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The Vitamin B6 Requirement of Breeder Hens1
THYROXINE SECRETION RATE
Mueller, W. J., and A. A. Amezcua, 1959. The relationship between certain thyroid characteristics of pullets and their egg production, body weight and environment. Poultry Sci. 38: 620-624. Pipes, G. W., B. N. Premachandra and C. W. Turner, 1958. Measurement of the thyroid hormone secretion rate of individual fowls. Poultry Sci. 37: 36-41. Premachandra, B. N., G. W. Pipes and C. W. Turner, 1958. Thyroxine secretion rates of two strains of New Hampshire chickens selected for high and low response to thiouracil. Poultry Sci. 37:399-405. Reineke, E. P., and O. N. Singh, 1955. Estimation
249
of thyroid hormone secretion rate of intact rat. Proc. Soc. Exp. Biol. Med. 88: 203-207. Snedecor, G. W., 1956. Statistical Methods (5th ed.). The Iowa State College Press, Ames. Turner, C. W., G. W. Pipes and B. N. Premachandra, 1959. A critique of indices of thyroid gland function. Proc. Oklahoma Conf. on Radioisotopes in Agric, pp. 97-103. U. S. Atomic Energy Comm. Publ. TID-7578. Wentworth, B. C, 1960. A radiochromatographic study of thyroxine and triiodothyronine in the thyroid gland and circulation of domestic birds. M. S. Thesis, University of Massachusetts.
The Vitamin B6 Requirement of Breeder Hens 1 H. L. FULLER, R. C. FIELD, 2 R. RONCALLI-AMICI, 3 W. S. DUNAHOO AND H. M. EDWARDS, JR.
Division of Poultry Husbandry, University of Georgia, Athens, Georgia (Received for publication May 6.1960)
T
HE need for vitamin B 6 for egg production and hatchability was established by Cravens et al. (1943). In a later report, they placed the requirement for maximum hatchability at 2.0 mg. per kg. of ration by adding crystalline pyridoxine HC1 to a purified diet of estimated vitamin B6 content (Cravens et al., 1946). In a study of the vitamin B6 requirement of chicks it was observed by Fuller and Dunahoo (1959) that the depletion of this vitamin occurred at a very early age when no dietary sources were provided. Reduction in weight gains, as compared with the control chicks receiving vitamin B6 was measurable at two days of age. This suggested a limited carryover of this vitamin from dam to chick and
1 Journal Paper No. 124 of the College Experiment Station, University of Georgia College of Agriculture Experiment Stations. 2 Present Address: Master Mix Feed Mills, Athens, Georgia. 8 Present Address: Hoffmann-La Roche, Inc., Nutley, New Jersey.
raised a question as to the adequacy of vitamin B6 in practical breeder rations. The vitamin B6 content of a simplified corn-soy ration assayed by the method of Atkins et al. (1943) and reported by Fuller and Kifer (1959) was approximately 5.5 mg. per kg. which should make further fortification with this vitamin unnecessary. EXPERIMENTAL PROCEDURE Two experiments were conducted in 1958 and 1959, the first using seven lots of 10 hens each and the second, six lots of 12 hens each in individual laying cages. A purified cerelose-soybean protein diet was used in both trials and supplemented with graded levels of pyridoxine HC1. This diet was assayed by the method of Atkins et al. (1943) and found to contain 0.34 mg. vitamin Be per kg. A corn-soy simplified diet estimated to contain 5 mg. vitamin B6 per kg. was fed to two groups in the first trial—one with and one without added pyridoxine. These diets are
250
FULLER, FIELD, RONCALLI-AMICI, DUNAHOO AND EDWARDS T A B L E 2.—Effect
T A B L E 1.—Basal diets
of pyridoxine
on egg
production
{Trial 1) Purified
Ingredient
Corn- Soy
%
%
Cerelose Drackett Protein C-l Non-nutrient fiber Salt mix 1 Vegetable fat Vigofac 2 Ground yellow corn Dehulled soybean meal Gr. limestone Defluorinated phosphate (34% Ca, 17% P) NaCl Trace minerals (Delamix) 3
68.7 20.0 2.0 6.0 3.0 0.3
.
