Introduction
Dietary selenium (Se) supplementation is increasingly discussed in modern fish feed formulations due to its low availability from plant raw materials and its essential role in the antioxidant system of fish (Fontagné-Dicharry et al., 2015). Thereby the effective biosynthesis of selenoproteins from organic Se is dependent on pyridoxal-phosphate as an enzymatic co-factor (Soda et al., 1999). A deficiency of vitamin B6 has been associated with impairments in the glutathione metabolism and antioxidant system (Taysi, 2005). Recent changes in the recommendation for dietary pyridoxine levels in plant-based aquafeed formulations (Hansen et al., 2015) raise the question, if the limited availability of the two micronutrients Se and pyridoxine may induce interactive effects in the glutathione metabolism of fish, especially when exposed to stress. Therefore, a feeding trial was conducted to investigate such possible interactive effects between dietary Se and pyridoxine supplementation in rainbow trout (Oncorhynchus mykiss).
Material and Methods
Four diets were designed: CTL, without any Se or pyridoxine supplementation; SEL, supplemented with 4 mg selenomethionine / kg diet; PYR, supplemented with 50 mg pyridoxine hydrochloride / kg diet and SEPY, co-supplemented with similar Se and pyridoxine levels. Groups of 50 juvenile rainbow trout (28 ± 3 g) were randomly distributed in triplicate tanks per treatment and fed one of the experimental diets for eleven weeks. At the end of the feeding period, 8 fish per tank were euthanized and tissue samples dissected. The remaining fish were exposed to oxygen stress (periodic hyperoxia) prior to sampling. Therefore, the dissolved oxygen (DO) level in the tanks was elevated for 8h (09:00-17:00h) per day from 8.5 ppm to 13 ppm (=168 %) DO over a one-week period. The liver samples collected of stressed and non-stressed fish were analyzed for metabolites and gene expression associated to the glutathione metabolism. The collected data was analyzed by repeated three-way ANOVA and is presented as mean ± standard error of means.
Results
Neither Se nor pyridoxine supplementation had any significant effect on fish growth performance (182 ± 3 g). Total liver glutathione levels were significantly lower in stressed compared to non-stressed fish (1351 ± 76 vs 927 ± 57 pg/mg protein). Fish fed diets supplemented with Se showed lower cysteine (Cys) levels in liver tissue compared to fish fed diets without Se supplementation (1522 ± 67 vs 1112 ± 57 pg/mg protein). Homocysteine (hCys) and total glutathione levels were observed to increase with pyridoxine supplementation (8.1 ± 0.7 vs 9.9 ± 0.9 and 1003 ± 66 vs 1275 ± 97 pg/mg protein). However, in stressed fish a significant interaction between Se and pyridoxine was detected as hCys levels were the highest in fish fed PYR, but the lowest when fed SEPY (12.9 ± 1.1 vs 5.7 ± 0.6). In addition, Cys levels were the lowest in stressed fish when fed CTL, but the highest with SEL (1587 ± 257 vs 806 ± 127). The gene expression of antioxidant enzyme glutathione peroxidase (Figure 1) and that of other selenoproteins was only affected by Se but not by pyridoxine supplementation.
Discussion and conclusion
The decrease in Cys levels detected with dietary Se supplementation might be a result of the co-metabolization of selenomethionine and methionine through the transsulfuration pathway (Dalto and Matte, 2020). On the contrary, dietary vitamin B6 supplementation seems to elevate transsulfuration as indicated through higher liver hCys and glutathione levels. This might relate to pyridoxal-phosphate being a co-factor of transsulfuration enzymes (Soda et al., 1999). Although vitamin B6 acts as a co-factor for the biosynthesis of selenoproteins through the delivery of Se to the translational machinery, in the present study, no effect of pyridoxine supplementation on selenoprotein gene expression was observed, suggesting that the pyridoxine levels in the non-supplemented treatments might have been sufficient to support selenoprotein synthesis. Nevertheless, interactive effects of Se and pyridoxine on transsulfuration metabolites in stressed fish indicate that both micronutrients play an important role to maintain glutathione homeostasis under stressful conditions similar to observations in mammals (Dalto et al., 2015).
Acknowledgement
This work was supported by the EU H2020 Research Innovation Program (AQUAEXCEL3.0, PID17737). This output reflects the author’s view. The EU cannot be held responsible for any use that may be made of the information contained therein.
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