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The Toe Ash as a Measure of Calcification in Chicks
RESEARCH NOTES
that the difference in shank length though still in favor of the Production line is no longer even questionably significant (P=0.09). ' In any case the natural population of the two lines is produced under conditions where the same factors (egg size and hatchability differentials) are operating. Hence from the standpoint of the original purpose of this note, the possible corrections of this nature are not entirely relevant. It then may be asserted that the conclusion previously reached by inference, that initial size differences are not, involved in the differentiation between the two lines, has now been confirmed by actual test. T , , T I. MICHAEL LERNER C. A. GUNNS University of California, Berkeley Received for publication May 29, 1944. REFERENCES
Lerner, I. M., 1943. Inheritance of size in SingleComb White Leghorns. J. Agr. Res. 67:447-457. , 1944. Ontogenetic size differentiation in Single-Comb White Leghorns. Growth 8:33^11.
THE TOE ASH AS A MEASURE OF CALCIFICATION IN CHICKS The usual procedure for studying calcification in growing chicks involves a determination of the percent ash in the tibiae of the chick. This procedure is time consuming and requires a considerable time after the animal experiment is finished before final results are obtained. Recently, Baird and MacMillan (1942) advocated the use of toes rather than tibiae for ashing in the A.O.A.C. chick method for estimating vitamin D. Evans and St. John (1944) further investigated the use of toes in the A.O.A.C. chick method for vitamin D. They found that the toe ash could be used in place of the tibia ash, and that very little difference
was observed in the results if the toes were extracted with alcohol and ether or were unextracted. I t appeared desirable to investigate the use of the toe ash as a measure of determining calcification in chicks on mineral experiments. Thirty-two groups of fifteen New Hampshire chicks per group were fed various levels of calcium and phosphorus and 60 A.O.A.C. chick units of vitamin D per 100 grams of diet. These chicks were kept on the various diets for six weeks. At six weeks of age the chicks were killed, and the middle toe of the left foot cut off as described by Baird and MacMillan (1942). The tibia of the left leg was removed and
Downloaded from http://ps.oxfordjournals.org/ at National Institute of Education Library, Serials Unit on June 18, 2015
tionship or as a result of the gradient in a natural-draft incubator, whereby the larger eggs would tend to be subjected to a somewhat higher temperature due to their greater proximity to the heating element. The Size-line is considerably more inbred than the Production line (see Lerner, 1943), and the observed embryonic mortality in it is somewhat greater. Thus during the 4-week hatching season immediately preceding the present test, embryonic mortality from 7 to 20 days was 24.4 percent for 409 Size-line embryos and only 19.9 percent for 512 Production-line embryos in the matings identical with those considered here. It is hence possible that more moribund embryos were included in the Size- than in the Productionline measurements., A crude correction for this difference may be made by truncating the frequency distributions of embryo weights of the two respective lines by the above percentages. When such a correction is applied to the data without eliminating the effects of egg size, it is found
351
352
RESEARCH NOTES
cleaned as described in the A.O.A.C. methods of analysis (1940). The tibiae were extracted with hot 95 percent ethyl alcohol for forty-eight hours. The percent ash of the tibiae was determined by combining the bones of all of the chicks in each group, drying at 100° C.
I
14.0
• ir
l 16
i 17
18
Peroont to* *sh
FIG. 1. Comparison of percent tibia ash and toe ash in New Hampshire chicks at eight weeks of age. Each point represents one group of chicks.
ROBERT JOHN EVANS
for approximately twenty hours, weighing to obtain the dry weight, and then ashing at 550° C. for approximately sixteen hours before weighing the ash. The toes were also dried and ashed by groups at the same temperatures and for the same time. In Figure 1 the percent bone ash is plotted against the percent toe ash for the thirty-two groups of chicks. Since there is not very much deviation from the straight line, considering the small difference between high and low ash, it appears that the agreement between bone ash and toe ash is very good. This is substantiated by a determination of the coefficient of cor-
J. S. CARVER
Divisions of Chemistry and Poultry Husbandry, Washington Agricultural Experiment Station, Pullman, Washington Received for publication May 30, 1944. REFERENCES
A.O.A.C. Methods of Analysis. 1940, p. 371-373. Baird, F. D., and M. J. MacMillan, 1942. Use of toes rather than tibiae in A.O.A.C. chick method of vitamin D determination. J. Assoc. Offic. Agr. Chem. 25:518-524. Evans, R. J., and J. L. St. John, 1944. Modifications of the A.O.A.C. chick method of vitamin D assay. J. Assoc. Offic. Agr. Chem. (In Press).
Downloaded from http://ps.oxfordjournals.org/ at National Institute of Education Library, Serials Unit on June 18, 2015
54
relation between these two values. This was +0.847, which is a highly significant correlation. I t appears from the data presented that the toe ash is a satisfactory method of determining calcification in growing chicks. This method has the advantage over the determination of tibiae ash in being much more rapid, since the final results for sixteen groups of chicks can be obtained within forty-eight hours after the time the chicks are killed. It also involves much less work than tibiae ash determinations. Baird and MacMillan (1942) and Evans and St. John (1944) suggested the possibility of not having to sacrifice chicks when the toe ash is used. Preliminary work with turkeys indicates that the toe can be satisfactorily removed without very harmful results to the poult. Although it was necessary to remove the toe at the second joint rather than the middle or third joint, there appeared to be good agreement between toe ash at six weeks of age and bone ash at eight weeks of age.
