Factors Affecting the Determination of
the Ash Content of the Tibiae
of Chicks*
R. M. BETHKE AND P. R. RECORD
Ohio Agricultural Experiment Station, Wooster
(Received for Publication March 14, 1933)
' I HE need for the standardization of
-*• the bone-ash method for determining
the extent or degree of calcification induced
by a particular ration or feed supplement
was recently pointed out by St. John,
Kempf, and Bond (1933). These investigators also reported variations in results due
to the method of preparation of the bones
for ashing. The need for standardization is
further necessitated because several state
feed control laboratories are using the boneash method for checking the potency of
the different vitamin D supplements that are
offered for sale within their state. Unless
there is uniformity in procedure, the results
from the different laboratories will vary and
the data will not be comparable.
A study of the procedures followed by
different investigators shows that many
variations of the general method are used.
Aside from the variations in the methods of
cleaning, extracting, and ashing of the
bones, the greatest discrepancies appear to
occur in the percentage of ash reported by
different investigators in the dried, extracted bones of normal birds of a certain
age. These differences between two laboratories are frequently greater than 5 percent.
While it is realized that bones from chicks
of the same age and on the same feed will
vary considerably in their ash content, it
does not appear reasonable that the average percentage ash in "normal" bones
•Published with the approval of the Director of
the Ohio Agricultural Experiment Station.
should vary so much as S percent, if the
procedure and method of expressing the ash
values were uniform.
In seeking for an explanation for the
variation in ash values between laboratories,
it occurred to us that the method of extraction, or the preparation of the bones for
extracting and ashing, might be responsible
factors. Some investigators use alcohol exclusively for defatting, while others use alcohol and ether. In cleaning the bones, some
laboratories leave the distal and proximal
cartilages in place, while some remove the
proximal cartilage.
To ascertain whether the method of extraction or the solvent used was a factor,
part of the tibiae from the several lots of
six-weeks-old White Leghorn chicks, which
had been fed a rachitic ration with varying
amounts of vitamin D, were crushed and
extracted with hot 95 percent ethyl alcohol
for 60 hours. The other tibiae were similarly
treated except that they were extracted for
24 hours with hot alcohol and then with
ether for a similar period. The ashing was
done under identical conditions. The results
are given in Table 1. The percentage ash is
expressed on a moisture-free and extracted
basis. It is apparent that the method of extraction did not affect the percentage of
bone ash.
To determine the effect of the removal of
the proximal cartilage on the percentage of
bone ash, the tibiae from each of five lots
of six-weeks-old chicks, with varying vita-
[29]
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V
30
POULTRY
SCIENCE
TABLE 1.—Ash in alcohol and alcohol-ether extracted tibiae
gms.
184
400
203
304
None
Cod liver oil
Irradiated ergosterol in corn oil
Irradiated yeast
Additions to basal ration
None
Cod liver oil
Irradiated ergosterol in corn oil
Irradiated ergosterol in corn oil
Irradiated ergosterol in corn oil
Alcohol
extracted
Alcohol-ether
extracted
percent
35.7 + 0.47
45.6 + 0.32
36.9 + 0.29
41.2 + 0.70
percent
34.8 + 0.41
46.2 + 0.23
35.9 + 0.53
40.1+0.61
cedure with that of other laboratories suggested that the basis on which the percentage of ash in the fat-free bone was expressed might be a factor. Some investigators report the percentage ash on a moisture-free and fat-free basis, while others
only state that the results are expressed on
the extracted bones, without making reference to the moisture content of the defatted
bones. To determine to what extent the
moisture content of the extracted bones influenced the final ash values, the percentage
ash in alcohol and ether extracted tibiae
from several lots of eight-weeks-old chicks,
which had been fed the same ration with
varying amount of fluorine, was determined
on an air-dry (laboratory) and moisturefree basis. All determinations were made on
the same bones from each lot. The tibiae
were placed, after extraction, in an open
dish in the laboratory for several days prior
to weighing. The weighed bones were then
placed for a minimum of 12 hours in an oven
operating at 100°C, after which their moisture-free weight was determined.
The results are recorded in Table 3. Each
of the removal of the proximal cartilage on the percentage bone asn
Average
weight at
6 weeks
gms.
186
473
332
390
393
Ash in tibiae
Proximal
cartilage
intact
Proximal
cartilage
removed
percent
37.4 + 0.35
46.9 + 0.42
41.9 + 0.59
46.9 + 0.29
46.1 + 0.53
percent
43.1 + 0.25
50.6 + 0.51
45.7 + 1.23
51.5 + 0.33
50.2+0.33
Difference
in ash
analysis
percent
5.7±0.43
3.7 + 0.66
3.8+1.37
4 . 6 + 0.44
4.1 + 0.62
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min D intakes, were divided into two groups
of ten pairs of tibiae each. One-half of the
tibiae were cleaned by leaving both cartilages in place, while in the others the
proximal cartilage was removed. The tibiae
were then dried, crushed, individually
wrapped in filter papers, extracted with hot
alcohol followed by ether, and ashed under
similar conditions. The percentage ash
(Table 2) is expressed on a moisture- and
fat-free basis. The removal of the proximal
cartilage made a significant difference in
the ash values. The order of the lots with
respect to degree of calcification was not
changed by the inclusion of the proximal
cartilage; however, the percentage of ash
in the tibiae would be misleading unless it
were stated whether or not the cartilage had
been left in place. The results show that
normal, well-calcified tibiae at six weeks
contain from 46 to 47 percent ash on a
moisture-free and fat-free basis, whereas if
the proximal cartilages were removed the
ash values would be approximately 50 percent.
