Raising Tads and Frogs
Petri Dish Culture
Download a copy of our published protocol here.
Stage 1 to Stage 43
We have 2 methods for raising embryos from a natural mating, as described below.
Method 1: Leave embryos in bucket to stage 30 - 32.
After ovulation, the mating bucket can be left undisturbed when raising embryos or collecting later stages. This allows production and fertilization of the maximum number of eggs. The number of fertilized embryos produced can be well over 3000. Allow embryos to develop undisturbed in the mating bucket at 24 – 25°C, to stage (28 - 32). Note there is no cysteine treatment in this method. The percentage of developing embryos is found to be improved when frogs are left in the bucket for up to 36 hours after boosting, instead of being removed the morning following boosting. The reason for this is unknown, but it is thought perhaps the movement of the frogs aids in water aeration . Embryos ready to be removed will be flat, may “flutter” when disturbed, but will return to resting; they are not actively swimming. They are often seen attached to the side of the mating bucket by the cement gland. At this point, embryos can easily be removed to a petri dish (Falcon, 150 mm) containing fresh 1/9X MR with a plastic pipette. Here they are raised to stage 43, then transferred to the main housing system for continued growth (see Free Swimming Tadpoles section). This method avoids the labor of de-jellying and sorting, and the embryos do quite well.
If contamination of embryos in the dishes becomes a problem, evidenced by fungal growth or disintegration of embryos, 1/9XMR media can be supplemented with gentamicin antibiotic at 100 µg/ml (Sigma, G3632). We keep a gentamicin 1000x stock at 4ºC.
Method 2: Remove from mating bucket and raise in pertri dish.
After fertilization, embryos can be removed from the mating bucket the day following ovulation and raised in Petri dishes in 1/9 X Modified Ringers solution until they are ready to go into the housing system. Gentamicin can be added at a dose of 100 µg/ml to the media to retard bacterial growth if necessary. We keep a gentamicin 1000x stock at 4ºC. Keep plenty of fluid in the dish to avoid evaporation.
Number per dish
The number of embryos in a dish is critical to survival. We keep 150-200 embryos per 150 mm petri dish. In 90 mm petri dishes, we keep no more than 75-100. In 60 mm petri dish, no more than 50-60. Over-crowding will kill embryos. Low density and the use of antibiotics in the media if needed will make the culturing of embryos simple and routine.
A light treatment of 2-3% cysteine will partially remove the jelly coat from the embryos and cause them to dissociate slightly. Usually we cysteine in a 250 ml erlenmeyer flask with gentle swirling for 30 seconds-1minute, or until the embryos separate and are no longer in clumps. Much of the jelly coat will remain, but the partial de-jelly will make sorting the embryos much easier. If you do wish to completely remove the jelly coat, then it is wise to coat the dish with agarose or 1XMBS+0.2% BSA. Embryos without a jelly coat will stick to the bottom of a petri dish.
It is important to remove the dead and dying embryos from the dish. Dying embryos will foul the water and kill others. Check for dead/sick embryos once a day until the larvae are moved from the dish to a tadpole rearing tank. The embryos are kept at 25-28ºC, depending on the desired rate of development, and hatch at Stage 24 to 26. Embryos can also be raised at 22ºC but we don't raise them at any lower temps (although after gastrulation they can tolerate 16-18ºC for 12 hours). We recommend changing the 1/9xMR+/- Gent daily as well.
At Stage 43, typically after 60 hours at 27-28ºC, the larvae are free swimming. They are brought to our tadpole nursery at this point.
Free Swimming TadpolesStage 41 to Stage 63
In general, raising embryos from fertilization to Stage 41 (Day 2 to 3 at 27ºC) is straightforward. The key is to keep the embryos at low density and perform daily media changes (gentamicin optional). We have been quite successful at these stages, provided dead and dying embryos are removed. The transition to free-swimming tadpoles can be more problematic. The first two weeks are an extremely fragile time for tadpoles, and they require great attention. Sudden, even minor, changes in the quality of the water can lead to widespread death.
