Elsevier

Fish & Shellfish Immunology

Volume 57, October 2016, Pages 293-300
Fish & Shellfish Immunology

Full length article
Effect of Shilajit enriched diet on immunity, antioxidants, and disease resistance in Macrobrachium rosenbergii (de Man) against Aeromonas hydrophila

https://doi.org/10.1016/j.fsi.2016.08.033Get rights and content

Highlights

  • THC and respiratory bursts activity significantly increased aal diets on weeks 2 and 4.

  • PO activity also was significantly increased all diets on fourth week.

  • Phagocytic activity significantly increased all diets on weeks 2 and 4.

  • SOD activity was significantly enhanced with any enriched diet during the experimental period.

  • GPx activity increased significantly with 2 and 4 g kg-1 enriched diet during the period.

Abstract

The effect of diet supplemented with Shilajit, a multi-component natural mineral substance on the antioxidant activity, immune response, and disease resistance in freshwater prawn, Macrobrachium rosenbergii (de Man) against Aeromonas hydrophila is reported. The total hemocyte count (THC) and phagocytic activity significantly increased with 2 g kg−1 supplemented diet on first week and with other enriched diets on weeks 2 and 4. The respiratory burst (RB) activity and glutathione peroxidase (GPx) activity were significantly increased with 2 g kg−1 supplemented diet on weeks 1 and 2 whereas 2 and 4 g kg−1 diets on week 4. The phenoloxidase (PO) activity increased significantly with 2 g kg−1 diet only on second week and with other enriched diets only on fourth week. The superoxide dismutase (SOD) activity increased significantly with any enriched diet during the experimental period except with 6 g kg−1 diets on first week. However, the glutathione reductase (GR) activity was enhanced significantly only with 2 g kg−1 enriched diets on weeks 2 and 4. The cumulative mortality of the prawn fed with 2 and 4 g kg−1 enriched diets was 10% and 15% whereas with 6 g kg−1 diet the mortality was 20%. The results suggest that diet enriched with Shilajit at 2 g kg−1 or 4 g kg−1 positively enhances the antioxidant activity, immunity, and disease resistance in M. rosenbergii against A. hydrophila.

Introduction

Freshwater prawn culture has been recognized as an eco-friendly alternative for sustainable prawn production. In many countries the freshwater prawn, Macrobrachium rosenbergii (de Man) is an economically important venture because of its high commercial value [1]. It is a highly priced elite food product in both domestic and export market; M. rosenbergii is euryhaline and survives in a wide range of salinities between 0 and 18‰ [2]. In many countries its farming has been expanding significantly during the last decade. However the juveniles and adults of M. rosenbergii suffer high mortalities especially in hatcheries [3], [4], [5] due to several disease outbreaks such as appendage deformity syndrome (ADS) [6] and bacterial pathogens such as Vibrio spp., Aeromonas spp., and Pseudomonas spp., and Lactococcus garviae [7], [8]. Among, these, Aeromonas spp. is considered to be the major threat in the commercial cultivation of M. rosenbergii in Taiwan [9] and Brazil [10] including India [11], [12].

In the prevention and control of prawn diseases large quantities of antibiotics and chemicals are applied; vaccines are another eco-friendly measure. However the traditional measures build up drug-resistance in pathogens; vaccines are pathogen specific and hence novel strategies to control bacterial diseases in aquaculture are needed. The effect of plant products on innate and adaptive immune response in fish and shellfish diseases was reviewed [13]. It is encouraging to note that in Penaeus monodon dietary administration of polysaccharide gel obtained from the fruit-rind of Durio zibethinus could significantly increase the immune response and disease resistance against Vibrio harveyi and white spot syndrome virus (WSSV) [14]. In M. rosenbergii administration of Withania somnifera and Eichhornia crassipes supplemented diets positively enhance the immunity and survival rate against A. hydrophila and L. garvieae [15], [16].