Purified Purified Purified Purified Purified
0 0.55 1.1 2.2 4.4
41 36 47 51 53
37 60 36 54 56
25 48 13 49 29
8 8 13 43 24
3 2 15 42 40
0 18 10 28 17
Corn-Soy Corn-Soy
0 4.4
53 37
53 44
50 50
47 53
50 25
50 53
diet
** 3.0 0.3 70.7 22.0 1.5 2.0 0.35 0.15
Added Vitamins and Amino Acids {per lb.) 4 Thiamine HC1 (mg.) Riboflavin (mg.) 4 2 Ca pantothenate (mg.) 8 4 Niacin (mg.) 40 20 Menadione sodium bisulfite (mg.) 1 0.5 Vitamin E (I.U.) 10 5 Folic acid (mg.) 1.5 1 Biotin (mg.) 0.1 Choline chloride (mg.) 800 300 Para amino benzoic acid (mg.) 36 Inositol (mg.) 225 Vitamin Bu (meg.) 10 10 Vitamin A (I.U.) 6,000 6,000 Vitamin D j (I.C.U.) 600 600 3,400 227 Methionine (mg.) Glycine (mg.) 2,270
—
•— —
•
— —
1
Salt mix supplied (% of ration): NaCl, 0.65: KjCsEUOr • H2O, 1.42; KtHPOi, 0.465; CaHP0 4 -2H 2 0, 2.13; CaCOj. 1.0; MgCOs, 0.25; FeCeHsOV3H20, 0.1; CuS04-5H2O, 0.0011; MnSO<, 0.007; KJAU(SO0J-24H2O, 0.00054; KI, 0.0003; C0CV6H2O, 0.00054;
ZnCoj, 0.0003; ZnS0<, 0.003; NaF, 0.000005 (hens fed oyster shells free choice). 2 Source of unidentified growth factors (Chas. Pfizer & Co., Inc., Brooklyn, N. Y.). * Delamix (Limestone Products Corp. of America, Inc., Newton, N. J.) contained: Mn 6%, Fe 2%, Ca 0.2%, I 0.12%, Co 0.02%, Ca 26.5%.
shown in Table 1. Pretest periods of one week in trial 1 and two weeks in trial 2 were employed during which the hens were fed a commercial breeder ration, and after which they received their respective experimental diets. The trials were of six and 10 weeks duration, respectively. Beginning seven days prior to the pretest period and every five days thereTABLE 3.—Effect
Egg production (%) (hen-day basis) Added PyrWoxine
Experimental (weeks)
(mg./kg.) p* e£
after, the hens were artificially inseminated with 0.1 cc. of pooled semen from a group of S. C. White Leghorn cockerels. Eggs were set in Jamesway model 252 incubators every two weeks. At the end of the second experiment, one day's production of eggs was assayed for vitamin B6. Records were kept of egg production, feed consumption, body weight, fertility, hatchability, and weight of chicks hatched (from the last two hatches in each experiment). In the second trial hatchability records were kept by individual hens to allow statistical treatment of hatchability data. These were analyzed by the analysis of variance of Snedecor (1956) and the multiple range test of Duncan (1955) as modified by Kramer (1956). RESULTS
Egg Production.—In trial 1 egg production declined in all lots receiving the purified diets but the decline was less se-
of pyridoxine on egg production, feed efficiency, and body weight {Trial 2)
E g g p r o d u c t i o n (%) (hen-day)
Added pyridoxine (mg./kg.) 0 1 2 3 4 5
Lbs. feed per Average change doz. eggs in body wt. (8 weeks) (8 weeks) (lbs.)
Experimental (weeks)
Pretest • (2 weeks)
1-2
3-4
S-6
7-8
87 85 77 84 84 90
84 84 77 82 89 83
48 82 74 89 91 90
19 63 80 88 93 78
1 27 71 75 79 81
5.17 4.78 4.03 3.79 3.73 3.69
-1.21 -0.25 +0.09 +0.03 +0.09 +0.21
251
VITAMIN Be REQUIREMENT OF H E N S
vere at the higher levels of pyridoxine (Table 2). It appeared that 2.2 mg. added pyridoxine per kg. was at least as good as the higher level. In trial 2 egg production continued to improve up to the highest level of vitamin B 6 fed or 5 mg. per kg. but the response to levels above 2 mg. per kg. was slight (Table 3). When the production rates for the 7-8 week period are compared with the pre-test production rates, it is obvious that the 2 mg./kg. level of added pyridoxine supported egg production as satisfactorily as did higher levels. When this was added to the 0.34 mg./kg. in the basal diet, the minimum requirement for satisfactory egg production became 2.34 mg./kg. In view of the sharp drop in production at the next lower level of this vitamin, it is felt that 2.34 mg./kg. may be borderline and that the next higher level or 3.34 mg./kg. is a better expression of the requirement for egg production. This is substantiated by feed efficiency data shown in Table 3 and by the vitamin Bj content of eggs shown in Table 6. Egg production was not improved by the addition of pyridoxine to the corn-soy diet in trial 1. Fertility and Hatchability.—Fertility was not affected by level of pyridoxine in the diet and was generally satisfactory at all levels where the number of eggs was sufficient to provide a valid measure. Hatchability dropped in all lots during the extremely cold weather experienced during most of the first trial, but remained near normal and recovered quickly in the group receiving the highest level of pyridoxine, 4.4 mg./kg. (Table 4). As shown in Table 5, trial 2 gave similar results, except that hatchability remained relatively high in this experiment. Note that hatchability during the experimental period should not be compared with pretest hatchability. In that instance fertility
TABLE 4.—Effect
of pyridoxine on
hatchability {Trial 1)
Basal diet
Added pyridoxine (mg.Ag.)
