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A Rapid Bioassay for the Comparison of Xanthophyll Availability from Various Sources
A Rapid Bioassay for the Comparison of Xanthophyll Availability from Various Sources D. F. MIDDENDORF, G. R. CHILDS, and W. W. CRAVENS Feed Research Department, Central Soya Company, Inc., Decatur, Indiana 46733 (Received for publication July 23, 1979)
1980 Poultry Science 59:1442-1450
INTRODUCTION N u m e r o u s m e t h o d s have been utilized to evaluate t h e pigmenting properties of various c o m p o u n d s . T h e most c o m m o n o n e has been visual, in which broilers are fed a ration containing a pigmenting source for several weeks, after which t h e broilers are sacrificed, t h e feathers removed, and pigmentation evaluated using an arbitrary scoring system. Occasionally, only shank pigmentation has been used as t h e test criterion. This subjective measure is difficult to identify and c o m m u n i c a t e . Hence, n u m e r o u s researchers have related t h e visual evaluation to a color reference. Formerly a color r o t o r described b y Heiman and Carver (1935) was frequently used, b u t most recent studies are based on either t h e Roche yolk color fan 1 or t h e Ralston Purina Poultry Skin Pigmentation Guide. F r y et al. (1969) introduced areflectance m e t h o d based u p o n three m e a s u r e m e n t s : d o m i n a n t wave length indicating h u e , luminosity measuring brightness, and excitation purity indicating color intensity at t h e d o m i n a n t wave length. Visual evaluations are often c o m p l e m e n t e d with chemical analyses. Heiman and Tighe ( 1 9 4 3 ) introduced a m e t h o d of measuring shank pigmentation b y determining carotenoid c o n c e n t r a t i o n of shank skin p u n c h e s . This was later modified to utilize t o e web p u n c h e s b y
1 Roche Chemical Division, Hoffman-La Inc., Nutley, NJ 07110.
Roche
Wilgus ( 1 9 5 4 ) and Day and Williams ( 1 9 5 8 ) . Wilson ( 1 9 5 6 ) utilized serum x a n t h o p h y l l levels t o identify nonlaying chickens while Grau and Klein ( 1 9 5 7 ) utilized it t o show t h a t t h e carotenoids from Cblorella and Scenedesmus were biologically available to t h e chick. Davis and Kratzer ( 1 9 5 8 ) found a correlation coefficient of .93 b e t w e e n serum x a n t h o p h y l l after one week of feeding a diet containing xanthophyll and shank pigmentation several weeks later. Dua et al. ( 1 9 6 7 ) reported highly significant correlations between serum x a n t h o p h y l l and dietary x a n t h o p h y l l and between serum x a n t h o p h y l l and skin x a n t h o p h y l l . Repeatability estimates of .92 - .98 for serum x a n t h o p h y l l were higher than t h e y were for skin, fat, and liver, leading S t o n e et al. (1971) to conclude that this m e a s u r e m e n t would be the best criterion for genetic selection. T h e correlation b e t w e e n serum and skin x a n t h o p h y l l may be slightly affected by the carotenoids which have provitamin A activity. While most are converted to vitamin A in t h e intestinal mucosa (Cheng and Deuel, 1950) small a m o u n t s escape conversion, enter the b l o o d stream (Ganguly et al, 195 3), and are deposted in yolks and skin (Gillam and Heilb r o n , 1 9 3 5 ; Ganguly et al., 1 9 5 3 ; Williams et al, 1 9 6 3 ; Nakaue
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ABSTRACT A rapid bioassay method for comparing xanthophyll availability from various sources or the pigmenting ability of genetic strains or crosses is proposed. Xanthophyll depleted, fasted birds are intubated with equal amounts of xanthophyll from various sources. Serum xanthophyll is then determined from blood samples obtained 14 to 24 hr following intubation. Results are expressed as the increase in serum xanthophyll (fig/ml) over the intubated controls per milligram xanthophyll intubated per kilogram body weight. Limitations of the bioassay are discussed.
XANTHOPHYLL AVAILABILITY BIOASSAY
MATERIALS AND METHODS Experiment 1. A commercial strain of broilers (Hubbard x Hubbard) received a commercial wheat-soybean meal ration in starter-grower cages. At 46 days of age they were randomly divided into seven groups of 12 birds each, 6 of each sex; they were weighed individually and the feed was removed although they continued to have access to water. After an 18 hr fast, one control group was intubated with dehulled soybean meal at 1% of body weight; another control group was not intubated. The intubation equipment consisted of a .5 in (1.27 cm) od stainless steel tube with a funnel welded at one end and a plunger. The tube was inserted into the crop and the test material was placed in the funnel. After all material had entered the tube, the plunger was used to assure that all of the material entered the crop. Blood samples via cardiac puncture were obtained from both control groups 6 and 24 hr after the one group was intubated. The remaining five groups were all fasted for 18 hr prior to being intubated with a corn gluten meal, dehulled soybean meal, bleached soybean oil mixture at 1% of body weight to provide xanthophyll 2.35 mg/kg body weight. The bleached soybean oil (5%) was included in the
mixture because previous work had indicated that it was very difficult to maintain uniformity of dry mixes. Blood samp \esvia cardiac puncture were obtained from a group every 2 hr starting 6 hr after intubation, and each group was sampled twice, with the second sample being obtained 10 hr after the first. The corn gluten meal contained xanthophyll 300.0 mg/kg and the bleached soybean oil, 13.6 mg/kg, when assayed by the AOAC method (1975) for total xanthophylls. After clotting, the blood samples were centrifuged and the serum was decanted. Using duplicate samples per bird, 1 ml of serum was then mixed with 7.5 ml of acetone to precipitate the proteins. After centrifugation, the serumacetone mixture was decanted and absorbance was read at 474 /im using a Spectronic-20. Serum xanthophyll was then calculated using a 0-carotene standard curve. For this reason, plus the background level found in the control birds, the results would be more accurately referred to as "serum carotenoids, /3-carotene equivalents", but the term "serum xanthophyll" conforms more to previously published terminology, so it will be used here. Furthermore, the term "xanthophyll" is used to refer to those carotenoids possessing pigmenting properties. Experiment 2. The procedure was the same as that for Experiment 1 except a mixture of marigold meal, dehulled soybean meal, and bleached soybean oil (5%) was used as the xanthophyll source. When intubated at 1% of body weight, this mixture provided xanthophyll 7.38 mg/kg body weight. The sample of marigold meal contained 11,525 mg/kg xanthophyll. The broilers used in this experiment were 53 days of age. Experiments 3 and 4. Thegeneral procedures of Experiments 1 and 2 were followed. Again, a control group was intubated with dehulled soybean meal at 1% of body weight; the treatment groups in Experiment 3 were intubated with a corn gluten meal, dehulled soybean meal mixture in which either0,5,or 10% of bleached soybean oil was substituted for an equivalent amount of the dehulled soybean meal. When intubated at 1% of body weight, these mixtures provided xanthophyll at 2.31, 2.35, and 2.17 mg/kg body weight, respectively. The design of Experiment 4 was identical to that of Experiment 3 except that marigold meal was used as the xanthophyll source and provided xanthophyll 7.95, 7.38, or 7.21 mg/kg body
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under numerous conditions, such as the shipment of international ingredients, the testing of experimental plant strains or crosses, or the studying of isolated carotenoids, which also present a problem of stability when mixed in experimental rations. Furthermore, poultry geneticists cannot sacrifice foundation lines to evaluate pigmentation if they elect to develop either a high pigmenting strain or one which has less bird-to-bird variation in pigmentation ability. Thomas et al. (1971) reported a correlation of .96 between serum xanthophyll levels of broilers fed different levels of xanthophyll from different sources for either two days or for four weeks. It, therefore, appeared possible to develop a bioassay to determine the xanthophyll availability of various sources based upon serum xanthophyll. This bioassay could be based upon intubating xanthophyll sources and measuring serum xanthophyll when it peaked. This would require only small amounts of time and test materials and would not necessitate sacrificing birds.