that the difference in shank length though still in favor of the Production line is no longer even questionably significant (P=0.09). ' In any case the natural population of the two lines is produced under conditions where the same factors (egg size and hatchability differentials) are operating. Hence from the standpoint of the original purpose of this note, the possible corrections of this nature are not entirely relevant. It then may be asserted that the conclusion previously reached by inference, that initial size differences are not, involved in the differentiation between the two lines, has now been confirmed by actual test. T , , T I. MICHAEL LERNER C. A. GUNNS University of California, Berkeley Received for publication May 29, 1944. REFERENCES
Lerner, I. M., 1943. Inheritance of size in SingleComb White Leghorns. J. Agr. Res. 67:447-457. , 1944. Ontogenetic size differentiation in Single-Comb White Leghorns. Growth 8:33^11.
THE TOE ASH AS A MEASURE OF CALCIFICATION IN CHICKS The usual procedure for studying calcification in growing chicks involves a determination of the percent ash in the tibiae of the chick. This procedure is time consuming and requires a considerable time after the animal experiment is finished before final results are obtained. Recently, Baird and MacMillan (1942) advocated the use of toes rather than tibiae for ashing in the A.O.A.C. chick method for estimating vitamin D. Evans and St. John (1944) further investigated the use of toes in the A.O.A.C. chick method for vitamin D. They found that the toe ash could be used in place of the tibia ash, and that very little difference
was observed in the results if the toes were extracted with alcohol and ether or were unextracted. I t appeared desirable to investigate the use of the toe ash as a measure of determining calcification in chicks on mineral experiments. Thirty-two groups of fifteen New Hampshire chicks per group were fed various levels of calcium and phosphorus and 60 A.O.A.C. chick units of vitamin D per 100 grams of diet. These chicks were kept on the various diets for six weeks. At six weeks of age the chicks were killed, and the middle toe of the left foot cut off as described by Baird and MacMillan (1942). The tibia of the left leg was removed and
Downloaded from http://ps.oxfordjournals.org/ at National Institute of Education Library, Serials Unit on June 18, 2015
tionship or as a result of the gradient in a natural-draft incubator, whereby the larger eggs would tend to be subjected to a somewhat higher temperature due to their greater proximity to the heating element. The Size-line is considerably more inbred than the Production line (see Lerner, 1943), and the observed embryonic mortality in it is somewhat greater. Thus during the 4-week hatching season immediately preceding the present test, embryonic mortality from 7 to 20 days was 24.4 percent for 409 Size-line embryos and only 19.9 percent for 512 Production-line embryos in the matings identical with those considered here. It is hence possible that more moribund embryos were included in the Size- than in the Productionline measurements., A crude correction for this difference may be made by truncating the frequency distributions of embryo weights of the two respective lines by the above percentages. When such a correction is applied to the data without eliminating the effects of egg size, it is found
351
352
RESEARCH NOTES
cleaned as described in the A.O.A.C. methods of analysis (1940). The tibiae were extracted with hot 95 percent ethyl alcohol for forty-eight hours. The percent ash of the tibiae was determined by combining the bones of all of the chicks in each group, drying at 100° C.
I
14.0
• ir
l 16
i 17
18
Peroont to* *sh
FIG. 1. Comparison of percent tibia ash and toe ash in New Hampshire chicks at eight weeks of age. Each point represents one group of chicks.
ROBERT JOHN EVANS
for approximately twenty hours, weighing to obtain the dry weight, and then ashing at 550° C. for approximately sixteen hours before weighing the ash. The toes were also dried and ashed by groups at the same temperatures and for the same time. In Figure 1 the percent bone ash is plotted against the percent toe ash for the thirty-two groups of chicks. Since there is not very much deviation from the straight line, considering the small difference between high and low ash, it appears that the agreement between bone ash and toe ash is very good. This is substantiated by a determination of the coefficient of cor-
J. S. CARVER
Divisions of Chemistry and Poultry Husbandry, Washington Agricultural Experiment Station, Pullman, Washington Received for publication May 30, 1944. REFERENCES
A.O.A.C. Methods of Analysis. 1940, p. 371-373. Baird, F. D., and M. J. MacMillan, 1942. Use of toes rather than tibiae in A.O.A.C. chick method of vitamin D determination. J. Assoc. Offic. Agr. Chem. 25:518-524. Evans, R. J., and J. L. St. John, 1944. Modifications of the A.O.A.C. chick method of vitamin D assay. J. Assoc. Offic. Agr. Chem. (In Press).
Downloaded from http://ps.oxfordjournals.org/ at National Institute of Education Library, Serials Unit on June 18, 2015
54
relation between these two values. This was +0.847, which is a highly significant correlation. I t appears from the data presented that the toe ash is a satisfactory method of determining calcification in growing chicks. This method has the advantage over the determination of tibiae ash in being much more rapid, since the final results for sixteen groups of chicks can be obtained within forty-eight hours after the time the chicks are killed. It also involves much less work than tibiae ash determinations. Baird and MacMillan (1942) and Evans and St. John (1944) suggested the possibility of not having to sacrifice chicks when the toe ash is used. Preliminary work with turkeys indicates that the toe can be satisfactorily removed without very harmful results to the poult. Although it was necessary to remove the toe at the second joint rather than the middle or third joint, there appeared to be good agreement between toe ash at six weeks of age and bone ash at eight weeks of age.