104 THE MAMMALIAN EGG 



though to do this without damaging the Fallopian tube requires 

 care, especially in small animals. Eggs that are still surrounded by 

 cumulus oophorus and grouped together in the extended part of the 

 ampulla are released simply by slitting the ampulla with an instru- 

 ment such as a Graefe knife, whereupon the cumulus masses gener- 

 ally emerge without further aid. Denuded eggs may be recovered 

 either by flushing or by manipulation. The flushing method is the 

 same in principle as that described for the rabbit Fallopian tube 

 except that the pipette used is necessarily of smaller dimensions. It 

 may be found helpful to make a small bulbous enlargement at the 

 tip of the pipette as this tends to retain it after it has been inserted 

 into the tube. The Fallopian tube can be flushed in either direction — 

 some authors prefer to insert the pipette into the lumen of the 

 isthmus, others pass it through the infundibulum. Recovery by 

 manipulation, on the other hand, involves the application of pressure 

 to the Fallopian tube in such a way as to drive the contents along 

 the tube and finally through the infundibulum, or, if preferred, 

 through an opening made in the wall of the tube. Pressure is applied 

 with a pair of dissecting needles. As a possible refinement, the 

 Fallopian tube may, on removal from the animal, be placed in 

 liquid paraffin in a Petri dish; this permits the eggs to be dissected 

 from the tube, and transferred to a microscope slide, while still 

 surrounded by their natural fluid medium. The method is perhaps 

 appropriate only in the murine rodents and when there is an appreci- 

 able accumulation of fluid in the tube, as is the case for a limited 

 period after ovulation. Phases in the fertilization of rat eggs were 

 found to continue in vitro more surely when the eggs had been 

 recovered in this way than when they were surrounded by artificial 

 medium (Austin, 1950a, 1951a). The method has also been applied 

 to hamsters (Ohnuki, 1959). 



If large numbers of follicle cells are still attached to the zona 

 pellucida they tend to obscure the finer details within the eggs when 

 these are examined with the higher powers of the microscope; 

 accordingly, the cumulus should first be removed by treatment 

 with solutions of hyaluronidase or trypsin. This procedure is 

 ineffective, however, with follicular oocytes, from which the 

 adherent cells must be removed by dissection. The corona radiata 

 of the rabbit egg is also resistant to removal by enzymes but can be 

 dislodged if the eggs are vigorously propelled into and out of a fine 

 pipette. 



