MATERIALS AND METHODS

A total of 216 day-old Arbor Acre broilers were used in a 42 days experiment. Broilers were allotted into three groups with six replicates per group (12 chicks/replicate). The three groups as follow: the first group was fed a Basal Diet (BD) without emulsifier; the second and third groups were fed basal diet supplemented with 0.05 (SL0.05) and 0.1% (SL0.10) of Soy Lecithin (SL), respectively. The basal diet was formulated to meet broiler requirements (Table 1). Each treatment group contained 50% male and 50% female. The chickens were given starter feed til 21 days and grower feed from 21-42 days. Both feed and water were accessible to the birds on an ad libitum basis in a controlled room (34-36°C) temperature during 1-14 days and later on decreased up to 26°C till the end of the trial with a 12 h light-dark cycle (06:00-18:00 h light).

Table 1:	Formulation and calculated composition of basal diets (as-fed basis)
Provided per kg of diet; Iron: 60 mg, Copper: 7.5 mg, Zinc: 65 mg, Manganese: 110 mg, Iodine: 1.1 mg, Selenium: 0.4 mg, Bacitracin zinc: 30 mg, Vitamin A: 4500 IU, Vitamin D3: 1,000 IU, Vitamin E: 20 mg, Vitamin K: 1.3 mg, Vitamin B1: 2.2 mg, Vitamin B2: 10 mg, Vitamin B3: 10 mg, Choline chloride: 400 mg, Vitamin B5: 50 mg, Vitamin B6: 4 mg, Biotin: 0.04 mg, Vitamin B11: 1 mg and Vitamin B12: 1.013 mg

The experiment was handled according to the guidelines of Animal Care and Use Committee of Nanjing Agricultural University.

Growth performance: On 21 and 42 days of age, broilers were weighed and Feed Intake (FI) was recorded to evaluate Average Daily Gain (ADG), Average Daily Feed Intake (ADFI) and Feed Conversion Ratio (FCR) for all phases of feeding. At the end of 3rd and 6th weeks of feeding trial, the birds were restricted from feed for 12 h but had free access to water, later on, one bird per replicate was randomly chosen, weighed and sacrificed by bleeding of jugular vein. After decapitation, the visceral organs (liver, thymus, bursa, pancreas and spleen) were collected and weighed. The relative weights of mentioned organs were immediately calculated and expressed as relative to BW (g of organ/kg of BW).

Serum measurements: At 42nd day of the trial, one bird per replicate was randomly selected and sacrificed to get blood. The samples were centrifuged at 1,500×g 4°C for 15 min to get the serum and frozen at -20°C for further analysis. The concentrations of serum triglyceride (TG), Total Cholesterol (TC), Low Density Lipoprotein Cholesterol (LDL-C) and High Density Lipoprotein Cholesterol (HDL-C) were determined by using the corresponding diagnostic kits obtained from Nanjing Jiancheng Bioengineering Institute, Nanjing, China, whereas the concentrations of total protein, glucose, urea and creatinine were calculated enzymatically by using an automatic biochemical analyzer (Olympus AU-800, Olympus, Tokyo, Japan).

Nutrient digestibility: On day 19-21, birds (6 chickens from each group) were kept into individual cages, trays were placed under each cage and 3-day total collection of excreta was performed. Before the collection, birds were restricted from feed for 12 h with free access to water and were fed with experimental diets ad libitum for 3 days. To avoid contamination, scales and feathers were carefully removed from the trays. The fecal samples were collected and stored at -20°C for each day. After 3 days excreta collection period, feed intakes were recorded and excreta of each cage were mixed. Mixture of samples were taken and dried in an oven at 65°C for 24 h and grounded with a laboratory mill fitted through 1 mm screen. On day 40-42, another 3 days total collection period for excreta was conducted in the same way. Samples of feed and excreta were analyzed for Gross Energy (GE), Dry Matter (DM), Crude Protein (CP) and Ether Extract (EE) according to standard method of AOAC⁹.

