Evaluation of Viligen® NE as a feed additive for pacific white shrimp reared in a biofloc system during the nursery phase

Evaluation of Viligen® NE as a feed additive for pacific white shrimp reared in a biofloc system during the nursery phase | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Evaluation of Viligen® NE as a feed additive for pacific white shrimp reared in a biofloc system during the nursery phase Camilla Souza Miranda, Ivanilson de Lima Santos, Ramon Felipe Siqueira Carneiro, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6974226/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 12 You are reading this latest preprint version Abstract The aim of this study was to evaluate Viligen® NE (Alltech®, USA) as a feed additive for Penaeus vannamei reared in a biofloc system during the nursery phase, focusing on growth performance, intestinal microbiological analysis, and resistance to thermal shock. Shrimp post-larvae (41 mg) were fed for six weeks with four experimental diets: (a) 0.5 g Viligen® NE kg⁻¹; (b) 1 g Viligen® NE kg⁻¹; (c) 2 g Viligen® NE kg⁻¹; and (d) a control diet (0 g Viligen® NE kg⁻¹). The experiment was conducted in quadruplicate, totaling 16 experimental tanks, with a stocking density of 2000 post-larvae m⁻³. Shrimp growth performance parameters were positively influenced by the inclusion of Viligen® NE in the diet. Polynomial regression indicated an optimal inclusion level of 1.17 g kg⁻¹ for maximizing specific growth rate and weight gain. Additionally, supplementation at 1 g kg⁻¹ enhanced shrimp resistance to thermal shock. However, no significant differences were observed in intestinal counts of total heterotrophic bacteria or Vibrio spp. among treatments. In conclusion, dietary supplementation with Viligen® NE at an optimal level of 1.17 g kg⁻¹ improved growth performance, and 1 g kg⁻¹ enhanced thermal shock resistance of nursery-phase shrimp reared in a biofloc system. Penaeus vannamei growth performance thermal shock final weight Figures Figure 1 Figure 2 Figure 3 1. INTRODUCTION The biofloc system is a sustainable technology developed for shrimp farming, offering high productivity with low water exchange. In this system, excess nitrogen compounds such as ammonia and nitrite can be controlled through a microbial community and the formation of aggregates composed of microalgae, protozoa, bacteria, yeast, and organic and inorganic matter, known as bioflocs (Avnimelech 2014; de Schryver et al. 2008 ). Benefits of this system include the ability to rear shrimp in smaller areas away from coastal regions, using lower water volumes, with high biosecurity (Hargreaves 2006 ), and allowing high stocking densities and organic matter input maintained through intense aeration (Browdy et al. 2001 ). The nursery phase is an intermediate stage between larviculture and grow-out, designed to acclimate post-larvae to grow-out conditions. Several studies have demonstrated the importance of the biofloc system during early rearing phases, resulting in a more controlled environment that supports optimal shrimp growth and health, ultimately improving performance during grow-out (Krummenauer et al. 2010 ; Wasielesky et al. 2013 ; Lorenzo et al. 2016 ). Moreover, combining the biofloc system with a nursery phase can help maintain suitable water quality parameters, even without water exchange (Xu et al. 2012 ). Despite the many benefits of the biofloc system, certain challenges remain. Due to the high organic matter and stocking densities, proper system balance and dimensioning are essential to avoid issues such as pathogenic bacterial outbreaks. Vibrio species are common in marine and estuarine environments and are part of the shrimp’s natural microbiota (Lightner 1996 ; Liu et al. 2011 ). However, they can become harmful when shrimp experience stress or immunosuppression, often due to poor water quality, suboptimal nutrition, or temperature fluctuations. Traditionally, harmful bacteria have been controlled with antibiotics. However, excessive or improper use of antibiotics can lead to the emergence of resistant strains and result in residues in both water and shrimp tissue (Weston 1996 ). Therefore, various alternative