Currently, our greatest success in the critical early period utilizes a recirculating aquaculture system as a nursery. We have found a stand-alone rack system to be inferior to a rack integrated into a larger recirculating system. The stand-alone rack does not have adequate waste filtering capability, resulting in large amounts of debris building up. Also, there is more potential for sudden changes in water quality, due to the smaller volume of water. The larger system with adequate filtering, such as a drum filter, does a much better job of waste management, and water quality tends to be more stable.
The water quality in our system at Yale is very stable. As a result, we have few problems raising large numbers of tadpoles. See our Environment section for information on parameters such as temperature and conductivity, and water quality issues in our facility.
There are a variety of recirculating systems available. See our Housing Systems section for more information on the systems we have, and our experiences with them. We recommend contacting a number of companies to see which would best suit your needs. Be sure to ask what modifications they have made to deal with the copious waste produced by the frogs.
The tadpoles can be introduced into the recirculating system when they are free swimming (st. 41). At earlier stages, the tadpoles seem to do poorly. We pour a petri dish containing the tadpoles in1/9X MR (+/- gent) directly into a tank filled with system water. An initial trial comparing sudden introduction (pouring the tads straight into a full tank) versus slowly introducing them to the water at this stage did not seem to make much difference (Grammer, TC unpublished observation). We replace the normal tank drain with a baffle lined with mesh to reduce the loss of tadpoles (mesh supplied by Aquatic EcoSystems). The image below shows a baffle with the mesh tadpole liner on the left, and without the liner on the right.
It is desirable to raise a clutch (sibling tadpoles from a single mating) in 2-3 tanks if space permits. If anything happens in one tank, the entire clutch is not lost. We do have occasional sudden and unexplained deaths in an entire tank when a neighboring tank with the same clutch of tadpoles is fine.
Concerning density, very small tadpoles at this stage have been shown to do better at higher densities. We use a 4 – 6 liter tank for up to 100 tadpoles. Numbers are thinned as they grow, to a final density of 20 - 25 tads per 6 liter tank (3-4 per liter) at 2-3 weeks of age. Thinning is done by selecting for the largest, fastest growing tadpoles.
The initial tank drip rate is set to 2-3 drops/second. The inflow hoses and valves should be monitored to ensure consistent flow. The drip rate is gradually increased, such that by 2 weeks it is a steady stream. The drip rate needs to be fast enough to clear the water, but not so fast the tadpoles appear to be suffering ill effects, such as floating and loss of equilibrium. They prefer stiller water over a river. If the water seems murky and stagnant, or the surface is covered with bubbles, increase the drip rate gradually over several hours, until the water is clean. Increasing the drip incrementally prevents shocking the tadpoles.
We monitor water level and change out the tube baffles as needed to prevent clogging and tank overflow. The tanks are changed every week, beginning one week after introducing the tadpoles to prevent excessive uneaten food and debris accumulating on the bottom of the tank. The change involves gently pouring the tank contents into a fresh tank. The tads do not appear to be negatively affected by this.Feeding
We have traditionally used a Sera Micron Solution (order from SeraNorthAmerica, 800-659-1970) as a sole diet for the first two weeks of life in the recirculating system. We have had great success with this high protein diet; the tadpoles thrive on it. It contains a variety of food sources such as spirulina, krill, shrimp, nettle, egg and cod liver oil, ground into a very fine powder.
Prepare the solution by suspending 1 tsp Sera Micron in 250 ml of system water. Shake well. (Note: Dry, unsuspended Sera Micron added directly to the tank does not disperse as well.) 10-15 cc of the solution is added to the tank as soon as the tadpoles are introduced, and the tadpoles are monitored. Normal tadpoles will be hungry, and should clear the water within 30 minutes. Tadpoles are fed twice daily. The volume of solution is increased by 10 ml each week, to a total of about 45 ml.
Other types of food including unmodified Nasco Tadpole Diet and ground rabbit chow have been tested on young tadpoles. These two foods led to a much higher mortality and so the trials were stopped. Sera Micron remains the diet of choice.(Trott, KA and Grammer TC unpublished observations).
Sifted Tadpole Diet
At 14 days of age, we begin adding a mixture we call Sifted Tadpole Diet, in addition to Sera Micron. The supplemented diet introduces a higher protein content and solid food for metamorphosing tadpoles and young froglets.
