• About Us
• Husbandry
• Methods
• Members
• Publications
• Database
• Reagent Request
• Transgenic Frogs
• Screens
• Meadia
• Affymetrix
• Links
• Contact Mead Lab

Mead Lab: Husbandry

Assorted tank sizes in the X. tropicalis room.

Aquanode, UV and drum filter controllers. The self-cleaning drum filter is housed inside large white fiberglass container on the left. Particulate and carbon filter units are shown in the foreground.

X. tropicalis room reservoir. The large heating element is seen on the bottom of the reservoir in the foreground. The biological filter is inside the center section. Pumps keep the biological filter material in constant circulation.

Reverse Osmosis (RO) system in the X. tropicalis room. The plastic RO water holding tank is on the right.

More racks of tanks in the X. tropicalis room.

Fertilization:

We, and others, have noticed a marked difference in the viability of X. tropicalis sperm compared to Xenopus laevis sperm. This can result in very poor fertilization rates in vitro. This is helped by using bovine serum albumin (BSA; 0.1% w/v) in all solutions and rinse glassware and plasticware with BSA containing solutions immediately prior to use. Not all batches of BSA are suitable for embryo handling - a fact we discovered in our early attempts at nuclear transfer. We recommend testing new lot numbers for toxicity prior to routine use in the lab. We now use L15 media plus 10% calf serum (instead of MMR or MBS) for homogenization of testes. Fertilization is performed in 0.1x MMR and the eggs are dejellied with 2% cysteine in 0.1x MMR pH 8.0. Use X. tropicalis testes as soon as possible after harvesting. The viability of sperm drops at a remarkable rate. If you keep these things in mind, fertilization rates are as good as in Xenopus laevis.

Note: Thanks to the Amaya, Grainger and Harland labs for tips on in vitro fertilizations of X. tropicalis.

For injection experiments using Xenopus tropicalis, we now exclusively use natural matings to harvest fertilized eggs. This saves time and money as the males can be reused after a two-week rest. A protocol for collecting one-cell fertilized eggs by natural matings can be found in our recent Methods paper: Yergeau et al. (2010) Transposon transgenesis in Xenopus.

Tadpole Husbandry:

Our method for raising tadpoles, both X. laevis and X. tropicalis, is to allow embryos to grow in static tanks until metamorphosis. Approximately two weeks after the embryos reach froglet stage they are transferred to our flow-through re-circulating system. Tadpoles are raised in plastic tanks in X. tropicalis system water (~900 uS marine salts, neutral pH). The tadpoles are raised in a separate nursery room kept at 27oC and on a 12/12 light/dark cycle. Each tank is aerated with a small airstone at a slow rate; enough to break the surface of the water but not high enough to generate a significant water current in the tank. We feed tadpoles at least three times a day, seven days/week, with a suspension of Sera Micron (HerpSupplies cat. # Sera-0720). As we have a large number of tanks, we make up a fresh suspension in a lab squeeze bottle to facilitate the speed of feeding. We add enough food to slightly cloud the water - the amount varies with the density and age of tadpoles. A good rule of thumb is that the tadpoles should clear the water in less than 20-30 minutes. We prepare the food fresh prior to each feeding using X. tropicalis system water. Any left over food is disposed of and is not saved for the next feeding. We perform a ~50% water change once a week with fresh sterile system water. The tanks will grow some algae. We do not clean the tanks as the algae keeps the ammonia levels in check, and the larger tadpoles seem to eat it.

Froglet Husbandry:

Nasco frog brittle
(post-metamorphic pellets)

Juvenile frogs are transferred to the recriculating system approximately two weeks after metamorphosis. We have found that transferring froglets too soon after metamorphosis results in significant mortality. We also allow that frogs to acclimate to the slightly lower temperature of the aquarium for several hours before adding them to the tank. To do this, we place the frogs in a large screw cap beaker containing frog system water (80oF) and leave them in the beaker in the frog room (76oF) prior adding the froglets to their tank. We feed postmetamorph frogs ZooMed Aquatic Frog & Tadpole Food (HerpSupplies cat. # SZMZM16) and as the frogs grow the food is gradually changed to NASCO frog brittle in the smallest pellet form. Juvenile frogs are fed at least once a day for maximum growth rate. Enough food is added so that the frogs will clear the tank in 5-10 minutes. The young froglets grow at a remarkable rate and one must be careful to sort frog according to size as the larger froglets will attempt to eat the smaller ones - this is more critical for Xenopus laevis than Xenopus tropicalis frogs.

Adult Frogs:

Adult Xenopus laevis frogs are feed twice a week (Tuesdays and Fridays) with NASCO frog brittle. Adult Xenopus tropicalis are fed three times a week (Monday, Wednesday and Friday). For Nasco Frog Brittle ingredients see http://www.enasco.com/action/Static?page=xen_brittle.

Females are primed in the evening prior to egg harvest and placed in a large static container filled with fresh system water. After egg harvest, the females are placed in a static tank again overnight before being reintroduced to the system. This allows the frogs to shed all their eggs before reentering the system. This practice helps to decrease the load on the mechanical and bio-filtration systems.

Frog Identification:

We use two methods for identifying individual frogs in our aquarium. Individual frogs can be housed in separate small (2-5 gallon) tanks. This method of identification is not ideal as the frogs are social animals and do not thrive in isolation for prolonged periods. This can be alleviated, in part, by housing, for example, an identified male with one or two wild type females. Individual housing is also not optimal as it does not maximize the use of space in the aquarium. We now implant microchips into identified animals and the chips can be read at any time using a RFID reader. The chips have a unique 16-character alphanumeric code. We routinely use the last four digits of the tag in the name for each frog. We have developed a web-based database based on the chip ID for each frog (see Database section for screen shots of our frog database). Each frog page contains all data for the individual frog; including date of birth, transgenes, all outcross and in cross information, where the animal is located in the system and a family tree. Information on the implanting of microchips can be found in the Methods section. For this simple surgical procedure we anesthetize the adult frogs with isoflurane rather than tricaine as it is faster acting and the recovery time is also shorter.

MeadLab Aquarium:

The frog systems were designed, built and maintained by Aquatic Habitats (see links). The frogs are housed in two 400 sq. ft. rooms in separate aquaria, one for Xenopus laevis and one for X. tropicalis. A 200 sq. ft. preparation/microscope room also serves as the tadpole nursery. All system parameters are constantly measured and adjusted by a central computer (Aquanode) in each room. The automatic controller performs all system functions including maintenance of system parameters and daily water changes. Each system room has it own reverse osmosis (RO) water purification system that feeds a 220 gal holding tank. Each day we perform a 10% water change. Each system has an in-line, self-cleaning 20 um drum filter, a 1,000 gal reservoir that houses the bio-filtration media, a 20 um particulate standing filter, an activated carbon filter and two serial 100 watt UV sterilizers. The Aquanodes constantly monitor the following water conditions; temperature, pH, salinity, dissolved oxygen (DO), oxidative reduction potential (ORP) and system water pressure. The Aquanodes adjust the temperature, pH and salinity by dosing from reservoirs and by turning off or on in-line heaters and chillers.

Frog capacity reflects numbers of adults housed in the system. In addition, we can house 250,000+ tadpoles/metamorphs at any given time.