Determination of hepatic antioxidant enzymes: About 0.3 g liver was taken to make homogenate. The minced liver tissue sample was homogenized in ice-cold 0.9% sodium chloride buffer (w/v, 1:9) using an Ultra-Turrax homogenizer and then centrifuged at 4000 rpm for 10 min to obtain the supernatant and was frozen at -80°C for further analysis. The activities of total antioxidant capacity (T-AOC), catalase (CAT), total superoxide dismutase (T-SOD) enzymes and the content of malondialdehyde (MDA) in the liver was determined by using kits (Nanjing Jiancheng Bioengineering Institute, China). The enzyme activities of liver T-SOD, T-AOC and CAT were expressed in U per milligram of protein and liver MDA content was expressed as Nano moles per milligram of protein^10,11. Bicinchoninic Acid Assay (BCA) was used to determine the protein concentration in the liver homogenates.

Statistical analysis: Data were analyzed by one-way ANOVA. Duncan±s test was used to identify significant difference among the means at p<0.05. All the analysis were executed using SPSS software (Version. 20). Cage means were used as experimental units in growth experiment, while individual observations were used as experimental units in other experiments.

RESULTS

Growth performance and relative organ weights: The effects of soy lecithin on broiler performance are summarized in Table 2. During starter (0-21 days), grower (21-42 days) and overall period (0-42 days) of chickens fed diets with soy lecithin has better (p<0.05) ADG and ADFI compared with control. However, FCR remained unaffected between the different treatments from days 0-21 and 21-42, while, during the overall period was improved (p<0.05) in SL0.10 compared to SL0.05 and control. Effect of soy lecithin on relative weight of liver, bursa, spleen, pancreas and thymus are shown in Table 3. During 21 and 42 day, SL0.10 recorded the highest relative liver weight compared to SL0.05 and control (p<0.05). Relative weights of bursa, spleen, pancreas and thymus were not affected among the different dietary treatments during days 21 and 42.

Table 2:	Effect of soy lecithin on growth performance of broilers (n = 6)
Means in the same row with different superscripts significantly (p<0.05). SEM: Standard error of mean, *Treatments: BD: Basal diet, SL0.05: BD+0.05% soy lecithin, SL2: BD+0.1% soy lecithin, ADG: Average daily gain, ADFI: Average daily feed intake and FCR: Feed conversion ratio

Table 3:	Effect of soy lecithin on relative weight of organs of broiler (n = 6)
Means in the same row with different superscripts significantly (p<0.05). SEM: Standard error of mean, *Treatments: BD: Basal diet, SL0.05: BD+0.05% soy lecithin and SL2: BD+0.1% soy lecithin

Table 4:	Effect of soy lecithin on nutrient digestibility of broiler (n = 6)
Means in the same row with different superscripts significantly (p<0.05). SEM: Standard error of mean, *Treatments: BD: Basal diet, SL0.05: BD+0.05% soy lecithin, SL2: BD+0.1% soy lecithin, GE: Gross energy, DM: Dry matter, CP: Crude protein and EE: Ether extract

Table 5:	Effect of soy lecithin on serum parameters of broiler at 42 days of age (n = 6)
Means in the same row with different superscripts differ (p<0.05). SEM: Standard error of mean, *Treatments: BD: Basal diet, SL0.05: BD+0.05% soy lecithin and SL2: BD+0.1% soy lecithin

Table 6:	Effect of soy lecithin on hepatic antioxidant enzymes of broiler (n = 6)
Means in the same row with different superscripts differ (p<0.05). SEM: Standard error of mean, *Treatments: BD: Basal diet, SL0.05: BD+0.05% soy lecithin, SL2: BD+0.1% soy lecithin, T-SOD: Total superoxide dismutase, T-AOC: Total antioxidant capacity, CAT: Catalase and MDA: Malondialdehyde

Effects on nutrients digestibility: The effects of soy lecithin on nutrients digestibility are shown in Table 4. On day 21, the digestibility of DM, GE, CP and ether extract in chicks fed with SL0.10 diet were increased (p<0.05) compared to SL1 and control groups. On day 42, utilization of GE and ether extract were relatively higher (p<0.05) compared to those fed with SL0.05 and control. However, there was no difference in DM and CP utilization on day 42.