Shilajit is a blackish-brown exudates of variable consistence obtained from the steep rocks of specific mountain regions of the world at altitudes between 0.6 and 5 km [17]. In India it is found in Himalayas region at altitudes between 1 and 5 km on the cave walls embedded in rocks or as rock exudates from Arunachal Pradesh in the East to Kashmir in the West [17], [18]. Though shilajit has similar physical properties and qualitative chemical composition, there is a regional variation in the ratio of individual components. Shilajit/humus consists of humic (80–85% of total organic mass) and non-humic (15–20%) substances [18], organic matter (60–80%), mineral matter (20–40%), and ∼5% trace elements. Shilajit contain more than 85 minerals in ionic form and humic substances (mainly fulvic and humic acid) [19]. It contains 14–20% humidity; 18–20% minerals; 13–17% proteins (with marked- α-amylase activity); 4–4.5% lipids; 3.3–6.5% steroids; 18–20% nitrogen-free compounds; 1.5–2% carbohydrates; and 0.05–0.08% alkaloids, and a number of amino acids [20]. Shilajit comprise 65 organic compounds namely, albumins, coumarins, free fatty acids, organic acids including adipic, succinic, citric, oxalic and tartaric, waxes, resins, polyphenols, essential oils and vitamins like B1, B12, etc. [21], [22]. Shilajit also contain a number of active constituents such as dibenzo-α-pyrones and related metabolites, such as tirucallane triterpenes, small peptides consisting of non-protein amino acids, some phenolic lipids, small tannoids, and FA [21].

In many countries Shilajit has been used for centuries as a traditional medicine [17] in treatment of genitourinary diseases, diabetes, digestive disorders, nervous diseases, tuberculosis, chronic bronchitis, asthma, jaundice, anemia, eczema, bone fractures, osteoporosis [23], [24], [25] kidney stones, edema, spondylitis, hemorrhoids, injured muscles, bone fractures, and diseases such as osteoporosis and other diseases; it is also used as a rejuvenator and an internal antiseptic [17], [26], [27]. Shilajit has potential use such as anti-inflammatory, anti-fungal, anti-ulcerogenic, anxiolytic activity, anti-allergic, analgesic, anti-diabetic, memory enhancer, and an antioxidant [18], [23], [28], [29], [30], [31]. For therapeutic applications it is administered in the form of an aqueous extract to activate phagocytosis and cytokine release by murine peritoneal macrophages [32], stimulate osteoblastic differentiation of mesenchymal stem cells [33], induce the proliferation of lymphocytes in the cortical thymus layer and increase migration of these cells into thymus-dependent zones of the lymph nodes and spleen [34]. In the aqueous extract of Shilajit humus comprise fulvic acid (FA) as the primary organic substance endowed with for many biological and medicinal properties [25], [29] effective in the treatment of disorders including gastritis, diarrhea, stomach ulcers, dysentery, colitis and diabetes mellitus [17], [25] and stimulate neutrophil and lymphocyte immune function [35], [36]. FA has broad spectrum antimicrobial property on a variety of bacteria, including P. gingivalis, F. nucleatum, S. mitis, A. actinomycetemcomitans, E. faecalis, S. mutans and also create a cytotoxic environment for cancer cells [37]. The complement system plays an essential role in innate immunity, contributing to inflammatory responses and the destruction and removal of pathogens. Likewise, the removal of complement by fixation has also been proposed to be a potential therapeutic strategy for treating inflammatory diseases [38]. It is also used in the form of an aqueous extract for therapeutic applications as an immuno stimulant and anabolic food additive [36]. To our knowledge this is the first study on the protective efficacy of dietary supplementation with Shilajit on antioxidant activities, innate immune function, and disease resistance in M. rosenbergii against A. hydrophila.

Section snippets

Diet

The basal diet (control) comprised mackerel meal, dehulled soybean meal, and corn gluten meal as the protein source; wheat flour, α-potato starch, and wheat gluten as carbohydrate and fish oil as lipid source in addition with vitamin and mineral premix (Table 1). The dietary Shilajit (Aravind Annai Herbals, Chennai, India) was incorporated with the basal diet at doses of 2, 4, and 6 g kg−1 by evenly mixing with the basal diet thoroughly. The enriched feeds were dried in a vacuum freeze drier

Hematology

The THC did not significantly increase in the infected prawn when fed with Shilajit-enriched diets on first week except with 2 g kg−1 diet as compared to control. However, THC significantly increased when fed with each enriched diets on weeks 2 and 4 (Fig. 1).

Respiratory bursts

The respiratory bursts activity did not significantly enhance in any Shilajit-enriched diet except with 2 g kg−1 diet on weeks 1 and 2 when compared to control; while it was significantly enhanced with 2 and 4 g kg−1 diets on fourth week (

Discussion

Globally especially in South East Asia, the freshwater giant prawn, M. rosenbergii is considered as a potential candidate species for sustainable aquaculture. However the production of prawns in countries including India has been vulnerable by many factors including diseases leading to significant economic loss in nursery and grow-out ponds. Among the bacterial pathogens, motile Aeromonas cause high mortality in prawn culture. In aquaculture, application of traditional medicine is

Acknowledgments

The authors express their sincere gratitude to the Dean of Scientific Research, the King Saud University, Riyadh, Saudi Arabia for funding this Research Group project no. RGP-1437 - 005.

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