Alteration in serum biochemical parameters due to garlic ( Allium sativum ) supplementation in broilers ’ diets

This study was undertaken to evaluate the effect of Allium sativum supplementation through drinking water and feed on some serum biochemical parameters of broiler chickens. For this purpose, a 42 day’s feeding trial was conducted in a completely randomized design (CRD) on growing broiler chicks. Garlic (Allium sativum) was supplemented at graded levels as juice (2.25 and 3%) through drinking water and as powder (0.75 and 1%) through feed to experimental treatment T2, T3, T4 and T5, respectively with three replicates of 10 broiler chicks each for a period of 42 days starting from one day of age. The serum glucose level, serum total cholesterol, serum glutamate oxaloacetate transaminase (SGOT) concentration, and serum glutamate pyruvate transaminase (SGPT) concentration decreased significantly (P≤0.05) due to A. sativum supplementation in different treatment group as compared to control group at 28th and 42nd days. Serum alkaline phosphatase (ALP) levels of the birds of different treatment did not reveal any significant impact of A. sativum supplementation on the 28th day but significant reduction was noted as compared to control group on the 42nd day.


INTRODUCTION
Poultry keeping in India is as old as its civilization.Red jungle fowl found in India and its neighbouring countries is considered to be the progenitor of all domestic breeds of fowl.According to Watt Executive Guide (2009 to 2010), poultry industry has emerged as the most dynamic and fastest expanding segment in animal husbandry sector with 47.4 billion eggs produced by 2.4 billion layers and 3 billion broilers giving 2.25 million metric tons of poultry meat and likely to grow up to 75.6 billion eggs and 5.21 million metric tons of poultry meat by year 2012.Recently, Narayana (2008) reported that India is the 3rd largest country in egg production and 4th in chicken meat production.Andhra Pradesh leads other states in poultry output and together with Tamil Nadu and Maharashtra account for 70% of the country's poultry production.The average per capita poultry meat consumption was also estimated to increase from 0.69 kg in the year 2000 to 1.28 kg during the year 2000 to 2004.Despite India's tremendous growth in poultry production, per capita consumption is still low that is, 40 eggs and 1.2 kg of poultry meat than per capita consumption of many development countries.Health conscious person avoid egg consumption because of high level of cholesterol, responsible for heart ailment.This problem can be solved by reducing the cholesterol level in poultry meat and egg by dietary modulation.There are several cholesterol reducing agents such as copper, garlic, linseed oil, soybean oil, etc.Out of them, garlic is selected for the proposed research because it is easily available and relatively cheaper.Garlic (A. sativum) belongs to the plant family Liliaceae.The main chemical components in the volatile form of isolated garlic seedling are diallyl disulphide (23.33%), 1,3-dithiane (18.34%) and dibutyl phthalate (6.30%) (Jin et al., 2007).Garlic and its preparations have been widely recognized as agents for prevention of various metabolic disorders such as atherosclerosis, hyperlipedemia, thrombosis, hypertension and diabetes.Several clinical reports have shown that garlic has cholesterol-lowering effect in animals due to the presence of sulphur-containing bioactive compounds in its homogenates (Neil et al., 1996;Chowdhury et al., 2002).When raw garlic bulb is chopped or crushed, the enzyme allinase activates alliin, a non-protein amino acid present in the intact garlic, to produce allicin.Other important sulphur-containing compounds present in garlic homogenates are allyl methyl thiosulphonate, l-propenyl allyl thiosulphonate and y-L-glutamyl-s-alkyl-L-cysteine (Banerjee and Maulik, 2002).Garlic products have become more popular in the last decade.Market research conducted in United States (1998) showed that garlic products were the most popular of all dietary supplements (Wyngate, 1998).Dozens of brands on store shelves can be classified into four groups: garlic oil, garlic oil macerate, garlic powder and aged garlic extract (AGE).A. sativum is a species in the onion family alliaceae.Epidemiological and medical studies suggest that individuals regularly consuming garlic have longer blood clotting times and show lower blood lipid levels which means a reduced risk of stroke and cardiovascular disease.In addition, garlic is reputed to have antibacterial, antiviral, antifungal, antioxidant and anti-inflammatory activity.Some other studies show that eating garlic regularly reduces risk of oesophageal, stomach, and colon cancer.Garlic has broad range of biological activity, including immune stimulation and anti-tumor activity (Riggs et al., 1997).In the view of the above observations, the present investigation was done to study the alteration of garlic juice and powder supplementation on certain serum biochemical parameters of broilers.

