Effects of Dietary Garlic on Cholesterol Metabolism in Laying Hens

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1 Effects of ary Garlic on Cholesterol Metabolism in Laying Hens S. R. Chowdhury,* S. D. Chowdhury, and T. K. Smith*,1 *Department of Animal and Poultry Science, University of Guelph, Ontario, Canada N1G 2W1; and Department of Poultry Science, Bangladesh Agricultural University, Mymensingh, Bangladesh ABSTRACT Experiments were conducted to evaluate ing levels of dietary garlic and differed among strains (P < the potential for dietary garlic to influence egg yolk cholesterol concentrations and overall performance in different layer strains. Thirty-six, 28-wk-old, Hisex Brown, Isa Brown, Lohmann, Starcross, Babcock, and Starcross-579 strains (six hens per strain) were fed diets supplemented with 0 (control), 2, 4, 6, 8, or 10% sun-dried garlic paste for 6 wk. There were no differences (P > 0.05) among diets or strains in egg weight, egg mass, feed consumption, feed efficiency, and BW gain as averaged over 6 wk. Yolk weight, however, responded quadratically with increas- 0.05). Egg production and yolk weights were significantly higher in the Babcock strain in comparison with other strains. Serum and egg yolk cholesterol concentrations decreased linearly (P < 0.05) with increasing levels of dietary garlic. Serum and egg yolk cholesterol concentrations also differed among different strains (P < 0.05). It was concluded that garlic paste in the diets of laying hens reduced serum and yolk cholesterol concentrations. It was also concluded that dietary garlic paste had no adverse effects on layer performance. (Key words: sun-dried garlic paste, cholesterol, yolk, serum, layer strain) 2002 Poultry Science 81: INTRODUCTION Garlic has acquired a reputation in the folklore of many cultures as a therapeutic agent (Amagase et al., 2001). According to folk medicine, garlic was used to treat cardiac disease (Essman, 1984). Several clinical reports, including meta-analyses, have described the hypocholesterolemic effect of garlic in humans (Silagy and Neil, 1994; Warshafsky et al., 1993). Some studies, however, suggested that commercial garlic oil, garlic powder, and commercially available garlic extract may not be hypocholesterolemic (Berthold et al., 1998; Isaacsohn et al., 1998; McCrindle et al., 1998). Although the reason for this is unknown, it likely relates to preparation methods, the stability of chemical components, and the duration of the study (Amagase et al., 2001). Many studies indicated that allicin was the potentially active component of garlic; however, it was observed that allicin was unstable and poorly absorbed from the digestive tract (Lawson et al., 1992). Animal studies suggest that garlic has potential hypolipidemic, hypotensive, hypoglycemic, hypothrombotic, and hypoatherogenic properties (Bordia et al., 1975; Shoetan et al., 1984). Garlic paste (3.8%), solvent fractions, or garlic oil equivalent to this amount reduced serum 2002 Poultry Science Association, Inc. Received for publication May 13, Accepted for publication July 23, To whom correspondence should be addressed: tsmith@ uoguelph.ca. cholesterol by 18 and 23% in broilers and 12-wk-old Leghorn pullets, respectively, when diets were fed for 4 wk (Qureshi et al., 1983b). Experiments in which garlic was fed to 5-wk-old male broilers for 3 wk and in vitro studies with chicken hepatocytes exposed to polar fractions of garlic powder (garlic equivalent to 1, 2, 4, 6 and 8% fresh garlic paste) showed a dose-dependent inhibition of hepatic β-hydroxy-β-methylglutaryl coenzyme A (HMG- CoA) reductase, cholesterol 7α-hydroxylase, and fatty acid synthetase (Qureshi et al., 1983a). Egg yolk cholesterol was reduced by the feeding of 1 or 3% garlic powder to laying hens for 3 wk (Sharma et al., 1979). Sklan et al. (1992) observed depressed hepatic cholesterol concentrations in chickens when 2% garlic was fed for 14 d. Birrenkott et al. (2000), however, reported that 3% garlic powder