MANIPULATION OF EGGS 123 



All these data constitute strong support for the claim that the eggs 

 investigated had indeed been fertilized in vitro, but it would have 

 been a much more convincing case had the authors transferred eggs 

 to recipients and recorded the birth of young. Curiously enough, 

 they do not appear to have tried transfer, and so it was left to Chang 

 (1959a) to take this important step and so provide what can reasonably 

 be regarded as proof. Having previously made several unsuccessful 

 attempts (see Chang, 1957a), he now followed the method used by 

 Dauzier and his associates, with minor modifications. Sperm 

 suspensions were made by flushing the uterine horns of rabbits 

 mated 12 hr beforehand with Krebs-Ringer bicarbonate solution 

 and placed in i-5-ml capacity Carrel flasks. Eggs were recovered 

 2 to 3 hr after ovulation with the same physiological solution and 

 placed in the sperm suspensions. The flasks were attached to a 

 rocking device within an incubator at 38°C and left for 3 to 4 hr. 

 After this time, the eggs were taken out and transferred to 8-ml 

 capacity Carrel flasks containing fresh homologous serum which 

 had earlier been heated to 55°C for 20 min. After incubation for 

 a further 18 hr, the eggs were removed and examined in the fresh 

 state. They were then transferred to recipient rabbits in which 

 ovulation had been artificially induced about 8 hr previously. 

 Chang reported that, when the eggs were examined in the fresh 

 state, 55 out of 266 (21 per cent) appeared to have undergone 

 normal cleavage into four cells. Of the fifty-five eggs, thirty-six 

 were transferred to six recipients. Two of the recipients did not 

 become pregnant, but the other four yielded fifteen living young. 



From the observations of these investigators, it is reasonable to 

 conclude that the fertilization of rabbit eggs in vitro can in fact be 

 procured, provided that the spermatozoa used have been recovered 

 from the female genital tract some hours after mating or artificial 

 insemination. Within limits, other conditions, such as the chemical 

 nature of a suspending medium, the oxygen partial pressure and the 

 redox potential, are evidently of minor significance compared to 

 the need for employing spermatozoa that have undergone capacita- 

 tion. This does not necessarily mean, however, that all reports 

 relating to the use of epididymal or ejaculated spermatozoa should 

 be doubted, for the experiments of Noyes, Walton and Adams 

 (1958) suggest that it is possible for capacitation to take place in vitro 

 under certain conditions. Of special interest in this connection is the 

 work of Smith (195 1) who maintained that sperm penetration took 



