Functional effects of natural silicate, yeast and saponins based product on the growth and health status of the Pacific white shrimp Litopenaeus vannamei Efek fungsional bahan berbasiskan silikat alamiah, ragi dan saponins terhadap pertumbuhan dan status kesehatan udang putih Litopenaeus vannamei

In this study, a commercial product of natural silicates and yeast (NSY) and saponins from Yucca schidigera and Quillaja saponaria plants (SBP) were used to evaluate their nutritional effects on growth and health status of pacific white shrimp Litopenaeus vannamei . Four isonitrogenous and iso-lipidic experimental diets were formulated to contain 0%, 0.3% NSY, 0.2% SBP and 0.6% NSY. After 60 days, shrimp were sampled and standard haemato-immunological parameters were measured. The growth performances of shrimp were significantly affected by the dietary inclusion of NSY and SBP, whereas the inclusion of NSY and SBP provide a better growth compared to the control group. Additionally, the inclusion of NSY and SBP significantly enhance the total haemocytes count and lysozyme activity in shrimp compared to control. Therefore, NSY and SBP can be regarded as the functional ingredients in shrimp diet to improve the growth and non-specific immune function of shrimp.


INTRODUCTION
For years, most of the diet formulation for shrimp was designed to include approximately 20% fish meal (FM) due to its excellent sources of nutrients and mineral content (Sookying & Davis, 2011;Suárez et al., 2009;Tacon & Akiyama, 1997). However, the combination of high prices and fluctuating supply of FM has resulted in higher feed costs; therefore it has grown the demand for the replacement of FM with plant-protein sources, which is relatively less expensive than FM (Novriadi & Davis, 2018;Hardy, 1996). Several alternative protein sources has been evaluated to replace FM and other animal proteins in shrimp feed diet formulation allowing increased levels of plant based proteins, including soybean meal and their advance products, cotton seed meal, and corn protein concentrate ( Van Nguyen et al. 2018;Jatobá, et al. 2017;Richardson et al 2016;Bauer et al. 2012;Daiyong et al. 2009). Among alternative protein sources, processed soybean (Glycine max) possesses many qualities, including favorable amino acid profile, reasonable prices, widely available and highly digestible to be used as the most promising alternatives to replace FM in formulated diets (Lech & Reigh, 2012;Suárez et al., 2009;Gatlin et al., 2007, Amaya et al., 2007Davis & Arnold, 2000).
However, wider use of soy protein may be hindered with deficient levels of indispensable amino acids, anti-nutritional factors and poor palatability problem (Davis & Arnold, 2000). In addition,  mentioned that due to the antigen protein, soy protein will likely induce a stress reaction in shrimp. Consequently, there is a need to properly adjust the diet composition or explore the use of functional dietary ingredients to enhance the efficacy of plant-based diets for shrimp. Functional ingredients could be defined as ingredients that provide healthy and economical benefits beyond the basic nutrition needs (Olmos et al., 2011). A study from Nonwachai et al. (2010) showed that the use of heterotrophic algae as a source of essential fatty acids in feed designed with 40% soy protein significantly affected the immune response of shrimp and improved the resistance of shrimp against Vibrio harveyi. Furthermore, some functional ingredients, such as protein hydrolysates, prebiotics and herbal compounds, have been reported to possess healthy and nutraceutical properties and have been used as an additives to enhance the growth and positively modulate the immune system of shrimp (Servin et al., 2021;Quinto et al., 2018;Nguyen et al., 2012). Under these circumstances, the use of functional ingredients could be considered as the most attractive innovation in aquafeed formulation to enhance the dietary nutrition of plant-based ingredients.
Other than formulated feed and ingredients used to produce the feed, healthy gut microbiota is essential to promote the use of plant-protein as the primary protein sources in aquafeed (Zhou et al., 2018;Ringø et al., 2016). The intestinal microbiota of shrimp may play a vital role in nutrient metabolism, pathogen resistance, supply vitamins and digestive enzymes when feeding with high carbohydrate diets (NRC, 2011). However, due to rapid passage rates, high load of plant-protein might prevent extensive microbial fermentation within the digestive system of shrimp (Shao et al., 2019;NRC, 2011). The dietary effects on the use of fermented soybean meal, with lower anti-nutritional factors and better nutritional values than traditional soybean meal, on the intestinal microbiota has been examined by Shao et al. (2019) showing the ability of fermented soy to enhance the growth performance, but not significantly affect the intestinal microbiota composition of shrimp. Thus, the search on the functional ingredients that can improve the growth performance and contributing to a healthy intestinal microbiota condition as well as the haemato-immunological parameters in shrimp when using low FM and high inclusion levels of plant-protein sources are needed.
Recently, the growth and gut microbiota composition of shrimp fed with different inclusion levels of proprietary combination of all natural silicates and yeast components (NSY, PAQ-Gro™ Phibro, Teaneck, USA) has been evaluated (Servin et al., 2021). The authors mentioned that the inclusion of 1% NSY into the diet formulated with 10% FM, 22% soybean meal (SBM), 18% poultry meal (PM) and 43% wheat flour (WF) as the primary ingredients were able to positively enhance the growth and modulating the gut microbiota of L.vannamei. Therefore, the purpose of this study is to focus on the functional effects of commercial NSY and SBP on the growth and haemato-immunological parameter of the Pacific white shrimp L.vannamei.

