MANIPULATION OF EGGS 117 



Better prospects are offered when eggs receive some protection 

 from the ill-effects of low temperatures by treatment with glycerol. 

 Fertilized (i-cell) rabbit eggs treated at 37°C with glycerol at final 

 concentrations of 10 to 20 per cent were subjected to various low 

 temperatures and then thawed, freed of glycerol and placed in 

 culture. More than half the eggs kept at — I5°C for 2 or 3 days, 

 and 10 to 30 per cent of those kept for 4 to 7 days, developed well 

 in culture. Out of about 600 eggs left for up to 2 days at — 79°C, 

 — i6o°C, or — ioo°C, however, only six passed through a few cleav- 

 age divisions in culture (Smith, 1952, 1953a). Mouse eggs (unferti- 

 lized) have so far proved to have little resistance to low temperatures 

 even with protection from glycerol, The eggs were handled in 

 a medium composed of Locke's solution, to which was added 

 some sodium citrate, together with glycerol at a concentration of 

 5 per cent. After chilling, they were transferred to mated recipient 

 mice. Of eggs kept at 5°C for ij to 2 hr, 22-8 per cent developed 

 to embryos that seemed normal at autopsy on the 19th day of 

 pregnancy, but only two eggs out of 276 survived storage for 24 hr, 

 and none storage for 3 days. Rapid cooling to — 21 °C, followed by 

 immediate rewarming, had no apparent effect on viability, but only 

 seven out of sixty eggs developed after being kept at — io°C for 

 3 \ hr, and four out of sixty-six at o°C for 6 hr (Lin, Sherman and 

 Willett, 1957; Sherman and Lin, 1958, 1959)- 



Most impressive are the results obtained by freezing follicular 

 oocytes within pieces of ovarian tissue, though these eggs cannot 

 be said to have been treated in vitro, in the strict sense of the term. 

 Observations based on the development of oocytes within sub- 

 cutaneous grafts of rat ovarian tissue have suggested that a few 

 oocytes (less than 10 per cent) are still viable after treatment with 

 15 per cent glycerol and freezing to — 79°C (Deanesly, i954> x 957; 

 Green, Smith and Zuckerman, 1956). Proof of viability was 

 supplied by results obtained with the technique of orthotopic 

 grafting in mice. Oocytes from ovaries frozen at — 79°C for as 

 long as 6 weeks have been found capable of subsequent development 

 into normal young (Parrott, 1958, i960; Parrott and Parkes, i960). 



Development in culture. Oocytes have been kept in vitro, under 

 tissue-culture conditions, to obtain their maturation prior to transfer 

 to recipient mated animals (Chang, 1955a, d) or prior to the 

 attempted induction of fertilization /'// vitro (Rock and Menkin, 

 1944; Menkin and Rock, 1948). In the great majority of investiga- 



