INDUCED BREEDING ^^ 



In general, the female glands are about twice as potent as those from the 

 male, but there is no qualitative difference. The suggested doses for Rana 

 pipiens are as follows: 



Male Pituitaries Female Pituitaries 



September to January 10 5 



January to February 8 4 



March 5 3 



April 4 2 



Injection of the hormone: For recipients, carefully select large and obviously 

 healthy feinales which have been recently received from hibernation. Such 

 females may be kept in the refrigerator at 4°C. in a small amount of water 

 for a number of weeks but should not be kept at laboratory temperatures for 

 more than a week if they are to be used for ovulation induction. Healthy fe- 

 males received from hibernation in January may thus be kept until June (i. e. , 

 at 4°C. ). At the laboratory temperatures the ovarian eggs deteriorate ra- 

 pidly. 



Before attaching the needle to the hypodermic syringe, draw up into its bar- 

 rel the requisite number of anterior pituitary glands. It will be best if the 

 glands are freed from their attached endolymphatic tissue and remain whole 

 as they pass into the syringe. There will be some loss if the glands are 

 damaged. Apply a large-bore hypodermic needle (#18) to the syringe, and 

 then insert the point of the needle through the skin and abdominal muscles of 

 the female frog, in the lower quadrant of the abdomen. Do not insert the 

 needle far enough to damage any of the vital organs and specifically avoid the 

 ventral abdominal and the subcutaneous veins. Inject downward, as the 

 glands are heavier than water. As the glands pass through the needle into 

 the abdominal cavity they will be broken up into a fine suspension, ready for 

 quick absorption. Absorption is probably accomplished largely through the 

 numerous ciliated peritoneal funnels on the ventral faces of the kidneys, 

 which funnels open directly into the venous sinuses. The female frog should 

 now be placed in a small amount of water in a wire-covered battery jar or 

 aquarium. If eggs are required within 24-48 hours, the female should be 

 kept at about 25°C. ; while if the eggs are not needed for 4 to 5 days the frog 

 may be kept at 10°C. and the eggs will be just as good. 



Test of ovulation : The presence of eggs in the uteri can be determined only 

 by "stripping", or squeezing of eggs from the uteri. It is not necessary to 

 sacrifice the frogs to get the eggs. Stripping is accomplished in the follow- 

 ing manner, without damage either to the frog or its eggs. 



The legs of the frog are grasped in the left hand so as to prevent body move- 

 ments on the part of the frog. The palm of the right hand is placed over the 

 back of the frog, and the fingers encircle the body just posterior to the fore- 

 limbs. By gentle closure of the right hand in the direction of the cloaca, eggs 

 will be forced from the uteri. The body may be bent at the pelvic region to 

 facilitate removal of the eggs. If jelly alone or fluid issues from the cloaca 

 the female should be replaced and tested again within 24 hours. It is general 

 practice to remove and discard the first few eggs that emerge because occa- 

 sionally cloacal fluid allows the swelling of the nearby jelly on uterine eggs 

 and this renders such eggs rather difficult of insemination. It is well also to 

 dry off the cloacal region of the frog prior to stripping. The fertilization 

 percentage will be higher if the eggs are allowed 24 hours for physiological 

 maturation in the uterus. 



Each sexually mature female of Rana pipiens should give about 2, 000 eggs 

 all in metaphase of the second maturation division, ready to be fertilized. 