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RESULTS AND DISCUSSION
In both Experiments 1 and 2, serum xanthophyll appeared to increase until 14 hr postintubation, after which time this increase either stopped or proceeded at a decreased rate (Table 1). This was confirmed through linear regression analysis after the data were divided into two time periods, control birds through 14
hr and 14 to 24 hr (Table 2). The variation among groups has two primary sources. Preliminary investigations in this laboratory indicate that the bird-to-bird coefficient of variation of serum xanthophyll within a flock receiving typical commercial broiler rations ranges from 15 to 20%. Second, the physiological limitation of blood sampling via cardiac puncture necessitated the use of five different groups of birds. Nonetheless, these data are sufficiently consistent to suggest that the absorption of the free xanthophylls in corn gluten meal and the esterfied xanthophylls in marigold meal follows approximately the same pattern following intubation. Hence, the principle of using a bioassay comparing concentrated sources of xanthophylls by measuring serum xanthophyll any time between 14 and 24 hr following intubation of the xanthophyll source is valid. This conclusion is supported by the correlation data in Table 3. Blood samples were obtained from the same birds at two different times following intubation in four of the eight experiments. While the serum xanthophyll was higher at the time of the second sampling in 11 of the 12 comparisons, a consistently significant correlation between the serum xanthophyll at the two different samplings occurred. Davis and Kratzer (1958) reported that it required four days for serum xanthophyll to reach its maximum level after being changed from a low to a high xanthophyll ration. Bartov and Bornstein (1969) estimated this to be nine days. In both reports, birds were fed ad libitum. In the studies reported here, birds received a single dose of xanthophyll via intubation so the time required for serum xanthophyll to reach a maximum level would be expected to vary from these literature values. The age of the bird used in the bioassay would not be expected to be a critical factor, although the data of Bartov and Bornstein (1969) indicate a decrease in serum xanthophyll from hatching up to three weeks of age, even in chicks from dams fed a low xanthophyll ration, although the amount of decrease is dependent upon the dietary xanthophyll level. All of the broilers used in this series of eight experiments ranged from 45 to 53 days of age and it is generally agreed that broilers pigment more readily during the finisher than the starter period. It is, therefore, suggested that broilers at least four weeks of age be used in the bioas-
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weight when the mixture contained 0, 5, or 10% of bleached soybean oil, respectively, and was intubated at 1% of body weight. Blood samples were obtained via cardiac puncture 24 hr after intubation. All intubating materials were from the same samples as used in Experiments 1 and 2. The broilers were 46 and 53 days of age for Experiments 3 and 4, respectively. Experiment 5. Again, the general procedure was used, but the purpose of this experiment was to determine the effect of levels of xanthophyll intubation upon serum xanthophyll. Marigold meal was assayed for xanthophyll and blended with dehulled soybean meal to contain xanthophyll 0, 4.0, 8.0, and 16.0 mg/10 g. The mixtures were then analyzed and the amount of material required to provide the desired xanthophyll levels was intubated. This approximated 1% of body weight. Twelve cockerels 53 days of age received each treatment and blood samples were obtained 18 and 28 hr after intubation. Experiment 6. This experiment was similar to Experiment 5 except that a commercial extract of marigold meal was used. It was blended with dehulled soybean meal and bleached soybean oil (5%) so that when the mixtures were intubated at 1% body weight they provided xanthophyll 0, 3.2, 7.8, 15.1, and 36.3 mg/kg body weight. Twelve 48-day old broilers, six of each sex, received each treatment and blood samples were obtained 18 and 24 hr after intubation. Experiments 7 and 8. Any assay must exhibit repeatability if it is to be valid. Hence, two identical experiments utilizing the same intubation mixtures were conducted during successive weeks. The broilers were 45 days of age in Experiment 7, while their flock mates were 52 days of age in Experiment 8. Eight birds of each sex were used per treatment. The xanthophyll intubation level was 2.30 mg/kg body weight for all treatments.
XANTHOPHYLL AVAILABILITY BIOASSAY
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TABLE 1. Effect of length of time following intubation upon serum xanthophyll level Serum xanthophyll Experiment 1 (Corn gluten meal)
Hours after intubation
Experiment 2 (Marigold Meal) /
i\
1.34 1.19 1.99 2.29 2.34 3.27 3.42 2.96 3.47 2.78 3.78 4.23
1.33 1.14 1.49 2.02 2.65 2.26 3.20 2.24 2.91 3.23 2.87 3.39
Average of samples taken 6 and 24 hours after control group was intubated.
say. This may result in more reliable and applicable data as well as facilitating intubation and blood sampling. Both Experiments 1 and 2 included a control group which was intubated with dehulled soybean meal at 1% of body weight and a nonintubated control group. Blood samples were obtained from both control groups at the same time, which was 6 and 24 hr after the one group was intubated. There was no difference in the serum xanthophyll between the two intubated and nonintubated control groups, but the serum xanthophyll was lower at the 24 hr postintubation sampling than at the 6 hr sampling (Table 4). In Experiment 1, this
decline was statistically significant, but not in Experiment 2. Because there was no significant difference in the serum xanthophyll of the intubated and nonintubated control groups in these first two experiments, only control groups intubated with a dehulled soybean meal and 5% bleached soybean oil mixture were used in the last six experiments. Blood samples were obtained only once for these groups in Experiments 3 and 4, but two samples at different times following intubation were obtained in Experiments 5 and 6. The data were conflicting again. In Experiment 5 the serum xanthophyll was significantly higher at 18 than at 28 hr after intubation
TABLE 2. Linear regression analysis of serum xanthophyll levels Characteristics of linear regression Experiment
% Variation explained
Correlation coefficient
92.80 82.25
.963** .907**
Significance from horizontal
Slope
(Control through 14 hr) 1% 5%
.157 .133
NS 3 NS
.083 .045
(14 to 24 hr) 34.39 16.91 P<.01. a
NS, not significant (P>.05).
