Effect of Dietary Rhodobacter capsulatus on Egg-Yolk Cholesterol and Laying Hen Performance U. Salma,* A. G. Miah,* K. M. A. Tareq,* T. Maki,† and H. Tsujii*1 *Laboratory of Animal Biotechnology, Interdisciplinary Graduate School of Science and Technology, Shinshu University, Minamiminowa-mura, Nagano 399-4598, Japan; and †Matsumoto Institute of Microorganisms Co., Ltd., 2904 Niimura, Matsomoto-city, Nagano 3901241, Japan genic index in serum. Yolk color was improved (P < 0.05) in the group fed the 0.04% R. capsulatus supplemented diet compared with the control group. Hepatic cholesterol and triglycerides were reduced (P < 0.05) by 0.04% R. capsulatus. Moreover, the supplementation of R. capsulatus in layer diets did not appear to cause any adverse effects on egg production, shell weight, shell thickness, Haugh unit, yolk index, and feed conversion efficiency compared with the same parameters for the control laying hens. It is postulated that known and unknown factors are present in R. capsulatus presumably responsible for the hypocholesterolemic effect on laying hens. Therefore, the dietary supplementation of R. capsulatus may lead to the development of low-cholesterol chicken eggs as demanded by health-conscious consumers.
ABSTRACT The present study was conducted to investigate the effects of dietary Rhodobacter capsulatus on the laying hen. A total of forty 23-wk-old Hy-Line Brown laying hens were randomly assigned into 4 treatment groups (10 laying hens/group) and fed diets supplemented with 0 (control), 0.01, 0.02, and 0.04% R. capsulatus during the 60-d feeding period. Dietary supplementation of R. capsulatus (0.04%) reduced (P < 0.05) cholesterol and triglycerides concentration in serum (15 and 11%), as well as in egg-yolk (13 and 16%) over a 60-d feeding period. Cholesterol and triglycerides concentrations in serum as well as egg-yolk were changed linearly in accordance with increasing levels of dietary R. capsulatus. Supplementation of R. capsulatus in diets increased high-density lipoprotein cholesterol level and decreased (P < 0.05) athero-
Key words: dietary Rhodobacter capsulatus, cholesterol, triglyceride, egg-yolk, laying hen 2007 Poultry Science 86:714–719
components to the inclusion of drugs and other agents (Noble, 1987; Hargis, 1988). But the high cost of most pharmaceuticals used in the treatment of patients with hypercholesterolemia currently limits the use of these drugs for investigating their efficacy in lowering eggyolk cholesterol. Thus the challenging task for scientists is to reduce cholesterol in egg-yolk or in egg-yolk and blood serum of chickens. Recently, animal biotechnologists have done some research on supplementation of chromium (Uyanik et al., 2002), cupric sulphate pentahydrate (Pesti and Bakalli, 1998), garlic (Chowdhury et al., 2002), and tamarind (Chowdhury et al., 2005) to the laying hen diet. As a probiotic, Rhodopseudomonas palustris has an important role in reducing serum cholesterol in rats fed a high cholesterol diet (Lee et al., 1990). We observed a marked reduction of serum cholesterol levels in rats too when fed diet supplemented with Rhodobacter capsulatus (Tsujii et al., 2007). However, as far as information available, no research work has yet been conducted to study the hypocholesterolemic effects of R. capsulatus on laying hens. Thus the current study was conducted to investigate the effect of R. capsulatus on cholesterol and triglyceride concentration in serum and egg-yolk of laying hens.
INTRODUCTION Chicken eggs are well established as an excellent source of all essential nutrients for persons of all ages. However, it is recommended that people should limit the consumption of eggs because of their high cholesterol (208 mg/egg) content (Kritchevsky and Kritchevsky, 2000). So, it would be beneficial for health to be able to provide a low-cholesterol egg, and research efforts should be directed toward this goal. Reduction of eggyolk cholesterol is a difficult task because cholesterol must be deposited by the hen to facilitate the proper development of the embryo. Genetic selection of chickens for low-cholesterol eggs could not bring significant success in lowering the cholesterol content. So, research with lowering egg-yolk cholesterol has centered mostly on diet and pharmacological intervention. Previous attempts to alter cholesterol level in the egg-yolk have ranged from the manipulation of the normal dietary
©2007 Poultry Science Association Inc. Received July 27, 2006. Accepted November 10, 2006. 1 Corresponding author:
[email protected]
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MATERIALS AND METHODS Culture and Preparation of Rhodobacter capsulatus Rhodobacter capsulatus cells were grown in outdoor culture under natural illumination as previously described (Kobayashi and Kurata, 1978). Briefly, the cells of R. capsulatus were collected by centrifugation and spraydried. The dried R. capsulatus powder was mixed with high soft mineral (MIM Co. Ltd., Matsumoto, Japan) and stored at 4°C. The dried R. capsulatus powder contained approximately 59.8% crude protein, 9.4% crude fat, 0.9% crude fiber, and 9.4% ash determined by the AOAC (1985).
