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The Utilization of Carotene and Vitamin A by Chicks during the First Week after Hatching12
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J. D. HARVEY, D. B. PARRISH, P. E. SANFORD AND J. S. HUGHES
REFERENCES Moreng, R. E., and C. S. Shaffner, 1951. Lethal internal temperatures for the chicken, from fertile egg to mature bird. Poultry Sci. 30: 225-266. Moreng, R. E., and R. L. Bryant, 1954a. Effects of sub-freezing temperature exposure on the chicken embryo. 1. Survival and subsequent growth up to time of hatch. Poultry Sci. 33: 855-863. Moreng, R. E., and R. L. Bryant, 1954b. Effects of
sub-freezing temperature exposure on the chicken embryo. 2. Hatchability, chick weight and survival to six weeks. Poultry Sci. 33: 987-991. Olsen, M. W., 1949. Effect of shipment on preincubated fertile eggs. Poultry Sci. 28: 731-738. Olsen, M. W., 1951. Effect of low temperature on the hatchability of eggs from various standard bred and crossbred chickens. Poultry Sci. 30: 180-183.
J. D. HARVEY, 3 D. B. PARRISH, P. E. SANFORD AND J. S. HUGHES Kansas State College, Manhattan (Received for publication March 31, 1955)
INTRODUCTION
W
ORK of Bolin et al. (1943) and Temperton and Dudley (1945) indicated that chicks absorbed dietary carotene and converted it to vitamin A, which was stored in the liver, by the end of the first week after hatching. However, based on the results of liver storage tests, Mann (1946) opined that young chicks could not utilize dietary carotene or vitamin A until they were about three weeks old. The report of Bolin et al. (1943), and also that of Rubin and Bird (1941), indicated that the liver was depleted of vitamin A during the first two weeks after hatching, unless dietary vitamin A was 1
Contribution No. 539, Department of Chemistry, and No. 224, Department of Poultry Husbandry, Kansas Agricultural Experiment Station. 2 Portion of a dissertation presented in partial fulfillment of the requirements for the degree Doctor of Philosophy in Biochemistry and Nutrition at Kansas State College. 3 Present address: Department of Nutrition, Ontario Agricultural College, Guelph, Ontario, Canada.
available in large quantities. Harvey et al. (1955b) commented on the difficulties of interpretation of liver storage data. It is possible that Mann was misled in interpretation of his data by depletion of liver vitamin A stores during the first few days after hatching. Since there is a paucity of information on absorption and utilization of vitamin A and carotene during the first week after hatching, the present authors undertook a study of this problem. MATERIALS AND METHODS
Survival times of chicks fed a basal diet free of vitamin A following experimental treatments, and determinations of liver vitamin A contents were used as measures of utilization of carotene and vitamin A. Balance studies were conducted to obtain data on extent of apparent absorption of carotene from the digestive tract and maximum amounts available within the body for metabolism and storage. White Leghorn chicks hatched from eggs laid by hens maintained on a diet
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The Utilization of Carotene and Vitamin A by Chicks during the First Week after Hatching1'2
UTILIZATION OF CAROTENE AND VITAMIN A
In carotene-balance experiments, chicks were placed in individual cages of a rat battery modified to provide heat when necessary by mounting a 15-mm.-diameter Pyrex tube carrying a coiled 100-watt electrical heating element between the backs of the cages on each level. Feed and water were supplied in small earthenware cups placed in galvanized iron containers so designed that waste from the cups was nearly all retained in the containers. Droppings were collected on trays made of folded wax paper, placed under each cage.
To ensure recovery of droppings without contamination from adjacent cages, the trays were made larger than bottoms of cages and alternate cages in the battery were left vacant. Deutectomies were made as described by Harvey et al. (1955a). Analyses of droppings, livers, and residual yolks for vitamin A were according to the method of Neff et al. (1949) for egg yolks, except that the emulsification procedure was omitted and the whole sample saponified. All analytical results were expressed as total content per organ or per total dropping of a given period. Carotene was determined by chromatographing an aliquot of the Skellysolve extract on a 1:1 column of magnesia and diatomaceous earth. Carotene color in the eluate was read on the photoelectric colorimeter at 440 imt. EXPERIMENTAL
Apparent Absorption of Carotene by Day-old Chicks. Twenty-six normal chicks were deutectomized on the day they hatched. The next day they were divided into seven groups, placed in individual cages of the modified rat battery, and given supplemented diets as shown in Table 1. Feed intakes were recorded over the ensuing 48 hours, and from them carotene intakes were calculated. Droppings were collected every two hours and frozen, the entire output of each chick during the test period being pooled for analysis of carotene. At the close of the 48-hour feeding period, each chick was given a few drops of a saturated aqueous solution of carmine from an eyedropper, and the experimental diets were replaced with the basal diet. When the dye appeared in the droppings (three to four hours after administration), collection of droppings was discontinued. Substantial quantities of carotene (65
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low in carotenoid pigments (Neff et al., 1949) were used to .avoid interference of carotenoids in vitamin A analyses of livers and yolks. Some of the chicks had low initial supplies of vitamin A (hereafter referred to as "deficient chicks" to differentiate them from "normal chicks") produced by restricting the maternal dietary vitamin A. In all but two experiments, chicks were raised in wire-floored, electrically heated brooder batteries in rooms thermostatically controlled at 75-80°F. Except where otherwise noted, chicks were given feed and water ad libitum. A basal all-mash starter, nearly devoid of carotenoid pigments and vitamin A (Harvey et al., 1955a), was used in formulating the test diets, which were supplemented with vitamin A-active materials as will be indicated. In some experiments, dehydrated alfalfa meal was fed alone for short periods, either ad libitum or by force. In the latter case, small portions were forced periodically into the gullet from the tip of a small nickel spatula bent into the shape of a trough, by a small glass rod. Liquid preparations were administered by volume-calibrated droppers inserted into the gullet. Oily solutions and aqueous emulsions of carotene and vitamin A, as well as placebos, were administered in this way.
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J. D. HARVEY, D. B. PARRISH, P. E. SANFORD AND J. S. HUGHES
TABLE 1.—Apparent absorption of carotene fed to newly hatched White Leghorn chicks as dehydrated alfalfa meal, aqueous emulsion or oil solution1 mixed with a basal diet deficient in vitamin A and carotene
Group
1 2 8
4 4 6 2 5 2 3
Kind of supplement
None (control) Dehydrated alfalfa meal Dehydrated alfalfa meal Emulsified carotene 2 Emulsified carotene 8 Carotene in oil3 - Carotene in oil 3
Vitamin A activity ( L U -/lb.feed) 0 500 2,000 500 2,000 500 2,000
Apparent absorption (percent) Mean Rang -0.3 80 73 100 81 77 66
- 0 . 8 to - 0 . 2 73 to 92 54 to 84 — 77 to 89 77 to 80 56 to 77
Supplements fed during period between 24 and 72 hours after hatching; no feeding prior to 24 hours. 90%(3-10%a-carotene emulsified in 5% aq. Tween 60 (Atlas Powder Co.). . . 90%/3-10%a-carotene in corn oil.
