800 



SPERM, OVA, AND PREGNANCY 



number of divisions. Eggs of guinea pigs, 

 cultured in vitro, rarely divide beyond the 

 first few blastomeres (Squier, 1932). Guinea 

 pig blastocysts, however, grow quite well in 

 a culture medium consisting of equal parts 

 Locke's solution (pH 7.5), serum from 

 guinea pigs pregnant from 20 to 24 days, and 

 embryo extract prepared from 19- to 20- 

 day-old guinea pig embryos (Blandau and 

 Rumery, 1957). As yet, no success has been 

 obtained with the very early fertilized eggs 

 of the hamster and rat (Wrba, 1956) . 



Hammond (1949.) cultured fertilized 

 mouse ova in dilute suspensions of whole 

 hen's egg in saline to which had been added 

 Ca, K, Mg, and glucose. No 2-cell ova de- 

 veloped beyond the 4-cell stage; 8-cell ova 

 ordinarily developed into blastocysts. Whit- 

 ten (1956) found that 8-cell mouse eggs de- 

 veloped into blastulae in an egg white-sa- 

 line mixture or in Krebs-Ringer bicarbonate 

 solution to which 0.003 m glycine had been 

 added. There seems to be some physiologic 

 difference between the 2- and 8-celled ova 

 in this animal because the 2-celled mouse 

 eggs are refractory to in vitro cultivation 

 unless calcium lactate replaces the calcium 

 chloride in the culture medium (Whitten, 

 1957). 



Considerable success has attended the in 

 vitro culture of embryos which are beyond 

 the blastocyst stage at the time of transfer 

 to tissue culture (Brachet, 1913; Wadding- 

 ton and Waterman, 1933; Jolly and Lieure, 

 1938; Nicholas, 1947; Moog and Lutwak- 

 Mann, 1958). Nicholas (1933) obtained bet- 

 ter growth in vitro when the embryos were 

 cultured in a circulating medium. 



Several investigators have studied the ef- 

 fects of cooling mammalian eggs in vitro. 

 Chang (1948a, b) found that rapid lowering 

 of the temperature of 2-celled rabbit ova 

 that had been suspended in a mixture of 

 equal parts of buffered Ringer's solution and 

 rabbit serum was harmful to subsequent 

 development. However, the important fac- 

 tor was not the rate of cooling but whether 

 the process was continued until +10°C. was 

 reached. Apparently, that is the optimal 

 temperature for the storage of fertilized 

 rabbit eggs. At this temperature eggs can 

 be kept in vitro up to 168 hours without loss 

 of viability. At +22°C. to +24°C. ova lived 

 for only 24 to 48 hours. Attempts to main- 



tain glycerol-treated rabbit ova at temper- 

 atures ranging from -79° to -190°C. have 

 so far been unsuccessful (Smith, 1953). 



C. INTRASPECIFIC EGG TRANSFER 



The technique for the transfer of unfer- 

 tilized and fertilized eggs between the mem- 

 bers of the same species was first described 

 by Heape (1890). He used this method in 

 rabbits to demonstrate that the genetical 

 characteristics of mammals are fixed at the 

 time of fertilization and are not influenced 

 by the intra-uterine environment of the 

 foster mother. Biedl, Peters and Hofstatter 

 (1922) and Pincus (1930) used Heape's 

 technique during investigations on fertility 

 and demonstrated that it is possible to 

 transplant fertilized rabbit eggs to pseudo- 

 pregnant does. 



In animal husbandry artificial insemina- 

 tion has been an important method for the 

 widespread distribution of desirable genes 

 by way of the spermatozoa. Similar geneti- 

 cal improvement through the egg has been 

 greatly limited in domestic farm animals 

 by the small number of offspring. A single 

 cow, for example, will produce 1 calf per 

 year and seldom more than 5 in a lifetime. 

 If transplantation of eggs could be per- 

 fected, the number of genetical experiments 

 could be increased at least 2-fold. That the 

 prospect is favorable, is indicated by the 

 fact that transfers which have resulted in 

 pregnancies have been reported for mice 

 (Bittner and Little, 1937; Fekete and Little, 

 1942; Fekete, 1947; Runner, 1951; Gates 

 and Runner, 1952; Runner and Palm, 1953; 

 McLaren and Michie, 1956; Tarkowski, 

 1959; McLaren and Riggers, 1958); rats 

 (Nicholas, 1933; Noyes, 1952); rabbits 

 (Heape, 1890; Bicdl, Peters and Hofstatter, 

 1922; Pincus, 1936, 1939; Chang, 1947, 

 1948a, b, 1949a, 1952b; Chang, Hunt and 

 Romanoff, 1958; Venge, 1953; Avis and 

 Sawin, 1951; Black, Otto and Casida, 1951; 

 Adams, 1953); sheep and goats (Warwick 

 and Berry, 1949; Averill and Rowson, 1958) ; 

 swine (Kvasnickii, 1951) ; and cows (Wil- 

 lett, Buckner and Larson, 1953). 



The majority of successful egg transfers 

 have been accomplished by exposing the 

 oviducts and cornua surgically and placing 

 the eggs within them (Fig. 14.2). Introduc- 

 ing fertilized eggs into the cornua by way of 



