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Effect of Trace Minerals and Other Dietary Ingredients upon Vitamin A Stability in Stored Poultry Diets*
STABILITY OF VITAMIN A IN STORED FEEDS
REFERENCES Carson, J. D., F. W. Lorenz and V. S. Asmundson, 1955a. Semen production in the turkey male. 1. Seasonal variation. Poultry Sci. 34: 336-343. Carson, J. D., F. W. Lorenz and V. S. Asmundson, 1955b. Semen production in the turkey males. 2. Age at sexual maturity. Poultry Sci. 34: 344347. Hazel, L. N., M. L. Baker and C. F. Reinmiller, 1943. Genetic and environmental correlations between the growth rate of pigs at different ages. J. Animal Sci. 2: 118-128.
Kosin, I. L., and M. S. Mitchell, 1953. Further data on the effect of ambient temperatures on fertility in turkey males. Poultry Sci. 32: 910. Lerner, I. M., 1950. Population Genetics and Animal Improvement, xviii+342 pages. Cambridge University Press, London. Lerner, I. M., V. S. Asmundson and D. M. Cruden, 1947. The improvement of New Hampshire fryers. Poultry Sci. 26: 515-524. Lorenz, F. W., 1950. Onset and duration of fertility in turkeys. Poultry Sci. 29: 20-26. McCartney, M. G., 1954. The physiology of reproduction in turkeys. Poultry Sci. 33: 390-391. Marshall, A. J., 1951. The refractory period of testes rhythm in birds and its possible bearing on breeding and migration. Wilson Bulletin, 63: 238-261. Parker, J. E., 1946. Semen production of Broad Breasted Bronze turkeys. Poultry Sci. 25: 65-68. Parker, J. E., 1949. Fertility in chickens and turkeys. In Fertility and Hatchability of Chicken and Turkey Eggs. L. W. Taylor, Editor. 95-149. J. Wiley and Sons, Inc., New York. Van Drimmelen, G. C , 1951. Artificial insemination of birds by the intra peritoneal route. Onderstepoort J. Vet. Res. Supplement No. 1: 3-212.
Effect of Trace Minerals and Other Dietary Ingredients upon Vitamin A Stability in Stored Poultry Diets* A. W. HALVERSON AND C. M.
HENDRICK
Experiment Station Biochemistry Department, South Dakota State College, College Station, South Dakota (Received for publication July 12, 1954)
T
HE problem of vitamin A stability in stored poultry feeds continues to cause concern, especially with the increased trend to add the free trace minerals to commercial mixed diets. The new vitamin A preparations which are being widely used also complicate interpretation of losses from previous data and make reappraisal with new studies essential. Previous researchers on vitamin A stability with cod liver oil or feeding oil
* Approved for publication by the Director of the South Dakota Agricultural Experiment Station as No. 326 of the Journal Series.
in poultry feeds have obtained considerable variation in losses during normal storage conditions. However, most of the data show that the added vitamin A is lost at a slow to moderate rate during normal storage (Bethke et al., 1939; Holder and Ford, 1939; Halpern et al., 1949; Halverson and Hart, 1950; and Siedler and Schweigert, 1954). A few investigators have shown that certain feed ingredients, including trace minerals, have a definite effect upon vitamin A stability in mixed feeds. Bethke et al. (1939) demonstrated that meat scraps caused increased vitamin A losses in practical diets.
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and between measurable sample and last plus. Heritability estimates were calculated. They ranged from intermediate to high. Those for semen volume tended to vary from 20 to 40 percent. The heritability estimates indicate that a substantial part of the total variation in semen volume as well as concentration and age at last measurable sample are determined by hereditary differences.
355
356
A. W. HALVERSON AND C M .
HENDRICK
such preparations were quite stable in feed diluents such as corn and soybean oil meal and that no marked superiority of any product was evident. Stephenson (1953) tested several dry or stabilized products by chick growth studies and found all feeds to support optimum growth even after 12 weeks of storage. Apparently the vitamin A in such products is readily available to the animal even though protected by wax and other inert materials. The present study which employed several different vitamin A sources and different diets, both with and without added trace minerals, should contribute needed information about the vitamin A stability problem.
The problem of the stability of dry, stabilized vitamin A preparations in mixed feeds has also been investigated to some extent in recent years. Burns and Quackenbush (1951) showed that all
EXPERIMENTAL Three practical diets which were supplemented with different vitamin A sources were prepared. The compositions of the diets are shown in Table 1. Diet
TABLE 1.—Composition of diets to which different vitamin A supplements were added
a
%
White corn 2 61.0 Soybean oil meal 3 35.0 Wheat bran Wheat middlings Oats Sorghum grain Dried buttermilk 0.5 Iodized salt 1.5 CaHPO. 2.0 CaCO, M e a t scraps Limestone Fish meal Steamed bone meal
+
Diet 3
Diet 2
Diet 1 Ingredients^ —
b
%
a
b
a
b
%
%
%
%
66.5 22.0
21.0 29.0 10.0 15.0 5.0 16.0
26.5 16.0 10.0 15.0 5.0 16.0
0.5
0.5 1.2 2.3
0.5
59.0 10.0 15.0 10.0 1.0 2.0 3.0
10.0 1.0
10.0 1.0
+_
+
4.0 38.0 10.0 15.0 10.0 1.0 10.0 4.0 7.0 1.0
+
.t.