Birds, Management, and Diets A total of 40 Hy-Line Brown ready-to-lay pullets with almost the same BW were collected from a local commercial flock. They were caged individually into wire cages (25 × 40 cm) with individual feed-trough and common water-trough. The birds were reared in accordance with university guidelines for animal experimentation. Birds were fed a balanced commercial layer diet as their daily requirement since 6 wk prior to commencement of the study for adaptation and reaching a standard level of egg production. The basal diet contained (in percentage): ground yellow corn, 56; soybean meal, 14.5; sesame meal, 7; corn gluten meal, 2.5; rice polish, 8; fish meal, 1; calcium carbonate, 7.68; dicalcium phosphate, 1.32; common salt, 0.45; animal fat, 1; and vitamin premix, 0.25. The determined and calculated (AOAC, 1985) nutrient composition of the basal diet was 2,935 kcal/kg of ME, 16.46% CP, 2.9% Ca, 0.34% available P, 0.54% Met + Cys, 0.71% Lys, and 86 g/g of cholesterol (enzymatically). The laying hens of 23-wk-old were assigned into 4 treatment groups under the completely randomized design, so that there were 10 laying hens in each group. The diets were supplemented with 4 levels of R. capsulatus; 0% as control group and 0.01, 0.02, and 0.04% as treatment groups. The diets were provided daily at 120 g per bird (considering 5% excess of requirement) during the 8-wk feeding period. Clean drinking water was supplied ad libitum, and birds were exposed to a 16-h incandescent light period.
Sample Collection The laying hens were weighed individually prior to blood collection at the beginning and at the end of each 15 d of the feeding period. Daily feed intake was recorded, and feed conversion efficiency (feed intake: mass production) was calculated during the 8-wk feeding period. Daily egg production and individual egg weight was recorded to determine the hen day egg production and the egg mass production (g/d/hen). Eggs from each group of laying hens were collected at the beginning and at the end of each 10 d of the feeding period for
Figure 1. Effect of dietary Rhodobacter capsulatus on serum cholesterol (A) and triglycerides (B) concentration in laying hens. Black circle (䊉), white circle (䊊), black square (䊏), and black cross (×) indicate 0 (control), 0.01, 0.02, and 0.04% of R. capsulatus supplemented in diets, respectively. Each line with error bar represents the means ± SEM for 10 hens per group. a,bData points without a common lowercase letter indicate significant differences (P < 0.05) among the groups at the same experimental period.
measuring egg quality (egg weight, shell weight, eggshell thickness, yolk weight, yolk index, yolk color, and Haugh unit), as well as for analysis of yolk cholesterol and triglycerides. Yolk index was calculated as the ratio of yolk height to yolk width. Yolk color was evaluated using a colorimetric fan (Roche), and color was scored according to their intensity. Haugh unit was calculated using the Haugh (1937) formula.
Blood Collection Blood samples (2.0 mL) from each individual layer were collected at the beginning and at the end of each 15 d of the feeding period. Blood was collected from the brachial wing vein using sterilized syringes and needles.
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1990). From each liver and muscle sample, 1 g was homogenized with 12-mL of chloroform-methanol 2:1 (by volume) and filtered directly into a 50-mL volumetric flask using a glass microfiber filter. Following rehomogenization and refiltration, the liver and muscle filtrates were diluted to a final volume of 50-mL with chloroformmethanol 2:1 (by volume). Then the obtained samples were stored at −80°C until further analysis.
Enzymatic Analysis Cholesterol, triglycerides, high-density lipoprotein (HDL) cholesterol, and glucose concentrations in the serum were determined enzymatically using commercially available reagent kits (Wako cholesterol 439-17501, Wako triglycerides 432-40201, Wako HDL-cholesterol 431-52501, and Wako glucose 439-90901, respectively, Wako Pure Chemical Industries Ltd., Tokyo, Japan). The atherogenic index was calculated as the ratio of lowdensity lipoprotein cholesterol to HDL cholesterol. Cholesterol and triglycerides concentrations in the egg-yolk, liver, and muscles (breast and thigh) were determined using the same reagent kits as those used for serum analysis.
