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Vitamin Profiles of Eggs as Indicators of Nutritional Status in the Laying Hen: Vitamin B12 Study1,2
Vitamin Profiles of Eggs as Indicators of Nutritional Status in the Laying Hen: Vitamin B12 Study1'2 MICHAEL W. SQUIRES and EDWARD C. NABER3 Department of Poultry Science, The Ohio State University, Columbus, Ohio 43210
1992 Poultry Science 71:2075-2082
conscious consumer. One such technique to assess the vitamin status of the laying New diagnostic techniques to assess hen might be through analysis of her eggs. laying flock vitamin status are needed due In a review of literature, Naber (1979) to dated requirement recommendations suggested that egg riboflavin, vitamin A, that may not apply to modern strains of and vitamin B12 could be markedly inlaying hens and the increasing demand for fluenced by dietary changes. Establishhigher quality eggs by the health- ment of critical egg vitamin B12 concentrations could be used to determine adequacy of dietary supplementation giving a direct measure of vitamin bioavailaReceived for publication June 3, 1992. bility that feed analysis does not yield. Accepted for publication August 28, 1992. Salaries and research support provided by state Data on the relationship between laying and federal funds appropriated to the Ohio Agricul- hen diet and egg vitamin B12 might be tural Research and Development Center, The Ohio used to establish egg quality standards State University. Manuscript Number 160-92. and diet to egg vitamin transfer costs. 2 Partial funding for this project was provided by a The vitamin B12 requirement for laying grant-in-aid sponsored by Sigma Xi. 3 To whom correspondence should be addressed. and breeding hens is 4 ug/kg of diet (National Research Council, 1984). Some INTRODUCTION
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ABSTRACT Hens of a type used for egg production were fed a corn and soybean meal diet supplemented with no vitamin B12 or with vitamin B12 levels to provide one, two, or four times the National Research Council (1984) breeding hen requirement of 4 ug/kg diet for 27 wk. All hens were placed on a recovery diet containing one and one-half times the requirement level of vitamin B12 from Weeks 27 through 30. Egg yolk vitamin B12 concentrations were determined frequently by radioisotope dilution analysis. Egg production records were kept continuously, and eggshell thickness, egg weight, hatchability of eggs, and hen body weights were measured at selected times. Although egg yolk vitamin B12 concentrations were high at the outset, they decreased markedly in 2 wk from hens fed the two lowest dietary levels. After 12 wk on the diets, egg concentrations of vitamin B12 stabilized and were proportional to the amount of vitamin added to the diet. Egg concentrations of vitamin B12 between 1.3 and 2.6 ug/100 g yolk appeared to be needed to support maximum hatchability and egg weight. Egg production was reduced after 12 wk on the diets in the hens fed the two lowest vitamin B12 levels. As vitamin B12 level increased, shell thickness decreased and egg weight, hen weight, and hatchability increased. Maximum egg production, egg weight, hen weight, and hatchability were obtained when the diet contained 8.0 M-g/kg of vitamin B12. Egg yolk vitamin B12 concentrations respond rapidly to dietary changes in the level of this vitamin and are indicative of the vitamin B12 status of the hen. (Key words: vitamin B12/ layers, nutritional status, egg yolk content, hatchability)
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ern requirement for breeding hens as being between 3.3 to 4.4 ug vitamin B12/ kg of diet using microbiologicial methods for vitamin analysis. Denton et al. (1954) showed that egg vitamin B12 concentrations decreased significantly 2 wk after supplementation was discontinued. This suggests that only moderate amounts of vitamin B12 are stored in the hen, although others have suggested that several months may be needed to deplete body reserves (Scott et al, 1982). The working hypothesis for the current study was that egg vitamin B12 concentrations can be used to assess the vitamin B12 status of the laying hen and the sufficiency of her diet for egg hatchability. The objectives were as follows: 1) to determine the effects of feeding practical rations containing graded levels of vitamin B12 on egg vitamin B12 content and important production variables of modern strains of layers during selected periods of time; 2) to establish the relationship between egg vitamin concentrations, diet vitamin levels, and decreases in important production variables; and 3) to determine minimum critical egg vitamin B12 concentrations needed for maximum reproductive function. MATERIALS AND METHODS Egg Yolk Vitamin B12 Extraction
The procedure used was an adaptation of that described by Casey et al. (1982). Sample preparation was done in subdued light. Two to 5 g of thawed egg yolk were weighed into a Virtis cup.4 Forty milliliters of extraction solvent (13 g anhydrous sodium phosphate, 12 g citric acid, and 10 g sodium metabisulfite/L of distilled water) were added to the cup. The sample was homogenized in a Virtis model Super 30 homogenizer4 equipped with metal blades for 15 s. The sample was poured into a 100-mL glass-stoppered volumetric flask. The cup was rinsed with 10 mL of extraction solvent and poured into the 100-mL flask. The samples were digested in a Barn4 The Virtis Co., Inc., Gardner, NY 12525. 5 2 5 Barnstead Thermoline Corp., Subsidiary of Sybron stead steam autoclave at 1.1 kg/cm for 10 min. After pressure had returned to zero, Corp., Dubuque, IA 52001.
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vitamin B12 can be obtained from microbial synthesis, but diets are usually in need of supplementation. Embryonic deficiency signs include edema, hemorrhages, perosis, beak shortening, myotrophy of legs, and fattiness of liver, heart, and kidneys (Mushett and Orr, 1949; Olcese et al, 1950; Ferguson and Couch, 1954). Chicks hatched without adequate stores may have increased mortality along with depressed growth, poor feathering, enlarged thyroid glands, and gizzard lesions. Hammond (1942) first noted an unidentified vitamin-like factor in cow manure that increased weight gain of chicks fed poor quality diets. He incorrectly assumed that the factor was riboflavin. Rubin and Bird (1946a,b) showed that this growth factor in cow manure was not any known nutrient and was transmitted through the egg to the chick. McGinnis et al. (1948) verified that vitamin B12 was the animal growth factor found in manure and that it was needed for proper amino acid utilization. Lillie et al. (1949) injected vitamin B12 into eggs and reported that hatchability of eggs from hens deficient in vitamin B12 increased with injection of the vitamin. This was the first evidence of a role for vitamin B12 in hatchability. Skinner et al. (1951) first noted that hens receiving diets low in vitamin B12 laid eggs that were smaller. Yacowitz et al. (1952a) suggested that chicks have the ability to store excess vitamin B12 in their liver, kidney, and pancreas. Milligan et al. (1952) demonstrated that increases in maternal dietary vitamin B12 caused increases in egg levels using both a chick and a microbiological assay. Efficiency of transmission of vitamin B12 to the egg, however, was variable and not related to dietary vitamin level. Yacowitz et al. (1952b) using a microbial assay estimated that 2.5 ng of vitamin Bi2/g of yolk is needed to ensure hatchability. This value seems to be very low when compared with more recent data. Peterson et al. (1953) established the mod-
EGGS INDICATING VITAMIN B12 STATUS IN THE LAYING HEN
Feed Vitamin B12 Extraction
The same procedure was used for feed as described for egg yolk. Some additional preparation of the samples was required however, prior to analysis. The feed was finely ground in a Brinkman Instruments Model ZM1 grinder7 equipped with a .2mm screen. Radioisotope Dilution Assay for Vitamin B12
Becton Dickinson vitamin B12 57Co radioassay kits8 (Catalog Number 262315) were used for assay. A total of 16 standard tubes and 48 sample tubes per assay run were employed. The kit reagents were: 1) A 5% dithiothreitol solution with stabilizer, which was mixed with vitamin B12 tracer solution containing [57Co] vitamin B12 in borate buffer, human serum albumen, dextran, potassium cyanide, nonintrinsic factor blocking agent, dye, and preservatives. This vitamin B12 tracer solution was stable for 1 wk under refrigeration. 2) A vitamin B12 binder containing porcine intrinsic factor, human serum albumen, dextran, and preservatives, which was mixed with binder diluent containing sodium borate, sodium chloride, dye, and preservative.