to 100 percent) disappeared from the ingesta during passage through the gastrointestinal tract when the carotene was ingested in alfalfa, aqueous emulsion or oil solution (Table-1). Apparent absorption of emulsified carotene was somewhat better than that of carotene in alfalfa or' in oil; ranges, however, overlapped somewhat among groups. Utilization of Carotene and Vitamin A Injected into the Residual Yolks of Day-old Chicks. Since previous work (Harvey et al., 1955b) has shown that vitamin A in the residual yolk at hatching was subjected to considerable movement within the chick body during the first week, it was attempted to determine whether carotene artificially introduced into the residual yolk after hatching would be converted to vitamin A in the body and subjected to similar distribution. Seventy-one newly hatched "deficient" chicks were divided into four groups. Group 1 (12 chicks) was sacrificed immediately for determinations of liver and yolk, contents of vitamin A and total carotenoids. Group 2 (22 chicks) served as untreated controls. Group 3 (14 chicks) were injected into the yolk with 500 I.U. of 90% /3-10%a-carotene emulsified in 0.1 ml. of -5 percent aqueous Tween 60.. Group 4 (23 chicks) were given similar in-
jections of 500 I.U. emulsified vitamin A alcohol. All chicks of groups 2, 3 and 4 were placed in brooder pens and given the basal vitamin A-deficient diet ad libitum. Representative chicks were sacrificed from groups 2, 3 and 4 at four, eight, twelve, seventeen and twenty-one days of age. Vitamin A, total carotenoids and carotene contents of their livers and residual yolks (if large enough to analyze) were determined, and survival times of the remaining chicks were recorded (Table 2). Chicks killed four days after injection of carotene (group 3) had an average liver vitamin A content more than 9 times that of controls, indicating substantial conversion of carotene to vitamin A and transport to the liver. The total carotenoids and carotene present in the liver were increased somewhat, suggesting that some of the injected carotene had been transferred per se to the liver and some of it altered to non-carotene carotenoids that also appeared in the liver. The contents of carotene, total carotenoids, and vitamin A in yolks of chicks of group 3 were elevated at four days. Only a limited number of chicks was available for analysis, and variations among the analytical results were such that data, obtained on groups 2 and 3 after four days revealed no definite trends.
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1 2 3 4 5 6 7
MJHI
UTILIZATION OF CAROTENE AND VITAMIN A
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TABLE 2.—Yolk and liver vitamin A, carotene and total carotenoid contents and mean survival times of White Leghorn chicks given intravitelline injections of vitamin A or carotene on-ihe day of hatching Ages of chicks in days
Group • No. 1 1 2 3 4
Numbers of chicks analyzed
. Yolk vitamin A, I.U.
Liver total carotenoids, meg.
Liver carotene, meg.
Liver vitamin A, I.U.
Mean survival times, days 1
17
21
2 2 2
2 1 2
1 ' 1 2
1 1 2
2
1.4 6.2 2.2
6.4
1.8 1.3
2.6 0.9 90.0 0/5
1.0
4.5 3.5
8.8
3.4 7.6
8.2 37.0 2.0 0.8 22.5
1 2 3 4
7.3
1 2 3 4
4.1
1 2 3 4
12
7.8
2 3 4
2 3 4
8
'
11.5 24.7 6.3
0 2.2 11.9
27.0
3.8 8.0 3.2
2.7 4.0 4.2
6.0 5.0 2.0
1.0 2.8 0.8
0.5
6.5 60.0 175.0
0 8.5 98.7
7.5 4.7 2.6
5.0
1.0 0.8
14.2
23 4
0 8.5 9.2
'
12.5 11.0 30.8
4.2
15.8, mean of 16 chicks 24.7, mean of 9 chicks 33.3, mean of 11 chicks
Group 1: Chicks sacrificed for analysis on the first day. Group 2: Untreated chicks used as controls. Group 3: Chicks given intravitelline injections of 500 I.U. of 90%/3-10a-carotene emulsified in 0.1 ml of 5 % aq. Tween 60 on the first day. Group 4: Chicks given intravitelline injections of 500 I.U. of vitamin A alcohol emulsified in 0.1 ml of 5 % aq. Tween 60 on the first day.
Chicks killed four days after the injection of vitamin A (group 4) had yolk vitamin A and total carotenoid contents smaller than those of controls. However, liver vitamin A content was 27 times that of controls, so that extensive transfer of injected vitamin A to the liver appeared to have occurred. Liver carotene and total carotenoids were not affected appreciably by injection of vitamin A. Liver vitamin A contents of vitamin A-injected chicks
continued to be larger than those of controls at eight, twelve and seventeen days, although there was a rapid decline from the four-day level. Yolk vitamin A content of chicks in group 4 was lower than in the controls at four days, but higher at eight and twelve days. Mean survival time of carotene-injected chicks was 9 days longer than that of the controls; that of the vitamin A-injected chicks was 18 days longer. These results
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Yolk carotene, meg.
4
12
1 2 3 4
Yolk total carotenoids, meg.
0
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J. D. HARVEY, D. B. PARRISH, P. E. SANFORD AND J. S. HUGHES
TABLE 3.—Survival times of deficient chicks deutectomized on the day of hatching and drenched with oil1 solutions of 400 I.U. of 90% 0-10% a-carotene or vitamin A natural esters at 2 days of age
Drench
No. of chicks
Mean survival time, days
1 2 3 4
none (controls) oil (controls) carotene, 400 I.U. vitamin A, 400 I.U.
23 S 12 4
6.9 6.2 19.0 25.2
1 A mixture of 1 part by volume corn oil and 2 parts of an oil separated from egg yolks. Drenched 4 times at 4-hour intervals between 40 and 52 hours after hatching.
indicate that the injections contributed vitamin A to the bodily stores of the chicks, which was used to maintain life. Utilization of Vitamin A and Carotene Given Orally During the First Week for Liver Storage and Maintenance of Life. Several experiments were conducted, in which vitamin A or carotene in various quantities and types of carriers was administered to either deficient or normal chicks per os shortly after hatching, and the effects of these treatments on either liver vitamin A content or total bodily stores of vitamin A (survival times) or both were determined. In some experiments deutectomized chicks were used. Chicks were divided into treatment groups at random and placed in brooder cages. In four of the experiments (Tables 3, 4, 5, 6)
The details of management in the various experiments are given in Tables 3 to 8, together with the experimental results. In experiment 1 (Table 3) mean survival times of chicks drenched with oil solutions of 400 I.U. of carotene or vitamin A natural esters were, respectively, about 3 and 4 times longer than those of the two control groups. In experiment 2 (Table 4), the liver
TABLE 4.—Vitamin A contents of yolks at hatching and livers and yolks at 5 days of age of deficient chicks drenched with corn oil solutions of 700 I.U. of 90% 0-10% a-carotene or vitamin A natural esters at 2 days of age1 Group
Drench
2,3 1
None 2 None (controls)
2 3
Carotene, 700 I.U. Vitamin A, 700 I.U.
1 2 8
No. of chicks
Organ analyzed
Time removed
Vitamin A content, I.U.
11 3
yolk yolk liver liver liver
1st day 5th day 5th day 5th day 5th day
9.1(H)3 5.8(3) 4.9(3) 14.1(6) 54.5(5)
6 5
Drenched 7 times at 2 or 3-hour intervals between 20 and 36 hours after hatching. Group 2 and 3 chicks were deutectomized on the day of hatching and the excised yolks were analyzed. Numbers of individual values in parentheses.
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Group
0.1 ml. of oily or aqueous preparations of carotene or vitamin A or of placebos was given by dropper to the chicks commencing at times ranging from twelve to forty hours after hatching, and repeated at intervals of two, three or four hours until the total scheduled dosages had been given. Concentrations of all the preparations were adjusted to 1,000 I.U. per ml. In three of the experiments (Tables 6, 7, 8) dehydrated alfalfa meal was the only feed given to certain groups of chicks for periods of thirty-four to fifty hours, commencing as soon as possible after hatching in two experiments and twelve hours after hatching in the third. In one experiment, dehydrated alfalfa meal was forcefed to appropriate chicks during the first twelve hours after hatching. In all experiments, the basal vitamin A-deficient diet was fed ad libitum to controls throughout the test periods, and to treated birds except at times when alfalfa feedings, either ad libitum or by force, were in progress.
UTILIZATION OF CAROTENE AND VITAMIN A
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TABLE 5.—Liver vitamin A contents at 5 and 26 days of age and mean survival time of deficient chicks deutectomized the day after hatching and drenched with aqueous emulsions of 800 I.U, of 90% (1-10% a-carotene or of vitamin A acetate or with placebos on the day after hatching Liver vitamin A (I.U.)
Drench1
Group
1st day
1 2 3
26th day
10.7(10) >200(9) >200(9)
2.1(3)
Mean survival time, days
11.8(11)'
None, killed at hatching placebos2 (controls) carotene, 800 I.U. vitamin A, 800 I.U.
