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Vitamin Profiles of Eggs as Indicators of Nutritional Status in the Laying Hen: Riboflavin Study1
METABOLISM AND NUTRITION Vitamin Profiles of Eggs as Indicators of Nutritional Status in the Laying Hen: Riboflavin Study1 MICHAEL W. SQUIRES and EDWARD C. NABER2 Department of Poultry Science, The Ohio State University, Columbus, Ohio 43210
1993 Poultry Science 72:483-494
INTRODUCTION Diagnostic techniques to assess laying flock vitamin status are needed due to dated requirement recommendations that may not apply to modern strains of laying hens and the increasing demand for higher quality eggs by the health-
Received for publication April 1, 1992. Accepted for publication November 20, 1992. Salaries and research support provided by State and Federal Funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University. Manuscript Number 94-92. 2 To whom correspondence should be addressed.
conscious consumer. One such technique to assess the vitamin status of the laying hen might be through the analysis of her eggs. In a review of the literature, Naber (1979) reported that egg riboflavin was markedly influenced by dietary changes. Establishment of critical egg riboflavin concentrations might be used to determine the adequacy of dietary supplementation, yielding a direct measure of vitamin bioavailability that feed analysis does not provide. Data on the relationship between laying hen diet and egg vitamin contents could be used to establish egg quality standards and diet to egg vitamin transfer efficiency.
483
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ABSTRACT Two experiments determined the effect of dietary riboflavin supplementation on egg yolk and albumen riboflavin concentrations, egg production, egg weight, shell thickness, hen weight, hatchability, incidence of clubbed down, and incidence of hemorrhagic embryos. In the first experiment, hens were fed rations containing 1.55,2.20,4.40, and 8.80 mg of riboflavin/kg of diet for 27 wk. Significant (P < .05) depressions in both yolk and albumen riboflavin concentrations were noted at the two lower riboflavin levels after 1 wk. Egg production, egg weight, hatchability, and hen weight were all significantly depressed by the two lower riboflavin levels later in the experiment when compared with the two higher levels. Results indicate that egg riboflavin concentrations are related to important production parameters that may be used to predict future dietary riboflavin inadequacies. In the second experiment, hens were fed either an unsupplemented diet or a riboflavin-adequate diet. Measurements of egg albumen riboflavin content, egg production, hatchability, and embryo abnormalities were made twice each week. Results showed depressed albumen riboflavin concentrations and hatchability and increased incidence of hemorrhagic embryos and clubbed down without changes in egg production during the 4- to 7-day period following feeding of the unsupplemented diet. These results show that low albumen riboflavin content immediately affect hatchability and embryonic development. The estimated minimum critical albumen riboflavin concentrations needed to support maximum reproductive function are between 1.9 and 2.9 jug of riboflavin/g of egg albumen. These critical values might be used to evaluate riboflavin status of laying and breeding flocks. (Key words: egg riboflavin content, diet, reproductive function, hatchability, nutritional status)
484
SQUIRES AND NABER
3
Fisher Scientific, Pittsburgh, PA 15219. Ivan Sorvall, Inc., Newtown, CT 06470.
4
riboflavin-binding protein (RFBP). This protein controls riboflavin transfer from plasma to the ovary and magnum and limits the amount of riboflavin in eggs. Amounts of dietary riboflavin were shown to have no effect on the amount of RFBP in the egg or plasma. The decreased amount of riboflavin in eggs from hens on deficient diets is due to riboflavin insufficiency for binding to the protein and not to lack of RFBP itself. One objective of the present study was to determine the effects of feeding practical rations containing graded levels of riboflavin on egg riboflavin content and important production parameters of modern strains of layers for selected periods of time. A second objective was to establish the time relationships between reductions in egg riboflavin contents and decreases in important production parameters to determine minimum critical egg riboflavin values needed for maximum egg production and reproductive function. MATERIALS AND METHODS Egg Yolk Riboflavin Extraction The extraction method is a modified procedure of the Association of Official Analytical Chemists (1980). The entire procedure was carried out under subdued light to protect the light-sensitive riboflavin. Ten grams (+ .02) of thawed liquid yolk and 50 mL of .1 N HC1 were placed in a 125-mL Erlenmeyer flask. The content was homogenized with a glass stir rod and placed in an autoclave at 121C (1.1 kg/cm) for 30 min. After cooling, 5 mL of 2 M sodium acetate were added to adjust the pH to between 4.5 and 5.0. Two milliliters of 50% (wt:vol) trichloroacetic acid were added to further adjust the pH to between 3.5 and 3.8 and to coagulate proteins. The content of the flask was filtered through 18.5-cm Whatman Number 1 Dacer3 and .1 N HC1 was used to bring the filtrate up to a 50-mL volume. If the filtrate was cloudy, it was centrifuged at 27,000 x g at 10 C for 15 min in a Sorvall RC2-B centrifuge equipped with a SS-34 rotor4 and then refiltered.
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Bethke et al. (1933) first noted a relationship between the diet of the laying hen, egg vitamin G levels (vitamin B complex), and the growth and livability of the resulting chicks. Lepkovsky and Jukes (1935) reported that chicks had an absolute requirement for vitamin G separate from the vitamin B complex and that the vitamin was probably a flavin compound. Bethke et al. (1937) identified riboflavin as the substance in the vitamin G complex responsible for the enhancement of hatchability of chicks. Lepkovsky et al. (1938) showed the relationship between laying hen diet, egg riboflavin concentrations, and various embryonic deficiency signs by measuring albumen riboflavin concentrations by a photometric assay. Hunt et al. (1939) showed that the riboflavin requirement for maximum hatchability was higher than that for egg production. Norris and Bauernfeind (1940) noted that there appeared to be an upper limit to the amount of riboflavin deposited in eggs. Stamberg et al. (1946) showed that 8 days were needed to bring egg riboflavin contents back to normal in deficient hens following the consumption of adequately supplemented diets. In 1947 (a,b), Petersen et al. established the modern riboflavin requirement (National Research Council, 1984) of 2.2 mg of riboflavin/kg of diet for egg production, with the required level for maximum hatchability being somewhat higher. In 1984, Onwudike and Adegbola demonstrated an 86% increase in the riboflavin requirement for laying hens and a 64% increase for breeding in the tropics. White et al. (1986) indicated that riboflavin in egg albumen depletes more rapidly than in yolk but only in hens fed very deficient diets. Normal egg yolk riboflavin concentrations are 4 to 5 /*g/g and egg albumen levels are 3 to 4 /tg/g. Dietary riboflavin levels influence egg riboflavin deposition in a linear fashion up to 4.2 mg/kg of diet; however, supplementation above this level does not increase the amount in egg appreciably. This ceiling on diet-to-egg transfer of riboflavin is due to
EGGS INDICATING RIBOFLAVIN STATUS IN THE LAYING HEN
485
Model 7120 sample injector,6 Perkin-Elmer Model 650-10LC fluorescence specThis extraction is a modified procedure trophotometer,7 and a Kipp and Zonen BDof Ashoor et ah (1983). The entire procedure 41 recorder.8 was carried out under subdued light. Ten A silicon Merck Lichrosorb Model RP-18 grams (± .02) of thawed liquid albumen and column9 (10-/an mesh, 25 cm x 4 mm) was 25 mL of a HPLC grade methanol-distilled used to separate and quantify the vitamin. water solution (1:1) were placed into a The solvent system was isocratic with an 50-mL polycarbonate centrifuge tube. After eluting solvent consisting by volume of stirring gently for no more than 5 min with HPLC grade methanol-distilled watera Fisher Dyna-Mix Model 143 variable glacial acetic acid (32:68:.l). The column speed mixer,3 5.0 mL glacial acetic acid was was not washed between runs, but was added dropwise with constant stirring to rinsed at the end of the day with methanoladjust the pH to 3 and to precipitate water (20:80). The flow rate was 2.0 mL/ proteins. The sample was then centrifuged min with a pressure of 140 to 210 kg/cm 2 . as described for yolk samples. After cen- The fluorescence detector was set at an trifugation, the supernatant was decanted excitation wavelength of 280 nm with into a foil-wrapped, 50-mL volumetric emission at 510 nm. flask. Ten milliliters of an HPLC grade methanol-distilled water-acetic acid solution (42:42:16) was added to the precipitate Design of Experiment 1 in the centrifuge tube, which was then Two hundred H and N Single Comb stirred, centrifuged, and decanted as before. White Leghorn hens,10 28 wk of age, were Extraction with the 10 mL methanol-water- divided into four treatment groups of 50 acetic acid was repeated, and the volumet- hens each. The hens received a basal diet ric flask was filled to volume with this same that was formulated to contain the requiresolution. Twenty-five drops of the yolk or ments of all nutrients except riboflavin albumen filtrate were passed through a .45- (National Research Council, 1984). The fi Millipore filter5 into a 4-mL sample vial. basal diet is shown in Table 1. The four The sample was stored in a freezer for later dietary treatments consisted of supHPLC analysis. plementing the basal diet with 0, .65, 2.85, and 7.25 mg of pure riboflavin/kg of diet.11 Total estimated dietary riboflavin and reHigh-Performance Liquid quirement levels for egg production and Chromatography Measurement hatchability are shown in Table 2. Hens in of Riboflavin all four treatments received ad libitum access This procedure is the same as that to feed and water. The hens were housed described by Ashoor et ah (1983) except for individually in an environmentally conthe column used (Wehling and Wetzel, trolled poultry house. Sixteen hours of light 1984) and the emission wavelength meas- were provided daily from 0500 to 2100 h. ured (Woodcock et ah, 1982). Twenty The experimental diets were fed for 27 wk. microliters of extract was injected into a All four treatment groups were placed on a HPLC apparatus consisting of an Altex recovery diet containing a riboflavin level Model 110A pump, 6 Altex Model 420 of 3.3 mg/kg (Table 1) for 3 wk after microprocessor-controller, 6 Rheodyne termination of the previous diet treatments. Hen-day production and mortality were calculated at 4-wk intervals for the duration of the experiment. Eggs from a 6-day 5 Millipore Corp., Bedford, MA 01730. 6 Beckman Instruments, Inc., Altex Division, San interval were weighed during the 16th wk and from a 5-day interval during the 27th Ramon, CA 94583. 7 Perkin-Elmer Corp., Instrument Division, Nor- wk of the experiment. Shell thicknesses of walk, CT 06856. 100 air-dried eggshells per treatment were 8 Kipp and Zonen, Bohemia, NY 11716. determined by a single measurement at the 9 Ranin Instrument Co., Inc., Woburn, MA 01801. 10 large end of the egg during the 18th wk of H and N International, Redmond, WA 98052. "United States Biochemical, Cleveland, OH 44122. the experiment. AH hens were weighed Egg Albumen Riboflavin Extraction
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486
SQUIRES AND NABER TABLE 1. Composition of experimental diets Experiment 2
Experiment 1 Ingredients and analysis
Basal diet
Recovery diet
Recovery diet
Basal diet FVi
54.5 6.5 26.5 2.6 8.4 .74 .04 .50 .20
2,797 17.8 .89 .32 .57 3.3 .50
53.4 10.2 2.5 20.1 3.0 9.0 1.0 .10 .50 .20
2,787 15.6 .75 .34 .56 3.6 .52
59.5
63.6
28.0 2.6 8.4 .74 .04 .50
2.5 20.1 3.0 9.0 1.0 .10 .50
.21
.21
2,791 18.0 .91 .32 .55 3.3 .50
2,807 15.2 .72 .33 .55 3.6 .52
1 Amount supplemented per kilogram of diet: manganese, 70 mg; zinc, 100 mg; iodized sodium chloride, 2,400 mg; selenium, .1 mg. 2 Amount supplemented per kilogram of diet: vitamin A, 2,000 IU; cholecalciferol, 1,000 ICU; vitamin E, 200IU; vitamin K, 1 mg; pantothenic acid, 15 mg; niacin, 15 mg; vitamin Bj2,5.5 /ig; Ethoxyquin (66%), 188 mg; Larvadex, 500 mg; and variable amounts of riboflavin (See Table 2). 3 Amount supplemented per kilogram of diet when the vitamin mix was added: vitamin A, 8,000 IU; cholecalciferol, 1,000 ICU; vitamin E, 20 IU; vitamin K, 1 mg; pantothenic acid, 15 mg; niacin, 15 mg; vitamin B^, 8 ftg; Ethoxyquin (66%), 188 mg; Larvadex, 500 mg; and variable amounts of riboflavin (See Table 2).