_+_
Trace minerals (None) MnSO.-HsO F e S 0 4 • 7H*0 CuSO. • SHsO CoSO. • 6HiO
— — — —
(None) — 0.02
— — — — — —
(None) 0.02 0.01 0.001 0.00001
—. — — —
(None) — 0.02
(None) 0.02 0.01 0.001
— — —. —. — — o.ooooi
— — — —
(None) — 0.08 — — — — — —
0.08 0.04 0.004 0.00004
1 The following amounts of vitamin and antibiotic ingredients were also included per 100 grams of diets 1 and 2: riboflavin, 0.3 mg.; niacin, 0.7 mg.; calcium pantothenate, 0.7 mg.; choline chloride, 40 mg.; vitamin D s , 100 AOAC chick units; vitamin Bit, 1.7 meg.; and procaine penicillin, 0.3 mg. Diet 3 received similar supplementation except at a 4-fold level. 2 Corn was freshly ground for each diet series. 3 Soybean oil meal prepared in laboratory from raw beans by grinding, hexane extraction and subsequent heat treatment with steam at 15 lbs. pressure for 30 minutes.
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Miller et at. (1942) showed that the addition of a high level of manganese sulfate to a bran-cod liver oil mixture increased vitamin A losses. Further, Halverson and Hart (1950) found that vitamin A losses increased greatly upon addition of moderately high levels of the common trace minerals, manganese, iron, copper and cobalt salts, to a white corn-cod liver oil mixture. Davies and Worden (1954) reported that fish or liver meal has a vitamin A stabilizing effect in mixed feeds as compared to oat or barley meal. These workers also confirmed the vitamin A destructive effect of the trace minerals. Thus, the work cited indicates that vitamin A stability with fish oils in mixed feeds appears to be much dependent upon the ingredient composition of the feed.
STABILITY OF VITAMIN A IN STORED FEEDS
with entire diets by addition in the form of a finely ground starch-mineral mix (approx. 3:1). The preparation and storage of ingredients prior to mixing involved special laboratory preparation of two constituents, white corn and soybean oil meal (see Table 1 footnotes), and then refrigeration at 0°C. of these and other ingredients until such time as all of the different diets were prepared. After completion of the diet preparation, the samples were placed in small Kraft paper sacks in anticipation of storage. All samples were refrigerated at — 17.5°C. until completion of the initial vitamin A analyses. Then the samples were placed in storage at a temperature of 37°+1.5°C. for 150 days, Vitamin A analyses were performed at the 30 day storage interval as well as at termination of storage. All analyses were in duplicate and the percentage losses were calculated from the average values. The maximum variation observed in duplicate analysis was less than 8 percent. All samples concerned with a given diet (e.g., diet 1 with three different vitamin A supplements) were stored simultaneously. The procedure employed in the vitamin A analysis is described as follows: 15.0 gm. samples of diet were Soxhlet-extracted for 4 hours with a 3:7 mixture of acetone and n-hexane. The extract was evaporated to a volume of 4-10 ml. and chromatographed by using a column composed of of a 1:1 ratio of magnesia (Micro brand No. 2642, Westvaco Corp.) and diatomaceous earth (Hyflo Supercel) and of 20 mm. diameter and 120 mm. length. Following elution of the column with 120 ml. of a 2:8 mixture of acetone and nhexane, the eluant was evaporated to dryness under vacuum and taken up in chloroform. Vitamin A was determined by the Carr-Price colorimetric reaction with 25% antimony trichloride. Blank analyses
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1 was a corn-soybean starting ration; diet 2 contained wheat bran, wheat middlings, oats and sorghum grain in partial replacement of the corn-soybean constituents; and diet 3 was a mash concentrate which contained high protein, vitamin and mineral levels. Each of the diets was similarily modified by the addition of meat scraps plus limestone (see b form of diets) and then by the further addition of manganese salt alone and with iron, copper and cobalt salts. Diet 3 also included fish meal and bone meal in the modified forms of the diet. Three different vitamin A sources were employed with each of the diets. A cod liver oil sample (Supplement 1—2,000A, 200D) and two dry, stabilized products were employed. One of the dry preparations (Supplement 2—12,200 U.S.P. units per gram) was of white powdery texture and consisted of fish oil in a fat carrier. The other preparation (Supplement 3— 12,900 U.S.P. units per gram) consisted of small yellow pellets which were composed of vitamin A feeding oil blended with wheat germ oil meal and soybean oil meal. With this latter preparation, the fat-soluble vitamins were sealed into the carrier by a process involving the use of microcrystalline wax. Supplements 1, 2 and 3 were added to the diets at the following levels: 1.5, 0.3 and 0.5 percent, respectively. In diet preparation, considerable attention was given to the mixing of the critical constituents such as the vitamin A supplements and the trace minerals with the diets. The procedure of storage and of preparation of the principal diet ingredients prior to mixing was also carefully controlled. The vitamin A supplements were dispersed in the diets by thoroughly premixing with a portion of diet before mixing with the entire diet contents. Trace minerals were later mixed
357
358
A. W. HALVERSON AND C. M. HENDRICK
However, the analytical loss with the actual diets should have been minimum as well as constant because of the predominance of the ester form in fish oils and because of the similarity of recovery of the vitamin among different diets even in the presence of added trace minerals.