Hatchability of fertile eggs (%) Weeks on test 0-1*
2-3
4-5
Purified Purified Purified Purified Purified
0 0.55 1.1 2.2 4.4
80 72 90 76 78
18 44 75 54 71
0 42 45 65 78
Corn-Soy Corn-Soy
0 4.4
81 89
61 54
70 77
* Includes the pretest week and first week of experimental period in order to provide uniform reporting periods.
was based on candled appearance only; whereas, in all subsequent hatches, apparent infertiles were broken out and classified more accurately as to infertility and early embryo mortality. This would have a tendency to lower the value for "percent hatch of fertile eggs," since much of the early mortality would be classified as "infertile" by the usual candling procedure. Each hen was considered a replicate for statistical treatment and percent hatchability values for individual hens were transformed into angles, whose sine is the square root of the percentage value, in order to avoid a binomial distribution (Snedecor, 1956). When these data were treated statistically with the analysis of variance, no significant difference was found among the hatches; however, a highly significant difference (P<.01) was present among treatments. Hatch X treatment interaction was also highly significant (P<.01). When the treatment data were further tested by the multiple range test of Duncan (1955) as modified by Kramer (1956), in order to have multiple comparisons, a significant difference was found among the treatments as shown at the bottom of Table 5.
252
FULLER, FIELD, RONCALLI-AMICI, DUNAHOO AND EDWARDS
TABLE 5.—Effect
Lot No.
Added pyridoxine (mg./kg.)
of pyridoxins on hatchability {Trial 2) Hatchability of fertile eggs (%)
Pretest* (2 weeks)
1-2
3-1
5-6
7-8
(89) (86) (78) (89) (98) (88)
39 74 77 80 80 76
5 79 79 81 82 81
0 57 62 74 88 75
42 67 69 83 77
Statistical results Lots: Average Treatment Means: (for entire 8 wk. period)
1
2
3
4
6
5
21.87
52.58
59.60
63.25
63.71
68.21
Means that are not underlined by the same lines are statistically different at the 5% level, based upon the method of Duncan (1955) as modified by Kramer (1956). * Fertility based on candled appearance only. For the remaining hatches eggs of doubtful fertility were broken out and examined.
It should be pointed out that these average treatment means represent average values for the entire eight-week test period; therefore, any differences due to treatment occurring during the latter part of the test period, when they would be expected to occur, would be minimized. Examining the data during the 7-8 week period reveals a continuous rise in percent hatch as pyridoxine is increased up to 4 mg./kg. This confirmed the results of trial 1 and indicates that approximately 4 mg./kg. (added) pyridoxine or a total of 4.34 mg. of vitamin B 6 /kg. were required to insure maximum hatchability TABLE 6.—Vitamin B$ content of eggs (Trial 2) Added pyridoxine (mg./kg.)
No. eggs*
Av. (and range) of B6 in eggs (mg./gm.)