Statistical Analysis
Figure 2. Effect of dietary Rhodobacter capsulatus on egg yolk cholesterol (A) and triglyceride (B) concentration. Black circle (䊉), white circle (䊊), black square (䊏), and black cross (×) indicate 0 (control), 0.01, 0.02, and 0.04% of R. capsulatus supplemented in diets, respectively. Each line with error bar represents the means ± SEM for 10 hens per group. a,b Data points without a common lowercase letter indicate significant differences (P < 0.05) among the groups at the same experimental period.
After 1 h standing at room temperature, serum was isolated by centrifugation at 1,150 × g for 10 min. Serum samples were stored at −80°C until further analysis.
Liver and Muscle Collection At the end of the 8-wk feeding period, the laying hens were decapitated, and liver, breast (pectoralis major), and thigh (biceps femoris) muscles were collected and washed with normal saline, blotted dry on filter paper, and weighed before freezing for storage.
Yolk, Liver, and Muscles Sample Preparation One gram of each egg yolk was homogenized with 15 mL of chloroform-methanol 2:1 (by volume), sonicated, and filtered as previously described (Elkin and Rogler,
The experiment was conducted under the completely randomized design. Data were analyzed for using the Fisher’s protected least significant difference test. The NCSS (Number Cruncher Statistical System, NCSS Statistical Software, Kaysville, UT) Version 5.01 computer software package was used for all statistical analysis. All data are expressed as means ± SEM. Differences were considered significant at the level of P < 0.05.
RESULTS The changes of cholesterol and triglycerides concentration in serum of the laying hens fed diets supplemented with 0, 0.01, 0.02, and 0.04% R. capsulatus are shown in Figure 1. After 45 d of feeding period, serum cholesterol concentration was significantly reduced (12 to 15%) in the layer group fed the 0.04% R. capsulatus supplemented diet compared with the control or all other groups. In accordance with increasing age, serum cholesterol concentration was decreased in the group fed 0.04% R. capsulatus supplemented diets, though increased in the groups fed 0, 0.01, 0.02% R. capsulatus supplemented diets. In contrast, the comparatively lowest concentration of triglycerides was exhibited in the serum of the group fed the 0.04% R. capsulatus supplemented diet after 45 d of feeding period. Effect of dietary R. capsulatus on HDL-cholesterol and atherogenic index in the serum of laying hens at 60 d of feeding period are shown in Table 1. HDL-cholesterol was significantly increased by supplementation of 0.04% R. capsulatus to layer diet compared with control diet. The atherogenic index was significantly lower in the
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DIETARY RHODOBACTER CAPSULATUS ON EGG-YOLK CHOLESTEROL Table 1. Effect of dietary Rhodobacter capsulatus on serum low-density and high-density lipoprotein (LDL and HDL) cholesterol and atherogenic index at 60 d of feeding period in laying hens1 Level of R. capsulatus (%) Parameter
0 (control) 2
LDL cholesterol (mg/dL) HDL cholesterol (mg/dL) Atherogenic index3
0.01
108.9 ± 3.4 42.2 ± 2.8b 2.6 ± 1.2a a
0.02
106.4 ± 2.8 42.7 ± 2.6b 2.5 ± 1.1a a
0.04
97.9 ± 1.3 46.8 ± 3.1ab 2.1 ± 0.4a ab
80.5 ± 0.8b 53.7 ± 2.4a 1.5 ± 0.3b
Means within a row without common superscripts differ significantly (P < 0.05). Values are means ± SEM for 10 laying hens per group. 2 Low-density lipoprotein (LDL) cholesterol = (total cholesterol − HDL cholesterol) − triglycerides/5 (Friedewald et al., 1972). 3 Atherogenic index: the ratio of LDL cholesterol to HDL cholesterol (LDL/HDL). a,b 1
serum of the group fed diet supplemented with 0.04% R. capsulatus than that of the other groups. The effects of dietary R. capsulatus on egg-yolk cholesterol and triglyceride concentration are shown in Figure 2. The average concentration of yolk cholesterol in the eggs laid at the 50 and 60 d of feeding period was significantly reduced 12 and 13%, respectively, in the 0.04% R. capsulatus group as compared with the control group. After the 50 d of feeding period, triglyceride concentration was also significantly lower in the egg-yolk from the group fed the 0.04% R. capsulatus supplemented diet than in the egg-yolk from the control group. The rate of hen day egg production, egg weight, egg mass, feed intake, and feed conversion efficiency changes over the 60-d feeding period were not significantly affected by R. capsulatus supplemented diets (Table 2). Effect of R. capsulatus on egg quality (yolk weight, shell weight, shell thickness, Haugh unit, yolk index, and yolk color) over the 60-d feeding period in laying hens is shown in Table 3. Yolk color was significantly improved in the group fed the 0.04% R. capsulatus supplemented diet than in the control group. Yolk color was markedly improved in the laying hens with increasing levels of R. capsulatus supplementation in diets. Yolk weight, shell weight, shell thickness, yolk index, and Haugh unit did not differ significantly among the laying hens fed diets containing different levels of R. capsulatus. Average weight, cholesterol, and triglyceride concentrations of liver were significantly lower in the group fed the 0.04% R. capsulatus supplemented diet than in
the control group (Table 4). It was observed that cholesterol and triglycerides concentrations in the liver of the laying hens were decreased with increasing level of R. capsulatus supplementation in diets. On the other hand, cholesterol concentrations in breast and thigh muscles were not significantly reduced among the layer groups fed diets supplemented with different levels of R. capsulatus (Table 4).