MATERIALS AND METHODS
The present investigation was conducted to observe the effect of supplementation of A. sativum on some serum biochemical parameters of broilers.The experiment was conducted on 150 birds, divided into five treatment groups with three replicates of 10 birds each in a six weeks period.Broilers of control treatment (T1) were provided basal feed and water while two different concentration of garlic juice were given viz.2.25% (T2) and 3% (T3) in drinking water and garlic powder 0.75% (T4) and 1% (T5) in feed, respectively.The biological experiment was conducted during the months of June and July 2008 at Student Practical Poultry Production Unit, College of Veterinary and Animal Sciences, G.B. Pant University of Agriculture and Technology, Pantnagar, Uttarakhand.Geographi-cally, the place is located at altitude of 243.48 m above in humid subtropical zone lying in the foot hills of Himalayas.

Experimental birds and their management
The birds used in this experiment were crossbred broiler chicks.A total of 150, day old broiler chicks were procured from the Venky's Hatchery, Dehradun.Vaccination against Marek's disease was done on the day of hatching through subcutaneous route by hatchery and vaccination against Newcastle disease (RDF strain) was done on 4th day of hatching.All the birds were individually weighed and randomly allocated into five different treatment groups (T 1, T 2, T 3, T 4 and T 5 ) with three replicates of 10 birds each.The birds were reared in deep litter system in a gable roofed, open sided house.Paddy husk was used as litter material with a thickness of 4 to 5 cm.Wet litter was replaced with dry and clean litter at weekly interval during experiment and stirring was done on alternate days.All the birds were provided with uniform floor, feederer and waterer space and were reared under standard managemental conditions throughout the six week period.The birds were fed ad-libitum, and clean drinking water was provided throughout the experiment.The waterers were cleaned twice daily to prevent picking of any infection.Care was taken to minimize spoilage and wastage of feed and water.

Design of experiment
The feeding trial was carried out in a completely randomized design (CRD) in which garlic (A.sativum) in the form of juice was supplemented at graded levels through drinking water and in the form of powder through feed to different groups of broiler chicks for a period of 42 days starting from the 4th day of age.
There were five treatments employed in the experiment.The treatment 1 served as control (T 1 ) in which no supplement was added to the water and feed, in treatment T 2 , garlic juice at 2.25% concentration was provide, in T 3 , garlic juice at 3% concentration was provided through drinking water , in T 4 and T 5 , garlic powder at 0.75 and 1% concentration, respectively was provided through feed.Garlic (A. sativum) was procured from local market of Pantnagar.Garlic feeding was done in two ways that is juice (1:1 with distilled water) and powder.Garlic powder was mixed with the feed and garlic juice was added to the drinking water.The powder and juice was prepared biweekly; powder was prepared by drying garlic in hot air oven at 65 to 70°C and juice was formed by using grinder.Chemical composition (g/kg) of garlic bulbs and their products are as shown in Table 1.

Blood sampling and serum separation
Blood sampling was done twice during 42 days trial for evaluation of biochemical parameters.First collection was done at 28 days and second collection was done at 42 days of experiment.Blood samples (approximately 3 ml) were collected from three birds of each replicate group aseptically from the wing vein in sterilized disposable syringes (24 gauge needle).Collected blood samples (2 ml) were transferred to clean, dry and sterilized glass tube and kept in slanted position at room temperature for 3 to 4 h for separation of serum.The serum samples were collected in sterile tubes and these were centrifuged at 4000 rpm for 20 min.The top layer was separated and stored at -20°C till further use.