Further comparisons of our regular proTABLE 2.—Effect
Ash in tibiae
Average
weight at
6 weeks
Additions to basal ration
JANUARY,
1934.
VOL.
XIII,
No. 1
TABLE 3.—Effect
Lot No.
1
2
3
4
5
6
7
stituted for the American product. Ration
4 consisted of 46 parts yellow corn, 20
parts wheat, 5 parts wheat bran, 20 parts
soybean oilmeal, 5 parts dried skimmilk,
3 parts steamed bone meal, and 1 part sodium chloride. This ration has been successfully used by this Station for calcification studies. Accurate records were kept of
the amount of feed and liquid milk consumed. At the end of 5 weeks, 10 representative chicks from each of the 8 lots were
killed for blood and bone analyses. The
chicks remaining in the four cod liver oil
lots were continued on experiment for three
more weeks, when 10 representative birds
of moisture taken up by the extracted bones on the percentage of bone ash
Average
weight at
8 weeks
Air-dry
Moisture-free
Difference
in ash
analysis
gms.
621
575
546
386
616
456
288
percent
45.8 + 0.31
48.7 + 0.27
47.8 + 0.40
45.5 + 0.32
47.3 + 0.32
47.2 + 0.33
44.5 + 0.33
percent
49.9 + 0.33
52.5 + 0.29
51.5 + 0.43
49.2 + 0.34
50.9 + 0.35
51.0 + 0.31
47.8 + 0.35
percent
4.1+0.45
3.8 + 0.40
3.7 + 0.59
3.7 + 0.46
3.6 + 0.47
3.8 + 0.46
3.3 + 0.48
Ash in tibiae
percent of a tested cod liver oil to 25 White
Leghorn chicks of the same parent stock.
The chicks were started on their respective
rations when a day old. All lots were
brooded in the same room in pens equipped
with wire screen floors.
Ration 1 consisted of 97 parts of yellow
corn, 2 parts of calcium carbonate, 1 part
of sodium chloride, and fresh separator
skimmilk ad libitum. Ration 2 was composed of 59 parts yellow corn, 25 parts
wheat middlings, 12 parts American casein,
1 part dried yeast, 1 part calcium carbonate, 1 part calcium phosphate (tribasic),
and 1 part sodium chloride. This ration is
recommended by Hart and associates
(1931) for calcification studies and is used
by several feed control laboratories. Ration
3, had the same composition as ration 2
except that an Argentine casein was sub-
were again taken from each lot for blood
and bone studies. Calcium and inorganic
phosphorus determinations were made on
the pooled serum from each lot.
The tibiae were carefully cleaned of adhering flesh—both cartilages being left in
place. They were then dried, immersed in
hot 95 percent alcohol for 48 hours, again
dried, crushed, and individually wrapped in
filter papers and extracted in a Soxhlet extractor for 48 hours with ether. The extracted ether-free bones were transferred
to an open dish in the laboratory. The airdry (laboratory) extracted tibiae were then
weighed into tared crucibles and dried in
the oven at 100°C. for a minimum of 12
hours. The crucibles and contents were removed to a desiccator and weighed as soon
as cool to determine the weight of the moisture-free extracted bones. The samples were
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lot represents an average of 10 birds. It is
evident that the moisture taken up by the
extracted bone is a significant factor in expressing the final ash percentage. The results show that well-calcified, eight-weeksold, fat-free tibiae contained from 46 to
48 percent ash on an air-dry (laboratory)
basis and from SO to 52 percent ash on a
moisture-free basis.
To verify the previous observations and
at the same time compare the effect of different rations on the percentage of bone
ash and their suitability for calcification
studies, three types of rations were used.
Each ration was fed with and without 1
31
POULTRY
32
TABLE 4.--Eject
Lot No.
Ration
Ration 1
2
Ration 1*
3
Ration 2
4
Ration 2*
5
Ration 3
6
Ration 3*
7
Ration 4
8
Ration 4*
of different rations on ash in tibiae (chicks 5 weeks old)
Ca and
P in
ration
Average
weight at
5 weeks
percent
Ca. 0.91
P . 0.44
Ca. 0.88
P. 0.64
Ca. 0.81
P . 0.64
Ca. 0.97
P. 0.84
Ash in tibiae
Blood analysis
Difference
in ash
analysis
Ca in
100 cc
serum
Pin
100 cc
serum
Air-dry
Moisturefree
gms.
208
mg.
