MATERIALS AND METHODS

Animals, treatments and experimental design: This study used 90 Lohmann Brown hens, 30 weeks of age, with a homogeneous body weight of 1654.36±14.85 g obtained from a commercial poultry farm. All chickens were given commercial feed specific for laying hens containing 2.750 kcal kg^–1 of Metabolizable Energy (ME), 18% of CP, 3.5% of Ca and available phosphor of 0.45%. For the treatments, hens were placed into three groups each containing 5 hens: (1) Hens were only given water as a drink, (2) Hens were given 5cc of sweet leaf extract in 100cc of drinking water and (3) Hens were given 5cc of garlic leaf extract in 100cc of drinking water. Each treatment was repeated 6 times for a total of 90 hens. Food and drinking liquid were given ad libitum. The individual hens were weighted weekly and food consumption and egg production was recorded daily.

Preparation of garlic (Allium sativum) and sweet leaf (Sauropus androgynus) extract: Fresh leaves of the sweet leaf and garlic were obtained from the local fresh food market. The leaves of both plants were blended and macerated overnight in distilled water (1:1, w/w)¹⁵. The blended extract was then filtered using a cheese cloth. This extract was used for the treatment.

Performance, egg quality metrics and laboratory analysis: Eggs were collected and labeled on a daily basis at 800 and 1400 h throughout the experimental period. The percentage of egg production was calculated. Once every two weeks, the eggs from three consecutive days were used to measure egg weight and quality. Yolk cholesterol content was analyzed for two consecutive weeks. Blood samples from 36 hens were allowed to clot and the serum were harvested to determine the total serum cholesterol content. Cholesterol levels were analyzed following the Liebermann-Burchard method¹⁶.

Statistical analysis: All data were analyzed with one-way ANOVA to determine the differences among treatments. If differences were found, then further analysis was performed with Duncan’s multiple range test.

RESULTS

The results of the present study showed that 5.0% Katuk leaf extract and 5.0% garlic leaf extract in drinking water significantly increased (p<0.05) the average number of eggs produced and the average total egg weight as well as the average hen-day production. However, no significant differences (p>0.05) in the efficiency of feed consumption, water consumption and egg weight per head (g head^–1) were noted among the treatments (Table 1). The average value of Feed Conversion Ratio (FCR) (feed consumption:total egg weight) over 10 weeks of observation in the control group was 3.37/head (Table 1). This was significantly different (p<0.05) from hens in treatment groups B and C an average of 10.39 and 8.61%, respectively. In addition, the results showed that an additional 5.0% (5cc/100cc) of Katuk and garlic leaves extract in drinking water resulted in a significant (p<0.05) decrease in levels of cholesterol in the serum and yolk of birds.

It was measured the effects of oral administration of Katuk or garlic leaf extract on egg production and yolk cholesterol levels in layer hens. The extract increased egg weight and mass, but there was no significant change in egg production. Serum and egg-yolk cholesterol content decreased significantly in the egg-laying hens administered Katuk or garlic leaves. Chowdhury et al.¹⁷ found that no effect on laying hens by mixing diets with 2-10% sun-dried garlic paste. It has been suggested the levels of garlic juice used in the present study were insufficient to influence egg yolk cholesterol¹. Syahruddin et al.¹³ reported that feed consumption and FCR were not affected by the levels of fermented Katuk leaf in the diet. However, the cholesterol content in broiler carcass was significantly affected by the dietary treatments. Another study found that garlic powder had no significant effects on broilers weight gain, Feed Intake (FI) and feed conversion¹⁸. Ao et al.⁷ reported that administration of 1-3% of fermented garlic powder in the diet had no effect on egg production and egg weight.

It was found that Katuk or garlic significantly increased the efficiency of feed (feed consumption:total egg weight). This could be the a result of phytochemical contained in Katuk leaves and garlic. Administration of garlic can markedly increase villus height and crypt depth¹. Thickness of the epithelium and the number of goblet cells in the duodenum, jejunum and ileum poultry decreased⁸. These features can increase nutrient absorption. They concluded that the morphological changes in the intestines of birds increases digestive capacity.