were made with all diets to determine whether non-vitamin A feed ingredients were affecting the reaction through interfering color development. However, the blank corrections were always low and thus did not appear to significantly affect the data. The reliability of the vitamin A method employed was further checked by several recovery determinations with the ester (acetate) and the alcohol forms of the vitamin. The vitamin A standards were added both before and after extraction of blank samples (15 gm.). Recoveries of the ester form amounted to 85% with extraction and to 95-97% without the extraction treatment. The alcohol form gave recoveries of 69-78% with extraction and 91-93% without. The results indicated that the 4-hour extraction as practiced in diet analysis caused considerable vitamin A destruction especially with the alcohol form of the vitamin. Hence, it is likely that some vitamin A destruction also occurred in analysis of supplemented diets.
RESULTS AND DISCUSSION
TABLE 2.—Vitamin A losses during storage of different diets containing different vitamin A supplements (Storage at 37°C.) Percent loss in 30 days storage
Percent loss in 150 days storage
Diet
Diet
Vitamin A supplements and diet modifications 1
2
3
1
2
3
Supplement No. 1 (cod liver oil) a b b + M n salt b-f-Mn, Fe, Cu and Co salts
4.2 8.7 5.7 20.6
8.4 8.3 7.9 19.5
7.6 7.9 11.6 36.9
52.1 56.8 57.2 72.9
45.9 47.6 47.4 61.9
56.8 50.6 56.1 93.0
Supplement No. 2 (dry-fat powder) a b b + M n salt b + M n , Fe, Cu and Co salts
10.6 10.4 15.3 17.9
7.7 7.6 7.6 19.2
12.8 9.4 17.7 31.7
48.6 51.0 52.6 71.1
49.3 52.4 50.5 66.6
44.9 53.5 60.8 88.2
00 00 00 00
10.1 2.7 7.9 10.1
3.1 7.2 2.2 4.8
17.2 15.1 18.9 25.5
21.7 23.7 24.0 28.3
5.7 19.8 23.0 34.4
Supplement No. 3 (wax-sealed pellets) a b b + M n salt b + M n , Fe, Cu and Co salts
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The vitamin A losses obtained with diets 1, 2 and 3 are shown in Table 2. In general, losses were small at the 30 day storage period but increased to as much as 50% or more by the 150 day period. Diet modification as by addition of meat scraps and limestone with diets 1 and 2 and by addition of meat scraps, fish meal, limestone and bone meal with diet 3 generally had no noticeable effect upon vitamin A stability with any of the vitamin A supplements (see b modifications). The further addition of manganese salt also did not materially affect the loss results. However, the addition of iron, copper and cobalt salts along with the other ingredi-
STABILITY OF VITAMIN A IN STORED FEEDS
The data are in accord with several previous investigators in regard to the overall low loss rate of vitamin A of various forms in mixed feeds (Holder and Ford, 1939; Burns and Quackenbush, 1951; and others). However, the destructive effects of meat scraps as observed by Bethke et al. (1939) and of manganese salt as observed by Miller et al. (1942) were not corroborated. Confirmation of previous reports by Halverson and Hart (1950) and by Davies and Worden (1954) on the destructive effect of the trace min-
erals toward the vitamin A of supplemented feeds was evident. Thus, the overall experimental results indicate the important role of the trace minerals in promoting vitamin A losses. Information as to the specific trace mineral or minerals involved in the vitamin A loss picture remains lacking, however, as does data on the relation of the oxidative state of diet ingredients to the response obtained from trace minerals. SUMMARY
Vitamin A storage losses were determined with three vegetable-type poultry diets which were supplemented with different vitamin A sources. Diet modifications which included the addition of meat scraps, limestone, manganese salt and iron, copper and cobalt salts were made with each of the diets to determine the effects of such ingredients upon vitamin A stability. The data obtained with storage at 31° C. for 30 and 150 day periods permit several conclusions: namely, (1) a fairly good degree of vitamin A stability was obtained with each diet and its modifications, (2) an overall uniformity in stability was observed with each vitamin A supplement among the different diets, (3) no definite changes in vitamin A stability were observed with diet modification except upon addition of all of the free trace minerals. (4) a greater vitamin A loss occurred when all of the trace minerals were added at a higher level as in a feed concentrate, and (5) a greater overall vitamin A stability was obtained with a wax-coated vitamin A supplement than with oil- or fat-type supplements. ACKNOWLEDGMENT
This investigation was supported in part by a grant-in-aid from the American Dehydrators Association.