6
Fisher Scientific, Pittsburgh, PA 15219. iBrinkman Instruments, Inc., Westbury, NY 11590. 8 Becton Dickinson Immunodiagnostics, Orangeburg, NY 10962. 911100138 Scientific, Swedesboro, NJ 08085. ^International Equipment Co., Division of Damon Corp., Needham Heights, MA 02194. "Beckman Instruments, Inc., Fullerton, CA 92634.
The vitamin B12 binder solution was stable for 8 wk under refrigeration. 3) Vitamin B12 standards in sodium borate, sodium chloride, dye, and preservative. 4) A dextran charcoal suspension containing charcoal, dextran, sodium chloride, sodium borate, and a pelleting aid. All samples and standards were run in duplicate in 12 x 75 mm polypropylene tubes. Two hundred microliters of sample filtrate was added to each sample tube and the same amount of the standards was added to Tubes 3 through 16 but not the first two tubes. The standard tubes contained 0, 100, 200, 400, 1,000, and 2,000 pg vitamin Bi2/mL. One milliliter of vitamin B12 tracer solution was added to each tube. The first two standard tubes, which provide data on total radioactivity counts, were pulled from the assay and held in the dark while all of the other tubes were mixed gently. One hundred microliters of the binder solution was added to standard Tube 5 through 16 and all of the sample tubes. After mixing gently by hand, both the standard and sample tubes were incubated at room temperature for 30 min in the dark. Four-tenths milliliter of dextran charcoal suspension was added to the tubes with a Cornwall syringe.9 All samples and standards including Tubes 1 and 2 were incubated in the dark at room temperature for 10 min. The sample and standard tubes were spun in an International PR-6 centrifuge™ equipped with a Number 276 rotor and Number 353 sample cup for 10 min at 1,000 x g (2,000 rpm) at 10 C. The clear supernatant was decanted into 6-mL Beckman mini POLY-Q vials containing 4 mL of Beckman Ready Sol MP or CP scintillation cocktail.11 This step was performed with plastic gloves and care was taken not to decant any of the charcoal from the sample tubes into the vials. The vials were capped and shaken vigorously and the sample tubes containing charcoal were discarded. Scintillation counting was carried out with a Beckman LS-5801 liquid scintillation spectrometer.11 This was possible because the 57Co gamma emissions resulted in the release of Auger and conversion electrons that caused scintillations that were easily counted. A radioimmunoassay program
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the flasks were removed from the autoclave and allowed to cool in the dark at room temperature. The samples were brought to 100 mL volume with phosphate buffer (12.1 g anhydrous potassium phosphate and 18.9 g anhydrous sodium phosphate/L of distilled water). The flasks were stoppered and mixed by inversion 10 times. The samples were filtered through Whatman Number 2 filter paper6 into 125-mL Erlenmeyer flasks to obtain 75 mL of filtrate. The filtrate was mixed by swirling, and a 4-mL aliquot was removed and stored in a small vial under refrigeration.
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SQUIRES AND NABER TABLE 1. Composition of vitamin B12 basal and recovery diets
Ingredients and analysis
55.5 5.5
Recovery diet
26.5 2.6 8.4 .74 .04 .50 .20
53.4 10.2 2.5 20.1 3.0 9.0 1.0 .10 .50 .20
2,800 17.7 .89 .32 .55 3.3 .50
2,787 15.6 .75 .34 .56 3.6 .52
iAmount supplemented per kilogram of diet: Mn, 70 mg; Zn, 100 mg; iodized NaCl, 2400 mg. 2 Amount supplemented per kilogram of diet: vitamin A, 2,000 IU; cholecalciferol, 1,000 ICU; vitamin E, 20IU; vitamin K, 1 mg; pantothenic acid, 15 mg; niacin, 15 mg; riboflavin, 1.75 mg; ethoxyquin (66%), 188 mg; Larvadex, 500 mg; and variable amounts of vitamin B 12 (see Table 2).
resident in the liquid scintillation spectrometer was used to calculate and tabulate the results. Design of the Experiment Two hundred Single Comb White Leghorn pullets12 were divided into four treatment groups of 50 birds each at 28 wk of age. The pullets received a basal diet that was formulated to contain the National Research Council (1984) requirements of all nutrients except vitamin B12. This basal diet is shown in Table 1. Vitamin B12 (.1% trituration of cyanocobalamin in mannitol)13 was used for dietary supplementation. The four treatment levels of vitamin B12 dietary supplementation and estimated requirement levels for hatchability are shown in Table 2. Assay of the diets for vitamin B12 agreed closely with the calculated values. Birds on all four treatments received ad libitum access to feed and water. Water was supplied by continuous flow in a
12
H and N International, Redmond, WA 98052. "United States Biochemical, Cleveland, OH 44122.
trough that was regularly cleaned to avoid microbial growth. The pullets were housed, one per cage, in an environmentally controlled poultry house. Sixteen hours of light were provided daily from 0500 until 2100 h. The experimental diets were fed for 27 wk. All four experimental groups were placed on a recovery diet containing 6.0 ug/kg of vitamin B12 for 3 wk after termination of the vitamin B12 treatments. Hen-day egg production on individual birds was calculated at 4-wk intervals for the duration of the experiment. Eggs were collected on 6 consecutive days during the 16th and 27th wk and weighed. Shell thickness of 100 air-dried eggshells per treatment were determined during the 22nd wk. All hens were weighed during the 1st and 27th wk of the experiment. Hatchability of eggs from 25 hens in each treatment was determined by setting eggs from the 20th and 21st wk. The hens were artificially inseminated 1 wk prior to each hatchability study with pooled semen from Athens Canadian randombred males. All unhatched eggs from the two settings were broken out to determine fertility or approximate day of death during incubation. Embryonic mortality was recorded by
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Ground yellow corn Ground wheat Alfalfa meal (17% CP) Soybean meal (44% CP) Hydrolyzed fat (animal and vegetable) Limestone (37% Ca) Dicalcium phosphate DL-methionine Mineral mix1 Vitamin mix2 Calculated analysis ME, kcal/kg Protein Lysine Methionine Methionine and cystine Calcium Total phosphorus
Basal diet
EGGS INDICATING VITAMIN B12 STATUS IN THE LAYING HEN
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TABLE 2. Dietary vitamin Bj 2 supplements to experimental and recovery diet ]Experinvental
Variable
.5
Vitamin Bi 2 supplement, Hg/kg diet Calculated total dietary vitamin B^, ug/kg diet Percentage of National Research Council requirement for breeding hens1 1
4.0
0 .5 12
3.5 4.0 100
diets
8.0 7.5 8.0 200
16.0
Recovery diet 6.0
15.5 16.0 400
5.5 6.0 150
The National Research Council (1984) requirement is 4.0 ug/kg of diet.
treatment and time period were analyzed. Vitamin B12 was corrected for recovery and reported as micrograms of vitamin B12 per 100 g of yolk. Statistical Analysis The data was subjected to a two-way ANOVA (Snedecor and Cochran, 1980) by the General Linear Models procedure using SAS® software (SAS Institute, 1982) and calculated as the mean and SEM for treatment, time, and treatment by time interaction. Statistical comparisons among treatment means at each time interval were made by the Least Squares Mean procedure (repeated t test) using SAS® software (SAS Institute, 1982) at a probability level of P < .05.
