Elsevier

Fish & Shellfish Immunology

Volume 106, November 2020, Pages 318-331
Fish & Shellfish Immunology

Full length article
Enzyme-treated soy protein supplementation in low protein diet enhanced immune function of immune organs in on-growing grass carp

https://doi.org/10.1016/j.fsi.2020.07.057Get rights and content

Highlights

  • Reducing dietary protein by 2% decreased immune function of fish immune organs.

  • ETSP supplementation enhanced antibacterial substances in fish immune organs.

  • ETSP supplementation alleviated inflammatory responses in fish immune organs.

  • ETSP supplementation regulated NF-κB and TOR signaling pathway.

Abstract

A 56 days feeding trial was conducted to investigate the effects of enzyme-treated soy protein (ETSP) supplementation in low protein diets on immune function of immune organs (head kidney, spleen and skin) in on-growing grass carp. A total of 540 on-growing grass carp (initial average weight: 325.72 ± 0.60 g) were fed six diets, which included a normal protein diet (28% crude protein) and five low protein diets (26% crude protein) supplemented with graded levels of ETSP (0.0, 0.8, 1.2, 1.6 and 2.0%). At the end of feeding period, a challenge test was performed by infection with Aeromonas hydrophila for two weeks. The results indicated that (1) reducing dietary protein content from 28 to 26% decreased antibacterial substances and aggravated inflammatory responses of above three immune organs; (2) under the condition of reducing protein level in diet, 0.8–1.2% ETSP supplementation reversed these above adverse effects on immune function of above three immune organs; (3) suitable ETSP supplementation-decreased inflammatory responses were partly associated with [IκB kinase β (IKKβ)/inhibitor of κBα (IκBα)/nuclear factor kappa B (NF-κB) p65 and p52 or NF-κB p65] signaling and [target of rapamycin (TOR)/(S6K1, 4E-BP)] signaling in above three immune organs. (4) On the basis of C3 content (head kidney), C4 content (spleen) and skin hemorrhage and lesion, the optimal ETSP supplementation levels in low protein diets were estimated to be 1.48%, 1.61% and 1.03%, respectively. In summary, ETSP supplementation in low protein diets improved immune function of head kidney, spleen and skin in on-growing grass carp.

Introduction

The shortage of dietary protein source in fish is one of the major issues in intensive aquaculture [1]. In this context, low protein diets have been concerned in aquaculture in recent years [2]. However, reducing dietary protein level led to the decrease of immune function in immune organs [such as systemic immune organs (head kidney and spleen) and mucosal immune organs (intestine and skin)] of fish [3,4]. Our previous studies showed that the suitable supplementation of functional feed additives in low protein diets could reverse the negative effects on immune function of immune organs in fish [5,6]. Indeed, ETSP is a kind of functional feed additives rich in bioactive peptides [7], and appropriate ETSP supplementation in low protein diets could reverse the adverse influences on intestinal immune function in Jian carp (Cyprinus carpio var. Jian) [5]. However, there is a lack of information about the effects of ETSP supplementation in low protein diets on immune function of systemic immune organs (head kidney and spleen) and mucosal immune organs (skin) in fish. It was reported that low molecular weight ETSP could get into the blood from the small intestine [8]. Funakoshi et al. [9] and Vale et al. [10] reported that head kidney, spleen and skin are composed of a highly complex vasculature in fish. Thus, low molecular weight ETSP may contact with head kidney, spleen and skin of fish through blood circulation. Moreover, dietary ETSP could increase intestinal arginine absorption in human [11]. Our previous studies revealed that dietary arginine improved the immune function of different immune organs (head kidney and spleen as well as intestine) relating to diverse signaling pathways (TOR/4E-BP and TLR4-Myd88 signalings, respectively) in Jian carp [12,13]. Hence, compared with the influences of ETSP supplementation in low protein diets on intestinal immune function, some diverse and interesting results may be obtained in systemic immune organs (head kidney and spleen) and mucosal immune organs (skin) of fish, which deserves further investigation.

