INDUCED BREEDING 



DEFINITION : The Induction of breeding activity by experimental means and under laboratory 

 conditions, outside of the normal breeding season. 



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

 times. 



MATERIAIg : 



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



Triturus, Amblystoma, Triton, or Eurycea adults (or other Urodela). 



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

 Dissecting instruments 

 Battery or aquarium Jars vrith weighted covers 



METHOD: (General description for Bana plplens; modifications for other forms appended.) 

 Precautions : 



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

 least 7*+ n™- and the males should measure at least 70 mm., from snout to anus. 



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

 directly from hibernation are the beet. 



5. Frogs should be kept in running cold water (below l8°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. 



k. Avoid red-leg contamination. ExEimlne frogs upon receipt. Bed-leg is not to be 

 confused with temporary rash brought on by sudden changes in temperature. 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 down. 



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

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

 al organs. Glands should be used fresh but may be preserved in alcohol, by 

 freezing in water, or by dissection. 



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. Efegs 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, adequate con- 

 trols for such an experiment would consist of the injection of another equivalent 

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

 trols have invariably given negative results. 



Procedure : (Description for Bana plplens; modifications for other forms appended.) 

 1. Bemoval 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 point 

 Just behind the tympanic membrane, then across the skull to the other side of the 

 head, and sever the skull from the body. Invert the Jawless head and push aside 

 the oral skin, thereby exposing the cross formed by the parasphenoidal and trans- 

 verse bones. Insert the sharp point of smaller scissors into the cranial cavity, 

 ventral to the exposed medulla, and cut through the floor of the cranium on 

 either side of the brain In an anterior direction. Avoid injury to the brain tis- 

 sue because in doing so the pituitary may be lost. The two parallel cuts should 

 extend well anterior to the transverse bone. With forceps, deflect this flap of 

 bone in a forward direction, thereby exposing the brain. The anterior pituitary 

 gland should be seen lying Just posterior to the optic chiasma and will appear as 

 a pinkish, kidney-shaped body surrounded to some extent by white endolymphatic 



- 102- 