Experimental diets
All diets were formulated to be similar in crude protein level (iso-nitrogenous) and crude lipid level (iso-lipidic) to contain 35% protein and 8% lipid as demonstrated in Table 1. In this growth trial, the control diet was designed with 10% fish meal (FM), 45% soybean meal (SBM), 17% wheat products (WP) and 8% corn protein concentrate (CPC) as the primary ingredients. Based on our internal assessment, diet 1 was produced by adding 0.3% of NSY, diet 2 was produced by incorporating 0.2% SBP, and diet 3 was produced by adding 0.6% of NSY into the control diet. All experimental diets were produced at the Main Center of Mariculture Development of Lampung using standard procedures for making shrimp feed and labeled as control diet, NSY 0.3; SBP 0.2; and NSY 0.6. Dry pellets were crumbled, packed in sealed bags, and stored in a freezer until use.

Growth trial and feeding management
The growth trials were conducted at the PT. Batam Dae Hae Seng research station (Batam, Indonesia). Pacific white shrimp post larvae (PL) were obtained from PT. Prima Akuakultur Lestari (Kalianda, Lampung, Indonesia) and were acclimatized to the culture system. Post-larvae were fed with a commercial feed (Evergreen Feed, Lampung, Indonesia) for three weeks until they reached the suitable size. Shrimp (4.24 ± 0.03 g initial mean weight) were randomly distributed into 24 tanks with size of 70 × 35 × 40 cm (98 L) per aquaria tank. Six replicate groups of shrimp were offered experimental diets using nutrition research standard protocol for 70 days and fed by hand four times daily at 07:00, 11:00, 15:00 and 20:00. Feed inputs were pre-programmed assuming the normal growth of shrimp and feed conversion ratio of 1.5. Daily allowances of feed were adjusted based on observed feed consumption, weekly counts of the shrimp and mortality. Uneaten feed, feces, and molts were removed by siphoning the aquaria tank prior to the first feeding.

Water quality and growth sampling
Dissolved oxygen (DO), pH, water temperature and salinity were measured four times daily using Aqua TROLL 500 Multiparameter Sonde instrument and connected to AquaEasy apps (Bosch, Singapore). Total ammonianitrogen (TAN), nitrate and nitrite were measured once a week by using absorption spectrophotometry (DR890, HACH, USA). At the end of feeding period, all shrimp were grouped and individually weighed to calculate the final biomass, final weight, percentage weight gain (PWG), feed conversion ratio (FCR), percentage survival (SR) and thermal unit growth coefficient (TGC) as follows: Where FBW is final body weight, IBW is initial body weight, T is water temperature ( 0 C) and D is number of trial days

Total haemocytes count
At the end of growth trial, hemolymph was sampled from two intermolt shrimp per tank or ten shrimp per treatment and total hemocytes count was determined. Hemolymph (100 μL) of individual shrimp was withdrawn from the pleopod base of the second abdominal segment with a sterile 1-mL syringe (25 G × 13 mm needle). Before hemolymph extraction, the syringe was loaded with a precooled (4°C) solution (10%-EDTA, Na2) used as an anticoagulant. The haemolymph with anti-coagulant solution was diluted in 150 μL of formaldehyde (4%) and then 20 μL was placed on a hemocytometer (Neubauer) to determine the total haemocytes count (THC) using an optical microscope (Olympus, DP72).