.586 .411
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Nonintubated control Intubated control 3 6 8 10 12 14 16 18 20 22 24
/
(Mg/ml)
3
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TABLE 3. Correlation between serum xanthophyll determined at two different times following Serum x a n t h o p h y l l at each sampling
Sampling t i m e after i n t u b a t i o n
between samplings
Experiment
First
Second
First
1
6 8 10 12 14
16 18 20 22 24
2.00 2.29 2.34 3.27 3.42
2.96 3.47 2.78 3.78 4.23
.71 .61 .76 .92 .76
2
6 8 10 12 14
16 18 20 22 24
1.49 2.02 2.65 2.26 3.20
2.24 2.91 3.23 2.87 3.39
.57 .80 .88 .94 .98
5
18
28
2.63
2.54
.96
24
3.40
3.91
.87
Second
intubation
Significance
18
NS, not significant.
whereas it was higher at 24 than at 18 hr after intubation in Experiment 6. These results indicate the necessity of sampling the control and treated birds at the same time following intubation. The addition of either 5% or 10% of bleached soybean oil to the intubating material to prevent segregation following mixing had no effect upon serum xanthophyll levels 24 hr after intubation when corn gluten meal was
the xanthophyll source (Experiment 3, Table 5). There was a rather large numerical increase in serum xanthophyll due to the addition of the oil when marigold meal supplied the xanthophyll (Experiment 4, Table 5), but this was not statistically significant. It is therefore concluded that fat per se did not affect xanthophyll absorption, which was unexpected. The xanthophylls are complexed with fatty acids prior to absorption (Scott et al, 1976) and the most
TABLE 4. Effect of time upon serum xanthophyll of control birds Serum xanthophyll Hours after intubation
Nonintubated controls 3
Intubated controls • (Mg/ml) -
Experiment 6hr 24 hr Experiment 6hr 24 hr Experiment 18 hr 28 hr Experiment 18 hr 24 hr
1 1.43** .94
1.57* 1.10
1.22 1.05
1.42 1.23
2 5 1.59** 1.31 6 .93 1.38**
Blood samples obtained at same time as intubated controls.
**
P<.01.
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6
( A)
5 NSa
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XANTHOPHYLL AVAILABILITY BIOASSAY TABLE 5. Effect of including bleached soybean oil in intubating mixture upon serum xanthophyll levels Serum xanthophyll Treatment
Experiment 3 (Corn gluten meal)
Controls 0% Bl soy oil 5% Bl soy oil 10% Bl soy oil
.94 a 4.24 b 3.85 b 3.92 b
Experiment 4 (Marigold meal) • (ug/ml)1.05 a 2.28 b 3.04 b 3.07 b
• Increase in serum xanthophyll 1 1.43 1.24 1.37
.15 .27 .28
a,b Means within the same column bearing different superscripts differ significantly (P<.05). Increase in serum xanthophyll Gjg/ml) over controls per milligram xanthophyll intubated per kilogram body weight.
active site of xanthophyll absorption (Littlefield et al, 1972) is the same as it is for fats (Renner, 1965). Furthermore, Quackenbush et al. (1965) reported that adding lard to rations in which corn supplied carotenols 8.8 mg/kg of ration increased both the total and the percentage of consumed xanthophyll found in toe web punches. Heath and Shaffner (1972) reported that soybean oil increased the amount of xanthophyll in the breast and back skin, the amount of body lipids, and the xanthophyll content of the lipids when marigold petal meal was the xanthophyll source. However, these workers apparently did not consider any xanthophyll which may have been in the soybean oil. Similarly, Day and Williams (1958) found an increase in xanthophyll utilization when 5% of stabilized beef tallow replaced white corn. Many reports in the literature have concluded that increasing dietary xanthophyll decreases xanthophyll utilization when expressed on a per xanthophyll unit basis. Hence, birds were intubated with varying levels of xanthophyll from marigold meal in Experiment 5 and varying levels of a commercial marigold meal extract in Experiment 6. Total serum xanthophyll was increased when the intubated level of xanthophyll increased up to 15 and 16 mg/kg body weight in both experiments (Tables 6 and 7). Regression analysis of the data from Experiment 6 indicates that this increase was curvilinear and that serum
xanthophyll would have reached maximum at 23.2 and 24.8 mg of intubated xanthophyll at 18 and 24 hr after intubation, respectively. When expressed as the increase in serum xanthophyll per milligram of xanthophyll intubated per kilogram body weight, xanthophyll utilization decreased markedly in three of the four instances. This means that when the biological availability of xanthophyll from different sources is being compared, it is
TABLE 6. Effect of xanthophyll intubation level upon serum xanthophyll levels (Experiment 5, marigold meal)
Xanthophyll intubated mg/kg body weight
0 4.0 8.0 16.0
Serum xanthophyll hours after intubation 18
28
1.59 2.17 2.65 3.13
1.31 1.86 2.60 3.13
Increase in serum xanthophyll 3 4.0 8.0 16.0
.145 .133 .096
.138 .161 .114
Increase in serum xanthophyll (jug/ml) over controls per milligram xanthophyll intubated per kilogram body weight.
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0% Bl soy oil 5% Bl soy oil 10% Bl soy oil
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TABLE 7. Effect of xanthophyll intubation level upon serum xanthophyll levels (Experiment 6, marigold extract)
Xanthophyll intubated mg/kg body •weight
Serum xanthophyll hours iifter intubation 18
24
/
(Mg/ml) •
.95 2.80 3.27 3.92 3.60
• •
1.38 3.17 3.71 4.33 4.44
I ncrease in serum xanthophyll 3 3.2 7.8 15.1 36.3
.578 .297 .197 .073
.559 .299 .195 .084
Increase in serum xanthophyll (;ug/ml) over controls per milligram xanthophyll intubated per kilogram body weight.
necessary to do so at the same xanthophyll level. Conducting two identical experiments one week apart but utilizing the same intubating materials and flock mates produced very similar results, thereby demonstrating the reproducability of the bioassay (Table 8). The xanthophyll content of the sources used in Experiments 7 and 8 was determined by the AOAC (1975) method for total xanthophylls; hence, the epoxide xanthophylls of dehydrated alfalfa are included. This invalidates relating its biological availability to the other test materials. When compared with corn gluten meal, the marigold based products had a relative biological value of 40 to 45%. The saponified marigold extract was supplied by a commercial manufacturer; the degree of saponification was not determined, but either saponification was not achieved or altering the chemical form of the marigold xanthophylls did not improve their utilization. This is not in agreement with Coon and Couch (1976), who obtained a significant improvement in utilization of xanthophyll from marigold meal following saponification. An equal number of pullets and cockerels received each treatment in seven of the eight experiments. The data of all xanthophyll intubated treatments were summarized by sex (Table 9). In two experiments the cockerels
The
results
of
these eight
experiments
TABLE 8. Repeatability of xanthophyll availability bioassay in two consecutive experiments Increase in serum xanthophyll 3
Corn gluten meal Dehydrated alfalfa Marigold meal Saponified marigold extract Marigold concentrate
Experiment 7
Experiment 8
1.25 .70 .54 .5 3
1.15 .70 .44 .51
.51
.53
Increase in serum xanthophyll (Mg/ml) over controls per milligram xanthophyll intubated per kilogram body weight.
