ARTIFICIAL PARTHENOGENESIS* 



PURPOSE : To repeat the earlier experiments^ using modern methods of inducing ovulation 

 and of equipment, in an attempt to initiate the development of the amphibian egg hy 

 artificial means (i.e., without benefit of spermatozoa). 



MATERIAIS : 



Biological : Uterine eggs from an ovulating anuran: Eana or Bufo. Blood fron a second 

 non-ovulating female anuran, same species. 



Technical : Slides, Petri dishes, finger howls, #2 Stenders, moist chamber, section 

 lifter, sharp-pointed (5 to 9 p) glass or platiniim (20 to JO n) needles, 

 and china marking pencil. 



METHOD : 



Precautions : 



1. All articles and female frogs must be kept sperm-sterile. The instruments and 

 glassware may be boiled for 5 minutes or immersed in 70^ alcohol and air dried. 



2. The female frog which produces the eggs for the experiment should be isolated 

 from all males for several days prior to the experiment and should be washed off 

 with tap water and dried before stripping. 



Controls : Two types of controls are necessary for this experiment. 



1. Some eggs are to remain untreated, but should be placed side-by-side with the 

 eggs experimentally treated. This provides identical environmental conditions 

 for the experlmentals and the controls, with but a single variable. 



2. The eggs should be tested to determine whether they are in fertilizable condi- 

 tion. Following the conclusion of the experiment, some of the uterine eggs 

 should be normally inseminated by frog spermatozoa, of the same species, in £Ui- 

 other laboratory. There must be no possible contamination of the experimental 

 eggs with spermatozoa. 



Procedure : 



1. Adjust a low-power microscope so that the heat-absorbed light will strike the 

 eggs from a ^+5° angle from above. A lantern slide cover glass might be used to 

 protect the microscope stage from water. 



2. Place 10 clean microscope slides, a slight distance apart, on clean paper towel- 

 ling. On the upper left hand comer of each mark "C" (for controls) and below 

 on the lower left hand corner of each slide mark "X" (for experlmentals), using 

 a china marking pencil. Number the slides in sequence. 



3. Strip a single row of eggs from the uteri of an ovulating female, placing them 

 along the length of the slide opposite "C" and then another opposite "X". Try 



to strip eggs in a single row so that they will not lie over each other, and will 

 adhere to the slide. Place all 10 slides of eggs in a moist chamber where they 

 may remain for an hour or more without deleterious effects. (The chamber should 

 have stood for at least an hour so that the contained air is completely saturated 

 with water vapor.) Such eggs will lose their CC^ and thereby facilitate their 

 physiological maturation (Bataillon &. Tchou-Su, 1930). 



k . Pith a non-ovulating female frog; lay it on some paper towelling; cut through the 

 leg muscles to prevent further reflex movements; open the abdomen and 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 coelomlc 

 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 coelomlc 

 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 partial coating of blood and 

 coelomlc fluid. 



* This laboratory procedure has been organized with the very generous help of Dr. C. 



Parmenter. 



-l85- 



