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Evaluation of Egg Injection of Folic Acid and Effect of Supplemental Folic Acid on Hatchability and Poult Weight
Evaluation of Egg Injection of Folic Acid and Effect of Supplemental Folic Acid on Hatchability and Poult Weight EDWARD J. ROBEL U.S. Department of Agriculture, Agricultural Research Service, Livestock and Poultry Sciences Institute, Germplasm and Gamete Physiology Laboratory, Beltsville, Maryland 20705-2350
ABSTRACT Two experiments were conducted with Large White turkey hens housed individually in cages in a conventional house. In Experiment 1, three dietary treatments were used: an unsupplemented practical corn-soybean meal basal diet; the basal diet supplemented with 2.64 mg folic acid/kg of diet; and the basal diet supplemented with 5.51 mg folic acid/kg of diet. Eggs from hens fed 2.64 mg folic acid/kg of diet were injected with folic acid in 20 injection trials over two production cycles. The response data from dietary supplemental folic acid were analyzed on a production period basis using all of the hens, and on a subset of hens producing eggs in each production period, for hatchability of fertile eggs and poult weight. The response patterns in each case were similar. Incremental dietary supplemental folic acid levels produced a positive linear response pattern on the transfer of folic acid in eggs, but did not result in a hatchability increase over the unsupplemented folic acid basal diet. Hatchability increase did not occur for eggs injected at 25 days of incubation with 19.3 fig folic acid per egg in 20 injection trials over two cycles of production. The results of the study indicate that hatchability is not increased in turkey eggs from hens fed supplemental folic acid or with egg folic acid injections. However, egg and poult weights were significantly increased (P < .05) in eggs containing 6 to 7 mg folic acid/g of dried egg, from hens fed 5.51 mg folic acid/kg of diet. (Key words: folic acid, turkeys, eggs, hatchability, embryonic development) 1993 Poultry Science 72:546-553
(1984) recommendation for turkey breeders is 1 mg folic acid/kg of diet. It is Selection for rapid growth and feed considered unlikely in the commercial efficiency in turkeys may have altered the turkey industry that folic acid could be requirement of folic acid for hatchability limiting for hatchability or embryonic and embryonic development. Amounts development, because commercial turkey and bioavailability of folic acid in feed breeder diets contain supplemental folic ingredients vary and are unreliable acid. This reasoning, however, fails to sources of the vitamin. Levels of sup- consider an important objective, which lies plemental folic acid required for em- in the transport of folic acid to the egg. bryonic survival are controversial, and for Hatchability and poult weight are related rapid embryonic development are unknown (Schweigert et ah, 1948; Kratzer et to folic acid in the egg (Schweigert et al, al., 1956; Ferguson et al., 1961; Lee et ah, 1948; Ferguson et al, 1961; Sirbu et al, 1965; Miller and Balloun, 1967; Couch and 1981). Because of the role of folic acid in Ferguson, 1972; Coleman, 1985; Froehli, cellular development, higher supplemen1987). The National Research Council's tal folic acid levels may be required for rapid embryonic development. Snetsinger et al. (1963) reported the importance of folic acid in poult livability and growth. The current study was conducted to Received for publication August 24, 1992. investigate the effect of supplemental folic Accepted for publication November 4, 1992. INTRODUCTION
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FOLIC ACID FOR HATCHABILITY AND POULT WEIGHT
acid on the transfer of folic acid in the egg as related to hatchability and embryonic development, and of folic acid egg injections on the hatchability of turkey eggs. MATERIALS AND METHODS Experimental Synopsis
Experiment 1 was conducted to determine the effect of dietary supplemental folic acid on the transfer of folic acid into the egg as related to hatchability and poult weight at hatch time. The treatments consisted of an unsupplemented practical corn-soybean meal basal diet (Table 1); the basal diet supplemented with 2.64 mg folic acid/kg of diet; and 5.51 mg folic acid/kg of diet. The response data were averaged across 5 or 6 wk and recorded as three grouped time periods over the fall-winter production cycle. The reproductive data among treatments were analyzed on a time period basis (0 to 5, 6 to 10, and 11 to 16 wk of production) using all hens in each treatment; and across time periods for hatchability and poult weight, using a subset of hens that produced eggs in the three time periods of the production cycle. Eggs from hens fed 2.64 mg folic acid/kg of diet were injected with folic acid in each of the three time periods of production to measure hatchability response. In Experiment 2, eggs from hens fed the 2.64 mg folic acid/kg of diet were injected with folic acid in each of three 3- or 4-wk time periods of production (0 to 3,4 to 7, and 8 to 10 wk) to measure hatchability response. Folic acid in diets and solution for egg injections was analyzed by a commercial laboratory using the Association of Official and Analytical Chemists (1990) method, and folic acid in egg yolk and albumen was analyzed by the method of the Association of Vitamin Chemists, Inc. (1966).
TABLE 1. Composition of the basal diet Ingredients and calculated analyses Ground yellow corn Soybean meal (49% CP) Alfalfa meal (17% CP) Meat and bone meal (50% CP) Soybean oil DL-methionine 1 Vitamin premix Trace mineral premix2 Iodized salt Selenium premix3 Antibiotic premix4 Ground limestone Dicalcium phosphate Total Calculated analyses Protein Calcium Phosphorus (total) ME, kcal/kg
Percentage 62.00 16.90 5.00 5.00 3.00 .10 .50 .10 .30 .10 .50 4.50 2.00 100.00 17.00 2.71 .76 2,960
Witamin premix supplied the following per kilogram of diet: riboflavin, 11 mg; ca-d-pantothenic acid, 37.4 mg; thiamine, 4.4 mg; niacin, 66 mg; d-biotin, .22 mg; pyridoxine, 6.6 mg; choline, 881 mg; vitamin K, 3.0 mg; d-a-tocopherol acetate, 66IU; vitamin Bi2/17.6 jig; vitamin A acetate, 19,823 IU; cholecalciferol, 6,608 IU; ethoxyquin, 150 mg. 2 Trace mineral premix supplied the following in milligrams per kilogram of diet: manganese (sulfate), 100; iron (sulfate, carbonate, oxide), 100; copper (oxide), 10; cobalt (carbonate), 1; iodine (potassium iodide), 3; zinc (sulfate), 100. 3 Selenium premix supplied .1 mg sodium selenite/ kg of diet. 4 Antibiotic premix supplied 110 mg chlortetracycline/kg of diet.