To make the Sifted Tadpole Diet, sift Nasco Tadpole Powder Diet to separate the fine powder from the small granules. (Reserve the granules for later as they can be useful to transition froglets to the larger size Nasco Post-metamorphic pellets.) Next, grind up some fish flake food almost into powder. The fish flakes should be ground up enough to be about 60% fine powder, and 40% small flakes. This can easily be done with your fingers in a dish or cup. We have been using a tropical fish type flake TetraCichlid Flakes (Tetra-Min, Aquatic Ecosystems) which seems to work fine. Mix approximately 1 part fine Nasco powder with 1 part fish flake.
The initial feeding is 1/16 tsp sifted diet twice a day. This is gradually increased to 1/8 tsp twice a day by the hindlimb bud stage. These amounts are guidelines based on density of 20 - 25 tads per 6 liter tank and should be adjusted for number of tadpoles and amount of food being consumed. Sera Micron continues to be fed as well.
Repeat feedings only if the last feeding has been eaten. A tank in which all the food has been eaten will have a clean bottom, clear water, and often the tadpoles will be actively skimming the bottom in search of food. These tadpoles need refeeding.
|These tadpoles have plenty of food in the water. The water is tinged green from Sera Micron, and the tadpoles are actively filtering food from the water in a typical tail up, head down, still position.||These tadpoles have eaten all the food and will be re-fed. The tank is clear, the bottom is cleaned of excess food, and the tadpoles are actively searching for food.|
In tanks with excess food, uneaten food will remain on the bottom and foul the water. You can see the food build up on the bottom as a yellow layer, and often the surface of the water will have bubbles on it and look scummy. This is an indication to change the tank, and feed less food.
Density and General Care
By 3 weeks of age, the tadpoles should be at a density of 3 to 4 per liter. We find lower density at this stage often results in faster growth and metamorphosis. Many researchers believe volume and/or surface area of water is as important to growth rate as density of tadpoles.
It is important to avoid sudden changes to the tadpole's environment. This includes rapid changes in pH, ammonia, temperature, and amount of debris in the tank. The temperature in the tadpole tanks is maintained between 24 - 26 degrees Celsius, and the pH around 6.5 - 7. We have found a sudden change in temperature or pH can be more detrimental to survival than a consistently low/high temperature or pH. In general, be sure to perform any changes gradually.
One issue to be aware of is possible super-saturation of water from having too many bubblers in the sumps. Bubblers help oxygenate and circulate the water. In a system where the sump is directly below the rack, such as in a free-standing system, too many bubblers may cause the water to become supersaturated. The tadpoles may become bloated with air, float at the surface of the water, and die. If you have a smaller recirculating system, consider using fewer bubblers than come with the system, and monitor your metamorphosing tadpoles for signs of gas problems. The gas disease should resolve after reducing the number of bubblers, or increasing the volume of water in the system (more sumps).
The importance of careful monitoring for changes in behavior cannot be over-stressed. The behavior of frogs and tadpoles is an important clue to the condition of the water. Adult frogs may be overly excitable or extremely lethargic. Tadpoles quickly display signs of distress including gasping, floating at the surface and loss of equilibrium. These signs can be due to a problem with water quality, lack of food, too much food, or a variety of other problems. Immediate troubleshooting is required when a problem arises. This is especially important with tadpoles, as all the tadpoles in a tank can die very quickly under unfavorable conditions. Having a Frog Technician who is dedicated to maintaining your system and experienced in animal behavior is well worth the investment.
It is worth noting we have found healthy tadpoles do sometimes lie on the bottom of the tank. The reason for this is not clear to us, but they do not seem to suffer from illness, and develop into normal frogs.
Tadpole Rearing without a Recirculating System
Attempting to raise tadpoles in a homemade drip system can be difficult. Facility water may lack adequate salts/buffering for young tads and continuously shocks them. The pH of the facility water can fluctuate from 5.5-9, depending on weather conditions and other factors. A commercial system equipped with monitoring and dosing equipment can properly maintain the pH, conductivity and other parameters crucial to tadpole survival.
Raising tads in small static tanks can be done but is a laborious task if you are trying to raise hundreds to thousands of tadpoles, due to the frequent water changes necessary to maintain water quality.