TABLE 3. Changes in egg yolk vitamin B12 over time for hens fed four dietary vitamin By levels1 Treatments (ug vitamin B^/kg of diet) Time (wk) 0 1 2 4 6 8 12 16 20 24 27 1 3 a_d
.5
4.0
10.1 ± .81 6.3 ± .41b 3.1 ± .17<* 2.0 ± .12d 1.7 ± .25= 1.1 ± .15c
11.2 6.5 3.6 3.2 2.7 1.5 1.1 1.3 1.5 1.2
8.0
16.0
fur/if
.3 ± .07°"
.5 ± .05d .4 ± .26= .3 ± .07°" 1.9 ± .40= 2.8 ± .07=
± ± ± ± ± ± ± ± ± ±
1.4 .43b .27<: .56= .25= .19= .40= .17= .26b .30=
2.4 ± .40b= 2.4 ± .07d
9.9 ± 1.1 6.9 ± .48b 5.1 ± .24b 4.1 ± .21b 4.0 ± .25b 2.4 ± .33b 2.8 ± .34b 2.4 ± .31b 2.5 ± .26b 2.7 ± .33b recovery diet 3.6 ± .40* 3.2 ± .07b
9.5 8.8 7.1 5.9 6.8 4.0 4.7 4.7 4.8 4.9
± ± ± ± ± ± ± ± ± ±
.63 .95" .34' .25' .25* .48" .42^ .48» .26* .38*
4.7 ± .40* 4.0 ± .07»
Means within rows with no common superscripts differ significantly (P < .05). Data reported as X ± SE (n = two to six pooled samples of four eggs each for each treatment and time observation). a
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dividing the 21-day incubation period into trimesters and calculating percentage mortality in each trimester. Dead embryos were examined for abnormalities and defects. Eggs were collected from each treatment during Weeks 0,1,2,4,6,8,12,16,20,24,28, and 30 for later isotope dilution analysis of vitamin Bj2 (described above). Twenty-four eggs per treatment were gathered during 1 day of the designated week and stored in an egg cooler at 13 C. Yolks were harvested by breaking out the eggs under subdued light within 3 days of laying. Excess albumen was removed from the yolks by rolling them on paper towels. Pooled yolk samples of four eggs each were placed into six plastic containers. These samples were placed in light-proof boxes and frozen at -20 C. Two to three yolk samples per
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TABLE 4. Changes in hen-day production of hens over time fed four dietary vitamin Bj2 levels1 Treatments (ng vitamin B 12 /kg of diet) Time (wk) 0 to 5 to 9 to 13 to 17 to 21 to 24 to
.5
8.0
16.0
93.0 ± 1.8 88.5 ± 1.8 88.8 ± 1.8 81.5 ± 1.8" 75.8 ± 1.8" 77.0 ± 1.8» 78.0 ± 2.1" to recovery diet 81.3 ± 2.1
92.8 89.8 85.3 85.3 75.3 77.8 78.3
4.0 f%l -
4 8 12 16 20 24 27
0 to 3
95.0 89.8 86.8 80.5 72.3 69.3 70.7
± ± ± ± ± ± ±
1.8 1.8 1.8 1.8*" 1.8* 1.8b 2.1b
77.0 ± 2.1
91.0 86.8 84.0 75.5 70.0 72.8 76.3
± ± ± ± ± ± ±
1.8 1.8 1.8 1.8b 1.8b 1.8ab 2.1ab
78.3 ± 2.1
\'°)
± 1.8 ± 1.8 ± 1.8 ± 1.8» ± 1.8" ± 1.8» ± 2.1a
81.3 ± 2.1
Means within rows with no common superscripts differ significantly (P < .05). !Data reported as x ± SE (n = three to four pooled observations of 7 days each for each treatment and time).
RESULTS
Differences in egg vitamin B12 content and hen-day egg production were significant (P < .05) for both treatment and time (Tables 3 and 4). The results presented here emphasize the interaction of treatment at various time intervals during the study. The data in Table 3 show that the vitamin B12 content of egg yolks was high at the outset of the experiment, indicating a substantial intake of the vitamin prior to the experiment. Significant differences (P < .05) in egg yolk vitamin B12 content appeared after 1 wk (Table 3). At this time hens fed the 16 ug/kg dietary treatment had significantly higher egg vitamin concentrations than eggs from hens on the other three treatments due to reductions in vitamin content of eggs from hens at lower treatment levels. At 2 wk, eggs from hens on all four dietary treatments were significantly different (P < .05) from each other and were proportional to dietary vitamin levels. At 12 wk, the vitamin B12 egg concentrations from hens fed the .5 ug/kg dietary treatment were 27% of those receiving the 4 ug/kg dietary treatment. The egg vitamin B12 concentrations in the latter treatment were 39% of those fed the 8 ug/kg treatment. The egg vitamin B12 content of the hens fed the 8 ug/kg dietary treatment was 60% of that from hens fed the 16 ug/kg treatment. This pattern of egg yolk vitamin B12 concentrations continued for the duration of the experiment.
Egg vitamin B12 content from hens fed the two lowest levels of vitamin B12 were still significantly lower than those that had received the two highest levels following a 3-wk recovery period (Table 3). However, egg vitamin B12 concentrations from hens fed the .5 and 4 ug/kg dietary treatments had returned to 87 and 75% of those from hens on the 8 ug/kg treatment after the 3-wk recovery. Much of the increase in egg yolk vitamin B12 occurred after 1st wk on the recovery diet. This is approximately the same time period needed for depletion of egg vitamin B12. Egg yolk vitamin B12 content stabilized after Week 12 in all of the treatments. The average egg yolk vitamin B12 content of the eggs for Weeks 12 through 24 were .4 ug/100 g yolk for the .5 ug/kg dietary treatment, 1.3 ug/100 g for the 4 ug/kg treatment, 2.6 ug/100 g for the 8 ug/kg treatment, and 4.8 ug/100 g of yolk for the 16 ug/kg treatment. A significant depression (P < .05) in egg production of hens fed the 4 ug/kg dietary treatment was apparent at Weeks 13 to 20 compared with the two higher dietary vitamin B12 levels (Table 4). At Weeks 21 to 27, the egg production of hens fed the .5 ug vitamin B^/kg of diet treatment was significantly depressed (P < .05) compared with the higher B12 levels. At Weeks 24 to 27 egg production of hens fed the .5 ug/kg treatment was 3% lower than that of hens fed the 4 ug/kg treatment, which in turn was 5% lower
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a_b
EGGS INDICATING VITAMIN B12 STATUS IN THE LAYING HEN
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TABLE 5. Effects of four dietary vitamin B 12 levels on shell thickness, egg weight, hen weight, hatchability, and embryonic mortality at selected weeks1 Treatments (ng vitamin B ^ / k g of diet) Variable Shell thickness, m x 100 Week 22 Egg weight, g Week 16 Week 27 Hen weight, kg Week 27 Hatchability, % Weeks 20 and 21
4.0
36.7 ± .33'
36.0 ± . 3 3 * 35.2
56.8 ± .38= 57.5 ± .29d
55.2 ± .38d 59.7 ± .29=
1.76 ± .02b
1.78 ±
.02b
16.0 ± .33b= 35.0 ± .33=
58.1 ± .38b 63.1 ± .29"
59.7 ± .38" 62.1 ± .29b
1.84 ± .02*
1.83 ± .02a
36.9 ± 1.9=
71.5 ± 1.9b
87.0 ± 1.9«
85.2 ± 1.9*
77.5* 4.1 18.4
69.0" 6.5 24.5
47.3b 7.9 44.8
52.1b 13.0 34.9
a_d
Means within rows with no common superscripts differ significantly (P < .05). Data reported as x ± SE (n = 100 observations for shell thickness; n = five to six pooled observations of 24 to 46 eggs for egg weight; n = 45 to 50 observations for hen weight; n = two pooled observations of 102 to 138 eggs for hatchability). 1