Large White turkey hens, 30 wk of age, were randomly assigned to individual wire-bottomed cages (61.25 cm deep x 45 cm wide x 60 cm high) equipped with cup waterers in a conventional house. Feed and water were available for ad libitum consumption. The control treatment consisted of feeding the basal diet without supplemental folic acid (Table 1), which conExperiment 1 tained .67 mg folic acid/kg based on A total of 120 British United Turkeys of analysis. The other two treatments conAmerica (BUTA) (commercial strain cross) sisted of supplementing the basal diet with two levels of exogenous folic acid:1 2.64 mg and 5.51 mg folic acid/kg, which contained 1 Folic acid, United States Pharmaiopoeia (USP) in a 2.73 and 7.37 mg folic acid/kg of diet based dry premix containing 2,205 >*g folic acid/g of premix; on analysis. The photoperiod was 14 h light: contribution of Hoffmann-La-Roche, Inc., Nutley, NJ 10 h dark. The hens were photostimulated 07110-1199.
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at 30 wk of age and placed on dietary treatments at that time. The hens were divided into three treatment groups containing 40 hens per group. The experiment extended over the fall and winter seasons. Eggs for folic acid assays were collected in each treatment for 4 days in the middle of each time period. Eggs were assayed from individual hens, for a total of 40 replications per treatment. Egg yolk and albumen from each replicate were separated, blended separately, and each was freeze-dried and assayed independently. Hatching variables were recorded at weekly intervals. Weekly data responses were averaged across 5 or 6 wk and recorded as three grouped time periods (0 to 5,6 to 10, and 11 to 16 wk) of production. Statistical Analyses
The data were analyzed separately for each period, whereby all hens were used, including those not laying eggs in all three production periods (Table 2). With data analyzed separately for each time period, a one-factor analysis of variance was done for each response variable, where treatment was the factor. If the response variable did not meet the analysis of variance assumptions, then the appropriate transformation was determined and the analysis was done on the transformed variable. The mean separation was done using pairwise comparisons on the least squares means with the Sidak adjustment to the significant level P to account for the three simultaneous comparisons. Means were not declared statistically different unless the P value was less than .017 for comparisons, which gives an overall experimental comparison rate of P < .05. For hatchability of fertile eggs and poult weight, the data were also analyzed on a restricted subset of hens—the hens that laid eggs in all three production periods (0 to 5,6 to 10, and 11 to 16 wk). This was done as an
2 Folic acid (pteroylglutamic acid) crystalline, Number F-7876, Sigma Chemical Co., St. Louis, MO 63178-9916. 3 Millipore Corp., Bedford, MA 01730. 4 LyphoMed, Inc., Rosemont, IL 60018. 5 Becton Dickinson, Inc., Rutherford, NJ 07070.
attempt to better isolate the folic acid effect, which might presumably be masked in the period by period production analysis. Because the results of the two analyses did not differ, the data are presented on a production period basis in Table 2 (SAS Institute, 1988). Egg Injection Trials and Solution Preparation Environment
Egg injections and folic acid solution preparation were completed under Gold Fluorescent nonultraviolet light in a particle-free environment with filtered high efficiency particulate air. Eggs were collected from hens fed the basal diet containing 2.64 mg supplemental folic acid/kg of diet. The eggs were injected on the 25th day of incubation in each of the three time periods of the production cycle (0 to 5, 6 to 10, and 11 to 16 wk); with three spanned injection trials equally spaced in time occurring from 0 to 5 wk, three from 6 to 10 wk, and four from 11 to 16 wk (Table 3). Folic Acid Solution
The folic acid solution was prepared by forming a mixture of 25 mg folic acid2 in 5 mL absolute ethanol. To the mixture was added 20 mL .01 N sodium hydroxide (reagent grade), and the contents were mixed. To this mixture was added 225 mL deionized water. The solution was mixed at approximately 25 C until its transparency was assured. Glacial acetic acid (reagent grade) was used to adjust the solution to pH 7. The solution was then brought to 250 mL volume with deionized water. A portion of the solution was withdrawn into a 60-mL sterile syringe. The syringe was then equipped with a .22-jim sterile nonpyrogenic filter unit (Millex-GV).3 The solution was then dispensed into 10-mL empty sterilized vials4 from which .2-mL aliquots were withdrawn using a microfine III, 28-gauge sterile needle locked to a 1-mL insulin syringe.5 For the egg injections, .2 mL of folic acid solution was injected per egg at 25 days of incubation, using a separate syringe equipped with needle for each individual egg. The folic acid solution contained 19.3 /*g folic acid/.2 mL of solution based on analysis.
231 ± 3 * 244 ± 5 a 225 ± 6 b
219 ± 4 223 ± 6 207 ± 6
185+3 183 ± 6 174 ± 4
01 2.64 5.51
11 to 16 w k O 1 2.64 5.51
75.6 76.5 77.9 79.4 79.6 82.0 81.4 81.0 85.7
± + ± ±
.6* .9 b 1.2b 1.0"
± .7*
± .6* ± .6b
.6b
(g) 45.4 47.5 48.2 51.7 53.1 54.9 53.8 53.2 58.4
+ ± ± ± + ± ± ± +
Poult weight .4b .5" .5 a .5b .5b .6= .8b 1.1b .8" 15.7 16.9 16.8 8.9 7.6 9.0
± ± ± + ± ±
(no.) 22.1 ± 22.0 ± 21.4 ± .5 .7 .8 1.0 1.0 1.0 1.7 1.5 1.6
Eggs per hen + ± + ± + ±
2.2 2.3 3.2 3.6 2.5 3.5
68.2 ± 7.3 71.3 + 5.6 83.0 ± 5.4
83.0 79.2 76.9 83.2 83.4 82.2
Hatchability of fertile eggs
2.1 2.3 1.3
0. .2 1.3 1 1
(%) .8 ± .9 ± 0. ±
7 days
Em
1
- Means + SE in a column within each production period with no common superscripts differ signific Basal diet (without supplemental folic acid) contained .67 mg folic acid/kg of diet based on analysis.
a c
6 to 10 wk
0 to 5 wk
(mg/kg) 01 2.64 5.51
Period
Feed consumption Supplemental per hen Egg folic acid per day weight
TABLE 2. Performance of turkey hens fed a basal diet supplemented with two levels of of the production cycle, Experiment 1
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ROBEL TABLE 3. Effect of folic acid egg injections on hatchability of fertile eggs, Experiment 1 Weeks of production1
Treatment
0 to 5*
6 to 103
11 to 164
Control (uninjected) Injected Folic acid in carrier solution Carrier solution Analysis of variance
83.6 ± 6.6
(%) 88.1 ± 2.9
87.0 ± 3.5
87.3 ± 5.5 78.9 ± 9.9 NS
89.1 ± 2.3 88.0 ± 2.3 NS
92.6 ± 3.5 88.6 ± 1.5 NS
iFall-winter season production cycle. 2 Means ±SE within the column represent three equally spaced injection trials (27 eggs set per treatment per trial). 3 Means ± SE within the column represent three equally spaced injection trials (22 eggs set per treatment per trial). 4 Means ± SE within the column represent four equally spaced injection trials (24 eggs set per treatment per trial).
