MATERIALS AND METHODS

Selenium sources: Nano red elemental selenium particles (Nano-Se) were synthesized as per the method described by Zhang et al. (2001). One milliliter of 25 mM sodium selenite was mixed with 4 mL of 25 mM GSH containing 15 mg of BSA for the Nano-Se preparations. The pH of the mixture was adjusted to 7.2 with 1.0 M sodium hydroxide, forming red elemental Se and oxidized GSH. The red suspension was dialyzed against double-distilled water for 96 h, with the water being changed every 24 h to separate the oxidized GSH from the Nano-Se. The final suspension containing Nano-Se and BSA was lyophilized and stored at room temperature. The size of the red elemental Se was 50-100 nm as determined by Dynamic Light Scattering (DLS) analysis using a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK) with the average size being 80 nm. Sodium selenite (Na₂SeO₃) was purchased from Sigma-Aldrich Co., USA.

Experimental birds: Commercial (BV 300) layer chicks (n = 300) were randomly allocated to six dietary treatments. Each treatment group had 2 replicates containing 25 chicks in each replicate. Earlier in the first phase, the experiment continued till 8th week of age (starter phase) (Mohapatra et al., 2014). This was a continuous study of the same treatment groups. The grower birds were provided 24 h free access to clean drinking water.

Experimental diets: Basal diet was formulated to meet nutrient requirements according to the Indian Standard 5672 (1992) except Se for the experimental feeding period of 9-20 weeks. Samples of the experimental feed were analyzed for dry matter, crude protein, ether extract, crude fiber, total ash and acid insoluble ashas per AOAC (1995). Calcium and phosphorus was measured according to the method modified by Talapatra et al. (1940).

Table 1:	Formulations of experimental diets

Table 2:	Concentration of selenium in experimental diets of different treated groups

The Se content of the feed samples was estimated by using atomic absorption spectrophotometer. The ingredient composition and proximate composition of the experimental ration is presented in Table 1. The dietary treatments of the experiment group were presented in Table 2.

Growth and feed consumption: Weights of all the individual grower birds in each group were determined at initial and at the end of experiment. At the same time, survival was also determined by counting the individuals in each group. The relative gain rate was calculated using the equation i.e:

The Feed Conversion Ratio (FCR) was expressed as:

Biochemical analysis: Blood and serum samples were collected at 20th weeks of post feeding for biochemical analysis. The serum biochemical indices determined were serum glucose, cholesterol, urea, alkaline phosphate (ALP), aspirate amino transferase (AST), alanine amino transferase (ALT) total protein, albumin, globulin, calcium (Ca) and phosphorus (P) were determined by using Crest biosystems (Goa, India) Kit.

Haematological parameters: Blood and serum samples were collected at 20th weeks of post feeding for haematological studies. The haemoglobin content and Packed Cell Volume (PCV) were determined as per methods described by Schalm et al. (1975) and Jain (1986), respectively. Total Erythrocyte Count (TEC) was estimated using Neubaur's hemocytometer.

Processing of organs: After 20th weeks of post feeding, 15 birds were randomly chosen from each treatment and slaughtered for collection of liver, breast muscles, pancreas, kidney, feathers, spleen and thymus. The birds were kept off fed overnight before bleeding and only water was provided. The live weight of the birds was recorded as pre slaughter weight. The birds were bled by modified Kosher’s method (Panda and Mohapatra, 1989). Spleen and thymus were clipped from the viscera with a pair of scissors by holding with a pair of forceps. Spleen and thymus were weighed in a top pan electronic balance. Feather samples were taken from the pectoral and ventral region from the same number of birds per group. Feathers were washed three times with acetone alternating with deionized water.

Selenium deposition in different tissues: The collected liver, breast muscles, pancreas, kidney and feathers samples were oven dried at 100°C for 24 h and finely ground. The Se content in the liver, breast muscles, pancreas, kidney and feathers samples were determined by digesting 0.5 g samples and 1 mL of serum samples at 120°C with 5 mL concentrate HNO₃ for 1 h using KEL plus digestion system. The digested samples were cooled and further digested with 30% H₂O₂ at 200°C. The process continued until the content appeared clear and colorless. The digested samples were filtered into a volumetric flask. The contents of digestion tubes were repeatedly washed with triple distilled water to obtain complete extract of the mineral.