than that of hens fed the two highest diet vitamin B12. During this same time period, levels of the vitamin. There were no when hatchability of eggs from the four significant differences in egg production dietary treatments was about 37, 72, 87, between treatments after being fed the and 85%, egg yolk vitamin B12 concentrations were .4, 1.3, 2.5, and 4.7 ug/100 g recovery diet for 3 wk. At Week 22, eggshell thickness was with increasing vitamin B12 dietary levels. The data on embryonic mortality for significantly different (P < .05), with the eggs from hens fed the higher vitamin each week during the incubation period, levels having thinner eggshells (Table 5). expressed as a percentage of total mortalAt Week 16, egg weight was increased by ity, shows that a greater proportion of increases in vitamin B12 supplementation embryos died during the 1st wk when the except for the 4 |Xg/kg dietary treatment. hens were fed the two lower levels of At Week 27, egg weight was increased by vitamin B12 (Table 5). Hemorrhages in the increases in vitamin B12 supplementation young embryos that died were the most except for the 16 (xg/kg dietary treatment. frequently observed defect in eggs from It seemed that average egg weight was hens fed the lower vitamin B12 levels. greater at the two highest levels of B12 supplementation when compared with the DISCUSSION two lower levels. The results demonstrate that dietary Weight of hens fed the two lower vitamin levels was significantly lower than vitamin B12 content affects egg concentrathose fed the two higher vitamin levels at tions of the vitamin after 1 to 2 wk. Egg the 27th wk of the experiment. At Week 20 vitamin amounts from hens fed the .5 and to 21, the average hatchability of eggs 4 Jig/kg treatment levels decreased 66% from hens fed from the .5 Hg/kg treatment during the first 2 wk of the study and was significantly lower than that of hens were repleted in about the same time. This receiving the 4 ug/kg treatment (Table 5). shows that vitamin B12 deficiency can The hatchability of eggs from hens fed the reduce egg vitamin content within a very 4 ug/kg treatment was significantly lower short time, which agrees with Denton et al. than that on the two highest levels of (1954). Although liver stores may not be
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Distribution of embryonic mortality, Percentage at Weeks 20 and 21 during incubation Week 1 Week 2 Week 3
8.0
.5
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SQUIRES AND NABER
REFERENCES Casey, P. J., K. R. Speckman, F. J. Ebert, and W. E. Hobbs, 1982. Radioisotope dilution technique for determination of vitamin Bj 2 in foods. J.
Assoc. Off. Anal. Chem. 65:85-88. Denton, C. A, W. L. Kellogg, J. R. Sizemore, and R. J. Lillie, 1954. Effect of injecting and feeding vitamin Bj2 to hens on content of the vitamin in the egg and blood. J. Nutr. 54:517-577. Ferguson, T. M., and J. R. Couch, 1954. Further observations on the B12 deficient chick embryo. J. Nutr. 54:361-370. Hammond, J. C, 1942. Cow manure as a source of certain vitamins for growing chickens. Poultry Sci. 21:554-559. Lillie, R. J., C. A. Denton, M. W. Olsen, and H. R. Bird, 1949. Vitamin B^. Assay methods and role in reproduction of chickens. Pages 37-38 in: Abstracts of papers, 116th Meeting. American Chemical Society, Washington, DC. McGinnis, J., P. T. Hsu, and W. D. Graham. 1948. Studies on an unidentified factor required by chicks for growth and protein utilization. Poultry Sci. 27:674.(Abstr.) Milligan, J. L., G. H. Arscott, and G. F. Combs, 1952. Vitamin Bj2 requirement of New Hampshires. 1. Requirement of laying pullets. Poultry Sci. 31: 595-603. Mushett, C. W., and W. H. Orr, 1949. Influence of crystalline vitamin B12 on gizzard erosion in chicks. Poultry Sci. 28:850-854. Naber, E. C, 1979. The effect of nutrition on the composition of eggs. Poultry Sci. 58:518-528. National Research Council, 1984. Nutrient Requirements of Poultry, 8th rev. ed. National Academy Press, Washington, DC. Olcese, O., J. R. Couch, J. H. Quisenberry, and P. B. Pearson, 1950. Congenital anomalies in the chick due to vitamin Bj2 deficiency. J. Nutr. 41: 423-431. Peterson, C. F., A. C. Wiese, G. E. Milne, and C E. Lampman, 1953. Vitamin B12 requirements for hatchability and production of high-quality chicks. Poultry Sci. 32:535-54. Rubin, M., and H. R. Bird, 1946a. A chick growth factor in cow manure. I. Its non-identity with chick growth factors previously described, J. Biol. Chem. 163:387-392. Rubin, M., and H. R. Bird, 1946b. A chick growth factor in cow manure, n. The preparation of concentrates and the properties of the factor. J. Biol. Chem. 163:393-400. SAS Institute, 1982. SAS® User's Guide: Statistics. SAS Institute Inc. Cary, NC. Scott, M. L., M. C. Nesheim, and R. J. Young, 1982. Nutrition of the Chicken, 3rd ed. M. L. Scott and Associates, Ithaca, NY. Skinner, J. L., J. H. Quisenberry, and J. R. Couch, 1951. High efficiency and APF concentrates in the ration of the laying fowl. Poultry Sci. 30: 319-324. Snedecor, G. W., and W. G. Cochran, 1980. StaHstical Methods, 7th ed. The Iowa State University Press, Ames, LA. Yacowitz, H., C. H. Hill, L. C. Norris, and G. F. Heuser, 1952a. Distribution of vitamin B12 in the organs and tissues of the chick. Proc. Soc. Exp. Biol. Med. 79:279-280. Yacowitz, H., R. F. Miller, L. C. Norris, and G. F. Heuser, 1952b. Vitamin B12 with the hen. Poultry Sci. 31:89-94.
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depleted for 12 wk (Scott et al, 1982), this does not imply that vitamin B12 status is adequate for that period of time. The present data indicate that egg vitamin B12 concentrations stabilize after 12 wk in response to the diets and are directly proportional to dietary levels within the range of 4 to 16 ug of vitamin Bi2/kg of diet. Hence, egg yolk vitamin B12 content is a good indicator of the nutritional status of the hen for this vitamin. Egg production was depressed at times in the lowest two treatments after 12 wk. This is surprising because decreases in egg production due to vitamin B12 deficiency have not been reported. Apparently, there is a very low requirement for vitamin B12 to support higher egg production. Hatchability, egg weight, and hen weight were significantly lower in the .5 and 4 M-g/kg dietary treatments. Shell thickness was inversely related to egg weight, as might be expected. These data along with egg vitamin content were used to predict that between 1.3 and 2.6 (Xg of vitamin B12/IOO g of egg yolk are needed to insure maximum hatchability and egg weight. It seems that egg vitamin B12 content may be used to predict problems in the vitamin B12 status of laying and breeding flocks. A decrease in egg weight due to vitamin B12 deficiency has been documented previously (Skinner et al, 1951). However, no such effects on egg production, shell thickness, or hen's weight have been reported. It is possible that the longterm nature of this study (27 wk) detected small changes in these variables that were not noted in studies of shorter duration. This suggests that a very low vitamin B12 requirement does exist for variables associated with egg laying. The decrease in hatchability has been noted previously along with various nonspecific deficiency signs such as hemorrhages, leg atrophy, and edema (Olcese et al, 1950; Ferguson and Couch, 1954). A shift toward earlier embryonic death was also noted.