Aseptic Egg Injections
Egg injections were performed under a sanitized clean-room particle-free environment using filtered high efficiency particulate air, and personnel were aseptically equipped. A 2% iodine tincture was applied to the injection site on the large end of the egg. A very slight indenture was made in the swabbed shell area, using a small, sharp, sterile steel punch. The force used to make the indenture was of a minor degree to avoid the formation of hairline cracks in the shell. The punch site exactly accommodated the microfine III, 28-gauge sterile needle that was locked to a sterile 1-mL insulin syringe. The needle was carefully inserted through the indenture site to a depth of 6 to 8 mm, and .2 mL of the folic acid solution was injected over the inner eggshell membrane. The puncture was sealed with a small drop of fast-drying Duco cement.6 Egg Injection Treatments
The treatments were the following: eggs injected with .2 mL of vitamin carrier solution containing 19.3 fig folic acid based on analysis, eggs injected with .2 mL of the vitamin carrier solution, and control (uninjected) eggs.
Statistical Analysis of Egg Injection Trials
Ten egg injection trials were completed, with three to four trials per period. Each period was analyzed separately. A onefactor analysis of variance with treatment as the factor was run on the percentage hatchability data. The data were also analyzed as a weighted least squares analysis of variance using categorical modeling on the frequencies of the response variable, hatchability (SAS Institute, 1988). Experiment 2. Egg Injection Trials
Eggs were collected over the springsummer seasons from a flock of 120 BUTA Large White turkey hens fed the basal diet supplemented with 2.64 mg folic acid/kg— the same diet used for egg injections of Experiment 1. The eggs were injected at 25 days of incubation in each of three time periods of the production cycle (0 to 3,4 to 7, and 8 to 10 wk); with three injection trials occurring at equal intervals within the 0- to 3-wk period, three in the 4- to 7-wk period, and four in the 8- to 10-wk period (Table 4). RESULTS AND DISCUSSION Experimental Synopsis
6
Devcon Corp., Wood Dale, IL 60191.
The reproductive data in Experiment 1 were analyzed within a single time period
551
FOLIC ACID FOR HATCHABILITY AND POULT WEIGHT TABLE 4. Effect of folic acid injections on hatchability of fertile eggs, Experiment 2 Weeks of production1 Treatment
0 to 32
4 to 73
8 to 104
Control (uninjected) Injected Folic acid in carrier solution Carrier solution Analysis of variance
88.6 ± 1.7
(%) 90.2 ± 2.8
87.4 ± 3.2
90.3 ±3.1 93.0 ± 2.2
89.3 ±2.6 87.4 ± 1.7
92.4 ± 4.2 90.5 ± 5.1
NS
NS
NS
Spring-summer season production cycle. Means ±SE within the column represent three equally spaced injection trials (37 eggs per treatment per trial). 3 Means ±SE within the column represent three equally spaced injection trials (36 eggs per treatment per trial). 4 Means ± SE within the column represent four equally spaced injection trials (24 eggs per treatment per trial). 2
of production (Table 2). With the analysis within each single time period of production (0 to 5,6 to 10, and 11 to 16 wk), dietary treatment comparisons were based on the relationship of dietary folic acid levels with reproductive response patterns. This system of analysis has the advantage of not being confounded with feed consumption, which changed with maternal age over the three periods of production in the production cycle. Reproductive response was, therefore, not affected by dietary intakes because the relationship of dietary intakes among treatments within each time period of production was similar. Data presented in this fashion can be of practical value to the commercial turkey industry, which can accommodate dietary folic acid levels, but cannot respond to physiological implications associated with feed consumption and depositions of folic acid in eggs. Analysis of the subset of hens that laid eggs in each production period was also completed for hatchability and poult weight. This analysis was done to investigate the effect of the treatment on hatchability and poult weight over time. Hatchability of fertile eggs and poult weight response data from dietary supplemental folic acid was similar in each case, when analyzed on a production period basis using all of the hens, and on a subset of hens producing eggs in each production period. The experimental data were based on caged hens, and not on hens from the floor, as is industry practice. Thomason et ah (1977) observed significantly lower (P < .05)
hatchability of caged hens as compared with hens housed in floor pens. Experiment 1
Feed consumption for the 5.51 mg folic acid/kg of diet treatment was the lowest in the three production periods, but was statistically significant (P < .05) only in the 0- to 5-wk period. This result does not have practical importance in relation to the reproductive parameters investigated. There were no significant differences (P < .05) in numbers of eggs per hen, hatchability of fertile eggs, early embryonic deaths (< 7 days incubation) or late embryonic deaths (> 7 days incubation) among dietary treatments in each production period (Table 2). Early embryonic mortality was low in all treatments in each production period. In this study, hatchability did not increase as egg levels of folic acid increased with incremental levels of supplemental folic acid over the basal dietary level, based on analysis (.67 mg folic acid/kg). Folic acid in the basal diet originated entirely from basic dietary ingredients from which the bioavailability of folic acid varies widely, and the analyzed level of folic acid was only 67% of the National Research Council's (1984) recommended level for turkey breeder hens. There appears, therefore, to be ample margin for hatchability increase with dietary supplemental folic acid treatments over the basal dietary treatment; however, this did not occur. Although the numerical average hatchability that in-
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creased across treatments from 11 to 16 wk seemed promising, it is atypical. The overall estimate of variability and use of the conservative Sidak method precluded from finding statistically significant differences among treatments. Egg weight increased within each treatment as production periods advanced. However, there was an additional treatment effect due to supplemental folic acid, which caused significant increases (P < .05) in egg and poult weights. The 5.51 mg folic acid/kg of diet treatment induced a 2 /xg folic acid/g dried egg increase, with resultant increases of 3.28% in poult weight and 2.93% in egg weight from 6 to 10 wk; and 8.90 and 5.37% increases, respectively, from 11 to 16 wk, in contrast to the 2.64 mg folic acid/kg treatment. The 8.9% poult weight increase with the supplemental folic acid treatment (5.51 mg folic acid/kg of diet), resulted in the deposition of 144 /*g folic acid/82 g of egg, in contrast to the 104 fig folic acid per egg by hens fed 2.64 mg folic acid/kg of diet. This suggests that greater egg weight provided more nutrients. It is possible that greater folic acid transfer to the egg advanced nutrient utilization by extending folic acid functions in intermediary amino acid metabolism and biosynthesis of nucleotides for greater embryonic cellular development.