12.9(10) 44.5(6) 50.5(6)
5.7(3)
Drenched 8 times at 2-hour intervals between 26 and 40 hours after hatching. Placebo: 10% aqueous Tween 60. Carotene and vitamin A emulsified in this medium. Numbers of individual values in parentheses.
TABLE 6.—Liver vitamin A contents at 5 days of age and mean survival times of deficient chicks drenched1 with 800 I.U. of 90% 0-10% a-carotene dissolved in Wesson oil or emulsified in 10% aqueous Tween 60, or fed only dehydrated alfalfa meal2 Liver vitamin A (I.U.) Group
Treatment 1st day
1 2 3 4 5
Killed at hatching None (controls) Drench, carotene in Wesson oil, 800 I.U. Drench, emulsified carotene 800 I.U. Alfalfa,2 carotene intake, 500 I.U.
5th day
Mean survival time, days
9(4)'
3.6(4) 52.5(4) 60.0(4) 22.8(4)
21 (3) 30 (6) 37.4(5) 27.7(3)
1
Drench 8 times at 3-hour intervals between 12 and 33 hours after hatching. Average alfalfa consumed per chick = 1.27 g=500 I.U. carotene. Fed ad lib. for the first 34 hours after hatching. 3 Numbers of individual values in parentheses. 2
TABLE 7.—Liver vitamin A contents and mean survival times of chicks fed only dehydrated alfalfa meal or basal diet ad lib. between 12 and 62 hours after hatching Liver vitamin A (I.U.) 1st day 1 2 3 1
Killed a t hatching Basal diet (controls) Alfalfa, 3400 I.U. carotene
7th day
11th day
15 th day
20th day
6.5(3) 90.5 (4)
0 (2) 57.5(3)
0(2) 0(2)
0(1) 3(2)
Mean survival time, days
4(2)
0(2)
15.8(16) 33.4(15)
Numbers of individual values in parentheses.
Initial Group vitamin A reserves
1 2
29th day
14(12)1
TABLE 8.—Mean survival times of unoperated and deutectomized1 normal and deficient chicks fed only dehydrated alfalfa meal during the first 48 hours after hatching
1 2 3 4 5 6 7 8
24th day
normal normal deficient deficient normal normal deficient deficient
Unoperated or deutectomized
Calculated carotene intake, I.U.2
Mean survival time, days
unoperated deutectomized unoperated deutectomized unoperated deutectomized unoperated deutectomized
0 0 0 0 788 680 574 131
16.6(14)3 12.1(14) 4.6(17) 4.3(19) 28.2(13) 19.5(11) 20.4(11) 12.6(6)
Deutectomized as soon as dry after hatching. Carotene intake groups 5-8 calculated from average alfalfa consumption per chick. ! Numbers of individual values in parentheses.
vitamin A contents of the chicks about 3 | days after final drenching with 700 I.U. of carotene or vitamin A natural esters in corn oil were, respectively, about 3 and 11 times that found in untreated controls. The apparent increases represent only 1.3 and 7 percent, respectively, of the quantities of carotene and vitamin A given. In experiment 3 (Table 5), drenching with 800 I.U. of carotene or vitamin A acetate in aqueous emulsion appeared to increase survival of chicks more than 3 times that of controls. Also, three days
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1 2 3 4
5th day
1354
J. D. HARVEY, D. B. PARRISH, P. E. SANFORD AND J. S. HUGHES
In experiment 5 (Table 7), chicks fed alfalfa meal between twelve and sixty-two hours after hatching lived more than two times longer than controls, and 4§ days after cessation of the alfalfa-feeding they had liver vitamin A contents 14 times larger. The apparent deposition of dietary vitamin A in the liver at this time represented only 2\ percent of the calculated intake of 3400 I.U. of carotene. On the eleventh day after hatching, liver vitamin A in the alfalfa-fed group was equal to 68 percent of that on the seventh day; on the fifteenth day and later, negligible quanti-
ties were found. No measurable vitamin A was found in livers of control chicks on the eleventh day or later. In experiment 6 (Table 8), survival times of all four types of chicks—unoperated or deutectomized and normal or deficient—were increased 60 to 400 percent by the ingestion of sufficient alfalfa meal during the first 48 hours to provide 131 to 788 I.U. of carotene per chick It appears from these data that a given quantity of provitamin A ingested early in life adds more days of survival time to vitamin A-deficient chicks than to those with normal initial vitamin A reserves, suggesting losses before physiologic use of the ingested and stored vitamin A or greater daily demand due to increased age by time initial reserves are used up or both. In another experiment, three chicks were force-fed alfalfa meal during the first twelve hours after hatching. They consumed an average of 682 I.U. of carotene. Liver vitamin A contents forty-eight hours after completion of the feeding averaged 23.3 I.U. compared with 2.7 I.U. in three control chicks. There appears to be little doubt that oral administration of carotene or vitamin A to chicks during the first 2\ days after hatching prolongs life on a vitamin A-deficient diet. Since in some experiments treatments were completed earlier (within 12 hours in one) without change in the qualitative nature of results, there is reason to believe what might be said of vitamin A metabolism of a 2 | day old chick might be true of a 12-hour-old chick. Also, if in sufficiently large quantities, this vitamin A ingested will result in increased liver vitamin A levels. However, the efficiency of transfer of the vitamin to the liver is generally low, and apparently influenced by the nature of the vitamin A preparation given.
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after treatment, liver vitamin A contents in carotene- and vitamin A-drenched groups were at least 20 times larger than that of controls. These data indicate that more than 25 percent of the vitamin or provitamin A given was moved to the liver. By the twenty-sixth day, the liver vitamin A contents again were low. In experiment 4 (Table 6), mean survival times of chicks drenched with 800 I.U. of carotene dissolved in Wesson oil or emulsified in 10 percent aqueous Tween 60 were, respectively, about If to 2 times longer than that of untreated controls. Liver vitamin A contents of chicks in the oil-, emulsion-, and alfalfa-fed groups about 3 | days after treatment were, respectively, 13, 15, and 6 times that in the untreated controls, indicating that about 6 , 1 \ and 2 | percents of the vitamin activities of drenches given were present in the liver. Chicks receiving 500 I.U. of carotene by consumption of dehydrated alfalfa meal ad libitum for thirty-four hours after hatching survived nearly as long as those given 800 I.U. of carotene in oil. The mean survival time of controls is longer than would be expected on the bases of the results of experiments 1 and 3, and of the low liver vitamin A contents at hatching and on the fifth day. No explanation can be given for the difference.
UTILIZATION OF CAROTENE AND VITAMIN A
DISCUSSION
In experiments in which carotene and vitamin A preparations were given to chicks shortly after hatching, and in which subsequent increases in liver vitamin A contents were observed, the efficiencies of utilization of the preparations for liver storage generally were considerably lower than in the case of 19-day-old chicks (Harvey and Parrish, 1953). While several groups of the young chicks had not been depleted of their vitamin A reserves prior to experimental treatment with vitamin A or carotene, the apparent mobilization of stored vitamin A during the first two weeks (Harvey et al., 1955b) might have similar effects upon efficiency of utilization of a dietary supplement for liver storage as would prior depletion in older chicks. The results indicate that the newly hatched chick has, or develops soon after hatching, the biochemical systems necessary to convert carotene to vitamin A and to use vitamin A. In the force-feeding experiment, alfalfa was not fed later than twelve hours after hatching. Probably the
residues of this material were voided within a few hours, since carmine given to chicks in the first balance trial was found in the droppings within three or four hours. Therefore, the extra vitamin A found in the livers of the chicks force-fed alfalfa and killed when forty-eight hours old probably was derived from the carotene of alfalfa digested and absorbed during the first sixteen hours after hatching. While treatments were not completed as soon after hatching in the other experiments as in the force-feeding experiment, the results were qualitatively-similar in all experiments. Without exception, carotene given during periods lasting not longer than sixty-two hours after hatching appeared to have been converted to vitamin A and stored in the liver, suggesting that the mechanism operative at that time and later in life differs little from those present during the first twelve hours. These results also confirm the findings of Bolin et al. (1943) and Rubin and Bird (1941) on chicks about one week old. As was found in the balance trial with newly hatched chicks, aqueous preparations of vitamin A or provitamin A were utilized better for survival and for liver storage of vitamin A than were oily solutions. This is similar to the findings of Frey and Wilgus (1949) and Halpern et al. (1947) with older birds. Preformed vitamin A always was more effective than carotene in any given similar preparation. Mean survival times of untreated deficient control chicks were 4 to 5 days. The prolongation of life of such chicks, when fed a vitamin A supplement during the first two days, indicates that the ingested material was used to support life as early as the fifth day after hatching. Probably it was used even earlier to correct the deficiencies which existed in the chick bodies some time before the average age at which untreated chicks died.