embryonic deficiency, such as clubbed down, were also noted. Incidence of meat and blood spots was determined by breakout of not less than 700 eggs per treatment from Weeks 17 through 19. Eggs were collected from each treatment during Weeks 0,1,2,4,6,8,12,16,20,24,28, and 30 for later HPLC riboflavin analysis. Twenty-four eggs per treatment were gathered during 1 day of the designated week and stored in an egg cooler maintained at 13 C. The eggs were broken out under subdued light within 3 days of collection and divided into yolk and albumen constituents. Excess albumen on the yolks was removed by rolling them on a paper towel. Pooled yolk and albumen samples from four eggs were placed into six plastic containers. These samples were placed in light-proof boxes and frozen at 12 Robbins Incubator Co., Denver, CO 80239. (Cur-20 C. Each of the six yolk and albumen rently serviced by Hawkhead International, Inc., samples per treatment for each time interOrange Park, FL 32073).
during the first and last weeks of the experiment. Hatchability of eggs from 25 hens in each treatment was determined by setting eggs in a Robbins Hatchomatic Incubator from the 15th and 16th wk after initiation of the experiment.12 The hens were artificially inseminated 1 wk prior to each hatchability study with pooled semen from Athens Canadian randombred males. Eggs were set randomly during the incubation phase but grouped by treatment during hatching. All unhatched eggs from the two settings were broken to determine fertility or approximate day of death during incubation. Embryonic mortality was recorded by dividing the 21-day incubation period into trimesters and reporting percentage mortality in each trimester. Signs of
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Ground corn Ground wheat Alfalfa meal (17% CP) Soybean meal (44% CP) Hydrolyzed fat (animal and vegetable) Limestone (37% Ca) Dicalcium phosphate DL-methionine Mineral mix 1 Vitamin mix 2 Vitamin mix 3 Calculated analysis ME, kcal/kg Protein Lysine Methionine Methionine and cystine Calcium Total phosphorus
487
EGGS INDICATING RIBOFLAVIN STATUS IN THE LAYING HEN TABLE 2. Dietary riboflavin treatments for Experiment 1 and 2 Riboflavin treatment (mg/kg of diet)i Variable
1.55 1 supplement, mg/kg dietary riboflavin, mg/kg of dietary requirement
1.58
0 1.55
.65 2.20
70 40 2 supplement, mg/kg diet dietary riboflavin, mg/kg diet of dietary requirement
2.20
100 58 0 1.58 72 42
4.40
4.43
2.85 4.40
8.80 7.25 8.80
200 116
400 232 2.85 4.43 201 117
1
National Research Council requirement (1984) is 2.2 mg/kg of diet. National Research Council requirement (1984) is 3.8 mg/kg of diet.
2
val were analyzed. Results were corrected for recovery and reported as micrograms of riboflavin per gram of yolk or albumen. Design of Experiment 2
determined daily during the course of the experiment. Hatchability of eggs from 25 hens in each treatment was determined by setting eggs from each day of the 4-wk experiment with data reported at twice weekly intervals. All unhatched eggs from the four settings were broken out to determine fertility or approximate day of death. Embryonic mortality was recorded by dividing the 21-day incubation period into trimesters and reporting percentage mortality in each trimester. Signs of embryonic deficiency, such as clubbed down and hemorrhagic embryos, were reported as percentage of all fertile eggs at twice weekly intervals. All newly hatched chicks were weighed in groups of 10 and an average weight per chick was calculated. Eggs were collected from each treatment at 2-day intervals for the 4 wk of the experiment for later HPLC analysis (described above). Sixteen eggs per treatment were gathered during each 2-day period and stored in an egg cooler at 13 C. Albumen was harvested from the eggs, pooled, and frozen as previously described. Results were corrected for recovery and reported as micrograms of riboflavin per gram of albumen at twice weekly intervals.
Two hundred H and N Single Comb White Leghorn hens,10 40 wk of age, were divided into two treatment groups of 100 hens each. The hens received a basal diet that was formulated to contain the requirements of all nutrients except riboflavin (National Research Council, 1984). The basal diet is shown in Table 1. One group of hens consumed an unsupplemented diet containing 1.58 mg riboflavin/kg of diet and the other group received a diet supplemented with pure riboflavin to contain a total of 4.43 mg riboflavin/kg of diet. These two treatments are shown in Table 2. The hens had received 10 h of light until the 32nd wk of age. At that time, the light was increased by 15 min every 2 wk up to a day length of 16 h. The experimental diets were fed for 3 wk. Both treatment groups were placed on a recovery diet (Table 1) for 1 wk after termination of the experimental diets. Other conditions were as described for Experiment 1. Hen-day production and mortality were calculated at weekly intervals. Eggs from Statistical Analysis each day were weighed through the entire 4 wk of the experiment. One measurement of The experimental data, except that for shell thicknesses from the large end of each hatchability, shell thickness, egg weight, egg in one-half the birds per treatment was and hen weight in Experiment 1, were
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Experiment Riboflavin Calculated Percentage Laying1 Breeding2 Experiment Riboflavin Calculated Percentage Laying1 Breeding2
488
SQUIRES AND NABER
TABLE 3. Changes in egg yolk riboflavin content from hens fed four dietary riboflavin treatments over time, Experiment l 1 Riboflavin treatment (mg/kg of diet) 1.55
(wk) 0 1 2 4 6 8 12 24 Pooled SEM 27 1 3 Pooled SEM
(pg/g) 7.2 8.0 5.9* 5.9* 6.1" 5.7* 6.3* 6.3* 7.0* 6.7* 4.8* 5.0* 4.2" 5.3* 5.0* 5.1* .32 — Return to recovery diet — 2.0b 2.4b 3.7* 4.3* 3.0 2.4 2.6 2.8 .28 7.2 3> 2.8C 3.0b 2.8b 2.0b 1.4b 1.6b
2.20
4.40
8.80
6.7 4.6b 3.9b 3.7b 3.9b 2.6b 2.0 b 2.2b
a_c Means in rows for both treatment and recovery periods with no common superscripts differ significantly (P < .05). ! Data reported as the mean of six observations for each treatment by time except Week 24 where n = 4. The two-way ANOVA showed riboflavin level significant at P < .0001, time effects significant at P < .0001, and the interaction of riboflavin level with time significant at P < .0001.
subjected to a two-way ANOVA (Snedecor and Cochran, 1980) using the General Linear Models procedure from SAS® software (SAS Institute, 1982). Means were calculated for the main effects, riboflavin level and time, as well as for the interaction of riboflavin levels at each time interval. Data for hatchability, shell thickness, egg weight, and hen weight from Experiment 1 were subjected to a one-way ANOVA (Snedecor and Cochran, 1980) and treatment means were calculated. Statistical comparisons among means were made by the Least Squares Mean procedure using SAS® software (SAS Institute, 1982) at a probability level of P < .05. RESULTS Experiment 1
Differences (P < .05) in egg yolk riboflavin content due to diet treatment appeared after 1 wk on experimental diets (Table 3).
TABLE 4. Changes in egg albumen riboflavin content from hens fed four dietary riboflavin treatments over time, Experiment l 1 Riboflavin treatment (mg/kg of diet) Time
1.55
2.20
(wk) 0 1 2 4 6 8 12 16 20 24
3.8 1.6b 1.1« l.Oc .9< .9C .7' .9' .6C .6C
3.5 2.0b 1.7b 1.6b 1.6b 1.6b 1.2b 1.9b 1.3b 1.1b
Pooled SEM 27
—
1 3 Pooled SEM
4.40
8.80
In
Khb' to'
3.7 3.7* 3.6* 3.9* 3.4* 3.3* 3.4* 2.9* 2.9* 2.9*
3.9 3.9* 3.6* 3.7* 3.7* 3.4* 3.6* 3.0* 3.1* 3.3*
.12 1.0b 1.0
]Return to recovery diet
1.0b 1.1
2.6* 1.3
— 2.7* 1.3
.11
'-"Means in rows for both treatment and recovery periods with no common superscripts differ significantly (P < .05). 1 Data reported as the mean of observations for each treatment by time. The two-way ANOVA showed riboflavin level significant at P < .001, time effects significant at P < .001, and the interaction of riboflavin level with time significant at P < .001.