compared with eggs injected with the vitamin carrier solution and control (uninjected) eggs. Injection of the folic acid carrier solution did not increase hatchability over the control (uninjected) eggs. Experiment 2
Eggs used in these injection trials were from hens fed the 2.64 mg supplemental folic acid/kg of diet. Table 4 illustrates treatment means for 10 folic acid egg injection trials over the production cycle. There were no significant differences (P > .05) in hatchability of fertile eggs injected with folic acid as compared with eggs injected with the vitamin carrier solution and control (uninjected) eggs. Injection of the folic acid carrier solution did not increase hatchability over control (uninjected) eggs. Folic acid levels of eggs from each of the dietary treatments were quite stable with maternal age (Table 2), and are not expected to have influenced the hatchability response data to folic acid egg injections. Eggs from Large White turkey hens, under similar conditions of housing and diet (2.1 mg folic acid/kg based on analysis) as in Experiment 1, also had relatively little folic acid change in eggs with maternal age (Robel, 1983). In conclusion, mere were no statistically significant differences in average hatchabilOver 99% of folic acid depositions in the ity of turkey eggs from hens fed the egg occurred in egg yolk. There was a practical corn-soybean meal basal diet with significant increase (P < .05) in folic acid of or without supplemental folic acid. The egg yolk with each increment of dietary bioavailability of folic acid in the basal diet folic acid (Table 2). Folic acid depositions in is expected to be less than .67 m g / k g based egg yolk from the basal diet and the basal on analysis, and is lower than the National diet containing supplemental folic acid Research Council's (1984) recommended 1 levels of 2.64 and 5.51 mg folic acid/kg of m g / k g level for breeder turkeys. This diet, increased linearly over the production suggests that 1 mg folic acid/kg of diet cycle, averaging 3.2, 5.0, and 6.7 /*g of folic recommended for turkey breeders is excesacid/g of dried egg, respectively, per sive. From the results, it is evident that folic treatment based on analysis. acid egg injections were instrumental in Eggs from hens fed the 2.64 mg sup- revealing folic acid to be nonlimiting in plemental folic acid/kg of diet were in- turkey eggs for hatchability. The egg injecjected with folic acid to determine whether tion results lend additional support to the folic acid in eggs was limiting on the basis of results of previous egg injection investigahatchability response data. The data of tions that determined biotin and vitamin Bg Table 3 illustrates hatchability response to be limiting in turkey eggs, as hatchability data of 10 folic acid egg injection trials over of eggs injected with biotin and vitamin B(, the production cycle. Regardless of the was 4% higher than that of eggs injected stage of egg production, there were no with vitamin carrier solution or that of significant differences (P > .05) in hatchabil- control (uninjected) eggs (Robel and ity of fertile eggs injected with folic acid, as Christensen, 1987, 1991; Robel, 1991).
FOLIC ACID FOR HATCHABILITY AND POULT WEIGHT
There was a dual beneficial effect in the use of dietary supplemental folic acid on the transfer of folic acid into eggs, which resulted in significantly larger (P < .05) eggs and greater poult weights. It is conjectured that the greater poult weight that resulted from dietary supplemental folic acid (5.51 mg/kg of diet) seems to aid in counterbalancing the deleterious result of intensive selection for rapid growth and feed efficiency in turkeys, which has resulted in smaller poult weight and poorer livability in commercial turkeys. In the commercial turkey industry, the heavier the newly hatched poult, the greater the poult vitality. The present experiment illustrates a positive relationship of heavier poult weight from eggs of hens fed the 5.51 mg folic acid/kg of diet supplemental level over that of the 2.64 mg folic acid/kg supplemental level. The 2.64 mg folic acid/kg supplemental level is generally used in commercial turkey breeder diets. Based on the current market price of folic acid (11.5 C/g), the cost differential between the 5.51 mg folic acid/ kg supplemental level and the 2.64 mg folic acid/kg level is 33 ^/metric ton of turkey breeder diet, which seems to be a relatively small investment for attaining heavier newly hatched poults. Because folic acid is essential for growth in birds, it is not difficult to anticipate a similar benefit with appropriate folic acid supplementation of broiler breeder hen diets. REFERENCES Association of Official Analytical Chemists, 1990. Method 944.12. Pages 1083-1084 in: Official Methods of Analysis. 14th ed. Vol. 2. Association of Official Analytical Chemists, Arlington, VA. Association of Vitamin Chemists, Inc., 1966. Methods of Vitamin Assay. 3rd ed. Interscience Publishers, New York, NY. Coleman, M. A., 1985. Mysterious "B complex" problem: What to do. Broiler Ind. Jan:57-60.
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Couch, J. R., and T. M. Ferguson, 1972. Effect of nutrition on embryonic development in chickens and turkeys. Pages 86-92 in: Cornell Nutrition Conference Feed Manufacturing, Buffalo, NY. Ferguson, T. M., C. H. Whiteside, C. R. Creger, M. L. Jones, R. L. Atkinson, and J. R. Couch, 1961. Bvitamin deficiency in the mature turkey hen. Poultry Sci. 40:1153-1159. Froehli, D. M., 1987. Importance of folic acid in turkey diets explored. Feedstuffs 59(17):26-29. Kratzer, F. H., P. N. Davis, and U. K. Abbott, 1956. The folic acid requirements of turkey breeder hens. Poultry Sci. 35:711-716. Lee, C. D., L. V. Belcher, and D. L. Miller, 1965. Field observations of folacin deficiency in poults. Avian Dis. 9:504-512. Miller, D. L., and S. L. Balloun, 1967. Folacin requirements of turkey breeder hens. Poultry Sci. 46:1502-1508. National Research Council, 1984. Nutrient Requirements of Poultry, 8th rev. ed. National Academy Press, Washington, DC. Robel, E. J., 1983. The effect of age of breeder hen on levels of vitamins and minerals in turkey eggs. Poultry Sci. 62:1751-1756. Robel, E. J., 1991. The value of supplemental biotin for increasing hatchability of turkey eggs. Poultry Sci. 70:1716-1722. Robel, E. J., and V. L. Christensen, 1987. Increasing hatchability of turkey eggs with biotin egg injections. Poultry Sci. 66:1429-1430. Robel, E. J., and V. L. Christensen, 1991. Increasing hatchability of turkey eggs by injecting eggs with pyridoxine. Br. Poult. Sci. 32:501-508. SAS Institute, 1988. SAS/STAT® Guide for Personal Computers. Version 6.03 Edition. SAS Institute Inc., Cary, NC. Schweigert, B. S., H. L. German, P. B. Pearson, and R. M. Sherwood, 1948. Effect of pteroylglutamic acid intake on the performance of turkeys and chickens. J. Nutr. 35:89-102. Sirbu, M., C. Damian, E. Rotunjeanu, and D. Turgu, 1981. Establishing the optimum level of folic acid in feeds for laying hens. Lucr. Stiint. Inst. Cerce. Pentru Nutr. Anim. 9/10:153-160. Snetsinger, D. C, P. E. Waibel, F. J. Siccardi, and B. S. Pomeroy, 1963. The effect of vitamin and antibiotic injections on early poult growth and mortality. Poultry Sci. 42:538-539. Thomason, D. M., A. T. Leighton, Jr., and J. P. Masson, Jr., 1977. Effect of temperature, environment and laying cages on the reproductive performance of turkeys. Poultry Sci. 56:42^434.