0 1 2 3 4 5
0 1 7 9 10 9
1.78(1.45-2.02) 3.13(2.38-4.00) 4.98(4.5 - 5 . 8 ) 4.95(4.5 - 5 . 6 ) 4.68(3.9-5.9 )
—
* 1-day production at termination of experiment.
under the conditions of these experiments. Whether or not practical breeder rations can be improved by supplementation with this vitamin, can only be determined by a great number of tests under many different conditions. For obvious reasons margins of safety should be provided for vitamin B6 as is commonly practiced with other vitamins. Vitamin B6 Content of Eggs.—The vitamin B6 content of eggs was directly related to the level of this vitamin in the diet up to the 3 mg. level (3.34 mg. total vitamin Be/kg. diet) where it reached a maximum of 4.98 mcg./gm. egg (Table 6). It was interesting to note the uniformity of values within treatments where no "overlapping" of values occurrred until the maximum was reached. From these values it would be reasonable to assume that no more than 3.34 mg. of vitamin B6 per kg. would be required for maximum hatchability; yet, in view of the hatchability data, it must be assumed that either (a) one day's supply of eggs was not a repre-
VITAMIN B 6 REQUIREMENT OF HENS
sentative sample or (b) a limitation of vitamin Be was manifested in some related fashion other than Be content of eggs per se.
253
eggs contained approximately 5 meg. of vitamin B6 per gm. ACKNOWLEDGEMENTS
Initial Weight of Chicks.—In trial 1 it appeared that the vitamin B6 level in the maternal diet influenced the weight of chicks hatched. In trial 2, therefore, all eggs and chicks were weighed in an effort to substantiate this relationship. Contrary to previous results, no relationship was found between egg weights or initial chick weights and level of dietary vitamin Be. It was concluded that no such relationship existed.
This work was supported by grants-inaid from Hoffmann-La Roche, Inc., Nutley, New Jersey, Dawes Laboratories, Chicago, Illinois, and Charles Pfizer and Co., Terre Haute, Indiana. Pyridoxine HC1, vitamins A and E, riboflavin, and biotin were supplied by Hoffmann-La Roche, Inc; vitamin B12, Terramycin, and Vigofac (source of unidentified growth factors) by Charles Pfizer and Co.; niacin and vitamin D 3 by Nopco Chemical Co., Harrison, New Jersey; choline chloride by Dawes Laboratories; menadione sodium bisulfite and arsanilic acid by Abbott Laboratories, North Chicago, Illinois. The authors wish to thank Dr. Jack Bauernfeind and Mr. J. Scheiner of Hoffmann-La Roche, Inc. for their assistance in providing the vitamin B6 assays of feed and eggs reported herein.
SUMMARY
REFERENCES
The vitamin B6 requirement of breeder hens was studied in two experiments using S.C. White Leghorn hens in individual laying cages. The hens were artificially inseminated with pooled semen for hatchability studies. Using a purified soy protein-cerelose diet of known vitamin Be content supplemented with pyridoxine HC1 at various levels, the minimum requirement for egg production was shown to be approximately 2.3 mg. and for hatchability, approximately 4.3 mg. per kg. of diet. Supplementing a corn-soy type diet containing an estimated S mg./kg. with pyridoxine HC1 did not improve egg production or hatchability in one trial. The vitamin Be content of eggs was directly related to the level in the diet up to 3 mg. (added) per kg. beyond which no increase occurred. At this dietary level,
Atkins, L. A., S. Schults, W. L. Williams and C. N . Frey, 1943. Yeast microbiological methods for determination of vitamins. Pyridoxine. Ind. Eng. Chem. (Anal. Ed.) 15: 141-144. Cravens, W. W., E. E. Sebesta, J. G. Halpin and E. B. Hart, 1943. Effect of vitamin B» on egg production and hatchability. Poultry Sci. 22: 94-95. Cravens, W. W., E. E. Sebesta, J. G. Halpin and E. B. Hart, 1946. Studies on the pyridoxin(e) requirements of laying and breeding hens. Poultry Sci. 25: 80-82. Duncan, D. B., 1955. Multiple range and multiple F tests. Biometrics, 11: 1-42. Fuller, H. L., and P. E. Kifer, 1959. The vitamin BB requirement of chicks. Poultry Sci. 38: 255260. Fuller, H. L., and W. S. Dunahoo, 1959. The effect of various drug additives on the vitamin Be requirement of chicks. Poultry Sci. 38: 1150-1154. Kramer, C. Y., 1956. Extension of multiple range test to group means with unequal number of replications. Biometrics, 12: 307-310. Snedecor, G. W., 1956. Statistical methods. Fifth Edition, Iowa State College Press, 534 pp.
Body Weight Changes.—The hens in trial 2 were unable to maintain their body weight at levels of pyridoxine below 2 mg./kg. diet (added). At 2 mg. or above, all groups gained slightly throughout the trial with no significant differences among groups. (Table 3).
