Folic Acid in Poultry Nutrition

Folic Acid in Poultry Nutrition I. The Critical Need for Folic Acid by Laying Pullets1 ROBERT J. LILLIE 2 , G. F. COMBS, AND G. M. BRIGGS 3 Poultry Department, University of Maryland, College Park (Received for publication August 6,1949)

AYLOR (1947) reported that 12 micrograms of folic acid4 per 100 grams of diet was an adequate level to support normal egg production in White Leghorn yearling hens fed a semi-purified diet. This amount, however, was not adequate for normal hatchability. Schweigert and associates (1948), in studies with White Leghorn pullets also fed a semipurified diet, found that 42 micrograms of folic acid4 per 100 grams of diet was adequate for normal egg production, hatchability, hemoglobin levels, and general appearance. Cravens and Halpin (1949), on the other hand, reported the folic acid requirement for normal hatchability in White Leghorn pullets to be approximately 50 to 100 micrograms per 100 grams of diet. These workers used a synthetic diet which did not favor intestinal 1

Scientific Paper No. A249. Contribution No 2184 of the Maryland Agricultural Experiment Station (Department of Poultry Husbandry). The authors are greatly indebted to Lederle Laboratories, Pearl River, New York, for synthetic folic acid; to Merck & Co., Inc., Rahway, New Jersey, for riboflavin and nicotinic acid; to Dr. Morley G. McCartney for his faithful assistance in artificial insemination of the pullets; and to Dr. Morley A. Jull for his valuable suggestions. 2 Now at the Agricultural Research Center, Bureau of Animal Industry, Beltsville, Maryland. 3 Now at the Division of Poultry Husbandry, University of Minnesota, St. Paul 1, Minnesota. 4 As determined by microbiological assay.

synthesis of this vitamin. A level of 25 micrograms of folic acid per 100 grams of ration was found to be adequate for normal egg production. Very little research concerning the importance of folic acid in poultry nutrition has been conducted in which practical rations have been used. This investigation was undertaken, therefore, to determine the critical need for folic acid by laying pullets fed a diet composed only of practical ingredients. EXPERIMENTAL PROCEDURE

Experiment 1. Four-month-old New Hampshire pullets were divided into 4 groups of 15 pullets each, and they were maintained in laying batteries for a period of 11 months. Group I received a practical ration (diet 1, as shown in Table 1) designed to be low in folic acid. Groups II and III received diet 1 with synthetic folic acid supplemented at a level of 200 and 400 micrograms per 100 grams of diet, respectively. Group IV received a typical all-purpose mash, hereinafter referred to as diet 3, shown in Table 1. Before selection, these pullets had been maintained on the Maryland growing mash designated as diet 4, also shown in Table 1. Feed and water were supplied ad libitum. Crushed oyster shell was fed twice weekly. Individual weighings were made at monthly intervals. Daily egg produc115

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T

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ROBERT J. LILLIE, G. F. COMBS AND G. M. BRIGGS

tion records and monthly feed consumption records were kept. Artificial insemination was performed twice weekly with semen from New Hampshire males fed diet 3. Eggs were pedigreed daily and stored in a refrigerator at 50° F. until setting time. All chicks that had hatched by the 22nd day of incubation were wing-

possible. However, diet 1 was formulated so that the folic acid activity of the diet was low without curtailing the balance of other nutrients in the ration. For each group of pullets, 50 pounds of feed were prepared at a time. The synthetic folic acid was added in dry form by mixing it thoroughly with a

TABLE 1.-—Composition of diets

Ground yellow corn Ground heavy oats Ground wheat Wheat std. middlings Wheat bran Soybean oil meal Corn gluten meal Alfalfa leaf meal Fish meal Meat scrap Dried whey Dried skim milk Fermentation solubles (80 meg. riboflavin/gm.) Oyster shell flour Rock phosphate, defluorinated Salt, iodized Vitamin A and D oil (400 units D/gm.) MnSOi Riboflavin, mg./lOO lbs. Nicotinic acid, gm./lOO lbs. Calculated analysis: Folic acid, mcg./lOO gms. Crude protein, %

Diet 1 Folic acid low

Diet 2 Folic acid low

Diet 3 Md. breeder mash

Diet 4 Md. starter mash

Folic acid activity of feedstuff

%

%

%

%

26.0 30.0

26.0 30.0 15.0

25.25 12.5

20.0 15.0

— — —

10.0 12.5 10.0 5.0 5.0 5.0 5.0 5.0

15.0 12.5 10.0 7.5 5.0 5.0 5.0 2.5

7.5

—

—

0.9 0.6

—

•

15.0

——.

10.0

10.0

—

—

— —

—• —.