did not have any significant effect on yolk and serum cholesterol concentrations when laying hens were fed diets for 8 mo. Reddy et al. (1991) reported that egg production, egg mass, BW, feed intake, and feed efficiency were not affected during the 8 wk that 0.02% garlic oil was fed to the Babcock B-300 strain. Egg yolk cholesterol concentrations have been shown to vary depending on the genetic strain of the laying hens (Han and Lee, 1992). The current study was conducted, therefore, to characterize the hypocholesterolemic effects of high levels of sun-dried garlic paste in the diets of different genetic strains of laying hens. MATERIALS AND METHODS Experimental Birds and s Thirty-six, 28-wk-old pullets of six different strains (six birds per strain; six birds per diet) were used in the current 1856

2 GARLIC IN LAYER DIETS 1857 TABLE 1. Composition of experimental diets (%) Garlic paste in diet (%) Ingredient Wheat Corn Rice polish Garlic paste Fish meal Sesame-oil cake Soybean oil Oyster shell Bone meal Common salt Vitamin-mineral mixture Vitamin C Calculated nutrient content ME, kcal/kg 2,730 2,739 2,799 2,804 2,813 2,804 Lysine Methionine Calcium Total phosphorus Analyzed nutrient content Dry matter Crude protein Crude fat Crude fiber Cholesterol concentration Provided per kilogram of diet: vitamin A (retinyl palmitate), 1,200 IU; cholecalciferol, 2,500 IU; vitamin E (dl-α-tocopheryl acetate), 20 IU; vitamin K 3, 4.0 mg; thiamin, 1.5 mg; riboflavin, 50.0 mg; pantothenic acid, 10.0 mg; niacin, 30 mg; pyridoxine, 4.0 mg; choline chloride, 250 mg; folic acid, 0.5 mg; biotin, 220 µg; vitamin B 12, 12 µg ; BHT, 250 mg; manganese, 48 mg; zinc, 40 mg; iron, 24 mg; copper, 16 mg; iodine, 0.6 mg; selenium, 0.12 mg; and DL-methionine, 50 mg. study. The strains were Hisex Brown, Isa Brown, Lohmann, Starcross, Babcock, and Starcross Hens were caged individually and provided with 14 h of light daily. All birds were fed isoenergetic and isonitrogenous mash diets for 6 wk. The diets were formulated to meet or exceed the nutrient requirements of laying hens (National Research Council, 1994). s were supplemented with 0 (control), 2, 4, 6, 8, or 10% sun-dried garlic in paste form. The composition of the experimental diets is shown in Table 1. Feed and water were provided ad libitum. Source and Preparation Methods for ary Garlic Locally produced garlic bulbs were purchased from the University Market, Mymensingh, Bangladesh. The garlic was harvested in April or May. The age of the bulbs, when dried, was 2 to 4 mo. Fresh garlic bulbs were ground with husk to produce paste. Fresh garlic paste was subsequently thinly spread on a mat in direct sunlight at 30 to 35 C. Drying process was continued for 8 to 10 h to ensure the appropriate consistency of the paste. s were prepared the following day and were stored at room temperature for a maximum of 6 wk. 2 Youth Training Center, Saver, Dhaka, Bangladesh. Experimental Parameters Measured Birds were randomly assigned to diets and fed daily. Body weights were measured weekly, and BW gain was recorded. Weekly feed consumption was recorded, and feed efficiency was calculated during the 6-wk experimental period. Daily egg production was recorded. Egg weights were determined weekly, and egg mass was calculated. Eggs were collected weekly from each bird for cholesterol analysis beginning at the 21 d of feeding. Eggs were subsequently hard-boiled to separate the yolk. Cooked yolk weights were recorded individually. Cooked yolk was extracted and subsequently analyzed for cholesterol concentration per yolk basis. Blood Collection Blood was also collected from each bird weekly beginning at the 21 d of feeding from the wing vein using sterilized syringes and needles. Serum was isolated 4 to 6 h after blood collection. Serum samples were maintained at 5 C for up to 2 d until cholesterol analysis. Extraction of Yolk and Lipid One gram of yolk was placed into a centrifuge tube. Fifteen milliliters of chloroform:methanol (2:1 v/v) was added, blended on a vortex mixture, and allowed to extract for 12 h. lipid was extracted by the same proce-