Mueller, W. J., and A. A. Amezcua, 1959. The relationship between certain thyroid characteristics of pullets and their egg production, body weight and environment. Poultry Sci. 38: 620-624. Pipes, G. W., B. N. Premachandra and C. W. Turner, 1958. Measurement of the thyroid hormone secretion rate of individual fowls. Poultry Sci. 37: 36-41. Premachandra, B. N., G. W. Pipes and C. W. Turner, 1958. Thyroxine secretion rates of two strains of New Hampshire chickens selected for high and low response to thiouracil. Poultry Sci. 37:399-405. Reineke, E. P., and O. N. Singh, 1955. Estimation
249
of thyroid hormone secretion rate of intact rat. Proc. Soc. Exp. Biol. Med. 88: 203-207. Snedecor, G. W., 1956. Statistical Methods (5th ed.). The Iowa State College Press, Ames. Turner, C. W., G. W. Pipes and B. N. Premachandra, 1959. A critique of indices of thyroid gland function. Proc. Oklahoma Conf. on Radioisotopes in Agric, pp. 97-103. U. S. Atomic Energy Comm. Publ. TID-7578. Wentworth, B. C, 1960. A radiochromatographic study of thyroxine and triiodothyronine in the thyroid gland and circulation of domestic birds. M. S. Thesis, University of Massachusetts.
The Vitamin B6 Requirement of Breeder Hens 1 H. L. FULLER, R. C. FIELD, 2 R. RONCALLI-AMICI, 3 W. S. DUNAHOO AND H. M. EDWARDS, JR.
Division of Poultry Husbandry, University of Georgia, Athens, Georgia (Received for publication May 6.1960)
T
HE need for vitamin B 6 for egg production and hatchability was established by Cravens et al. (1943). In a later report, they placed the requirement for maximum hatchability at 2.0 mg. per kg. of ration by adding crystalline pyridoxine HC1 to a purified diet of estimated vitamin B6 content (Cravens et al., 1946). In a study of the vitamin B6 requirement of chicks it was observed by Fuller and Dunahoo (1959) that the depletion of this vitamin occurred at a very early age when no dietary sources were provided. Reduction in weight gains, as compared with the control chicks receiving vitamin B6 was measurable at two days of age. This suggested a limited carryover of this vitamin from dam to chick and
1 Journal Paper No. 124 of the College Experiment Station, University of Georgia College of Agriculture Experiment Stations. 2 Present Address: Master Mix Feed Mills, Athens, Georgia. 8 Present Address: Hoffmann-La Roche, Inc., Nutley, New Jersey.
raised a question as to the adequacy of vitamin B6 in practical breeder rations. The vitamin B6 content of a simplified corn-soy ration assayed by the method of Atkins et al. (1943) and reported by Fuller and Kifer (1959) was approximately 5.5 mg. per kg. which should make further fortification with this vitamin unnecessary. EXPERIMENTAL PROCEDURE Two experiments were conducted in 1958 and 1959, the first using seven lots of 10 hens each and the second, six lots of 12 hens each in individual laying cages. A purified cerelose-soybean protein diet was used in both trials and supplemented with graded levels of pyridoxine HC1. This diet was assayed by the method of Atkins et al. (1943) and found to contain 0.34 mg. vitamin Be per kg. A corn-soy simplified diet estimated to contain 5 mg. vitamin B6 per kg. was fed to two groups in the first trial—one with and one without added pyridoxine. These diets are
250
FULLER, FIELD, RONCALLI-AMICI, DUNAHOO AND EDWARDS T A B L E 2.—Effect
T A B L E 1.—Basal diets
of pyridoxine
on egg
production
{Trial 1) Purified
Ingredient
Corn- Soy
%
%
Cerelose Drackett Protein C-l Non-nutrient fiber Salt mix 1 Vegetable fat Vigofac 2 Ground yellow corn Dehulled soybean meal Gr. limestone Defluorinated phosphate (34% Ca, 17% P) NaCl Trace minerals (Delamix) 3
68.7 20.0 2.0 6.0 3.0 0.3
.
