Introduction
The progressive replacement of fishmeal and fish oil by plant-based ingredients in marine fish diets has altered their micronutrient composition, increasing the need for vitamin supplementation, including vitamin D. Traditionally, cholecalciferol (vitamin D3) has been used as the dietary source, which must undergo two sequential hydroxylations to be converted first into calcifediol and subsequently into calcitriol, its biologically active form.
Direct dietary supplementation with calcifediol (25 OH D3) bypasses the first hepatic hydroxylation step, potentially enhancing vitamin D bioavailability and biological efficacy. In terrestrial vertebrates and some cold-water aquaculture species, such as salmonids, calcifediol supplementation has been associated with improved growth, mineral metabolism and physiological health (Rider et al., 2023; 2024; 2025). In contrast, information regarding its application in Mediterranean marine species remains scarce, with only one recent study conducted in juvenile gilthead seabream (Sparus aurata) (Dom��nguez et al., 2025). Therefore, the present study aims to provide new evidence on the effects of calcifediol supplementation in juvenile European sea bass, contributing to the nutritional knowledge base of Mediterranean aquaculture.
Materials and Methods
A total of 600 juvenile European sea bass (D. labrax; 62.0 ± 0.8 g) were randomly distributed among 15 tanks following a completely randomized design, with five experimental diets tested in triplicate. Diets were identical except for calcifediol supplementation levels (<2.0; 21.6; 53.5; 102.6 and 169.8 ppb). Fish were fed to apparent satiation three times daily for 113 days under a natural photoperiod.
On day 35, samples were collected for gene expression analyses, and a subgroup of fish was subjected to a Vibrio harveyi challenge, with survival monitored for 23 days. At the end of the trial, growth performance parameters (FCR and SGR) were calculated, and samples were collected for body composition, somatic indices, vitamin content, gene expression, enzymatic activity, digestive histology, and skeletal anomaly assessment. Data were analyzed using ANOVA and linear regression.
Results and Discussion
At the end of the feeding trial, no significant differences were observed in final body weight, total length, or other growth-related parameters among dietary treatments. Fillet and liver biochemical composition did not differ between groups, nor did skeletal anomalies, although a tendency toward increased anomaly frequency was observed at the highest calcifediol level without a specific anatomical pattern. The study incorporates a broad set of complementary analyses currently in progress, including mineral and vitamin retention and digestive tract histology, which will further support the interpretation of these results.
Table 1. Production parameters and skeletal anomalies after 113 days of calcifediol supplementation
Analyzed caclifediol in feed (ppb)
<2.0
21.6
53.5
102.6
169.8 p-value
Final mean weight (g)
247.4±22.7
246.1±27.9
253.2±23.1
250.9±26.0
252.9±25.0
0.265
Final mean total length (g)
26.3±0.9
26.4±1.0
26.4±1.0
26.7±0.9
26.6±1.0
0.126
FCR
1.66±0.15
1.69±0.07
1.64±0.10
1.63±0.09
1.59±0.05
0.909
Skeletal anomaly frequencies (%)
3.0 ± 5.3
0.0 ± 0.0
2.6 ± 4.4
4.2 ± 5.9
13.9 ± 4.8
0.103
Survival following Vibrio harveyi challenge exhibited a non-linear, treatment-dependent pattern, with the highest protective effect observed at intermediate dietary calcifediol levels (21.6 and 53.5 ppb). This response was followed by a progressive reduction in survival at both lower and higher supplementation levels. Higher calcifediol concentrations (102.6 and particularly 169.8 ppb), as well as the absence of calcifediol supplementation, appeared to negatively affect the host response to infection, potentially linked to physiological stress, disruptions in homeostatic balance, or interference with immune regulatory mechanisms. Ongoing analyses of gene expression and enzymatic activity form part of the study and will support a more integrated understanding of the mechanisms underlying the responses observed during the V. harveyi challenge.
Figure 1. Survival curve after a 23���day challenge with V. harveyi.
Figure 1. Survival curve after a 23���day challenge with V. harveyi.
Funding
The funding was conducted by Huvepharma NV (Belgium) under a contract for research purposes between Huvepharma NV and the University of Las Palmas de Gran Canaria.
References
Dominguez, D., ��� & Montero, D. (2025). Use of calcifediol in gilthead seabream (Sparus aurata): Effects on growth performance, biochemical composition, skeletal anomalies and histology. Aquaculture Reports, 43, 103006.
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Rider, S., ... & Dantagnan, P. (2024). Dietary calcifediol reduces mesenteric adiposity to the benefit of carcass growth independently of circulating vitamin D hormone in juvenile Atlantic salmon. Aquaculture, 585, 740687.
Rider, S., ... & Santigosa, E. (2025). Limited in vivo conversion of dietary D3 to 25-OH-D3 in rainbow trout and undetectable endogenous photo-production of D3 in indoor-raised fish. Aquaculture, 607, 742645.