3 1858 CHOWDHURY ET AL. dure using a 5-g sample with 40 ml of chloroform:methanol. Analysis of Cholesterol and ary Nutrients Serum, extracted yolk, and diet samples were analyzed for cholesterol according to the colorimetric method of Abell et al. (1952). s were analyzed for dry matter, crude protein, crude fiber, and crude fat according to the Association of Official Analytical Chemists (1980). Statistical Analyses Data were analyzed by analysis of variance using the General Linear Models procedure of SAS for a factorial design (two factor analysis; diet and strain) (Kuehl, 1994; SAS Institute, 2000). Orthogonal polynomial contrasts were used to determine the nature of the response to diets. Differences among strains, when significant, were also ordered using Tukey s test (Kuehl, 1994). Statements of statistical significance were based on P < RESULTS AND DISCUSSION It was observed that cholesterol concentrations in all diets were very low and similar to those found in the control diet (Table 1). The first 2 wk that garlic was added as a supplement to the diet allowed the birds to be acclimatized with the experimental diet. Layer Performance Egg production, egg weight, egg mass, feed consumption, feed efficiency, and BW gain were not affected by diet (P > 0.05) as averaged over the 6-wk period (Table 2). In agreement with the current study, Reddy et al. (1991) reported that egg production, egg mass, BW, feed intake, and feed efficiency were not affected during the 8 wk that 0.02% garlic oil was fed to the Babcock B- 300 strain. It was observed in the current study that there were no (P > 0.05) differences among strains in egg weight, egg mass, feed consumption, feed efficiency, and BW gain. Egg production was, however, higher in the Babcock strain than in other strains (P < 0.05), which was likely due to the genetic potential of the Babcock strain. Serum Cholesterol Serum cholesterol concentrations responded quadratically (P < 0.01) with increasing levels of sun-dried dietary garlic paste in Weeks 3, 4, and 6. In Week 5, however, serum cholesterol decreased linearly (Table 3). Plasma cholesterol was reduced by 30% when rats were fed diets supplemented with 2 or 3% garlic powder (Myung et al., 1982). In the current study, 2, 4, 6, or 8% garlic paste reduced serum cholesterol concentrations on average by 15, 28, 33, and 43%, respectively. There was no further reduction in serum cholesterol concentration when layers were fed 10% garlic paste. Qureshi et al. (1983a) reported that diets equivalent to 1, 2, 4, 6, and 8% garlic paste reduced serum cholesterol by 18, 21, 21, 24, and 25%, respectively, in male broiler chickens. The cholesterol concentrations in laying hens fed the control diet in the current study were higher than those reported by McNaughton (1978), who observed 165 mg/100 ml plasma cholesterol in White Leghorn hens when fed similar levels of crude fiber as the current study. This result may be due to differences in analytical techniques and strain of the TABLE 2. Effects of dietary garlic and different genetic strains on performance of laying hens 1 Egg Egg Egg Feed production weight mass 2 consumption Feed BW gain (%) (g) (g/d per hen) (g/d per hen) efficiency 3 (g/wk) 0% garlic paste % garlic paste % garlic paste % garlic paste % garlic paste % garlic paste Linear NS 4 NS NS NS NS NS Quadratic NS NS NS NS NS NS Pooled SD Hisex Brown 83 a 53 a 44 a 114 a 2.64 a 58 a Isa Brown 82 a 54 a 45 a 117 a 2.65 a 125 a Lohmann 82 a 55 a 45 a 115 a 2.59 a 66 a Starcross 84 a 52 a 44 a 114 a 2.63 a 100 a Babcock 88 b 54 a 48 a 116 a 2.47 a 142 a Starcross a 53 a 44 a 111 a 2.71 a 113 a a,b Means with the same superscript within a column are not significantly different. 1 Data are reported as least squares means as averaged over 6 wk; n = 6. 2 Egg mass = (egg production egg weight)/ Feed efficiency = feed intake:egg mass (g:g). 4 P > 0.05.

