MANIPULATION OF EGGS 119 



eggs. But if the recipient has been brought into a suitable state by 

 prior mating with a vasectomized male, there is the obvious danger 

 that the vasectomy was incompletely effective and that the male 

 was still ejaculating spermatozoa. Clearly, a better procedure is to 

 prepare the recipient by appropriate hormone treatment. Again, 

 eggs transferred after treatment with spermatozoa may be accom- 

 panied by free spermatozoa which later effect fertilization within 

 the recipient female tract — fertilization either of the transferred eggs 

 or of the recipient's eggs. This could happen even if the eggs under 

 test are carefully washed immediately before transfer, for it is 

 extremely difficult to remove adherent or accompanying sperma- 

 tozoa altogether. The danger that the recipient's eggs may be 

 fertilized can be taken into account by the use of genetic markers. 

 Probably the best way to circumvent the risk that the transferred 

 eggs are fertilized in this way is to transfer them only after they have 

 been kept in culture until the occurrence of cleavage (or fragmenta- 

 tion) indicates that the stage of fertilization is past. (There are several 

 other possible sources of error, in addition to those just described, 

 and these arise chiefly from the production of artefacts during 

 preparation of the eggs for histological study and from the mis- 

 interpretation of objects seen in histological sections. These points 

 have been discussed on several occasions: Chang and Pincus, 195 1; 

 Smith, 195 1 ; Austin and Bishop, 1957b; Chang, 1957a; Austin and 

 Walton, i960; Austin, 1961c.) 



In view of the difficulties of establishing conclusively the occur- 

 rence of fertilization /'// vitro, it is not surprising that the great 

 majority of the claims for success, the more detailed of which are 

 shown in Table 5, are far from convincing. For various reasons, the 

 claims that seem to merit the most serious consideration are those 

 of Dauzier and his colleagues (Dauzier, Thibault and Wintenberger, 

 1954; Thibault, Dauzier and Wintenberger, 1954; Dauzier and 

 Thibault, 1956, 1959; Thibault and Dauzier, i960), of Moricard 

 (1954a, b) and of Chang (1959a). 



Dauzier and his associates recovered eggs from rabbits soon after 

 artificially induced ovulation and held them under conditions that 

 were considered unlikely to provoke parthenogenetic development, 

 in the light of Thibault's (1949) earlier experience with this pheno- 

 menon. The eggs were maintained in Locke's solution in short 

 lengths of glass tubing. Spermatozoa in suspension were obtained 

 by flushing the tubal, uterine or vaginal lumina of rabbits mated 