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ents caused an increase in losses with all diets especially at the 150 day storage period. A great difference in vitamin A stability was observed between the wax-sealed supplement and the oil and fat supplements. This difference was evident with all three diets with losses being about onethird to one-half as great with the waxsealed preparation as with the other two. The wax-sealed product was also less sensitive to complete diet modification which included all of the trace minerals. The similarity of stability between two of the vitamin A supplements, namely, cod liver oil (No. 1) and stabilized fish oil in dry fat (No. 2), was of considerable interest. Both products were almost equal in stability both within and among diets. Further, the other supplement (No. 3) also had a similar stability pattern among different diets even though it was much superior to the other supplements in overall stability. These observations suggest that the replacement of a cereal ingredient with others as well as with small amounts of meat scraps, limestone and manganese salt has no definite effect upon vitamin A stability. However, it is evident that the interpretation of equal vitamin A stability among a variety of diets applies only to controlled conditions such as were employed in the present experiments.
359
360
E. M. D E N I N G T O N AND A. M.
REFERENCES
liver oil in cereal feeds. J. Nutrition, 40:415-428. Holder, R. C, and S. K. Ford, 1939. The stability of vitamin A from cod liver oil in mixed feeds. Poultry Sci. 18: 345-349. Miller, M. W., V. Joukovsky and N. Hokenstad, 1942. The effect of manganese sulphate on the stability of vitamins A and D of cod liver oil when stored in mixed feeds. Poultry Sci. 21: 200202. Siedler, A. J., and B. S. Schweigert, 1954. Effect of added stabilized animal fats on stability of vitamin A in feeds. J. Agr. Food Chem. 2: 193195. Stephenson, E. L., 1953. Stability of vitamin A in poultry feeds. Arkansas Farm Research, Vol. 2, No. 3.
Blood Technics for Chickens EFFIE
M.
D E N I N G T O N AND A L F R E D M .
Regional Poultry Research Laboratory,^
East Lansing,
LUCAS* Mich.
(Received for publication July 13. 1954)
A
N A T O M I C Energy Project under• taken at this Laboratory involved numerous blood determinations made over a two-year period and, prelininary to the actual work, a comparative study of technics suitable for avian blood was made. T h e technics selected have been applied to over 2,500 blood samples and have given results which appear to be reliable. During the period when work was in progress, inquires were received conconcerning the technics which had been selected and the development of modifications which had been found useful. In order to make this information generally available, a report covering the following subjects has been prepared: (1) description of a unit tray for handling large numbers of blood samples, (2) determination of avian hemoglobin, (3) accuracy of * Research conducted under a grant from the Atomic Energy Commission to this Laboratory. f Agricultural Research Service, United States Department of Agriculture.
hemoglobin pipettes, (4) hematocrit with the Van Allen pipettes, (5) composition of the buffy coat, (6) a shaker for red blood cell pipettes, (7) variability in Samples of Wright's stain, (8) a comparison of three types of methods for making total white cell counts, and (9) an indirect method for making thrombocyte counts. All studies were based on Single Comb White Leghorn chickens. Most of them were females and some of them had been irradiated with x-rays at low dosages of between 50 and 300 r. RESULTS A unit tray for transporting blood samples.—The t r a y was designed to handle tubes for hemoglobin, hematocrit and red and white blood cell determinations. T h e box and the contained tubes are shown in Fig. 1. In addition, clean slides and an infra red lamp for warming the slides used in making blood smears were part of the equipment. I t was necessary to have a
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Bethke, R. M., P. R. Record and O. H. M. Wilder, 1939. The stability of carotene and vitamin A in a mixed ration. Poultry Sci. 18: 179-187. Burns, M. J., and F. W. Quackenbush, 1951: Stability of dry vitamin A concentrates. Ind. Eng. Chem. 43: 1592-1593. Davies, A. W., and A. N. Worden, 1954. The stability of vitamin A in animal feeding stuffs. J. Sci. Food Agr. 5: 107-112. Halpern, G. R., B. March and J. Biely, 1949. Stability and utilization of vitamin A emulsions in mixed feeds. Poultry Sci. 28: 168-172. Halverson, A. W., and E. B. Hart, 1950. Factors affecting the stability of the vitamin A from cod
LUCAS
REFERENCES Carson, J. D., F. W. Lorenz and V. S. Asmundson, 1955a. Semen production in the turkey male. 1. Seasonal variation. Poultry Sci. 34: 336-343. Carson, J. D., F. W. Lorenz and V. S. Asmundson, 1955b. Semen production in the turkey males. 2. Age at sexual maturity. Poultry Sci. 34: 344347. Hazel, L. N., M. L. Baker and C. F. Reinmiller, 1943. Genetic and environmental correlations between the growth rate of pigs at different ages. J. Animal Sci. 2: 118-128.
