MATERIALS AND METHODS

Selenium sources: Nano red elemental selenium particles (nano-Se) were synthesized 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.

Animals’ diet and experimental procedures: Vaccinated day old sexed 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. The chicks were randomly distributed so as to eliminate any significant difference between treatments with respect to body weight. The chicks were protected against Newcastle and infectious bursal diseases by routine vaccination. The chicks were provided 24 h free access to clean drinking water. From 0-4 weeks of age, artificial light was provided to chicks to achieve brooding temperature and further the day length was the photoperiod for the birds during chick and grower stage.

Basal diet was formulated to meet nutrient requirements according to the National Research Council (1994) except Se for the experimental feeding period of 0-8 weeks. Samples of the experimental feed were analyzed for dry matter, crude protein, ether extract, crude fiber, total ash and acid insoluble ash. Calcium and phosphorus was measured according to the method modified by Talapatra et al. (1940). 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 chicks in each group were determined at initial and at the end of experiment.

Table 1:	Formulations of experimental diets

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

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 8th 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 8th week 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 8th week of post feeding, 15 birds were randomly chosen from each treatment and slaughtered for collection of liver, breast muscles, pancreas, kidney, feathers, spleen, bursa of fabricius 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 chicks were bled by modified Kosher’s method (Panda and Mohapatra, 1989). Spleen, bursa of fabricius and thymus were clipped from the viscera with a pair of scissors by holding with a pair of forceps. Spleen, bursa of fabricius and thymus were weighed in a top pan electronic balance.

Selenium content 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 8th weeks of post feeding, 5 birds (in duplicate) in each treated groups were injected intra dermally in the comb with 100 μg 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 measure of humoral immunity. At 8th weeks of post feeding, chicks 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 SRBCs 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 SRBCs was measured using haemagglutination technique with microtitre plate U shape of 96 wells plates according to Bachman and Mashaly (1986) and Kai et al. (1988). All SRBCs antibody titers were expressed as log₂ of the reciprocal of the highest serum dilution causing agglutination of SRBCs.

Preparation of erythrocyte pellet: Five milliliter 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 3000xg for 10 min at 4°C, plasma and buffy coats were separated. The resulting erythrocyte pellet was washed thrice with phosphate buffer saline (PBS, pH 7.4). RBC diluted to 1:1 in PBS was used for the estimation of haemoglobin. For the estimation of catalase, SOD, lipid peroxidation (LPO) and glutathione peroxidase (GSH-Px), 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 by the method of Paglia and Valentine (1967) Super Oxide Dismutase (SOD) activity of RBCs were measured using NBT assays by Masayasu and Hiroshi (1979) and catalase was assayed in erythrocytes by the method of Bergmayer (1983).

Statistical analysis: The SAS (1991) software (version 6.12) was used to analyze the data.

RESULTS

Body weight and FCR: The weekly average body weight and FCR of layer chicks under different treated groups upto 8th week of post feeding were presented in Table 3. At the beginning upto 3rd week of post feeding, no significant difference was observed in the initial weight between different treated groups and the control. However, there was significant difference (p<0.05) in RGR and final weight of all different treated groups (T₁, T₂, T_3, T₄ and T₅) as compared to control group after 8th weeks of post feeding. Moreover, RGR and final weight of T₂, T₃ and T₄ group were significantly increased as compared to T₁ and T₆ group. However, RGR and final weight in T₆ group was higher as compared to T₁. Survival rate and FCR were not affected by the dietary treatments after 8th week of post feeding.

Serum biochemical parameters: Serum biochemical parameters viz., glucose, cholesterol, triglycerides, total protein, albumin, globulin, urea, SGPT, SGOT, ALP, calcium and phosphorus at eight weeks of age of layer chicks are presented in the Table 4. Serum glucose, total protein, globulin, SGOT, Urea levels were increased linearly and quadratically (p<0.05) starting from control to T₁, T₂, T₃, T₄ and T₅ groups. Whereas, serum cholesterol, triglyceride, A/G ratio, ALP decreased linearly (p<0.05) starting from control to T₁, T₂, T₃, T₄ and T₅ groups. In addition to that, serum albumin, Ca and p-level of the layer chicks showed no significant difference (p>0.05) in all the treated groups along with the control group.

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 chicks.

Table 3:	Growth performance and feed utilization of layer chicks supplemented with different Se sources (nano-Se and sodium selenite of different concentration) and without Se (control)
RGR: Relative gain rate, FCR: Feed conversion ratio. Results were presented as Mean±SE of triplicate observations. Means in the same row with different letters were significantly different (p<0.05)

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

Table 5:	Immunity status of layer 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 layer birds in different treated groups
Values bearing different superscripts in a row differ significantly (p<0.05)

Table 7:	Antioxidant enzyme activities in different treated groups supplemented with different selenium sources of layer chicks
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 layer chicks
Mean values within a row with different superscripts differ significantly (p<0.05)

The antibody titers (log₂) against SRBCs immunization of 8th week chicks were significantly higher (p<0.05) in T₂, T₃, T₄ and T₅ groups as compared to T₁ and control group. The CBH response was found to be significantly higher (p<0.05) in T₂, T₃, T₄ and T₅ groups as compared to T₁ and control group. However, T₁ group showed higher antibody response as compared to control against both SRBCs and CBH immunizations. The average weights of lymphoid organs viz., spleen, bursa and thymus was expressed as percentage of live body weight after 8th week of post feeding, showed no significant (p>0.05) difference among different treated groups.

