What if a feed additive could help sea bass eat more, grow better, and reshape the gut microbiome under hot, low-oxygen conditions? This study tests whether yeast-derived nucleotides and nucleic acids can do exactly that. Mediterranean aquaculture needs better feed, but aquafeed can account for up to seventy percent of running expenses and remains mainly tied to fish meal, an unsustainable protein source for carnivorous fish.

Replacing fish meal with crop-derived ingredients can create anti-nutritional and nutrient-requirement problems, so tailored functional aquafeeds are being developed to support growth, feed utilization, health, and stress resistance. Nucleotides and nucleic acids became interesting functional ingredients because they can act as alternative nitrogen sources and health promoters, while fish meal is one of the richest natural feed sources of nucleotides.

A central unresolved question is how much dietary nucleotide supplementation is appropriate for each species and for each specific life stage. Although fish can synthesize nucleotides endogenously, that process is metabolically costly, so dietary nucleotides may help, especially during early development and rapid growth.

Nucleotides are organic molecules involved in life-supporting pathways and are the building blocks of nucleic acids, which serve as genetic repositories. When fish meal is seriously limited, fast-growing carnivorous fish might fall below minimum nucleotide requirements, which motivates testing supplements in European sea bass juveniles.

The trial compared ten and twenty percent fish meal diets, each with five hundred milligrams per kilogram of yeast-originated nucleotides or nucleic acids, over eighty days. Afterward, the fish experienced one week of suboptimal temperature and dissolved oxygen conditions so the study could examine immune response and gut microbiome alteration.

Table one lays out six diets combining ten or twenty percent fish meal with either no additive, five hundred ppm nucleotide, or five hundred ppm nucleic acid. The formulation rows show how plant ingredients and fish-derived ingredients were adjusted between fish-meal levels, while the proximate analysis reports moisture, crude protein, crude lipid, and ash for each treatment.

This matters because it documents the intended dietary comparisons and confirms that the diets had closely matched protein and lipid compositions. Six diets were formulated with two low fish meal levels, ten and twenty percent, as control treatments, while all diets were isonitrogenous at forty-five point sixteen percent protein and isolipidic at seventeen point forty-three percent lipids.

Nucleotides and nucleic acids were then added at the same dose of five hundred milligrams per kilogram of feed. The experimental diets were produced by extrusion as two-millimeter pellets by the Danish Technological Institute in Taastrup, Denmark. The researchers randomly allocated sixty fish per tank, with an initial weight of fourteen point thirty-three plus or minus zero point eighteen grams, across eighteen eight-hundred-liter tanks.

The diets were assigned randomly in triplicate, and the animals were fed for eighty days in a closed recirculation system using natural seawater. Fish were fed freely twice a day for six days each week, using an overfeeding approach, and uneaten pellets were collected and weighed to calculate feed intake.

Fish were weighed at the beginning and end of the eighty-day feeding trial, and the study calculated specific growth rate, feed intake, and feed conversion ratio. The researchers also measured carcass composition and calculated protein and lipid efficiency indices, including protein efficiency ratio and lipid efficiency ratio.

For immune measurements, fish were sampled at the beginning, after feeding, and after suboptimal rearing conditions to assess inflammatory and immune-response gene expression in the distal intestinal mucosa. Fecal samples from six fish per diet were collected after the feeding and stress periods for gut bacterial community characterization.

The suboptimal conditions were designed to simulate an unfavorable Mediterranean summer condition. Within twenty-four hours, temperature increased at two degrees per day while oxygen concentration decreased from eight point zero plus or minus one point zero to four point six plus or minus zero point six milligrams per liter.

Temperature and oxygen were continuously measured using an automatic system regulated by software. At the end of the growth period, final body weight was higher in the twenty-percent fish meal diets with nucleotides or nucleic acids than in the twenty-percent fish meal control.

The twenty-percent fish meal control had the lowest weight gain, while specific growth rate was lower in that control than in the supplemented twenty-percent fish meal diets. Feed intake was lower in the unsupplemented ten- and twenty-percent fish meal diets than in the corresponding nucleotide-supplemented diets, and survival did not differ significantly among treatments.

Table three summarizes European sea bass performance after eighty days on six experimental diets, combining growth, body indices, composition, and nutritional efficiency. Growth differed significantly for final body weight, weight gain, specific growth rate, feed conversion, and voluntary feed intake, while survival and all somatometric indices showed no significant differences.

Crude lipid, moisture, and each nutritional index also varied among treatments, whereas crude protein and ash did not, giving the authors a broad view of how diet formulation affected both production and fish composition. Gross protein efficiency was higher in the twenty-percent fish meal diet with nucleic acids than in the ten-percent fish meal diets with nucleic acids or nucleotides.

Lipid efficiency ratio was higher in the twenty-percent fish meal diet with nucleic acids than in the other treatment groups, while gross lipid efficiency was higher than in the twenty-percent control and both supplemented ten-percent fish meal diets. At the end of feeding, fish meal level significantly affected interleukin one beta and transforming growth factor beta, and there was also an interaction between fish meal level and supplementation for transforming growth factor beta.

Interleukin one beta was upregulated in the ten-percent fish meal diets supplemented with nucleic acids or nucleotides compared with the ten-percent control, with a significant increase in the nucleic acid group. Transforming growth factor beta was downregulated in the supplemented ten-percent fish meal diets compared with the ten-percent control, with a significant decrease in the nucleotide group.