Kosin, I. L., and M. S. Mitchell, 1953. Further data on the effect of ambient temperatures on fertility in turkey males. Poultry Sci. 32: 910. Lerner, I. M., 1950. Population Genetics and Animal Improvement, xviii+342 pages. Cambridge University Press, London. Lerner, I. M., V. S. Asmundson and D. M. Cruden, 1947. The improvement of New Hampshire fryers. Poultry Sci. 26: 515-524. Lorenz, F. W., 1950. Onset and duration of fertility in turkeys. Poultry Sci. 29: 20-26. McCartney, M. G., 1954. The physiology of reproduction in turkeys. Poultry Sci. 33: 390-391. Marshall, A. J., 1951. The refractory period of testes rhythm in birds and its possible bearing on breeding and migration. Wilson Bulletin, 63: 238-261. Parker, J. E., 1946. Semen production of Broad Breasted Bronze turkeys. Poultry Sci. 25: 65-68. Parker, J. E., 1949. Fertility in chickens and turkeys. In Fertility and Hatchability of Chicken and Turkey Eggs. L. W. Taylor, Editor. 95-149. J. Wiley and Sons, Inc., New York. Van Drimmelen, G. C , 1951. Artificial insemination of birds by the intra peritoneal route. Onderstepoort J. Vet. Res. Supplement No. 1: 3-212.
Effect of Trace Minerals and Other Dietary Ingredients upon Vitamin A Stability in Stored Poultry Diets* A. W. HALVERSON AND C. M.
HENDRICK
Experiment Station Biochemistry Department, South Dakota State College, College Station, South Dakota (Received for publication July 12, 1954)
T
HE problem of vitamin A stability in stored poultry feeds continues to cause concern, especially with the increased trend to add the free trace minerals to commercial mixed diets. The new vitamin A preparations which are being widely used also complicate interpretation of losses from previous data and make reappraisal with new studies essential. Previous researchers on vitamin A stability with cod liver oil or feeding oil
* Approved for publication by the Director of the South Dakota Agricultural Experiment Station as No. 326 of the Journal Series.
in poultry feeds have obtained considerable variation in losses during normal storage conditions. However, most of the data show that the added vitamin A is lost at a slow to moderate rate during normal storage (Bethke et al., 1939; Holder and Ford, 1939; Halpern et al., 1949; Halverson and Hart, 1950; and Siedler and Schweigert, 1954). A few investigators have shown that certain feed ingredients, including trace minerals, have a definite effect upon vitamin A stability in mixed feeds. Bethke et al. (1939) demonstrated that meat scraps caused increased vitamin A losses in practical diets.
Downloaded from http://ps.oxfordjournals.org/ at Universitat Autònoma de Barcelona on October 28, 2014
and between measurable sample and last plus. Heritability estimates were calculated. They ranged from intermediate to high. Those for semen volume tended to vary from 20 to 40 percent. The heritability estimates indicate that a substantial part of the total variation in semen volume as well as concentration and age at last measurable sample are determined by hereditary differences.
355
356
A. W. HALVERSON AND C M .
HENDRICK
such preparations were quite stable in feed diluents such as corn and soybean oil meal and that no marked superiority of any product was evident. Stephenson (1953) tested several dry or stabilized products by chick growth studies and found all feeds to support optimum growth even after 12 weeks of storage. Apparently the vitamin A in such products is readily available to the animal even though protected by wax and other inert materials. The present study which employed several different vitamin A sources and different diets, both with and without added trace minerals, should contribute needed information about the vitamin A stability problem.
The problem of the stability of dry, stabilized vitamin A preparations in mixed feeds has also been investigated to some extent in recent years. Burns and Quackenbush (1951) showed that all
EXPERIMENTAL Three practical diets which were supplemented with different vitamin A sources were prepared. The compositions of the diets are shown in Table 1. Diet
TABLE 1.—Composition of diets to which different vitamin A supplements were added
a
%
White corn 2 61.0 Soybean oil meal 3 35.0 Wheat bran Wheat middlings Oats Sorghum grain Dried buttermilk 0.5 Iodized salt 1.5 CaHPO. 2.0 CaCO, M e a t scraps Limestone Fish meal Steamed bone meal
+
Diet 3
Diet 2
Diet 1 Ingredients^ —
b
%
a
b
a
b
%
%
%
%
66.5 22.0
21.0 29.0 10.0 15.0 5.0 16.0
26.5 16.0 10.0 15.0 5.0 16.0
0.5
0.5 1.2 2.3
0.5
59.0 10.0 15.0 10.0 1.0 2.0 3.0
10.0 1.0
10.0 1.0
+_
+
4.0 38.0 10.0 15.0 10.0 1.0 10.0 4.0 7.0 1.0
+
.t.