7.5 7.5

—

7.5

—

—

meg./gram 0.3 0.2 0.4 0.9 2.1 7.7 0.2 6.0 0.0

—

—

2.0 1.0 0.5

1.25 2.0 0.75 0.75

—

5.1

2.0 1.0 0.5

1.25 0.5 0.5

—. — —

0.5 0.012

0.5 0.012

0.5 0.012

0.2 0.012

— —

. . —

—

150 2 33.8 18.4

pedigreed. These chicks were used in experiments to be described in a second paper. Those eggs that did not hatch were examined to determine the time of embryonic mortality. On the basis of folic acid activity of feedstuffs (Lillie and Briggs, 1947), diets 1 and 3 were calculated to contain approximately 34 and 164 micrograms per 100 grams of diet, respectively. Since cereal grains contain traces of folic acid, the formulating of a special practical ration completely free of folic acid was not

150 2 26.3 18.1

— 164.2 20.4

159.5 21.6

small amount of ground yellow corn. This premix was then incorporated in the basal diet. Experiment 2. In this experiment, the basal diet was modified by substituting ground wheat for wheat middlings. This modified basal diet is designated diet 2 and is shown in Table 1. This basal ration was calculated to contain approximately 26 micrograms of folic acid activity per 100 grams, as compared to 34 micrograms per 100 grams of diet 1. New Hampshire pullets 10 months old, which had been

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Ingredient

FOLIC ACID IN PRACTICAL RATIONS

maintained on diet 3, were fed diet 2 for a 4-week period to further deplete any folic acid reserves. At the end of this depletion period, the pullets were divided into 2 groups of 8 each on the basis of egg production records. The first group (group V) was continued on basal diet 2, while the second group (group VI) reTABLE 2.—Effect

Total hen. days

groups I and II is not clear. In experiment 2, the egg production of the hens fed the basal diet, which contained 26 micrograms of folic acid per 100 grams, was slightly less than that of the hens fed the folic acid supplemented diet. This difference, however, was small and is not considered significant.

of met upon egg production and feed consumption of New Hampshire pullets

Total eggs laid

Average egg production per hen

Total lbs. feed consumed

Average lbs. feed consumed per hen

Lbs. of feed per dozen

I II III IV

Experiment 1,15 hens per group. (Aug. 1,1947 to July 1, 1948) 4159 1823 146.8 1188.5 95.7 3839 1088 94.9 1036.5 90.4 4303 1585 123.4 1168.5 90.9 4167 1502 120.8 1221.3 98.2

7.9 11.4 8.9 9.8

V VI

Experiment 2, 8 hens per group. {Mar. 13, 1941 to July 30, 1948) 1120 465 58.1 294.5 36.9 1120 542 67.8 293.3 36.6

7.6 6.5

' Group Group Group Group Group Group

I—Diet II—Diet III—Diet IV—Diet V—Diet VI—Diet

1. 1 plus 200 meg. folic acid per 100 grams of diet. 1 plus 400 meg. folic acid per 100 grams of diet. 3. 2. 2 plus 200 meg. folic acid per 100 grams of diet.

ceived diet 2 supplemented with 200 micrograms of synthetic folic acid per 100 grams of diet. Subsequently, the experimental procedure was precisely the same as that described in experiment 1, except that chicks were not saved for further experiments. RESULTS

Egg production and feed consumption records as influenced by the dietary level of folic acid in a practical ration are summarized in Table 2. In experiment 1, the average egg production of the hens in group I was somewhat greater than that of the other three groups. These results clearly indicate that a level of 34 micrograms of folic acid per 100 grams of diet can sustain normal egg production. The reason for the large difference between

Hatchability studies, as shown in Table 3, showed no differences in the 4 groups in experiment 1; however, additional folic acid was apparently required for normal hatchability when diet 2 was used in experiment 2. In both experiments, the fertility data revealed no apparent differences. Embryonic mortality data are summarized in Table 3. At hatching time, all the progeny from the 6 groups of dams appeared normal with respect to down color, size, and weight. The mean body weights for experiment 1 are presented in Figure 1. In this experiment the group II hens maintained a slightly higher mean body weight than did the unsupplemented group. However, the mean body weight of group III hens, which received an even higher level of

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Group*

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ROBERT J. LILLIE, G. F. COMBS AND G. M. BRIGGS

TABLE 3.—Effect

Group*

No. eggs set

of diet upon fertility and hatchability of New Hampshire pullets

No. fertile eggs

Dead germs** 1

2

3

I "ipped eggs

898 459 721 711

829 422 608 642

V VI

459 527

407 478

59 44

Fertility

Hatchability of fertile eggs

%

%

33 24 14 13

650 332 474 513

92.3 91.9 84.3 90.3

78.4 78.5 77.9 79.9

Experiment 2 (4% months) 18 22 25 13 9 11

283 401

88.7 90.7

69.5 83.9

* Diets are described in Table 2. ** 1—Dead germs the first seven days of incubation. 2—Dead germs between the 7th and 18th day of incubation. 3—Dead germs between the 19th and 21st day of incubation.