In the twenty-percent fish meal diets with nucleic acids or nucleotides, interleukin one beta was not upregulated and was less expressed than in the supplemented ten-percent fish meal diets. Figure one measures intestinal expression of four immune and inflammatory genes—IL-one beta, TNF-alpha, TGF-beta, and IL-ten—in seabass given ten or twenty percent fish meal, with control, nucleotide, or nucleic-acid supplementation.

It compares the initial feeding period, T-one, with conditions after the suboptimal rearing period, T-two, using six fish per diet and significance markers. The authors report significant effects of fish-meal level for IL-one beta and TGF-beta at T-one, plus an interaction between fish-meal level and supplementation for TGF-beta, showing that immune responses depended on both diet formulation and timing.

The gut microbiome was assessed using sixteen S ribosomal RNA gene sequencing, with thirty-six gut contents analyzed at each time point, after feeding and after suboptimal rearing conditions. The samples were evaluated at phylum, family, and genus levels, corresponding to Figures 2, 3, and 4.

At the first time point, Corynebacteriaceae abundance was affected by supplementation and fish meal level, with a significant interaction between them. Corynebacteriaceae abundance was higher in the ten-percent fish meal nucleotide diet than in the ten-percent control, ten-percent nucleic acid diet, and twenty-percent nucleotide diet.

After suboptimal conditions, Leuconostocaceae abundance was affected by supplementation and fish meal level, with higher abundance in the ten-percent fish meal nucleotide diet than in the ten-percent control and twenty-percent nucleotide diet. Paenibacillaceae abundance was higher in the twenty-percent fish meal nucleic acid diet than in the twenty-percent control, while Clostridiaceae abundance was lower in the ten-percent fish meal nucleotide diet than in its control.

Figure two shows stacked bar plots of distal-gut microbiota at the phylum level in European seabass, comparing ten- and twenty-percent fish-meal diets with nucleotide or nucleic-acid supplementation at two timepoints: after eighty days, T1, and after suboptimal rearing, T2. Across both timepoints, Firmicutes, Proteobacteria, Cyanobacteria, Actinobacteria, and Bacteroidetes are the main reported phyla, while the chart also displays less abundant groups.

This matters because the sequencing comparison provides a community-level view of how diet formulation and rearing conditions relate to gut bacterial composition. Figure three compares the relative abundance of bacterial families in seabass distal-gut contents across fish-meal levels and additive treatments, shown after eighty days at T one and following a suboptimal rearing period at T two.

The stacked bars highlight dominant groups including Leuconostocaceae, Clostridiaceae, Streptococcaceae, and Lactobacillaceae, while also displaying less abundant families. This matters because the authors use these community-level differences, alongside significant treatment interactions for families such as Corynebacteriaceae at T one and Leuconostocaceae at T two, to assess how diet composition and additives shape gut microbiota.

At the phylum level, the sea bass gut community was mainly Firmicutes, followed by Proteobacteria, Cyanobacteria, and Actinobacteria. After suboptimal conditions, nucleotides and nucleic acids in the ten-percent fish meal diet promoted beneficial taxa such as Weissella and Leuconostoc, while a significant separation in principal coordinates analysis indicated an overall impact on gut microbiome layout.

Dietary nucleotides also increased Bacillus abundance in the low fish meal diet and counteracted Clostridiaceae abundance compared with its control after the suboptimal conditions. Figure four shows stacked bar charts of distal-gut microbiota at the genus level for European seabass.

The four diet combinations compare ten or twenty percent fish meal with nucleotide or nucleic-acid supplementation, measured after eighty days and again following suboptimal rearing conditions. Dominant genera include Streptococcus, Weissella, Leuconostoc, Lactobacillus, Propionibacterium, and Corynebacterium, while the authors identify treatment-dependent differences in taxa such as Weissella and Corynebacterium at T1 and Weissella and Leuconostoc at T2.

Figure seven compares bacterial alpha diversity in European seabass gut contents after eighty days and after a suboptimal rearing period. Boxplots show Shannon, Simpson, and Fisher diversity at phylum, family, and genus levels, across ten or twenty percent fish-meal diets supplemented with nucleotides or nucleic acids.

The authors report no significant phylum-level differences at T1, but at T2 all three phylum-level indices show a fish-meal-by-supplement interaction, highlighting that microbiome responses depend on both diet composition and rearing conditions. At five hundred parts per million, nucleotide and nucleic acid inclusion enhanced voluntary feed intake in vegetable-based diets containing ten or twenty percent dietary fish meal.

Fish fed twenty-percent fish meal showed further improvement in feed efficiency and the highest overall growth-performance parameters, while the combination with nucleotides or nucleic acids improved lipid efficiency. With twenty-percent fish meal, the supplements increased anti-inflammatory transforming growth factor beta and decreased pro-inflammatory interleukin one beta in the intestinal mucosa.

With ten-percent fish meal, the supplements showed prebiotic properties by stimulating beneficial taxa such as Weissella and Leuconostoc after elevated temperature and low oxygen. The conclusion is that five hundred parts per million of nucleotides or nucleic acids promoted growth and lipid efficiency when included with twenty-percent fish meal, while a higher nutritional nucleotide requirement may apply with ten-percent fish meal.

With ten-percent fish meal, the supplements upregulated pro-inflammatory interleukin one beta, downregulated anti-inflammatory transforming growth factor beta, and favored beneficial gut bacterial taxa after high temperature and low oxygen exposure. The strongest pattern is context-dependent: at twenty percent fish meal, the additives improved growth and lipid efficiency, while at ten percent fish meal they altered intestinal immune signals and favored beneficial bacterial taxa after stress.