_+_
Trace minerals (None) MnSO.-HsO F e S 0 4 • 7H*0 CuSO. • SHsO CoSO. • 6HiO
— — — —
(None) — 0.02
— — — — — —
(None) 0.02 0.01 0.001 0.00001
—. — — —
(None) — 0.02
(None) 0.02 0.01 0.001
— — —. —. — — o.ooooi
— — — —
(None) — 0.08 — — — — — —
0.08 0.04 0.004 0.00004
1 The following amounts of vitamin and antibiotic ingredients were also included per 100 grams of diets 1 and 2: riboflavin, 0.3 mg.; niacin, 0.7 mg.; calcium pantothenate, 0.7 mg.; choline chloride, 40 mg.; vitamin D s , 100 AOAC chick units; vitamin Bit, 1.7 meg.; and procaine penicillin, 0.3 mg. Diet 3 received similar supplementation except at a 4-fold level. 2 Corn was freshly ground for each diet series. 3 Soybean oil meal prepared in laboratory from raw beans by grinding, hexane extraction and subsequent heat treatment with steam at 15 lbs. pressure for 30 minutes.
Downloaded from http://ps.oxfordjournals.org/ at Universitat Autònoma de Barcelona on October 28, 2014
Miller et at. (1942) showed that the addition of a high level of manganese sulfate to a bran-cod liver oil mixture increased vitamin A losses. Further, Halverson and Hart (1950) found that vitamin A losses increased greatly upon addition of moderately high levels of the common trace minerals, manganese, iron, copper and cobalt salts, to a white corn-cod liver oil mixture. Davies and Worden (1954) reported that fish or liver meal has a vitamin A stabilizing effect in mixed feeds as compared to oat or barley meal. These workers also confirmed the vitamin A destructive effect of the trace minerals. Thus, the work cited indicates that vitamin A stability with fish oils in mixed feeds appears to be much dependent upon the ingredient composition of the feed.
STABILITY OF VITAMIN A IN STORED FEEDS
with entire diets by addition in the form of a finely ground starch-mineral mix (approx. 3:1). The preparation and storage of ingredients prior to mixing involved special laboratory preparation of two constituents, white corn and soybean oil meal (see Table 1 footnotes), and then refrigeration at 0°C. of these and other ingredients until such time as all of the different diets were prepared. After completion of the diet preparation, the samples were placed in small Kraft paper sacks in anticipation of storage. All samples were refrigerated at — 17.5°C. until completion of the initial vitamin A analyses. Then the samples were placed in storage at a temperature of 37°+1.5°C. for 150 days, Vitamin A analyses were performed at the 30 day storage interval as well as at termination of storage. All analyses were in duplicate and the percentage losses were calculated from the average values. The maximum variation observed in duplicate analysis was less than 8 percent. All samples concerned with a given diet (e.g., diet 1 with three different vitamin A supplements) were stored simultaneously. The procedure employed in the vitamin A analysis is described as follows: 15.0 gm. samples of diet were Soxhlet-extracted for 4 hours with a 3:7 mixture of acetone and n-hexane. The extract was evaporated to a volume of 4-10 ml. and chromatographed by using a column composed of of a 1:1 ratio of magnesia (Micro brand No. 2642, Westvaco Corp.) and diatomaceous earth (Hyflo Supercel) and of 20 mm. diameter and 120 mm. length. Following elution of the column with 120 ml. of a 2:8 mixture of acetone and nhexane, the eluant was evaporated to dryness under vacuum and taken up in chloroform. Vitamin A was determined by the Carr-Price colorimetric reaction with 25% antimony trichloride. Blank analyses
Downloaded from http://ps.oxfordjournals.org/ at Universitat Autònoma de Barcelona on October 28, 2014
1 was a corn-soybean starting ration; diet 2 contained wheat bran, wheat middlings, oats and sorghum grain in partial replacement of the corn-soybean constituents; and diet 3 was a mash concentrate which contained high protein, vitamin and mineral levels. Each of the diets was similarily modified by the addition of meat scraps plus limestone (see b form of diets) and then by the further addition of manganese salt alone and with iron, copper and cobalt salts. Diet 3 also included fish meal and bone meal in the modified forms of the diet. Three different vitamin A sources were employed with each of the diets. A cod liver oil sample (Supplement 1—2,000A, 200D) and two dry, stabilized products were employed. One of the dry preparations (Supplement 2—12,200 U.S.P. units per gram) was of white powdery texture and consisted of fish oil in a fat carrier. The other preparation (Supplement 3— 12,900 U.S.P. units per gram) consisted of small yellow pellets which were composed of vitamin A feeding oil blended with wheat germ oil meal and soybean oil meal. With this latter preparation, the fat-soluble vitamins were sealed into the carrier by a process involving the use of microcrystalline wax. Supplements 1, 2 and 3 were added to the diets at the following levels: 1.5, 0.3 and 0.5 percent, respectively. In diet preparation, considerable attention was given to the mixing of the critical constituents such as the vitamin A supplements and the trace minerals with the diets. The procedure of storage and of preparation of the principal diet ingredients prior to mixing was also carefully controlled. The vitamin A supplements were dispersed in the diets by thoroughly premixing with a portion of diet before mixing with the entire diet contents. Trace minerals were later mixed
357
358
A. W. HALVERSON AND C. M. HENDRICK
However, the analytical loss with the actual diets should have been minimum as well as constant because of the predominance of the ester form in fish oils and because of the similarity of recovery of the vitamin among different diets even in the presence of added trace minerals.