DISCUSSION In this study, supplementation of R. capsulatus into laying hen diets has been shown to reduce not only serum cholesterol but also egg-yolk cholesterol concentration. The reduction of egg-yolk cholesterol by 0.04% R. capsulatus feeding is greater than the 6 to 7% decrease by including oat bran or cotton seed hulls (Lirette et al., 1993); the 5 to 7% decrease by feeding probucol (Waldroup et al., 1986); or the 5% decrease by feeding probucol (Naber et al., 1982). Sharma et al. (1979) reported that egg-yolk cholesterol was reduced by 4.1 or 5.5% when laying hens were fed 1 or 3% garlic powder for 21 d. The reduction of serum cholesterol by 0.04% R. capsulatus feeding is quite similar to those obtained by Chowdhury et al. (2005): 12 to 14% by feeding 2, 4, 6, or 8% ovendried and crushed tamarind. Shim et al. (2004) reported that serum cholesterol was reduced by 11.7% when broiler were fed 0.50% Codonopsis lanceolata root. The present results are in agreement with our previous results; Tsujii et al. (2007) reported significant reduction of serum cholesterol in rats fed a R. capsulatus supple-
Table 2. Effect of dietary Rhodobacter capsulatus on egg production, egg weight, egg mass, feed intake, and feed efficiency in laying hens during the 60-d feeding period1 Level of R. capsulatus (%) Parameter
0 (control)
Egg production (%) Egg weight (g) Egg mass2 (g/d/hen) Feed intake (g/d/hen) Feed conversion efficiency3
98.4 ± 59.1 ± 58.1 ± 116.9 ± 2.03
1.9 1.2 0.9 0.9
0.01 97.9 ± 58.6 ± 57.4 ± 116.8 ± 2.03
0.02 1.6 0.8 1.1 2.1
97.9 ± 58.5 ± 57.3 ± 115.8 ± 2.02
0.04 1.7 0.5 1.2 2.8
98.0 ± 58.4 ± 57.2 ± 115.5 ± 2.01
All measurements were done as fresh basis; values are means ± SEM for 10 laying hens per group. Egg mass = (egg production × egg weight)/100. 3 Feed conversion efficiency = feed intake: egg mass (g:g). 1 2
2.6 0.4 1.4 2.7
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SALMA ET AL. Table 3. Effect of dietary Rhodobacter capsulatus on egg quality (yolk weight, shell weight, shell thickness, Haugh unit, yolk index, and yolk color) during the 60-d feeding period in laying hens1 Level of R. capsulatus (%) Parameter Yolk weight (g) Shell weight (g) Shell thickness (mm) Haugh unit Yolk index2 Yolk color
0 (control) 15.3 7.1 0.37 78.5 0.43 8.9
± ± ± ± ± ±
0.5 0.3 0.02 4.2 0.01 0.25b
0.01 15.0 7.3 0.34 70.9 0.42 10.3
± ± ± ± ± ±
0.6 0.2 0.01 3.4 0.03 0.28ab
0.02 14.8 6.7 0.35 71.8 0.41 10.7
± ± ± ± ± ±
0.6 0.1 0.03 3.6 0.02 0.25ab
0.04 ± ± ± ± ± ±
14.9 7.0 0.36 73.2 0.42 12.2
0.7 0.3 0.01 4.1 0.01 0.26a
Means within a row without common superscripts differ significantly (P < 0.05). All measurements were done as fresh basis; values are means ± SEM for 10 laying hens per group. 2 Yolk index = yolk height: yolk width. a,b 1
mented diet. Lee et al. (1990) also reported similar results in rats fed Rhodopseudomonas palustris supplemented diet. In the present study, after 60-d feeding of a 0.04% R. capsulatus supplemented diet, triglyceride concentrations in egg-yolk and serum were reduced (16 and 11%, respectively), a significantly decrease compared with the control diet. Our previous study (Tsujii et al., 2007) also demonstrated that triglyceride concentrations in rat serum tended to decrease in the R. capsulatus supplemented diet after the 42 d of feeding period. In the study, the hypocholesterolemic effect of R. capsulatus was expressed after the 4 wk of feeding period in serum first, and then in egg-yolk; a certain period may be needed to accumulate hypocholesterolemic factors from R. capsulatus into blood stream. Higher level of carotenoids 4.2% (Kobayashi and Kurata, 1978) in R. capsulatus improved egg-yolk color where yolk-color was increased depending on the quantity of supplemented R. capsulatus in diets. It might be speculated that carotenoids from R. capsulatus can be well absorbed and transfer into yolk and markedly increase yolk color. The hypocholesterolemic and other beneficiary effects of R. capsulatus demonstrated in this study were not clearly understood. However, R. capsulatus contains many known and unknown factors, which are speculated to be associated directly or indirectly with the hypocholesterolemic and other beneficiary performances of laying hens. In fact, R. capsulatus is a rich source of