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Utilization of dietary vitamin A and carotene by newly hatched chicks would depend upon the existence of mechanisms for the digestion of the feed components containing the vitamin A-active material and for the absorption of the free vitamin and carotene. The carotene-balance trial carried out with day-old chicks indicated a high apparent absorption of carotene from oil solution, aqueous emulsion or alfalfa in the diet. Probably destruction in the digestive tract accounts for some of the carotene that appeared to have been absorbed in these experiments. As has been found also by Frey and Wilgus (1949) with laying hens and by Halpern et al. (1947) with older chicks, the aqueous preparation was absorbed better than the oily.
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J. D. HARVEY, D. B. PARRISH, P. E. SANFORD AND J. S. HUGHES
It is interesting to speculate on the site of conversion of the injected carotene, for there should be no need for it to pass through the intestinal tissues, which generally is accepted now as the site of conversion of dietary carotene (Matterson el al., 1950; Thompson el al., 1950), on the way to the liver. If intestinal tissues were involved, it would seem that it could be only as the result of carotene being carried to that site by the arterial route. The liver was regarded as the site of conversion in earlier work (Ahmad, 1931; Olcott and McCann, 1931; Rea and Drummond, 1932). One also might suspect the yolk-sac. SUMMARY
Between 65 and 100 percent of carotene fed as oily solution, aqueous emulsion or alfalfa meal to newly hatched chicks in balance trials was removed from the ingesta during passage through the gastrointestinal tract. Aqueous or oily drenches of carotene or vitamin A, and ad libitum or force-feeding of alfalfa meal during periods terminated
as early as twelve hours and never longer than sixty-two hours after hatching resulted in increased liver vitamin A contents at four days and prolonged survival times in chicks fed a diet deficient in vitamin A and carotenoid pigments after treatments. Injections of vitamin A or carotene emulsions into the residual yolks of newly hatched chicks also resulted in increased liver vitamin A contents at four days of age and prolonged survival times. Injected vitamin A and carotene were removed rapidly from the yolk and conversion of carotene to vitamin A appeared to have occurred without necessarily having passed through the intestinal tissues on the way to the liver. In addition to the liver, the yolk-sac might be suspected as site of conversion. It is concluded that the chick converted carotene to vitamin A as early as during the first day, and that prolongation of life in vitamin A-deficient chicks beyond a mean of four or five days by early treatment with carotene or vitamin A indicate that dietary vitamin A was used to support physiological functions involving vitamin A at least by the fifth day and probably earlier. These results advance to earlier ages the contentions of Bolin el al. (1943), Rubin and Bird (1941) and Temperton and Dudley (1945) that carotene and vitamin A could be utilized by chicks one week old. The report of Mann (1946) that these materials could not be utilized until three weeks or more after hatching is not substantiated. REFERENCES Ahmad, B., 1931. The fate of carotene after absorption in the animal organism. Biochem. J. 25: 1195-1204. Bolin, D. W., C. E. Lampman and L. R. Berg, 1943. The influence of carotene intake as supplied by dehydrated alfalfa on the storage of vitamin A and pigments in the livers of the young chick. Poultry Sci. 22: 348-353.
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The presence of systems for the conversion of carotene to vitamin A in newly hatched chicks was indicated also by the experiment on intravitelline injections. There is a rapid transport of vitamin A from the residual yolk to the rest of the chick body during the first week (Harvey el al., 1955b). In the present study, it was found that, if the yolk vitamin A content were increased by the injection of 500 I.U. of emulsified vitamin A alcohol just after hatching, most of this additional vitamin A was removed within four days, about 35 percent of-it appearing in the liver. When similar quantities of emulsified carotene were injected, most of the carotene also disappeared from the yolk by the fourth day and liver vitamin A content increased by an amount equivalent to 12 percent of the carotene injected.
HAEMATOLOGICAL CHANGES WITH Frey, P. R., and H. S. Wilgus, 1949. The utilization of carotene from different sources by laying chickens. J. Nutrition, 39:517-528. Halpern, G. R., J. Biely and F. Hardy, 1947. Utilization of vitamin A in water emulsion. Science, 106: 40-41. Harvey, J. D., and D. B. Parrish, 1953. Unpublished data.
Harvey, J. D., D. B. Parrish and P. E. Sanford, 1955b. The application of deutectomy for reducing initial vitamin A reserves in newly hatched chicks, and some observations on the apparent consumption of these reserves. Poultry Sci. 34: 1314-1321. Mann, T. B., 1946. Chick rearing, ix. Studies on the storage of vitamin A and carotenoid pigments by chicks from birth to six weeks, using cyclized vitamin A, vitamin A-alcohol, vitamin A-ester (cod liver oil) and the pigments of green
AND CORTISONE
1357
food and maize as criteria. J. Agric. Sci. 36: 289300. Matterson, L. D., J. R. Carson and L. Griswald, 1950. Studies on the conversion of /3-carotene to vitamin A in vitro and in vivo. Poultry Sci. 29: 769. Neff, A. N., D. B. Parrish, J. S. Hughes and L. F. Payne, 1949. The state of vitamin A in eggs. Arch. Biochem. 21: 315-320. Olcott, H. S., and D. C. McCann, 1931. Carotenase. The transformation of carotene to vitamin A in vitro. J. Biol. Chem. 94: 185-193. Rea, J. L., and J. C. Drummond, 1932. Formation of carotene from vitamin A in the animal organism. Zeit. Vitaminforsch. 1:177-183. Rubin, M., and H. R. Bird, 1941. Some experiments on the physiology of vitamin A storage in the chick. Poultry Sci. 20:291-297. Temperton, H., and F. J. Dudley, 1945. The use made by the chick of dietary sources of carotene and vitamin A during the first month of life. Harper Adams Util. Poultry J. 30, N. S. 4:57-84. Thompson, S. Y., M. E. Coates and S. K. Kon, 1950. The conversion of carotene to vitamin A in the intestine of the chick. Biochem J. 46: xxx.
Haematological Changes in Cockerels after A C T H and Cortisone-Acetate Treatment* JAN HUBLE Laboratory for Animal Morphology, University of Ghent, Belgium (Received for publication April 4, 1955)
L
ITTLE is known about the avian respone to ACTH or adrenal steroids. In his review Sturkie (1954) lists only very limited, partly contradictory data: a single injection of adrenal cortical extract in adult fowl causes a transient lymphopenia and leucocytosis (Shapiro and Schechtman, 1949); ACTH injected for several days had no effect upon the number of
* Aided by grants from the Centre National de Recherches sur la Croissance normale et pathologique, Brussels. ACTH and Cortisone generously put at our disposal by N.V. Organon, Oss, Holland.
eosinophils (Stamler et al., 1950). Therefore we found it worthwhile to publish this preliminary report on haematological changes induced by injection of ACTH (Organon) or crystalline Cortisone-acetate (Organon Labs.). MATERIAL AND METHODS
Pure bred Rhode Island Red cockerels varying in age from 2 to 4 weeks at time of blood analysis were used. Blood was taken from the ulnar vein and diluted with a special fluid adapted for counting avian blood cells (Natt and Herrick, 1952). Collecting of blood was done in the
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Harvey, J. D., D. B. Parrish and P. E. Sanford, 1955a. Improvements in the technique of deutectomy of newly hatched chicks, and the effects of the operation on their subsequent development. Poultry Sci. 43:3-8.