were made with all diets to determine whether non-vitamin A feed ingredients were affecting the reaction through interfering color development. However, the blank corrections were always low and thus did not appear to significantly affect the data. The reliability of the vitamin A method employed was further checked by several recovery determinations with the ester (acetate) and the alcohol forms of the vitamin. The vitamin A standards were added both before and after extraction of blank samples (15 gm.). Recoveries of the ester form amounted to 85% with extraction and to 95-97% without the extraction treatment. The alcohol form gave recoveries of 69-78% with extraction and 91-93% without. The results indicated that the 4-hour extraction as practiced in diet analysis caused considerable vitamin A destruction especially with the alcohol form of the vitamin. Hence, it is likely that some vitamin A destruction also occurred in analysis of supplemented diets.
RESULTS AND DISCUSSION
TABLE 2.—Vitamin A losses during storage of different diets containing different vitamin A supplements (Storage at 37°C.) Percent loss in 30 days storage
Percent loss in 150 days storage
Diet
Diet
Vitamin A supplements and diet modifications 1
2
3
1
2
3
Supplement No. 1 (cod liver oil) a b b + M n salt b-f-Mn, Fe, Cu and Co salts
4.2 8.7 5.7 20.6
8.4 8.3 7.9 19.5
7.6 7.9 11.6 36.9
52.1 56.8 57.2 72.9
45.9 47.6 47.4 61.9
56.8 50.6 56.1 93.0
Supplement No. 2 (dry-fat powder) a b b + M n salt b + M n , Fe, Cu and Co salts
10.6 10.4 15.3 17.9
7.7 7.6 7.6 19.2
12.8 9.4 17.7 31.7
48.6 51.0 52.6 71.1
49.3 52.4 50.5 66.6
44.9 53.5 60.8 88.2
00 00 00 00
10.1 2.7 7.9 10.1
3.1 7.2 2.2 4.8
17.2 15.1 18.9 25.5
21.7 23.7 24.0 28.3
5.7 19.8 23.0 34.4
Supplement No. 3 (wax-sealed pellets) a b b + M n salt b + M n , Fe, Cu and Co salts
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The vitamin A losses obtained with diets 1, 2 and 3 are shown in Table 2. In general, losses were small at the 30 day storage period but increased to as much as 50% or more by the 150 day period. Diet modification as by addition of meat scraps and limestone with diets 1 and 2 and by addition of meat scraps, fish meal, limestone and bone meal with diet 3 generally had no noticeable effect upon vitamin A stability with any of the vitamin A supplements (see b modifications). The further addition of manganese salt also did not materially affect the loss results. However, the addition of iron, copper and cobalt salts along with the other ingredi-
STABILITY OF VITAMIN A IN STORED FEEDS
The data are in accord with several previous investigators in regard to the overall low loss rate of vitamin A of various forms in mixed feeds (Holder and Ford, 1939; Burns and Quackenbush, 1951; and others). However, the destructive effects of meat scraps as observed by Bethke et al. (1939) and of manganese salt as observed by Miller et al. (1942) were not corroborated. Confirmation of previous reports by Halverson and Hart (1950) and by Davies and Worden (1954) on the destructive effect of the trace min-
erals toward the vitamin A of supplemented feeds was evident. Thus, the overall experimental results indicate the important role of the trace minerals in promoting vitamin A losses. Information as to the specific trace mineral or minerals involved in the vitamin A loss picture remains lacking, however, as does data on the relation of the oxidative state of diet ingredients to the response obtained from trace minerals. SUMMARY
Vitamin A storage losses were determined with three vegetable-type poultry diets which were supplemented with different vitamin A sources. Diet modifications which included the addition of meat scraps, limestone, manganese salt and iron, copper and cobalt salts were made with each of the diets to determine the effects of such ingredients upon vitamin A stability. The data obtained with storage at 31° C. for 30 and 150 day periods permit several conclusions: namely, (1) a fairly good degree of vitamin A stability was obtained with each diet and its modifications, (2) an overall uniformity in stability was observed with each vitamin A supplement among the different diets, (3) no definite changes in vitamin A stability were observed with diet modification except upon addition of all of the free trace minerals. (4) a greater vitamin A loss occurred when all of the trace minerals were added at a higher level as in a feed concentrate, and (5) a greater overall vitamin A stability was obtained with a wax-coated vitamin A supplement than with oil- or fat-type supplements. ACKNOWLEDGMENT
This investigation was supported in part by a grant-in-aid from the American Dehydrators Association.