ABSTRACT Two experiments were conducted with Large White turkey hens housed individually in cages in a conventional house. In Experiment 1, three dietary treatments were used: an unsupplemented practical corn-soybean meal basal diet; the basal diet supplemented with 2.64 mg folic acid/kg of diet; and the basal diet supplemented with 5.51 mg folic acid/kg of diet. Eggs from hens fed 2.64 mg folic acid/kg of diet were injected with folic acid in 20 injection trials over two production cycles. The response data from dietary supplemental folic acid were analyzed on a production period basis using all of the hens, and on a subset of hens producing eggs in each production period, for hatchability of fertile eggs and poult weight. The response patterns in each case were similar. Incremental dietary supplemental folic acid levels produced a positive linear response pattern on the transfer of folic acid in eggs, but did not result in a hatchability increase over the unsupplemented folic acid basal diet. Hatchability increase did not occur for eggs injected at 25 days of incubation with 19.3 fig folic acid per egg in 20 injection trials over two cycles of production. The results of the study indicate that hatchability is not increased in turkey eggs from hens fed supplemental folic acid or with egg folic acid injections. However, egg and poult weights were significantly increased (P < .05) in eggs containing 6 to 7 mg folic acid/g of dried egg, from hens fed 5.51 mg folic acid/kg of diet. (Key words: folic acid, turkeys, eggs, hatchability, embryonic development) 1993 Poultry Science 72:546-553
(1984) recommendation for turkey breeders is 1 mg folic acid/kg of diet. It is Selection for rapid growth and feed considered unlikely in the commercial efficiency in turkeys may have altered the turkey industry that folic acid could be requirement of folic acid for hatchability limiting for hatchability or embryonic and embryonic development. Amounts development, because commercial turkey and bioavailability of folic acid in feed breeder diets contain supplemental folic ingredients vary and are unreliable acid. This reasoning, however, fails to sources of the vitamin. Levels of sup- consider an important objective, which lies plemental folic acid required for em- in the transport of folic acid to the egg. bryonic survival are controversial, and for Hatchability and poult weight are related rapid embryonic development are unknown (Schweigert et ah, 1948; Kratzer et to folic acid in the egg (Schweigert et al, al., 1956; Ferguson et al., 1961; Lee et ah, 1948; Ferguson et al, 1961; Sirbu et al, 1965; Miller and Balloun, 1967; Couch and 1981). Because of the role of folic acid in Ferguson, 1972; Coleman, 1985; Froehli, cellular development, higher supplemen1987). The National Research Council's tal folic acid levels may be required for rapid embryonic development. Snetsinger et al. (1963) reported the importance of folic acid in poult livability and growth. The current study was conducted to Received for publication August 24, 1992. investigate the effect of supplemental folic Accepted for publication November 4, 1992. INTRODUCTION
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FOLIC ACID FOR HATCHABILITY AND POULT WEIGHT
acid on the transfer of folic acid in the egg as related to hatchability and embryonic development, and of folic acid egg injections on the hatchability of turkey eggs. MATERIALS AND METHODS Experimental Synopsis
Experiment 1 was conducted to determine the effect of dietary supplemental folic acid on the transfer of folic acid into the egg as related to hatchability and poult weight at hatch time. The treatments consisted of an unsupplemented practical corn-soybean meal basal diet (Table 1); the basal diet supplemented with 2.64 mg folic acid/kg of diet; and 5.51 mg folic acid/kg of diet. The response data were averaged across 5 or 6 wk and recorded as three grouped time periods over the fall-winter production cycle. The reproductive data among treatments were analyzed on a time period basis (0 to 5, 6 to 10, and 11 to 16 wk of production) using all hens in each treatment; and across time periods for hatchability and poult weight, using a subset of hens that produced eggs in the three time periods of the production cycle. Eggs from hens fed 2.64 mg folic acid/kg of diet were injected with folic acid in each of the three time periods of production to measure hatchability response. In Experiment 2, eggs from hens fed the 2.64 mg folic acid/kg of diet were injected with folic acid in each of three 3- or 4-wk time periods of production (0 to 3,4 to 7, and 8 to 10 wk) to measure hatchability response. Folic acid in diets and solution for egg injections was analyzed by a commercial laboratory using the Association of Official and Analytical Chemists (1990) method, and folic acid in egg yolk and albumen was analyzed by the method of the Association of Vitamin Chemists, Inc. (1966).
TABLE 1. Composition of the basal diet Ingredients and calculated analyses Ground yellow corn Soybean meal (49% CP) Alfalfa meal (17% CP) Meat and bone meal (50% CP) Soybean oil DL-methionine 1 Vitamin premix Trace mineral premix2 Iodized salt Selenium premix3 Antibiotic premix4 Ground limestone Dicalcium phosphate Total Calculated analyses Protein Calcium Phosphorus (total) ME, kcal/kg
Percentage 62.00 16.90 5.00 5.00 3.00 .10 .50 .10 .30 .10 .50 4.50 2.00 100.00 17.00 2.71 .76 2,960
Witamin premix supplied the following per kilogram of diet: riboflavin, 11 mg; ca-d-pantothenic acid, 37.4 mg; thiamine, 4.4 mg; niacin, 66 mg; d-biotin, .22 mg; pyridoxine, 6.6 mg; choline, 881 mg; vitamin K, 3.0 mg; d-a-tocopherol acetate, 66IU; vitamin Bi2/17.6 jig; vitamin A acetate, 19,823 IU; cholecalciferol, 6,608 IU; ethoxyquin, 150 mg. 2 Trace mineral premix supplied the following in milligrams per kilogram of diet: manganese (sulfate), 100; iron (sulfate, carbonate, oxide), 100; copper (oxide), 10; cobalt (carbonate), 1; iodine (potassium iodide), 3; zinc (sulfate), 100. 3 Selenium premix supplied .1 mg sodium selenite/ kg of diet. 4 Antibiotic premix supplied 110 mg chlortetracycline/kg of diet.
Large White turkey hens, 30 wk of age, were randomly assigned to individual wire-bottomed cages (61.25 cm deep x 45 cm wide x 60 cm high) equipped with cup waterers in a conventional house. Feed and water were available for ad libitum consumption. The control treatment consisted of feeding the basal diet without supplemental folic acid (Table 1), which conExperiment 1 tained .67 mg folic acid/kg based on A total of 120 British United Turkeys of analysis. The other two treatments conAmerica (BUTA) (commercial strain cross) sisted of supplementing the basal diet with two levels of exogenous folic acid:1 2.64 mg and 5.51 mg folic acid/kg, which contained 1 Folic acid, United States Pharmaiopoeia (USP) in a 2.73 and 7.37 mg folic acid/kg of diet based dry premix containing 2,205 >*g folic acid/g of premix; on analysis. The photoperiod was 14 h light: contribution of Hoffmann-La-Roche, Inc., Nutley, NJ 10 h dark. The hens were photostimulated 07110-1199.