The tadpoles we raise take four to eight weeks to reach metamorphosis. Metamorphosis is a delicate stage. The froglets can die by drowning or exhaustion as they try to stay at the surface to breathe, especially in tanks with no fixtures (drains) to hang on to.The picture below shows froglets hanging on to the drain baffle. They can stay at the surface without the support, but it requires much more effort to stay there, using valuable energy needed for growth. It is important to include items such as floating plastic mesh squares for this purpose (Sefar), if there are not interior fixtures in the tank.
Metamorphs also eat less, as their digestive system is undergoing significant changes. Ensuring the tadpoles are well fed prior to metamorphosis seems to result in healthier, more vigorous froglets, which begin to eat sooner and have a lower mortality rate.
Once a tadpole has completely metamorphosed (st. 66), we refer to it as a froglet. As froglets appear, they are transferred to a separate froglet tank. Density in the froglet tanks is 3 - 4/liter. We have a number of different sized tanks in our facility, and adjust the number of froglets accordingly. It is important to have enough froglets in the tank to encourage feeding behavior. Solitary froglets or those kept at low density may not be adequately stimulated to feed.
At the froglet stage, a diet consisting of a blend of 3 parts the larger chunks of Nasco Tadpole Powder Diet (reserved when making the Sifted Tadpole Diet), and 2 parts 1.2 mm size Bio-Vita pellets from Bio-Oregon.
We feed new froglets 3–5 pellets per froglet twice a day. This amount is gradually increased as the froglets grow. We generally increase the amount of food when the froglets are eating the first feeding within about 30 minutes.
At about 4-5 months of age, the froglets transition to a blend of 3 parts larger BioVita pellets (2.5 mm) with 2 parts Nasco Post metamorphic Frog Brittle (Frog Brittle for Post-metamorphic Xenopus, Catalog #: SB29028(LM)M). They are fed as much as they will eat in 15 minutes, twice a day, 5 days a week.
The frogs are maintained on this diet for their adult life. At sexual maturity, they are transitioned to an every other day feeding regime. Sexual maturity is judged by egg production in the female (usually about 6-7 months of age), and appearance of nuptial pads in the male (usually about 4 months of age). The guideline amount is 1/16th tsp. every other day. This is adjusted with number of frogs in the tank, as well as sex of frog. Female frogs are given an extra feeding once or twice a week to encourage production of many, high quality eggs.
Note: We greatly prefer a diet called HBH Frog and Tadpole Bites purchased from Pondside Herp Supply in place of the Bio-Vita pellets. We found the smallest sized HBH pellet to be a superior diet for small froglets. It is high in fat and protein, and very palatable. Unfortunately, this size pellets does not seem to be available anymore.
From the age of 4 to 5 months on, the healthy adult frogs require very little care. We maintain a frog density of 3 frogs per liter. Our very important screening frogs are housed in smaller "library rack" tanks, (pictured below) on our wet rack. The tanks are changed every month. The water flow keeps them quite clean in the interim.
Note: The flow to our small size "library tank" racks is turned off during feeding, as the food tends to wash out of the tanks. The flow to other tanks in the system is not turned off.
We conducted a trial raising and feeding brine shrimp to the tadpoles and froglets. Tadpoles did not seem to be interested in eating the shrimp, and we did not notice any change in growth or health. The froglets did seem to eat the shrimp, but this did not justify the amount of effort and time involved in raising the shrimp. As a result, we do not use the shrimp at this point.
We have experimented with feeding California Blackworms (Aquatic Foods, email: email@example.com) to skinny or otherwise unhealthy frogs and froglets two to three times weekly. The frogs love the worms, and an anorexic frog may be encouraged to eat. We have found the large scale feeding of worms is not economically feasible. In addition, the frogs may become "fixated" on the worms, and refuse to eat a prepared diet. We find the number of sick frogs needing supplements negligible, and the effort in maintaining the worms outweighs the benefits for us. If you want to experiment with them, Aquatic Foods has information on care of the worms.
For information on treatment of sick frogs, please refer to the disease section.
Contributed by Maura Lane, K. Trott, Tim Grammer, and Mustafa Khokha
Updated by Michael Slocum and Maura Lane, 02/2017.
Many thanks to L. Zimmerman and N. Hirsch, and the Grainger web site for help with raising tads.