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ents caused an increase in losses with all diets especially at the 150 day storage period. A great difference in vitamin A stability was observed between the wax-sealed supplement and the oil and fat supplements. This difference was evident with all three diets with losses being about onethird to one-half as great with the waxsealed preparation as with the other two. The wax-sealed product was also less sensitive to complete diet modification which included all of the trace minerals. The similarity of stability between two of the vitamin A supplements, namely, cod liver oil (No. 1) and stabilized fish oil in dry fat (No. 2), was of considerable interest. Both products were almost equal in stability both within and among diets. Further, the other supplement (No. 3) also had a similar stability pattern among different diets even though it was much superior to the other supplements in overall stability. These observations suggest that the replacement of a cereal ingredient with others as well as with small amounts of meat scraps, limestone and manganese salt has no definite effect upon vitamin A stability. However, it is evident that the interpretation of equal vitamin A stability among a variety of diets applies only to controlled conditions such as were employed in the present experiments.
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360
E. M. D E N I N G T O N AND A. M.
REFERENCES
liver oil in cereal feeds. J. Nutrition, 40:415-428. Holder, R. C, and S. K. Ford, 1939. The stability of vitamin A from cod liver oil in mixed feeds. Poultry Sci. 18: 345-349. Miller, M. W., V. Joukovsky and N. Hokenstad, 1942. The effect of manganese sulphate on the stability of vitamins A and D of cod liver oil when stored in mixed feeds. Poultry Sci. 21: 200202. Siedler, A. J., and B. S. Schweigert, 1954. Effect of added stabilized animal fats on stability of vitamin A in feeds. J. Agr. Food Chem. 2: 193195. Stephenson, E. L., 1953. Stability of vitamin A in poultry feeds. Arkansas Farm Research, Vol. 2, No. 3.
Blood Technics for Chickens EFFIE
M.
D E N I N G T O N AND A L F R E D M .
Regional Poultry Research Laboratory,^
East Lansing,
LUCAS* Mich.
(Received for publication July 13. 1954)
A
N A T O M I C Energy Project under• taken at this Laboratory involved numerous blood determinations made over a two-year period and, prelininary to the actual work, a comparative study of technics suitable for avian blood was made. T h e technics selected have been applied to over 2,500 blood samples and have given results which appear to be reliable. During the period when work was in progress, inquires were received conconcerning the technics which had been selected and the development of modifications which had been found useful. In order to make this information generally available, a report covering the following subjects has been prepared: (1) description of a unit tray for handling large numbers of blood samples, (2) determination of avian hemoglobin, (3) accuracy of * Research conducted under a grant from the Atomic Energy Commission to this Laboratory. f Agricultural Research Service, United States Department of Agriculture.
hemoglobin pipettes, (4) hematocrit with the Van Allen pipettes, (5) composition of the buffy coat, (6) a shaker for red blood cell pipettes, (7) variability in Samples of Wright's stain, (8) a comparison of three types of methods for making total white cell counts, and (9) an indirect method for making thrombocyte counts. All studies were based on Single Comb White Leghorn chickens. Most of them were females and some of them had been irradiated with x-rays at low dosages of between 50 and 300 r. RESULTS A unit tray for transporting blood samples.—The t r a y was designed to handle tubes for hemoglobin, hematocrit and red and white blood cell determinations. T h e box and the contained tubes are shown in Fig. 1. In addition, clean slides and an infra red lamp for warming the slides used in making blood smears were part of the equipment. I t was necessary to have a
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Bethke, R. M., P. R. Record and O. H. M. Wilder, 1939. The stability of carotene and vitamin A in a mixed ration. Poultry Sci. 18: 179-187. Burns, M. J., and F. W. Quackenbush, 1951: Stability of dry vitamin A concentrates. Ind. Eng. Chem. 43: 1592-1593. Davies, A. W., and A. N. Worden, 1954. The stability of vitamin A in animal feeding stuffs. J. Sci. Food Agr. 5: 107-112. Halpern, G. R., B. March and J. Biely, 1949. Stability and utilization of vitamin A emulsions in mixed feeds. Poultry Sci. 28: 168-172. Halverson, A. W., and E. B. Hart, 1950. Factors affecting the stability of the vitamin A from cod
LUCAS