4 GARLIC IN LAYER DIETS 1859 TABLE 3. Effects of dietary garlic and different genetic strains on serum cholesterol concentrations in laying hens (mg/100 ml) 1 0% garlic paste % garlic paste % garlic paste % garlic paste % garlic paste % garlic paste Linear Quadratic NS Pooled SD Hisex Brown 181 ab 181 a 166 a 181 b Isa Brown 157 a 197 b 150 a 157 ab Lohmann 160 a 171 a 152 a 129 a Starcross 165 a 183 ab 183 ab 148 a Babcock 193 b 203 bc 223 bc 145 a Starcross ab 211 bc 195 b 156 a a,b,c Means with the same superscript within a column are not significantly different. 1 Data are reported as least squares means; n = 6 birds. Serum cholesterol concentrations differed significantly among strains. The Babcock layer strain had the highest serum cholesterol concentrations in Weeks 3, 4, and5(p < 0.05). variation in cholesterol concentrations might be due to inherent genetic differences. Egg Yolk Weight Yolk weight responded quadratically (P < 0.05) in Weeks 3 and 4 with increasing levels of sun-dried dietary garlic paste (Table 4). The Babcock strain had the highest yolk weight in comparison with other strains after 6 wk of feeding. To the best of our knowledge, the effect of dietary garlic on yolk weight has not been previously reported in the literature. Egg Yolk Cholesterol (mg/g) Cholesterol concentrations per gram of yolk decreased linearly (P < 0.01) with increasing levels of sun-dried dietary garlic paste (Table 5). ary garlic paste at 2, 4, 6, 8, or 10% reduced egg yolk cholesterol, on average, by 5, 9, 14, 20, and 24%, respectively. 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 3 wk. There were no significant differences among strains in per gram yolk cholesterol concentrations in Weeks 3 and 6. Cholesterol concentrations in the Babcock, Lohmann, and Starcross-579 strains were significantly higher than those of other strains at the end of Week 4. Cholesterol concentration in the Babcock strain was TABLE 4. Effects of dietary garlic and different genetic strains on cooked egg yolk weight (g) 1 0% garlic paste % garlic paste % garlic paste % garlic paste % garlic paste % garlic paste Linear 0.01 NS 2 NS NS Quadratic NS NS Pooled SD Hisex Brown 13.2 a 12.3 a 13.4 a 13.0 a Isa Brown 13.5 a 12.8 a 13.7 a 13.2 a Lohmann 13.3 a 12.0 a 13.5 a 12.8 a Starcross 13.0 a 12.6 a 12.7 a 13.0 a Babcock 14.5 a 12.9 a 13.4 a 15.2 b Starcross a 11.9 a 13.2 a 13.1 a a,b Means with the same superscript within a column are not significantly different. 1 Data are reported as least squares means; n = 6.

5 1860 CHOWDHURY ET AL. TABLE 5. Effects of dietary garlic and different genetic strains on egg yolk cholesterol concentrations (mg/g) 1 0% garlic paste % garlic paste % garlic paste % garlic paste % garlic paste % garlic paste Linear Quadratic NS 2 NS NS NS Pooled SD Hisex Brown 11.8 a 12.3 a 12.2 ac 11.7 a Isa Brown 11.7 a 12.4 a 12.2 ac 11.9 a Lohmann 11.8 a 12.6 b 11.8 bc 12.1 a Starcross 11.6 a 12.4 a 12.2 ac 12.0 a Babcock 11.9 a 12.6 b 12.5 a 11.8 a Starcross a 12.7 b 11.9 bc 11.8 a a,b,c Means with the same superscript within a column are not significantly different. 1 Data are reported as least squares means; n = 6. significantly higher than in the Lohmann and Starcross- 579 strains in Week 5. The high yolk cholesterol concentrations in the Babcock strain might be genetic differences among strains. Han and Lee (1992) observed significantly lower cholesterol concentrations in hens laying brown eggs. Yolk cholesterol values found in the current study, when hens were fed control diets, were comparable with those of Jiang et al. (1991), who observed 14.6 mg/g yolk using the same procedure in White Leghorn hens. Total Yolk Cholesterol Total yolk cholesterol decreased linearly (P < 0.01) with increasing levels of dietary garlic paste throughout the experimental period (Table 6). Total yolk cholesterol was higher (P < 0.05) in the Babcock strain than in other strains at the end of Week 6. The effect of dietary garlic paste on yolk size was inconsistent. Becker et al. (1977) also reported that the correlation of yolk cholesterol with yolk weight was inconsistent. Total yolk cholesterol values found in the current study, when hens were fed control diets, were lower than those found by Jiang et al. (1991), who used the same colorimetric method and observed 243 mg cholesterol per yolk in White Leghorn hens. This result was due to the higher yolk weight (16.7 g) reported by Jiang et al. (1991). Cholesterol Metabolism The reduction of serum and egg yolk cholesterol observed when garlic paste was fed might be due to the reduction of synthetic enzyme activity. Qureshi et al. TABLE 6. Effects of dietary garlic and different genetic strains on total egg yolk cholesterol (mg) 1 0% garlic paste % garlic paste % garlic paste % garlic paste % garlic paste % garlic paste Linear Quadratic NS 2 NS NS NS Pooled SD Hisex Brown 155 a 151 a 162 a 152 a Isa Brown 157 a 159 a 167 a 158 a Lohmann 155 a 150 a 158 a 154 a Starcross 150 a 156 a 154 a 155 a Babcock 172 a 162 a 167 a 181 b Starcross a 150 a 157 a 155 a a,b Means with the same superscript within a column are not significantly different. 1 Data are reported as least squares means; n = 6.

