Introduction
Atlantic cod ( Gadus morhua ) farming has recently resurged in Norway , but larval nutrition remains a major challenge (Nardi et al., 2021), and no commercial diets are specifically tailored to cod larvae. Lipids are a key dietary component , providing both energy and essential fatty acids (Hamre et al., 2013) . Cod larvae are thought to have limited ability to digest dietary lipids due to low endogenous bile salt (BS) levels (Olsen et al., 1991) . Adding BS to the diet could improve lipid digestion, as seen in other fish species (Wang et al., 2023). After absorption in enterocytes, lipids are transported to peripheral tissues in lipoproteins, but this process may be restricted in larvae due to low de novo synthesis of phospholipids (PLs ) (Teshima et al., 1987), an essential component of lipoproteins. F ish larvae therefore need more dietary PL than juvenile and adult fish (Tocher et al. 2008), yet the specific requirements in cod larvae remain unclear.
This study aimed to assess how dietary PL level, BS supplementation, and their interaction (PL×BS) affect growth, survival, and development in cod larvae. We hypothesized that larval performance would be improved by (1) a higher dietary PL level and (2) BS supplementation , and (3) that the effect of BS would be more pronounced at low PL levels , where the diets contained more neutral lipids to maintain an isolipidic formulation.
Materials and methods
Atlantic cod larvae were reared in 200 L tanks and fed one of four formulated diets from 17 to 61 days post-hatching (dph), using a 2×2 factorial design with n = 3 tanks per group. The d iets differed in PL level – high (7 % dry matter, dm) or low (6 % dm) – and BS supplementation – 0.04 % dm or 0 % dm – yielding the following treatment groups: HPL (high PL), HPL-BS (high PL with BS), LPL (low PL), and LPL-BS (low PL with BS).
Larvae were sampled throughout the experiment to measure dry weight (DW) and standard length (SL) , and for histological analysis of the liver. At 61 dph, samples were taken for bone staining to assess skeletal ossification and anomalies , as well as biochemical analyses of proximate composition, lipid classes, and fatty acid composition. Survival from 20 to 60 dph was calculated by daily counts of dead larvae.
Results
No significant differences in larval growth (DW and SL) were observed among the groups at the end of the experiment. Nonetheless, the HPL group had significantly higher DW compared to the LPL-BS and LPL groups at 38 and 45 dph, respectively. Larvae receiving high-PL diets also showed a modest but significant increase (6 %) in specific growth rate (SGR) compared to larvae receiving low-PL diets between 15 and 52 dph. Survival at 60 dph ranged from 42 to 60 % across the treatment groups, with no significant differences.
Liver histology showed a decrease in hepatocyte nucleus size following 15 dph, with a subsequent increase towards 61 dph. Notably, LPL-BS larvae maintained significantly smaller nuclei at the end of the trial. Hepatic energy storage increased markedly between 45 and 61 dph, with a tendency for higher storage in high-PL groups (22%) compared to low-PL groups (12%) at 52 dph. Energy storage was closely associated with larval size and followed a Gompertz growth model (sigmoid curve), with no differences between the dietary treatments.
Bone staining revealed significantly more ossified dorsal and anal fins in the HPL and HPL-BS groups compared to the LPL-BS group. Additionally, larvae fed the HPL-BS diet showed a greater number of fully ossified vertebrae compared to the LPL group. Model comparisons using Akaike’s Information Criterion (AIC) indicated that these differences in skeletal development were associated with dietary PL level rather t han larval size. The diets showed no consistent effects on skeletal anomalies, though the frequency of kyphosis was significantly higher in the LPL group (15 %) than in the HPL group (1 %).
No significant diet effects were observed in larval proximate and lipid class compositions. The fatty acid composition of the PL fraction also showed no differences across diets . In contrast, the level of n-3 polyunsaturated fatty acids (PUFAs) in the triacylglycerol (TAG) fraction was significantly higher among larvae fed low-PL diets (13 %) compared to high-PL diets (9 %).
Conclusions
Our findings suggest that increasing dietary PL levels enhances skeletal development and, to a lesser extent, supports larval growth in Atlantic cod. These benefits may be linked to more efficient utilization of n-3 PUFAs when supplied through the dietary PL fraction rather than the TAG fraction. BS supplementation did not improve larval performance , suggesting that endogenous BS levels were sufficient to ensure effective lipid digestion under the tested dietary conditions.
References
Hamre K, Yufera M, Rønnestad I, Boglione C, Conceição LE, Izquierdo M (2013) Fish larval nutrition and feed formulation: knowledge gaps and bottlenecks for advances in larval rearing. Rev Aquac 5:S26-S58. https://doi.org/10.1111/j.1753-5131.2012.01086.x
Nardi G, Prickett R, van der Meeren T, Boyce D, Moir J (2021) Atlantic cod aquaculture: Boom, bust, and rebirth?. J World Aquac Soc 52:672-690. https://doi.org/10.1111/jwas.12811
Olsen RE, Henderson RJ, Pedersen T (1991) The influence of dietary lipid classes on the fatty acid composition of small cod Gadus morhua L. juveniles reared in an enclosure in northern Norway. J Exp Mar Biol Ecol 148:59-76. https://doi.org/10.1016/0022-0981(91)90147-O
Teshima SI, Kanazawa A, Horinouchi K, Yamasaki S, Hirata H (1987) Phospholipids of the Rotifer, Prawn and Larval Fish. Nippon Suisan Gakkaishi 53:609-615. https://doi.org/10.2331/suisan.53.609
Tocher DR, Bendiksen EÅ, Campbell PJ, Bell JG (2008) The role of phospholipids in nutrition and metabolism of teleost fish. Aquaculture 280:21-34. https://doi.org/10.1016/j.aquaculture.2008.04.034
Wang L, Sagada G, Wang C, Liu R, Li Q, Zhang C, Yan Y (2023) Exogenous bile acids regulate energy metabolism and improve the health condition of farmed fish. Aquaculture 562:738852. https://doi.org/10.1016/j.aquaculture.2022.738852