Hernandez et al.¹⁹ reported that plant extract supplementation improved apparent whole tract digestibility of the nutrients. Adibmoradi et al.²⁰ reported that garlic administration enhanced villus height and crypt depth and decreased epithelial thickness and goblet cell numbers in duodenum, jejunum and ileum of birds and similar results were also have been shown by Nusairat⁸. Rao et al.²¹ reported that garlic supplementation probably enhanced the activities of the pancreatic enzymes and provided micro-environment for better nutrient utilization in rats.

In more recent studies, Yalcin et al.²² found that supplementing garlic powder at 5 or 10 g kg^–1 increased layer production. Khan et al.²³ reported that laying hens fed on dried garlic (2-8%) showed higher egg-production intensity.

Table 1:	Effect of garlic (Allium sativum) and sweet leaf or Katuk (Sauropus androgynus) leaf water extract added in drinking water and administered to 30-40 weeks aged of egg laying hens to the egg production and yolk cholesterol level
*A: Drinking water without garlic or Katuk leaves extract as control, B: Drinking water with 5cc/100cc garlic water extract and C: Drinking water with 5cc/100cc Katuk water extract, respectively, SEM: Standard error of treatment means, Means with different superscripts within raw values are significantly different (p<0.05)

This result contrasts with previous studies showing supplementation of 1% garlic powder caused a higher thigh yield the poorest thigh yield occurred with 3% garlic powder²⁴. Groups with 1% garlic powder had significantly higher breast yield than others²⁴.

Ao et al.⁷ reported that administration of 1-3% of fermented garlic powder in the diet increased egg yolk color compared with controls. Beta-carotene and vitamin E are natural antioxidants and antioxidants have an important role in inhibiting and scavenging free radicals. Therefore, they protect humans against infections and degenerative diseases. In addition, Sauropus androgynus leaves were also rich in flavonoids, vitamin C²⁵ and other phenolic compounds²⁶, these are natural antioxidants.

Andarwulan et al.²² reported that supplementing garlic powder at 5 or 10 g kg^–1 increased in hen egg production with a significant increase in egg weight similar to our results. Khan et al.²³ also reported that laying hens fed on dried garlic (2-8%) showed higher egg production intensity and increases in egg mass, however, the egg weight did not change significantly when compared to the control group. The diversity of garlic preparation and administration methods makes it harder to contrast our results with the literature.

Cholesterol levels in serum and egg yolks declined markedly by administration of Katuk leaves and leaves of garlic extract in drinking water. The decrease in plasma cholesterol via garlic powder supplementation might be due to the reduction of synthetic enzymes¹⁸. Significant decreases in hepatic 3-hydroxy-3-methylglutaryl-CoA reductase, cholesterol 7α-hydroxylase, fatty acid synthetase and pentose phosphate pathway activities were seen in various fractions of garlic (petroleum ether, methanol and water-soluble fractions). Results of the present study are consistent with the findings of Yalcin et al.²⁷ who reported that garlic powder supplementation significantly reduced the plasma cholesterol concentration when laying hens were fed 0.5 and 1.0% garlic powder. This decreased total cholesterol and triglycerides in diabetic rats²⁸.

Chowdhury et al.¹⁷ and Ao et al.⁷ reported a reduction in yolk cholesterol contents due to the use of garlic. Birrenkott et al.²⁹ reported that diet supplementation with garlic oil at 0.02% had no effect on yolk cholesterol or other lipid components even when fed for up to 8 months.

Chowdhury et al.¹⁷ reported that plasma cholesterol concentrations decreased on average by 15, 28, 33 and 43% with increasing levels of dietary garlic paste of 2, 4, 6 and 8%, respectively. Prasad et al.³⁰ reported similar findings for total cholesterol and triglycerides. These were significantly decreased by garlic supplementation in chickens up to 8 weeks of age in comparison to a control group. Khan et al.²³ reported that dried garlic powder in the diets of commercial laying hens reduced serum and yolk cholesterol concentrations and skewed the layer performance upwards significantly. However, Birrenkott et al.²⁹ also reported that supplementing the diet with 3% garlic powder is not effective in lowering cholesterol yolk (mg g^–1) or other lipid components of serum of laying hens even when given a diet for 8 months. Yalcin et al.²² reported that total cholesterol was not affected by the yellow garlic supplements.