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at 30 wk of age and placed on dietary treatments at that time. The hens were divided into three treatment groups containing 40 hens per group. The experiment extended over the fall and winter seasons. Eggs for folic acid assays were collected in each treatment for 4 days in the middle of each time period. Eggs were assayed from individual hens, for a total of 40 replications per treatment. Egg yolk and albumen from each replicate were separated, blended separately, and each was freeze-dried and assayed independently. Hatching variables were recorded at weekly intervals. Weekly data responses were averaged across 5 or 6 wk and recorded as three grouped time periods (0 to 5,6 to 10, and 11 to 16 wk) of production. Statistical Analyses
The data were analyzed separately for each period, whereby all hens were used, including those not laying eggs in all three production periods (Table 2). With data analyzed separately for each time period, a one-factor analysis of variance was done for each response variable, where treatment was the factor. If the response variable did not meet the analysis of variance assumptions, then the appropriate transformation was determined and the analysis was done on the transformed variable. The mean separation was done using pairwise comparisons on the least squares means with the Sidak adjustment to the significant level P to account for the three simultaneous comparisons. Means were not declared statistically different unless the P value was less than .017 for comparisons, which gives an overall experimental comparison rate of P < .05. For hatchability of fertile eggs and poult weight, the data were also analyzed on a restricted subset of hens—the hens that laid eggs in all three production periods (0 to 5,6 to 10, and 11 to 16 wk). This was done as an
2 Folic acid (pteroylglutamic acid) crystalline, Number F-7876, Sigma Chemical Co., St. Louis, MO 63178-9916. 3 Millipore Corp., Bedford, MA 01730. 4 LyphoMed, Inc., Rosemont, IL 60018. 5 Becton Dickinson, Inc., Rutherford, NJ 07070.
attempt to better isolate the folic acid effect, which might presumably be masked in the period by period production analysis. Because the results of the two analyses did not differ, the data are presented on a production period basis in Table 2 (SAS Institute, 1988). Egg Injection Trials and Solution Preparation Environment
Egg injections and folic acid solution preparation were completed under Gold Fluorescent nonultraviolet light in a particle-free environment with filtered high efficiency particulate air. Eggs were collected from hens fed the basal diet containing 2.64 mg supplemental folic acid/kg of diet. The eggs were injected on the 25th day of incubation in each of the three time periods of the production cycle (0 to 5, 6 to 10, and 11 to 16 wk); with three spanned injection trials equally spaced in time occurring from 0 to 5 wk, three from 6 to 10 wk, and four from 11 to 16 wk (Table 3). Folic Acid Solution
The folic acid solution was prepared by forming a mixture of 25 mg folic acid2 in 5 mL absolute ethanol. To the mixture was added 20 mL .01 N sodium hydroxide (reagent grade), and the contents were mixed. To this mixture was added 225 mL deionized water. The solution was mixed at approximately 25 C until its transparency was assured. Glacial acetic acid (reagent grade) was used to adjust the solution to pH 7. The solution was then brought to 250 mL volume with deionized water. A portion of the solution was withdrawn into a 60-mL sterile syringe. The syringe was then equipped with a .22-jim sterile nonpyrogenic filter unit (Millex-GV).3 The solution was then dispensed into 10-mL empty sterilized vials4 from which .2-mL aliquots were withdrawn using a microfine III, 28-gauge sterile needle locked to a 1-mL insulin syringe.5 For the egg injections, .2 mL of folic acid solution was injected per egg at 25 days of incubation, using a separate syringe equipped with needle for each individual egg. The folic acid solution contained 19.3 /*g folic acid/.2 mL of solution based on analysis.
231 ± 3 * 244 ± 5 a 225 ± 6 b
219 ± 4 223 ± 6 207 ± 6
185+3 183 ± 6 174 ± 4
01 2.64 5.51
11 to 16 w k O 1 2.64 5.51
75.6 76.5 77.9 79.4 79.6 82.0 81.4 81.0 85.7
± + ± ±
.6* .9 b 1.2b 1.0"
± .7*
± .6* ± .6b
.6b
(g) 45.4 47.5 48.2 51.7 53.1 54.9 53.8 53.2 58.4
+ ± ± ± + ± ± ± +
Poult weight .4b .5" .5 a .5b .5b .6= .8b 1.1b .8" 15.7 16.9 16.8 8.9 7.6 9.0
± ± ± + ± ±
(no.) 22.1 ± 22.0 ± 21.4 ± .5 .7 .8 1.0 1.0 1.0 1.7 1.5 1.6
Eggs per hen + ± + ± + ±
2.2 2.3 3.2 3.6 2.5 3.5
68.2 ± 7.3 71.3 + 5.6 83.0 ± 5.4
83.0 79.2 76.9 83.2 83.4 82.2
Hatchability of fertile eggs
2.1 2.3 1.3
0. .2 1.3 1 1
(%) .8 ± .9 ± 0. ±
7 days
Em
1
- Means + SE in a column within each production period with no common superscripts differ signific Basal diet (without supplemental folic acid) contained .67 mg folic acid/kg of diet based on analysis.
a c
6 to 10 wk
0 to 5 wk
(mg/kg) 01 2.64 5.51
Period
Feed consumption Supplemental per hen Egg folic acid per day weight
TABLE 2. Performance of turkey hens fed a basal diet supplemented with two levels of of the production cycle, Experiment 1
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ROBEL TABLE 3. Effect of folic acid egg injections on hatchability of fertile eggs, Experiment 1 Weeks of production1
Treatment
0 to 5*
6 to 103
11 to 164
Control (uninjected) Injected Folic acid in carrier solution Carrier solution Analysis of variance
83.6 ± 6.6
(%) 88.1 ± 2.9
87.0 ± 3.5
87.3 ± 5.5 78.9 ± 9.9 NS
89.1 ± 2.3 88.0 ± 2.3 NS
92.6 ± 3.5 88.6 ± 1.5 NS
iFall-winter season production cycle. 2 Means ±SE within the column represent three equally spaced injection trials (27 eggs set per treatment per trial). 3 Means ± SE within the column represent three equally spaced injection trials (22 eggs set per treatment per trial). 4 Means ± SE within the column represent four equally spaced injection trials (24 eggs set per treatment per trial).
