ing time (fig. 307 j, hut by the 21st day (fig. 230, 

 cells 7 and 8) cells having this appearance can be 

 considered to be truly definitive and mature. 



Cells occasionally found in circulating 

 blood of the enibyro 



Granulocytes may be found in the circulating 

 blood during the last week of embryonic life and 

 occasionally during the second week. Their oc- 

 currence, however, is sporadic, and in many 

 smears none were present; yet a study of hemato- 

 poietic organs during this same period — and even 

 of organs like the pancreas, which is not a 

 hematopoietic organ in adult life — shows that 

 tremendous numbers of granulocytes are being 

 produced, but apparently they are being held in 

 storage and normally are not lil^erated until after 

 hatching, so that the typical blood smear even for 

 a few hours after hatching shows only eryth- 

 rocytes and thrombocytes (fig. 230). 



When granulocytes do appear in the circulat- 

 ing blood of the embryo, this question comes to 

 mind: Is it rough handling, or is it infection of 

 the egg, that caused these granulocytes to appear 

 in some embryos and not in others? Wlien the 

 egg is opened, the embryo with its membranes is 

 slid into a bowl of warm saline and then is lifted 

 to a filter paper in a flat Petri dish. Here the 

 amnion is opened, and often in the moving proc- 

 ess the other membranes are ruptured or torn 

 away. If the ventral body wall has closed over, 

 the tissues in this region are laid apart in order to 

 expose the heart and then the cannula is inserted. 

 No matter how dexterously these steps are ex- 

 ecuted, there is still enough roughness to dislodge 

 some granulocytes from embryonic depots. 



On the other hand, approximately 8 percent 

 of hens' eggs have been found to carry a bacterial 

 contaminant after all precautions have been 

 taken to prevent entrance of organisms from out- 

 side the shell. From indirect evidence viruses 

 also can be transmitted from the hen to the egg 

 (Cottral, 1950 and 1952). Probably our best 

 evidence comes from work done at this Labora- 

 tory — evidence that lymphomatosis, a virus dis- 

 ease, is transmitted through the egg (Cottral, 

 Burmester, and Waters, 1954, and Burmester, 

 Gentry, and Waters, 1955) . If some eggs carry 

 bacterial or virus infections, and if phagocytic 

 cells are already on hand, it seems entirely pos- 



sible that the chick could be stinmlated to give a 

 defensive response just as well before hatching 

 as after hatching. 



Chick embryos have been inoculated with so 

 many different viruses and bacteria that it is 

 probably safe to say that growth or serial passage 

 of every well-known disease has been attempted 

 on this medium. In nearly every case, the em- 

 bryo was used merely as a test tube because a 

 better, synthetic one has not yet been devised. 

 Embryos have been used as a culture medium 

 without nuicli regard for possible contamination 

 of the egg. The studies of embryo reactions are 

 often based on grossly visible lesions, which do 

 not tell the full story. Studies of egg-borne dis- 

 eases must be carried back to the cells of the 

 embryo. These cells must be searched for in- 

 conspicuous deviations from the normal, some 

 of which may represent defense reactions to 

 pathogens. The meaning of the presence or ab- 

 sence of heterophils in the circulating blood is a 

 case in point; there is certainly no a priori reason 

 why the chick embryo could not be studied in 

 health and in disease just as scientifically as the 

 adult fowl. Zuckerman (1946) has provided 

 us with an example of blood response to infection 

 in the embryo. Introduction of Plasmodium 

 gallinaceum stimulated the heterophils to become 

 almost as numerous as thrombocytes. In the 

 differential count on one embryo, there were 44.5 

 percent thrombocytes; 19.0 percent mature 

 heterophils, and 24.0 percent heterophil myelo- 

 cytes, a total of 43.0 percent. In addition there 

 were 1.0 percent macrophages, 8.0 percent mono- 

 cytes, and 3.5 percent lymphocytes. 



Roberts, Severens, and Card (1939) found the 

 white cells of the embryo composed of neutro- 

 phils and lymphocytes. During the last week 

 of hatching there was a fairly constant number 

 of each of these cell types. Their values were 

 averages and the variability among individual 

 birds was not indicated. No mention was made 

 of the thrombocytes, which in the embryo often 

 look like lympocytes. Whether some, or all, 

 of the white cell types are constant components 

 of embiyo circulating blood can be decided 

 definitely only by additional investigations. 



The macrophage is another cell sporadically 

 found in smears of blood taken from the embryo 

 heart. The large cell shown in figure 308 came 

 from blood drawn from the dorsal aorta and 

 differs in appearance from all the other cells 



133 