Purified Purified Purified Purified Purified
0 0.55 1.1 2.2 4.4
41 36 47 51 53
37 60 36 54 56
25 48 13 49 29
8 8 13 43 24
3 2 15 42 40
0 18 10 28 17
Corn-Soy Corn-Soy
0 4.4
53 37
53 44
50 50
47 53
50 25
50 53
diet
** 3.0 0.3 70.7 22.0 1.5 2.0 0.35 0.15
Added Vitamins and Amino Acids {per lb.) 4 Thiamine HC1 (mg.) Riboflavin (mg.) 4 2 Ca pantothenate (mg.) 8 4 Niacin (mg.) 40 20 Menadione sodium bisulfite (mg.) 1 0.5 Vitamin E (I.U.) 10 5 Folic acid (mg.) 1.5 1 Biotin (mg.) 0.1 Choline chloride (mg.) 800 300 Para amino benzoic acid (mg.) 36 Inositol (mg.) 225 Vitamin Bu (meg.) 10 10 Vitamin A (I.U.) 6,000 6,000 Vitamin D j (I.C.U.) 600 600 3,400 227 Methionine (mg.) Glycine (mg.) 2,270
—
•— —
•
— —
1
Salt mix supplied (% of ration): NaCl, 0.65: KjCsEUOr • H2O, 1.42; KtHPOi, 0.465; CaHP0 4 -2H 2 0, 2.13; CaCOj. 1.0; MgCOs, 0.25; FeCeHsOV3H20, 0.1; CuS04-5H2O, 0.0011; MnSO<, 0.007; KJAU(SO0J-24H2O, 0.00054; KI, 0.0003; C0CV6H2O, 0.00054;
ZnCoj, 0.0003; ZnS0<, 0.003; NaF, 0.000005 (hens fed oyster shells free choice). 2 Source of unidentified growth factors (Chas. Pfizer & Co., Inc., Brooklyn, N. Y.). * Delamix (Limestone Products Corp. of America, Inc., Newton, N. J.) contained: Mn 6%, Fe 2%, Ca 0.2%, I 0.12%, Co 0.02%, Ca 26.5%.
shown in Table 1. Pretest periods of one week in trial 1 and two weeks in trial 2 were employed during which the hens were fed a commercial breeder ration, and after which they received their respective experimental diets. The trials were of six and 10 weeks duration, respectively. Beginning seven days prior to the pretest period and every five days thereTABLE 3.—Effect
Egg production (%) (hen-day basis) Added PyrWoxine
Experimental (weeks)
(mg./kg.) p* e£
after, the hens were artificially inseminated with 0.1 cc. of pooled semen from a group of S. C. White Leghorn cockerels. Eggs were set in Jamesway model 252 incubators every two weeks. At the end of the second experiment, one day's production of eggs was assayed for vitamin B6. Records were kept of egg production, feed consumption, body weight, fertility, hatchability, and weight of chicks hatched (from the last two hatches in each experiment). In the second trial hatchability records were kept by individual hens to allow statistical treatment of hatchability data. These were analyzed by the analysis of variance of Snedecor (1956) and the multiple range test of Duncan (1955) as modified by Kramer (1956). RESULTS
Egg Production.—In trial 1 egg production declined in all lots receiving the purified diets but the decline was less se-
of pyridoxine on egg production, feed efficiency, and body weight {Trial 2)
E g g p r o d u c t i o n (%) (hen-day)
Added pyridoxine (mg./kg.) 0 1 2 3 4 5
Lbs. feed per Average change doz. eggs in body wt. (8 weeks) (8 weeks) (lbs.)
Experimental (weeks)
Pretest • (2 weeks)
1-2
3-4
S-6
7-8
87 85 77 84 84 90
84 84 77 82 89 83
48 82 74 89 91 90
19 63 80 88 93 78
1 27 71 75 79 81
5.17 4.78 4.03 3.79 3.73 3.69
-1.21 -0.25 +0.09 +0.03 +0.09 +0.21
251
VITAMIN Be REQUIREMENT OF H E N S
vere at the higher levels of pyridoxine (Table 2). It appeared that 2.2 mg. added pyridoxine per kg. was at least as good as the higher level. In trial 2 egg production continued to improve up to the highest level of vitamin B 6 fed or 5 mg. per kg. but the response to levels above 2 mg. per kg. was slight (Table 3). When the production rates for the 7-8 week period are compared with the pre-test production rates, it is obvious that the 2 mg./kg. level of added pyridoxine supported egg production as satisfactorily as did higher levels. When this was added to the 0.34 mg./kg. in the basal diet, the minimum requirement for satisfactory egg production became 2.34 mg./kg. In view of the sharp drop in production at the next lower level of this vitamin, it is felt that 2.34 mg./kg. may be borderline and that the next higher level or 3.34 mg./kg. is a better expression of the requirement for egg production. This is substantiated by feed efficiency data shown in Table 3 and by the vitamin Bj content of eggs shown in Table 6. Egg production was not improved by the addition of pyridoxine to the corn-soy diet in trial 1. Fertility and Hatchability.—Fertility was not affected by level of pyridoxine in the diet and was generally satisfactory at all levels where the number of eggs was sufficient to provide a valid measure. Hatchability dropped in all lots during the extremely cold weather experienced during most of the first trial, but remained near normal and recovered quickly in the group receiving the highest level of pyridoxine, 4.4 mg./kg. (Table 4). As shown in Table 5, trial 2 gave similar results, except that hatchability remained relatively high in this experiment. Note that hatchability during the experimental period should not be compared with pretest hatchability. In that instance fertility
TABLE 4.—Effect
of pyridoxine on
hatchability {Trial 1)
Basal diet
Added pyridoxine (mg.Ag.)
