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Effect of Bacillus subtilis probiotics (live vs. pressure-killed) on growth performance, immune function, and biochemical indices in the rainbow trout, Oncorhynchus mykiss Cover

Effect of Bacillus subtilis probiotics (live vs. pressure-killed) on growth performance, immune function, and biochemical indices in the rainbow trout, Oncorhynchus mykiss

Open Access
|Jun 2026

Figures & Tables

Figure 1.

Effects of the experimental supplements on serum biochemicals of the rainbow trout, Oncorhynchus mykiss over 30 and 60 days supplementation. Control: probiotic-paraprobiotic free commercial diet; PRO: diet containing 1.5 × 106 Bacillus subtilis (probiotic) CFU/g diet; PAR: diet containing 1.5 × 106 CFU/g diet inactivated Bacillus subtilis (paraprobiotic); PRO + PAR: diet containing 1.5 × 106 CFU/g diet probiotic + 1.5 × 106 CFU/g diet paraprobiotic. ALB: albumin, TG: triglyceride, GLU: glucose, AST: aspartate aminotransferase, ALT: alanine transaminase, CR: creatinine. Differences between the experimental treatments at day 30 are shown in lowercase subscripts (P<0.01). Differences between the experimental treatments at day 60 are shown in uppercase subscripts (P<0.01). Same symbols indicate no difference between day 30 and day 60 in each treatment (P<0.01)

Figure 2.

Effects of the experimental supplements on antioxidant enzyme activities and total antioxidant capacity in the rainbow trout, Oncorhynchus mykiss over 30 and 60 days supplementation. Control: probiotic-paraprobiotic free commercial diet; PRO: diet containing 1.5 × 106 Bacillus subtilis (probiotic) CFU/g diet; PAR: diet containing 1.5 × 106 CFU/g diet inactivated Bacillus subtilis (paraprobiotic); PRO + PAR: diet containing 1.5 × 106 CFU/g diet probiotic + 1.5 × 106 CFU/g diet paraprobiotic. CAT: catalase, GPx: gluthatione peroxidase, SOD: superoxide dismutase, TAC: total antioxidant capacity. Differences between the experimental treatments at day 30 are shown in lowercase subscripts (P<0.01). Differences between the experimental treatments at day 60 are shown in uppercase subscripts (P<0.01). Same symbols indicate no difference between day 30 and day 60 in each treatment (P<0.01)

Figure 3.

Effects of the experimental supplements on the immune-related gene expression in the rainbow trout, Oncorhynchus mykiss over 30 and 60 days supplementation. Control: probioticparaprobiotic free commercial diet; PRO: diet containing 1.5 × 106 Bacillus subtilis (probiotic) CFU/g diet; PAR: diet containing 1.5 × 106 CFU/g diet inactivated Bacillus subtilis (paraprobiotic); PRO + PAR: diet containing 1.5 × 106 CFU/g diet probiotic + 1.5 × 106 CFU/g diet paraprobiotic. CD4: clusters of differentiation 4, IL-β: interleukin-β, MHC-I: major histocompatibility complex class I. Differences between the experimental treatments at day 30 are shown in lowercase subscripts (P<0.01). Differences between the experimental treatments at day 60 are shown in uppercase subscripts (P<0.01). Same symbols indicate no difference between day 30 and day 60 in each treatment (P<0.01)

Figure 4.

The intestinal bacterial load in the rainbow trout, Oncorhynchus mykiss over 30 and 60 days supplementation. Control: probiotic-paraprobiotic free commercial diet; PRO: diet containing 1.5 × 106 Bacillus subtilis (probiotic) CFU/g diet; PAR: diet containing 1.5 × 106 CFU/g diet inactivated Bacillus subtilis (paraprobiotic); PRO + PAR: diet containing 1.5 × 106 CFU/g diet probiotic + 1.5 × 106 CFU/g diet paraprobiotic. Differences between the experimental treatments at day 30 are shown in lowercase subscripts (P<0.01). Differences between the experimental treatments at day 60 are shown in uppercase subscripts (P<0.01). Same symbols indicate no difference between day 30 and day 60 in each treatment (P<0.01)

Primers used for detection of target genes

Gene namePrimer sequence
GAPDH-FACAAGGGTGAGGTTAAGGCAG
GAPDH-RCCCTTAATGTGAGCAGAAGCC
CD4-FGGTGGAAGTGAATCTGTGCTG
CD4-RTCCATCTCTTTCCTGTCCACC
IL1B-FAAGGAGACTACGACGACCTCA
IL1B-RCCGGCTTTCGTTGCTGATTTG
MHC1-FTGGATCAGACAGAATGAGGGG
MHC1-RCACAGCCCCATACATCACCTGAA

