Introduction
Commercial aquafeeds are usually high in lipids, since this ingredient is the main source of dietary energy and essential fatty acids for fish. However, an excess of fat can generate an increase in the amount of lipid bodies and adipose tissue, which has a negative impact on animal welfare, as well as in terms of the fillet quality (Salmerón et al., 2018). Since bile salts (BS) act as lipid emulsifiers and contribute to the digestion and absorption of fat (Swann et al., 2011), they have been tested as additives in fish diets, showing an improvement in growth performance, in efficiency of feed utilization and in the oxidative state (Ding et al., 2020; Mansour et al., 2020). Their supplementation in diets has also contributed to elucidate their regulatory role on lipid digestibility, lipase activity, adiposity and, cholesterol and bile acid (BA) levels in fish (Gu, Bai & Kortner, 2017). Likewise, in a nutritional trial conducted by Xiong et al. (2018), grass carp were fed diets supplemented with different BS and, as a result, it was observed that, especially the ones containing primary BS (i. e., sodium taurocholate), could modify the BA profiles in the gall bladder and change gut microbiome. These variations in the BA profile were attributed to a change in the intestinal epithelium permeability induced by gut microbiota, which would lead to a higher reabsorption (Xiong et al., 2018). Previous studies have revealed that BA can act as regulators of gut microbiome community structure, but also that the intestinal microbiota would be responsible for the regulation of the BA pool size and composition through mechanisms such as BS deconjugation (Ridlon et al., 2014). Based on this information, the objective of this work was to test whether by adding bile salts in diets with a high saturated fats content it was possible to improve the health and quality of gilthead sea bream (Sparus aurata), and its regulating effect on growth performance, feed utilization, oxidative stress, adiposity, fatty acids profile, and gut microbiome.
Materials and Methods
A 90-day feeding trial was carried out in which juvenile S. aurata (initial body weight: 44.05 ± 4.08 g) were fed with three experimental isoproteic (44 %), isolipidic (22.6 %) and isoenergetic (21.4 MJ/kg) diets: a “control”, with a high content of saturated fats, and two others with a composition similar to the previous one but supplemented with a blend of BS (sodium taurocholate and deoxycholic acid) at different levels of inclusion (0.06 % and 0.12 %). During the trial, monthly sampling to monitor fish growth in terms of weight (g) and standard length (SL) was performed. At the end, the specific growth rate (SGR) and the feed conversion ratio (FCR) were calculated, as well as the hepatosomatic index and the perivisceral fat index. Oxidative stress in liver was measured through its total antioxidant capacity (TAC), lipid peroxidation (LPO) (through the concentration of MDA), and enzymatic activity of catalase (CAT), glutathione reductase (GR) and superoxide dismutase (SOD). Microscopic observation of histological sections of the liver and foregut allowed to evaluate the overall condition and accumulation of fat in these tissues. In addition, the villi height, thickness of the musculature, height of enterocytes and density of goblet cells in the intestinal mucosa was evaluated, as well as its level of inflammation. The serum levels of cholesterol, triglycerides, alkaline phosphatase, GTP transaminase, GOT transaminase, albumin, globulins, and total proteins of the fish were measured. Fatty acid profiles of muscle and liver were analysed using gas chromatography. From DNA extracted from scraping the interior walls of the intestine, Next-Generation Sequencing (Illumina - MiSeq platform) using specific primers for the V3-V4 hypervariable regions of 16S rRNA genes was performed and RDP database was used for classifying the OTUs, identifying the composition of the gut microbial communities.
Results and Discussion
At the end of the nutritional trial, growth results showed that fish fed the diets with a BS inclusion of 0.06 % and 0.12 % were respectively 2.4 % and 2.1 % heavier than those fed the control diet (P < 0.05). In addition, a trend towards an increase in SGR was observed in both groups with respect to the control, obtaining significant differences in those fed the diet with 0.06 % BS (P < 0.05). This is in accordance with the results obtained by Ding et al. (2020). Furthermore, the inclusion of BS in diets reduced the level of perivisceral fat. In particular, PVI was 3.01 ± 0.28 % in the control group and 2.58 ± 0.19 % and 2.67 ± 0.19 % in fish fed the diets with 0.06 and 0.12 % BS, respectively (P < 0.05). On the other hand, the addition of BS in the diet did not affect the hepatosomatic index and FCR values (P > 0.05). Although a priori the antioxidant status of the liver did not vary between the groups, a lower CAT activity was detected in the diet with an inclusion of 0.06 % of BS (63.76 ± 15.87 nmol min-1 mg protein-1) with respect to the control group (96.88 ± 5.62 nmol min-1 mg protein-1) (P < 0.05). This could be part of a response to less oxidative stress in fish fed these diets. Histological results showed lower levels of hepatic and intestinal fat accumulation in fish fed with the 0.06 % BS diet. These results are in accordance with data from the perivisceral fat index; both results showing that the addition of BS reduces adiposity, possibly thanks to their role as fat emulsifier and in lipid metabolism (Swann et al., 2011). There were no differences in the rest of the histological and serum parameters, which remained within the range of the normal values for the species, indicating an optimal health condition. In conclusion, the addition of BS in diets with high saturated fats content is capable of modulating growth efficiency, accumulation of perivisceral, hepatic and intestinal fat and, in part, oxidative stress in Sparus aurata, which would lead to an improvement in the health and quality of the fish. Results of fatty acid profiles, BA profiles and gut microbiota will also be discussed.
References
Ding, T., Xu, N., Liu, Y., Du, J., Xiang, X., Xu, D., ... & Ai, Q. (2020). Effect of dietary bile acid (BA) on the growth performance, body composition, antioxidant responses and expression of lipid metabolism-related genes of juvenile large yellow croaker (Larimichthys crocea) fed high-lipid diets. Aquaculture, 518, 734768.
Gu, M., Bai, N., & Kortner, T. M. (2017). Taurocholate supplementation attenuates the changes in growth performance, feed utilization, lipid digestion, liver abnormality and sterol metabolism in turbot (Scophthalmus maximus) fed high level of plant protein. Aquaculture, 468, 597-604.
Mansour, A. T., El-Feky, M. M., El-Beltagi, H. S., & Sallam, A. E. (2020). Synergism of Dietary Co-Supplementation with Lutein and Bile Salts Improved the Growth Performance, Carotenoid Content, Antioxidant Capacity, Lipid Metabolism, and Lipase Activity of the Marbled Spinefoot Rabbitfish, Siganus rivulatus. Animals, 10(9), 1643.
Ridlon, J. M., Kang, D. J., Hylemon, P. B., & Bajaj, J. S. (2014). Bile acids and the gut microbiome. Current opinion in gastroenterology, 30(3), 332.
Salmerón, C (2018). Adipogenesis in fish. Journal of Experimental Biology, 221(Suppl_1), jeb161588.
Swann, J. R., Want, E. J., Geier, F. M., Spagou, K., Wilson, I. D., Sidaway, J. E., ... & Holmes, E. (2011). Systemic gut microbial modulation of bile acid metabolism in host tissue compartments. Proceedings of the National Academy of Sciences, 108(Suppl_1), 4523-4530.
Xiong, F., Wu, S. G., Zhang, J., Jakovlić, I., Li, W. X., Zou, H., ... & Wang, G. T. (2018). Dietary bile salt types influence the composition of biliary bile acids and gut microbiota in grass carp. Frontiers in microbiology, 9, 2209
Acknowledgments: This project has been funded by the Ministry of Science, Innovation and Universities (with reference RTI2018-095653).