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Time
At 2 wk, the egg yolk riboflavin content from the 1.55 mg/kg dietary treatment was significantly lower than that from the 2.2 mg/kg treatment. At this same time, the riboflavin egg yolk content from the 2.2 mg/kg treatment was significantly lower than those from both of the 4.4 and 8.8 mg/ kg treatments, which were not significantly different from each other. This pattern continued for the duration of the experiment. Egg yolk riboflavin concentrations were similar in all treatment groups after 3 wk on a recovery diet. This same pattern of riboflavin content change occurred in egg albumen (Table 4) but with a faster depletion rate than for egg yolk. Albumen depletion was essentially complete at Week 2 with eggs from the 1.55 mg/kg dietary treatment stabilizing at an average of .84 ng riboflavin/g albumen and from the 2.2 mg/ kg dietary treatment at 1.5 /xg/g- Yolk depletion was complete at 12 wk from the 1.55 mg/kg dietary treatment, stabilizing at
EGGS INDICATING RIBOFLAVIN STATUS IN THE LAYING HEN
TABLE 5. Changes in hen-day egg production of hens fed four dietary riboflavin treatments over time, Experiment l 1 Riboflavin
treatment
(mg/kg of diet) Time
1.55
(wk) 0 to 4 5 to 8 9 to 12 13 to 16 17 to 20 21 to 24 25 to 27 Pooled SEM 0 to 3 Pooled SEM
2.20
4.40
8.80
(%) 91.5 87.8 74.0b 63.8" 59.5b
91.8 87.8 83.0* 70.0b 70.5* 75.0b 73.3bc
65.8<= 68.3 C
78.0
91.3 86.5 86.0* 84.8* 75.8* 83.5* 81.0a 2.1
91.0 87.0 85.5* 84.3a 74.3a 80.0*b 79.0 ab
Return to recovery diet 78.0 79.3 76.3 2.4
a_c Means in rows with no common superscripts differ significantly (P < .05). !Data reported as the mean of three to four pooled observations of 7 days for each treatment by time. The two-way ANOVA showed riboflavin level significant at P < .0001, time effects significant at P < .0001, and the interaction of riboflavin level with time significant at P < .0001.
from hens fed the 2.2 mg/kg treatment was significantly lower than that of hens from the 4.4 and 8.8 mg/kg treatments. A shift toward earlier embryonic mortality was observed in dead embryos from hens fed the 1.55 mg/kg treatment. The incidence of blood spots appeared to be slightly higher
TABLE 6. Effects of four dietary riboflavin treatments on hatchability shell thickness, egg weight, and hen weight at selected weeks, Experiment l 1 Riboflavin treatment (mg/kg of diet) Variable Hatchability, % Week 16 Shell thickness, mm/100 Week 18 Egg weight, g Week 16 Week 27 Hen weight, kg Week 27 c
1.55
2.20
4.40
8.80
SEM
0<
56.8b
88.2 a
89.1 a
4.8
37.8 a
36.6b
35.3c
35.2c
.32
54.7c 56.9c
56.4b 60.3b
58.5* 62.0*
59.2* 62.5*
.48 .20
1.70b
1.84*
1.82*
1.86*
.03
*- Means in rows with no common superscripts differ significantly (P < .05). !Data reported as the mean of two pooled observations of 87 to 143 eggs for hatchability; 100 observations for shell thickness; five to six pooled observations of 21 to 47 eggs for egg weight; and 45 to 50 observations for hen weight. The one-way ANOVA showed significant effects of riboflavin level at P < .0005 for hatchability, P < .0001 for shell thickness, P < .0001 for egg weight at both times, and P < .0003 for hen weight.
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an average of 1.5 ng riboflavin/g yolk and from the 2.2 mg/kg dietary treatment at 2.1 Mg/gIn addition to the effects of dietary riboflavin level on both egg yolk and albumen riboflavin content at various times during the experiment, there were also significant main effects of treatment and time. Egg riboflavin concentrations decreased with time or age and the extent of the decrease in egg riboflavin content was dependent on dietary level of the vitamin (P < .0001). A decrease (P < .05) in egg production (Table 5) was noted for hens fed the 1.55 mg/kg dietary treatment compared with the three higher riboflavin-containing diets (2.2, 4.4, and 8.8 mg/kg) in the 9- to 12-wk period and through the remainder of the experiment. Following the 12th wk, egg production of hens fed the 2.2 mg/kg diet was usually (except Week 17 to 20) significantly lower than for those hens fed the two higher riboflavin levels (4.4 and 8.8 mg/kg). Egg production of hens from all treatments was not significantly different after 3 wk on the recovery diet. There were also reductions in egg production with time and changes in egg production due to diet treatment over time (P < .0001). At Week 16, the hatchability of eggs from hens fed the 1.55 mg/kg treatment was lower (P < .05) than that from hens fed from the 2.2 mg/kg treatment (Table 6). The hatchability of eggs
489
490
SQUIRES AND NABER TABLE 7. Effect of either a riboflavin-supplemented or unsupplemented diet on riboflavin albumen content and hatchability, Experiment 21
Time
1 to 3 4 to 7 Pooled I SEM
Hatchability
Riboflavin treatment (mg/kg diet)
Riboflavin treatment (mg/kg diet)
1.58
4.43
1.58
2.76 2.35a 2.34» 2.68* 2.65* 2.98*
92.5 63.5b 19.0b .8b 5.0b 4.8b
u W hi 2.44 1.3>>
.8" .82 b
.8b .92b
4.43 - v«y 87.5 85.5* 83.3* 84.0" 71.0* 73.5a 6.8
.15 Return to recovery diet — 2.59a 33.0b 87.0 3.10"
1.49 b 2.65 b
.14
74.7* 83.7 7.5
ab
' Means in rows for each variable with no common superscripts differ significantly (P < .05). 1 Data reported as the mean of five to eight pooled observations of four eggs for riboflavin albumen content at each treatment time and three to four pooled observations of 19 to 51 fertile eggs for hatchability at each treatment time. The two-way ANOVA showed riboflavin level significant at P < .0001, time effects significant at P < .0001, and the interaction of riboflavin level with time significant at P < .0001 for albumen riboflavin content and hatchability.
feeding and never returned to those of eggs from hens fed the supplemented diet, even after 7 days on a recovery diet (Table 7). Hatchability of eggs from hens fed the unsupplemented treatment was depressed (P < .05) during the 4- to 7-day period and remained significantly lower than that from the hens on the supplemented dietary treatment until 7 days after return to the recovery diet. The data show that a 40% decrease in the egg albumen riboflavin content on the unsupplemented treatment occurred at Day 4 to 7, where a minimum egg albumen content of approximately .8 fig riboflavin/g was established by Day 10. A 26% depression in hatchability on the unsupplemented treatment occurred from 4 to 7 days, and hatchability dropped to near 0 by Day 14. The albumen riboflavin values in eggs from the unsupplemented treatment during the period of depressed hatchability dropped from 1.30 |tg/g for Days 4 to 7 to a low of .8 ftg/g for Days 15 to 17. Experiment 2 There was also an increase (P < .05) in the Albumen riboflavin contents in eggs incidence of hemorrhagic embryos (hemorfrom hens fed the unsupplemented diet rhages within the embryo or the extrawere depressed (P < .05) after 4 to 7 days of embryonic circulatory system) from the
in the lowest treatments (1.55 and 1.73%) when compared with the two highest riboflavin treatments (.92 and .69%). Data for embryonic mortality and blood spot incidence was not analyzed statistically. At Week 18, the shell thickness of eggs from hens fed the 1.55 mg/kg dietary treatment was greater (P < .05) than eggs from the 2.2 mg/kg diet treatment (Table 6). The shell thickness of eggs from hens fed the 2.2 mg/kg dietary treatment was significantly greater than that for both the 4.4 mg/kg and 8.8 mg/kg treatments. At both Week 16 and 27, weight of eggs from hens fed the 1.55 mg/kg treatment was lower (P < .05) than from the 2.2 mg/kg treatment. The weight of eggs from hens fed the 2.2 mg/kg treatment was significantly lower than both that from the 4.4 and 8.8 mg/kg treatments. Hen weight at the end of the experiment was lower (P < .05) in hens fed 1.55 mg/kg treatment than in the other three treatments.
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(days) 0 to 3 4 to 7 8 to 10 11 to 14 15 to 17 18 to 21 Pooled I SEM
Albumen riboflavin content
EGGS INDICATING RIBOFLAVIN STATUS IN THE LAYING HEN
DISCUSSION The results of Experiment 1 demonstrate that dietary riboflavin deficiency affects egg concentrations of the vitamin after 1 wk. In the deficient diets (1.55 and 2.2 m g / k g of diet), both albumen and yolk riboflavin contents decreased markedly during the first 2 wk. This is rather surprising because yolk material takes u p to 2 wk to be deposited in the follicle, whereas albumen is deposited in the egg in a matter of hours. It was thought that
egg albumen would reflect dietary changes in a much shorter time than yolk. As this is not the case, most of the riboflavin must be deposited in yolk during the last several days of follicular maturation. It is also possible that RFBP riboflavin transfer to the follicle operates more efficiently under restricted availability than the albumen transfer mechanism. The albumen riboflavin in eggs from hens fed deficient diets decreased to a minimum level and stabilized after 2 wk. The initial decrease in yolk riboflavin content was large, but did not decrease to a minimum and stabilize until 12 wk. In a shorter term study, White et al. (1986) reported that riboflavin in albumen depletes more rapidly than yolk, but only for hens on very deficient diets. It is interesting to note that yolk riboflavin concentrations stabilized at about the same time that egg production in the 1.55 /xg/kg treatment started to decrease. Apparently, minimum egg yolk riboflavin contents are correlated closely with egg production. Because albumen riboflavin concentrations decrease to a minimum stable level long before egg production is adversely affected, albumen riboflavin analysis might be used to predict future decline in this important economic factor. Also, egg albumen riboflavin analysis is less complicated than the yolk riboflavin analysis due to the absence of lipid. The time for return of egg riboflavin concentrations to equilibrium levels after being placed on an adequate diet was shown to be between 1 to 3 wk. This is approximately the same time needed to deplete the eggs in the first place, indicating that the riboflavin transport system is not impaired by long-term deficiency. In Experiment 2, with more frequent sampling, egg albumen riboflavin contents were reduced within 4 to 7 days after hens were fed the deficient diet and repletion occurred during the same time interval after return to the recovery diet. In Experiment 1, hatchability of eggs from hens fed 1.55 and 2.2 mg of riboflavin/kg of diet was depressed at Week 16 (and probably at an earlier time but such data were not obtained in the first experiment). There was also a shift toward early embryonic mortality with a
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unsupplemented treatment compared with the supplemented treatment by Days 8 to 10 that continued through Day 3 of the recovery period (Table 8). In addition there was a significant increase in embryos displaying clubbed down during Days 4 to 7, in the unsupplemented treatment, which continued through Day 3 after being fed a recovery diet. In the unsupplemented dietary treatment, a numerical average of 27% of all fertile eggs displayed clubbed down after Day 7, and 11% showed signs of hemorrhaging. Sporadic incidence of clubbed down and hemorrhagic embryos also occurred on the riboflavin supplemented diet at the same time (1.2 and .2%, respectively). Embryonic mortality in eggs from hens fed the unsupplemented diet shifted from the last 7 days of incubation to the middle 7 days during the 2nd and 3rd wk of the experiment. There were no differences (P > .05) in egg production, chick weight, and egg weight between the supplemented and unsupplemented dietary treatments for the 21-day duration of the experiment and the 7-day recovery period. Shell thickness of eggs was numerically lower in the unsupplemented treatment, but only four nonconsecutive time periods had significant differences (P < .05) between treatments. In addition to the above diet effects at individual time intervals in Experiment 2, there was also a significant main effect of time or age on decreases in egg albumen riboflavin content. Hatchability decreased and incidence of both hemorrhagic embryos and clubbed down increased with time or age (P < .05). An interaction of time on dietary riboflavin level was found for each of the above parameters (P < .05).
