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Published in final edited form as: Methods Mol Biol. 2023;2562:27–39. doi: 10.1007/978-1-0716-2659-7_2

Establishing a New Research Axolotl Colony

Anastasia S Yandulskaya 1, James R Monaghan 1
PMCID: PMC10948202  NIHMSID: NIHMS1875896  PMID: 36272066

Abstract

The field of regenerative biology has taken a keen interest in the Mexican axolotl (Ambystoma mexicanum) over the past few decades, as this salamander successfully regenerates amputated limbs and injured body parts. Recent progress in research tool development has also made possible axolotl genetic manipulation and single-cell analysis, which will help understand the molecular mechanisms of complex tissue regeneration. To support the growing popularity of this model, we describe how to set up a new axolotl housing facility at a research laboratory. We also review husbandry practices for raising axolotls and using them in biological research, with a focus on diet, water quality, breeding, and anesthesia.

Keywords: Axolotl, Husbandry, Housing, Aquatic

1. Introduction

The Mexican axolotl (Ambystoma mexicanum) is an aquatic salamander used in biomedical research to study development, evolution, and regeneration. Axolotls were first collected from the Lake Xochimilco System, Mexico, in 1863, making them the oldest laboratory animal model [1, 2]. The use of axolotls has considerably grown in the past decades, partly due to an increased interest in stem cells and regeneration, as well as technical advances in transcriptomic and transgenic approaches. The axolotl is an appealing laboratory animal because it is relatively hardy, thrives in near room-temperature freshwater, and breeds year-round. Axolotls also have a striking ability to regenerate organs and appendages after injury. Organ regeneration occurs in the brain, spinal cord, retina, heart, ovaries, liver, and teeth [39]. One of the more striking examples of regeneration is that of the limb [10, 11], which has motivated several new groups to adopt the axolotl system.

This chapter focuses on creating a new axolotl breeding colony for laboratory research. Also, we encourage readers to reference other reviews on the topic of axolotl housing and maintenance practices [1215] and the Ambystoma Genetic Stock Center (https://ambystoma.uky.edu/genetic-stock-center/).

2. Materials

2.1. Housing

  1. A large colony requires a recirculating aquatic system in a windowless or a blacked-out room. Our laboratory has two Aquatic Habitats® Z-Hab Systems (Pentair Aquatic Eco-systems, Inc.), but newer models such as the Iwaki LAb-REED systems will serve the same purpose. Each system houses ~100 adult axolotls, which are sufficient for establishing a breeding colony.

  2. Food-grade plastic storage containers, at least 4.5 L in volume, for housing adult animals off the recirculating system.

  3. Food-grade Choice 8 oz. clear deli containers (500/case) (Item # 127DM8BULK, Webstaurantstore.com), for housing young juveniles.

  4. Food-grade Choice 2 oz. clear plastic portion cups (2500/case) (Item # 127P2C, Webstaurantstore.com), for housing larvae individually.

  5. Cafeteria trays for storing dishes and cups with young animals.

  6. Insulation tape for sticking onto bottoms of housing trays.

  7. MetroMax I open grid cart with rubber casters 24″ × 48″ (Item # 461X556BGX3, Webstaurantstore.com), for storing containers and trays with animals.

  8. Vented Brute® 32-gallon trash can, with a lid and a dolly, for storing housing water (SKU: FG263200GRAY, FG263100GRAY, FG263100GRAY, Rubbermaid Commercial Products).

  9. Scotch-Brite® Little Handy scrubbers, for cleaning water barrels.

  10. Stainless steel tube brushes for cleaning containers and dishes.

  11. Fishnets (8″, 5″, and 3″).

  12. Room temperature controller (Schneider Electric).

  13. Janitorial bucket 26 quarts with a side press wringer (Lavex®).

  14. A floor mop, such as Unger SmartColor RoughMop String Mops Heavy Duty, with a handle.

  15. A squeegee broom, such as Unger AquaDozer® Heavy Duty, with a mop handle.

  16. A dustpan with a broom.

  17. A disinfectant cleaner.

  18. A stainless sink (Aero Aerospec Sink 1-bowl 20″—2F1–2020-30LR).

2.2. Water Supply

  1. In-house reverse osmosis deionized (RO/DI) water will provide the best source for clean water, which can be used to make salamander housing water at pH 7.0, 1000 μS/cm.

  2. City water can be used depending upon local water quality. We triple filter Boston City water with one in-line 20″ sediment and two 20″ carbon filters.

