Introduction
Manganese (Mn) is an essential micromineral involved in several biological processes such as protein metabolism, bone mineralization, immuno-modulator, and energy metabolism (Aschner and Aschner, 2005). Moreover, Mn plays an important role in antioxidant defence system, a Mn-dependent superoxide dismutase (manganese superoxide dismutase, Mn-SOD) which functions against oxidative damage in mitochondria. The deficiency of dietary Mn in juvenile fish caused poor mineralization, increased skeletal abnormalities, growth reduction, low Mn content in body tissue, and reduced liver Mn-SOD activity (Yamamoto et al., 1983, Antony Jesu Prabhu et al., 2016). In fact, the reduction of MnSOD caused by deficiency of Mn may alter the mitochondrial function resulting in mitochondrial dysfunction, ultimately increasing the production of Reactive Oxygen Species (ROS) and leading to oxidative stress (Li and Yang, 2018). The excessive ROS production may disrupt the balance between MSC (Mesenchymal stem cells)-mediated bone formation and HSC (Hematopoietic stem cells)-mediated bone resorption and thus leads to bone disease (Li et al., 2021). However, high Mn supplementation in diet (1000 mg/kg) reduce feed efficiency and body Fe, Ca, P concentration in grouper (Ye et al., 2009). Therefore, it is needed to define the optimum dietary Mn level to fulfil the Mn requirement level for fish.
Mn requirement studies have been carried out in several of juvenile fish (Antony Jesu Prabhu et al., 2016, Dominguez et al., 2020), the requirement levels vary from 2.4 – 24.9 mg/kg bases on the growth performance, vertebrae Mn and Liver Mn-SOD level. However, the effects of dietary Mn on growth, skeletal development and antioxidant activity have been scarcely studied in marine fish larvae. Copepods are part of the natural food for marine fish larvae, Mn content in copepods range between 8-25 mg/kg (Hamre et al., 2008). Hence, this study aimed to investigate the effect of dietary Mn level below 8 and beyond 25 mg/kg in growth, skeletal development, and antioxidant activity in gilthead seabream larvae. As, gilthead seabream larvae are prone to present skeletal anomalies during the early developmental stage (Boglione and Costa, 2011), this study would help in bridging the knowledge gap on Mn and its effect on skeletal development in gilthead seabream larvae.
Materials and methods
Diets. Six experimental microdiets based on squid meal and casein and containing different levels of Mn at the levels of 6.3, 39, 75, 87, 200 and 270 mg/kg were tested. Manganese sulfate monohydrate (MnSO4.H2O) was used as the Mn source. Larvae (dry weight: 0.29 ± 0.06 mg, total length 7.02 ± 0.71 mm, 23 dph) were randomly distributed into eighteen tanks (1800 individuals/ 200 L tank) and hand fed every 45 minutes from 8:00 am to 8:00 pm until 48 dph. Larvae dry weight and length were measured at different points: initial (23 dph), intermediates (29, 38, 43 dph) and final (48 dph). At the final sampling, larvae were collected for whole mount stain and gene expression, and the remaining larvae for mineral analysis. Daily mortality was calculated for survival rate. All data were tested for normality and homogeneity of variances and means compared by Tukey’s test and p values were estimated using regression (P < 0.05). Quadratic and linear regressions were used to establish a relation between dietary Mn level and their effect on the different variables.
Results and Discussions
After 26 days of feeding, fish larvae fed increasing dietary Mn levels showed an increase in body weight and total length, followed by the linear regression (Fig.1 Left) and significantly (P<0.05) increased the Mn levels in larvae whole body content from 2.03 to 7.67 mg/kg (Fig.1Right). Noteworthy, in the previous study, Mn content at 7.5 mg/kg in the red seabream (Pagrus major) larvae fed with enriched artemia significantly increased the growth (Satoh et al., 2008). This suggesting the 7.67 mg Mn/kg in larvae whole body consumed from the diets may be one of the reasons causing an increase in the larval growth in the present study. This reflects the importance of Mn supplementation in the diet to maintain the normal growth in fish. Larvae survivals were not significantly affected by dietary Mn levels, average survival rate was 37%. The mnsod expression of larvae (48 dph) showed a significant (P<0.05) up regulation with increasing dietary Mn levels (Fig.2 Left). A similar trend was found in the expression of runx2 (Fig.2 Right), whereas oc genes showed the significantly (P<0.05) highest expression in larvae containing 7.67 mg Mn/kg fed the 270 mg Mn/kg diet thus resulted in a higher mineralization.
Conclusions
In summary, larvae fed the non-supplemented diet (6.3 mg Mn/kg) showed symptoms of Mn deficiency, including low growth, oc expression, Mn content in larvae and reduced bone mineralization. Whereas larvae fed with highest amount of dietary Mn level at 270 mg/kg didn’t show signs of toxicity in gilthead seabream larvae at 48 dph.
Acknowledgment
This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 766347.This output reflects the views only of the authors, and the European Union cannot be held responsible for any use which may be made of the information contained therein.
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