Introduction
Aquaculture expansion increases demand for sustainable feeds that reduce reliance on fish oil (FO). However, reducing dietary FO in aquafeeds is challenging, as FO is a major source of n-3 LC-PUFA, namely EPA and DHA, which marine fish require. Promising alternatives to FO have emerged, driven by their fatty acid composition, cholesterol content, and lipid-soluble vitamin profile. Among these are oil from Hermetia illucens larvae (HIO), rich in medium-chain fatty acids, and fatty acid ethyl ester oil (FAEE), a FO-derived by-product of omega-3 concentration production for human consumption, containing approximately 5–8% EPA and DHA[1,2]. This study evaluated the effects of dietary FO replacement by HIO and FAEE on juvenile European seabass (Dicentrarchus labrax) growth performance, feed utilization, fillet fatty acid composition, plasma immune parameters, blood cell counts, and survival following a bacterial challenge.
Materials and Methods
Four isoproteic and isolipidic diets containing 48% crude protein and 15% crude lipids were formulated, varying only in lipid source via different combinations of FO, FAEE, and HIO. The positive control (diet D1) contained 78.2% FO and 21.8% HIO, whereas the negative control (diet D2) contained 21.8% FO and 78.2% HIO. Diets D3 and D4 were FO-free and included HIO and FAEE at different proportions (56.4%/43.6% and 21.8%/78.2%, respectively). A feeding trial was performed with European seabass (initial body weight: 25 g) in a thermo-regulated recirculating aquaculture system comprising 12 tanks of 200 L capacity (15 fish per tank) at 23.0 ± 1.0 °C. Each experimental diet was tested in triplicate, and fish were fed until apparent visual satiation for 84 days. At the end of the trial, 3 fish per tank were euthanized by anesthetic overdose, bulk weighed, and blood, plasma, and muscle were sampled. Total red blood cell (RBC) and white blood cell (WBC) counts were determined. After the feeding trial, 20 fish per diet were intraperitoneally injected with 100 μL of 3 × 107 CFU mL-1 Vibrio anguillarum (VA). A control group was injected with 100 μL of phosphate-buffered saline solution (PBS). Fish survival was monitored for 9 days. Dead or moribund fish were daily removed from each tank. Nine additional fish from each diet were also injected as above, and blood and plasma samples were collected 24 hours after injection for RBC and WBC counts and plasma immune parameter assessment. Statistical analyses were conducted using one-way ANOVA, followed by Tukey's test for pairwise comparisons (P < 0.05).
ResultsNo differences in growth performance or feed efficiency were found in fish fed the experimental diets. Plasmatic proteases, anti-proteases, and peroxidase activities were not affected by dietary treatments. Nevertheless, plasma lisozyme activity was higher in fish fed D1 compared to fish fed D2 and D3, but not to those fed D4. Fish fed D3 and D4 showed lower WBC counts than those fed D2, and fish fed D3 had lower RBC counts compared to fish fed D4. The fatty acid profile of the fish fillets is still under analysis. Upon bacterial challenge, no differences in WBC or RBC counts were observed between dietary treatments, and plasmatic immunological parameters are still under analysis. However, fish fed D3 showed higher survival against VA than the other groups (Figure 1).
Conclusion
In conclusion, the present results showed that dietary replacement of FO with HIO and FAEE oil maintained growth and feed efficiency, and that a balanced combination of HIO and FAEE in the diets enhanced disease resistance following a VA infection.
Figure 1 – Kaplan-Meier survival curves during the 9-day bacterial challenge of European seabass fed the experimental diets. Data are represented as mean (n=2) and standard deviation. Significant differences are denoted by asterisks * (p-value = 0.0371).
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Acknowledgment
RM was financed by a CEEC Individual contract (ref: 2023.09082.CEECIND/CP2848/CT0017, and DOI https://doi.org/10.54499/2023.09082.CEECIND/CP2848/CT0017). This work was funded by the "OmegaGreen23" project, funded by the Portuguese Foundation for Science and Technology (Ref: 2023.13017.PEX and DOI: https://doi.org/10.54499/2023.13017.PEX).
References1.Lutfi Royo, E., Noor, A., Nilsson, A., Haugen, J.-E., Kousoulaki, K., Berge, G. M., ��stbye, T.-K. K., & Ruyter, B. (2024). Nutritional value of ethyl ester oils in fish diets: A review on their potential impact on growth and health (Report No. 17/2024, 14 pp.). Nofima AS. https://hdl.handle.net/11250/3152953.2.Barragan-Fonseca, K.B., M. Dicke, and J.J.A. van Loon, Nutritional value of the black soldier fly (Hermetia illucens L.) and its suitability as animal feed – a review. Journal of Insects as Food and Feed, 2017. 3(2): p. 105-120.