ACTH
J. D. HARVEY, D. B. PARRISH, P. E. SANFORD AND J. S. HUGHES
REFERENCES Moreng, R. E., and C. S. Shaffner, 1951. Lethal internal temperatures for the chicken, from fertile egg to mature bird. Poultry Sci. 30: 225-266. Moreng, R. E., and R. L. Bryant, 1954a. Effects of sub-freezing temperature exposure on the chicken embryo. 1. Survival and subsequent growth up to time of hatch. Poultry Sci. 33: 855-863. Moreng, R. E., and R. L. Bryant, 1954b. Effects of
sub-freezing temperature exposure on the chicken embryo. 2. Hatchability, chick weight and survival to six weeks. Poultry Sci. 33: 987-991. Olsen, M. W., 1949. Effect of shipment on preincubated fertile eggs. Poultry Sci. 28: 731-738. Olsen, M. W., 1951. Effect of low temperature on the hatchability of eggs from various standard bred and crossbred chickens. Poultry Sci. 30: 180-183.
J. D. HARVEY, 3 D. B. PARRISH, P. E. SANFORD AND J. S. HUGHES Kansas State College, Manhattan (Received for publication March 31, 1955)
INTRODUCTION
W
ORK of Bolin et al. (1943) and Temperton and Dudley (1945) indicated that chicks absorbed dietary carotene and converted it to vitamin A, which was stored in the liver, by the end of the first week after hatching. However, based on the results of liver storage tests, Mann (1946) opined that young chicks could not utilize dietary carotene or vitamin A until they were about three weeks old. The report of Bolin et al. (1943), and also that of Rubin and Bird (1941), indicated that the liver was depleted of vitamin A during the first two weeks after hatching, unless dietary vitamin A was 1
Contribution No. 539, Department of Chemistry, and No. 224, Department of Poultry Husbandry, Kansas Agricultural Experiment Station. 2 Portion of a dissertation presented in partial fulfillment of the requirements for the degree Doctor of Philosophy in Biochemistry and Nutrition at Kansas State College. 3 Present address: Department of Nutrition, Ontario Agricultural College, Guelph, Ontario, Canada.
available in large quantities. Harvey et al. (1955b) commented on the difficulties of interpretation of liver storage data. It is possible that Mann was misled in interpretation of his data by depletion of liver vitamin A stores during the first few days after hatching. Since there is a paucity of information on absorption and utilization of vitamin A and carotene during the first week after hatching, the present authors undertook a study of this problem. MATERIALS AND METHODS
Survival times of chicks fed a basal diet free of vitamin A following experimental treatments, and determinations of liver vitamin A contents were used as measures of utilization of carotene and vitamin A. Balance studies were conducted to obtain data on extent of apparent absorption of carotene from the digestive tract and maximum amounts available within the body for metabolism and storage. White Leghorn chicks hatched from eggs laid by hens maintained on a diet
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The Utilization of Carotene and Vitamin A by Chicks during the First Week after Hatching1'2
UTILIZATION OF CAROTENE AND VITAMIN A
In carotene-balance experiments, chicks were placed in individual cages of a rat battery modified to provide heat when necessary by mounting a 15-mm.-diameter Pyrex tube carrying a coiled 100-watt electrical heating element between the backs of the cages on each level. Feed and water were supplied in small earthenware cups placed in galvanized iron containers so designed that waste from the cups was nearly all retained in the containers. Droppings were collected on trays made of folded wax paper, placed under each cage.
To ensure recovery of droppings without contamination from adjacent cages, the trays were made larger than bottoms of cages and alternate cages in the battery were left vacant. Deutectomies were made as described by Harvey et al. (1955a). Analyses of droppings, livers, and residual yolks for vitamin A were according to the method of Neff et al. (1949) for egg yolks, except that the emulsification procedure was omitted and the whole sample saponified. All analytical results were expressed as total content per organ or per total dropping of a given period. Carotene was determined by chromatographing an aliquot of the Skellysolve extract on a 1:1 column of magnesia and diatomaceous earth. Carotene color in the eluate was read on the photoelectric colorimeter at 440 imt. EXPERIMENTAL
Apparent Absorption of Carotene by Day-old Chicks. Twenty-six normal chicks were deutectomized on the day they hatched. The next day they were divided into seven groups, placed in individual cages of the modified rat battery, and given supplemented diets as shown in Table 1. Feed intakes were recorded over the ensuing 48 hours, and from them carotene intakes were calculated. Droppings were collected every two hours and frozen, the entire output of each chick during the test period being pooled for analysis of carotene. At the close of the 48-hour feeding period, each chick was given a few drops of a saturated aqueous solution of carmine from an eyedropper, and the experimental diets were replaced with the basal diet. When the dye appeared in the droppings (three to four hours after administration), collection of droppings was discontinued. Substantial quantities of carotene (65
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low in carotenoid pigments (Neff et al., 1949) were used to .avoid interference of carotenoids in vitamin A analyses of livers and yolks. Some of the chicks had low initial supplies of vitamin A (hereafter referred to as "deficient chicks" to differentiate them from "normal chicks") produced by restricting the maternal dietary vitamin A. In all but two experiments, chicks were raised in wire-floored, electrically heated brooder batteries in rooms thermostatically controlled at 75-80°F. Except where otherwise noted, chicks were given feed and water ad libitum. A basal all-mash starter, nearly devoid of carotenoid pigments and vitamin A (Harvey et al., 1955a), was used in formulating the test diets, which were supplemented with vitamin A-active materials as will be indicated. In some experiments, dehydrated alfalfa meal was fed alone for short periods, either ad libitum or by force. In the latter case, small portions were forced periodically into the gullet from the tip of a small nickel spatula bent into the shape of a trough, by a small glass rod. Liquid preparations were administered by volume-calibrated droppers inserted into the gullet. Oily solutions and aqueous emulsions of carotene and vitamin A, as well as placebos, were administered in this way.
1349
1350
-
J. D. HARVEY, D. B. PARRISH, P. E. SANFORD AND J. S. HUGHES
TABLE 1.—Apparent absorption of carotene fed to newly hatched White Leghorn chicks as dehydrated alfalfa meal, aqueous emulsion or oil solution1 mixed with a basal diet deficient in vitamin A and carotene
Group
1 2 8
4 4 6 2 5 2 3
Kind of supplement
None (control) Dehydrated alfalfa meal Dehydrated alfalfa meal Emulsified carotene 2 Emulsified carotene 8 Carotene in oil3 - Carotene in oil 3
Vitamin A activity ( L U -/lb.feed) 0 500 2,000 500 2,000 500 2,000
Apparent absorption (percent) Mean Rang -0.3 80 73 100 81 77 66
- 0 . 8 to - 0 . 2 73 to 92 54 to 84 — 77 to 89 77 to 80 56 to 77
Supplements fed during period between 24 and 72 hours after hatching; no feeding prior to 24 hours. 90%(3-10%a-carotene emulsified in 5% aq. Tween 60 (Atlas Powder Co.). . . 90%/3-10%a-carotene in corn oil.