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0 3.2 7.8 15.1 36.3
had slightly higher, but insignificantly different serum xanthophyll levels than the pullets. In the other five experiments the pullets had the higher serum xanthophyll level; this ranged from small numerical differences to one which was statistically significant. Also, when the data from all seven experiments were combined and analyzed by a paired t test, the difference was statistically significant. Although these data are not completely consistent, it appears advisable to utilize the same sex distribution when conducting the bioassay. This suggestion is supported by the report of Hinton et al. (1973) who obtained greater pigmentation in pullets when one strain of broilers was used in one experiment but did not obtain a difference between sexes when a different strain was used in a different experiment. The assay method for determining serum xanthophyll as outlined by Wilson (1956) utilized a serum to acetone ratio of 1:15. Comparatively low serum xanthophyll levels were anticipated in this bioassay, so a more concentrated solution was desired to maintain the spectrophotometric readings in the most sensitive range. Hence a pooled serum sample was obtained from broilers which had received a commercial ration; serum xanthophyll was determined using different serum to acetone ratios and five replicates were assayed at each ratio (Table 10). There was no consistent effect of this ratio upon the average serum xanthophyll or the standard deviation. It is concluded that any serum-to-acetone ratio within the range of 1:5.0 to 1:20.0 may be used without materially affecting the results.
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XANTHOPHYLL AVAILABILITY BIOASSAY TABLE 9. Effect of sex upon serum xanthophyll level
TABLE 10. Effect of serum to acetone ratios upon serum xanthophyll assay results
Serum xanthophyll Experiment
Males
Females
Acetone ml/ml serum
(/ug/ml)
• (Mg/ml) 3.09 2.46 3.78 2.83 3.29 2.23 2.33 2.79
3.05 2.76 4.26 2.75 3.85* 2.39 2.42 3.00*
5.0 7.5 10.0 12.5 15.0 17.5 20.0
11.01 ± 10.75 ± 10.64 ± 10.73 ± 10.74 ± 10.79 ± 11.19 ±
.09 .06 .03 .05 .06 .14 .06
P<.05.
indicate the feasibility of using a bioassay to compare the biological availability of xanthophyll from various sources. A stepwise description of this bioassay is as follows: 1. Maintain chickens on a low xanthophyll ration for at least two weeks so the base serum xanthophyll is low. 2. Fast the chickens for approximately 18 hr prior to intubating the xanthophyll source(s). 3. Use the same sex distribution for all treatments. 4. A control group should be intubated with a xanthophyll free material at 1% of body weight. Treatment groups should be intubated with equal amounts of xanthophyll from the test materials. This may be easily accomplished by blending the xanthophyll source with a xanthophyll-free material so the total amount of material intubated equals 1% of body weight. Adding 5% of a xanthophyll-free vegetable oil to the material to be intubated will help minimize separation of the blended ingredients. 5. Blood samples may be obtained any time between 14 and 24 hr postintubation, but the interval between the two procedures must be the same for each bird. 6. After clotting, decant the serum, centrifuge it and precipitate the serum proteins with acetone. The ratio of serum to acetone may vary from 1:5.0 to 1:20.0, but it should be constant within any given experiment. 7. Determine absorbance at 474 jum and calculate serum xanthophyll Oug/ml/J-caro-
tene equivalents) from a 0-carotene standard curve. 8. Express results as increase in serum xanthophyll, mcg/ml, over controls per milligram xanthophyll intubated per kilogram body weight. There are several limitation to this bioassay. Efforts to intubate quantities of material much above 1% of body weight failed due to the size of the crop. Hence, a sufficient amount of corn or other comparatively low xanthophyll product could not be intubated to obtain a meaningful increase in serum xanthophyll. Also, it does not consider hues. Individual carotenoids range from pale yellow to deep red in color and the carotenoids deposited in the skin closely resemble those in the diet (Livingston et al., 1969). Some give objectional color tones and some carotenoid combinations have complementary effects (Marusich and Bauernfeind, 1970). Yet, it would be possible to use this bioassay to determine the relative biological activity of a completely unknown xanthophyll source and then feed it to a minimum number of broilers. This would be adequate to at least determine any objectionable hue while still requiring much smaller amounts of a test material than past procedures. Furthermore, any procedure which requires feather removal is subjected to increased error since both scald temperature and time affect feather tract and skin xanthophyll content and pigmentation color (Heath and Thomas, 1973, 1974), making a study of complementary effect of xanthophyll sources very difficult. Visual evaluations are also subject to other errors. Herrick et al. (1970) reported that skin pigmented faster than shanks whereas Hinton et al. (1973) reported that shanks pigmented better than skin. Davies
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1 2 3 4 6 7 8 Combined
Serum xanthophyll Average ± SD
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et al. ( 1 9 6 9 ) used reflectance m e a s u r e m e n t s t o show t h a t foot p a d and shank color were n o t correlated with breast or thigh skin color. REFERENCES
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Association of Official Agricultural Chemists, 1975. Pages 822—823 in Official methods of analysis. 12th ed. AOAC, Washington, DC. Bartov, I., and S. Bornstein, 1969. Depletion and repletion of body xanthophyll reserves as related to broiler pigmentation. Poultry Sci. 48:495— 504. Cheng, A. L. S., and H. J. Deuel, Jr., 1950. Studies on carotenoid metabolism. X. The site of the conversion of carotene to vitamin A in the chick. J. Nutr. 41:619-627. Coon, C. N., and J. R. Couch, 1976. Effect of storage and fatty acid esters on the utilization of xanthophyll from marigold meal by laying hens. Poultry Sci. 55:841-847. Davies, R. E., M. L. Jones, and H. Yacowitz, 1969. Direct instrumental measurement of skin color in broilers. Poultry Sci. 48:1800. Davis, P. N., and F. H. Kratzer, 1958. The relation of serum xanthophyll in chickens to the pigmentation of their shanks. Poultry Sci. 37:851—854. Day, E. J., and W. P. Williams, Jr., 1958. A study of certain factors that influence pigmentation in broilers. Poultry Sci. 37:1373-1381. Dua, P. N., E. J. Day, J. E. Hill, and C. O. Grogan, 1967. Utilization of xanthophylls from natural sources by the chick. J. Agr. Food Chem. 15: 324-328. Fry, J. L., E. M. Ahmed, G. M. Herrick, and R. H. Harms, 1969. A reflectance method of determining skin and shank pigmentation. Poultry Sci. 48:1127-1129. Ganguly, J., J. W. Mehl, and H. J. Deuel, 1953. Studies on carotenoid metabolism. XII. The effect of dietary carotenoids on the carotenoid distribution in the tissues of chickens. J. Nutr. 50:59—72. Gillam, A. D., and I. M. Heilbron, 1935. Vitamin A active substances in egg yolk. Biochem. J. 29: 1064-1067. Grau, C. R., and N. W. Klein, 1957. Sewage-grown algae as a feedstuff for chicks. Poultry Sci. 36:1046-1051. Heath, J. L., and C. S. Shaffner, 1972. The effect of dietary soybean oil on the deposition of xanthophyll in broiler skin. Poultry Sci. 51:502— 506. Heath, J. L., and O. P. Thomas, 1973. The xanthophyll content and color of broiler skin after scalding. Poultry Sci. 52:967-971. Heath, J. L., and O. P. Thomas, 1974. The effect of feather removal during processing on the xantho-