  3. Ninety-gallon storage tank and distribution system (Pentair).

2.3. Diet

  1. A simple brine shrimp hatchery can be constructed out of a sizeable upright square cooler (>45 cm in height). A small fluorescent light bulb can be used to generate enough heat (~30 °C) and light to trigger egg hatching.

  2. A hatchery cone with a stand for hatching brine shrimp (Brine Shrimp Direct).

  3. A Tetra Whisper Aquarium Air Pump.

  4. A rotifer sieve for collecting brine shrimp (Brine Shrimp Direct).

  5. Artemia cysts (such as INVE Aquaculture).

  6. California blackworms (Eastern Aquatics).

  7. Rangen 3/16″ Soft Moist Salmon Pellets for feeding small axolotls (Aquatic Foods and Blackworm Company).

  8. Rangen 1/8″ Soft Moist Salmon Pellets for feeding adult axolotls (Aquatic Foods and Blackworm Company).

2.4. Water Quality

  1. Sea salt for making salamander housing solution (Instant Ocean, Pentair).

  2. Baking soda for making salamander housing solution and cleaning tanks (such as Arm & Hammer).

  3. eXact® Eco-Check® Photometer Kit, for monitoring water quality (Industrial Test Systems, Inc.).

  4. Kordon AmQuel Plus, for removing ammonia products from housing water (#AM75P, Pentair).

  5. Kordon NovAqua Plus, for removing heavy metals from housing water (#NA5P, Pentair).

  6. Fisherbrand polypropylene rectangular carboy with a spigot, 20 L, for storing conductivity solution (03–007-648, Fisher Scientific).

  7. Carbon filters for the flow-through system (gpe50–20, Pentair).

  8. Pad filters for the flow-through system (PF11P4-A, Pentair).

2.5. Anesthetic

  1. Salamander housing water.

  2. 95–100% EtOH.

  3. Benzocaine powder.

3. Methods

3.1. Housing

Just a few adult male and female animals are needed to establish a new colony, which can be kept in free-standing tanks of at least 4.5-L capacity if the water is changed every other day. Axolotls should be housed in a temperature-controlled, windowless room on a 12:12 light/dark cycle.

To grow the colony, mate adult animals and raise the resulting larvae in individual dishes to prevent overcrowding and cannibalism (see Note 1). Larvae and juveniles should be transferred to larger containers when they start outgrowing their dishes.

When the number of adult axolotls in free-standing tanks becomes challenging to manage, it is time to establish a housing system with automated water exchange. Recirculating systems for zebrafish (without heating element) are well suited to hold axolotl tanks because they control the number of contaminants in the water [16]. However, axolotls are sensitive to strong water flow, so only adult animals should be housed in a standard recirculating system. Even then, axolotls are most comfortable if new water is introduced to their tanks as a slow drip. Ten percent daily water exchange is optimal. A forceful stream can be turned on to flush out foul water but return to low flow as soon as possible. However, the best way to routinely clean system tanks is to remove waste and uneaten food with a serological pipette. Water flow can injure or upset smaller animals, so juveniles and larvae should reside in size-appropriate free-standing containers with salamander housing solution.

Stress and subpar water quality are the chief drivers of disease in axolotls (see Note 2). The best way to ensure the colony’s health is to closely monitor the temperature in the animal facility, handle the animals with care, and check the water quality regularly. Axolotls thrive in water cooler than room temperature; the ideal water temperature is 16–18 °C (see Note 3). Installing chillers for the flow-through systems will help maintain the optimal temperature of the system’s water. The ambient room temperature should be kept below 24 °C at all times.

If the number of staff is limited, such as during holidays or quarantine, the animals can be fed and their water changed twice a week. This excludes larvae, who still need to be fed brine shrimp and have their water changed daily or every other day. If operating on this reduced schedule, it is vital to continuously monitor animal health for signs of stress or disease. Any ill-looking animals should be removed from the recirculating system and housed in free-standing containers (see Note 4). Tanks should be cleaned if they are dirty, regardless of the schedule.

Often, washing filthy tanks with water is not enough to remove the slimy yellow residue. However, no soap or detergents should ever come into contact with any of the axolotl equipment or cleaning tools because they are toxic to the animals. Food-grade sodium bicarbonate (baking soda) is a suitable replacement for soap when scrubbing housing containers, and it also possesses mild antimicrobial properties [17].

In a recirculating system, tanks are filled with water from tubes that are connected to supply lines. Over time, the insides of those tubes accumulate dirt. A short burst of forceful water flow removes some of this debris. To remove some of the remaining debris, put the tubes in a container with a concentrated sodium bicarbonate solution and leave it on a magnetic stirrer with a stir bar for several hours. Rinse the tubes well before replacing them.

