Maintaining Adult Frogs
The most convenient method of “holding” adult frogs (Rana pipiens) until needed is to render them inactive by reducing their metabolic rate. This method makes feeding unnecessary, reduces waste build-up, and reduces the occurrence of various frog diseases. In addition this method will maintain the frog in a specific physiological state (e.g., maturation, or pre- or post-ovulation, etc.)
The metabolic rate is lowered by refrigerating the frogs. If the frogs are to be held for 1– 3 days, they may be placed in a loosely covered pan with1 –2″ of water (tap water may be used). The pan should then be placed in a refrigerator at 0° – 5°C (32° – 41°F). For longer holding periods (1– 3 weeks) slight modifications are necessary. Cool the warm frogs for several hours in the refrigerator 0° – 5°C (32° – 41°F) before unpacking. Have a container of water (tap water may be used) 6 – 12″ deep, cooling at this time. This container must be well aerated at all times through the use of an airstone and air pump. Frogs may be placed in the container as soon as the above temperatures are reached. Change water at least twice a week with fresh water that has been pre-cooled to holding temperature.
Induced Ovulation
Pituitary Extract contains the lyophilized pituitary glands of seven female Ranid frogs in addition to 6 mg progesterone. Only gravid northern Rana pipiens should be used, and not the “southern” sympatric species which is not physiologically suited for induced ovulation protocols. The addition of the hormone progesterone has been found to be more effective in producing consistent ovulation success.
Since seasonal changes in pituitary potency and female sensitivity occur, the following table will serve as a general guide on the use of either collected pituitaries or pituitary extract.
Please consult the separate product literature sheet accompanying Ward’s Pituitary Extract for re-hydration instructions. Please note that you will need a syringe with a #20 gauge needle to inject the female. Syringes and needles can be obtained at a local pharmacy with a prescription or through WARD’S.
Injection procedure for inducing ovulation is as follows: Carefully, so as not to injure the ventral blood vessel or internal organs, insert the needle just through the skin and underlying muscle wall of the lower abdomen and inject the pituitary-progesterone suspension. Be certain to choose a gravid female to inject. Females have reduced nuptial pads on the “thumbs” of the forelegs. Gravid females will be more plump than their non-gravid counterparts because the abdomen will be full of eggs.
Place injected females in water approximately 11⁄2 –2″ deep and keep animals at a temperature between 18 –21°C (64 – 70°F). Injection should be done 48 – 62 hours before scheduled artificial insemination.
After 48 hours, check the enclosure for evidence of egg release. Eggs may require longer periods to “ripen” and observation of released eggs is the best indication that ovulation is proceeding. You may also “test” for ovulation by gently squeezing the abdomen towards the cloaca. To ensure a longer yield of mature eggs, do not extract them until 24 hours after the start of ovulation. Mature R. pipiens should yield between 1,000 and 2,000 eggs for fertilization.
Artificial Insemination
To obtain active sperm, either pith a male frog or overdose the animal using WARD’S Biocalm, an amphibian anesthetizing/euthanizing agent. Remove both testes, dissect away adhering tissues, and wash testes free of blood. Macerate in 10 – 20 ml of either pond water, dechlorinated tap water, or 10% Holtfreter’s Solution*. A convenient way to do this is to force the testes through a syringe with an #18 gauge needle. Large clumps may be broken by aspiration. Wait 15 –25 minutes for full sperm activity, which can be determined by examination under a compound microscope. The sperm suspension may be diluted to a total volume of 100 ml, if necessary, for very large egg masses. However, better results (e.g., more consistent rates of fertilization) are obtained by using the sperm suspension as prepared, without further dilution. Note that polyspermy may occur in concentrated sperm solutions with Octobercaught or long-held animals
Following confirmation of sperm activity, eggs are stripped from the female by holding her and applying pressure to the abdomen with the force directed towards the cloaca, thus squeezing the eggs from the ovisacs into a dry Petri dish. This is accomplished by bringing the legs of the female forward parallel to the abdomen. This assures that pressure is not dissipated laterally. The third and fourth finger are applied firmly over the throat and thoracic region to avoid dissipating pressure anteriorly. Remaining fingers are used to “milk” the abdomen toward the cloaca. Initially, rather sharp pressure may be needed to open the cloaca sphincter muscle; subsequently, gentle pressure will suffice to aid egg release.
Eggs are expressed in a circular pattern on the dry Petri dish. Due care must be exercised so as not to cause rectal prolapse by trying to obtain every “last” egg.
Expressed eggs are inseminated by pipetting the sperm suspension over them. Make sure that each egg comes in contact with the sperm suspension, although moistening is all that is required. Application of sperm suspension must be done prior to egg jelly swelling. After 10 –15 minutes, flood the eggs with medium. Use of a dry dish is recommended because eggs stick to its surface. This facilitates changing the medium. The spiral or circular pattern prevents eggs from clumping. After an additional 15 minutes, pour off the medium plus sperm and replace with fresh medium. Set the eggs aside for 30 –50 minutes. They then may be scraped free from the Petri dish and transferred to culture bowls. A single-edged razor blade, tissue section lifter, or scalpel should be used. Do not attempt to pull eggs free. About one hour after fertilization, those eggs successfully inseminated will rotate so that the black animal hemisphere is uppermost. After several hours, remove the unfertilized eggs to prevent decay. At optimum development temperature 18 – 25°C (64 – 77°F) cleavage will begin about 21⁄2 hours after fertilization, muscular response will be observed in about 96 hours, and hatching will occur in 5 – 7 days.
* Holtfreter’s Solution: Prepare a stock solution by adding 3.5 g NaCl, 0.2 g NaHCO3, 0.1 g CaCl2, and 0.05 g KLC to 1 L of glass-distilled water. Dilute this stock solution to 10% with distilled water before use.
Raising Tadpoles
About 3 –5 days after hatching, the tadpoles are ready to feed. They should be placed in an aquarium filled with tap water that has been standing overnight (to dechlorinate) or fresh pond water. Place an airstone in the water and keep the air pump constantly running. The number of tadpoles per liter of water may be as high as 100 immediately after hatching. As metamorphosis approaches (2 –3 months) the number must be gradually reduced to about five tadpoles per liter of water.
Since tadpoles are omnivorous, many types of food have proved suitable. Prepared fish food works well as does ground dry dog food, mashed hard boiled egg, chopped liver (raw or cooked), and boiled lettuce leaves. The diet should be varied from time to time. Only those foods which the tadpoles readily consume should be given. As with any aquarium set up, overfeeding must always be avoided. If the normally clear water starts to turn cloudy the tadpoles are either being overfed or have not consumed the particular type of food they are being fed. This is a dangerous condition. Be sure that the food given is being eaten and use only that amount of food which can be consumed in a short time.
As metamorphosis proceeds, a provision must be made so that the young frogs (now air-breathing) may climb from the water. This occurs very shortly after the front legs erupt from under skin flaps that have been concealing them during their development. For the remainder of its life the frog will be basically a “meat-eater”.
Feeding of Adult Grassfrogs
At metamorphosis, R. pipiens shift from an omnivorous to a carnivorous diet comprised of food that must be moving. They differ from R. catesbeiana and R. clamitans in that they do not or cannot take food while submerged. If animals are not held in hypothermic conditions, they should be fed 10 – 12 full-grown live crickets (2 – 3 times per week). Other food that is recommended include sowbugs, beetles, moths, and earthworms. Fly maggots are not recommended, since such ingested maggots may destroy the frog.
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