Cellular immunity: At 20th weeks of post feeding, five birds (in duplicate) in each treated groups were injected intradermally in the comb with 100 micro gram of Phytohaemaglutinin-P (PHAP) in 0.1 mL of normal saline to measure the cellular immune response by Cutaneous Basophilic Hypersensitivity (CBH) test (Edelman et al., 1986). The thickness of comb was measured using digital caliper before inoculation and 24 h post inoculation and CBH response was calculated using the equation:

Humoral immunity: The measure of humoral immunity was carried out as per the method described by Abdallah et al. (2009). Sheep Red Blood Cells (SRBCs) were used as test antigens to quantitatively analyze specific antibody response as a measure of humoral immunity. At 20th week, birds from each groups (in triplicate) were immunized intravenously via., a wing vein with 0.07 mL packed RBC mixed with 0.93 mL physiological saline (0.9% NaCl) for measurement of primary response. The SRBC were obtained in heparin solution from local sheep (reared at Instructional Livestock Farm, Bhubaneswar, Odisha) and washed three times in physiological saline. Seven days following the antigen challenge, blood samples were collected and serum samples were used to measure humoral immunity. Antibody production to SRBC was measured using haemagglutination technique with microtitre plate U shape of 96 wells according to Bachman and Mashaly (1986) and Kai et al. (1988). All the SRBC antibody titers were expressed as log₂ of the reciprocal of the highest serum dilution causing agglutination of SRBCs.

Preparation of erythrocyte pellet: The 5 mL of whole blood was collected into sterilized micro-centrifuge tube containing 0.75 mL of Acid Citrate Dextrose (ACD; citric acid 8.0 g: Sodium citrate 22.0 g and dextrose 25.0 g and volume made to 1 L in distilled water) as anticoagulant. The blood samples were centrifuged at 3000 x g for 10 min at 4°C, plasma and buffy coats were separated. The resulting erythrocyte pellet was washed thrice with Phosphate Buffer Saline (PBS, pH7.4). For the estimation of catalase, glutathione peroxidase (GSH-Px) and SOD, 1 mL of the 1:1 diluted RBCs in PBS were mixed with 9 mL distilled water to prepare a haemolysate of 1:20 dilution.

Estimation of antioxidant enzymes: Different antioxidant enzymatic activities such as Glutathione peroxidase (GSH-Px) activity was assayed by the method of Paglia and Valentine (1967); Super Oxide Dismutase (SOD) activity of RBCs was measured using NBT assays by Masayasu and Hiroshi (1979) and catalase was assayed in erythrocytes by the method of Bergmayer (1983).

Statistical analysis: The Statistical Analysis System (SAS) software (version 6.12) was used to analyze the data.

RESULTS

Body weight and FCR: The growth performance and feed utilization of grower birds supplemented with different Se sources are presented in Table 3. The initial body weights of the experimental birds were significantly different. This experiment was continued in two phases: Starter phase (0-8 weeks) and grower phase (9-20 weeks), the body weight that achieved at the end of starter phase is reported as the initial body weight at grower phase. The final weight at 20th week of all the treated groups and the control group differed significantly (p<0.05) with highest body weight was recorded in T₄ group. The best FCR of 4.73 was shown by treatment T₄ group which showed no significant difference with T₅. The FCR of other groups recorded significantly higher than that of T₄ and T₅ groups.

Serum biochemical parameters: Serum biochemical parameters viz., glucose, cholesterol, triglycerides, total protein, albumin, globulin, urea, SGPT, SGOT, ALP, calcium and phosphorus at twenty weeks of age of layer grower birds are presented in the Table 4. Serum glucose, total protein and SGOT levels were increased significantly (p<0.05) starting from control (T₁) to T₂, T₃, T₄, T₅ and T₆ groups, whereas, significantly (p<0.05) lower serum cholesterol, A/G ratio and SGPT in T₆ groups than that of control group.

Table 3:	Growth performance and feed utilization of grower birds supplemented with different Se sources (Nano-Se and sodium selenite of different concentration) and without Se (control)
Results were presented as Means±SE of triplicate observations. Means in the same row with different letters were significantly different (p<0.05)

Table 4:	Serum biochemical profile of grower birds supplemented with different dietary treatments
Values bearing different superscripts in a row differ significantly (p<0.05)

Table 5:	Immunity status of grower birds under different dietary treatments
Values bearing different superscripts in a row differ significantly (p<0.05)

Table 6:	Lymphoid organs (% of live weight) of grower birds in different treated groups
Values bearing different superscripts in a row differ significantly (p<0.05)

Immunity status: Antibody titer against SRBC and CBH response (Table 5) and weight of lymphoid organs (Table 6) were used as measures to study the immunity status of the layer grower birds. The antibody titers (log₂) against SRBC immunization of 20th week grower birds were significantly higher (p<0.05) in Nano-Se supplemented groups as compared to control (T₁) group. The CBH response was found to be significantly higher (p<0.05) in T₄ and T₅ groups. The average weights of lymphoid organs viz., spleen and thymus, expressed as percentage of live body weight at 20th week, showed no significant (p>0.05) difference among different treated groups.