Axolotls of all sizes can jump out of their tanks or containers and desiccate to death if not discovered in time, so it is best to keep them covered with lids or trays. However, young animals may suffocate if their dishes are full to the brim and covered very tightly, such as with other trays in a stack. To prevent sealing of containers in a tray stack and ensure air exchange for animals, attach insulation tape to the bottom side of the trays (Fig. 1).

Fig. 1.

Fig. 1

Housing juvenile axolotls. (a) Young axolotls (up to 1 cm in length) can be housed in food-grade plastic dishes and stored on stacked cafeteria trays. (b) Insulation tape on the bottom of trays prevents animals on the tray below from suffocating

Axolotls’ sudden bouts of agility are best kept in mind when designing the animal room and planning the placement of recirculating systems. Even large axolotls sometimes manage to escape their tanks. Cleaning is especially risky since it requires sliding off lids to allow in serological pipettes and animals are agitated by the commotion. If a salamander does disappear in the system’s innards, partially disassemble the system to rescue. This is easiest if all sides of the system are readily accessible and not against a wall. To keep animals from jumping during cleaning, avoid touching them with the serological pipette and replace their lids the moment they start thrashing around their tanks. They will calm down in a minute and not object to resuming the cleaning.

Even if an agitated axolotl does not jump out, it may knock a water supply tube out of its tank. If not discovered within an hour or two, this tube will flood the room. In anticipation of an inevitable deluge, a well-designed animal room will have a drain in the floor, and the floor will be slanted toward it and away from any doors. A squeegee broom is also useful for quickly drying the floor. After a flood, monitor the pH and salinity of the system’s water, as they may drop due to the large water loss. To prevent flooding, check for water drips from tanks and for puddles on the floor after cleaning and ensure that no water supply tubes are out of the tanks.

3.2. Water Quality

Water quality is measured by its pH, salinity, mineral levels, toxic contaminants like chlorine, and waste products. All these parameters must be monitored regularly, at least monthly. pH and conductivity should be checked daily.

Housing water should be at neutral pH; the acceptable range is 7.0–7.5. If the pH is too low and the recirculating housing system lacks a pH adjustment tool, the pH of the water can be increased by slowly introducing a solution of sodium bicarbonate (20 g to 1 L RO/DI water) into the recirculating system, such as by pouring it into an empty tank and setting the drip to low. Never add any solutions directly to occupied tanks because sudden water changes will distress axolotls.

Axolotls are content with water of varying conductivity, from 500 to 2300 microsiemens (μS)/cm [14, 15]. In our experience, maintaining adult housing water salinity between 850 and 1000 μS/cm works well. Flow-through system salinity is adjusted with a high salinity solution by filling a 20 L carboy with deionized water and dissolving 500 g of sea salt in it. Conductivity solution should be introduced either through a recirculating system-specific installed salinity adjustment bucket or an unoccupied tank with drip set to low.

Some of the biggest threats to axolotl wellbeing are ammonia and its products, nitrite and nitrate. Ammonia is especially toxic. It is released by animal waste and decomposing uneaten food, which is why any debris should be removed from tanks and dishes as soon as possible. Young animals are particularly sensitive to dirty water. AmQuel Plus and NovAqua Plus conditioners help control ammonia levels and its products. These can be added directly to the flow-through system according to water volume in the system. In free-standing water, add each conditioner according to manufacturer directions to every 32 gallons of salamander housing solution. Conditioning housing water may change its pH and conductivity, and regular monitoring of water quality is advised.

Recirculating systems also contain biological filters, where bacteria convert ammonia into nitrite and then relatively safe nitrate. However, those bacteria require a period of adjustment in a newly established recirculating system, and ammonia levels should be monitored daily in the beginning [14]. Target levels for ammonia and nitrite in the housing water are zero.

Recirculating systems can operate on municipal tap water depending upon water quality, which is usually treated with chlorine or chloramines. These should be removed from water before it reaches the system. We filter tap water in one 20″ sediment filter and two 20″ carbon filters before storing it in a 90-gallon recirculating storage tank. To dechlorinate free-standing salamander housing solution, it can be prepared from deionized or distilled water with chlorine, treated with AmQuel Plus, and allowed to rest for 24 h before use. We find that carbon filters can be changed once a month and pad filters switched out once a week. Systems relying on carbon or reverse osmosis filtration require regular monitoring of water for chlorine to ensure proper filtration.

