96 



INDUCED BREEDING 



The sperm suspension may be divided between several finger bowls, Petri 

 dishes or large slenders so that in each there will be a thin film of suspension 

 on the bottom of the container. Eggs are stripped directly into this sperm 

 suspension in such a manner that all eggs are exposed. If this is not accom- 

 plished it will be necessary to pipette some of the suspension onto the eggs. 

 The inseminated eggs should stand for about 5 minutes and then should be 

 flooded with the same water used to make up the sperm suspension. The eggs 

 should be barely covered with this water. In about 20 minutes pour off this 

 first water and add enough fresh water to again cover the eggs. It is the ex- 

 posed surface rather than the volume of water that is important for respira- 

 tion of the eggs. If the eggs are successfully inseminated they should all ro- 

 tate so that the animal hemisphere is uppermost within about an hour and by 

 2-2 hours the eggs should be in the 2-cell stage at laboratory temperature of 



23^ 



25"C. 



(a) Egg of Rana pipiens at the 

 moment of insemination. 



(b) Egg of Rana pipiens 29 



minutes after insemination, 

 showing grey crescent. 



Care of the material : As the jelly membranes swell (by imbibition) the egg 

 mass will expand and it may become necessary to add some water to cover. 

 The jelly mass generally sticks to the bottom of the container but may be 

 separated by means of a stiff, clean section lifter. This may be done as 

 soon as 1 hour after insemination. The jelly should be allowed to swell to its 

 maximum and then the egg mass should be cut up into small groups of eggs. 

 This should be done before the first cleavage. The optimum ratio is about 

 25 eggs per finger bowl of 500 cc. water. 



The optimum temperature for the normal development of the eggs of Rana 

 pipiens is 18° - 25°C. Development can be slowed down without the production 

 of abnormalities if the eggs are kept at the lower temperatures, but never 

 lower than 10°C. By retarding the developmental rate of some eggs it is 

 possible to have various stages of development available at all times. Re- 

 member, however, that when eggs are removed from a cold to a warm en- 

 vironment, sufficient time must be allowed for the adjustment before the 

 eggs are to be used for experimental procedures. Eggs and embryos should 

 never be transferred suddenly from one temperature level to another. 