folic acid, was no greater than that of the hens fed the unsupplemented diet. This diet contained 34 micrograms of folic acid per 100 grams. The somewhat lower level of egg production by the hens of group II may explain their greater mean body weight. The results of experiment 2, on the other hand, indicate that the addition of 3400

3200

3000

;2400

2000

IBOO AUG

SEP

OCT

NOV

DEC

JAN

FEB

MAR

1947

APR

MAY

OUN

1948 DATE OF WEIGHING

FIG. 1. Effect of diet upon body weights of New Hampshire pullets in experiment 1.

folic acid to the basal diet improved the maintenance of body weight (Figure 2). Since the basal diet used in this experiment contained 26 micrograms of folic acid per 100 grams, it appears that the minimum amount of folic acid required for the maintenance of normal body weight was greater than 26 but less than 34 micrograms per 100 grams of diet. Hemoglobin studies with the 4 groups of dams in experiment 1 showed some small differences, as shown in Table 4. The method used in these hemoglobin determinations was described in detail by Evelyn (1936). On both dates, the number of grams of hemoglobin per 100 cc. of whole blood was the lowest in group I dams. In all instances, the hemoglobin level was higher in the second than in the first test. Biological determinations of the folic acid activity of egg yolks from eggs laid by the 4 groups in experiment 1 are summarized in Table 5. The chick assay used for expressing the total folic acid content of the sample, including both the free and conjugated forms, is explained in detail by Lillie and Briggs (1947). None of the chicks fed any of the egg yolks added to the basal diet in the raw state grew as

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I II III IV

Experiment 1 111 months) 87 17 42 28 15 23 74 16 30 69 14 33

Total number chicks

FOLIC ACID IN PRACTICAL RATIONS

119

plemented with folic acid than in yolks of eggs from hens fed basal diet 1.

2700

2650

DISCUSSION - 2600

! 2550

2500

2450 MAY 8

JUN. 5

DATE OF WEIGHING

FIG. 2. Effect of diet upon body weights of New Hampshire pullets in experiment 2.

well as those fed 25 micrograms of folic acid per 100 grams of diet. The growth rate of chicks fed egg yolks from group II dams was greater than that of chicks fed egg yolks from group I dams. This finding indicates a greater amount of folic acid in the yolks of eggs from hens fed diet 1 SUPTABLE 4.—Hemoglobin studies with New Hampshire pullets (Experiment 1) Date of blood testing

Group I*

Group II*

Group III*

Group IV*

September 1, 1947 July 25, 1948

8.23* 9.45

9.14 9.91

8.61 9.75

8.90 9.74

* Diets are described in Table 2. ** All figures represent the number of grams of hemoglobin per 100 cc. of whole blood.

TABLE 5.—Folic acid activity of egg yolks from eggs laid by New Hampshire pullets in Experiment 1 Supplement to synthetic basal ration 110*

At start

At 4 wks.

Average weights at 4 wks.**

None 25 meg. folic acid/100 grams 50 meg. folic acid/100 grams 100 meg. folic acid/100 grams 200 meg. folic acid/100 grams 2 egg yolks (Group I) /kilogram 2 egg yolks (Group II)/kilogram 2 egg yolks (Group III)/kilogram 2 egg yolks (Group IV)/kilogram

20 20 20 20 20 20 20 20 20

5 17 16 18 16 10 9 4 5

79.3 245.9 247.8 253.5 278.9 122.4 157.1 152.3 169.9

* Diet is described by Lillie and Briggs (1947). '* Average of 2 duplicate experiments.

No. chicks

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1949

From these data, it is evident that the folic acid requirement for normal egg production of New Hampshire pullets fed a practical diet low in folic acid is below 34 micrograms per 100 grams of diet. This is in general agreement with the data of Schweigert et al. (1948) who reported that normal egg production was maintained on a folic acid-low ration containing 42 micrograms of folic acid per 100 grams of diet. This also agrees favorably with the conclusions of Taylor (1947) and Cravens et al. (1949) who found that very low amounts of acid were needed for egg production. In the present study, the lowered egg production, which resulted when a practical diet containing 26 micrograms of folic acid per 100 grams was fed, may have been due to variability among the pullets rather than to the dietary level of folic acid. The minimum folic acid requirement for normal hatchability in New Hampshire pullets fed a practical diet appears to be within the range of 26 to 34 micrograms per 100 grams of diet. Cravens et al. (1949) estimated a level of SO to 100 micrograms of folic acid per 100 grams of diet to be