Hatchability of fertile eggs (%) Weeks on test 0-1*
2-3
4-5
Purified Purified Purified Purified Purified
0 0.55 1.1 2.2 4.4
80 72 90 76 78
18 44 75 54 71
0 42 45 65 78
Corn-Soy Corn-Soy
0 4.4
81 89
61 54
70 77
* Includes the pretest week and first week of experimental period in order to provide uniform reporting periods.
was based on candled appearance only; whereas, in all subsequent hatches, apparent infertiles were broken out and classified more accurately as to infertility and early embryo mortality. This would have a tendency to lower the value for "percent hatch of fertile eggs," since much of the early mortality would be classified as "infertile" by the usual candling procedure. Each hen was considered a replicate for statistical treatment and percent hatchability values for individual hens were transformed into angles, whose sine is the square root of the percentage value, in order to avoid a binomial distribution (Snedecor, 1956). When these data were treated statistically with the analysis of variance, no significant difference was found among the hatches; however, a highly significant difference (P<.01) was present among treatments. Hatch X treatment interaction was also highly significant (P<.01). When the treatment data were further tested by the multiple range test of Duncan (1955) as modified by Kramer (1956), in order to have multiple comparisons, a significant difference was found among the treatments as shown at the bottom of Table 5.
252
FULLER, FIELD, RONCALLI-AMICI, DUNAHOO AND EDWARDS
TABLE 5.—Effect
Lot No.
Added pyridoxine (mg./kg.)
of pyridoxins on hatchability {Trial 2) Hatchability of fertile eggs (%)
Pretest* (2 weeks)
1-2
3-1
5-6
7-8
(89) (86) (78) (89) (98) (88)
39 74 77 80 80 76
5 79 79 81 82 81
0 57 62 74 88 75
42 67 69 83 77
Statistical results Lots: Average Treatment Means: (for entire 8 wk. period)
1
2
3
4
6
5
21.87
52.58
59.60
63.25
63.71
68.21
Means that are not underlined by the same lines are statistically different at the 5% level, based upon the method of Duncan (1955) as modified by Kramer (1956). * Fertility based on candled appearance only. For the remaining hatches eggs of doubtful fertility were broken out and examined.
It should be pointed out that these average treatment means represent average values for the entire eight-week test period; therefore, any differences due to treatment occurring during the latter part of the test period, when they would be expected to occur, would be minimized. Examining the data during the 7-8 week period reveals a continuous rise in percent hatch as pyridoxine is increased up to 4 mg./kg. This confirmed the results of trial 1 and indicates that approximately 4 mg./kg. (added) pyridoxine or a total of 4.34 mg. of vitamin B 6 /kg. were required to insure maximum hatchability TABLE 6.—Vitamin B$ content of eggs (Trial 2) Added pyridoxine (mg./kg.)
No. eggs*
Av. (and range) of B6 in eggs (mg./gm.)