Antioxidant enzyme activities: The antioxidant enzyme activities of grower birds are presented in Table 7. Erythrocyte catalase activity was significantly (p<0.05) higher in T₂, T₃, T₄, T₅ and T₆ as compared to control (T₁) group and highest level was observed in T₆. Glutathione peroxidase (GPX) activities were significantly (p<0.05) highest in T₆. Nano-Se supplemented groups showed better GPX activity than sodium selenite and control groups.

Table 7:	Antioxidant enzyme activities in different treated groups supplemented with different selenium sources of grower birds
Values bearing different superscripts in a row differ significantly (p<0.05)

Table 8:	Effect of different dietary selenium sources and levels on different haematological parameters of grower birds
Mean values within a row with different superscripts differ significantly (p<0.05)

Table 9:	Effect of different dietary selenium sources and level on selenium concentration in serum and tissues of grower birds
Mean values within a row with different superscripts differ significantly (p<0.05)

Similarly, Super Oxide Dismutase (SOD) activity were significantly (p<0.05) higher in T₆ group as compared to other treated groups.

Hematological parameters: Different hematological parameters of different treated groups are presented in Table 8. The haemoglobin content, TEC and PCV values showed no significant (p>0.05) difference among different treated groups.

Bioavailability of selenium: The bioavailability of Se in different tissues of layer grower birds in different dietary treated groups is presented in Table 9. The Se levels in serum, liver, breast muscle, pancreas, kidney and feathers were significantly higher (in increasing order with respect to increasing selenium concentration in the diet) in all the Nano-Se treated groups (T₃, T₄, T₅ and T₆) than the untreated control (T₁) and sodium selenite treated group (T₂).

DISCUSSION

The present study showed that the growth performance of grower birds was affected by dietary Se level. The improvement in the live body weight of birds fed selenium could be attributed to some of its biological function such as its role on enzymatic oxidation-reduction, nucleic acid metabolism and in promoting the activity of easily oxidized substances as carotenoides and vitamin A. In addition, improved feather growth may be due to improved growth performance (BWG) or may be due to increase of protein and water in cells (Tayeb and Quader, 2012). In contrast to current results, some reports demonstrated no effect of Se source or Se level on daily gain, feed intake or gain: feed ratio (Payne and Southern, 2005; Yoon et al., 2007). The differences were possibly due to the background Se in the feedstuffs. Ryu et al. (2005) also reported that feeding even higher concentrations (1-8 ppm) of dietary Se from an inorganic source did not affect the BW of broilers. However, in the present study, increased growth performance was observed when 0.6 mg kg^-1 of supplemental Nano-Se was fed. This suggests that the addition of 0.6 mg kg^-1 of Nano-Se was acceptable in avian feeding. The results indicated that the range between optimal and toxic dietary levels of Nano-Se was wider than that of sodium selenite. Improved FCR of broilers fed organic Se supplemented diet could be related to the increased concentrations of the active form of thyroid hormone in the serum of chickens supplemented with Se as well as to the immunomodulating properties of Se (Zelenka and Fajmonova, 2005; Surai, 2006; Hoffmann, 2007; Ozkan et al., 2007).

Serum glucose, total protein and SGOT levels were increased significantly (p<0.05) starting from control (T₁) to T₂, T₃, T₄, T₅ and T₆ groups. Whereas, significantly (p<0.05) lower serum cholesterol, A/G ratio and SGPT in T₆ groups than that of control group were observed. Similarly, the findings of Yang et al. (2012) revealed that the aspartate amino transferase, alkaline phosphatase (ALP), globulin, total bilirubin, urea and high density lipoprotein levels were observed to be non-significant between control and Se supplemented group in chicks. The rise in blood glucose may indicate disrupted carbohydrate metabolism due to enhanced breakdown of liver glycogen, possibly mediated by an increase in adrenocorticotrophic and glucagon hormones and/or reduced insulin activity (Raja et al., 1992).