Biochemical parameters
Serum glucose: Estimation of glucose was done by enzymatic glucose oxidase-peroxidase (GOD-POD) method with the help of  Span Diagnostic Kit at 505 nm wavelength against blank reagent (Sacks, 1998).Concentration of serum glucose was expressed in mg/dl.
Serum cholesterol: Serum cholesterol concentration was estimated spectrophotometrically using Span Diagnostic Kit with enzymatic CHOD-PAP method at 505 nm wavelength (Tietz, 1998).Concentration of serum cholesterol was expressed in mg/dl.

Serum glutamate pyruvate transaminase (SGPT):
Plasma SGPT concentration was estimated by DNPH colorimetric method with the help of Span Diagnostic Kit at 505 nm wavelength (Tietz, 1970).Concentration of SGOT was expressed in IU/L.

Serum alkaline phosphatase (ALP):
Serum ALP concentration was estimated by Kind and King's method with the help of Span diagnostic Kit at 510 nm wavelength (Verley, 1975).

Statistical analysis
Statistical analysis of the data was done using analysis of variance (ANOVA) technique according to the method described by Snedecor and Cochran (1994).Comparisons among the treated groups were made.Statistically significant difference was considered at 5% level.

Glucose
The data comprising of mean serum glucose concentration (mg/dl) in different treatment groups recorded at the end of the 28th and 42nd days study are summarized in Tables 2 and 3.The serum glucose concentration in control, T 2 , T 3, T 4 and T 5 groups of broilers were 235. 25 ± 2.96, 226.15 ± 3.49, 225.11 ± 2.45, 215.53 ± 1.94 and 211.92±0.19mg/dl, respectively at the end of 28 days (Figure 2).In the present investigation, serum glucose concentration in T 5 group was significantly (P<0.05)lower than the serum glucose level in T 1 , T 2 and T 3 groups but not significant with T 4 group.However, T 2 and T 3 groups showed significant decrease in serum glucose levels and T 4 group showed no significant decrease in serum glucose levels.Maximum and mini-mum glucose concentrations were observed in T 1 and T 5 groups, respectively.
The result obtained at the 42nd day of study revealed that garlic supplemented groups T 3, T 4, T 5 showed significantly (P < 0.05) lower serum glucose concentration compared to the control, the broilers of groups T 4 showed lowest glucose concentration (206.90 ± 0.59 mg/dl) followed by T 5 (209.02± 0.35), T 3 (211.77± 4.99), T 2 (219.11± 1.29) and T 1 (223.87± 1.70) groups.However, there was no significant difference among T 3, T 4 and T 5 groups.The present study reports that there was reduction in the serum glucose concentration in garlic supple-   (2006), Ibrahiem et al. (1995) and Kamal and Daoud (2003) whom observed significant reduction in serum glucose concentration due to garlic supplementation.The significant reduction in glucose due to A. sativum supplementation in diets might be due to allicin and sulfur compounds of garlic.In addition, several ginger components exhibit serotonin receptor-blocking activity (Huang et al., 1991;Abdel-Aziz et al., 2005).This hypoglycaemic action of ginger may be due to these effects involving serotonin receptors, an increase in pancreatic secretion of insulin from beta cells or release of bound insulin.