3.3. Diet

Animals living in recirculating systems thrive on large salmon pellets. A serving of 5–7 pellets 2–3 times a week is sufficient for an adult axolotl. Juveniles that are almost large enough to move into the systems (about 10 cm long) can also be fed 3–4 large pellets at the same intervals. It is critical to avoid overfeeding animals with pellets because any uneaten ones will soon decay and contaminate the housing water. If leftover pellets are routine, reduce portions.

Smaller juveniles (about 5 cm long and larger) can eat small salmon pellets (5–7 at a time), and their diet can be supplemented with brine shrimp (Artemia). However, juvenile axolotls grow fastest on California blackworms (L. variegatus) [18]. For animals too small to swallow an entire worm, the worms can be cut up with scissors or a transfer pipette. Overfeeding with blackworms is acceptable, and any leftover worms can be reused for the next feeding.

California blackworms should be stored in shallow levels of salamander housing solution at 4 °C. There is no need to feed them. They crawl up the walls of their container, so it should be covered but not airtight. The worms should be washed daily with copious changes of salamander housing solution until the water runs clear and most floating dead worms are gone. White clumps of decayed dead worms should be removed with a transfer pipette. If there are any leeches attached to the container’s bottom, they should be wiped away with a paper towel. Avoid feeding dead worms and leeches to axolotls.

When axolotl larvae lose their yolk, which is indicated by the loss of dark pigmentation, they can be introduced to brine shrimp. Add generous amounts of hatched brine shrimp into their dishes with a pipette and let the larvae feed for at least 1 h. When they are full of shrimp, their stomachs become round and orange. Very young larvae should not be left in the water with brine shrimp overnight, as the shrimp die in fresh water and can be toxic. Older larvae are more tolerant. Young axolotls can live on this diet until they are big enough to eat cut up blackworms. If the supply of blackworms runs out and some juveniles are still too small for salmon pellets, they can survive off brine shrimp or even crushed salmon pellets, although most young axolotls refuse them.

Brine shrimp (Artemia) should be hatched from cysts daily according to the manufacturer’s instructions. A clear hatching cone works very well, as it allows to easily discard most unhatched cysts floating at the water surface and dead shrimp at the bottom. Because brine shrimp hatch in water with high salt content, they should be thoroughly washed with salamander housing solution after being harvested. Allow the collected shrimp to sit in a container full of salamander housing solution for at least 10 min before serving them to axolotl larvae so that any dead shrimp sink and any unhatched ones float. The layer of live shrimp will be in the middle, and they also swim toward light. Larvae should only be fed live shrimp.

Sick adult axolotls ignoring salmon pellets may instead accept blackworms. If an axolotl is so ill that it does not hunt its worms, dangling them in front of its face from a transfer pipette may entice it to eat. This deluxe diet may help the animal recover.

3.4. Anesthesia

It is imperative to properly sedate and anesthetize axolotls before carrying out any surgeries, injections, or other procedures. The best way to administer anesthesia is to take advantage of the axolotls’ permeable skin and bathe them in an anesthetic solution.

Benzocaine is an efficient and economical choice of anesthetic. To prepare stock benzocaine solution, fully dissolve 1 g of benzocaine powder in 30 mL of absolute ethanol and add 970 mL of deionized water. For temporary anesthesia, prepare a 0.01% solution by diluting one part of stock benzocaine in nine parts of housing solution (by volume). For terminal anesthesia, prepare a 0.05% solution by combining stock benzocaine and housing solution in equal volumes. To ensure death after terminal anesthesia, animals must also undergo pithing. It is important to remember that prolonged exposure (several hours) to 0.01% benzocaine can also kill axolotls, so avoid leaving animals in anesthetic for longer than necessary.

Other possible anesthetics, with varying concentrations and delivery methods, are propofol, tricaine sulfonate, butorphanol, and buprenorphine [1921].

The time for axolotls to become anesthetized depends on their size. Juveniles typically are fully sedated after 15–20 min in the anesthetic, while large adults can take 45 min to an hour. Monitoring animals is critical. An anesthetized animal will not react to a gentle poke or protest if flipped upside down. However, anesthesia will start wearing off after the animal is removed from the solution. To prolong sedation during a surgery, soak a thin, lint-free piece of tissue in the anesthetic solution and cover the animal, especially its gills. This precaution will also protect the animal’s skin from desiccation.

After the surgery, return the animal to its housing tank or allow to regain mobility in shallow water under the covers of wet thin tissue.

