BIOLOGY OF EGGS AND IMPLANTATION 



799 



In the cow relatively large quantities of 

 l)hysiologic solutions are used to flush out 

 tlie cornu on the side on which the corpus 

 luteum has been detected by rectal palpa- 

 tion (Rowson and Bowling, 1949). The 

 recovered fluid is poured into a series of 

 French separatory funnels and allowed to 

 stand for 20 minutes. Ordinarily, this inter- 

 val is long enough for the ovum to gravitate 

 to the bottom. A few milliliters of fluid are 

 removed from each funnel and the egg 

 searched for. By this method, Dracy and 

 Petersen reported the recovery of 10 fer- 

 tilized ova from a single cow which had 

 been superovulated. 



3. Recovery of Attached Embryos 



The techniques devised by Dr. Chester 

 Heuser, thus far unsurpassed in the degree 

 of their perfection, provide the safest 

 method of obtaining blastocysts or early 

 implanting embryos. Uteri of man or other 

 primates which have been removed by hys- 

 terectomy are completely immersed in 

 Locke's solution. The uterus is cut coronally 

 into dorsal and ventral halves. The sur- 

 face of the mucosa can then be examined 

 under a binocular dissecting microscope in 

 order to locate the site of the implanting 

 embryo (Heuser and Streeter, 1941 ; Hertig 

 and Rock, 1951 ) . 



A somewhat similar procedure can be fol- 

 lowed in observing and recovering implant- 

 ing embryos of the guinea pig, rat, and 

 rabbit. The cornu is cut longitudinally along 

 the mesometrial border with iridectomy 

 scissors and the entire cornu laid open as 

 a book. The mucosa of the antimesometrial 

 area is examined under a binocular dissect- 

 ing microscope in order to find the implant- 

 ing embryos and, when they are found, 

 fixatives can be added directly and only a 

 small segment of the uterus removed for 

 sectioning (Blandau, 1949b; Boving, per- 

 sonal communication). 



B. EGG CULTURE AND PRESERVATION 

 IN VITRO 



Studies of the effects of various environ- 

 mental conditions on mammalian eggs and 

 zygotes are of more than academic interest. 

 The possibility of applying such knowledge 

 to artificial insemination and intergeneric 



and reciprocal transplantation of eggs is of 

 economic importance, especially in animal 

 husbandry. Consequently, for years special 

 attention has been given to the problem of 

 finding satisfactory media for the successful 

 culture and transplantation of eggs. 



Gates and Runner (1952) compared Or- 

 tho-bovine semen-diluter containing egg 

 yolk with regular Locke's solution as a me- 

 dium for transplanting mouse ova and con- 

 cluded that the semen diluter was the more 

 satisfactory medium. Many other media 

 have proved successful. These include, to 

 list only a few, Ringer-Locke solution with 

 an equal volume of homologous blood serum 

 (Pincus, 1936), Krebs' solution (Black, 

 Otto and Casida, 1951), phosphate-buffered 

 Ringer-Dale solution mixed with an equal 

 volume of homologous plasma (Chang, 

 1952b), and Krebs-Ringer bicarbonate con- 

 taining 1 mg. per ml. glucose and 1 mg. per 

 ml. crystalline bovine plasma albumin (Ar- 

 mour) (McLaren and Biggers, 1958). 



Rabbit eggs have been used most often 

 as test objects in the evaluation of media. 

 The eggs of this animal are particularly 

 hardy during manipulation and storage in 

 vitro, a condition which may be related to 

 the presence of the mucous coat. Aqueous 

 humor from sheep's eyes has been used 

 successfully for the transfer of eggs from 

 sheep to sheep (Warwick and Berry, 1949) . 

 Willett, Buckner and Larson (1953) ob- 

 tained pregnancies in cows from eggs sus- 

 pended in homologous blood serum during 

 transfer. 



Except when the rabbit was used, at- 

 tempts at growing fertilized eggs in vitro 

 in the same media used for their transfer 

 have not been successful. The pioneering 

 work on the cultivation of mammalian eggs 

 under conditions of tissue culture must be 

 attributed to Brachet (1913), Long (1912), 

 Lewis and Gregory (1929), Pincus (1930), 

 and Nicholas and Hall (1942). Lewis and 

 Gregory recorded their notable success in 

 culturing fertilized rabbit ova in homologous 

 blood scrum in vitro by means of cine- 

 microphotography. Fertilized rabbit ova 

 will cleave regularly in vitro up to and be- 

 yond the initial stages of blastocyst expan- 

 sion (Pincus and Werthessen, 1938). Lewis 

 and Hartman (1933) succeeded in culturing 

 the fertilized eggs of Macacus rhesus for a 