phyll content of broiler skin. Poultry Sci. 53: 291-295. Heiman, V., and J. S. Carver, 1935. The yolk color index. U. S. Egg-Poultry Magazine 41:40—41. Heiman, V., and L. W. Tighe, 1943. Observations on the shank pigmentation of chicks. Poultry Sci. 22:102-107. Herrick, G. M., S. L. Frey, and R. H. Harms, 1970. Repletion and depletion of xanthophyll in the skin and shanks of broilers. Poultry Sci. 49: 1396-1397. Hinton, C. F., J. L. Fry, and R. H. Harms, 1973. Subjective and colormetric evaluation of the xanthophyll utilization of natural and synthetic pigments in broiler diets. Poultry Sci. 52:2169— 2180. Littlefield, L. H., J. K. Bletner, H. V. Shirley, and O. E. Goff, 1972. Locating the site of absorption of xanthophylls in the chicken by a surgical technique. Poultry Sci. 51:1721-1725. Livingston, A. L., D. D. Kuzmicky, R. E. Knowles, and G. O. Kohler, 1969. The nature and deposition of the carotenoids from alfalfa and corn gluten meal in chicken skin. Poultry Sci. 48: 1678-1683. Marusich, W. L., and J. C. Bauernfeind, 1970. Oxycarotenoids in poultry pigmentation. 2. Broiler studies. Poultry Sci. 49:1566-1579. Nakaue, H. S., A. A. Kurnick, B. J. Hulett, and B. L. Reid, 1966. Effect of ethoxyquin on carotenoid stability and utilization. Poultry Sci. 45:478— 483. Quackenbush, F. W., S. Kvakovszky, T. Hoover, and J. C. Rogler, 1965. Deposition of individual carotenoids in avian skin. J. Assoc. Offic. Agr. Chem. 48:1241-1244. Renner, R., 1965. Site of fat absorption in the chick. Poultry Sci. 44:861-864. Scott, M. L., M. C. Nesheim, and R. J. Young, 1976. Nutrition of the chicken. 2nd ed. M. L. Scott and Associates, Ithaca, NY. Stone, H. A., W. M. Collins, and W. E. Urban, Jr., 1971. Evaluation of carotenoid concentration in chicken tissues. Poultry Sci. 50:675—681. Thomas, O. P., P. V. Twining, Jr., E. H. Bossard, and P. G. Lund, 1971. Serum xanthophyll as a means of evaluating different dietary sources of xanthophyll. Poultry Sci. 50:1636. Wilgus, H. S., 1954. Effect of DPPD on utilization of different sources of carotenoid pigment. Experiment B854 Xan. Peter Hand Foundation. Chicago, IL. Williams, W. P., R. E. Davies, and J. R. Couch, 1963. The utilization of carotenoids by the hen and chick. Poultry Sci. 42:691-699. Wilson, W. O., 1956. Identifying nonlaying chicken hens. Poultry Sci. 35:226-227.
1980 Poultry Science 59:1442-1450
INTRODUCTION N u m e r o u s m e t h o d s have been utilized to evaluate t h e pigmenting properties of various c o m p o u n d s . T h e most c o m m o n o n e has been visual, in which broilers are fed a ration containing a pigmenting source for several weeks, after which t h e broilers are sacrificed, t h e feathers removed, and pigmentation evaluated using an arbitrary scoring system. Occasionally, only shank pigmentation has been used as t h e test criterion. This subjective measure is difficult to identify and c o m m u n i c a t e . Hence, n u m e r o u s researchers have related t h e visual evaluation to a color reference. Formerly a color r o t o r described b y Heiman and Carver (1935) was frequently used, b u t most recent studies are based on either t h e Roche yolk color fan 1 or t h e Ralston Purina Poultry Skin Pigmentation Guide. F r y et al. (1969) introduced areflectance m e t h o d based u p o n three m e a s u r e m e n t s : d o m i n a n t wave length indicating h u e , luminosity measuring brightness, and excitation purity indicating color intensity at t h e d o m i n a n t wave length. Visual evaluations are often c o m p l e m e n t e d with chemical analyses. Heiman and Tighe ( 1 9 4 3 ) introduced a m e t h o d of measuring shank pigmentation b y determining carotenoid c o n c e n t r a t i o n of shank skin p u n c h e s . This was later modified to utilize t o e web p u n c h e s b y
1 Roche Chemical Division, Hoffman-La Inc., Nutley, NJ 07110.
Roche
Wilgus ( 1 9 5 4 ) and Day and Williams ( 1 9 5 8 ) . Wilson ( 1 9 5 6 ) utilized serum x a n t h o p h y l l levels t o identify nonlaying chickens while Grau and Klein ( 1 9 5 7 ) utilized it t o show t h a t t h e carotenoids from Cblorella and Scenedesmus were biologically available to t h e chick. Davis and Kratzer ( 1 9 5 8 ) found a correlation coefficient of .93 b e t w e e n serum x a n t h o p h y l l after one week of feeding a diet containing xanthophyll and shank pigmentation several weeks later. Dua et al. ( 1 9 6 7 ) reported highly significant correlations between serum x a n t h o p h y l l and dietary x a n t h o p h y l l and between serum x a n t h o p h y l l and skin x a n t h o p h y l l . Repeatability estimates of .92 - .98 for serum x a n t h o p h y l l were higher than t h e y were for skin, fat, and liver, leading S t o n e et al. (1971) to conclude that this m e a s u r e m e n t would be the best criterion for genetic selection. T h e correlation b e t w e e n serum and skin x a n t h o p h y l l may be slightly affected by the carotenoids which have provitamin A activity. While most are converted to vitamin A in t h e intestinal mucosa (Cheng and Deuel, 1950) small a m o u n t s escape conversion, enter the b l o o d stream (Ganguly et al, 195 3), and are deposted in yolks and skin (Gillam and Heilb r o n , 1 9 3 5 ; Ganguly et al., 1 9 5 3 ; Williams et al, 1 9 6 3 ; Nakaue
1442
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ABSTRACT A rapid bioassay method for comparing xanthophyll availability from various sources or the pigmenting ability of genetic strains or crosses is proposed. Xanthophyll depleted, fasted birds are intubated with equal amounts of xanthophyll from various sources. Serum xanthophyll is then determined from blood samples obtained 14 to 24 hr following intubation. Results are expressed as the increase in serum xanthophyll (fig/ml) over the intubated controls per milligram xanthophyll intubated per kilogram body weight. Limitations of the bioassay are discussed.