Cholesterol
Mean serum cholesterol concentration (mg/dl) in different treatment groups at 28th day of experiment is illustrated in Table 2. Total serum cholesterol concentrations in T 4 , T 5, T 3 and T 2 groups were 141.43 ± 0.16, 142.18 ± 0.03, 142.59 ± 0.5503 and 147.80 ± 1.58 mg/dl, respectively which were significantly (P<0.05)lower compared to the control group (154.31±0.69 mg/dl) (Figure 1).Further, there was a significant (P <0.05) decrease noticed in T 4 , T 5, T 3 and T 2 groups with value 130.14± 1.17, 131.55 ± 1.83, 134.51±0.09and 137.73±0.85mg/dl, respectively when compared with T 1 at the 42nd day.Highest and lowest values of cholesterol were observed in T 1 and T 4 group of broilers.Lower cholesterol concentrations were significantly (P < 0.05) obtained among garlic supplemented groups compared to the control.Further, cholesterol concentration recorded at 28th day was found to be significantly reduced in the 42nd day for all treatment groups.In this study, total serum cholesterol showed a significant (P < 0.05) reduction between control and garlic supplemented groups.These findings are in agreement with earlier reports of Choudhury et al. ( 2002) who observed a marked decrease in total serum cholesterol and yolk cholesterol concentration due to supplementation of A. sativum in diet of broilers (Lijuan, 2001).Chen and Li (2006) also observed that the addition of 1 to 2% garlic reduced the serum and muscle cholesterol in broilers similarly.Mottaghitalab and Taraz, (2004)   synthesis (Myung et al.,1981;Qureshi et al., 1983).

Serum alkaline phosphatase (ALP)
Table 2 furnishes the serum ALP concentration (KA Units) in different treatment groups.The serum ALP concentration was found to be non-significantly different among garlic supplemented groups and control group at the 28th day of study with the value of 11.75 ± 2.96 KA Units in T1, 11.62 ± 0.29 KA Units in T2, 11.96 ± 0.27KA Units in T3, 12.33±0.1527KAUnits in T 4 and 11.83 ± 0.05 KA Units in T 5 (Figure 3.).There were significant decrease in T 3, T 2, T 4 and T 5 as compared to the control group of serum ALP concentration (KA Units) in different treatment groups at the 42nd day of study (Table 3).The mean values of ALP concentration in T1, T2, T3, T4 and T5 groups were 12.25 ± 0.13, 11.34 ± 0.11, 11.18 ± 0.02, 11.7 ± 0.23 and 11.44±0.12KA, respectively on the 42nd day.Maximum value of serum ALP concentration was observed in T 1 group.However, treatment groups did not differ significantly among themselves.Present research reveals that up to 28th day of supplementation of A. sativum, there were no significant changes in ALP concentration.Our finding is in accordance with Elhaster et al. (1997) who also found that broilers fed with 5% did not affect ALP activity.Augusti et al. (2005) observed that there was significant decrease in ALP in serum of rats.These results can be attributed to A. sativum, which may cause stabilized cell membrane and protect the liver against deleterious agents and free radical-mediated toxic damages to the liver cells.This is reflected in the reduction of liver enzymes.A. sativum helps the liver to maintain its normal function by accelerating the regenerative capacity of its cells.

Serum glutamate oxaloacetate transaminase (SGOT)
The data regarding serum GOT concentration (IU/L) in different treatment groups recorded at the 28th and 42nd day of study is summarized in Tables 2 and 3.The SGOT concentration was found to be minimum in T 5 group (164.69 ± 0.23 IU/L) followed by T 2 (165.06±0.60IU/L), T 3 (165.14±0.33IU/L) and T 4 (165.57±0.15IU/L) in comparison to the control group (176.85±1.41IU/L) at the 28th day of study (Figure 4).There was significant difference between the treatment groups as compared to the control group.At the 42nd day of the study, a similar trend in serum GOT concentration was observed as that of 28th  Ibrahim et al. (2000) supplemented garlic in rabbit and observed that SGOT decreased significantly.Ganiyu (2006) observed the effect of garlic supplementation on SGOT of rat and found that there was significant decrease (P < 0.05) in SGOT of rats fed diet containing 4% garlic.Salahy (2003) reported that when fish was orally given garlic and onion with feed, there was decrease in SGOT.These results can be attributed to A. sativum, which may cause stabilized cell membrane and protect the liver against deleterious agents and free radical-  mediated toxic damages to the liver cells.This is reflected in the reduction of liver enzymes.A. sativum helps the liver to maintain its normal function by accelerating the regenerative capacity of its cells.

