Background/Objectives: The effect of sodium butyrate (NaB), β-glucan (βG) and vitamins in the diet on gut microbiome, cortisol level, lysozyme activity and growth parameters of juvenile hybrid sturgeon (Acipenser gueldenstaedtii♀ × Acipenser baerii♂) was determined. Methods: Sturgeon hybrids (n = 144) were divided into three groups with enriched feeding (mg/kg of feed): FQV1 (50 NaB; 20 βG; const. vitamins), FQV2 (150 NaB; 20 βG; const. vitamins), FQV3 (50 NaB; 60 βG; const. vitamins) and control (not supplemented), each group in triplicate, 12 fish in each repetition. Rearing was carried out for 30 days in controlled conditions. Gut microbiome was characterized using Next Generation Sequencing (NGS) of DNA samples isolated from intestinal content. Cortisol level was determined using the ELISA test. Lysozyme activity was measured by turbidimetric test. Results: Based on data obtained from NGS, it was determined that the FQV1 group is characterized by the highest values of diversity indices (Shannon, Simpson and Chao-1) and the largest number of ASVs (Amplicon Sequence Variants). The highest abundance of probiotic bacteria (Lactobacillus, Lactococcus) was determined in the FQV1 group. The highest cortisol concentration was determined in the control (33.26 ng/mL), while the lowest was in FQV3 (27.75 ng/mL). The highest lysozyme activity was observed in FQV1 (154.64 U/mL), and the lowest in FQV2 (104.39 U/mL) and control (121.37 U/mL) (p < 0.05). FQV2 was characterized by significantly more favorable values of breeding indicators (p < 0.05). Conclusions: The obtained results prove that an appropriate composition of NaB, βG and vitamins can be used in the commercial breeding of juvenile hybrid sturgeons.
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