Antioxidant enzyme activities: The antioxidant enzyme activities of layer chicks were presented in Table 7. Erythrocyte catalase activity were significantly (p<0.05) higher in T₃, T₄, T₅ and T₆ as compared to T₁ and control group. Glutathione peroxidase (GPX) activities were significantly (p<0.05) higher in T₃, T₄, T₅ and T₆ as compared to T₁ and control. However, both GPX and catalase activities were significantly higher in T₁ as compared to untreated control group. Similarly super oxide dismutase activity were significantly (p<0.05) higher in T₄, T₅ and T₆ group as compared to other treated and control groups.

Hematological parameters: Different hematological parameters of different treated groups are presented in Table 8. The haemoglobin content, TEC and PVC values were significantly (p>0.05) higher in all the treated groups (T₁, T₂, T₃, T₄ and T₅) than the control group. However, there is no significance difference among different treated groups.

Bioavailability of selenium: The bioavailability of Se in different tissues of layer chicks 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 (T₂, T₃, T₄ and T₅) than the untreated control and sodium selenite treated group (T₁). However, T1 group showed significantly higher selenium deposition in liver, pancreas and kidney than the untreated control group.

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

DISCUSSION

The supplementation of feed with selenium is usually limited to selenides such as sodium selenite and selenium containing organic compounds. It was found that nano-Se had similar or higher bioavailability and much less toxicity in mice, rat, broiler and goat compared with selenite (Zhang et al., 2001, 2005; Gao et al., 2002; Jia et al., 2005; Wang et al., 2007; Shi et al., 2011; Zhou and Wang, 2011). The present study showed that the growth performance of chicks was affected after 3rd weeks of post feeding by dietary Se level. 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 of Se in the feedstuffs. A concentration of 0.15 mg Se/kg diet is recommended for broiler chickens (National Research Council, 1994). However, the basal diet used in this experiment contained only 0.075 mg Se/kg diet which was far lower than the requirements. Poultry diets deficient in selenium resulted in poor growth and development, increased mortality and reduced egg production, decreased hatchability (Kim and Mahan, 2003). The present result proved this point and the group not supplemented with any forms of selenium showed the symptoms of selenium deficiency such as lower weight gain and RGR values. On the other hand, dietary Se levels exceeding 0.5 mg kg^-1 might impair the growth while clinical symptoms of Se toxicity appear above 3-5 mg Se/g of diet (Kirchgessner et al., 1997). 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.

It was obvious that the Se contents in layer chick tissues such as breast muscle, liver, kidney, pancreas and feathers were markedly increased with the addition of dietary nano-Se in treated groups than the sodium selenite and untreated control groups. Moreover, increased selenium deposition was found with increasing nano-Se content in the diet. In general, animal study trials demonstrated that bioavailability of nano-Se was higher than that of inorganic forms (sodium selenite) (Zhang et al., 2001). Thus, Se bioavailability depended not only on its absorption by the intestine but also on its conversion to a biologically active form (Foster and Sumar, 1995). There are several reports of Se supplementation to increase the breast, liver, or plasma Se levels (Downs et al., 2000; Spears et al., 2003).

Selenium has a number of biological functions in animals and the most important action is its antioxidant effect (Levander and Burk, 1994). The results of present study also showed higher GSH-Px activity, SOD and catalase activities in erythrocytes of layered chicks as compared to the sodium selenite and untreated control groups. This suggested that serum GSH-Px activity seemed to be reflective of its dietary Se level but additional dietary Se did not stimulated further activity of the enzyme. The present finding indicated 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). Increasing the Se content of food for human consumption by manipulating source and level of Se supplementation to livestock has been area of interest to food scientists (Zhan et al., 2007; Wang and Xu, 2008). The results indicated that nano-Se was more greatly accumulated in breast muscle than the sodium selenite.

An improved antioxidant system of the chick may also enhance immune system function which is extremely important at this point in physiological development. 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 higher antibody response than that of sodium selenite and untreated control groups. However, different dietary selenium sources did not affect weight of different lymphoid organs and different haematological parameters such as haemoglobin content, TEC and PVC values. In addition to that, different physiological parameters such as serum glucose, total protein, globulin, SGOT, urea levels were increased linearly and quadratically (p<0.05) with increase in concentration of nano-Se in diet. However, there was no effect on serum cholesterol, triglyceride, A/G ratio, ALP due to dietary administrations of nano-Se in layered chicks. Similarly, the findings of Yang et al. (2012) revealed that the aspartate amino transferase, alkaline phosphatase (ALP), total protein, globulin, total bilirubin, glucose, urea, total cholesterol, triglyceride and high density lipoprotein levels were observed to be non-significant between control and Se supplemented group in chicks.

The different physiological effects of nano-Se and sodium selenite were 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 (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, improved intestinal absorption and tissue depositions.

CONCLUSION

The present study had demonstrated that dietary administration of nano-Se could improve the final weight, relative gain rate, antioxidant status (GSH-Px activities, SOD and erythrocyte catalase activities) and selenium deposition in several tissues especially breast muscle of layer chicks. Moreover, nano-Se appeared to be more effective (p<0.05) for than that of inorganic sodium selenite in increasing different biochemical parameters, haematological parameters, cellular and humoral immunity. The results also showed that the range of optimum dietary levels of nano-Se was wider than that of sodium selenite and nano-Se of 0.3 mg kg^-1 of dry diet is the optimum dose for getting better physiological effects in layer chicks. Further supportive study is needed to explore absorption mechanism, metabolic pathways, ideal dose and form of nano-Se that should be fed to poultry under commercial conditions.

ACKNOWLEDGMENTS

Authors are highly thankful to All India coordinated research project on “Improvement of feed resources and nutrient utilization in raising animal production” of ICAR, New Delhi, INSPIRE Fellowship under INSPIRE Program of the Department of Science and Technology (DST), New Delhi and National Fellow scheme of ICAR, New Delhi, for providing all financial support and other facilities to carry out this study.