Aseptic Egg Injections
Egg injections were performed under a sanitized clean-room particle-free environment using filtered high efficiency particulate air, and personnel were aseptically equipped. A 2% iodine tincture was applied to the injection site on the large end of the egg. A very slight indenture was made in the swabbed shell area, using a small, sharp, sterile steel punch. The force used to make the indenture was of a minor degree to avoid the formation of hairline cracks in the shell. The punch site exactly accommodated the microfine III, 28-gauge sterile needle that was locked to a sterile 1-mL insulin syringe. The needle was carefully inserted through the indenture site to a depth of 6 to 8 mm, and .2 mL of the folic acid solution was injected over the inner eggshell membrane. The puncture was sealed with a small drop of fast-drying Duco cement.6 Egg Injection Treatments
The treatments were the following: eggs injected with .2 mL of vitamin carrier solution containing 19.3 fig folic acid based on analysis, eggs injected with .2 mL of the vitamin carrier solution, and control (uninjected) eggs.
Statistical Analysis of Egg Injection Trials
Ten egg injection trials were completed, with three to four trials per period. Each period was analyzed separately. A onefactor analysis of variance with treatment as the factor was run on the percentage hatchability data. The data were also analyzed as a weighted least squares analysis of variance using categorical modeling on the frequencies of the response variable, hatchability (SAS Institute, 1988). Experiment 2. Egg Injection Trials
Eggs were collected over the springsummer seasons from a flock of 120 BUTA Large White turkey hens fed the basal diet supplemented with 2.64 mg folic acid/kg— the same diet used for egg injections of Experiment 1. The eggs were injected at 25 days of incubation in each of three time periods of the production cycle (0 to 3,4 to 7, and 8 to 10 wk); with three injection trials occurring at equal intervals within the 0- to 3-wk period, three in the 4- to 7-wk period, and four in the 8- to 10-wk period (Table 4). RESULTS AND DISCUSSION Experimental Synopsis
6
Devcon Corp., Wood Dale, IL 60191.
The reproductive data in Experiment 1 were analyzed within a single time period
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FOLIC ACID FOR HATCHABILITY AND POULT WEIGHT TABLE 4. Effect of folic acid injections on hatchability of fertile eggs, Experiment 2 Weeks of production1 Treatment
0 to 32
4 to 73
8 to 104
Control (uninjected) Injected Folic acid in carrier solution Carrier solution Analysis of variance
88.6 ± 1.7
(%) 90.2 ± 2.8
87.4 ± 3.2
90.3 ±3.1 93.0 ± 2.2
89.3 ±2.6 87.4 ± 1.7
92.4 ± 4.2 90.5 ± 5.1
NS
NS
NS
Spring-summer season production cycle. Means ±SE within the column represent three equally spaced injection trials (37 eggs per treatment per trial). 3 Means ±SE within the column represent three equally spaced injection trials (36 eggs per treatment per trial). 4 Means ± SE within the column represent four equally spaced injection trials (24 eggs per treatment per trial). 2
of production (Table 2). With the analysis within each single time period of production (0 to 5,6 to 10, and 11 to 16 wk), dietary treatment comparisons were based on the relationship of dietary folic acid levels with reproductive response patterns. This system of analysis has the advantage of not being confounded with feed consumption, which changed with maternal age over the three periods of production in the production cycle. Reproductive response was, therefore, not affected by dietary intakes because the relationship of dietary intakes among treatments within each time period of production was similar. Data presented in this fashion can be of practical value to the commercial turkey industry, which can accommodate dietary folic acid levels, but cannot respond to physiological implications associated with feed consumption and depositions of folic acid in eggs. Analysis of the subset of hens that laid eggs in each production period was also completed for hatchability and poult weight. This analysis was done to investigate the effect of the treatment on hatchability and poult weight over time. Hatchability of fertile eggs and poult weight response data from dietary supplemental folic acid was similar in each case, when analyzed on a production period basis using all of the hens, and on a subset of hens producing eggs in each production period. The experimental data were based on caged hens, and not on hens from the floor, as is industry practice. Thomason et ah (1977) observed significantly lower (P < .05)
hatchability of caged hens as compared with hens housed in floor pens. Experiment 1
Feed consumption for the 5.51 mg folic acid/kg of diet treatment was the lowest in the three production periods, but was statistically significant (P < .05) only in the 0- to 5-wk period. This result does not have practical importance in relation to the reproductive parameters investigated. There were no significant differences (P < .05) in numbers of eggs per hen, hatchability of fertile eggs, early embryonic deaths (< 7 days incubation) or late embryonic deaths (> 7 days incubation) among dietary treatments in each production period (Table 2). Early embryonic mortality was low in all treatments in each production period. In this study, hatchability did not increase as egg levels of folic acid increased with incremental levels of supplemental folic acid over the basal dietary level, based on analysis (.67 mg folic acid/kg). Folic acid in the basal diet originated entirely from basic dietary ingredients from which the bioavailability of folic acid varies widely, and the analyzed level of folic acid was only 67% of the National Research Council's (1984) recommended level for turkey breeder hens. There appears, therefore, to be ample margin for hatchability increase with dietary supplemental folic acid treatments over the basal dietary treatment; however, this did not occur. Although the numerical average hatchability that in-
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creased across treatments from 11 to 16 wk seemed promising, it is atypical. The overall estimate of variability and use of the conservative Sidak method precluded from finding statistically significant differences among treatments. Egg weight increased within each treatment as production periods advanced. However, there was an additional treatment effect due to supplemental folic acid, which caused significant increases (P < .05) in egg and poult weights. The 5.51 mg folic acid/kg of diet treatment induced a 2 /xg folic acid/g dried egg increase, with resultant increases of 3.28% in poult weight and 2.93% in egg weight from 6 to 10 wk; and 8.90 and 5.37% increases, respectively, from 11 to 16 wk, in contrast to the 2.64 mg folic acid/kg treatment. The 8.9% poult weight increase with the supplemental folic acid treatment (5.51 mg folic acid/kg of diet), resulted in the deposition of 144 /*g folic acid/82 g of egg, in contrast to the 104 fig folic acid per egg by hens fed 2.64 mg folic acid/kg of diet. This suggests that greater egg weight provided more nutrients. It is possible that greater folic acid transfer to the egg advanced nutrient utilization by extending folic acid functions in intermediary amino acid metabolism and biosynthesis of nucleotides for greater embryonic cellular development.
