4. INDUCED BREEDING 



DEFINITION: The induction of breeding activity by experimental means and under labor- 

 atory conditions, outside of the normal breeding season. 



PURPOSE: To produce eggs and embryos suitable for laboratory experimentation at 

 specified times. 



MATERIALS: 



Biological : Rana, Bufo, or Xenopus adults (or other Anura). 



Triturus, Ambly stoma, Triton, or Eurycea adults (or other Urodela). 



Technical : Hypodermic syringe (2 cc. capacity) and #18 needle 

 Dissecting instruments 



Battery or aquarium jars with weighted covers 

 Spring or pond water 



METHOD: (General description for Rana pipiens; modifications for other forms appended. 

 Precautions : 



1. Animals must be sexually mature. The females should have a body length of 

 at least 74 mm. and the males should measure at least 70 mm. , from snout 

 to anu s. 



2. Animals should be well fed and in pre-breeding condition. Those animals 

 taken directly from hibernation are the best. 



3. Frogs should be kept in running cold water (below 1 S'-'C. ) in which case they 

 will be satisfactory for several weeks. Feeding is unnecessary during this 

 laboratory confinement, if they were well fed before capture. 



4. Avoid red-leg contamination. Examine frogs upon receipt. Red-leg is not 

 to be confused with temporary rash brought on by sudden changes in temper- 

 ature. The fungus condition known as red-leg is very contagious and tanks 

 with infected animals should be sterilized with permanganate. A copper 

 penny in the frog tank will give off enough metallic ions to keep the red-leg 

 under control. 



5. The fresh pituitary glands are injected into the abdominal cavity. Avoid in- 

 jury to the median ventral abdominal vein, the sub-cutaneous veins, and to 

 the internal organs. Glands should be used fresh but may be preserved in 

 alcohol, or by freezing in water. They are always larger and heavier. 



6. Female glands are twice as potent per gland as are the male glands. The 

 number of glands necessary to induce complete ovulation varies with the 

 season. 



7. Eggs should be allowed to accumulate in the uteri before stripping any of 

 them. Vigorous stripping is apt to damage the eggs. 



8. Pre-check the medium in which the sperm suspension is made to be certain 

 that the spermatozoa will survive. Spring or pond water are best. 



Control : It will not be necessary to run controls for this experiment in as much as 

 the major purpose is to secure eggs and developing embryos. However, ade- 

 quate controls for such an experiment would consist of the injection of another 

 equivalent endocrine gland, such as an equivalent number of adrenals or thyroids. 

 Such controls have invariably given negative results. 



Procedure : (Description for Rana pipiens; modifications for other forms appended. ) 

 1- Removal of the anterior pituitary gland : Insert large, sharp-pointed scissors 

 into the mouth of the donor, at the angle of the jaw. Cut posteriorly to a 



