to obtain uniform distribution of sperm, and the 

 finger bowl set aside for 5 hours, half submerged 

 in running sea water. At the expiration of this 

 period a sample was taken for examination and 

 cleaved and uncleaved eggs were counted. In 

 each case 300 eggs of the sample wei-e examined. 

 All tests were made in water of 31 to 32°/oo 

 salinity and 20.8° to 21.4° C. During the first 4 

 hours of aging the percentages of cleaved eggs 

 declined from 90 to 70. After 5 or 6 hours the 

 percentages dropped to 60. Then the fertilizabil- 

 ity decreased to about 20 percent in 10 hours, and 

 only a few eggs cleaved normally after 12 and 24 

 hom's of aging. 



It is common knowledge among embryologists 

 that the fertilizing power of spermatozoa is not 

 decreased if sperm is kept at 10° to 12° C. in a 

 concentrated suspension in a tightly closed con- 

 tainer. This is also true for oyster sperm. Its 

 fertilizing power is affected by dilution and in- 

 creased temperature. At room temperature in a 

 dilute suspension, the spermatozoa lose their 

 fertilizing ability within 4 to 5 hours. However, 

 in a concentrated suspension, protected from 

 evaporation, and stored in a refrigerator at about 

 10° C. the sperm remains active and retains its 

 full fertilizing power for 24 hours and possibly 

 longer. 



The effect of cold storage on the fertilizability 

 of eggs is not known. On several occasions ripe 

 females with intact shells were kept for 3 to 4 

 days in a refrigerator (about 10° C) and after 

 that were successfully used in spawning experi- 

 ments. The effect of cold storage on eggs of the 

 excised ovary has not been studied. 



POLARITY OF EGG 



The polarity of all moUuscan eggs apparently 

 is determined while they are still attached to the 

 wall of the ovarian follicle. Presumably the side 

 on which the food reaches the growing ovocyte 

 becomes the vegetative pole of the mature egg 

 (Raven, 1958). 



The metabolic gradient along the egg axis is 

 indicated by a concentration of cytochrome oxidase 

 which Kobayashi (1959) detected with M-Nadi 

 reaction ; the activity of the enzyme was observed 

 using Graff's modification of this method. (The 

 reader not familiar with the reaction and its sig- 

 nificance in cytochemical research is referred to 

 the publications of Danielli (1958), Deane, Barr- 

 nett, and Seligman (1960), and to a review by 



NovikoflF (1961a, p. 308).) In brief, the localiza- 

 tion of oxidative enzymes and their presence in 

 mitochondria can be determined by the staining 

 reactions. The rate of respiration of eggs of C. 

 mrginica increases with fertilization by a factor of 

 1.4 (Ballentine, 1940) but in the eggs of the 

 Sydney rock oyster, C. commercialis, the rate of 

 respiration increases only at the onset of the first 

 cleavage (Cleland, 1950). 



CLEAVAGE 



The spermatozoon may enter the oyster egg 

 anywhere. Its path inside the egg cytoplasm 

 toward the nucleus has not been described, and 

 cytological details of the process leading to the 

 fusion of the female and male pronuclei have not 

 been studied. It is probable that the major fea- 

 tures of these events are not different from those 

 found in other mollusks. Maturation divisions 

 occur in the oyster egg after the elevation of the 

 fertilization membrane. The germinal vesicle 

 (ovocyte nucleus) breaks down and moves toward 

 the egg's periphery. At temperatures of 22° to 

 24° C. the first polar body is formed within 25 to 

 50 minutes after the addition of sperm. The re- 

 duction of the number of chi'omosomes probably 

 takes place during the first meiotic division. This 

 is to a certain extent corroborated by an exami- 

 nation of fertilized eggs of C. mrginica stained in 

 toto with Feulgen reagent or with acetic orcein. 

 Unfortunately the results are not consistent enough 

 to draw a final conclusion and the question remains 

 unanswered, awaiting a complete cytological study. 



The second polar body is formed shortly after 

 the first, within 45 to 70 minutes after fertilization 

 (at 22.5° to 24° C). The two polar bodies remain 

 attached to the surface of the egg (fig. 319) until 

 the completion of cleavage and emergence of the 

 trochophore. 



The fu'st cleavage following the formation of 

 the second polar body divides the egg meridionally 

 into two unecjual cells designated as AB and CD 

 (fig. 320). The inequality of the blastomeres is 

 due to the occurrence of a polar lobe. Because 

 the egg appears to consist of three cells this stage 

 received the name trefoil. 



The plane of the second division, also meridional, 

 is at a right angle to the first. Both blastomeres 

 divide synchronously and separate into the four 

 quadrants. In fig. 321, drawn from a photograph 

 of a cleaving egg taken from the animal pole, the 

 position of the spindles indicates the plane of new 



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FISH AND WILDLIFE SERVICE 



