Land-based aquaculture systems can have an elevated content of metabolically-derived carbon dioxide (CO2) in the water. Interestingly elevated CO2 levels have been suggested to induce nephrocalcinosis and to increase the bone mineral content [1,2]. This raises the question if dietary phosphorus (P), one of the main components of bone minerals, can be reduced in systems with elevated CO2 levels. The optimisation of dietary P levels is desirable, as excess of dietary P can cause water pollution . Using mono-ammonium phosphate (MAP) as a dietary P supplement, this study aims to analyse if the requirements of dietary P are reduced with an elevated water CO2 level.
Materials and Methods
Atlantic salmon post-smolt (start weight 200 g) were fed MAP-supplemented diets with a low (0.28%) (0.5P), regular (0.56%) (1P), or very high (2.34%) (3P) estimated available P for 13 weeks. Animals from all diet groups were either subjected to water with an increased CO2 level (20 mg/L) (20CO2) or to standard control levels of CO2 level (5 mg/L) (5CO2). Animals from all experimental groups were reared in duplicates. Analytics included radiography, whole mount Alizarin red S staining, mineralised and non-demineralised histology, gene expression analysis, and plasma, and mineral content analysis.
Results and Discussion
The growth of animals fed the 3P diet, 5CO2 and 20CO2, was reduced compared to 5CO2 animals fed either 0.5P or 1P . 5CO2 animals fed 0.5P diet showed a known low-mineralised vertebrae phenotype characterised by extended areas of non-mineralised bone; osteomalacia. Plasma P levels in 0.5P animals were reduced. Animals fed 1P and 3P diet animals had fully mineralised vertebrae irrespective of the CO2 level. The 3P diet had no further effect on bone mineralisation. Unexpectedly, 50% of the 20CO2/0.5P animals showed a moderate increase of vertebral centra mineralisation, on x-ray images, whole mount Alizarin red S stained specimens, and histological sections (Fig. 1B,E,E’). This indicates that animals were able to use the dietary P more efficiently when reared in water with high CO2 levels. Fibroblasts growth factor (fgf23) expression, a hormone synthetised by osteoblasts and osteocytes which increases renal phosphate release  was downregulated in 0.5P animals. This suggests that more phosphate is retained under levels of increased water CO2 and reduced dietary P intake.
Figure 1. Representative vertebral centra of Atlantic salmon fed a low P diet and reared under low CO2 (5 mg/L) (A,D,D’) or high CO2 (20 mg/L) conditions visualised on x-ray images (A-C, scale bar = 1.5 cm), whole mount stained with Alizarin red S (D-F), and on non-demineralised histological sections stained by von Kossa/Van Gieson (D’-F’) (scale bar D-F’ = 1 mm). (A,D,D’) Characteristic reduced mineralisation is observed in animals fed a low P diet, characterised by the increased size of radiolucent spaces between the vertebral bodies (A) and by extended areas of non-mineralised bone at zygapophyses (arrowhead in D) and vertebral body endplates (arrowhead in D’). (B,E,E’) A moderate increase of mineralisation is observed in animals fed a low P diet and reared at increased water CO2 levels, characterised by the reduction of radiolucent spaces (B) and reduced areas of non-mineralised bone (E-E’). (C,F,F’) A regularly mineralised vertebral bodies in low P animals reared in high CO2 characterised by a further reduction of the radiolucent spaces (C) and areas of non-mineralised bone (F-F’).
The current study indicates that the requirements for dietary P in seawater stages of Atlantic salmon can be reduced when animals are reared in water with elevated CO2 levels. This offers a possibility to lower the P content in salmon feeds for animals reared in systems prone to accumulate CO2.
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