Serum glutamate pyruvate transaminase (SGPT)
Tables 2 and 3 represent the SGPT concentration in different treatment groups.The serum GPT concentration was found to be lowest in T 4 group (23.13 ± 0.24 IU/L) followed by T 3 (23.66± 0.46 IU/L), T 2 group (23.73 ± 0.29 IU/L) and T 5 group (24.53 ± 0.17) in comparison to the control group (28.3±0.49IU/L) at the 28th day of study (Figure 5).At the 42nd day of study, mean values of SGPT in T 5, T 4, T 3, T 2 and T 1 groups were 21.4±0.11,22.13±0.17,22.93 ± 0.29, 23.33±0.24and 28.53 ± 0.52 IU/L, respectively.During the 28th and 42nd day, there was significant difference between the treatment groups as compared to the control group.This study demonstrates that the activities of SGPT were much influenced by dietary garlic.Lower values of SGPT were observed in treatment which indicates A. sativum up to 3% concentration has no adverse effect on liver.These findings are in accordance with those of Elhaster et al. (1997) who found that 5% A. sativum in rat diet significantly decreased SGPT.Augusti et al. (2005) also observed that A. sativum supplementation decreased SGPT significantly in serum of rat.Ibrahim et al. (2000) supplemented garlic in rabbit and observed that SGPT decreased significantly.Ganiyu (2006) observed the effect of garlic supplementation on SGPT of rat and found that there was significant decrease (P < 0.05) in SGPT of rats fed diet containing 4% garlic.Salahy (2003) reported that when fish was orally given garlic and onion with feed, there was decrease in SGPT.These results can be attributed to A. sativum, which may cause stabilized cell membrane and protect the liver against deleterious agents and free radical-mediated toxic damages to the liver cells.This is reflected in the reduction of liver enzymes.A. sativum helps the liver to maintain its normal function by accelerating the regenerative capacity of its cells.

Conclusion
The results on serum biochemical parameters indicate that there was significant reduction in glucose, cholesterol, SGOT and SGPT concentration due to A. sativum supplementation in broiler checkens diets.The study demonstrates that A. sativum administration did not have effect on serum alkaline phosphatase concentration at the 28th day however at 42 days of age, it was also significantly reduced in garlic supplemented groups and the presented study reveals that A. sativum supplementation, through drinking water and feed, reduced glucose, cholesterol, SGPT and SGOT.Finally, from the obtained results, it could be recommended that garlic (A.sativum) may be used as a growth promoter and antibiotic for the treatment or prevention of diseases and for enhancing chicken tolerance to environmental stress; therefore garlic powder should be added to the diets of poultry.

Figure 1 .
Figure 1.Effect of A. sativum supplementation on serum cholesterol (mg/dl) of broilers at 28th and 42nd day of study reported that feeding of garlic powder and tylosin reduced serum and egg yolk serum concentration.Similar observations were also observed by Yun et al. (2005) in chicken and Sakine and Onbaslar (2006) in laying hen.Garlic extracts exhibited hypocholesterolemic effects; possible reason for this may be mainly because of the inhibition of the key enzymes, such as hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, cholesterol 7α-hydroxylase, and fatty acid synthetase in cholesterol and lipid

Figure 2 .
Figure 2. Effect of A. sativum supplementation on serum glucose (mg/dl) of broilers at the 28th and 42nd day of study.

Figure 3 .
Figure 3.Effect of A. sativum supplementation on serum ALP (KA units) of broilers at the 28th and 42nd day of study.

Figure 4 .
Figure 4. Effect of A. sativum supplementation on SGOT (IU/L) of broilers at 28th and 42nd day of study.

Figure 5 .
Figure 5.Effect of A. sativum supplementation on SGPT (IU/L) of broilers at the 28th and 42nd day of study.

Table 1 .
Chemical composition (g/kg) of garlic bulbs and their products.

Table 2 .
Effect of A. sativum supplementation on serum enzymes of broilers at 28 days of study (mean ±S.E).

Table 3 .
Effect of A. sativum supplementation on serum enzymes of broilers at 42 days study (mean ±S.E.).