1992 Poultry Science 71:2075-2082
conscious consumer. One such technique to assess the vitamin status of the laying New diagnostic techniques to assess hen might be through analysis of her eggs. laying flock vitamin status are needed due In a review of literature, Naber (1979) to dated requirement recommendations suggested that egg riboflavin, vitamin A, that may not apply to modern strains of and vitamin B12 could be markedly inlaying hens and the increasing demand for fluenced by dietary changes. Establishhigher quality eggs by the health- ment of critical egg vitamin B12 concentrations could be used to determine adequacy of dietary supplementation giving a direct measure of vitamin bioavailaReceived for publication June 3, 1992. bility that feed analysis does not yield. Accepted for publication August 28, 1992. Salaries and research support provided by state Data on the relationship between laying and federal funds appropriated to the Ohio Agricul- hen diet and egg vitamin B12 might be tural Research and Development Center, The Ohio used to establish egg quality standards State University. Manuscript Number 160-92. and diet to egg vitamin transfer costs. 2 Partial funding for this project was provided by a The vitamin B12 requirement for laying grant-in-aid sponsored by Sigma Xi. 3 To whom correspondence should be addressed. and breeding hens is 4 ug/kg of diet (National Research Council, 1984). Some INTRODUCTION
2075
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ABSTRACT Hens of a type used for egg production were fed a corn and soybean meal diet supplemented with no vitamin B12 or with vitamin B12 levels to provide one, two, or four times the National Research Council (1984) breeding hen requirement of 4 ug/kg diet for 27 wk. All hens were placed on a recovery diet containing one and one-half times the requirement level of vitamin B12 from Weeks 27 through 30. Egg yolk vitamin B12 concentrations were determined frequently by radioisotope dilution analysis. Egg production records were kept continuously, and eggshell thickness, egg weight, hatchability of eggs, and hen body weights were measured at selected times. Although egg yolk vitamin B12 concentrations were high at the outset, they decreased markedly in 2 wk from hens fed the two lowest dietary levels. After 12 wk on the diets, egg concentrations of vitamin B12 stabilized and were proportional to the amount of vitamin added to the diet. Egg concentrations of vitamin B12 between 1.3 and 2.6 ug/100 g yolk appeared to be needed to support maximum hatchability and egg weight. Egg production was reduced after 12 wk on the diets in the hens fed the two lowest vitamin B12 levels. As vitamin B12 level increased, shell thickness decreased and egg weight, hen weight, and hatchability increased. Maximum egg production, egg weight, hen weight, and hatchability were obtained when the diet contained 8.0 M-g/kg of vitamin B12. Egg yolk vitamin B12 concentrations respond rapidly to dietary changes in the level of this vitamin and are indicative of the vitamin B12 status of the hen. (Key words: vitamin B12/ layers, nutritional status, egg yolk content, hatchability)
2076
SQUIRES AND NABER
ern requirement for breeding hens as being between 3.3 to 4.4 ug vitamin B12/ kg of diet using microbiologicial methods for vitamin analysis. Denton et al. (1954) showed that egg vitamin B12 concentrations decreased significantly 2 wk after supplementation was discontinued. This suggests that only moderate amounts of vitamin B12 are stored in the hen, although others have suggested that several months may be needed to deplete body reserves (Scott et al, 1982). The working hypothesis for the current study was that egg vitamin B12 concentrations can be used to assess the vitamin B12 status of the laying hen and the sufficiency of her diet for egg hatchability. The objectives were as follows: 1) to determine the effects of feeding practical rations containing graded levels of vitamin B12 on egg vitamin B12 content and important production variables of modern strains of layers during selected periods of time; 2) to establish the relationship between egg vitamin concentrations, diet vitamin levels, and decreases in important production variables; and 3) to determine minimum critical egg vitamin B12 concentrations needed for maximum reproductive function. MATERIALS AND METHODS Egg Yolk Vitamin B12 Extraction
The procedure used was an adaptation of that described by Casey et al. (1982). Sample preparation was done in subdued light. Two to 5 g of thawed egg yolk were weighed into a Virtis cup.4 Forty milliliters of extraction solvent (13 g anhydrous sodium phosphate, 12 g citric acid, and 10 g sodium metabisulfite/L of distilled water) were added to the cup. The sample was homogenized in a Virtis model Super 30 homogenizer4 equipped with metal blades for 15 s. The sample was poured into a 100-mL glass-stoppered volumetric flask. The cup was rinsed with 10 mL of extraction solvent and poured into the 100-mL flask. The samples were digested in a Barn4 The Virtis Co., Inc., Gardner, NY 12525. 5 2 5 Barnstead Thermoline Corp., Subsidiary of Sybron stead steam autoclave at 1.1 kg/cm for 10 min. After pressure had returned to zero, Corp., Dubuque, IA 52001.
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vitamin B12 can be obtained from microbial synthesis, but diets are usually in need of supplementation. Embryonic deficiency signs include edema, hemorrhages, perosis, beak shortening, myotrophy of legs, and fattiness of liver, heart, and kidneys (Mushett and Orr, 1949; Olcese et al, 1950; Ferguson and Couch, 1954). Chicks hatched without adequate stores may have increased mortality along with depressed growth, poor feathering, enlarged thyroid glands, and gizzard lesions. Hammond (1942) first noted an unidentified vitamin-like factor in cow manure that increased weight gain of chicks fed poor quality diets. He incorrectly assumed that the factor was riboflavin. Rubin and Bird (1946a,b) showed that this growth factor in cow manure was not any known nutrient and was transmitted through the egg to the chick. McGinnis et al. (1948) verified that vitamin B12 was the animal growth factor found in manure and that it was needed for proper amino acid utilization. Lillie et al. (1949) injected vitamin B12 into eggs and reported that hatchability of eggs from hens deficient in vitamin B12 increased with injection of the vitamin. This was the first evidence of a role for vitamin B12 in hatchability. Skinner et al. (1951) first noted that hens receiving diets low in vitamin B12 laid eggs that were smaller. Yacowitz et al. (1952a) suggested that chicks have the ability to store excess vitamin B12 in their liver, kidney, and pancreas. Milligan et al. (1952) demonstrated that increases in maternal dietary vitamin B12 caused increases in egg levels using both a chick and a microbiological assay. Efficiency of transmission of vitamin B12 to the egg, however, was variable and not related to dietary vitamin level. Yacowitz et al. (1952b) using a microbial assay estimated that 2.5 ng of vitamin Bi2/g of yolk is needed to ensure hatchability. This value seems to be very low when compared with more recent data. Peterson et al. (1953) established the mod-
EGGS INDICATING VITAMIN B12 STATUS IN THE LAYING HEN
Feed Vitamin B12 Extraction
The same procedure was used for feed as described for egg yolk. Some additional preparation of the samples was required however, prior to analysis. The feed was finely ground in a Brinkman Instruments Model ZM1 grinder7 equipped with a .2mm screen. Radioisotope Dilution Assay for Vitamin B12
Becton Dickinson vitamin B12 57Co radioassay kits8 (Catalog Number 262315) were used for assay. A total of 16 standard tubes and 48 sample tubes per assay run were employed. The kit reagents were: 1) A 5% dithiothreitol solution with stabilizer, which was mixed with vitamin B12 tracer solution containing [57Co] vitamin B12 in borate buffer, human serum albumen, dextran, potassium cyanide, nonintrinsic factor blocking agent, dye, and preservatives. This vitamin B12 tracer solution was stable for 1 wk under refrigeration. 2) A vitamin B12 binder containing porcine intrinsic factor, human serum albumen, dextran, and preservatives, which was mixed with binder diluent containing sodium borate, sodium chloride, dye, and preservative.