Katuk leaf can lower cholesterol levels because of the content of beta-carotene¹⁴. Beta-carotene can decrease the cholesterol associated with hydroxy methyl glutaril enzyme-CoA (HMG)³¹. This enzyme plays a role in the formation of mevalonic in the biosynthesis of cholesterol. Cholesterol synthesis and synthesis of beta-carotene are derived from acetyl CoA along with mevalonic. If the consumption of beta-carotene is greater than that of saturated fatty acid, then it makes the biosynthesis process by enzyme HMGCoA directed at beta-carotene. Thus, the saturated fatty acids are not converted into cholesterol³². The lowest cholesterol level was obtained by feeding the chickens with diets containing 14% fermented Katuk leaf¹³.

More beta-carotene in hen diets lowers cholesterol in the carcass because beta-carotene can inhibit enzyme HMG-CoA reductase (hydroxy methyl glutaryl-CoA). This plays a role in the formation of mevalonic acid. Mevalonic is required for cholesterol synthesis by inhibition of the enzyme that blocks formation of cholesterol^13,33. Oka et al.¹¹ reported that the antioxidant content in Katuk leaf extract is 1411.06 mg L^–1, which is much higher than the antioxidant content of Moringa leaves. However, its vitamin C content is 8.73 mg/100 mL, which is still lower than the Moringa leaf extract (19.13 mg/100 mL). Fermented Sauropus androgynus leaves offered the best broiler meat quality as indicated by lower fat and cholesterol with higher vitamin A, beta-carotene, protein, iron contents with better amino acid and fatty acid content¹². Sauropus androgynus leaves also reduced cholesterol in broiler meat³⁴ and eggs².

Issa et al.¹⁸ reported that garlic powder significantly decreased the levels of triglycerides. Garlic powder decreased triglyceride levels compared to the control group. This effect can be explained by the possible inhibition of the acetyl CoA synthetase enzyme, which is necessary for the biosynthesis of fatty acids. This might explain the hypocholesterolemic and hypolipidemic action of garlic products that depresses the hepatic activities of lipogenic and cholesterogenic enzymes, such as malic enzyme, fatty acid synthase and glucose-6-phosphatase dehydrogenase. Compounds that lower cholesterol in serum and egg yolk include drinking water with 5cc/100cc garlic water extract and drinking water with 5cc/100cc Katuk extract, respectively. These might contain alkaloids and non-alkaloids³⁵, saponins³⁶, flavonoid³⁷ and polyphenol³⁸. The reduction of cholesterol by alkaloids is due to reduction of lipogenic enzyme activities and increased bile acid excretion in the feces³⁹. Garlic powder supplementation significantly reduced the plasma cholesterol concentration when laying hens were fed 0.5 and 1.0% garlic powder²⁷.

These results are consistent with the beneficial effects of garlic on cholesterol metabolism in human health. Birrenkott et al.²⁹ reported that diet supplementation with 3% powdered garlic was not effective in lowering yolk cholesterol (mg g^–1) or other lipid components in the serum of laying hen seven when fed for up to 8 months and on broiler chickens¹⁰. More recently, Yalcin et al.²² reported that total yolk cholesterol was not affected by garlic supplementation. Similar findings were reported in rats-garlic powder failed to influence the lipid profiles in rats⁴⁰. Raeesi et al.²⁴ reported that 1 and 3% garlic had no significant effects on relative weights of carcass, fat pad or digestive organs among different treatments except for the small intestine. The different commercial garlic products might explain these contradictory results¹⁷.

CONCLUSION

It is concluded that 5.0% Katuk (Sauropus androgynus) or garlic (Allium sativum) water extract in drinking water improved egg production and may decrease both serum and yolk cholesterol in laying hens up to 30 weeks of age.

ACKNOWLEDGMENTS

The authors would like to thank to staff of laboratory attendants at the Nutrition Laboratory, Udayana University for their assistance in chemical analysis of the samples. We also would like to thank the Head of Research and Public Service Department and Rector of Udayana University for their support during this study including research funding.