XANTHOPHYLL AVAILABILITY BIOASSAY
MATERIALS AND METHODS Experiment 1. A commercial strain of broilers (Hubbard x Hubbard) received a commercial wheat-soybean meal ration in starter-grower cages. At 46 days of age they were randomly divided into seven groups of 12 birds each, 6 of each sex; they were weighed individually and the feed was removed although they continued to have access to water. After an 18 hr fast, one control group was intubated with dehulled soybean meal at 1% of body weight; another control group was not intubated. The intubation equipment consisted of a .5 in (1.27 cm) od stainless steel tube with a funnel welded at one end and a plunger. The tube was inserted into the crop and the test material was placed in the funnel. After all material had entered the tube, the plunger was used to assure that all of the material entered the crop. Blood samples via cardiac puncture were obtained from both control groups 6 and 24 hr after the one group was intubated. The remaining five groups were all fasted for 18 hr prior to being intubated with a corn gluten meal, dehulled soybean meal, bleached soybean oil mixture at 1% of body weight to provide xanthophyll 2.35 mg/kg body weight. The bleached soybean oil (5%) was included in the
mixture because previous work had indicated that it was very difficult to maintain uniformity of dry mixes. Blood samp \esvia cardiac puncture were obtained from a group every 2 hr starting 6 hr after intubation, and each group was sampled twice, with the second sample being obtained 10 hr after the first. The corn gluten meal contained xanthophyll 300.0 mg/kg and the bleached soybean oil, 13.6 mg/kg, when assayed by the AOAC method (1975) for total xanthophylls. After clotting, the blood samples were centrifuged and the serum was decanted. Using duplicate samples per bird, 1 ml of serum was then mixed with 7.5 ml of acetone to precipitate the proteins. After centrifugation, the serumacetone mixture was decanted and absorbance was read at 474 /im using a Spectronic-20. Serum xanthophyll was then calculated using a 0-carotene standard curve. For this reason, plus the background level found in the control birds, the results would be more accurately referred to as "serum carotenoids, /3-carotene equivalents", but the term "serum xanthophyll" conforms more to previously published terminology, so it will be used here. Furthermore, the term "xanthophyll" is used to refer to those carotenoids possessing pigmenting properties. Experiment 2. The procedure was the same as that for Experiment 1 except a mixture of marigold meal, dehulled soybean meal, and bleached soybean oil (5%) was used as the xanthophyll source. When intubated at 1% of body weight, this mixture provided xanthophyll 7.38 mg/kg body weight. The sample of marigold meal contained 11,525 mg/kg xanthophyll. The broilers used in this experiment were 53 days of age. Experiments 3 and 4. Thegeneral procedures of Experiments 1 and 2 were followed. Again, a control group was intubated with dehulled soybean meal at 1% of body weight; the treatment groups in Experiment 3 were intubated with a corn gluten meal, dehulled soybean meal mixture in which either0,5,or 10% of bleached soybean oil was substituted for an equivalent amount of the dehulled soybean meal. When intubated at 1% of body weight, these mixtures provided xanthophyll at 2.31, 2.35, and 2.17 mg/kg body weight, respectively. The design of Experiment 4 was identical to that of Experiment 3 except that marigold meal was used as the xanthophyll source and provided xanthophyll 7.95, 7.38, or 7.21 mg/kg body
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under numerous conditions, such as the shipment of international ingredients, the testing of experimental plant strains or crosses, or the studying of isolated carotenoids, which also present a problem of stability when mixed in experimental rations. Furthermore, poultry geneticists cannot sacrifice foundation lines to evaluate pigmentation if they elect to develop either a high pigmenting strain or one which has less bird-to-bird variation in pigmentation ability. Thomas et al. (1971) reported a correlation of .96 between serum xanthophyll levels of broilers fed different levels of xanthophyll from different sources for either two days or for four weeks. It, therefore, appeared possible to develop a bioassay to determine the xanthophyll availability of various sources based upon serum xanthophyll. This bioassay could be based upon intubating xanthophyll sources and measuring serum xanthophyll when it peaked. This would require only small amounts of time and test materials and would not necessitate sacrificing birds.
1443
MIDDENDORF ET AL.
1444
RESULTS AND DISCUSSION
In both Experiments 1 and 2, serum xanthophyll appeared to increase until 14 hr postintubation, after which time this increase either stopped or proceeded at a decreased rate (Table 1). This was confirmed through linear regression analysis after the data were divided into two time periods, control birds through 14
hr and 14 to 24 hr (Table 2). The variation among groups has two primary sources. Preliminary investigations in this laboratory indicate that the bird-to-bird coefficient of variation of serum xanthophyll within a flock receiving typical commercial broiler rations ranges from 15 to 20%. Second, the physiological limitation of blood sampling via cardiac puncture necessitated the use of five different groups of birds. Nonetheless, these data are sufficiently consistent to suggest that the absorption of the free xanthophylls in corn gluten meal and the esterfied xanthophylls in marigold meal follows approximately the same pattern following intubation. Hence, the principle of using a bioassay comparing concentrated sources of xanthophylls by measuring serum xanthophyll any time between 14 and 24 hr following intubation of the xanthophyll source is valid. This conclusion is supported by the correlation data in Table 3. Blood samples were obtained from the same birds at two different times following intubation in four of the eight experiments. While the serum xanthophyll was higher at the time of the second sampling in 11 of the 12 comparisons, a consistently significant correlation between the serum xanthophyll at the two different samplings occurred. Davis and Kratzer (1958) reported that it required four days for serum xanthophyll to reach its maximum level after being changed from a low to a high xanthophyll ration. Bartov and Bornstein (1969) estimated this to be nine days. In both reports, birds were fed ad libitum. In the studies reported here, birds received a single dose of xanthophyll via intubation so the time required for serum xanthophyll to reach a maximum level would be expected to vary from these literature values. The age of the bird used in the bioassay would not be expected to be a critical factor, although the data of Bartov and Bornstein (1969) indicate a decrease in serum xanthophyll from hatching up to three weeks of age, even in chicks from dams fed a low xanthophyll ration, although the amount of decrease is dependent upon the dietary xanthophyll level. All of the broilers used in this series of eight experiments ranged from 45 to 53 days of age and it is generally agreed that broilers pigment more readily during the finisher than the starter period. It is, therefore, suggested that broilers at least four weeks of age be used in the bioas-
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weight when the mixture contained 0, 5, or 10% of bleached soybean oil, respectively, and was intubated at 1% of body weight. Blood samples were obtained via cardiac puncture 24 hr after intubation. All intubating materials were from the same samples as used in Experiments 1 and 2. The broilers were 46 and 53 days of age for Experiments 3 and 4, respectively. Experiment 5. Again, the general procedure was used, but the purpose of this experiment was to determine the effect of levels of xanthophyll intubation upon serum xanthophyll. Marigold meal was assayed for xanthophyll and blended with dehulled soybean meal to contain xanthophyll 0, 4.0, 8.0, and 16.0 mg/10 g. The mixtures were then analyzed and the amount of material required to provide the desired xanthophyll levels was intubated. This approximated 1% of body weight. Twelve cockerels 53 days of age received each treatment and blood samples were obtained 18 and 28 hr after intubation. Experiment 6. This experiment was similar to Experiment 5 except that a commercial extract of marigold meal was used. It was blended with dehulled soybean meal and bleached soybean oil (5%) so that when the mixtures were intubated at 1% body weight they provided xanthophyll 0, 3.2, 7.8, 15.1, and 36.3 mg/kg body weight. Twelve 48-day old broilers, six of each sex, received each treatment and blood samples were obtained 18 and 24 hr after intubation. Experiments 7 and 8. Any assay must exhibit repeatability if it is to be valid. Hence, two identical experiments utilizing the same intubation mixtures were conducted during successive weeks. The broilers were 45 days of age in Experiment 7, while their flock mates were 52 days of age in Experiment 8. Eight birds of each sex were used per treatment. The xanthophyll intubation level was 2.30 mg/kg body weight for all treatments.
