190 APPENDIX 



D. Artificial Ovulation (Exercise 45, Frog) 



Ovulation in the frog can be artificially induced late in the 

 autumn and with increasing ease as the winter advances. 

 Better results are obtained if large, mature females are 

 selected for stimulation. Hypophyses should be obtained 

 from large female donors beheaded after pithing. Insert 

 the point of scissors into the foramen magnum, cut forward, 

 and remove the floor of the brain case. The hypophysis will 

 be seen lying against the brain somewhat posterior to the 

 optic chiasma or will be found adhering to the removed 

 bone. Take two glands in about 1 c.c. of water and draw 

 into a hypodermic syringe. Attach a short needle with a 

 bore adequate for passage of the glands and inject them into 

 the posterior ccelom of the host female; the viscera are less 

 likely to be damaged if the needle is inserted obliquely. If 

 only a small bore needle is available the glands can be 

 mashed with the end of the syringe to form a coarse sus- 

 pension which can be injected. Repeat the dose after 

 twenty-four hours. Ovulation will occur on the third day. 

 The eggs can be obtained by "stripping," or gentle pressure 

 with thumb and forefinger downward from the region of 

 the forelegs as the female is held vertically. 



When eggs are known to be available a sperm suspension is 

 prepared by teasing two testes from a mature, recently 

 killed male in about 10 c.c. of pond water or an amount 

 which will just cover a single layer of eggs in the dish which 

 is to be used. Water from a pond where frog eggs are known 

 to be laid and normally fertilized is the best medium for the 

 sperm suspension and as a medium for early development. 

 However, other water may by test be found to be entirely 

 satisfactory. The sperm suspension is allowed to stand for 

 about thirty minutes in order that the sperm may become 

 fully active. Then the eggs are stripped into the dish until 

 a single layer covers the bottom. When the eggs have ro- 

 tated until the pigmented hemispheres are uppermost, which 

 is about thirty minutes after insemination, the dish is filled 

 one-half to two-thirds full of water. At normal room tem- 

 perature cleavage should occur in two to three hours. 