compared with eggs injected with the vitamin carrier solution and control (uninjected) eggs. Injection of the folic acid carrier solution did not increase hatchability over the control (uninjected) eggs. Experiment 2
Eggs used in these injection trials were from hens fed the 2.64 mg supplemental folic acid/kg of diet. Table 4 illustrates treatment means for 10 folic acid egg injection trials over the production cycle. There were no significant differences (P > .05) in hatchability of fertile eggs injected with folic acid as compared with eggs injected with the vitamin carrier solution and control (uninjected) eggs. Injection of the folic acid carrier solution did not increase hatchability over control (uninjected) eggs. Folic acid levels of eggs from each of the dietary treatments were quite stable with maternal age (Table 2), and are not expected to have influenced the hatchability response data to folic acid egg injections. Eggs from Large White turkey hens, under similar conditions of housing and diet (2.1 mg folic acid/kg based on analysis) as in Experiment 1, also had relatively little folic acid change in eggs with maternal age (Robel, 1983). In conclusion, mere were no statistically significant differences in average hatchabilOver 99% of folic acid depositions in the ity of turkey eggs from hens fed the egg occurred in egg yolk. There was a practical corn-soybean meal basal diet with significant increase (P < .05) in folic acid of or without supplemental folic acid. The egg yolk with each increment of dietary bioavailability of folic acid in the basal diet folic acid (Table 2). Folic acid depositions in is expected to be less than .67 m g / k g based egg yolk from the basal diet and the basal on analysis, and is lower than the National diet containing supplemental folic acid Research Council's (1984) recommended 1 levels of 2.64 and 5.51 mg folic acid/kg of m g / k g level for breeder turkeys. This diet, increased linearly over the production suggests that 1 mg folic acid/kg of diet cycle, averaging 3.2, 5.0, and 6.7 /*g of folic recommended for turkey breeders is excesacid/g of dried egg, respectively, per sive. From the results, it is evident that folic treatment based on analysis. acid egg injections were instrumental in Eggs from hens fed the 2.64 mg sup- revealing folic acid to be nonlimiting in plemental folic acid/kg of diet were in- turkey eggs for hatchability. The egg injecjected with folic acid to determine whether tion results lend additional support to the folic acid in eggs was limiting on the basis of results of previous egg injection investigahatchability response data. The data of tions that determined biotin and vitamin Bg Table 3 illustrates hatchability response to be limiting in turkey eggs, as hatchability data of 10 folic acid egg injection trials over of eggs injected with biotin and vitamin B(, the production cycle. Regardless of the was 4% higher than that of eggs injected stage of egg production, there were no with vitamin carrier solution or that of significant differences (P > .05) in hatchabil- control (uninjected) eggs (Robel and ity of fertile eggs injected with folic acid, as Christensen, 1987, 1991; Robel, 1991).
FOLIC ACID FOR HATCHABILITY AND POULT WEIGHT
There was a dual beneficial effect in the use of dietary supplemental folic acid on the transfer of folic acid into eggs, which resulted in significantly larger (P < .05) eggs and greater poult weights. It is conjectured that the greater poult weight that resulted from dietary supplemental folic acid (5.51 mg/kg of diet) seems to aid in counterbalancing the deleterious result of intensive selection for rapid growth and feed efficiency in turkeys, which has resulted in smaller poult weight and poorer livability in commercial turkeys. In the commercial turkey industry, the heavier the newly hatched poult, the greater the poult vitality. The present experiment illustrates a positive relationship of heavier poult weight from eggs of hens fed the 5.51 mg folic acid/kg of diet supplemental level over that of the 2.64 mg folic acid/kg supplemental level. The 2.64 mg folic acid/kg supplemental level is generally used in commercial turkey breeder diets. Based on the current market price of folic acid (11.5 C/g), the cost differential between the 5.51 mg folic acid/ kg supplemental level and the 2.64 mg folic acid/kg level is 33 ^/metric ton of turkey breeder diet, which seems to be a relatively small investment for attaining heavier newly hatched poults. Because folic acid is essential for growth in birds, it is not difficult to anticipate a similar benefit with appropriate folic acid supplementation of broiler breeder hen diets. REFERENCES Association of Official Analytical Chemists, 1990. Method 944.12. Pages 1083-1084 in: Official Methods of Analysis. 14th ed. Vol. 2. Association of Official Analytical Chemists, Arlington, VA. Association of Vitamin Chemists, Inc., 1966. Methods of Vitamin Assay. 3rd ed. Interscience Publishers, New York, NY. Coleman, M. A., 1985. Mysterious "B complex" problem: What to do. Broiler Ind. Jan:57-60.
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Couch, J. R., and T. M. Ferguson, 1972. Effect of nutrition on embryonic development in chickens and turkeys. Pages 86-92 in: Cornell Nutrition Conference Feed Manufacturing, Buffalo, NY. Ferguson, T. M., C. H. Whiteside, C. R. Creger, M. L. Jones, R. L. Atkinson, and J. R. Couch, 1961. Bvitamin deficiency in the mature turkey hen. Poultry Sci. 40:1153-1159. Froehli, D. M., 1987. Importance of folic acid in turkey diets explored. Feedstuffs 59(17):26-29. Kratzer, F. H., P. N. Davis, and U. K. Abbott, 1956. The folic acid requirements of turkey breeder hens. Poultry Sci. 35:711-716. Lee, C. D., L. V. Belcher, and D. L. Miller, 1965. Field observations of folacin deficiency in poults. Avian Dis. 9:504-512. Miller, D. L., and S. L. Balloun, 1967. Folacin requirements of turkey breeder hens. Poultry Sci. 46:1502-1508. National Research Council, 1984. Nutrient Requirements of Poultry, 8th rev. ed. National Academy Press, Washington, DC. Robel, E. J., 1983. The effect of age of breeder hen on levels of vitamins and minerals in turkey eggs. Poultry Sci. 62:1751-1756. Robel, E. J., 1991. The value of supplemental biotin for increasing hatchability of turkey eggs. Poultry Sci. 70:1716-1722. Robel, E. J., and V. L. Christensen, 1987. Increasing hatchability of turkey eggs with biotin egg injections. Poultry Sci. 66:1429-1430. Robel, E. J., and V. L. Christensen, 1991. Increasing hatchability of turkey eggs by injecting eggs with pyridoxine. Br. Poult. Sci. 32:501-508. SAS Institute, 1988. SAS/STAT® Guide for Personal Computers. Version 6.03 Edition. SAS Institute Inc., Cary, NC. Schweigert, B. S., H. L. German, P. B. Pearson, and R. M. Sherwood, 1948. Effect of pteroylglutamic acid intake on the performance of turkeys and chickens. J. Nutr. 35:89-102. Sirbu, M., C. Damian, E. Rotunjeanu, and D. Turgu, 1981. Establishing the optimum level of folic acid in feeds for laying hens. Lucr. Stiint. Inst. Cerce. Pentru Nutr. Anim. 9/10:153-160. Snetsinger, D. C, P. E. Waibel, F. J. Siccardi, and B. S. Pomeroy, 1963. The effect of vitamin and antibiotic injections on early poult growth and mortality. Poultry Sci. 42:538-539. Thomason, D. M., A. T. Leighton, Jr., and J. P. Masson, Jr., 1977. Effect of temperature, environment and laying cages on the reproductive performance of turkeys. Poultry Sci. 56:42^434.