Effects of the experimental supplements on serum immune parameters of the rainbow trout, Oncorhynchus mykiss over 30 and 60 days supplementation_ Control: probioticparaprobiotic free commercial diet; PRO: diet containing 1_5 × 106 Bacillus subtilis (probiotic) CFU/g diet; PAR: diet containing 1_5 × 106 CFU/g diet inactivated Bacillus subtilis (paraprobiotic); PRO + PAR: diet containing 1_5 × 106 CFU/g diet probiotic + 1_5 × 106 CFU/g diet paraprobiotic_ Differences between the experimental treatments at day 30 are shown in lowercase subscripts (P<0_01)_ Differences between the experimental treatments at day 60 are shown in uppercase subscripts (P<0_01)_ Same symbols indicate no difference between day 30 and day 60 in each treatment (P<0_01)

Treatments
ControlPROPARPRO + PAR
Serum componentsDay 30Day 60Day 30Day 60Day 30Day 60Day 30Day 60
Lysozyme activity U/ml)27.2±5.2 c *31.5±7.2 C*41.8±6.5 b55.3±5.1 B45.1±5.5 b#52.8±6.1 B#59.3±5.8 a71.5±7.5 A
ACH50 activity U/ml)10.2±3.1 b*12.5±2.2 C*15.8±2.4 b#20.5±2.5 B#19.1±2.3 a24.5±1.8 A23.1±2.1 a+27.4±2.2 A+
Total Ig (mg/ml)1.5±0.15 c*1.7±0.2 D*2.1±0.12 b2.4 ±0.1 C2.5±0.2 a3.4 ±0.15 B2.7±0.1 a3.9 ±0.2 A
Two-way ANOVA (P-value)
Lysozyme activityACH50 activityTotal Ig
PRO0.0020.0040.003
PAR0.0060.0020.005
PRO × PAR0.0070.0060.009

Effects of the experimental supplements on growth parameters of the rainbow trout, Oncorhynchus mykiss over 30 and 60 days supplementation_ Control: probiotic-paraprobiotic free commercial diet; PRO: diet containing 1_5 × 106 Bacillus subtilis (probiotic) CFU/g diet; PAR: diet containing 1_5 × 106 CFU/g diet inactivated Bacillus subtilis (paraprobiotic); PRO + PAR: diet containing 1_5 × 106 CFU/g diet probiotic + 1_5 × 106 CFU/g diet paraprobiotic_ IW: initial weight, FW: final weight, WG: weight gain, SGR: specific growth rate, FCR: feed conversion ratio, SR: survival rate

Treatments
ControlPROPARPRO + PAR
IW (g)178.3±5.1181.1±4.5179.5±4.8181.9±5.2
Day 30Day 60Day 30Day 60Day 30Day 60Day 30Day 60
FW (g)251.5±5.5 c325.9±7.5 C265.6±6.7 b378.9±8.2 A271.5±7.2 ab342.5±6.4 B282.5±7.4 a390.8±12.3 A
WG (%)29.1±2.5 b*82.5±4.2 C**31.8±2.2 ab#108.9±5.3 A##33.8±2.4 ab+91.5±6.2 B++35.6±3.5 a×115.4±6.5 A××
SGR (%/day)1.02±0.02 b1.01±0.07 C1.35±0.05 a1.23±0.09 A1.28±0.03 a*1.08±0.07 B**1.35±0.4 a1.28±0.06 A
FCR1.38±0.02 a*1.22±0.05 A**1.28±0.03 b1.25±0.04 B1.28±0.02 b1.31±0.03 B1.14±0.01 c1.12±0.02 C
SR (%)97. 8±2.498. 5±2.5 A99. 5±1.596.8±2.3 A100%97.5±2.5 A100%96.4±2.6 A
Two-way ANOVA (P-value)
IWFWWGSGRFCR
PRO0.230.0060.0040.0060.002
PAR0.510.0020.0030.0080.004
PRO × PAR0.320.0080.0050.110.13
DOI: https://doi.org/10.2478/aoas-2025-0124 | Journal eISSN: 2300-8733 | Journal ISSN: 1642-3402
Language: English
Submitted on: Jul 6, 2025
Accepted on: Nov 7, 2025
Published on: Jun 5, 2026
In partnership with: Paradigm Publishing Services
Publication frequency: 4 issues per year
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© 2026 Neda Behmagham, Maryam Tajabadi Ebrahimi, Mehdi Soltani, Behin Omidi, published by National Research Institute of Animal Production
This work is licensed under the Creative Commons Attribution 4.0 License.

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