XANTHOPHYLL AVAILABILITY BIOASSAY
1445
TABLE 1. Effect of length of time following intubation upon serum xanthophyll level Serum xanthophyll Experiment 1 (Corn gluten meal)
Hours after intubation
Experiment 2 (Marigold Meal) /
i\
1.34 1.19 1.99 2.29 2.34 3.27 3.42 2.96 3.47 2.78 3.78 4.23
1.33 1.14 1.49 2.02 2.65 2.26 3.20 2.24 2.91 3.23 2.87 3.39
Average of samples taken 6 and 24 hours after control group was intubated.
say. This may result in more reliable and applicable data as well as facilitating intubation and blood sampling. Both Experiments 1 and 2 included a control group which was intubated with dehulled soybean meal at 1% of body weight and a nonintubated control group. Blood samples were obtained from both control groups at the same time, which was 6 and 24 hr after the one group was intubated. There was no difference in the serum xanthophyll between the two intubated and nonintubated control groups, but the serum xanthophyll was lower at the 24 hr postintubation sampling than at the 6 hr sampling (Table 4). In Experiment 1, this
decline was statistically significant, but not in Experiment 2. Because there was no significant difference in the serum xanthophyll of the intubated and nonintubated control groups in these first two experiments, only control groups intubated with a dehulled soybean meal and 5% bleached soybean oil mixture were used in the last six experiments. Blood samples were obtained only once for these groups in Experiments 3 and 4, but two samples at different times following intubation were obtained in Experiments 5 and 6. The data were conflicting again. In Experiment 5 the serum xanthophyll was significantly higher at 18 than at 28 hr after intubation
TABLE 2. Linear regression analysis of serum xanthophyll levels Characteristics of linear regression Experiment
% Variation explained
Correlation coefficient
92.80 82.25
.963** .907**
Significance from horizontal
Slope
(Control through 14 hr) 1% 5%
.157 .133
NS 3 NS
.083 .045
(14 to 24 hr) 34.39 16.91 P<.01. a
NS, not significant (P>.05).
.586 .411
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Nonintubated control Intubated control 3 6 8 10 12 14 16 18 20 22 24
/
(Mg/ml)
3
MIDDENDORF ET AL.
1446
TABLE 3. Correlation between serum xanthophyll determined at two different times following Serum x a n t h o p h y l l at each sampling
Sampling t i m e after i n t u b a t i o n
between samplings
Experiment
First
Second
First
1
6 8 10 12 14
16 18 20 22 24
2.00 2.29 2.34 3.27 3.42
2.96 3.47 2.78 3.78 4.23
.71 .61 .76 .92 .76
2
6 8 10 12 14
16 18 20 22 24
1.49 2.02 2.65 2.26 3.20
2.24 2.91 3.23 2.87 3.39
.57 .80 .88 .94 .98
5
18
28
2.63
2.54
.96
24
3.40
3.91
.87
Second
intubation
Significance
18
NS, not significant.
whereas it was higher at 24 than at 18 hr after intubation in Experiment 6. These results indicate the necessity of sampling the control and treated birds at the same time following intubation. The addition of either 5% or 10% of bleached soybean oil to the intubating material to prevent segregation following mixing had no effect upon serum xanthophyll levels 24 hr after intubation when corn gluten meal was
the xanthophyll source (Experiment 3, Table 5). There was a rather large numerical increase in serum xanthophyll due to the addition of the oil when marigold meal supplied the xanthophyll (Experiment 4, Table 5), but this was not statistically significant. It is therefore concluded that fat per se did not affect xanthophyll absorption, which was unexpected. The xanthophylls are complexed with fatty acids prior to absorption (Scott et al, 1976) and the most
TABLE 4. Effect of time upon serum xanthophyll of control birds Serum xanthophyll Hours after intubation
Nonintubated controls 3
Intubated controls • (Mg/ml) -
Experiment 6hr 24 hr Experiment 6hr 24 hr Experiment 18 hr 28 hr Experiment 18 hr 24 hr
1 1.43** .94
1.57* 1.10
1.22 1.05
1.42 1.23
2 5 1.59** 1.31 6 .93 1.38**
Blood samples obtained at same time as intubated controls.
**
P<.01.
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6
( A)
5 NSa
1447
XANTHOPHYLL AVAILABILITY BIOASSAY TABLE 5. Effect of including bleached soybean oil in intubating mixture upon serum xanthophyll levels Serum xanthophyll Treatment
Experiment 3 (Corn gluten meal)
Controls 0% Bl soy oil 5% Bl soy oil 10% Bl soy oil
.94 a 4.24 b 3.85 b 3.92 b
Experiment 4 (Marigold meal) • (ug/ml)1.05 a 2.28 b 3.04 b 3.07 b
• Increase in serum xanthophyll 1 1.43 1.24 1.37
.15 .27 .28
a,b Means within the same column bearing different superscripts differ significantly (P<.05). Increase in serum xanthophyll Gjg/ml) over controls per milligram xanthophyll intubated per kilogram body weight.
active site of xanthophyll absorption (Littlefield et al, 1972) is the same as it is for fats (Renner, 1965). Furthermore, Quackenbush et al. (1965) reported that adding lard to rations in which corn supplied carotenols 8.8 mg/kg of ration increased both the total and the percentage of consumed xanthophyll found in toe web punches. Heath and Shaffner (1972) reported that soybean oil increased the amount of xanthophyll in the breast and back skin, the amount of body lipids, and the xanthophyll content of the lipids when marigold petal meal was the xanthophyll source. However, these workers apparently did not consider any xanthophyll which may have been in the soybean oil. Similarly, Day and Williams (1958) found an increase in xanthophyll utilization when 5% of stabilized beef tallow replaced white corn. Many reports in the literature have concluded that increasing dietary xanthophyll decreases xanthophyll utilization when expressed on a per xanthophyll unit basis. Hence, birds were intubated with varying levels of xanthophyll from marigold meal in Experiment 5 and varying levels of a commercial marigold meal extract in Experiment 6. Total serum xanthophyll was increased when the intubated level of xanthophyll increased up to 15 and 16 mg/kg body weight in both experiments (Tables 6 and 7). Regression analysis of the data from Experiment 6 indicates that this increase was curvilinear and that serum
xanthophyll would have reached maximum at 23.2 and 24.8 mg of intubated xanthophyll at 18 and 24 hr after intubation, respectively. When expressed as the increase in serum xanthophyll per milligram of xanthophyll intubated per kilogram body weight, xanthophyll utilization decreased markedly in three of the four instances. This means that when the biological availability of xanthophyll from different sources is being compared, it is
TABLE 6. Effect of xanthophyll intubation level upon serum xanthophyll levels (Experiment 5, marigold meal)
Xanthophyll intubated mg/kg body weight
0 4.0 8.0 16.0
Serum xanthophyll hours after intubation 18
28
1.59 2.17 2.65 3.13
1.31 1.86 2.60 3.13
Increase in serum xanthophyll 3 4.0 8.0 16.0
.145 .133 .096
.138 .161 .114
Increase in serum xanthophyll (jug/ml) over controls per milligram xanthophyll intubated per kilogram body weight.