491
492
SQUIRES AND NABER
TABLE 8. Effect of either a riboflavin-supplemented or unsupplemented diet on incidence of hemorrhagic embryos and clubbed down, Experiment 21 Hemorrhagic embryos
Clubbed down
Riboflavin treatment (mg/kg diet)
Riboflavin treatment (mg/kg diet)
Time
1.58
4.43
0 to 3 4 to 7 8 to 10 11 to 14 15 to 17 18 to 21 Pooled SEM
0 1.0 6.3* 15.5* 12.0* 12.5*
.3
1.58
4.43
0 11.0* 19.7* 31.8* 30.7* 22.8*
.30 0b 0b 0b 5.0b .8b
(%)
3.4
1.5 - Return to recovery diet 0b 13.7* 1.7 0
6.0* 0
1.6
0b 0 3.7
a b
- Means in rows for each variable with no common superscripts differ significantly (P < .05). iData reported as mean of three to four pooled observations of 19 to 51 fertile eggs for hemorrhagic embryos and clubbed down at each treatment by time. The two-way ANOVA showed riboflavin level significant at P < .0001, time effects significant at P < .0001, and the interaction of riboflavin level with time significant at P < .001 for both hemorrhagic embryos and clubbed down.
greater percentage of embryos dying during the first 2 wk of incubation. This suggests that some riboflavin is needed for early embryonic development, which conflicts with the findings of Maw (1954) that little riboflavin is needed until the 13th day of incubation. This could be tested by measuring oxygen consumption. This early embryonic death suggests that certain riboflavin-requiring, energysynthesizing pathways such as the electron transport system are limiting before the 13th day of incubation. Increasing severity of the deficiency causes more embryonic death at an earlier time period. Clubbed down, a common sign of riboflavin deficiency, was not detected until the 3rd wk of incubation, which means that this lesion does not have an opportunity to be expressed during severe deficiency. In Experiment 2, hatchability, clubbed down, and the incidence of hemorrhagic embryo were very closely related to egg riboflavin albumen contents. By Day 7, hatchability of the eggs from hens fed the unsupplemented dietary treatment was 26% lower than from the supplemented control, in which as clubbed down was 71% higher in the unsupplemented treat-
ment. By Day 10, incidence of hemorrhagic embryos was 75% higher in eggs from hens fed the unsupplemented diet. The egg albumen riboflavin content of the unsupplemented treatment averaged 1.3 Mg/g by Day 7. Therefore, the minimum critical riboflavin albumen level to support maximum hatchability and associated parameters is above 1.3 /*g/gHatchability and egg albumen riboflavin values of eggs from hens fed the unsupplemented diet in Experiment 2 returned to those of hens fed the supplemented diet 7 days after being placed on a recovery diet. The 7-day depletion is shorter than the 14-day period reported by Stamberg et al. (1946). This might be due to a genetic influence on riboflavin egg deposition in modern strains of layers. Genetic influence of riboflavin egg deposition was reported 35 yr ago in several popular breeds (Mayfield et al, 1955). Breed differences in the response of White Leghorns and Rhode Island Reds to riboflavin deficiency on egg production have been reported (Leeson et al., 1979). Another reason for the earlier appearance of problems associated with riboflavin deficiency might be due to the higher plane of
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1 to 3 4 to 7 Pooled SEM
0 0" 0" 1.0b 0b
EGGS INDICATING RIBOFLAVIN STATUS IN THE LAYING HEN
493
shown to increase the riboflavin requirement of laying hens (Onwudike and Adegbola, 1984). The incidence of hemorrhages either in extra-embryonic circulation or the embryo itself is commonly associated with embryonic death, especially early in development. The present research shows that riboflavin deficiency causes a large increase in hemorrhagic embryos. This observation, coupled with the previously documented shift to earlier embryonic mortality, reemphasizes the significant role of riboflavin in early development. The results of Experiment 2 indicate that 3 wk is not ample time for riboflavin deficiency to affect egg production or egg weight. As previously noted, egg albumen riboflavin concentrations can be used to predict future problems with egg production and weight, because the egg contents decrease long before these production parameters are affected. On the other hand, hatchability closely follows egg albumen riboflavin content. Riboflavin deficiency probably has no effect on shell thickness during a 3-wk period, but the data were inconclusive.
The short time needed to produce a riboflavin deficiency may explain why low level incidence of clubbed down in embryos exists in flocks that seem nutritionally sound. Hawes and Buss (1965) clearly demonstrated the presence of a mutant allele that can cause clubbed down. However, the current results, as well as other research (Coles and Cumber, 1955), have shown a greater influence of dietary riboflavin on incidence of clubbed down. It is possible that individual birds may experience marginal riboflavin deficiency caused by depressed feed intake over a period of several days. This depressed intake could be due to heat stress, disease, or social pressure from other birds higher in the peck order. Heat stress has been
Ashoor, S. H., G. J. Seperich, W. C. Monte, and J. Welty, 1983. HPLC determination of riboflavin in eggs and dairy products. J. Food Sci. 48: 92-95. Association of Official Analytical Chemists, 1980. Official Methods of Analysis, 13 ed. Association of Official Analytical Chemists, Washington, DC. Bethke, R. M., P. R. Record, and D. C. Kennard, 1933. Relationship of the vitamin G complex to hatchability and nutritive value of eggs. Poultry Sci. 12:332. Bethke, R. M., P. R. Record, and O.H.M. Wilder, 1937. Further studies on vitamin G in chick nutrition. Poultry Sci. 16:175-182. Coles, R., and F. Cumber, 1955. Observations on the relationship between riboflavin, hatchability and clubbed down. J. Agric. Sci. 46:191-198. Hawes, R. O., and E. G. Buss, 1965. The use of the riboflavinless gene (rd) in determining the cause of clubbed down. Poultry Sci. 44:773-778. Hunt, C. H., A. R. Winter, and R. M. Bethke, 1939.
ACKNOWLEDGMENT The authors would like to express their appreciation to Rene Waldman for her technical assistance. REFERENCES
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nutrition needed to support greater egg production. At Week 18 in Experiment 1, shell thickness appeared to actually increase at lower dietary riboflavin levels. However, at this same time, egg production and egg weight of hens fed deficient diets were reduced. Shell thickness and egg weight are known to be negatively correlated. Therefore, the increased shell thickness observed in eggs from hens on deficient diets was probably due to decreased egg production and egg weight and not to dietary riboflavin treatment. Egg weight at Week 16 and 27 decreased with decreasing dietary riboflavin levels. This has not been previously noted, but suggests another important economic parameter that might be monitored by egg riboflavin analysis. The weight of hens fed the 1.55 m g / k g diet was less than that of hens fed the higher riboflavin levels. This may demonstrate impairment of energy metabolism in hens fed deficient diets over the long term, and agrees with the findings of Petersen et al. (1947a,b). Hatchability, egg weight, and egg production were all shown to be depressed in hens fed both the 1.55 and 2.2 m g / k g dietary treatments at 16 wk. Therefore, after 16 wk levels of 1.9 /xg riboflavin/g albumen in eggs from hens fed the 2.2 m g / k g dietary treatment were associated with reduced reproductive function. At the two highest dietary treatment levels, egg albumen riboflavin levels of 2.9 Mg/g were associated with maximum reproduction.
494
SQUIRES AND NABER production and reproduction in the humid tropics. Trop. Agric. 61:205-207. Petersen, C. F., C. E. Lampman, and O. E. Stamberg, 1947a. Effect of riboflavin intake on egg production and riboflavin content of eggs. Poultry Sci. 26:180-186. Petersen, C. F., C. E. Lampman, and O. E. Stamberg, 1947b. Effect of riboflavin intake on hatchability of eggs from battery confined hens. Poultry Sci. 26:187-191. SAS Institute, 1982. SAS® User's Guide: Statistics. SAS Institute Inc., Cary, NC. Snedecor, G. W., and W. G. Cochran, 1980. Statistical Methods. 7th ed. The Iowa State University Press, Ames, IA. Stamberg, O. E., C. F. Peterson, and C. E. Lampman, 1946. Effect of riboflavin intake on the content of egg whites and yolks from individual hens. Poultry Sci. 25:320-326. Wehling, R. L., and D. L. Wetzel, 1984. Simultaneous determination of pyridoxine, riboflavin, and thiamin in fortified cereal products by high performance liquid chromatography. J. Agric. Food Chem. 32:1326-1331. White, H. B., m, J. Armstrong, and C. C. Whitehead, 1986. Riboflavin-binding protein concentration and fractional saturation in chicken eggs as a function of dietary riboflavin. Biochem. J. 238: 671-675. Woodcock, E. A., J. J. Warthesen, and T. P. Labuza, 1982. Riboflavin photochemical degradation in pasta measured by high performance liquid chromatography. J. Food Sci. 47:545-555.
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Further studies on the riboflavin requirements of the chicken. Poultry Sci. 18:330-336. Leeson, S., B. S. Reinhart, and J. D. Summers, 1979. Response of White Leghorn and Rhode Island Red breeder hens to dietary deficiencies of synthetic vitamins. 2. Egg production, hatchability and chick growth. Can. J. Anim. Sci. 59: 561-567. Lepkovsky, S., and T. H. Jukes, 1935. The vitamin G requirements of the chick. J. Biol. Chem. Ill: 119-131. Lepkovsky, S., L. W. Taylor, T. H. Jukes, and H. J. Almquist, 1938. The effect of riboflavin and the filtrate factor on egg production and hatchability. Hilgardia 11:559-591. Maw, A.J.G., 1954. Inherited riboflavin deficiency in chicken eggs. Poultry Sci. 33:216-217. Mayfield, H. L., R. R. Roehm, and A. F. Beeckler, 1955. Riboflavin and thiamine content of eggs from New Hampshire and White Leghorn hens fed diets containing condensed fish or dried whole solubles. Poultry Sci. 34:1106-1111. 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. Norris, L. C, and J. C. Bauernfeind, 1940. Effect of level of dietary riboflavin upon quality stored in eggs and rate of storage. Food Res. 5:521-532. Onwudike, O. C, and A. A. Adegbola, 1984. Riboflavin requirement of laying hens for egg
1993 Poultry Science 72:483-494
INTRODUCTION Diagnostic techniques to assess laying flock vitamin status are needed due to dated requirement recommendations that may not apply to modern strains of laying hens and the increasing demand for higher quality eggs by the health-
Received for publication April 1, 1992. Accepted for publication November 20, 1992. Salaries and research support provided by State and Federal Funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University. Manuscript Number 94-92. 2 To whom correspondence should be addressed.
conscious consumer. One such technique to assess the vitamin status of the laying hen might be through the analysis of her eggs. In a review of the literature, Naber (1979) reported that egg riboflavin was markedly influenced by dietary changes. Establishment of critical egg riboflavin concentrations might be used to determine the adequacy of dietary supplementation, yielding a direct measure of vitamin bioavailability that feed analysis does not provide. Data on the relationship between laying hen diet and egg vitamin contents could be used to establish egg quality standards and diet to egg vitamin transfer efficiency.
