lOU INDUCED BREEDING 



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 sub- 

 cutaneous 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 ac- 

 complished 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 2^-1+8 hours, the female 

 should be kept at about 25°C;.; while if the eggs are not needed for i* to 5 days 

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



5. 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 sacri- 

 fice the frogs to get the eggs. Stripping Is accomplished in the following man- 

 ner, 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 forellmbe. 

 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 2U hours. It is general practice to 

 remove and discard the first few eggs that emerge because occasionally 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 2U hours for physiological maturation in the 

 uterus. 



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

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



h. Artificial fertilization : Male frogs need not be injected with the pituitary 

 hormone unless it is desired to show amplexus. In this case, inject the male at 

 the same time the female is Injected, with the same dose of pituitary glands, and 

 place them together in the same container. Amplexus, at laboratory temperatures, 

 will be achieved in about 2k hours and fertilized eggs will be layed in the aquar- 

 ium, generally early in the morning. Use water in which embryos are known to 

 develop. 



From September until April mature, functional spermatozoa can be secured 

 from Sana pipiens males of body length of more than 70 mm. simply by dissecting 

 the testes in Spring or Pond Water, or any medium in which they are known to be 

 viable. Generally two pairs of testes are dissected in about 10 cc. of water and 

 are allowed to stand for 5-10 minutes at laboratory temperatures In order to al- 

 low the spermatozoa to become active. 



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

 dishes or large etenders 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 sus- 

 pension in such a manner that all eggs are exposed. If this is not accomplished 

 it will be necessary to pipette some of the suspension onto the eggs. The in- 

 seminated 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 exposed surface rather 

 than the volume of water that is important for respiration of the eggs. If the 

 eggs are successfully inseminated they should all rotate bo that the animal heml- 