6
Fisher Scientific, Pittsburgh, PA 15219. iBrinkman Instruments, Inc., Westbury, NY 11590. 8 Becton Dickinson Immunodiagnostics, Orangeburg, NY 10962. 911100138 Scientific, Swedesboro, NJ 08085. ^International Equipment Co., Division of Damon Corp., Needham Heights, MA 02194. "Beckman Instruments, Inc., Fullerton, CA 92634.
The vitamin B12 binder solution was stable for 8 wk under refrigeration. 3) Vitamin B12 standards in sodium borate, sodium chloride, dye, and preservative. 4) A dextran charcoal suspension containing charcoal, dextran, sodium chloride, sodium borate, and a pelleting aid. All samples and standards were run in duplicate in 12 x 75 mm polypropylene tubes. Two hundred microliters of sample filtrate was added to each sample tube and the same amount of the standards was added to Tubes 3 through 16 but not the first two tubes. The standard tubes contained 0, 100, 200, 400, 1,000, and 2,000 pg vitamin Bi2/mL. One milliliter of vitamin B12 tracer solution was added to each tube. The first two standard tubes, which provide data on total radioactivity counts, were pulled from the assay and held in the dark while all of the other tubes were mixed gently. One hundred microliters of the binder solution was added to standard Tube 5 through 16 and all of the sample tubes. After mixing gently by hand, both the standard and sample tubes were incubated at room temperature for 30 min in the dark. Four-tenths milliliter of dextran charcoal suspension was added to the tubes with a Cornwall syringe.9 All samples and standards including Tubes 1 and 2 were incubated in the dark at room temperature for 10 min. The sample and standard tubes were spun in an International PR-6 centrifuge™ equipped with a Number 276 rotor and Number 353 sample cup for 10 min at 1,000 x g (2,000 rpm) at 10 C. The clear supernatant was decanted into 6-mL Beckman mini POLY-Q vials containing 4 mL of Beckman Ready Sol MP or CP scintillation cocktail.11 This step was performed with plastic gloves and care was taken not to decant any of the charcoal from the sample tubes into the vials. The vials were capped and shaken vigorously and the sample tubes containing charcoal were discarded. Scintillation counting was carried out with a Beckman LS-5801 liquid scintillation spectrometer.11 This was possible because the 57Co gamma emissions resulted in the release of Auger and conversion electrons that caused scintillations that were easily counted. A radioimmunoassay program
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the flasks were removed from the autoclave and allowed to cool in the dark at room temperature. The samples were brought to 100 mL volume with phosphate buffer (12.1 g anhydrous potassium phosphate and 18.9 g anhydrous sodium phosphate/L of distilled water). The flasks were stoppered and mixed by inversion 10 times. The samples were filtered through Whatman Number 2 filter paper6 into 125-mL Erlenmeyer flasks to obtain 75 mL of filtrate. The filtrate was mixed by swirling, and a 4-mL aliquot was removed and stored in a small vial under refrigeration.
2077
2078
SQUIRES AND NABER TABLE 1. Composition of vitamin B12 basal and recovery diets
Ingredients and analysis
55.5 5.5
Recovery diet
26.5 2.6 8.4 .74 .04 .50 .20
53.4 10.2 2.5 20.1 3.0 9.0 1.0 .10 .50 .20
2,800 17.7 .89 .32 .55 3.3 .50
2,787 15.6 .75 .34 .56 3.6 .52
iAmount supplemented per kilogram of diet: Mn, 70 mg; Zn, 100 mg; iodized NaCl, 2400 mg. 2 Amount supplemented per kilogram of diet: vitamin A, 2,000 IU; cholecalciferol, 1,000 ICU; vitamin E, 20IU; vitamin K, 1 mg; pantothenic acid, 15 mg; niacin, 15 mg; riboflavin, 1.75 mg; ethoxyquin (66%), 188 mg; Larvadex, 500 mg; and variable amounts of vitamin B 12 (see Table 2).
resident in the liquid scintillation spectrometer was used to calculate and tabulate the results. Design of the Experiment Two hundred Single Comb White Leghorn pullets12 were divided into four treatment groups of 50 birds each at 28 wk of age. The pullets received a basal diet that was formulated to contain the National Research Council (1984) requirements of all nutrients except vitamin B12. This basal diet is shown in Table 1. Vitamin B12 (.1% trituration of cyanocobalamin in mannitol)13 was used for dietary supplementation. The four treatment levels of vitamin B12 dietary supplementation and estimated requirement levels for hatchability are shown in Table 2. Assay of the diets for vitamin B12 agreed closely with the calculated values. Birds on all four treatments received ad libitum access to feed and water. Water was supplied by continuous flow in a
12
H and N International, Redmond, WA 98052. "United States Biochemical, Cleveland, OH 44122.
trough that was regularly cleaned to avoid microbial growth. The pullets were housed, one per cage, in an environmentally controlled poultry house. Sixteen hours of light were provided daily from 0500 until 2100 h. The experimental diets were fed for 27 wk. All four experimental groups were placed on a recovery diet containing 6.0 ug/kg of vitamin B12 for 3 wk after termination of the vitamin B12 treatments. Hen-day egg production on individual birds was calculated at 4-wk intervals for the duration of the experiment. Eggs were collected on 6 consecutive days during the 16th and 27th wk and weighed. Shell thickness of 100 air-dried eggshells per treatment were determined during the 22nd wk. All hens were weighed during the 1st and 27th wk of the experiment. Hatchability of eggs from 25 hens in each treatment was determined by setting eggs from the 20th and 21st wk. The hens were artificially inseminated 1 wk prior to each hatchability study with pooled semen from Athens Canadian randombred males. All unhatched eggs from the two settings were broken out to determine fertility or approximate day of death during incubation. Embryonic mortality was recorded by
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Ground yellow corn Ground wheat Alfalfa meal (17% CP) Soybean meal (44% CP) Hydrolyzed fat (animal and vegetable) Limestone (37% Ca) Dicalcium phosphate DL-methionine Mineral mix1 Vitamin mix2 Calculated analysis ME, kcal/kg Protein Lysine Methionine Methionine and cystine Calcium Total phosphorus
Basal diet
EGGS INDICATING VITAMIN B12 STATUS IN THE LAYING HEN
2079
TABLE 2. Dietary vitamin Bj 2 supplements to experimental and recovery diet ]Experinvental
Variable
.5
Vitamin Bi 2 supplement, Hg/kg diet Calculated total dietary vitamin B^, ug/kg diet Percentage of National Research Council requirement for breeding hens1 1
4.0
0 .5 12
3.5 4.0 100
diets
8.0 7.5 8.0 200
16.0
Recovery diet 6.0
15.5 16.0 400
5.5 6.0 150
The National Research Council (1984) requirement is 4.0 ug/kg of diet.
treatment and time period were analyzed. Vitamin B12 was corrected for recovery and reported as micrograms of vitamin B12 per 100 g of yolk. Statistical Analysis The data was subjected to a two-way ANOVA (Snedecor and Cochran, 1980) by the General Linear Models procedure using SAS® software (SAS Institute, 1982) and calculated as the mean and SEM for treatment, time, and treatment by time interaction. Statistical comparisons among treatment means at each time interval were made by the Least Squares Mean procedure (repeated t test) using SAS® software (SAS Institute, 1982) at a probability level of P < .05.