6 GARLIC IN LAYER DIETS 1861 (1983a) reported a dose-dependent inhibition of hepatic HMG-CoA reductase, cholesterol 7α-hydroxylase, and fatty acid synthetase when chickens were fed polar fractions of garlic powder equivalent to 1, 2, 4, 6, and 8% fresh garlic paste. Konjufca et al. (1997) reported that feeding 3% commercial garlic power reduced the activities of HMG-CoA reductase and cholesterol 7α-hydroxylase by 40%. Reddy et al. (1991), however, reported that feeding 0.02% garlic oil did not have any effect on plasma and yolk cholesterol in the Babcock B-300 strain. Reddy et al. (1991) concluded that the feeding of 0.02% garlic oil might be insufficient to inhibit HMG-CoA reductase in White Leghorn hens. Hypocholesterolemic Effect of Different Garlic Products The hypocholesterolemic effect of dietary sun-dried garlic paste to laying hens in the current study confirmed previous reports (Essman, 1984; Warshafsky et al., 1993; Silagy and Neil, 1994). The contradictory results in the literature were due to use of different commercial garlic products. Some studies have suggested that commercial garlic oil, garlic powder, and commercially available garlic extract may not be hypocholesterolemic (Berthold et al., 1998; Isaacsohn et al., 1998; McCrindle et al., 1998). The different garlic products that are commercially available may be divided into allicin-rich products and nonallicin-rich products. The former are made from raw garlic, and the latter are made from processed garlic. All may differ significantly in the substances they contain (Kasuga et al., 2001). Questions arise, therefore, as to the equivalence of garlic products with respect to their hypocholesterolemic effects. The active components in garlic have not yet been determined, although many studies have indicated that allicin is the potentially active component. It has been observed that allicin is unstable and poorly absorbed from the digestive tract (Lawson et al., 1992). Garlic preparations that are produced by heat or solvent extraction processes are known to contain alliin, but also to be devoid of allinase; hence, no allicin may be formed. Garlic oil and steam-distilled products are rich in secondary metabolites such as ajoene. It is, however, unclear as to what extent these secondary compounds are formed in the body following the ingestion of garlic and whether their effects can be compared with those of fresh garlic (Newall et al., 1995). The use of sun-dried garlic paste in the current study maximized the possibilities of the presence of active components. Further studies are needed to confirm the active components of garlic and the chemical characteristics of these components. Organoleptic studies are also required to evaluate the effect of dietary garlic paste on color and flavor of eggs. Birrenkott et al. (2000), however, reported no differences in color and flavor in eggs from hens consuming up to 3% dietary garlic powder. It was concluded that supplemental sun-dried garlic paste decreased serum and yolk cholesterol concentrations. Significant differences exist among strains with respect to egg production, yolk weight, and cholesterol concentration. Sun-dried dietary garlic paste up to 8% can be used as a hypocholesterolemic agent in practical layer diets. ACKNOWLEDGMENTS The authors are grateful to P. K. Roy, Bangladesh Agricultural University, Mymensingh, Bangladesh for technical assistance. S. R. Chowdhury is grateful to the Ministry of Science and Technology, Government of the Peoples Republic of Bangladesh for awarding him a fellowship. REFERENCES Abell, L. L., B. B. Levy, B. B. Brodie, and F. E. Kendall A simplified method for the estimation of total cholesterol in serum and demonstration of its specificity. J. Biol. Chem. 195: Amagase, H., B. L. Petesch, H. Matsuura, S. 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