483
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ABSTRACT Two experiments determined the effect of dietary riboflavin supplementation on egg yolk and albumen riboflavin concentrations, egg production, egg weight, shell thickness, hen weight, hatchability, incidence of clubbed down, and incidence of hemorrhagic embryos. In the first experiment, hens were fed rations containing 1.55,2.20,4.40, and 8.80 mg of riboflavin/kg of diet for 27 wk. Significant (P < .05) depressions in both yolk and albumen riboflavin concentrations were noted at the two lower riboflavin levels after 1 wk. Egg production, egg weight, hatchability, and hen weight were all significantly depressed by the two lower riboflavin levels later in the experiment when compared with the two higher levels. Results indicate that egg riboflavin concentrations are related to important production parameters that may be used to predict future dietary riboflavin inadequacies. In the second experiment, hens were fed either an unsupplemented diet or a riboflavin-adequate diet. Measurements of egg albumen riboflavin content, egg production, hatchability, and embryo abnormalities were made twice each week. Results showed depressed albumen riboflavin concentrations and hatchability and increased incidence of hemorrhagic embryos and clubbed down without changes in egg production during the 4- to 7-day period following feeding of the unsupplemented diet. These results show that low albumen riboflavin content immediately affect hatchability and embryonic development. The estimated minimum critical albumen riboflavin concentrations needed to support maximum reproductive function are between 1.9 and 2.9 jug of riboflavin/g of egg albumen. These critical values might be used to evaluate riboflavin status of laying and breeding flocks. (Key words: egg riboflavin content, diet, reproductive function, hatchability, nutritional status)
484
SQUIRES AND NABER
3
Fisher Scientific, Pittsburgh, PA 15219. Ivan Sorvall, Inc., Newtown, CT 06470.
4
riboflavin-binding protein (RFBP). This protein controls riboflavin transfer from plasma to the ovary and magnum and limits the amount of riboflavin in eggs. Amounts of dietary riboflavin were shown to have no effect on the amount of RFBP in the egg or plasma. The decreased amount of riboflavin in eggs from hens on deficient diets is due to riboflavin insufficiency for binding to the protein and not to lack of RFBP itself. One objective of the present study was to determine the effects of feeding practical rations containing graded levels of riboflavin on egg riboflavin content and important production parameters of modern strains of layers for selected periods of time. A second objective was to establish the time relationships between reductions in egg riboflavin contents and decreases in important production parameters to determine minimum critical egg riboflavin values needed for maximum egg production and reproductive function. MATERIALS AND METHODS Egg Yolk Riboflavin Extraction The extraction method is a modified procedure of the Association of Official Analytical Chemists (1980). The entire procedure was carried out under subdued light to protect the light-sensitive riboflavin. Ten grams (+ .02) of thawed liquid yolk and 50 mL of .1 N HC1 were placed in a 125-mL Erlenmeyer flask. The content was homogenized with a glass stir rod and placed in an autoclave at 121C (1.1 kg/cm) for 30 min. After cooling, 5 mL of 2 M sodium acetate were added to adjust the pH to between 4.5 and 5.0. Two milliliters of 50% (wt:vol) trichloroacetic acid were added to further adjust the pH to between 3.5 and 3.8 and to coagulate proteins. The content of the flask was filtered through 18.5-cm Whatman Number 1 Dacer3 and .1 N HC1 was used to bring the filtrate up to a 50-mL volume. If the filtrate was cloudy, it was centrifuged at 27,000 x g at 10 C for 15 min in a Sorvall RC2-B centrifuge equipped with a SS-34 rotor4 and then refiltered.
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Bethke et al. (1933) first noted a relationship between the diet of the laying hen, egg vitamin G levels (vitamin B complex), and the growth and livability of the resulting chicks. Lepkovsky and Jukes (1935) reported that chicks had an absolute requirement for vitamin G separate from the vitamin B complex and that the vitamin was probably a flavin compound. Bethke et al. (1937) identified riboflavin as the substance in the vitamin G complex responsible for the enhancement of hatchability of chicks. Lepkovsky et al. (1938) showed the relationship between laying hen diet, egg riboflavin concentrations, and various embryonic deficiency signs by measuring albumen riboflavin concentrations by a photometric assay. Hunt et al. (1939) showed that the riboflavin requirement for maximum hatchability was higher than that for egg production. Norris and Bauernfeind (1940) noted that there appeared to be an upper limit to the amount of riboflavin deposited in eggs. Stamberg et al. (1946) showed that 8 days were needed to bring egg riboflavin contents back to normal in deficient hens following the consumption of adequately supplemented diets. In 1947 (a,b), Petersen et al. established the modern riboflavin requirement (National Research Council, 1984) of 2.2 mg of riboflavin/kg of diet for egg production, with the required level for maximum hatchability being somewhat higher. In 1984, Onwudike and Adegbola demonstrated an 86% increase in the riboflavin requirement for laying hens and a 64% increase for breeding in the tropics. White et al. (1986) indicated that riboflavin in egg albumen depletes more rapidly than in yolk but only in hens fed very deficient diets. Normal egg yolk riboflavin concentrations are 4 to 5 /*g/g and egg albumen levels are 3 to 4 /tg/g. Dietary riboflavin levels influence egg riboflavin deposition in a linear fashion up to 4.2 mg/kg of diet; however, supplementation above this level does not increase the amount in egg appreciably. This ceiling on diet-to-egg transfer of riboflavin is due to
EGGS INDICATING RIBOFLAVIN STATUS IN THE LAYING HEN
485
Model 7120 sample injector,6 Perkin-Elmer Model 650-10LC fluorescence specThis extraction is a modified procedure trophotometer,7 and a Kipp and Zonen BDof Ashoor et ah (1983). The entire procedure 41 recorder.8 was carried out under subdued light. Ten A silicon Merck Lichrosorb Model RP-18 grams (± .02) of thawed liquid albumen and column9 (10-/an mesh, 25 cm x 4 mm) was 25 mL of a HPLC grade methanol-distilled used to separate and quantify the vitamin. water solution (1:1) were placed into a The solvent system was isocratic with an 50-mL polycarbonate centrifuge tube. After eluting solvent consisting by volume of stirring gently for no more than 5 min with HPLC grade methanol-distilled watera Fisher Dyna-Mix Model 143 variable glacial acetic acid (32:68:.l). The column speed mixer,3 5.0 mL glacial acetic acid was was not washed between runs, but was added dropwise with constant stirring to rinsed at the end of the day with methanoladjust the pH to 3 and to precipitate water (20:80). The flow rate was 2.0 mL/ proteins. The sample was then centrifuged min with a pressure of 140 to 210 kg/cm 2 . as described for yolk samples. After cen- The fluorescence detector was set at an trifugation, the supernatant was decanted excitation wavelength of 280 nm with into a foil-wrapped, 50-mL volumetric emission at 510 nm. flask. Ten milliliters of an HPLC grade methanol-distilled water-acetic acid solution (42:42:16) was added to the precipitate Design of Experiment 1 in the centrifuge tube, which was then Two hundred H and N Single Comb stirred, centrifuged, and decanted as before. White Leghorn hens,10 28 wk of age, were Extraction with the 10 mL methanol-water- divided into four treatment groups of 50 acetic acid was repeated, and the volumet- hens each. The hens received a basal diet ric flask was filled to volume with this same that was formulated to contain the requiresolution. Twenty-five drops of the yolk or ments of all nutrients except riboflavin albumen filtrate were passed through a .45- (National Research Council, 1984). The fi Millipore filter5 into a 4-mL sample vial. basal diet is shown in Table 1. The four The sample was stored in a freezer for later dietary treatments consisted of supHPLC analysis. plementing the basal diet with 0, .65, 2.85, and 7.25 mg of pure riboflavin/kg of diet.11 Total estimated dietary riboflavin and reHigh-Performance Liquid quirement levels for egg production and Chromatography Measurement hatchability are shown in Table 2. Hens in of Riboflavin all four treatments received ad libitum access This procedure is the same as that to feed and water. The hens were housed described by Ashoor et ah (1983) except for individually in an environmentally conthe column used (Wehling and Wetzel, trolled poultry house. Sixteen hours of light 1984) and the emission wavelength meas- were provided daily from 0500 to 2100 h. ured (Woodcock et ah, 1982). Twenty The experimental diets were fed for 27 wk. microliters of extract was injected into a All four treatment groups were placed on a HPLC apparatus consisting of an Altex recovery diet containing a riboflavin level Model 110A pump, 6 Altex Model 420 of 3.3 mg/kg (Table 1) for 3 wk after microprocessor-controller, 6 Rheodyne termination of the previous diet treatments. Hen-day production and mortality were calculated at 4-wk intervals for the duration of the experiment. Eggs from a 6-day 5 Millipore Corp., Bedford, MA 01730. 6 Beckman Instruments, Inc., Altex Division, San interval were weighed during the 16th wk and from a 5-day interval during the 27th Ramon, CA 94583. 7 Perkin-Elmer Corp., Instrument Division, Nor- wk of the experiment. Shell thicknesses of walk, CT 06856. 100 air-dried eggshells per treatment were 8 Kipp and Zonen, Bohemia, NY 11716. determined by a single measurement at the 9 Ranin Instrument Co., Inc., Woburn, MA 01801. 10 large end of the egg during the 18th wk of H and N International, Redmond, WA 98052. "United States Biochemical, Cleveland, OH 44122. the experiment. AH hens were weighed Egg Albumen Riboflavin Extraction
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486
SQUIRES AND NABER TABLE 1. Composition of experimental diets Experiment 2
Experiment 1 Ingredients and analysis
Basal diet
Recovery diet
Recovery diet
Basal diet FVi
54.5 6.5 26.5 2.6 8.4 .74 .04 .50 .20
2,797 17.8 .89 .32 .57 3.3 .50
53.4 10.2 2.5 20.1 3.0 9.0 1.0 .10 .50 .20
2,787 15.6 .75 .34 .56 3.6 .52
59.5
63.6
28.0 2.6 8.4 .74 .04 .50
2.5 20.1 3.0 9.0 1.0 .10 .50
.21
.21
2,791 18.0 .91 .32 .55 3.3 .50
2,807 15.2 .72 .33 .55 3.6 .52
1 Amount supplemented per kilogram of diet: manganese, 70 mg; zinc, 100 mg; iodized sodium chloride, 2,400 mg; selenium, .1 mg. 2 Amount supplemented per kilogram of diet: vitamin A, 2,000 IU; cholecalciferol, 1,000 ICU; vitamin E, 200IU; vitamin K, 1 mg; pantothenic acid, 15 mg; niacin, 15 mg; vitamin Bj2,5.5 /ig; Ethoxyquin (66%), 188 mg; Larvadex, 500 mg; and variable amounts of riboflavin (See Table 2). 3 Amount supplemented per kilogram of diet when the vitamin mix was added: vitamin A, 8,000 IU; cholecalciferol, 1,000 ICU; vitamin E, 20 IU; vitamin K, 1 mg; pantothenic acid, 15 mg; niacin, 15 mg; vitamin B^, 8 ftg; Ethoxyquin (66%), 188 mg; Larvadex, 500 mg; and variable amounts of riboflavin (See Table 2).
