510 THE TOCOPHEROLS 



2. The 4- to 5-day estrous cycles, having been regular before fertiliza- 

 tion, as determined by vaginal smears, should cease. 



3. Implantation should be demonstrated by the placental sign or vaginal 

 blood leak about the twelfth day of pregnancy. 



4. On the twentieth or twenty-first day, the animal casts a litter if vita- 

 min E supplies have been adequate, or her weight declines steeply but not 

 abruptly, indicating resorption of the young. If there is any question of the 

 fertility of the animal as such, an adequate dose of vitamin E should be 

 given after another positive mating, and it is also essential to recognize 

 a pseudopregnancy. 



Because of the possibility of initial or first litter fertility due to vitamin 

 E stores, a resorption gestation was once considered a necessary step be- 

 fore any animal was used for test. It was demonstrated, however, that the 

 animal was a less sensitive indicator after a resorption gestation, and the 

 practice first recommended by Mason and Bryan^^ is now generally fol- 

 lowed: the rats to be used for assay purposes are reared on a vitamin E- 

 deficient diet beginning in their nursling period. Animals whose vitamin E 

 storage is limited to that obtained through placental and mammary trans- 

 fer, on such a regime, never show first litter fertility. In any case, placental 

 transfer is very limited, whereas mammary transfer, especially after ad- 

 ministration of large doses of tocopherol, has been demonstrated in lactat- 

 ing rats and in other species^^' ^^ to be considerable, particularly in colos- 

 trum. ^■'' ^^ Tocopherols in early human milk are reported as 0.13 to 3.6 

 mg. per 100 ml., with an average of 0.14 mg. in later milk.^^ A later study" 

 confirmed the mammary transfer of tocopherol; premature infants on for- 

 mulas low in vitamin E demonstrated a rapid decline in serum tocopherol 

 levels. During the first six days after birth, the figures for breast-fed infants 

 increased much more rapidly than those for bottle-fed. 



A further improvement in the direction of standardization^^ was the ex- 

 amination of the uterus at the sixteenth day by laparotomy, a positve re- 

 sponse being the presence of two or more viable fetuses with at least four 

 implantation sites. The presence of placental scars or of resorbing fetuses 

 (or of both) testifies to the positive character of the mating and the in- 



" K. E. Mason and W. L. Bryan, Biochetn. /. 32, 1785 (1938). 

 " K. E. Mason and W. L. Bryan, /. Nutrition 20, 501 (1940). 

 " A. M. Pappenheimer, H. Kaunitz, and C. Schogoleff, Proc. Soc. Exptl. Biol. Med. 



55, 229 (1944). 

 54 F. Whiting and J. K. Loosli, /. Nutrition 36, 721 (1948). 

 85 D. B. Parrish, G. H. Wise, and J. S. Hughes, J. Dairy Sci. 30, 849 (1947) ; Ann. N. Y. 



Acad. Sci. 52, 251 (1949). 

 66 M. L. Quaife, /. Biol. Chem. 169, 513 (1947). 



" S. W. Wright, L. J. Filer, Jr., and K. E. Mason, Pediatrics 7, 386 (1951). 

 68 K. E. Mason, /. Nutrition 23, 59 (1942). 



