Introduction
Skeletal development in hatchery reared gilthead seabream larvae (Sparus aurata) is a crucial process, where fish often present several anomalies such as lordosis, kyphosis, cranial malformations, and other vertebral anomalies (Izquierdo et al, 2019). This onset of skeletal anomalies lowers the survival rate by affecting the larval growth, swimming behaviour, and other physiological functions (Boglione and Costa, 2011). To overcome this issue, studies have tried to improve the larval production by altering the biotic and abiotic factors affecting larval skeletal development (Izquierdo et al., 2016). In accordance with that, nutrition is one of the major factors involved in skeletal development during early larval stages (Izquierdo et al. 2000). Hence, a series of trials focused on studying the effects of essential fat-soluble micronutrient vitamins D and K on gilthead seabream skeletal development during larval and juvenile stages were conducted. In addition, fish vertebra-derived cell lines were also used to understand the action of these vitamins on bone cell mineralization.
Vitamin D and K are essential micronutrients as they can’t be synthesised de novo in fish (Lock et al., 2010, Krossoy 2011). They are involved in bone formation by regulating calcium metabolism and bone biomarkers in fish (Graff et al., 2016). They work synergistically to synthesise bone regulators such as osteocalcin (oc) and matrix Gla protein (mgp), which function in bone formation, mineralization, and soft tissue calcification, respectively (Graff et al., 2016). Deficiency of these vitamins in diet causes skeletal anomalies, growth reduction and increase mortality in fish (Udagawa 2000, Dominguez et al.,2021,2022). Thus, the aim of the study was to determine the dietary requirement of vitamin D and K and its physiological role on growth and skeletal development of gilthead seabream larvae, juvenile and skeletal derived-bone cells.
Materials and methods
Four different in vivo experiments were conducted with dietary vitamin D and vitamin K, in which two were focused solely on vitamin D or K, while other two were aimed at understanding the interactions between both vitamins. Additionally, a similar model was designed for an in vitro cell culture study (Table 1).
Gilthead seabream larvae and juvenile were obtained from Aquaculture research group (GIA), ECOAQUA Institute, University of las Palmas de Gran Canaria, Spain. For larval study, they were initially fed with enriched (ORIGREEN, Skretting, Norway) rotifer (Brachinous plicatilis) and randomly distributed into experimental tanks at a similar density. For the juvenile study, fish were screened for abnormal body shape, which were considered to present skeletal anomalies, were removed and the fish with normal body shape and a similar body weight were homogenously distributed in experimental tanks. At the end of the experimental period, fish were sampled for growth performance, skeletal anomalies, gene expression and other biochemical analysis. For the cell culture study, VSa13 bone derived cell lines from CCMAR (Centro de Ciências do Mar, Faro, Portugal) were used. The cells were regularly observed under the microscope and growth media (contains different dilution of vitamin) was changed at regular time intervals to understand the effect of vitamins in cells. Cultured cells were analysed for extracellular mineralisation (ECM), cell toxicity and gene expression.
Results and Discussion
Larvae fed increasing dietary vitamin D3 showed an increase in complete vertebral mineralization with increase in severe skeletal anomalies and a reduced survival rate. The bmp2 and alp gene expression were affected with the highest dietary group. Meanwhile larvae fed increasing dietary vitamin K3 showed a reduction in complete vertebral mineralization (Figure 1a) with increasing abdominal kyphosis and reduced survival rate. Heatmap showed a reduced expression of osteocalcin (oc) gene expression with increase in dietary vitamin K3. On the other hand, when larvae were fed with different combination of vitamin D and K there were no significant differences with skeletal anomalies among the groups and there was a tendency to reduce the larval survival at the group fed with the highest dietary vitamin D and K combination. The vitamin K-dependent proteins such as oc and mgp were highly expressed during day 7 and tended to reduce at day 21. The other bone biomarker gene such as bmp2 were also affected on day 21 along with calcium regulators on both day 7 and day 21. However, when a different combination of dietary vitamin D and K were fed to gilthead seabream juvenile, no significant differences were observed among growth parameters and skeletal anomalies between the groups. Despite that, the combination of vitamin D and K upregulated the bmp2 and alp gene expression and calcium regulators in gilthead seabream juveniles. In the case of, in vitro cell culture, the cells treated separately with vitamin D and vitamin K showed no significant difference in extra cellular mineralization (ECM). However, the combined effect of vitamin D and K showed significant reduction in ECM when treated with higher dilution of vitamin K with different vitamin D level (Figure 1 b).
Conclusion
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”).
References
Boglione C, Costa C. (2011). Skeletal deformities and juvenile quality. In: Pavlidies MA, Mylonas CC, editors. Sparidae. Biology and Aquaculture of Gilthead Seabream and other Species. Wiley-Blackwell Chichester. pp. 233– 294.
Dominguez, D., et al., (2021). Effects of vitamin D3 supplementation in gilthead seabream (Sparus aurata) juveniles fed diets high in plant-based feedstuffs. Aquaculture 543.
Dominguez, D., et al., (2022). Effects of Menadione Sodium Bisulphite (Vitamin K3) Supplementation of the Diets Based on Plant Feed Ingredients on Growth and Bone Health of Gilthead Seabream (Sparus aurata) Fingerlings. Aquaculture Nutrition, 2022.
Graff, I.E., Øyen, J., Kjellevold, M., Frøyland, L., Gjesdal, C.G., Almås, B., Rosenlund, G. and Lie, Ø., (2016). Reduced bone resorption by intake of dietary vitamin D and K from tailor-made Atlantic salmon: a randomized intervention trial. Oncotarget, 7(43), p.69-200.
Izquierdo, M., Domínguez, D., Jiménez, J.I., Saleh, R., Hernández-Cruz, C.M., Zamorano, M.J., Hamre, K., (2019). Interaction between taurine, vitamin E and vitamin C in microdiets for gilthead seabream (Sparus aurata) larvae. Aquaculture 498, 246–253.
Izquierdo, M.S., Ghrab, W., Roo, J., Hamre, K., Hernández-Cruz, C.M., Bernardini, G., Terova, G., Saleh, R. (2016) Organic, inorganic and nanoparticles of Se, Zn and Mn in early weaning diets for gilthead seabream (Sparus aurata; Linnaeus, 1758). Aquaculture Research 1–16.
Izquierdo, M.S., Socorro, J., Arantzamendi, L., Hern, C.M., (2000). Recent advances in lipid nutrition in fish larvae. Fish Physiology and Biochemistry, 22(2), pp.97-107.
Krossøy, C., Waagbø, R., Ørnsrud, R. (2011). Vitamin K in fish nutrition. Aquaculture Nutrition 17; 585–594.
Lock, E-J., Waagbø, R., Bonga, S.W., Flick, G. (2010) The Significance of vitamin D for fish: A review. Aquaculture nutrition 16; 100–116.
Udagawa, M., (2000). Physiological role of vitamin K in fish-review. JARQ, Japan Agricultural Research Quarterly, 34(4), pp.279-284.