be analyzed in a "blind trial" before analyzing a series of birds. This 
provides the investigator with an indication of any variation that may be 
encountered between species. It is mandatory that the plasma extract be con- 
centrated, and that as much of the concentrated extract as possible be added 
to the silica gel column, in order to obtain enough steroid to be identified 
by visualization and charring procedures. 
While uniform success was attained in distinguishing the sexes of all 
species examined, present techniques of extraction, separation, and elution 
must be improved before positive identification of individual steroid hormones 
can be obtained. 
Authentication of Separatory Procedures 
The recovery of testosterone (38.8 percent) was 2,3-fold greater than that 
for B-estradiol (15.5 percent). The very high lipid levels in the plasma of 
females resulted in emulsions when chloroform was added, and probably decreased 
the efficiency of the extraction procedures. Almost all of the radioactivity 
present on the columns resided in the bands corresponding to authentic testos- 
terone or B-estradiol, indicating these steroid hormones separated well in 
the solvent system employed. This also shows that the R, values for testoster- 
one and 8-estradiol are authentic and reproducible. 
Circulating Steroid Hormones in Females 
It appears that fB-estradiol was present in all of the females of these 
species, since the Rm values, color after sulfuric acid charring, and appear- 
ance under ultraviolet light of the separated component, corresponded to that 
of authentic B-estradiol. However, Furr (see Gilbert, 1971) was reportedly 
unable to detect estrogens in the blood, and concluded their concentration must 
be less than 2 nanograms per ml. Gilbert (1971) cited others who found much 
higher concentrations, but cautioned that the methods may have been less 
specific. Blood levels of progesterone between 0.88 and 17.21 nanograms /ml 
were also reported (Gilbert, 1971). It is possible that the steroid band 
tentatively identified as ®-estradiol in this study is in reality a combination 
of several steroids in the estrane series, since in the solvent system employed 
B-estradiol, estrone, and progesterone had similar Rm values (32, 34, and 36 
mn). Cholesterol was present in both male and female extracts, and was always 
in the highest concentration of all the steroids detected. 
Circulating Steroid Hormones in Males 
The reddish-purple band in extracts from males emitted a brilliant blue 
fluorescence, but did not migrate with authentic testosterone; its identity is 
unknown. Another band that stained with iodine and did chromatograph with 
testosterone did not char with sulfuric acid. The plasma levels of testoster- 
one reportedly ranged from 84 - 783 nanograms/100 ml in domestic fowl, less 
than 10 - 120 nanograms/100 ml in quail, 65 - 270 nanograms/100 ml in ducks, 
and 15 - 98 nanograms/100 ml in pigeons (Lake and Furr, 1971). Perhaps testos- 
terone was the only steroid of the androstane series present in the species 
examined; if so, the levels extracted were evidently too low to be charred 
with sulfuric acid. 