Effects on serum parameters: Data presented in Table 5 show that serum total cholesterol and triglyceride concentrations were significantly (p<0.05) decreased in SL0.05 and SL0.10 treatments compared with control, while LDL cholesterol decreased (p<0.05) in SL0.10. However, no difference was seen in HDL cholesterol on d 42 day. Furthermore, elevated (p<0.05) serum glucose was observed in SL0.10 compared to SL0.05 and control groups. In addition, concentrations of total protein, albumin and total bilirubin were unaffected among different treatments.

Effect on hepatic antioxidant enzymes: Table 6 shows data of hepatic antioxidant enzymes and lipid peroxidation in broilers at day 42. Compared with control and SL0.05, chicken diet supplemented with SL0.10 resulted in the decreased MDA content and increased hepatic enzyme activities of CAT, T-SOD and TAOC (p<0.05).

DISCUSSION

Growth performance and relative organs weights: Average daily gain and ADFI of broilers fed with the diet containing 0.1% soy lecithin were improved during starter (0-21 day), grower (21-42 days) and overall (0-42 days) periods, but FCR was improved during the overall period only. Results of our study coincided with the findings of Roy et al.³ and Zosangpuii et al.¹² with exogenous emulsifiers were reported having beneficial effects on BW, feed intake and FCR in broilers. The positive effects with the addition of emulsifier to the diets on growth performance might be due to improved palatability which leads to higher feed and energy intake¹³. On the contrary, Azman and Ciftci⁸ demonstrated that the body weight was not affected between the groups supplemented with lecithin and control diet at 21 and 35 days of age. Zhao et al.⁷ reported that emulsifiers enhanced digestibility of nutrients in animals fed a diet with low energy using beef tallow as fat source. No impact of emulsifiers on feed intake was previously reported by Aguilar et al.¹⁴. Therefore, inconsistent results on growth performance of the birds fed with palm oil and emulsifier might be associated with fatty-acid composition of fat source and its effects on fat digestion and absorption so that the birds extracted more nutrients and sustained their growth rate even under low energy density diet.

In birds, liver is principal place involved in lipids metabolism of the body which accounts for 95% of the de novo fatty acid synthesis¹⁵. In present study, relative liver weight was improved in the broiler fed with 0.1% soy lecithin diet on days 21 and 42, respectively. Current results are in line with the previous findings of Huang et al.¹⁶ and Nagargoje et al.¹⁷ who observed better weight of liver by adding soy lecithin into the diet of broilers. Therefore, improved weight of liver possibly associated with the increased lipid metabolism within the organ.

Serum parameters: Blood measurements (protein and its fractions, lipid indices, renal and hepatic enzymes) are usually related to health status and they are good indicators of the nutritional status of the animals. Jones et al.¹⁸ observed lower content of serum triglycerides in weaning piglets given tallow with emulsifiers (lecithin or lysolecithin) compared to those fed without emulsifiers. This is according to our findings that triglycerides were lower in broilers fed SL compared to those groups without SL. Similarly, De Rodas et al.¹⁹ found decreasing trends of serum triglycerides by supplementing emulsifiers into the diet of pigs. Soy stanol-lecithin powder reduces cholesterol absorption²⁰ and increased fecal sterol excretion was observed with addition of polyunsaturated phosphatidylcholine (PC) to diet²¹, which is consistent with our findings that serum cholesterol and LDL were decreased in broiler diet supplemented with SL as compared to diet without SL. On the contrary, Guerreiro Neto et al.²² found non-significant differences on serum total cholesterol, LDL or triglyceride levels with emulsifier addition. The reduction mechanism of serum cholesterol by soy lecithin is still not clear, but this action may be returned to increase digestibility of fat as a consequence of which chylomicrons are rapidly cleared from the blood or secreted into the blood at a slower speed¹⁸. In the present study, serum glucose was increased with the birds fed SL0.10 diet. Such improvement in serum glucose might be reflected that sufficiently glucose was consumed by SL0.10 supplemented group for growth and performance.

