The Effects of Dietary Supplementation with Different Organic Selenium Sources on Oxidative Stress in Broilers

The role of Se in poultry nutrition has been reviewed extensively (Surai, 2002a,b). Selenium is involved in the control of several physiological functions such as growth and immunocompetence. Se, as a constituent of selenoproteins, has structural and enzymatic roles, which impact on the antioxidant status and thyroid secretion of the bird (Sevcikova et al,. 2006). Also, Se is a component of the cell enzyme glutathione peroxidase which has a protective function in relation to tissue damage from oxidative stress. Usually, inorganic Se is incorporated into broiler diets as a constituent of the vitamin-trace mineral premix. However, evidence is emerging that organic Se may be superior to inorganic Se sources (Rutz et al., 2003; Wang et al., 2011a,b). For this reason two organic Se sources were compared in the present study. Furthermore, the form of the organic Se was evaluated in order to explore possible functional differences in vivo. 

The feeding study consisted of four dietary treatments based on wheat, soybean meal and canola meal. Starter diets were offered to birds from 1-21 days-post-hatch and finisher diets from 22-42 days post-hatch. The composition and the nutrient specifications of the diets are reported in the companion paper (Selle et al., 2013). The control diet contained a custom vitamin-trace mineral premix prepared by International Animal Health Products (Huntingwood, NSW) to Aviagen recommendations for Ross 308 birds. The premix did not contain any selenium. The experimental diets contained either BiOnyc® Tor-Sel (primarily composed of selenohomolanthionine; SH) or Alltech Sel PlexTM (primarily composed of selenomethionine; SM) individually, or in combinations of the two (50% SC:50% SM), so as to provide at total of 0.3 mg organic Se per kg of diet. Each of the four dietary treatments were offered to 12 replicates of 6 birds per cage from 1-42 days post-hatch as "starter" (1-14 days) and "finisher" diets (15-42 days). The parameters assessed included growth performance, nutrient utilisation and oxidative stress. Growth performance and nutrient utilisation data were compiled by following standard procedures as outlined by Selle et al. (2003) and are presented in the companion paper (Selle et al., 2013). At 42 days post-hatch, one bird from each cage was bled. Blood samples were collected from the jugular vein and were centrifuged (1000 × g for 15 minutes) and plasma was stored at - 80°C until assayed for reactive oxygen metabolites (d-ROMs), biological antioxidant potential (BAP), glutathione (GSH), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and advance oxidative protein products (AOPP). After blood was collected, birds were euthanised by cervical dislocation and breast muscle and liver tissues were collected, placed on liquid nitrogen and stored at (- 80°C) in order to determine a range of oxidative stress parameters until assayed for total antioxidant capacity (TAC), GSH-Px and SOD. ROMs and BAP were determined by commercial kits (Diacron, Grosseto, Italy) by FRAS4 (H & D, Parma, Italy). The results of the d-ROMs test were expressed in arbitrary units called Carratelli units' (U. Carr.), where 1 U. Carr. corresponds to 0.08 mg/100 mL The results of the BAP test are expressed in μmol/L of reduced iron. The degree of oxidative stress was estimated by the ratio of ROMs/BAP (U. Carr./μmol/L) * 100 = Oxidative Stress Index, given that the relationship between the level of oxidative stress and pathology is higher when ROMs and BAP measurements are so combined (Celi, 2011). Advanced oxidation protein products (AOPP) are novel markers of protein oxidation that were first described and characterised in plasma of uremic patients (Witko-Sarsat et al., 1996). Commercial kits (Cayman) were used for the determination of GSH-Px and SOD activities and GSH and TAC concentrations.

Experimental data were analysed by univariate analyses of variance using the general linear models procedure of the IBM®, SPSS® Statistics 20 program. A probability level of less than 5% was deemed to be statistically significant. The feeding study was conducted so as to comply fully with the specific guidelines for the "evaluation of dietary feed ingredients for broiler chickens" [N00/10-2011/2/5612] as stipulated by the Animal Ethics Committee of Sydney University. 

The effects of Se supplementation on parameters of oxidative status in plasma are shown in Table 1. Se supplementation did not influence (P > 0.65) reactive oxygen metabolites (ROMs), biological antioxidant potential (BAP), oxidative stress index (OSI) or advanced oxidative protein products (AOPP).