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ROBERT J. LILLIE, G. F. COMBS AND G. M. BRIGGS

The significance of the results presented in this paper indicates that a folic acid deficiency can occur under farm conditions if an unusual combination of feedstuffs low in folic acid is used. The cereal grains and most animal protein supplements, including milk, are quite low in folic acid, and it is conceivable that some home-

mixed rations could be prepared from these feedstuffs alone. This is not likely, however, because of the common usage of alfalfa leaf meal and soybean oil meal, both of which are good sources of folic acid. Since commercial manufactured feeds contain ingredients which supply appreciable amounts of folic acid, there is no apparent danger of a folic acid deficiency occurring from the use of such rations for chickens. It is possible, however, as can be seen from the results of this study, that the poor hatchability of eggs, retarded growth of chicks, and faulty feather pigmentation observed in chicks and turkeys under practical conditions may be due, in some cases, to a partial deficiency of folic acid in the maternal diet. Nevertheless, further proof of this is required under controlled conditions. SUMMARY AND CONCLUSIONS

Studies were made with New Hampshire pullets fed practical diets containing varying amounts of folic acid. 1. When a practical ration containing approximately 26 micrograms of folic acid per 100 grams of diet was fed to New Hampshire pullets, the addition of synthetic folic acid improved hatchability and body weight maintenance. Fertility was not affected and the differences in egg production were not consistent. 2. A practical ration which contained approximately 34 micrograms of folic acid per 100 grams of diet was adequate for the maintenance of normal egg production, fertility, hatchability, and body weight when fed to New Hampshire pullets. On the other hand, the addition of folic acid to this ration was found to increase hemoglobin levels and to raise the folic acid content of the egg yolks. 3. The minumum folic acid requirements of New Hampshire pullets for nor-

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required for normal hatchability. This discrepancy may be explained on the basis of breed differences and types of diet used since their semi-purified diet was not favorable to the intestinal synthesis of this vitamin. Apparently, the dietary requirement is somewhat less when pullets are fed a ration composed of practical feedstuffs. Although a practical ration containing only 34 micrograms of folic acid per 100 grams of diet was adequate for normal egg production, fertility, and hatchability, additional folic acid slightly increased the hemoglobin levels. Since Schweigert et al. (1948) reported no differences in hemoglobin levels of laying pullets fed a semipurified ration containing 42 micrograms of folic acid per 100 grams, it appears that the folic acid requirement for optimum hemoglobin levels is between 34 and 42 micrograms per 100 grams of ration. A definite requirement for folic acid in practical rations for laying hens may be difficult to establish since Luckey, Moore, Elvehjem, and Hart (1946) stated that the response of chicks to a given amount of folic acid depended upon the types of ration used. For example, high fat diets or diets containing glucose, sucrose, or starch as the sole carbohydrate supported a lower growth response than diets containing high protein, low fat, or corn meal and dextrin as the carbohydrates when folic acid was incorporated in the diets. Hemoglobin responses also were better on diets containing dextrin or corn meal than diets containing other carbohydrates.

BOOK REVIEWS

mal hatchability has not been determined but appears to be between 26 and 34 micrograms per 100 grams of diet. 4. No apparent differences were noted in down color, size, and weight of progeny at hatching time as a result of the low folic acid content of the maternal diet. REFERENCES

colorimeter with light filters. J. Biol. Chem. 115: 63-75. Lillie, R. J., and G. M. Briggs, 1947. Biological assay of folic acid activity in common feedstuffs. Poultry Sci. 26: 289-294. Luckey, T. D., P. R. Moore, C. A. Elvehjem, and E. B. Hart, 1946. Effect of diet on the response of chicks to folic acid. Proc. Soc. Exp. Biol. Med. 62: 307-312. Schweigert, B. S., H. L. German, P. B. Pearson, and R. M. Sherwood, 1948. Effect of the pteroylglutamic acid intake on the performance of turkeys and chickens. J. Nutrition 35: 89-102. Taylor, L. W., 1947. The effect of folic acid on egg production and hatchability. Poultry Sci. 26: 372-376.

Book Reviews (Continued from page 114)

study until they have acquired a background from other sources. The republication of four of the classical papers of Sewall Wright is, indeed, a most welcome undertaking. As is known to all workers in the field, they form the very bones and sinews of the corpus of

population genetics as applied to animal breeding. The graduate students in Dr. Lush's laboratory should be most heartily congratulated for their enterprise in making possible personal ownership of this out of print material. I. M. L.

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Cravens, W. W., and J. G. Halpin, 1949. The effect of pteroylglutamic acid and an unidentified factor on egg production and hatchability. J. Nutrition 37: 127-138. Evelyn, K. A., 1936. A stabilized photoelectric

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