embryonic deficiency, such as clubbed down, were also noted. Incidence of meat and blood spots was determined by breakout of not less than 700 eggs per treatment from Weeks 17 through 19. Eggs were collected from each treatment during Weeks 0,1,2,4,6,8,12,16,20,24,28, and 30 for later HPLC riboflavin analysis. Twenty-four eggs per treatment were gathered during 1 day of the designated week and stored in an egg cooler maintained at 13 C. The eggs were broken out under subdued light within 3 days of collection and divided into yolk and albumen constituents. Excess albumen on the yolks was removed by rolling them on a paper towel. Pooled yolk and albumen samples from four eggs were placed into six plastic containers. These samples were placed in light-proof boxes and frozen at 12 Robbins Incubator Co., Denver, CO 80239. (Cur-20 C. Each of the six yolk and albumen rently serviced by Hawkhead International, Inc., samples per treatment for each time interOrange Park, FL 32073).
during the first and last weeks of the experiment. Hatchability of eggs from 25 hens in each treatment was determined by setting eggs in a Robbins Hatchomatic Incubator from the 15th and 16th wk after initiation of the experiment.12 The hens were artificially inseminated 1 wk prior to each hatchability study with pooled semen from Athens Canadian randombred males. Eggs were set randomly during the incubation phase but grouped by treatment during hatching. All unhatched eggs from the two settings were broken to determine fertility or approximate day of death during incubation. Embryonic mortality was recorded by dividing the 21-day incubation period into trimesters and reporting percentage mortality in each trimester. Signs of
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Ground corn Ground wheat Alfalfa meal (17% CP) Soybean meal (44% CP) Hydrolyzed fat (animal and vegetable) Limestone (37% Ca) Dicalcium phosphate DL-methionine Mineral mix 1 Vitamin mix 2 Vitamin mix 3 Calculated analysis ME, kcal/kg Protein Lysine Methionine Methionine and cystine Calcium Total phosphorus
487
EGGS INDICATING RIBOFLAVIN STATUS IN THE LAYING HEN TABLE 2. Dietary riboflavin treatments for Experiment 1 and 2 Riboflavin treatment (mg/kg of diet)i Variable
1.55 1 supplement, mg/kg dietary riboflavin, mg/kg of dietary requirement
1.58
0 1.55
.65 2.20
70 40 2 supplement, mg/kg diet dietary riboflavin, mg/kg diet of dietary requirement
2.20
100 58 0 1.58 72 42
4.40
4.43
2.85 4.40
8.80 7.25 8.80
200 116
400 232 2.85 4.43 201 117
1
National Research Council requirement (1984) is 2.2 mg/kg of diet. National Research Council requirement (1984) is 3.8 mg/kg of diet.
2
val were analyzed. Results were corrected for recovery and reported as micrograms of riboflavin per gram of yolk or albumen. Design of Experiment 2
determined daily during the course of the experiment. Hatchability of eggs from 25 hens in each treatment was determined by setting eggs from each day of the 4-wk experiment with data reported at twice weekly intervals. All unhatched eggs from the four settings were broken out to determine fertility or approximate day of death. Embryonic mortality was recorded by dividing the 21-day incubation period into trimesters and reporting percentage mortality in each trimester. Signs of embryonic deficiency, such as clubbed down and hemorrhagic embryos, were reported as percentage of all fertile eggs at twice weekly intervals. All newly hatched chicks were weighed in groups of 10 and an average weight per chick was calculated. Eggs were collected from each treatment at 2-day intervals for the 4 wk of the experiment for later HPLC analysis (described above). Sixteen eggs per treatment were gathered during each 2-day period and stored in an egg cooler at 13 C. Albumen was harvested from the eggs, pooled, and frozen as previously described. Results were corrected for recovery and reported as micrograms of riboflavin per gram of albumen at twice weekly intervals.
Two hundred H and N Single Comb White Leghorn hens,10 40 wk of age, were divided into two treatment groups of 100 hens each. The hens received a basal diet that was formulated to contain the requirements of all nutrients except riboflavin (National Research Council, 1984). The basal diet is shown in Table 1. One group of hens consumed an unsupplemented diet containing 1.58 mg riboflavin/kg of diet and the other group received a diet supplemented with pure riboflavin to contain a total of 4.43 mg riboflavin/kg of diet. These two treatments are shown in Table 2. The hens had received 10 h of light until the 32nd wk of age. At that time, the light was increased by 15 min every 2 wk up to a day length of 16 h. The experimental diets were fed for 3 wk. Both treatment groups were placed on a recovery diet (Table 1) for 1 wk after termination of the experimental diets. Other conditions were as described for Experiment 1. Hen-day production and mortality were calculated at weekly intervals. Eggs from Statistical Analysis each day were weighed through the entire 4 wk of the experiment. One measurement of The experimental data, except that for shell thicknesses from the large end of each hatchability, shell thickness, egg weight, egg in one-half the birds per treatment was and hen weight in Experiment 1, were
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Experiment Riboflavin Calculated Percentage Laying1 Breeding2 Experiment Riboflavin Calculated Percentage Laying1 Breeding2
488
SQUIRES AND NABER
TABLE 3. Changes in egg yolk riboflavin content from hens fed four dietary riboflavin treatments over time, Experiment l 1 Riboflavin treatment (mg/kg of diet) 1.55
(wk) 0 1 2 4 6 8 12 24 Pooled SEM 27 1 3 Pooled SEM
(pg/g) 7.2 8.0 5.9* 5.9* 6.1" 5.7* 6.3* 6.3* 7.0* 6.7* 4.8* 5.0* 4.2" 5.3* 5.0* 5.1* .32 — Return to recovery diet — 2.0b 2.4b 3.7* 4.3* 3.0 2.4 2.6 2.8 .28 7.2 3> 2.8C 3.0b 2.8b 2.0b 1.4b 1.6b
2.20
4.40
8.80
6.7 4.6b 3.9b 3.7b 3.9b 2.6b 2.0 b 2.2b
a_c Means in rows for both treatment and recovery periods with no common superscripts differ significantly (P < .05). ! Data reported as the mean of six observations for each treatment by time except Week 24 where n = 4. The two-way ANOVA showed riboflavin level significant at P < .0001, time effects significant at P < .0001, and the interaction of riboflavin level with time significant at P < .0001.
subjected to a two-way ANOVA (Snedecor and Cochran, 1980) using the General Linear Models procedure from SAS® software (SAS Institute, 1982). Means were calculated for the main effects, riboflavin level and time, as well as for the interaction of riboflavin levels at each time interval. Data for hatchability, shell thickness, egg weight, and hen weight from Experiment 1 were subjected to a one-way ANOVA (Snedecor and Cochran, 1980) and treatment means were calculated. Statistical comparisons among means were made by the Least Squares Mean procedure using SAS® software (SAS Institute, 1982) at a probability level of P < .05. RESULTS Experiment 1
Differences (P < .05) in egg yolk riboflavin content due to diet treatment appeared after 1 wk on experimental diets (Table 3).
TABLE 4. Changes in egg albumen riboflavin content from hens fed four dietary riboflavin treatments over time, Experiment l 1 Riboflavin treatment (mg/kg of diet) Time
1.55
2.20
(wk) 0 1 2 4 6 8 12 16 20 24
3.8 1.6b 1.1« l.Oc .9< .9C .7' .9' .6C .6C
3.5 2.0b 1.7b 1.6b 1.6b 1.6b 1.2b 1.9b 1.3b 1.1b
Pooled SEM 27
—
1 3 Pooled SEM
4.40
8.80
In
Khb' to'
3.7 3.7* 3.6* 3.9* 3.4* 3.3* 3.4* 2.9* 2.9* 2.9*
3.9 3.9* 3.6* 3.7* 3.7* 3.4* 3.6* 3.0* 3.1* 3.3*
.12 1.0b 1.0
]Return to recovery diet
1.0b 1.1
2.6* 1.3
— 2.7* 1.3
.11
'-"Means in rows for both treatment and recovery periods with no common superscripts differ significantly (P < .05). 1 Data reported as the mean of observations for each treatment by time. The two-way ANOVA showed riboflavin level significant at P < .001, time effects significant at P < .001, and the interaction of riboflavin level with time significant at P < .001.