TABLE 3. Changes in egg yolk vitamin B12 over time for hens fed four dietary vitamin By levels1 Treatments (ug vitamin B^/kg of diet) Time (wk) 0 1 2 4 6 8 12 16 20 24 27 1 3 a_d
.5
4.0
10.1 ± .81 6.3 ± .41b 3.1 ± .17<* 2.0 ± .12d 1.7 ± .25= 1.1 ± .15c
11.2 6.5 3.6 3.2 2.7 1.5 1.1 1.3 1.5 1.2
8.0
16.0
fur/if
.3 ± .07°"
.5 ± .05d .4 ± .26= .3 ± .07°" 1.9 ± .40= 2.8 ± .07=
± ± ± ± ± ± ± ± ± ±
1.4 .43b .27<: .56= .25= .19= .40= .17= .26b .30=
2.4 ± .40b= 2.4 ± .07d
9.9 ± 1.1 6.9 ± .48b 5.1 ± .24b 4.1 ± .21b 4.0 ± .25b 2.4 ± .33b 2.8 ± .34b 2.4 ± .31b 2.5 ± .26b 2.7 ± .33b recovery diet 3.6 ± .40* 3.2 ± .07b
9.5 8.8 7.1 5.9 6.8 4.0 4.7 4.7 4.8 4.9
± ± ± ± ± ± ± ± ± ±
.63 .95" .34' .25' .25* .48" .42^ .48» .26* .38*
4.7 ± .40* 4.0 ± .07»
Means within rows with no common superscripts differ significantly (P < .05). Data reported as X ± SE (n = two to six pooled samples of four eggs each for each treatment and time observation). a
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dividing the 21-day incubation period into trimesters and calculating percentage mortality in each trimester. Dead embryos were examined for abnormalities and defects. Eggs were collected from each treatment during Weeks 0,1,2,4,6,8,12,16,20,24,28, and 30 for later isotope dilution analysis of vitamin Bj2 (described above). Twenty-four eggs per treatment were gathered during 1 day of the designated week and stored in an egg cooler at 13 C. Yolks were harvested by breaking out the eggs under subdued light within 3 days of laying. Excess albumen was removed from the yolks by rolling them on paper towels. Pooled yolk samples of four eggs each were placed into six plastic containers. These samples were placed in light-proof boxes and frozen at -20 C. Two to three yolk samples per
2080
SQUIRES AND NABER
TABLE 4. Changes in hen-day production of hens over time fed four dietary vitamin Bj2 levels1 Treatments (ng vitamin B 12 /kg of diet) Time (wk) 0 to 5 to 9 to 13 to 17 to 21 to 24 to
.5
8.0
16.0
93.0 ± 1.8 88.5 ± 1.8 88.8 ± 1.8 81.5 ± 1.8" 75.8 ± 1.8" 77.0 ± 1.8» 78.0 ± 2.1" to recovery diet 81.3 ± 2.1
92.8 89.8 85.3 85.3 75.3 77.8 78.3
4.0 f%l -
4 8 12 16 20 24 27
0 to 3
95.0 89.8 86.8 80.5 72.3 69.3 70.7
± ± ± ± ± ± ±
1.8 1.8 1.8 1.8*" 1.8* 1.8b 2.1b
77.0 ± 2.1
91.0 86.8 84.0 75.5 70.0 72.8 76.3
± ± ± ± ± ± ±
1.8 1.8 1.8 1.8b 1.8b 1.8ab 2.1ab
78.3 ± 2.1
\'°)
± 1.8 ± 1.8 ± 1.8 ± 1.8» ± 1.8" ± 1.8» ± 2.1a
81.3 ± 2.1
Means within rows with no common superscripts differ significantly (P < .05). !Data reported as x ± SE (n = three to four pooled observations of 7 days each for each treatment and time).
RESULTS
Differences in egg vitamin B12 content and hen-day egg production were significant (P < .05) for both treatment and time (Tables 3 and 4). The results presented here emphasize the interaction of treatment at various time intervals during the study. The data in Table 3 show that the vitamin B12 content of egg yolks was high at the outset of the experiment, indicating a substantial intake of the vitamin prior to the experiment. Significant differences (P < .05) in egg yolk vitamin B12 content appeared after 1 wk (Table 3). At this time hens fed the 16 ug/kg dietary treatment had significantly higher egg vitamin concentrations than eggs from hens on the other three treatments due to reductions in vitamin content of eggs from hens at lower treatment levels. At 2 wk, eggs from hens on all four dietary treatments were significantly different (P < .05) from each other and were proportional to dietary vitamin levels. At 12 wk, the vitamin B12 egg concentrations from hens fed the .5 ug/kg dietary treatment were 27% of those receiving the 4 ug/kg dietary treatment. The egg vitamin B12 concentrations in the latter treatment were 39% of those fed the 8 ug/kg treatment. The egg vitamin B12 content of the hens fed the 8 ug/kg dietary treatment was 60% of that from hens fed the 16 ug/kg treatment. This pattern of egg yolk vitamin B12 concentrations continued for the duration of the experiment.
Egg vitamin B12 content from hens fed the two lowest levels of vitamin B12 were still significantly lower than those that had received the two highest levels following a 3-wk recovery period (Table 3). However, egg vitamin B12 concentrations from hens fed the .5 and 4 ug/kg dietary treatments had returned to 87 and 75% of those from hens on the 8 ug/kg treatment after the 3-wk recovery. Much of the increase in egg yolk vitamin B12 occurred after 1st wk on the recovery diet. This is approximately the same time period needed for depletion of egg vitamin B12. Egg yolk vitamin B12 content stabilized after Week 12 in all of the treatments. The average egg yolk vitamin B12 content of the eggs for Weeks 12 through 24 were .4 ug/100 g yolk for the .5 ug/kg dietary treatment, 1.3 ug/100 g for the 4 ug/kg treatment, 2.6 ug/100 g for the 8 ug/kg treatment, and 4.8 ug/100 g of yolk for the 16 ug/kg treatment. A significant depression (P < .05) in egg production of hens fed the 4 ug/kg dietary treatment was apparent at Weeks 13 to 20 compared with the two higher dietary vitamin B12 levels (Table 4). At Weeks 21 to 27, the egg production of hens fed the .5 ug vitamin B^/kg of diet treatment was significantly depressed (P < .05) compared with the higher B12 levels. At Weeks 24 to 27 egg production of hens fed the .5 ug/kg treatment was 3% lower than that of hens fed the 4 ug/kg treatment, which in turn was 5% lower
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a_b
EGGS INDICATING VITAMIN B12 STATUS IN THE LAYING HEN
2081
TABLE 5. Effects of four dietary vitamin B 12 levels on shell thickness, egg weight, hen weight, hatchability, and embryonic mortality at selected weeks1 Treatments (ng vitamin B ^ / k g of diet) Variable Shell thickness, m x 100 Week 22 Egg weight, g Week 16 Week 27 Hen weight, kg Week 27 Hatchability, % Weeks 20 and 21
4.0
36.7 ± .33'
36.0 ± . 3 3 * 35.2
56.8 ± .38= 57.5 ± .29d
55.2 ± .38d 59.7 ± .29=
1.76 ± .02b
1.78 ±
.02b
16.0 ± .33b= 35.0 ± .33=
58.1 ± .38b 63.1 ± .29"
59.7 ± .38" 62.1 ± .29b
1.84 ± .02*
1.83 ± .02a
36.9 ± 1.9=
71.5 ± 1.9b
87.0 ± 1.9«
85.2 ± 1.9*
77.5* 4.1 18.4
69.0" 6.5 24.5
47.3b 7.9 44.8
52.1b 13.0 34.9
a_d
Means within rows with no common superscripts differ significantly (P < .05). Data reported as x ± SE (n = 100 observations for shell thickness; n = five to six pooled observations of 24 to 46 eggs for egg weight; n = 45 to 50 observations for hen weight; n = two pooled observations of 102 to 138 eggs for hatchability). 1