3.5. Courtship and Mating

Axolotls reach sexual maturity around the age of 12 months. Males develop dark nails, cloacal bulges, and deep costal grooves; females grow broad and round bellies due to an internal egg supply [12, 15]. Prior to the onset of sexual maturity, a genotyping assay is the only way to distinguish males from females [22].

The axolotl breeding season begins in midwinter and ends in late spring. Axolotls are reluctant to procreate midsummer to late fall, although an unvarying artificial light schedule minimizes this seasonality. Males can be mated once a month, and females once every 2–3 months. To breed axolotls, put a male and a female together in a large tank that is lined with foil on the outside to protect them from light. Place terracotta clay saucers or flat natural rocks on the bottom of the tank so that the male can attach his spermatophores to a textured surface. Put several reusable ice packs in the tank as well, so that the water stays cool. Ensure a sufficient supply of housing water and leave them together overnight. There is no need to provide food.

Soon after being introduced, the male deposits spermatophores and begins a courtship dance. He walks in front of the female, prods her with his snout, and waves his tail, secreting pheromones from his cloaca [23]. Responding to his odorants, the female follows him and takes up the spermatophores with her cloaca, storing them [24]. After the female takes up the spermatophores, remove the male, put one or two mesh nets into the breeding tank, change out the ice packs, and let the female rest in solitude.

The female will usually begin to spawn within 12–24 h. Eggs leave her oviducts and enter the cloaca, where they encounter the spermatozoa from the stored spermatophores and become fertilized [15]. The female coats the fertilized eggs in a thick, sticky layer of clear jelly. She then climbs into a mesh net and lays the eggs in strings. Avoid disturbing her as much as possible. When she takes a break, collect the eggs from the net with a cutoff transfer pipette or a razor blade. This process can be delayed by putting the female into a 4 °C refrigerator for several hours or overnight. Refrigeration will slow down the spawning and the development of eggs. If the female does not begin to lay on the same day but it is important to know exactly when the eggs are fertilized or to work on recently laid embryos, it may help to set up a tablet in front of the breeding tank and use baby-monitoring software to observe the female remotely overnight (Fig. 2).

Fig. 2.

Fig. 2

Remote surveillance of an egg-laying female. The female is preparing to spawn in a temperature-controlled incubator, and a tablet is being used to monitor the process during the night

Within one spawning, a single female can lay from 200 to 1000 eggs. Ensure that she is done laying before transferring her back to her housing tank. It may be useful to keep track of successful breeders to facilitate future spawnings.

Occasionally, breedings fail. Some potential couples ignore each other in the breeding tank and the male does not lay any spermatophores even over the course of several nights. If natural spawning is difficult to achieve, embryos can be generated via in vitro fertilization.

Adult axolotls can be induced to breed with hormonal treatment. Females lay eggs after being injected with follicle-stimulating hormone [25], and an injection of human chorionic gonadotrophin can induce both spermiation in males and ovulation in females. Gentle massage of the animals’ abdomens can promote deposition of gametes. In vitro fertilization is carried out by mixing sperm with eggs and then adding fertilization saline solution. High pH of the solution improves the hatching rate [26]. Axolotl sperm can be refrigerated and remain viable for up to 28 days [27].

4. Notes

  1. Largest juveniles nip limbs and tails of their smaller siblings.

  2. Even though axolotls are usually resilient against bacterial infection, chronic stress (caused by poor water quality, high temperature, or a physical injury) may undermine this resistance. Axolotls are vulnerable to fungal and viral infections. Albino larvae may be especially at risk, possibly due to their weaker immune systems [28].

  3. Axolotls living at a constantly elevated water temperature (greater than 18 °C) may be at risk of swelling of the body cavity. Exact causes of this common amphibian disorder are unknown, and it may also be a symptom of other diseases such as flavobacteriosis [29, 30].

  4. Ill or stressed axolotls typically float at the water surface, refuse food, shed skin, and/or have curled tails. The bright red gill branches may grow paler and smaller. The animals may also become pale in color themselves (except for the dark wild-type axolotls).

Acknowledgments

We would like to thank the Ambystoma Genetic Stock Center for providing all the animals and methodologies these methods have been built upon. We also thank Jackson Griffiths for taking the pictures and Michelle Lim for lending the camera.

Grant Sponsor

NSF grants 1558017 and 1656429 to JRM. NIH grant HD099174 to JRM. Material and information obtained from the Ambystoma Genetic Stock Center funded through NIH grant: P40-OD019794.

Footnotes

Competing Interests Authors declare no competing interests.

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