184 ARTIFICIAL PARTHENOGENESIS 



expose the heart. With the frog on its back cut off the tip end of its ventricle 

 and allow the blood to flow freely into the body cavity, mixing there with the 

 coelomic fluid. Keep the abdomen closed until ready to use the blood. 



5. Remove one slide from the moist chamber. Take a small strip of abdominal 

 muscle from the non-ovulating female, draw it through the mixture of blood 

 and coelomic fluid, and gently pass it over each of the two rows of eggs. 

 Avoid any pressure on the eggs but see that each egg is provided with a par- 

 tial coating of blood and coelomic fluid. 



6. Cortical stimulation . Gently but firnnly prick each egg with a sharp point of 

 a glass or platinum needle. The puncture should be applied within the 

 animal hemisphere but not in its exact center where the second maturation 

 spindle is likely to be located. Leave the eggs in row "C" untouched as 

 controls. 



7. Immediately after pricking the experimental row of eggs, immerse the slide 

 in Spring Water or Standard Solution in which normal development is known 

 to occur. It is best to use Petri dishes and only about 2 cm. depth of me- 

 dium to cover. 



8. Repeat the above procedure with 4 other slides from the moist chamber. 



9. Follow the above procedure with the remaining 5 slides from the moist cham- 

 ber but limit the pricking to the vegetal hemisphere of the egg. Mark these 

 slides to indicate location of stinnulation. 



If time permits, the same procedure should be followed with single variations 

 which might increase the incidence of successful stimulation. Such variables 

 are as follows: 



a. Allow the eggs to remain within the uterus at 1 C. for 5 days before 

 stripping (Zorzoli and Rugh, 1941). Such aged eggs must be allowed to 

 come to the laboratory temperature before stimulation. 



b. Keep the female at refrigerator {4 C. ) temperature, and in the moist 

 channber provided with ice cubes, to determine whether a lower tempera- 

 ture alone would increase the sensitivity of the egg to artificial stimula- 

 tion. 



c. Omit the use of blood or serum (Guyer, 1907; Bataillon, 1911 and 1919). 



d. Vary the depth of cortical injury, deep or shallow pricking. 



e. Allow the eggs to dry (partially) on the slide before pricking. 



f. Allow the jelly to swell in water to various degrees, before stimulating. 

 The cortical pricking will be a bit more difficult through swollen jelly. 



g. Follow the artificial stimulation of the egg by immersion in media of 

 various osmotic conditions. 



h. Determine the role of the presence and the absence of calcium (using 

 oxalates and citrates) in the response to parthenogenetic stiniulation. 



OBSERVATIONS AND TABULATION OF DATA: 



Record the data from your experiment in tabular form on the following page. 



Total experimentals should include all eggs stimulated. The pseudo-cleavages include 

 irregular cleavages and superficial indications of attempts at cleavage. Along with these 

 data, include a statement regarding the exact method of stimulation, instrument used, 

 and any variations in the prescribed technique. If, perchance, you achieve an unusually 

 high percentage of cleavages, you will want to be able to repeat the procedure in every 

 detail. 



There are qualitative aspects of the problem which should be recorded under: 



1. Pattern of cleavage when it is not normal. Is the injury point in any way related 

 to the position of the cleavage furrow or the position of the grey crescent? 