than that of hens fed the two highest diet vitamin B12. During this same time period, levels of the vitamin. There were no when hatchability of eggs from the four significant differences in egg production dietary treatments was about 37, 72, 87, between treatments after being fed the and 85%, egg yolk vitamin B12 concentrations were .4, 1.3, 2.5, and 4.7 ug/100 g recovery diet for 3 wk. At Week 22, eggshell thickness was with increasing vitamin B12 dietary levels. The data on embryonic mortality for significantly different (P < .05), with the eggs from hens fed the higher vitamin each week during the incubation period, levels having thinner eggshells (Table 5). expressed as a percentage of total mortalAt Week 16, egg weight was increased by ity, shows that a greater proportion of increases in vitamin B12 supplementation embryos died during the 1st wk when the except for the 4 |Xg/kg dietary treatment. hens were fed the two lower levels of At Week 27, egg weight was increased by vitamin B12 (Table 5). Hemorrhages in the increases in vitamin B12 supplementation young embryos that died were the most except for the 16 (xg/kg dietary treatment. frequently observed defect in eggs from It seemed that average egg weight was hens fed the lower vitamin B12 levels. greater at the two highest levels of B12 supplementation when compared with the DISCUSSION two lower levels. The results demonstrate that dietary Weight of hens fed the two lower vitamin levels was significantly lower than vitamin B12 content affects egg concentrathose fed the two higher vitamin levels at tions of the vitamin after 1 to 2 wk. Egg the 27th wk of the experiment. At Week 20 vitamin amounts from hens fed the .5 and to 21, the average hatchability of eggs 4 Jig/kg treatment levels decreased 66% from hens fed from the .5 Hg/kg treatment during the first 2 wk of the study and was significantly lower than that of hens were repleted in about the same time. This receiving the 4 ug/kg treatment (Table 5). shows that vitamin B12 deficiency can The hatchability of eggs from hens fed the reduce egg vitamin content within a very 4 ug/kg treatment was significantly lower short time, which agrees with Denton et al. than that on the two highest levels of (1954). Although liver stores may not be
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Distribution of embryonic mortality, Percentage at Weeks 20 and 21 during incubation Week 1 Week 2 Week 3
8.0
.5
2082
SQUIRES AND NABER
REFERENCES Casey, P. J., K. R. Speckman, F. J. Ebert, and W. E. Hobbs, 1982. Radioisotope dilution technique for determination of vitamin Bj 2 in foods. J.
Assoc. Off. Anal. Chem. 65:85-88. Denton, C. A, W. L. Kellogg, J. R. Sizemore, and R. J. Lillie, 1954. Effect of injecting and feeding vitamin Bj2 to hens on content of the vitamin in the egg and blood. J. Nutr. 54:517-577. Ferguson, T. M., and J. R. Couch, 1954. Further observations on the B12 deficient chick embryo. J. Nutr. 54:361-370. Hammond, J. C, 1942. Cow manure as a source of certain vitamins for growing chickens. Poultry Sci. 21:554-559. Lillie, R. J., C. A. Denton, M. W. Olsen, and H. R. Bird, 1949. Vitamin B^. Assay methods and role in reproduction of chickens. Pages 37-38 in: Abstracts of papers, 116th Meeting. American Chemical Society, Washington, DC. McGinnis, J., P. T. Hsu, and W. D. Graham. 1948. Studies on an unidentified factor required by chicks for growth and protein utilization. Poultry Sci. 27:674.(Abstr.) Milligan, J. L., G. H. Arscott, and G. F. Combs, 1952. Vitamin Bj2 requirement of New Hampshires. 1. Requirement of laying pullets. Poultry Sci. 31: 595-603. Mushett, C. W., and W. H. Orr, 1949. Influence of crystalline vitamin B12 on gizzard erosion in chicks. Poultry Sci. 28:850-854. Naber, E. C, 1979. The effect of nutrition on the composition of eggs. Poultry Sci. 58:518-528. National Research Council, 1984. Nutrient Requirements of Poultry, 8th rev. ed. National Academy Press, Washington, DC. Olcese, O., J. R. Couch, J. H. Quisenberry, and P. B. Pearson, 1950. Congenital anomalies in the chick due to vitamin Bj2 deficiency. J. Nutr. 41: 423-431. Peterson, C. F., A. C. Wiese, G. E. Milne, and C E. Lampman, 1953. Vitamin B12 requirements for hatchability and production of high-quality chicks. Poultry Sci. 32:535-54. Rubin, M., and H. R. Bird, 1946a. A chick growth factor in cow manure. I. Its non-identity with chick growth factors previously described, J. Biol. Chem. 163:387-392. Rubin, M., and H. R. Bird, 1946b. A chick growth factor in cow manure, n. The preparation of concentrates and the properties of the factor. J. Biol. Chem. 163:393-400. SAS Institute, 1982. SAS® User's Guide: Statistics. SAS Institute Inc. Cary, NC. Scott, M. L., M. C. Nesheim, and R. J. Young, 1982. Nutrition of the Chicken, 3rd ed. M. L. Scott and Associates, Ithaca, NY. Skinner, J. L., J. H. Quisenberry, and J. R. Couch, 1951. High efficiency and APF concentrates in the ration of the laying fowl. Poultry Sci. 30: 319-324. Snedecor, G. W., and W. G. Cochran, 1980. StaHstical Methods, 7th ed. The Iowa State University Press, Ames, LA. Yacowitz, H., C. H. Hill, L. C. Norris, and G. F. Heuser, 1952a. Distribution of vitamin B12 in the organs and tissues of the chick. Proc. Soc. Exp. Biol. Med. 79:279-280. Yacowitz, H., R. F. Miller, L. C. Norris, and G. F. Heuser, 1952b. Vitamin B12 with the hen. Poultry Sci. 31:89-94.
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depleted for 12 wk (Scott et al, 1982), this does not imply that vitamin B12 status is adequate for that period of time. The present data indicate that egg vitamin B12 concentrations stabilize after 12 wk in response to the diets and are directly proportional to dietary levels within the range of 4 to 16 ug of vitamin Bi2/kg of diet. Hence, egg yolk vitamin B12 content is a good indicator of the nutritional status of the hen for this vitamin. Egg production was depressed at times in the lowest two treatments after 12 wk. This is surprising because decreases in egg production due to vitamin B12 deficiency have not been reported. Apparently, there is a very low requirement for vitamin B12 to support higher egg production. Hatchability, egg weight, and hen weight were significantly lower in the .5 and 4 M-g/kg dietary treatments. Shell thickness was inversely related to egg weight, as might be expected. These data along with egg vitamin content were used to predict that between 1.3 and 2.6 (Xg of vitamin B12/IOO g of egg yolk are needed to insure maximum hatchability and egg weight. It seems that egg vitamin B12 content may be used to predict problems in the vitamin B12 status of laying and breeding flocks. A decrease in egg weight due to vitamin B12 deficiency has been documented previously (Skinner et al, 1951). However, no such effects on egg production, shell thickness, or hen's weight have been reported. It is possible that the longterm nature of this study (27 wk) detected small changes in these variables that were not noted in studies of shorter duration. This suggests that a very low vitamin B12 requirement does exist for variables associated with egg laying. The decrease in hatchability has been noted previously along with various nonspecific deficiency signs such as hemorrhages, leg atrophy, and edema (Olcese et al, 1950; Ferguson and Couch, 1954). A shift toward earlier embryonic death was also noted.