The immune function of immune organs in fish is always related to antibacterial compounds [such as lysozyme (LZ) and immunoglobulin M (IgM)], antimicrobial peptides (such as β-defensin-1) and cytokines [including pro-inflammatory cytokines (such as interleukin (IL) −6) and anti-inflammatory cytokines (such as IL-10 and TGF-β)] [14,15]. Moreover, cytokines could be regulated by nuclear factor-κB (NF-κB) [16] and target of rapamycin (TOR) [17] in fish. However, until now, there was little study concerning the effects of ETSP supplementation in low protein diets on antibacterial substances and cytokines as well as related regulation mechanisms in systemic immune organs (head kidney and spleen) and mucosal immune organs (skin) of fish. In growing broilers, dietary ETSP could increase intestinal phosphorus digestibility [18]. Our previous study demonstrated that dietary phosphorus enhanced LZ activity and IgM concentration in the gill of grass carp [19]. In 3T3-L1 cells, ETSP supplementation could up-regulate PPARγ protein expression [20]. The increased protein expression of PPARγ could enhance β-defensin-1 expression in rat colon [21]. Moreover, dietary ETSP could increase threonine level in the muscle of starry flounder [22]. It was reported that in juvenile grass carp, dietary threonine could decrease IL-6 gene expression and increase IL-10 and TGF-β gene expressions in the intestine [23]. In addition, in pigs, dietary ETSP supplementation could elevate serum insulin level [24]. It was reported that insulin could activate mTOR signaling in HEK293 cells [25] and inhibit NF-κB signaling in mononuclear cells in human [26]. These observations indicated that ETSP supplementation in low protein diets may affect antibacterial substances and cytokines (relating to NF-κB and TOR signallings) in the systemic immune organs (head kidney and spleen) and mucosal immune organs (skin) of fish, and this deserves further investigation.

This present study used the same growth trial as our previous study, and was a larger investigation involved in determining the effects of ETSP supplementation in low protein diets on growth performance of fish [27]. Here, we hypothesized that ETSP supplementation may reverse the adverse effects on immune function of systemic immune organs (head kidney and spleen) and mucosal immune organs (skin) by low protein diets in fish. To confirm this assumption, we firstly investigated the effects of ETSP supplementation in low protein diets on antibacterial substances and cytokines as well as underlying mechanisms (NF-κB and TOR signalings) in head kidney, spleen and skin of fish.

Section snippets

Diets and feeding trial

The formulation of the diets in this study is consistent with that in our previous study (as shown in Table S1) [27]. Dietary protein sources were provided by fish meal, dehulled soybean meal, cottonseed meal and rapeseed meal; dietary lipid sources were supplied by fish oil, soybean oil and coconut oil. Moreover, 28% and 26% crude protein (CP) diets were the normal and low protein diets, respectively. The experimental diets were formulated according to ETSP supplementation: 0.0% (28% CP, the

Skin hemorrhage and lesions morbidity

As shown in Fig. 1, no significant difference was found in skin hemorrhage and lesions morbidity for fish fed 28% CP diets and 26% CP diets (un-supplemented ETSP) (P > 0.05). Moreover, skin hemorrhage and lesions morbidity significantly decreased with increasing ETSP supplementation levels in 26% CP diets up to 1.6%, and significantly increased thereafter (P < 0.05).

As shown in Fig. 2, skin hemorrhage and lesions morbidity for fish fed 0.8% ETSP supplementation diet was significantly lower than

Discussion

The present research used the identical animal trial as our previous investigation [27], which showed that suitable ETSP supplementation in low protein diets enhanced growth performance thereby sparing dietary protein in on-growing grass carp. Fish growth is closely connected with the immune function of systemic and mucosal immune organs [4,30]. Thus, the current study firstly explored the effects of ETSP supplementation in low protein diets on immune function of systemic immune organs (head

Conclusions

In summary, based on previous research in our lab that ETSP supplementation improved the growth of on-growing grass carp, the present investigation first investigated the effects of ETSP supplementation in low protein diets on immune function in head kidney, spleen and skin of fish. Firstly, reducing dietary protein content from 28 to 26% decreased immune function in head kidney, spleen and skin; secondly, under the condition of reducing dietary protein level, 0.8–1.2% ETSP supplementation

CRediT authorship contribution statement

Yan Song: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Software, Visualization, Writing - original draft, Writing - review & editing. Liang-Chao Yan: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Software, Visualization. Wei-Wei Xiao: Conceptualization, Investigation, Methodology, Software, Visualization. Lin Feng: Conceptualization, Funding acquisition, Project administration, Supervision, Validation. Wei-Dan Jiang:

Declaration of competing interest

The authors declare no competing or financial interests.

Acknowledgements

This research was financially supported by National Key R&D Program of China (2019YFD0900200), the Earmarked Fund for China Agriculture Research System (CARS-45), Outstanding Talents and Innovative Team of Agricultural Scientific Research (Ministry of Agriculture), Foundation of Sichuan Youth Science and Technology Innovation Research Team (2017TD0002), Key Research and Development Plan in Sichuan Province (2018NZ0007), the 111 project (D17015) and Supported by Sichuan Science and Technology

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