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Time
At 2 wk, the egg yolk riboflavin content from the 1.55 mg/kg dietary treatment was significantly lower than that from the 2.2 mg/kg treatment. At this same time, the riboflavin egg yolk content from the 2.2 mg/kg treatment was significantly lower than those from both of the 4.4 and 8.8 mg/ kg treatments, which were not significantly different from each other. This pattern continued for the duration of the experiment. Egg yolk riboflavin concentrations were similar in all treatment groups after 3 wk on a recovery diet. This same pattern of riboflavin content change occurred in egg albumen (Table 4) but with a faster depletion rate than for egg yolk. Albumen depletion was essentially complete at Week 2 with eggs from the 1.55 mg/kg dietary treatment stabilizing at an average of .84 ng riboflavin/g albumen and from the 2.2 mg/ kg dietary treatment at 1.5 /xg/g- Yolk depletion was complete at 12 wk from the 1.55 mg/kg dietary treatment, stabilizing at
EGGS INDICATING RIBOFLAVIN STATUS IN THE LAYING HEN
TABLE 5. Changes in hen-day egg production of hens fed four dietary riboflavin treatments over time, Experiment l 1 Riboflavin
treatment
(mg/kg of diet) Time
1.55
(wk) 0 to 4 5 to 8 9 to 12 13 to 16 17 to 20 21 to 24 25 to 27 Pooled SEM 0 to 3 Pooled SEM
2.20
4.40
8.80
(%) 91.5 87.8 74.0b 63.8" 59.5b
91.8 87.8 83.0* 70.0b 70.5* 75.0b 73.3bc
65.8<= 68.3 C
78.0
91.3 86.5 86.0* 84.8* 75.8* 83.5* 81.0a 2.1
91.0 87.0 85.5* 84.3a 74.3a 80.0*b 79.0 ab
Return to recovery diet 78.0 79.3 76.3 2.4
a_c Means in rows with no common superscripts differ significantly (P < .05). !Data reported as the mean of three to four pooled observations of 7 days for each treatment by time. The two-way ANOVA showed riboflavin level significant at P < .0001, time effects significant at P < .0001, and the interaction of riboflavin level with time significant at P < .0001.
from hens fed the 2.2 mg/kg treatment was significantly lower than that of hens from the 4.4 and 8.8 mg/kg treatments. A shift toward earlier embryonic mortality was observed in dead embryos from hens fed the 1.55 mg/kg treatment. The incidence of blood spots appeared to be slightly higher
TABLE 6. Effects of four dietary riboflavin treatments on hatchability shell thickness, egg weight, and hen weight at selected weeks, Experiment l 1 Riboflavin treatment (mg/kg of diet) Variable Hatchability, % Week 16 Shell thickness, mm/100 Week 18 Egg weight, g Week 16 Week 27 Hen weight, kg Week 27 c
1.55
2.20
4.40
8.80
SEM
0<
56.8b
88.2 a
89.1 a
4.8
37.8 a
36.6b
35.3c
35.2c
.32
54.7c 56.9c
56.4b 60.3b
58.5* 62.0*
59.2* 62.5*
.48 .20
1.70b
1.84*
1.82*
1.86*
.03
*- Means in rows with no common superscripts differ significantly (P < .05). !Data reported as the mean of two pooled observations of 87 to 143 eggs for hatchability; 100 observations for shell thickness; five to six pooled observations of 21 to 47 eggs for egg weight; and 45 to 50 observations for hen weight. The one-way ANOVA showed significant effects of riboflavin level at P < .0005 for hatchability, P < .0001 for shell thickness, P < .0001 for egg weight at both times, and P < .0003 for hen weight.
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an average of 1.5 ng riboflavin/g yolk and from the 2.2 mg/kg dietary treatment at 2.1 Mg/gIn addition to the effects of dietary riboflavin level on both egg yolk and albumen riboflavin content at various times during the experiment, there were also significant main effects of treatment and time. Egg riboflavin concentrations decreased with time or age and the extent of the decrease in egg riboflavin content was dependent on dietary level of the vitamin (P < .0001). A decrease (P < .05) in egg production (Table 5) was noted for hens fed the 1.55 mg/kg dietary treatment compared with the three higher riboflavin-containing diets (2.2, 4.4, and 8.8 mg/kg) in the 9- to 12-wk period and through the remainder of the experiment. Following the 12th wk, egg production of hens fed the 2.2 mg/kg diet was usually (except Week 17 to 20) significantly lower than for those hens fed the two higher riboflavin levels (4.4 and 8.8 mg/kg). Egg production of hens from all treatments was not significantly different after 3 wk on the recovery diet. There were also reductions in egg production with time and changes in egg production due to diet treatment over time (P < .0001). At Week 16, the hatchability of eggs from hens fed the 1.55 mg/kg treatment was lower (P < .05) than that from hens fed from the 2.2 mg/kg treatment (Table 6). The hatchability of eggs
489
490
SQUIRES AND NABER TABLE 7. Effect of either a riboflavin-supplemented or unsupplemented diet on riboflavin albumen content and hatchability, Experiment 21
Time
1 to 3 4 to 7 Pooled I SEM
Hatchability
Riboflavin treatment (mg/kg diet)
Riboflavin treatment (mg/kg diet)
1.58
4.43
1.58
2.76 2.35a 2.34» 2.68* 2.65* 2.98*
92.5 63.5b 19.0b .8b 5.0b 4.8b
u W hi 2.44 1.3>>
.8" .82 b
.8b .92b
4.43 - v«y 87.5 85.5* 83.3* 84.0" 71.0* 73.5a 6.8
.15 Return to recovery diet — 2.59a 33.0b 87.0 3.10"
1.49 b 2.65 b
.14
74.7* 83.7 7.5
ab
' Means in rows for each variable with no common superscripts differ significantly (P < .05). 1 Data reported as the mean of five to eight pooled observations of four eggs for riboflavin albumen content at each treatment time and three to four pooled observations of 19 to 51 fertile eggs for hatchability at each treatment time. The two-way ANOVA showed riboflavin level significant at P < .0001, time effects significant at P < .0001, and the interaction of riboflavin level with time significant at P < .0001 for albumen riboflavin content and hatchability.
feeding and never returned to those of eggs from hens fed the supplemented diet, even after 7 days on a recovery diet (Table 7). Hatchability of eggs from hens fed the unsupplemented treatment was depressed (P < .05) during the 4- to 7-day period and remained significantly lower than that from the hens on the supplemented dietary treatment until 7 days after return to the recovery diet. The data show that a 40% decrease in the egg albumen riboflavin content on the unsupplemented treatment occurred at Day 4 to 7, where a minimum egg albumen content of approximately .8 fig riboflavin/g was established by Day 10. A 26% depression in hatchability on the unsupplemented treatment occurred from 4 to 7 days, and hatchability dropped to near 0 by Day 14. The albumen riboflavin values in eggs from the unsupplemented treatment during the period of depressed hatchability dropped from 1.30 |tg/g for Days 4 to 7 to a low of .8 ftg/g for Days 15 to 17. Experiment 2 There was also an increase (P < .05) in the Albumen riboflavin contents in eggs incidence of hemorrhagic embryos (hemorfrom hens fed the unsupplemented diet rhages within the embryo or the extrawere depressed (P < .05) after 4 to 7 days of embryonic circulatory system) from the
in the lowest treatments (1.55 and 1.73%) when compared with the two highest riboflavin treatments (.92 and .69%). Data for embryonic mortality and blood spot incidence was not analyzed statistically. At Week 18, the shell thickness of eggs from hens fed the 1.55 mg/kg dietary treatment was greater (P < .05) than eggs from the 2.2 mg/kg diet treatment (Table 6). The shell thickness of eggs from hens fed the 2.2 mg/kg dietary treatment was significantly greater than that for both the 4.4 mg/kg and 8.8 mg/kg treatments. At both Week 16 and 27, weight of eggs from hens fed the 1.55 mg/kg treatment was lower (P < .05) than from the 2.2 mg/kg treatment. The weight of eggs from hens fed the 2.2 mg/kg treatment was significantly lower than both that from the 4.4 and 8.8 mg/kg treatments. Hen weight at the end of the experiment was lower (P < .05) in hens fed 1.55 mg/kg treatment than in the other three treatments.
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(days) 0 to 3 4 to 7 8 to 10 11 to 14 15 to 17 18 to 21 Pooled I SEM
Albumen riboflavin content
EGGS INDICATING RIBOFLAVIN STATUS IN THE LAYING HEN
DISCUSSION The results of Experiment 1 demonstrate that dietary riboflavin deficiency affects egg concentrations of the vitamin after 1 wk. In the deficient diets (1.55 and 2.2 m g / k g of diet), both albumen and yolk riboflavin contents decreased markedly during the first 2 wk. This is rather surprising because yolk material takes u p to 2 wk to be deposited in the follicle, whereas albumen is deposited in the egg in a matter of hours. It was thought that
egg albumen would reflect dietary changes in a much shorter time than yolk. As this is not the case, most of the riboflavin must be deposited in yolk during the last several days of follicular maturation. It is also possible that RFBP riboflavin transfer to the follicle operates more efficiently under restricted availability than the albumen transfer mechanism. The albumen riboflavin in eggs from hens fed deficient diets decreased to a minimum level and stabilized after 2 wk. The initial decrease in yolk riboflavin content was large, but did not decrease to a minimum and stabilize until 12 wk. In a shorter term study, White et al. (1986) reported that riboflavin in albumen depletes more rapidly than yolk, but only for hens on very deficient diets. It is interesting to note that yolk riboflavin concentrations stabilized at about the same time that egg production in the 1.55 /xg/kg treatment started to decrease. Apparently, minimum egg yolk riboflavin contents are correlated closely with egg production. Because albumen riboflavin concentrations decrease to a minimum stable level long before egg production is adversely affected, albumen riboflavin analysis might be used to predict future decline in this important economic factor. Also, egg albumen riboflavin analysis is less complicated than the yolk riboflavin analysis due to the absence of lipid. The time for return of egg riboflavin concentrations to equilibrium levels after being placed on an adequate diet was shown to be between 1 to 3 wk. This is approximately the same time needed to deplete the eggs in the first place, indicating that the riboflavin transport system is not impaired by long-term deficiency. In Experiment 2, with more frequent sampling, egg albumen riboflavin contents were reduced within 4 to 7 days after hens were fed the deficient diet and repletion occurred during the same time interval after return to the recovery diet. In Experiment 1, hatchability of eggs from hens fed 1.55 and 2.2 mg of riboflavin/kg of diet was depressed at Week 16 (and probably at an earlier time but such data were not obtained in the first experiment). There was also a shift toward early embryonic mortality with a
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unsupplemented treatment compared with the supplemented treatment by Days 8 to 10 that continued through Day 3 of the recovery period (Table 8). In addition there was a significant increase in embryos displaying clubbed down during Days 4 to 7, in the unsupplemented treatment, which continued through Day 3 after being fed a recovery diet. In the unsupplemented dietary treatment, a numerical average of 27% of all fertile eggs displayed clubbed down after Day 7, and 11% showed signs of hemorrhaging. Sporadic incidence of clubbed down and hemorrhagic embryos also occurred on the riboflavin supplemented diet at the same time (1.2 and .2%, respectively). Embryonic mortality in eggs from hens fed the unsupplemented diet shifted from the last 7 days of incubation to the middle 7 days during the 2nd and 3rd wk of the experiment. There were no differences (P > .05) in egg production, chick weight, and egg weight between the supplemented and unsupplemented dietary treatments for the 21-day duration of the experiment and the 7-day recovery period. Shell thickness of eggs was numerically lower in the unsupplemented treatment, but only four nonconsecutive time periods had significant differences (P < .05) between treatments. In addition to the above diet effects at individual time intervals in Experiment 2, there was also a significant main effect of time or age on decreases in egg albumen riboflavin content. Hatchability decreased and incidence of both hemorrhagic embryos and clubbed down increased with time or age (P < .05). An interaction of time on dietary riboflavin level was found for each of the above parameters (P < .05).
