PHYSIOLOGY: L. S. PALMER 585 
between fecundity and the fading of the yellow pigmentation of 
the shanks, ear lobes, etc. The fact that the carotinoid-free hens 
exhibited normal fecundity enhanced greatly their value for the 
investigation. 
The question was attacked in two ways, first, by observing the histo- 
logical changes in the shank skin when carotinoid-free food was fed 
to non-laying pigmented birds, and second, by observing the effects 
on the tissue and skin pigmentation of feeding carotinoid-rich food to 
laying carotinoid-free hens. The birds used for the histological studies 
comprise several yellow shanked White Leghorn cockerels, the specific 
source of whose pigmentation was not known, and one cockerel from 
the carotinoid-free flock whose feed was changed from the carotinoid- 
free ration to one composed principally of yellow corn. The visible 
skin parts of the latter bird took on a yellow color very rapidly after 
the introduction of the yellow corn until at the end of 42 days his 
plumage had a rich creamy appearance and the shanks, beak, ear lobes 
and vent a deep yellow color. Each of the pigmented birds was placed 
on a carotinoid-free ration and histological studies made on vertical 
frozen sections of the shank skin of individuals from time to time as 
the pigment gradually faded. 
As the result of these studies the observation of Barrows^^ was con- 
firmed that the yellow pigment of the shank skin is confined chiefly to 
the Malphigian layer of the epidermis, with some pigment in the corium. 
Especially instructive were the sections after staining with Nile blue. 
The sample of this dye which was used was found to be dichromatic 
with respect to fat and pigment, fat staining red and carotinoid pigment 
deep blue. By this means it was determined that carotinoid pigment 
exists free in granular condition in the shank epidermis, which is con- 
trary to the results reported by Barrows, who concluded that the 
lipochrome of the shank skin is dissolved in fat. The failure of Sudan 
III to color the visible skin parts of fowls, as observed by Blakeslee,^^ 
and confirmed by m^e, is explained readily by the observation that the 
Malphigian layer of both the pigmented and non-pigmented skin lacks 
appreciable am^ounts of stainable fat. 
The histological studies of the shank skin as the xanthophyll gradually 
faded on the carotinoid-free ration showed first a disappearance of pig- 
ment from the corium, then a disappearance from the outer layer of the 
corium which gradually extended to the rete of Malphigi, the last 
pigment to disappear being the xanthophyll at the base of the Mal- 
phigian layer. These observations are interpreted to mean that when 