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0% Bl soy oil 5% Bl soy oil 10% Bl soy oil
MIDDENDORF ET AL.
1448
TABLE 7. Effect of xanthophyll intubation level upon serum xanthophyll levels (Experiment 6, marigold extract)
Xanthophyll intubated mg/kg body •weight
Serum xanthophyll hours iifter intubation 18
24
/
(Mg/ml) •
.95 2.80 3.27 3.92 3.60
• •
1.38 3.17 3.71 4.33 4.44
I ncrease in serum xanthophyll 3 3.2 7.8 15.1 36.3
.578 .297 .197 .073
.559 .299 .195 .084
Increase in serum xanthophyll (;ug/ml) over controls per milligram xanthophyll intubated per kilogram body weight.
necessary to do so at the same xanthophyll level. Conducting two identical experiments one week apart but utilizing the same intubating materials and flock mates produced very similar results, thereby demonstrating the reproducability of the bioassay (Table 8). The xanthophyll content of the sources used in Experiments 7 and 8 was determined by the AOAC (1975) method for total xanthophylls; hence, the epoxide xanthophylls of dehydrated alfalfa are included. This invalidates relating its biological availability to the other test materials. When compared with corn gluten meal, the marigold based products had a relative biological value of 40 to 45%. The saponified marigold extract was supplied by a commercial manufacturer; the degree of saponification was not determined, but either saponification was not achieved or altering the chemical form of the marigold xanthophylls did not improve their utilization. This is not in agreement with Coon and Couch (1976), who obtained a significant improvement in utilization of xanthophyll from marigold meal following saponification. An equal number of pullets and cockerels received each treatment in seven of the eight experiments. The data of all xanthophyll intubated treatments were summarized by sex (Table 9). In two experiments the cockerels
The
results
of
these eight
experiments
TABLE 8. Repeatability of xanthophyll availability bioassay in two consecutive experiments Increase in serum xanthophyll 3
Corn gluten meal Dehydrated alfalfa Marigold meal Saponified marigold extract Marigold concentrate
Experiment 7
Experiment 8
1.25 .70 .54 .5 3
1.15 .70 .44 .51
.51
.53
Increase in serum xanthophyll (Mg/ml) over controls per milligram xanthophyll intubated per kilogram body weight.
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0 3.2 7.8 15.1 36.3
had slightly higher, but insignificantly different serum xanthophyll levels than the pullets. In the other five experiments the pullets had the higher serum xanthophyll level; this ranged from small numerical differences to one which was statistically significant. Also, when the data from all seven experiments were combined and analyzed by a paired t test, the difference was statistically significant. Although these data are not completely consistent, it appears advisable to utilize the same sex distribution when conducting the bioassay. This suggestion is supported by the report of Hinton et al. (1973) who obtained greater pigmentation in pullets when one strain of broilers was used in one experiment but did not obtain a difference between sexes when a different strain was used in a different experiment. The assay method for determining serum xanthophyll as outlined by Wilson (1956) utilized a serum to acetone ratio of 1:15. Comparatively low serum xanthophyll levels were anticipated in this bioassay, so a more concentrated solution was desired to maintain the spectrophotometric readings in the most sensitive range. Hence a pooled serum sample was obtained from broilers which had received a commercial ration; serum xanthophyll was determined using different serum to acetone ratios and five replicates were assayed at each ratio (Table 10). There was no consistent effect of this ratio upon the average serum xanthophyll or the standard deviation. It is concluded that any serum-to-acetone ratio within the range of 1:5.0 to 1:20.0 may be used without materially affecting the results.
1449
XANTHOPHYLL AVAILABILITY BIOASSAY TABLE 9. Effect of sex upon serum xanthophyll level
TABLE 10. Effect of serum to acetone ratios upon serum xanthophyll assay results
Serum xanthophyll Experiment
Males
Females
Acetone ml/ml serum
(/ug/ml)
• (Mg/ml) 3.09 2.46 3.78 2.83 3.29 2.23 2.33 2.79
3.05 2.76 4.26 2.75 3.85* 2.39 2.42 3.00*
5.0 7.5 10.0 12.5 15.0 17.5 20.0
11.01 ± 10.75 ± 10.64 ± 10.73 ± 10.74 ± 10.79 ± 11.19 ±
.09 .06 .03 .05 .06 .14 .06
P<.05.
indicate the feasibility of using a bioassay to compare the biological availability of xanthophyll from various sources. A stepwise description of this bioassay is as follows: 1. Maintain chickens on a low xanthophyll ration for at least two weeks so the base serum xanthophyll is low. 2. Fast the chickens for approximately 18 hr prior to intubating the xanthophyll source(s). 3. Use the same sex distribution for all treatments. 4. A control group should be intubated with a xanthophyll free material at 1% of body weight. Treatment groups should be intubated with equal amounts of xanthophyll from the test materials. This may be easily accomplished by blending the xanthophyll source with a xanthophyll-free material so the total amount of material intubated equals 1% of body weight. Adding 5% of a xanthophyll-free vegetable oil to the material to be intubated will help minimize separation of the blended ingredients. 5. Blood samples may be obtained any time between 14 and 24 hr postintubation, but the interval between the two procedures must be the same for each bird. 6. After clotting, decant the serum, centrifuge it and precipitate the serum proteins with acetone. The ratio of serum to acetone may vary from 1:5.0 to 1:20.0, but it should be constant within any given experiment. 7. Determine absorbance at 474 jum and calculate serum xanthophyll Oug/ml/J-caro-
tene equivalents) from a 0-carotene standard curve. 8. Express results as increase in serum xanthophyll, mcg/ml, over controls per milligram xanthophyll intubated per kilogram body weight. There are several limitation to this bioassay. Efforts to intubate quantities of material much above 1% of body weight failed due to the size of the crop. Hence, a sufficient amount of corn or other comparatively low xanthophyll product could not be intubated to obtain a meaningful increase in serum xanthophyll. Also, it does not consider hues. Individual carotenoids range from pale yellow to deep red in color and the carotenoids deposited in the skin closely resemble those in the diet (Livingston et al., 1969). Some give objectional color tones and some carotenoid combinations have complementary effects (Marusich and Bauernfeind, 1970). Yet, it would be possible to use this bioassay to determine the relative biological activity of a completely unknown xanthophyll source and then feed it to a minimum number of broilers. This would be adequate to at least determine any objectionable hue while still requiring much smaller amounts of a test material than past procedures. Furthermore, any procedure which requires feather removal is subjected to increased error since both scald temperature and time affect feather tract and skin xanthophyll content and pigmentation color (Heath and Thomas, 1973, 1974), making a study of complementary effect of xanthophyll sources very difficult. Visual evaluations are also subject to other errors. Herrick et al. (1970) reported that skin pigmented faster than shanks whereas Hinton et al. (1973) reported that shanks pigmented better than skin. Davies
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1 2 3 4 6 7 8 Combined
Serum xanthophyll Average ± SD
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et al. ( 1 9 6 9 ) used reflectance m e a s u r e m e n t s t o show t h a t foot p a d and shank color were n o t correlated with breast or thigh skin color. REFERENCES
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