Lysozyme activity analysis
Lysozyme activity was measured using a lysozyme detection kit (Sigma-Aldrich, Cat. no. LY0100) according to the manufacturer's instruction. The results of lysozyme activity were defined by the lysis of the Micrococcus lysodeikticus cells. The reactions were conducted at 25 °C and absorbance at 450 nm was measured on the ultraviolet/visible spectrophotometer (Perkin Elmer, Lambda XLS, USA) df = dilution factor 0.001 = ΔA450 as per the unit definition 0.03 = Volume (in milliliters) of enzyme solution

Whole-body protein level and protein retention rate
Upon termination of the trial, four shrimp from each tank or twenty-four shrimp per treatment were randomly sampled and stored at -60 0 C for body composition analysis. Prior to the protein analysis, dried whole shrimp were rigorously blended and chopped in a mixer according to the standard methods established by Association of Official Analytical Chemists (AOAC, 1990). Protein contents of whole shrimp body were analyzed by using Kjeldahl method and conducted at PT. Angler BioChem Lab (Surabaya, East Java, Indonesia). The protein retention rate (%) were calculated using formula as follows:

Statistical analysis
All data were analyzed using one-way analysis of variance to determine the significant difference (P<0.05) among the treatment means followed by the Tukey's multiple comparison test to determine difference between treatments means in each trial. The pooled standard errors were used across all the growth parameters as the variance of each treatment is the same. Statistical analyses were conducted using SAS system (V9.4, SAS Institute, Cary, NC, USA).

Growth performance
Based on the growth performance and survival of Pacific white shrimp L. vannamei fed with the experimental diet summarized in Table 3, the use of NSY and SBP were able to significantly enhance the growth of L. vannamei compared to the control group. With respect to individual growth, both treatments displayed significantly higher final mean weight with better FCR compared to the control group. Despite lack of statistical significance, numerically, the survival of shrimp treated with NSY and SBP increased by 5.06-6.33% compared to the control group. The thermal growth coefficient (TGC) and weight gain (WG) of shrimp treated both with NSY and SBP were significantly higher than control group (P<0.05). Protein level and protein retention (%) of pacific white shrimp, Litopenaeus vannamei fed diets containing different levels of NSY and SBP are displayed in Figure 1. Supplementation of NSY and SBP did not affect the protein level of whole body of the shrimp. However, biologically, diet supplemented with 0.2% of SBP showed a higher protein level and protein retention rate (%) compared to other treatments which is also correlates with the growth of the shrimp.

Hemato-immunological response
The effect of adding NSY and SBP on the haemato-immunological parameters of shrimp are displayed in Figure 2 and Figure 3. The group of shrimp treated with 0.2% SBP and 0.6% NSY had the highest total haemocytes count (THC) compared to the control group. This also correlates with the lysozyme activity, whereas the highest activity was found in shrimp treated with 0.2% SBP followed with the supplementation of 0.3 and 0.6% NSY.