carotenoids (Kobayashi and Kurata, 1978); some of the carotenoids are known as hypocholesterolemic agent. Beta-carotene has a hypocholesterolemic effect in rats and seems to displace cholesterol in the transport of lipoproteins (Amen and Lachance, 1974). Yeum and Russell (2002) reported that carotenoid-rich diets are associated with reducing serum cholesterol concentration. Dietary carotenoids are absorbed by intestinal cells and incorporated into triglyceride-rich lipoproteins (chylomicrons) and released into the circulation. Triglycerides are depleted from circulating chylomicrons through the activity of lipoprotein lipase, resulting in the formation of chylomicron remnants. Another mechanism through which R. capsulatus may exert its hypocholesterolemic action is via bile acids. The cholic and deoxycholic bile acids are produced from cholesterol by hepatocytes and are conjugated with glycine and taurine, respectively. These acids enter the small intestine, where they are absorbed and directed to the liver, and a decrease in bile acid recycling would ultimately result in a lowering of serum cholesterol concentration because cholesterol is used for bile acid synthesis (St-Onge et al., 2000). However, this study did not measure the amount of bile acid synthesis to support this speculation. Rhodobacter capsulatus may stimulate binding of cholesterol with bile acids, and inhibition of micelle formation combined with the effect of fermentation on short chain fatty acids production are mechanisms that have been proposed to explain the potential choles-
Table 4. Effect of dietary Rhodobacter capsulatus on liver weight, cholesterol, and triglyceride concentrations in liver and cholesterol concentrations in breast (pectoralis major) and thigh (biceps femoris) muscles of laying hens at 60 d of feeding period1 Level of R. capsulatus (%) Parameter Liver Weight (g/100 g of BW) Cholesterol (mg/g) Triglycerides (mg/g) Muscle cholesterol Breast (mg/100 g) Thigh (mg/100 g)
0 (control)
0.01
0.02
0.04
2.3 ± 0.15a 4.3 ± 0.46a 14.6 ± 0.7a
2.3 ± 0.08a 3.9 ± 0.40a 12.6 ± 1.4ab
2.0 ± 0.13ab 3.3 ± 0.15ab 10.2 ± 1.5b
1.9 ± 0.06b 2.3 ± 0.09b 10.0 ± 1.2b
41.7 ± 2.3 46.2 ± 3.3
41.1 ± 2.21 45.9 ± 2.3
40.5 ± 2.6 45.0 ± 2.1
39.6 ± 2.0 44.5 ± 1.9
Means within a row without common superscripts differ significantly (P < 0.05). All measurements were done on an as fresh basis; values are means ± SEM for 10 laying hens per group.
a,b 1
DIETARY RHODOBACTER CAPSULATUS ON EGG-YOLK CHOLESTEROL
terol-lowering effects (Jenkins et al., 1991). Rhodobacter capsulatus may change enzymes, which are associated in regulating cholesterol synthesis, oxidation, or elimination for lowering cholesterol in laying hens. In conclusion, dietary supplementation of R. capsulatus decreases cholesterol and triglyceride concentrations in serum and egg-yolk as well as increasing HDL-cholesterol concentration in serum and markedly improving yolk color. The supplementation of R. capsulatus in layer diets under the present study did not appear to cause any adverse effects on egg production and egg quality compared with the same parameters for the control laying hens. It is postulated that the known and unknown factors present in R. capsulatus are presumably responsible for the hypocholesterolemic effect on laying hens. Therefore, the dietary supplementation of R. capsulatus may lead to the development of low-cholesterol chicken eggs as demanded by health-conscious consumers.
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