to 100 percent) disappeared from the ingesta during passage through the gastrointestinal tract when the carotene was ingested in alfalfa, aqueous emulsion or oil solution (Table-1). Apparent absorption of emulsified carotene was somewhat better than that of carotene in alfalfa or' in oil; ranges, however, overlapped somewhat among groups. Utilization of Carotene and Vitamin A Injected into the Residual Yolks of Day-old Chicks. Since previous work (Harvey et al., 1955b) has shown that vitamin A in the residual yolk at hatching was subjected to considerable movement within the chick body during the first week, it was attempted to determine whether carotene artificially introduced into the residual yolk after hatching would be converted to vitamin A in the body and subjected to similar distribution. Seventy-one newly hatched "deficient" chicks were divided into four groups. Group 1 (12 chicks) was sacrificed immediately for determinations of liver and yolk, contents of vitamin A and total carotenoids. Group 2 (22 chicks) served as untreated controls. Group 3 (14 chicks) were injected into the yolk with 500 I.U. of 90% /3-10%a-carotene emulsified in 0.1 ml. of -5 percent aqueous Tween 60.. Group 4 (23 chicks) were given similar in-
jections of 500 I.U. emulsified vitamin A alcohol. All chicks of groups 2, 3 and 4 were placed in brooder pens and given the basal vitamin A-deficient diet ad libitum. Representative chicks were sacrificed from groups 2, 3 and 4 at four, eight, twelve, seventeen and twenty-one days of age. Vitamin A, total carotenoids and carotene contents of their livers and residual yolks (if large enough to analyze) were determined, and survival times of the remaining chicks were recorded (Table 2). Chicks killed four days after injection of carotene (group 3) had an average liver vitamin A content more than 9 times that of controls, indicating substantial conversion of carotene to vitamin A and transport to the liver. The total carotenoids and carotene present in the liver were increased somewhat, suggesting that some of the injected carotene had been transferred per se to the liver and some of it altered to non-carotene carotenoids that also appeared in the liver. The contents of carotene, total carotenoids, and vitamin A in yolks of chicks of group 3 were elevated at four days. Only a limited number of chicks was available for analysis, and variations among the analytical results were such that data, obtained on groups 2 and 3 after four days revealed no definite trends.
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1 2 3 4 5 6 7
MJHI
UTILIZATION OF CAROTENE AND VITAMIN A
1351
TABLE 2.—Yolk and liver vitamin A, carotene and total carotenoid contents and mean survival times of White Leghorn chicks given intravitelline injections of vitamin A or carotene on-ihe day of hatching Ages of chicks in days
Group • No. 1 1 2 3 4
Numbers of chicks analyzed
. Yolk vitamin A, I.U.
Liver total carotenoids, meg.
Liver carotene, meg.
Liver vitamin A, I.U.
Mean survival times, days 1
17
21
2 2 2
2 1 2
1 ' 1 2
1 1 2
2
1.4 6.2 2.2
6.4
1.8 1.3
2.6 0.9 90.0 0/5
1.0
4.5 3.5
8.8
3.4 7.6
8.2 37.0 2.0 0.8 22.5
1 2 3 4
7.3
1 2 3 4
4.1
1 2 3 4
12
7.8
2 3 4
2 3 4
8
'
11.5 24.7 6.3
0 2.2 11.9
27.0
3.8 8.0 3.2
2.7 4.0 4.2
6.0 5.0 2.0
1.0 2.8 0.8
0.5
6.5 60.0 175.0
0 8.5 98.7
7.5 4.7 2.6
5.0
1.0 0.8
14.2
23 4
0 8.5 9.2
'
12.5 11.0 30.8
4.2
15.8, mean of 16 chicks 24.7, mean of 9 chicks 33.3, mean of 11 chicks
Group 1: Chicks sacrificed for analysis on the first day. Group 2: Untreated chicks used as controls. Group 3: Chicks given intravitelline injections of 500 I.U. of 90%/3-10a-carotene emulsified in 0.1 ml of 5 % aq. Tween 60 on the first day. Group 4: Chicks given intravitelline injections of 500 I.U. of vitamin A alcohol emulsified in 0.1 ml of 5 % aq. Tween 60 on the first day.
Chicks killed four days after the injection of vitamin A (group 4) had yolk vitamin A and total carotenoid contents smaller than those of controls. However, liver vitamin A content was 27 times that of controls, so that extensive transfer of injected vitamin A to the liver appeared to have occurred. Liver carotene and total carotenoids were not affected appreciably by injection of vitamin A. Liver vitamin A contents of vitamin A-injected chicks
continued to be larger than those of controls at eight, twelve and seventeen days, although there was a rapid decline from the four-day level. Yolk vitamin A content of chicks in group 4 was lower than in the controls at four days, but higher at eight and twelve days. Mean survival time of carotene-injected chicks was 9 days longer than that of the controls; that of the vitamin A-injected chicks was 18 days longer. These results
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Yolk carotene, meg.
4
12
1 2 3 4
Yolk total carotenoids, meg.
0
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J. D. HARVEY, D. B. PARRISH, P. E. SANFORD AND J. S. HUGHES
TABLE 3.—Survival times of deficient chicks deutectomized on the day of hatching and drenched with oil1 solutions of 400 I.U. of 90% 0-10% a-carotene or vitamin A natural esters at 2 days of age
Drench
No. of chicks
Mean survival time, days
1 2 3 4
none (controls) oil (controls) carotene, 400 I.U. vitamin A, 400 I.U.
23 S 12 4
6.9 6.2 19.0 25.2
1 A mixture of 1 part by volume corn oil and 2 parts of an oil separated from egg yolks. Drenched 4 times at 4-hour intervals between 40 and 52 hours after hatching.
indicate that the injections contributed vitamin A to the bodily stores of the chicks, which was used to maintain life. Utilization of Vitamin A and Carotene Given Orally During the First Week for Liver Storage and Maintenance of Life. Several experiments were conducted, in which vitamin A or carotene in various quantities and types of carriers was administered to either deficient or normal chicks per os shortly after hatching, and the effects of these treatments on either liver vitamin A content or total bodily stores of vitamin A (survival times) or both were determined. In some experiments deutectomized chicks were used. Chicks were divided into treatment groups at random and placed in brooder cages. In four of the experiments (Tables 3, 4, 5, 6)
The details of management in the various experiments are given in Tables 3 to 8, together with the experimental results. In experiment 1 (Table 3) mean survival times of chicks drenched with oil solutions of 400 I.U. of carotene or vitamin A natural esters were, respectively, about 3 and 4 times longer than those of the two control groups. In experiment 2 (Table 4), the liver
TABLE 4.—Vitamin A contents of yolks at hatching and livers and yolks at 5 days of age of deficient chicks drenched with corn oil solutions of 700 I.U. of 90% 0-10% a-carotene or vitamin A natural esters at 2 days of age1 Group
Drench
2,3 1
None 2 None (controls)
2 3
Carotene, 700 I.U. Vitamin A, 700 I.U.
1 2 8
No. of chicks
Organ analyzed
Time removed
Vitamin A content, I.U.
11 3
yolk yolk liver liver liver
1st day 5th day 5th day 5th day 5th day
9.1(H)3 5.8(3) 4.9(3) 14.1(6) 54.5(5)
6 5
Drenched 7 times at 2 or 3-hour intervals between 20 and 36 hours after hatching. Group 2 and 3 chicks were deutectomized on the day of hatching and the excised yolks were analyzed. Numbers of individual values in parentheses.
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Group
0.1 ml. of oily or aqueous preparations of carotene or vitamin A or of placebos was given by dropper to the chicks commencing at times ranging from twelve to forty hours after hatching, and repeated at intervals of two, three or four hours until the total scheduled dosages had been given. Concentrations of all the preparations were adjusted to 1,000 I.U. per ml. In three of the experiments (Tables 6, 7, 8) dehydrated alfalfa meal was the only feed given to certain groups of chicks for periods of thirty-four to fifty hours, commencing as soon as possible after hatching in two experiments and twelve hours after hatching in the third. In one experiment, dehydrated alfalfa meal was forcefed to appropriate chicks during the first twelve hours after hatching. In all experiments, the basal vitamin A-deficient diet was fed ad libitum to controls throughout the test periods, and to treated birds except at times when alfalfa feedings, either ad libitum or by force, were in progress.
UTILIZATION OF CAROTENE AND VITAMIN A
1353
TABLE 5.—Liver vitamin A contents at 5 and 26 days of age and mean survival time of deficient chicks deutectomized the day after hatching and drenched with aqueous emulsions of 800 I.U, of 90% (1-10% a-carotene or of vitamin A acetate or with placebos on the day after hatching Liver vitamin A (I.U.)
Drench1
Group
1st day
1 2 3
26th day
10.7(10) >200(9) >200(9)
2.1(3)
Mean survival time, days
11.8(11)'
None, killed at hatching placebos2 (controls) carotene, 800 I.U. vitamin A, 800 I.U.