Nutrient digestibility: Some previous studies indicated that the activities of lipase increase with age²³. On the other hand, bile salts also play important role as a limiting factor for the digestion of lipids during the first week after hatching²⁴. The digestibility of fat is limited in young chickens when secretion of lipase is not enough. In current study, the digestibility of DM, GE, CP and EE were increased on day 21, while utilization of GE and EE were higher in the chickens fed SL0.10 vs. SL0.05 and control diets. The same results were reported by Huang et al.¹⁶ who stated that increased nutrient utilization in chickens fed diets supplemented with soy-oil and soy-lecithin in a proportion of 25/75 during 19-21 day. Findings of our study are also supported by the earlier reports^25-28 who speculated that increased nutrient digestibility in the broilers fed diet incorporated with fat emulsifier and low dietary fat level. On the contrary, linear decrease in the ATTD of DM, CP or GE was also reported¹³ and with LPL supplementation in weanling piglets²⁹. Inconsistency in the obtained results can be regarded due to different sources of lipids and various levels of emulsifiers used in the diet^14,30. Furthermore, vegetable oil are more digestible than animal oils documented by Tan et al.³¹ and Li et al.³², which may be one of causes that no effects were seen in proceeding research conducts.

Hepatic antioxidant enzyme activities: Oxidative stress is harmful to animals that may alleviate their immune response and affects their performance by generating Reactive Oxygen Species (ROS). Obtained results indicated that supplementation of SL0.10 decreased hepatic MDA content and increased TAOC, T-SOD and CAT enzymes activities compared to those fed control and SL0.05. Outcomes in our study are accordance with Attia and Kamel³³ who pointed out that activities of GSH, GPx, SOD and GST was increased, but TBARS decreased in blood and seminal plasma with increasing soy lecithin level in rabbits. Such improvements in current study are also in line with Al-Daraji et al.³⁴ and Das et al.³⁵. Similarly, soy lecithin have antioxidant and neuroprotective properties and it decreases liver injuries and improves oxidative strength^36-38. It can improve the oxidative stability of oils and fats and its effect may be due to phospholipids, the main components of lecithin. According to King et al.³⁹ this property was characterized to the fact that phospholipids can contribute a hydrogen atom from amino group which triggering the oxidized phenolic molecule of the true antioxidant. Furthermore, Judde et al.⁴⁰ depicted that antioxidant effect of lecithin rely on the type of oil, tocopherol and fatty acids composition. Therefore, this can imagine that the effect of soy lecithin occurred with the γ- and δ-forms of tocopherols or tocotrienols that are naturally found in palm oil. From the previous findings, it was found that soy lecithin is a natural source, superior feed ingredient that is an important source of dietary phospholipids. Supplementation of lecithin in poultry or animal feed improves feed utilization and growth rates.

SIGNIFICANCE STATEMENTS

CONCLUSION

Soy lecithin can be used as fat replacer in poultry diet; supplementation of soy lecithin (0.10%) led to improvement in growth performance and nutrient digestibility. In addition, broilers fed diet supplemented with 0.10% of soy lecithin had a lower lipid profile or lipid peroxidation rate in serum. Furthermore, dietary soy lecithin improved the activities of hepatic antioxidant enzymes and has ability to protect against oxidative stress.

ACKNOWLEDGEMENT

This study was supported by the Jiangsu province Government (China) through the program "Twelve Five" Rural areas research of national science and technology (2013BAD10B03). This study was also supported by Jiangsu Science and Technology Project (BY2013074-01).