Table 1 - Effect of selenium supplementation on broiler plasma oxidative status

However, there were significant responses to Se supplementation observed for total glutathione (GSH), glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) plasma concentrations. Relative to the negative control, plasma levels of GSH in birds offered Se-supplemented diets were, on average, increased by 25.1% (26.21 versus 20.95 μM; P < 0.02). SH increased GSH by 24.2%; whereas, SM increased GSH by 23.4%. Similarly, relative to the negative control, plasma levels of GSH-Px in birds offered Sesupplemented diets were, on average, increased by 230% (188 versus 57 nmol/min.mL; P < 0.005). SH increased GSH-Px by 235%; whereas, SM increased GSH-Px by 188%. Again, relative to the negative control, plasma levels of SOD in birds offered Se-supplemented diets were, on average, increased by 35.2% (12.11 versus 8.96 U/ml; P < 0.005). SH increased SOD by 37.2%; whereas, SM increased SOD by 27.1%.

Table 2 - Effect of selenium supplementation on broiler breast muscle oxidative status

The effects of Se supplementation on parameters of oxidative status in the liver are shown in Table 3. Se supplementation did not influence (P > 0.10) the total antioxidant capacity (TAC) in the liver. However, relative to the negative control, average hepatic levels of glutathione peroxidase in birds offered Se-supplemented diets were significantly higher by 33% (55.14 versus 41.38 nmol/min.mg protein; P < 0.05). SH increased GSH-Px by 21%; whereas, SM increased GSH-Px by 37%. Also, relative to the negative control, average hepatic levels of superoxide dismutase in birds offered Se-supplemented diets were significantly higher by 52% (9.9 versus 6.5 U/mg protein; P < 0.03). SH increased SOD by 65%; whereas, SM increased SOD by 83%.

Table 3 - Effect of selenium supplementation on broiler liver oxidative status

The effects of Se supplementation on parameters of oxidative status in breast muscle are shown in Table 2. Se supplementation did not influence (P > 0.40) the total antioxidant capacity (TAC) in breast muscle. However, relative to the negative control, average breast muscle levels of GSH-Px in birds offered Se-supplemented diets were significantly higher by 101% (30.76 versus 15.30 nmol/min.mg protein; P < 0.005). SH increased GSH-Px by 108%; whereas, SM increased GSH-Px by 93%. Also, relative to the negative control, average breast muscle levels of SOD in birds offered Se-supplemented diets tended to be significantly higher by 80% (9.47 versus 5.25 U/mg protein; P < 0.10). SH increased SOD by 57%; whereas, SM increased SOD by 70%. 

The observed range of ROMs in this study is lower than those reported in ruminants (Celi et al., 2012). The BAP values reported in this study are consistent with the ranges reported in dairy cattle (Celi and Raadsma, 2010; Celi et al., 2012). In a study conducted in heat stressed sheep, supra-physiological levels of selenium were able to reduce ROMs levels by 10% and increase BAP levels by 5% (Chauhan et al. 2012); therefore the lack of changes in ROMs and BAP after selenium supplementation observed in this study, may be indicative that the level of selenium supplementation was not adequate to induce changes in these biomarkers. There was a trend toward an increase in AOPP concentrations in the majority of treatment groups however this was not significant. The lack of effects on AOPP may reflect the gut health was not compromised in this study. AOPP are pro-inflammatory mediators associated with a number of inflammatory conditions, are considered an indicator of oxidative stress and are associated with embryonic losses in dairy cows (Celi et al., 2011). GSH-Px activity is considered an indicator of oxidative stress and is also related to plasma lipid peroxide content (Di Trana et al., 2006). It is noteworthy that SH significantly increased GSH-Px concentrations in plasma, breast muscle and liver by 235, 108 and 21%, respectively. Alternatively, SM increased GSH-Px concentrations in plasma, breast muscle and liver by 188, 93 and 37%, respectively. The results gathered in this study suggest that Se improved the antioxidant property of broilers by elevating activities of antioxidant enzymes in plasma and tissues and there may be functional differences between SH and SM in broilers. 

ACKNOWLEDGMENTS: This study was funded by BiOnyc Pty Ltd. 

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