12.9(10) 44.5(6) 50.5(6)
5.7(3)
Drenched 8 times at 2-hour intervals between 26 and 40 hours after hatching. Placebo: 10% aqueous Tween 60. Carotene and vitamin A emulsified in this medium. Numbers of individual values in parentheses.
TABLE 6.—Liver vitamin A contents at 5 days of age and mean survival times of deficient chicks drenched1 with 800 I.U. of 90% 0-10% a-carotene dissolved in Wesson oil or emulsified in 10% aqueous Tween 60, or fed only dehydrated alfalfa meal2 Liver vitamin A (I.U.) Group
Treatment 1st day
1 2 3 4 5
Killed at hatching None (controls) Drench, carotene in Wesson oil, 800 I.U. Drench, emulsified carotene 800 I.U. Alfalfa,2 carotene intake, 500 I.U.
5th day
Mean survival time, days
9(4)'
3.6(4) 52.5(4) 60.0(4) 22.8(4)
21 (3) 30 (6) 37.4(5) 27.7(3)
1
Drench 8 times at 3-hour intervals between 12 and 33 hours after hatching. Average alfalfa consumed per chick = 1.27 g=500 I.U. carotene. Fed ad lib. for the first 34 hours after hatching. 3 Numbers of individual values in parentheses. 2
TABLE 7.—Liver vitamin A contents and mean survival times of chicks fed only dehydrated alfalfa meal or basal diet ad lib. between 12 and 62 hours after hatching Liver vitamin A (I.U.) 1st day 1 2 3 1
Killed a t hatching Basal diet (controls) Alfalfa, 3400 I.U. carotene
7th day
11th day
15 th day
20th day
6.5(3) 90.5 (4)
0 (2) 57.5(3)
0(2) 0(2)
0(1) 3(2)
Mean survival time, days
4(2)
0(2)
15.8(16) 33.4(15)
Numbers of individual values in parentheses.
Initial Group vitamin A reserves
1 2
29th day
14(12)1
TABLE 8.—Mean survival times of unoperated and deutectomized1 normal and deficient chicks fed only dehydrated alfalfa meal during the first 48 hours after hatching
1 2 3 4 5 6 7 8
24th day
normal normal deficient deficient normal normal deficient deficient
Unoperated or deutectomized
Calculated carotene intake, I.U.2
Mean survival time, days
unoperated deutectomized unoperated deutectomized unoperated deutectomized unoperated deutectomized
0 0 0 0 788 680 574 131
16.6(14)3 12.1(14) 4.6(17) 4.3(19) 28.2(13) 19.5(11) 20.4(11) 12.6(6)
Deutectomized as soon as dry after hatching. Carotene intake groups 5-8 calculated from average alfalfa consumption per chick. ! Numbers of individual values in parentheses.
vitamin A contents of the chicks about 3 | days after final drenching with 700 I.U. of carotene or vitamin A natural esters in corn oil were, respectively, about 3 and 11 times that found in untreated controls. The apparent increases represent only 1.3 and 7 percent, respectively, of the quantities of carotene and vitamin A given. In experiment 3 (Table 5), drenching with 800 I.U. of carotene or vitamin A acetate in aqueous emulsion appeared to increase survival of chicks more than 3 times that of controls. Also, three days
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1 2 3 4
5th day
1354
J. D. HARVEY, D. B. PARRISH, P. E. SANFORD AND J. S. HUGHES
In experiment 5 (Table 7), chicks fed alfalfa meal between twelve and sixty-two hours after hatching lived more than two times longer than controls, and 4§ days after cessation of the alfalfa-feeding they had liver vitamin A contents 14 times larger. The apparent deposition of dietary vitamin A in the liver at this time represented only 2\ percent of the calculated intake of 3400 I.U. of carotene. On the eleventh day after hatching, liver vitamin A in the alfalfa-fed group was equal to 68 percent of that on the seventh day; on the fifteenth day and later, negligible quanti-
ties were found. No measurable vitamin A was found in livers of control chicks on the eleventh day or later. In experiment 6 (Table 8), survival times of all four types of chicks—unoperated or deutectomized and normal or deficient—were increased 60 to 400 percent by the ingestion of sufficient alfalfa meal during the first 48 hours to provide 131 to 788 I.U. of carotene per chick It appears from these data that a given quantity of provitamin A ingested early in life adds more days of survival time to vitamin A-deficient chicks than to those with normal initial vitamin A reserves, suggesting losses before physiologic use of the ingested and stored vitamin A or greater daily demand due to increased age by time initial reserves are used up or both. In another experiment, three chicks were force-fed alfalfa meal during the first twelve hours after hatching. They consumed an average of 682 I.U. of carotene. Liver vitamin A contents forty-eight hours after completion of the feeding averaged 23.3 I.U. compared with 2.7 I.U. in three control chicks. There appears to be little doubt that oral administration of carotene or vitamin A to chicks during the first 2\ days after hatching prolongs life on a vitamin A-deficient diet. Since in some experiments treatments were completed earlier (within 12 hours in one) without change in the qualitative nature of results, there is reason to believe what might be said of vitamin A metabolism of a 2 | day old chick might be true of a 12-hour-old chick. Also, if in sufficiently large quantities, this vitamin A ingested will result in increased liver vitamin A levels. However, the efficiency of transfer of the vitamin to the liver is generally low, and apparently influenced by the nature of the vitamin A preparation given.
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after treatment, liver vitamin A contents in carotene- and vitamin A-drenched groups were at least 20 times larger than that of controls. These data indicate that more than 25 percent of the vitamin or provitamin A given was moved to the liver. By the twenty-sixth day, the liver vitamin A contents again were low. In experiment 4 (Table 6), mean survival times of chicks drenched with 800 I.U. of carotene dissolved in Wesson oil or emulsified in 10 percent aqueous Tween 60 were, respectively, about If to 2 times longer than that of untreated controls. Liver vitamin A contents of chicks in the oil-, emulsion-, and alfalfa-fed groups about 3 | days after treatment were, respectively, 13, 15, and 6 times that in the untreated controls, indicating that about 6 , 1 \ and 2 | percents of the vitamin activities of drenches given were present in the liver. Chicks receiving 500 I.U. of carotene by consumption of dehydrated alfalfa meal ad libitum for thirty-four hours after hatching survived nearly as long as those given 800 I.U. of carotene in oil. The mean survival time of controls is longer than would be expected on the bases of the results of experiments 1 and 3, and of the low liver vitamin A contents at hatching and on the fifth day. No explanation can be given for the difference.
UTILIZATION OF CAROTENE AND VITAMIN A
DISCUSSION
In experiments in which carotene and vitamin A preparations were given to chicks shortly after hatching, and in which subsequent increases in liver vitamin A contents were observed, the efficiencies of utilization of the preparations for liver storage generally were considerably lower than in the case of 19-day-old chicks (Harvey and Parrish, 1953). While several groups of the young chicks had not been depleted of their vitamin A reserves prior to experimental treatment with vitamin A or carotene, the apparent mobilization of stored vitamin A during the first two weeks (Harvey et al., 1955b) might have similar effects upon efficiency of utilization of a dietary supplement for liver storage as would prior depletion in older chicks. The results indicate that the newly hatched chick has, or develops soon after hatching, the biochemical systems necessary to convert carotene to vitamin A and to use vitamin A. In the force-feeding experiment, alfalfa was not fed later than twelve hours after hatching. Probably the
residues of this material were voided within a few hours, since carmine given to chicks in the first balance trial was found in the droppings within three or four hours. Therefore, the extra vitamin A found in the livers of the chicks force-fed alfalfa and killed when forty-eight hours old probably was derived from the carotene of alfalfa digested and absorbed during the first sixteen hours after hatching. While treatments were not completed as soon after hatching in the other experiments as in the force-feeding experiment, the results were qualitatively-similar in all experiments. Without exception, carotene given during periods lasting not longer than sixty-two hours after hatching appeared to have been converted to vitamin A and stored in the liver, suggesting that the mechanism operative at that time and later in life differs little from those present during the first twelve hours. These results also confirm the findings of Bolin et al. (1943) and Rubin and Bird (1941) on chicks about one week old. As was found in the balance trial with newly hatched chicks, aqueous preparations of vitamin A or provitamin A were utilized better for survival and for liver storage of vitamin A than were oily solutions. This is similar to the findings of Frey and Wilgus (1949) and Halpern et al. (1947) with older birds. Preformed vitamin A always was more effective than carotene in any given similar preparation. Mean survival times of untreated deficient control chicks were 4 to 5 days. The prolongation of life of such chicks, when fed a vitamin A supplement during the first two days, indicates that the ingested material was used to support life as early as the fifth day after hatching. Probably it was used even earlier to correct the deficiencies which existed in the chick bodies some time before the average age at which untreated chicks died.