6.3
mg.
5.4
percent
32.3 + 0.17
percent
34.6 + 0.18
percent
2.3 + 0.25
242
11.0
9.4
44.1±0.18
47.1±0.22
3.0 + 0.29
189
5.3
6.1
30.3 + 0.24
32.4 + 0.22
2.1 + 0.33
296
11.4
7.4
44.3±0.17
47.3±0.18
3.0 + 0.25
173
7.1
6.3
30.1 + 0.42
32.3 + 0.46
2.2 + 0.63
231
10.4
7.7
43.5±0.16
46.6±0.16
3.1±0.23
198
7.0
6.5
31.9 + 0.34
34.2 + 0.23
2.3 + 0.41
341
11.3
7.5
44.9 + 0.12
47.9 + 0.12
3.0 + 0.17
* Plus cod liver oil.
then ashed in an electric muffle furnace at
7S0°C.
The essential data are given in Tables
4 and 5. It is evident that all four rations
were rachitic; however, the bone-ash values show that rations 2 and 3 (corn-casein)
were significantly more rachitic than rations
1 and 4. Rations 2 and 4 supplemented with
cod liver oil gave significantly better growth
than ration 1 (corn-skimmilk) and ration
3 (corn-Argentine casein) fortified with
vitamin D. It was noted that several chicks
in lots 5 and 6 (ration 3) showed the characteristic leg paralysis, due to a partial deficiency of the vitamin-G complex, as reported by Norris and associates (1930) and
Bethke et al (1931). This partial deficiency
was undoubtedly a limiting factor in the
growth of these lots. We have repeatedly
observed, with rats and chicks, that the
Argentine casein is relatively free from vitamin G in contrast to the average commercial casein manufactured in this country.
The vitamin-G content of the casein is a
factor which must be taken into consideration when the yellow corn-casein ration is
used for experimental purposes.
The ash values show similar significant
differences when expressed on an air-dry
and moisture-free basis as in the previous
trial (Table 3). The blood analyses show
the significantly lowered calcium and slight-
TABLE 5.—Effect of different rations on the ash in tibiae (chicks 8 weeks old)
Lot No.
2
4
6
8
Ration
Ration 1*
Ration 2*
Ration 3*
Ration 4*
* Plus cod liver oil.
Average
weight at
8 weeks
gms.
458
580
443
601
Blood analysis
Ash in tibiae
Ca in
100 cc.
serum
Pin
100 cc.
serum
Air-dry
Moisture-free
Difference
in ash
analysis
mg.
12.4
11.5
11.4
10.6
mg.
6.6
6.4
6.8
6.6
percent
46.1+0.29
47.0 + 0.26
45.3 + 0.26
48.3 + 0.23
percent
49.1 + 0.31
49.9 + 0.23
48.2 + 0.25
51.5 + 0.25
percent
3.0 + 0.42
2.9 + 0.35
2.9 + 0.36
3.2 + 0.34
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1
SCIENCE
JANUARY,
1934.
VOL.
X I I I , No. 1
SUMMARY
It was observed (a) that bones can be
as effectively extracted with hot 95 percent ethyl alcohol as with alcohol followed
by ether; (b) that the removal of the proxi-
mal cartilage causes a significant increase
in the percentage of ash in the tibiae; (c)
that the moisture taken up by the extracted
bones also significantly affects the ash percentage.
The need for the standardization of the
procedure of determining ash in bone is
discussed. It is suggested that a more or
less uniform procedure of preparing the
bones for ashing be followed and that the
percentage of ash be expressed on a moisture-free and fat-free basis.
REFERENCES
Bethke, R. M., P. R. Record, and D. C. Kennard,
1931. A type of nutritional leg paralysis affecting chicks. Poul. Sci., 10:355-368.
Hart, E. B., 0 . L. Kline, and J. A. Keenan, 1931.
A ration for the production of rickets in chicks.
Science 73:710.
Norris, L. C , G. F. Heuser, and H. S. Wilgus, Jr.,
1930. Is the chief value of milk for feeding
poultry due to the presence of a new vitamin?
Poul. Sci. 9:133-140.
St. John, J. L., Clayton Kempf, and Leonard
Bond, 1933. Observations on the bone ash method of determining effectiveness of vitamin D
supplements. Poul. Sci. 12 :34-36.
Downloaded from http://ps.oxfordjournals.org/ at Penn State University (Paterno Lib) on September 11, 2016
ly reduced inorganic phosphorus that is
generally experienced on Vitamin D deficient rations. We are unable to account
for the high (9.4) inorganic phosphorus
value in case of lot 2. repeated analysis of
the serum gave the same results.
The data, in general, show the need for
the standardization of the bone-ash method
if results from different laboratories are to
be compared. It is necessary not only to
have a more or less uniform procedure of
preparing the bones for ashing, but the calculations of the percentage of ash should
be made on the same basis. Since the moisture taken up by extracted bones is dependent upon the humidity and temperature of the air, it would seem that calculations based on the moisture-free and fatfree bone would be preferable.
33