In the present study, dietary supplementation of Nano-Se increased both humoral and cellular immunity as measured by antibody titer against SRBC, CBH responses and gave better antibody response than that of sodium selenite and untreated control groups. An increased antibody titre against SRBCs was found when selenium and vitamin E were included at higher levels in the diet (Marsh et al., 1981; Gore and Qureshi, 1997; Larsen et al., 1997; Leshchinsky and Klasing, 2001; Nageswara et al., 2003). On the contrary, Friedman et al. (1998) observed depression in antibody production against E. coli and Newcastle disease virus vaccination in both chicken and turkey when vitamin E supplementation in the diet was increased from 10 to 150 mg kg^-1. Selenium supplementation might have influenced intracellular transmission of signals necessary to initiate proliferation of lymphocytes (Schumacher et al., 1990) leading to increase in the antibody titre.

The results of this study also showed higher GSH-Px activity, SOD and catalase activities in erythrocytes of grower birds as compared to the sodium selenite and untreated control groups. Increased glutathione peroxidase activity was found in groups fed Nano-Se compared with the control group in serum and tissue. Feeding Nano-Se could improve the glutathione peroxidase activities and the elevation of the glutathione peroxidase activities in serum, liver and muscle may be optimized with the supplementation of 0.3 mg kg^-1 of Nano-Se (Cai et al., 2012). The differences in GSH-Px activity due to Se source are in agreement with the results presented by Cantor et al. (1982), Hassan et al. (1988) and Spears et al. (2003), who reported that Se supplementation increased plasma GPx3 activity.

The present finding stated that the Nano-Se had higher Se retention in liver, pancreas and breast muscle and was consistent with previously published results in goats (Shi et al., 2011). There are several reports of Se supplementation increasing breast, liver or plasma Se levels (Scott and Thompson, 1971; Cantor et al., 1982; Echevarria et al., 1988a, b; Downs et al., 2000; Spears et al., 2003). Furthermore, Cantor et al. (1982) and Spears et al. (2003) both indicated that organic Se increased tissue Se levels more than inorganic Se or a diet with no supplemental Se. Choct et al. (2004) found that an increasing supplementation rate of Se from 0.1-0.25 mg kg^-1 increased the breast muscle selenium concentration from 0.232-0.278 mg kg^-1 and both selenium source (organic and inorganic) and concentration significantly influenced (p≤0.05) the selenium content of the excreta at day 28. Se supplementation increased plasma Se levels in either turkey poultry’s or broilers (Cantor et al., 1975a, b; Cantor et al., 1982; Echevarria et al., 1988b; Spears et al., 2003). The results indicated that Nano-Se was more accumulated in muscle than that of sodium selenite. Elemental selenium nanoparticles have higher bioavailability compared to other selenium compounds, as it was proven in broiler chicken and goat compared with selenite (Shi et al., 2011; Zhou and Wang, 2011). These differences may be the result of the differences in lipophylic properties and metabolic pathways of the species (Zhang et al., 2008). The higher retention of Nano-Se in muscle may effectively reduce the available Se for inducing the selenosis response (Kim and Mahan, 2001). This may be partly the reason for the lower toxicity of Nano-Se compared with selenite. The different retention of Nano-Se and sodium selenite was probably related to the different absorption process and metabolic pathways. It has been reported that nanoparticle show new characteristics of transport and uptake and exhibit higher absorption efficiencies (Davda and Labhasetwar, 2002; Chithrani and Chan, 2007; Zha et al., 2008; Liao et al., 2010). They suggested that the superior performance of nanoparticles may be attributed to their smaller particle size and larger surface area, increased mucosal permeability and improved intestinal absorption due to the formation of nanoemulsion droplets.

CONCLUSION

The present study showed that dietary supplementation of Nano-Se improved the body weight, feed consumption ratio, antioxidant status, immunity and tissue Se concentrations of grower birds. Considering improvements in all the parameters studied, 0.3 mg Nano-Se per kg (on dry matter basis) of grower ratio is optimum. Further studies related to absorptive mechanism, metabolic pathway, ideal dose required for commercial poultry farming should be needed.

ACKNOWLEDGMENTS

Authors are highly thankful to (1) National Fellow project on “Nanotechnology in aquaculture: An alternative approach for fish health management and water remediation”, ICAR, New Delhi, (2) All India coordinated research project on “Improvement of feed resources and nutrient utilization in raising animal production” of ICAR, New Delhi and (3) INSPIRE Fellowship under INSPIRE Program of the Department of Science and Technology (DST), New Delhi for providing all financial support and other facilities to carry out this study.