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Utilization of dietary vitamin A and carotene by newly hatched chicks would depend upon the existence of mechanisms for the digestion of the feed components containing the vitamin A-active material and for the absorption of the free vitamin and carotene. The carotene-balance trial carried out with day-old chicks indicated a high apparent absorption of carotene from oil solution, aqueous emulsion or alfalfa in the diet. Probably destruction in the digestive tract accounts for some of the carotene that appeared to have been absorbed in these experiments. As has been found also by Frey and Wilgus (1949) with laying hens and by Halpern et al. (1947) with older chicks, the aqueous preparation was absorbed better than the oily.
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J. D. HARVEY, D. B. PARRISH, P. E. SANFORD AND J. S. HUGHES
It is interesting to speculate on the site of conversion of the injected carotene, for there should be no need for it to pass through the intestinal tissues, which generally is accepted now as the site of conversion of dietary carotene (Matterson el al., 1950; Thompson el al., 1950), on the way to the liver. If intestinal tissues were involved, it would seem that it could be only as the result of carotene being carried to that site by the arterial route. The liver was regarded as the site of conversion in earlier work (Ahmad, 1931; Olcott and McCann, 1931; Rea and Drummond, 1932). One also might suspect the yolk-sac. SUMMARY
Between 65 and 100 percent of carotene fed as oily solution, aqueous emulsion or alfalfa meal to newly hatched chicks in balance trials was removed from the ingesta during passage through the gastrointestinal tract. Aqueous or oily drenches of carotene or vitamin A, and ad libitum or force-feeding of alfalfa meal during periods terminated
as early as twelve hours and never longer than sixty-two hours after hatching resulted in increased liver vitamin A contents at four days and prolonged survival times in chicks fed a diet deficient in vitamin A and carotenoid pigments after treatments. Injections of vitamin A or carotene emulsions into the residual yolks of newly hatched chicks also resulted in increased liver vitamin A contents at four days of age and prolonged survival times. Injected vitamin A and carotene were removed rapidly from the yolk and conversion of carotene to vitamin A appeared to have occurred without necessarily having passed through the intestinal tissues on the way to the liver. In addition to the liver, the yolk-sac might be suspected as site of conversion. It is concluded that the chick converted carotene to vitamin A as early as during the first day, and that prolongation of life in vitamin A-deficient chicks beyond a mean of four or five days by early treatment with carotene or vitamin A indicate that dietary vitamin A was used to support physiological functions involving vitamin A at least by the fifth day and probably earlier. These results advance to earlier ages the contentions of Bolin el al. (1943), Rubin and Bird (1941) and Temperton and Dudley (1945) that carotene and vitamin A could be utilized by chicks one week old. The report of Mann (1946) that these materials could not be utilized until three weeks or more after hatching is not substantiated. REFERENCES Ahmad, B., 1931. The fate of carotene after absorption in the animal organism. Biochem. J. 25: 1195-1204. Bolin, D. W., C. E. Lampman and L. R. Berg, 1943. The influence of carotene intake as supplied by dehydrated alfalfa on the storage of vitamin A and pigments in the livers of the young chick. Poultry Sci. 22: 348-353.
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The presence of systems for the conversion of carotene to vitamin A in newly hatched chicks was indicated also by the experiment on intravitelline injections. There is a rapid transport of vitamin A from the residual yolk to the rest of the chick body during the first week (Harvey el al., 1955b). In the present study, it was found that, if the yolk vitamin A content were increased by the injection of 500 I.U. of emulsified vitamin A alcohol just after hatching, most of this additional vitamin A was removed within four days, about 35 percent of-it appearing in the liver. When similar quantities of emulsified carotene were injected, most of the carotene also disappeared from the yolk by the fourth day and liver vitamin A content increased by an amount equivalent to 12 percent of the carotene injected.
HAEMATOLOGICAL CHANGES WITH Frey, P. R., and H. S. Wilgus, 1949. The utilization of carotene from different sources by laying chickens. J. Nutrition, 39:517-528. Halpern, G. R., J. Biely and F. Hardy, 1947. Utilization of vitamin A in water emulsion. Science, 106: 40-41. Harvey, J. D., and D. B. Parrish, 1953. Unpublished data.
Harvey, J. D., D. B. Parrish and P. E. Sanford, 1955b. The application of deutectomy for reducing initial vitamin A reserves in newly hatched chicks, and some observations on the apparent consumption of these reserves. Poultry Sci. 34: 1314-1321. Mann, T. B., 1946. Chick rearing, ix. Studies on the storage of vitamin A and carotenoid pigments by chicks from birth to six weeks, using cyclized vitamin A, vitamin A-alcohol, vitamin A-ester (cod liver oil) and the pigments of green
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food and maize as criteria. J. Agric. Sci. 36: 289300. Matterson, L. D., J. R. Carson and L. Griswald, 1950. Studies on the conversion of /3-carotene to vitamin A in vitro and in vivo. Poultry Sci. 29: 769. Neff, A. N., D. B. Parrish, J. S. Hughes and L. F. Payne, 1949. The state of vitamin A in eggs. Arch. Biochem. 21: 315-320. Olcott, H. S., and D. C. McCann, 1931. Carotenase. The transformation of carotene to vitamin A in vitro. J. Biol. Chem. 94: 185-193. Rea, J. L., and J. C. Drummond, 1932. Formation of carotene from vitamin A in the animal organism. Zeit. Vitaminforsch. 1:177-183. Rubin, M., and H. R. Bird, 1941. Some experiments on the physiology of vitamin A storage in the chick. Poultry Sci. 20:291-297. Temperton, H., and F. J. Dudley, 1945. The use made by the chick of dietary sources of carotene and vitamin A during the first month of life. Harper Adams Util. Poultry J. 30, N. S. 4:57-84. Thompson, S. Y., M. E. Coates and S. K. Kon, 1950. The conversion of carotene to vitamin A in the intestine of the chick. Biochem J. 46: xxx.
Haematological Changes in Cockerels after A C T H and Cortisone-Acetate Treatment* JAN HUBLE Laboratory for Animal Morphology, University of Ghent, Belgium (Received for publication April 4, 1955)
L
ITTLE is known about the avian respone to ACTH or adrenal steroids. In his review Sturkie (1954) lists only very limited, partly contradictory data: a single injection of adrenal cortical extract in adult fowl causes a transient lymphopenia and leucocytosis (Shapiro and Schechtman, 1949); ACTH injected for several days had no effect upon the number of
* Aided by grants from the Centre National de Recherches sur la Croissance normale et pathologique, Brussels. ACTH and Cortisone generously put at our disposal by N.V. Organon, Oss, Holland.
eosinophils (Stamler et al., 1950). Therefore we found it worthwhile to publish this preliminary report on haematological changes induced by injection of ACTH (Organon) or crystalline Cortisone-acetate (Organon Labs.). MATERIAL AND METHODS
Pure bred Rhode Island Red cockerels varying in age from 2 to 4 weeks at time of blood analysis were used. Blood was taken from the ulnar vein and diluted with a special fluid adapted for counting avian blood cells (Natt and Herrick, 1952). Collecting of blood was done in the
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Harvey, J. D., D. B. Parrish and P. E. Sanford, 1955a. Improvements in the technique of deutectomy of newly hatched chicks, and the effects of the operation on their subsequent development. Poultry Sci. 43:3-8.
ACTH