Discussion
In intensive culture system, more attention is being given to functional ingredients, additives and supplements to provide nutritional support for defense mechanisms enhancement, response to the degradation of water quality, maintain the energy requirement and optimize the metabolism function of shrimp (Delgado et al., 2021;Servin et al., 2021;Niu et al., 2019;Zhu et al., 2019;Cruz-Suárez et al., 2009;Li et al., 2007;Ochoa-Solano et al., 2006). At the same time, the use of  functional ingredients to partially or completely replace the use of fish meal (FM) could provide economic benefits with well-balanced and less expensive diets, as well as increasing the productivity of shrimp farms (Olmos et al., 2011;Bautista-Teruel et al., 2003). The present study evaluated the effect of commercial natural silicates and yeast (NSY) and saponins from Yucca schidigera and Quillaja saponaria plants (SBP) as feed additives on the growth performance and health condition of shrimp.
Based on the growth performance and survival of Pacific white shrimp L. vannamei fed with the experimental diet summarized in Table 3, the use of a combination of all-natural silicates and yeast components (NSY) and natural feed additive containing saponin blend (SBP) significantly increase final biomass (FB), average individual weight (AIW), thermal growth coeffcient (TGC) and weight gain (WG). With reference to the use of NSY, the growth results are in line with the recent study from Servin et al. (2021) in which shrimp displayed significantly better AIW, WG and FB with the administration of 1.0 % NSY into the diet (10% FM, 22% SBM and 43% WF) in comparison to the group of shrimp that were not receiving NSY into their diet. The same author observes that the direct stimulation of the immune system by the polysaccharides-rich feed contained in 1.0 % NSY diet appears to be directly related to the superior growth of shrimp in this group compared with lower dose of NSY (0.5 %) and control group. Interestingly, in this study, the inclusion level of NSY also played a significant role in enhancing the growth of shrimp. Despite no significant statistical differences, numerically, it was observed that FB, AIW, TGC, and WG improved with the use of 0.6% NSY compared to 0.3% NSY. The synergistic effect between various prebiotics and immunostimulant contain within the product could influence the physiological function of shrimp and eventually able to support the optimum growth of shrimp.
The addition of 0.2% SBP had the most influence on the growth of the shrimp. Studies on the use of natural compounds extracted from Yucca schidigera and Quillaja saponaria has been evaluated resulting in better growth performance of striped catfish, Pangasianodon hypophthalmus (Güroy et al., 2016), nile tilapia Oreochromis niloticus (Angeles et al., 2017) and shrimp, Litopenaeus vannamei (Hernández- Acosta et al., 2016). According to Acosta et al. (2019), saponins extracted from Yucca schidigera and Quillaja saponaria have proven to be very promising ingredients for aquafeed as natural growth promoters. Furthermore, studies using high levels of steroidal compounds from branches and leaves of Yucca schidigera have shown promising activity in the adsorption of harmful compounds such as ammonia (Fayed et al., 2019). Thus, the use of SBP as a feed additive during shrimp production could provide economic benefits in terms of productivity and effciency.
Other than growth performance, this study evaluated the protein retention rate and protein level in the whole body of the shrimp as the effect of NSY and SBP addition into the diet. Despite no significant differences in terms of statistical analysis, the findings showed higher numerical value of protein level and retention rate (%) in shrimp treated with NSY and SBP compared to the control treatment. A similar effect was also found in a study with striped catfish Pangasianodon hypophthalmus, where the dietary inclusion of Yucca schidigera and Quillaja saponaria at all levels investigated did not affect whole body proximate composition of the fish (Güroy et al., 2016). Rates and effciencies in nutrient deposition are governed by dietary factors, including nutrient balance and nutrient utilization, as well as biological factors such as genetics and the physiological state of the animal (NRC, 2011). Since all shrimps were maintained in similar culture environments and the diets were designed to be iso-nitrogenous in this study, this could explain the comparable protein levels in the whole body composition of the shrimp among the dietary treatments.
The potential application of saponins from Yucca schidigera and Quillaja saponaria to enhance the passive immunization response has been well described in Cheeke (2000). The strong immunoadjuvant activity of Q. saponaria and its derivatives makes them ideal substances for the development of vaccines (Rajput et al., 2007;Sjölander et al., 2001;Cheeke, 2000) In aquaculture, the presence of yucca in the diet positively affects the hematological and immunological response of European seabass, Dicentrarchus labrax (Fayed et al., 2019). Furthermore, the lysozyme activity of juvenile nile tilapia Oreochromis niloticus increases as the inclusion of dietary yucca increases up to 0.1 % in the diet formulation (Njagi et al., 2017). However, the same authors also reported that the use of dietary yucca higher than 0.1% did not show any further increase in lysozyme activity.
In this study, the haemocytes counts and lysozyme activity were higher in the group of shrimp that fed with commercial NSY and SBP. The elevation of total haemocytes counts and lysozyme activity in shrimp might be due to the immunomodulatory function of commercial NSY and SBP as well as the presence of saponins that are able to enhance the non-specific immune system of shrimp. Increased percentage the use of 0.2% SBP provided better results for lysozyme activity compared to the control group. Invertebrates lack the complexity of the adaptive immune system compared to vertebrates. This means that invertebrates, due to the absence of "true" lymphocytes and functional antibodies rely solely on the innate immune system that are largely based on haemocytes (Smith, 1991). There are three types of haemocytes in shrimp, namely: hyaline cells, semi-granular cells, and granular cells, mainly responsible for phagocytic activity and synthesis of melanin which is a powerful antimicrobial component for shrimp (Söderhäll & Cerenius, 1998;1992;Söderhäll & Smith, 1983).

CONCLUSION
Results in this study confirm the beneficial effects of all-natural silicates and yeast components and saponins obtained from Yucca schidigera and Quillaja saponaria as a functional property in shrimp feed formulation. Under the conditions of the present study, the inclusion of 0.3 and 0.6% commercial complex of all-natural silicates and yeast components and 0.2 % saponin blend extracted from Yucca schidigera and Quillaja saponaria could be used to enhance the growth and health status of Pacific white shrimp L. vannamei.