to 1–2 additional tanks can be started.

  9. The levels of NO2− and NO3− should begin to increase.

  10. Keep monitoring NH3/NH4+ and NO2− levels. Once both fall to 0.5 ppm, additional frogs can be safely added to the system.

    This process can take 2 weeks to 2 months. Nitrifying bacteria can thrive when water parameters are consistent. If dosing reservoirs are not present, alkaline buffers can be used to stabilize pH. Once NH3/NH4+ and NO2− levels reach the desired level, gradually add frogs to the system while monitoring water quality parameters.

Daily Water Assessment and Maintenance Required for Proper Operation

  1. Record system readings.

    This allows the user to deduce trends and identify potential issues that may be occurring.

  2. Inspect the UV bulb.

  3. Regularly scrape internal sides of the tanks to remove any algae buildup.

    Algal buildup will vary depending on several factors including lighting and dissolved nutrients. Excessive buildup should be removed as needed.

  4. In flood and flush systems shake the stand pipes to clear them. Tanks with only an overflow bulkhead will require daily active removal of detritus to keep them clean.

  5. Exchange 10% of the system water.

  6. Measure pH and conductivity dosing reservoirs daily.

    Use sodium bicarbonate to buffer and regulate pH and sea salt to control conductivity.

  7. Observe carbon and mechanical filters for buildup of waste and replace as needed once passage of water through them is significantly impeded.

  8. Observe individual tanks for accumulation of waste and film on the bottom and sides and replace ones that are particularly dirty.

    To clean tanks: scrub and rinse with Type II water, spray with 10% bleach and let sit for 1 h, rinsing again with Type II water, spray with 70% ethanol and let sit for 1 h, rinse once more with Type II water, and allow to air dry.

Weekly Water Assessment and Maintenance Required for Proper Operation

  1. Assess NH3/NH4+, NO2−, and NO3− levels with API test.

    Acceptable measurements: NH3/NH4+ = 0 ppm (0–0.5 ppm), NO2− < 1 ppm, NO3− < 40 ppm, alkalinity >40 ppm. If levels are out of range perform a 25% water exchange

  2. Test pH and conductivity with a reliable external probe as a reference for internal system probes.

    If the external and internal probes are not in agreement, clean the system probe and check that it is correctly reading calibration solutions. Recalibrate the probe if necessary.

  3. Measure temperature with an external thermometer.

    Heaters and chillers are used to keep the temperature stable.

Annual Water Assessment and Maintenance Required for Proper Operation

  1. Replace UV bulbs and check to ensure that the quartz sleeve is intact

  2. Replace pH electrodes.

  3. Remove and clean return pipes

  4. Replace all rubber tubing.

Water quality parameters for Xenopus tropicalis

The water quality parameters and animal stacking density necessary for optimal growth will vary dependent on the life history stage of the animal (Green 2009; Hilken et al. 1995). These parameters are listed in the table below giving the optimal conditions, as well as the allowable tolerance range given in parentheses.

Age Water Temperature (°C) pH Conductivity (μS)
 Density
Tadpole/Froglet 27 (25–27.5) 7.0 (6.8–7.2) 1000 (900–1100) 2–4 per 1 L
Juvenile 26 (25–27) 7.0 (6.8–7.2) 1200 (1000–1300) 1 per 1–2 L
Adult 25 (24–26) 7.0 (6.8–7.2) 1200 (1000–1300) 1 per 1 L
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System sterilization

  1. Disable all system components including the water pump, biomedia agitators, and probes.

  2. Discard biomedia or sterilize in a 10% bleach solution.

  3. Scrub the inside of the sump and associated parts to remove all detritus.

  4. Remove and discard filter pads, cartridges, and carbon from the system.

  5. Disassemble and scrub all the tanks and accessories.

  6. Rinse the tanks with water, coat with 10% bleach and let stand for 1 hour.

  7. Rinse the tanks with water, coat with 70% ethanol and let stand for 1 hour.

  8. Rinse the tanks with Type II water and let air dry.

  9. Soak accessories in 10% bleach for 1 hour.

  10. Soak accessories in 10 g/L solution of Na2S2O3 in RO water for 1 hour.

  11. Soak accessories in 70% ethanol for 10 minutes, rinse, and air dry.

  12. Drain 75% of the sump volume.

  13. Reconstruct the system but keep the carbon filter empty.

  14. Start the system and add bleach.

    Ensure the final concentration of NaClO in the system is 0.06%.

  15. Reduce the effluent rate to its lowest setting.

    Do not reduce the daily water exchange rate to less than 1%.

  16. Wait 2–3 days then increase the daily water exchange rate to approximately 50% to begin washing the bleach out.

  17. After a week, begin testing the pH.

  18. Add Virkon to the system once the system pH matches the influent water pH.

    The concentration of Virkon should be 3.2 g per 1 m2 of estimated internal surface area of the entire system.

  19. Reduce the effluent rate back to its lowest setting.

    Virkon can cause foam accumulation that may over flow. To help mitigate this, limit agitation of the water and spray 70% ethanol directly on the foam to help disperse bubbles.

  20. Wait 7 days then increase the daily water exchange rate to approximately 50% to wash Virkon out of the system.

  21. Allow the system to run for 2–3 days and replace all rubber tubing.

    The system is now sterile, follow the protocol for establishing a new recirculating system, described above, before using it to house frogs.

Discussion

Optimal eggs and embryos are produced by healthy laboratory population of Xenopus tropicalis. Regular monitoring of water quality parameters, as well as daily monitoring and visual inspection of the animals is crucial for prevention and early identification of developing conditions that may be detrimental to overall colony health. Use of tap water is discouraged and starting with a good water source providing Type II water will aid in maintenance of optimal water parameters as well as limit the chances of introducing pathogens and toxic contaminants. Municipal tap water may also be basic and thus may add the additional complication of needing to use acid buffers to aid in stabilizing the pH. The stability of the parameters will also be affected by the animal maintenance system configuration, with recirculating systems being more cost effective and efficient than flow-through systems and also requiring much less maintenance than static systems. Furthermore, animal health is best when the accumulation of nitrogenous waste is low, thus the initial effort made to establish a stable biofilter in a recirculating system is absolutely necessary. The Nitrosomonas and Nitrobacter bacteria in the biofilter respectively oxidize the toxic ammonia into toxic nitrite and then the nitrite into nontoxic nitrate (Hem et al. 1994). Following establishment of a stable ammonia/nitrogen cycle regular maintenance and tests of water parameter levels will aid in maintaining the animals in good health (McNamara et al. 2018). Nonetheless, sterilization of the entire system may be necessary if addition of new frogs to the system also introduces pathogens that negatively affect their quality of life. Once sterilized, the system can be put back into operation by again following the steps for establishing a functional and stable biofilter.

References

  1. International ASTM. 2018. ASTM Standard D1193 – 06 (2018) . “Standard Specification for Reagent Water.” West Conshohocken, PA. [Google Scholar]
  2. Green SL. 2009. The Laboratory Xenopus sp. CRC Press. [Google Scholar]
  3. Hem LJ, Rusten B, Ødegaard H. 1994. Nitrification in a moving bed biofilm reactor. Water Research 28: 1425–1433. [Google Scholar]
  4. Hilken G, Dimigen J, Iglauer F. 1995. Growth of Xenopus laevis under different laboratory rearing conditions. Lab Anim 29: 152–162. [DOI] [PubMed] [Google Scholar]
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