491
492
SQUIRES AND NABER
TABLE 8. Effect of either a riboflavin-supplemented or unsupplemented diet on incidence of hemorrhagic embryos and clubbed down, Experiment 21 Hemorrhagic embryos
Clubbed down
Riboflavin treatment (mg/kg diet)
Riboflavin treatment (mg/kg diet)
Time
1.58
4.43
0 to 3 4 to 7 8 to 10 11 to 14 15 to 17 18 to 21 Pooled SEM
0 1.0 6.3* 15.5* 12.0* 12.5*
.3
1.58
4.43
0 11.0* 19.7* 31.8* 30.7* 22.8*
.30 0b 0b 0b 5.0b .8b
(%)
3.4
1.5 - Return to recovery diet 0b 13.7* 1.7 0
6.0* 0
1.6
0b 0 3.7
a b
- Means in rows for each variable with no common superscripts differ significantly (P < .05). iData reported as mean of three to four pooled observations of 19 to 51 fertile eggs for hemorrhagic embryos and clubbed down at each treatment by time. The two-way ANOVA showed riboflavin level significant at P < .0001, time effects significant at P < .0001, and the interaction of riboflavin level with time significant at P < .001 for both hemorrhagic embryos and clubbed down.
greater percentage of embryos dying during the first 2 wk of incubation. This suggests that some riboflavin is needed for early embryonic development, which conflicts with the findings of Maw (1954) that little riboflavin is needed until the 13th day of incubation. This could be tested by measuring oxygen consumption. This early embryonic death suggests that certain riboflavin-requiring, energysynthesizing pathways such as the electron transport system are limiting before the 13th day of incubation. Increasing severity of the deficiency causes more embryonic death at an earlier time period. Clubbed down, a common sign of riboflavin deficiency, was not detected until the 3rd wk of incubation, which means that this lesion does not have an opportunity to be expressed during severe deficiency. In Experiment 2, hatchability, clubbed down, and the incidence of hemorrhagic embryo were very closely related to egg riboflavin albumen contents. By Day 7, hatchability of the eggs from hens fed the unsupplemented dietary treatment was 26% lower than from the supplemented control, in which as clubbed down was 71% higher in the unsupplemented treat-
ment. By Day 10, incidence of hemorrhagic embryos was 75% higher in eggs from hens fed the unsupplemented diet. The egg albumen riboflavin content of the unsupplemented treatment averaged 1.3 Mg/g by Day 7. Therefore, the minimum critical riboflavin albumen level to support maximum hatchability and associated parameters is above 1.3 /*g/gHatchability and egg albumen riboflavin values of eggs from hens fed the unsupplemented diet in Experiment 2 returned to those of hens fed the supplemented diet 7 days after being placed on a recovery diet. The 7-day depletion is shorter than the 14-day period reported by Stamberg et al. (1946). This might be due to a genetic influence on riboflavin egg deposition in modern strains of layers. Genetic influence of riboflavin egg deposition was reported 35 yr ago in several popular breeds (Mayfield et al, 1955). Breed differences in the response of White Leghorns and Rhode Island Reds to riboflavin deficiency on egg production have been reported (Leeson et al., 1979). Another reason for the earlier appearance of problems associated with riboflavin deficiency might be due to the higher plane of
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1 to 3 4 to 7 Pooled SEM
0 0" 0" 1.0b 0b
EGGS INDICATING RIBOFLAVIN STATUS IN THE LAYING HEN
493
shown to increase the riboflavin requirement of laying hens (Onwudike and Adegbola, 1984). The incidence of hemorrhages either in extra-embryonic circulation or the embryo itself is commonly associated with embryonic death, especially early in development. The present research shows that riboflavin deficiency causes a large increase in hemorrhagic embryos. This observation, coupled with the previously documented shift to earlier embryonic mortality, reemphasizes the significant role of riboflavin in early development. The results of Experiment 2 indicate that 3 wk is not ample time for riboflavin deficiency to affect egg production or egg weight. As previously noted, egg albumen riboflavin concentrations can be used to predict future problems with egg production and weight, because the egg contents decrease long before these production parameters are affected. On the other hand, hatchability closely follows egg albumen riboflavin content. Riboflavin deficiency probably has no effect on shell thickness during a 3-wk period, but the data were inconclusive.
The short time needed to produce a riboflavin deficiency may explain why low level incidence of clubbed down in embryos exists in flocks that seem nutritionally sound. Hawes and Buss (1965) clearly demonstrated the presence of a mutant allele that can cause clubbed down. However, the current results, as well as other research (Coles and Cumber, 1955), have shown a greater influence of dietary riboflavin on incidence of clubbed down. It is possible that individual birds may experience marginal riboflavin deficiency caused by depressed feed intake over a period of several days. This depressed intake could be due to heat stress, disease, or social pressure from other birds higher in the peck order. Heat stress has been
Ashoor, S. H., G. J. Seperich, W. C. Monte, and J. Welty, 1983. HPLC determination of riboflavin in eggs and dairy products. J. Food Sci. 48: 92-95. Association of Official Analytical Chemists, 1980. Official Methods of Analysis, 13 ed. Association of Official Analytical Chemists, Washington, DC. Bethke, R. M., P. R. Record, and D. C. Kennard, 1933. Relationship of the vitamin G complex to hatchability and nutritive value of eggs. Poultry Sci. 12:332. Bethke, R. M., P. R. Record, and O.H.M. Wilder, 1937. Further studies on vitamin G in chick nutrition. Poultry Sci. 16:175-182. Coles, R., and F. Cumber, 1955. Observations on the relationship between riboflavin, hatchability and clubbed down. J. Agric. Sci. 46:191-198. Hawes, R. O., and E. G. Buss, 1965. The use of the riboflavinless gene (rd) in determining the cause of clubbed down. Poultry Sci. 44:773-778. Hunt, C. H., A. R. Winter, and R. M. Bethke, 1939.
ACKNOWLEDGMENT The authors would like to express their appreciation to Rene Waldman for her technical assistance. REFERENCES
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nutrition needed to support greater egg production. At Week 18 in Experiment 1, shell thickness appeared to actually increase at lower dietary riboflavin levels. However, at this same time, egg production and egg weight of hens fed deficient diets were reduced. Shell thickness and egg weight are known to be negatively correlated. Therefore, the increased shell thickness observed in eggs from hens on deficient diets was probably due to decreased egg production and egg weight and not to dietary riboflavin treatment. Egg weight at Week 16 and 27 decreased with decreasing dietary riboflavin levels. This has not been previously noted, but suggests another important economic parameter that might be monitored by egg riboflavin analysis. The weight of hens fed the 1.55 m g / k g diet was less than that of hens fed the higher riboflavin levels. This may demonstrate impairment of energy metabolism in hens fed deficient diets over the long term, and agrees with the findings of Petersen et al. (1947a,b). Hatchability, egg weight, and egg production were all shown to be depressed in hens fed both the 1.55 and 2.2 m g / k g dietary treatments at 16 wk. Therefore, after 16 wk levels of 1.9 /xg riboflavin/g albumen in eggs from hens fed the 2.2 m g / k g dietary treatment were associated with reduced reproductive function. At the two highest dietary treatment levels, egg albumen riboflavin levels of 2.9 Mg/g were associated with maximum reproduction.
494
SQUIRES AND NABER production and reproduction in the humid tropics. Trop. Agric. 61:205-207. Petersen, C. F., C. E. Lampman, and O. E. Stamberg, 1947a. Effect of riboflavin intake on egg production and riboflavin content of eggs. Poultry Sci. 26:180-186. Petersen, C. F., C. E. Lampman, and O. E. Stamberg, 1947b. Effect of riboflavin intake on hatchability of eggs from battery confined hens. Poultry Sci. 26:187-191. SAS Institute, 1982. SAS® User's Guide: Statistics. SAS Institute Inc., Cary, NC. Snedecor, G. W., and W. G. Cochran, 1980. Statistical Methods. 7th ed. The Iowa State University Press, Ames, IA. Stamberg, O. E., C. F. Peterson, and C. E. Lampman, 1946. Effect of riboflavin intake on the content of egg whites and yolks from individual hens. Poultry Sci. 25:320-326. Wehling, R. L., and D. L. Wetzel, 1984. Simultaneous determination of pyridoxine, riboflavin, and thiamin in fortified cereal products by high performance liquid chromatography. J. Agric. Food Chem. 32:1326-1331. White, H. B., m, J. Armstrong, and C. C. Whitehead, 1986. Riboflavin-binding protein concentration and fractional saturation in chicken eggs as a function of dietary riboflavin. Biochem. J. 238: 671-675. Woodcock, E. A., J. J. Warthesen, and T. P. Labuza, 1982. Riboflavin photochemical degradation in pasta measured by high performance liquid chromatography. J. Food Sci. 47:545-555.
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Further studies on the riboflavin requirements of the chicken. Poultry Sci. 18:330-336. Leeson, S., B. S. Reinhart, and J. D. Summers, 1979. Response of White Leghorn and Rhode Island Red breeder hens to dietary deficiencies of synthetic vitamins. 2. Egg production, hatchability and chick growth. Can. J. Anim. Sci. 59: 561-567. Lepkovsky, S., and T. H. Jukes, 1935. The vitamin G requirements of the chick. J. Biol. Chem. Ill: 119-131. Lepkovsky, S., L. W. Taylor, T. H. Jukes, and H. J. Almquist, 1938. The effect of riboflavin and the filtrate factor on egg production and hatchability. Hilgardia 11:559-591. Maw, A.J.G., 1954. Inherited riboflavin deficiency in chicken eggs. Poultry Sci. 33:216-217. Mayfield, H. L., R. R. Roehm, and A. F. Beeckler, 1955. Riboflavin and thiamine content of eggs from New Hampshire and White Leghorn hens fed diets containing condensed fish or dried whole solubles. Poultry Sci. 34:1106-1111. 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. Norris, L. C, and J. C. Bauernfeind, 1940. Effect of level of dietary riboflavin upon quality stored in eggs and rate of storage. Food Res. 5:521-532. Onwudike, O. C, and A. A. Adegbola, 1984. Riboflavin requirement of laying hens for egg