Introduction
A group of potentially interesting immunomodulatory feed additives are marine sulphated polysaccharides (MSP) isolated from cell wall matrices of marine macroalgae
. MSP are highly complex sulphated polysaccharides, named ulvans when extracted from green macroalgae. Previously, the effects of different extracts from green macroalgae (Ulva sp.) and red macroalgae (Solieria sp.) both rich in MSP, were studied for direct antibacterial properties and induction of innate immune activity in vitro . Inhibition of bacterial growth , induction of reactive oxygen species (ROS), nitric oxide (NO) and immune gene expression were studied in primary head kidney leukocytes as read-out parameters in rainbow trout and Nile tilapia (Petit et al., 2024). The earlier in vitro studies led to a in vivo study in which the effects of green algae (Ulva sp.) derived MSP-rich extracts on fish health and growth performance were explored.
Materials and methods
Rainbow trout (Oncorhynchus mykiss ) were reared at 14.6–16.2 °C, with a 12-12h light-dark cycle. The fish were fed a trout specific research diet twice per day. Prior to the start of the experiment, fish were starved for 24 hours, caught from a common batch and randomly distributed over t hree different dietary treatments: Control, 3 g kg-1 of crude processed Ulva sp . concentrate (UC) and 3 g kg-1 of crude processed and heat-treated Ulva sp. concentrate (2 hours at 80 °C) (UC-T). Fish were randomly distributed in four tanks per dietary treatment at a stocking density of 30 fish per tank. The duration of the feeding trial was 42 days.
On day 0, f ish were batch weighed and 10 fish per dietary treatment were sampled for proximal analysis and 5 fish per dietary treatment for baseline gene expression of pro-inflammatory cytokines: tumour necrosis factor (TNFα), interleukin-1 (IL1b), interleukin-12 (IL12p40) and type II interferon (IFNγ); the anti-inflammatory cytokine interleukin-10 (IL 10), and two tight junction genes; occludin and marvelD2 (Tricellulin). On day 42, fish were batch weighed and 5 fish/tank were sampled for proximal analysis and 2 fish/tank sampled for gene expression of cytokines and tight junctions.
On day 48, 5 fish per dietary treatment were intra-peritoneally injected with 200 μl PBS or 200 μl of the viral mimic synthetic double-stranded RNA (Poly (I:C), P1530 Sigma-Aldrich) at 5 μg /gram fish, under mild sedation. At 24h post-injection, fish were euthanized, and posterior intestines were collected and stored in RNAlater at -20°C until RNA isolation for gene expression analysis of cytokines, tight junction and antiviral associated genes (viperin and pkr) .
Total RNA from posterior intestinal tissue were stored at −80°C. Prior to cDNA synthesis, total RNA was treated with DNase I, Amplification Grade (Invitrogen), and cDNA was synthesized using random primers (300 ng) and Superscript III First-Strand Synthesis for RT-PCR (Invitrogen). cDNA samples were diluted in nuclease-free water prior to real-time quantitative PCR (RT-qPCR) analysis. Gene expression was measured with RT-qPCR using ABsolute qPCR SYBR Green Mix (Thermo Scientific) in a Rotor-Gene Q (Qiagen), and fluorescence data were analysed using Rotor-Gene Analysis software version 1.7. The relative expression ratio (R) of each sample was calculated according to the Pfaffl method based on the take-off deviation of sample versus each of the PBS controls and normalized relative to elongation factor 1α (elf1α) as reference gene.
A one-way ANOVA was used to test for significance the effects of dietary supplementation with MSP-rich extracts from green (Ulva sp) algae on growth performance and apparent digestibility . When significant effects were found, results were checked with a Tukey HSD test. Gene expression data presented as fold-changes were transformed with natural logarithm. Subsequently, transformed data was tested for normality by using a Q-Q plot and performing a Shapiro-Wilk test. Data was then analysed using a one-way ANOVA, followed by a least significant difference (LSD) post hoc. All statistical analysis was performed in IBM SPSS statistical data editor version 26.
Results
Rainbow trout fed with diets with MSP-rich extracts (UC and UC-T) showed no significant differences in terms of growth performance and apparent digestibility when compared to the control group. On day 42, significant differences in baseline gene expression coding for cytokines (TNFα , IL1b , IL10 , IL12p40 , IFNγ) and tight junction proteins (occludin and marvelD2 ) were observed in both UC- and UC-T-enriched diets when compared to the control group but differed in the extent of their effect. Moreover, the immune challenge with poly (I:C) triggered a modulation of the gene expression of cytokines in diets with MSP-rich extracts (UC and UC-T) . The expression of antiviral related genes viperin and pkr was significantly up-regulated i n control group and groups fed with MSP-rich extracts (UC AND UC-T). MSP thermal processing during extraction had some effect on the parameters for immune modulation, but not on growth performance.
Conclusion
Overall, in this in vivo study, both, maintenance of growth performance and modulation of the immune responses suggest that MSP-rich extracts derived from green algae (Ulva sp.) can modulate physiological responses of rainbow trout.