0 1 2 3 4 5
0 1 7 9 10 9
1.78(1.45-2.02) 3.13(2.38-4.00) 4.98(4.5 - 5 . 8 ) 4.95(4.5 - 5 . 6 ) 4.68(3.9-5.9 )
—
* 1-day production at termination of experiment.
under the conditions of these experiments. Whether or not practical breeder rations can be improved by supplementation with this vitamin, can only be determined by a great number of tests under many different conditions. For obvious reasons margins of safety should be provided for vitamin B6 as is commonly practiced with other vitamins. Vitamin B6 Content of Eggs.—The vitamin B6 content of eggs was directly related to the level of this vitamin in the diet up to the 3 mg. level (3.34 mg. total vitamin Be/kg. diet) where it reached a maximum of 4.98 mcg./gm. egg (Table 6). It was interesting to note the uniformity of values within treatments where no "overlapping" of values occurrred until the maximum was reached. From these values it would be reasonable to assume that no more than 3.34 mg. of vitamin B6 per kg. would be required for maximum hatchability; yet, in view of the hatchability data, it must be assumed that either (a) one day's supply of eggs was not a repre-
VITAMIN B 6 REQUIREMENT OF HENS
sentative sample or (b) a limitation of vitamin Be was manifested in some related fashion other than Be content of eggs per se.
253
eggs contained approximately 5 meg. of vitamin B6 per gm. ACKNOWLEDGEMENTS
Initial Weight of Chicks.—In trial 1 it appeared that the vitamin B6 level in the maternal diet influenced the weight of chicks hatched. In trial 2, therefore, all eggs and chicks were weighed in an effort to substantiate this relationship. Contrary to previous results, no relationship was found between egg weights or initial chick weights and level of dietary vitamin Be. It was concluded that no such relationship existed.
This work was supported by grants-inaid from Hoffmann-La Roche, Inc., Nutley, New Jersey, Dawes Laboratories, Chicago, Illinois, and Charles Pfizer and Co., Terre Haute, Indiana. Pyridoxine HC1, vitamins A and E, riboflavin, and biotin were supplied by Hoffmann-La Roche, Inc; vitamin B12, Terramycin, and Vigofac (source of unidentified growth factors) by Charles Pfizer and Co.; niacin and vitamin D 3 by Nopco Chemical Co., Harrison, New Jersey; choline chloride by Dawes Laboratories; menadione sodium bisulfite and arsanilic acid by Abbott Laboratories, North Chicago, Illinois. The authors wish to thank Dr. Jack Bauernfeind and Mr. J. Scheiner of Hoffmann-La Roche, Inc. for their assistance in providing the vitamin B6 assays of feed and eggs reported herein.
SUMMARY
REFERENCES
The vitamin B6 requirement of breeder hens was studied in two experiments using S.C. White Leghorn hens in individual laying cages. The hens were artificially inseminated with pooled semen for hatchability studies. Using a purified soy protein-cerelose diet of known vitamin Be content supplemented with pyridoxine HC1 at various levels, the minimum requirement for egg production was shown to be approximately 2.3 mg. and for hatchability, approximately 4.3 mg. per kg. of diet. Supplementing a corn-soy type diet containing an estimated S mg./kg. with pyridoxine HC1 did not improve egg production or hatchability in one trial. The vitamin Be content of eggs was directly related to the level in the diet up to 3 mg. (added) per kg. beyond which no increase occurred. At this dietary level,
Atkins, L. A., S. Schults, W. L. Williams and C. N . Frey, 1943. Yeast microbiological methods for determination of vitamins. Pyridoxine. Ind. Eng. Chem. (Anal. Ed.) 15: 141-144. Cravens, W. W., E. E. Sebesta, J. G. Halpin and E. B. Hart, 1943. Effect of vitamin B» on egg production and hatchability. Poultry Sci. 22: 94-95. Cravens, W. W., E. E. Sebesta, J. G. Halpin and E. B. Hart, 1946. Studies on the pyridoxin(e) requirements of laying and breeding hens. Poultry Sci. 25: 80-82. Duncan, D. B., 1955. Multiple range and multiple F tests. Biometrics, 11: 1-42. Fuller, H. L., and P. E. Kifer, 1959. The vitamin BB requirement of chicks. Poultry Sci. 38: 255260. Fuller, H. L., and W. S. Dunahoo, 1959. The effect of various drug additives on the vitamin Be requirement of chicks. Poultry Sci. 38: 1150-1154. Kramer, C. Y., 1956. Extension of multiple range test to group means with unequal number of replications. Biometrics, 12: 307-310. Snedecor, G. W., 1956. Statistical methods. Fifth Edition, Iowa State College Press, 534 pp.
Body Weight Changes.—The hens in trial 2 were unable to maintain their body weight at levels of pyridoxine below 2 mg./kg. diet (added). At 2 mg. or above, all groups gained slightly throughout the trial with no significant differences among groups. (Table 3).