and preventive approaches are under investigation to control pathogens and enhance shrimp health. Among these alternatives, feed additives such as sodium butyrate, hydrolyzed yeast, and zinc proteinate have shown promising results. Regarding commercial additives, Viligen® NE (Alltech®, USA) is a zootechnical acidifier used as an alternative to antibiotics to promote growth in aquaculture (Lückstädt 2008 ). Acidifiers may improve productivity, general health, immune function, and intestinal integrity, while also supporting a balanced gut microbiota. In feed, acidifiers inhibit microbial growth, enhance quality, and reduce nutrient loss during storage (Ng and Koh 2016 ). In the digestive tract, they improve digestion by increasing nutrient availability and suppress the growth of pathogenic bacteria (Ng and Koh 2016 ). Additionally, acidifiers provide a source of energy due to their high gross energy values (Lückstädt 2008 ). In Penaeus vannamei , the use of sodium butyrate has been shown to improve growth, palatability, feed intake, nutrient digestibility, and survival (Silva et al. 2013 , 2016 ). In another study with P. vannamei post-larvae, supplementation with the marine yeast Meyerozyma guilliermondii in a super-intensive biofloc system reduced solid accumulation and increased survival (Ferreira et al., 2023 ). Zinc proteinate has also been reported to enhance shrimp growth and immunity (Lin et al. 2013 ). Based on this evidence, individual feed additives can support the growth performance of P. vannamei shrimp reared in both clear-water and biofloc systems. Therefore, the present study aimed to evaluate the effects of Viligen® NE at three concentrations (0.5, 1, and 2 g kg⁻¹) and a control treatment (without Viligen® NE) on growth performance, intestinal microbiota, and thermal shock resistance in Pacific white shrimp reared in a biofloc system during the nursery phase. 2. METHODOLOGY The experiment was conducted at the Marine Shrimp Laboratory (LCM), and the experimental diets were produced at the Laboratory of Aquatic Species Nutrition (LABNUTRI), both located at the Federal University of Santa Catarina (UFSC), in Florianópolis, SC, Brazil. 2.1 ANIMALS Post-larval Penaeus vannamei with 20 days (PL20), obtained from the commercial hatchery Aquatec, were acclimated in a biofloc system at the Marine Shrimp Laboratory (LCM/UFSC) until reaching an average weight of approximately 41 mg. 2.2 PREPARATION OF EXPERIMENTAL DIETS Four experimental diets were formulated to provide Viligen® NE at 0.5, 1, or 2 g kg⁻¹, along with a non-supplemented control diet (0 g kg⁻¹). The formulations were developed using Optimal Formula 2000® software, based on the nutritional requirements for Penaeus vannamei (Gong et al. 2000 ; NRC 2011 ; Zhou et al. 2012 ). Viligen® NE was included by replacing kaolin in the formulation. Dry ingredients were pre-ground and sieved through a 600 µm mesh. Micro-ingredients were homogenized and then mixed with the macro-ingredients. Oils, soy lecithin, and water were subsequently added. The resulting mixture was pelletized using a microextruder (Inbramaq MX-40), dried in an oven at 40°C, and stored under refrigeration at 4°C until use. The ingredients and proximate composition, analyzed following AOAC ( 1999 ) guidelines, are presented in Table 1 . Table 1 Formulation of the Experimental Diet containing 0.5, 1, and 2 g kg − 1 of Viligen® NE and the Control Diet 0 g kg − 1 , without the product addition. Ingredients (g kg − 1 Dry Weight) Amount in g kg − 1 of Viligen® NE 0 0.5 1.0 2.0 Wheat meal a 100.00 100.00 100.00 100.00 Soybean meal b 350.00 350.00 350.00 350.00 Fish byproduct meal c 200.00 200.00 200.00 200.00 Poultry viscera meal d 80.00 80.00 80.00 80.00 Carboxymethyl cellulose 5.00 5.00 5.00 5.00 Soy lecithin 20.00 20.00 20.00 20.00 Monocalcium phosphate 25.00 25.00 25.00 25.00 Soybean oil e 5.00 5.00 5.00 5.00 Vitamin C 0.70 0.70 0.70 0.70 Vitamin premix f 4.00 4.00 4.00 4.00 Mineral premix g 17.00 17.00 17.00 17.00 Magnesium sulfate 15.00 15.00 15.00 15.00 Kaolin 80.00 75.00 70.00 60.00 Sodium Chloride 10.00 10.00 10.00 10.00 Salmon byproduct meal 51.30 51.30 51.30 51.30 Potassium Chloride 10.00 10.00 10.00 10.00 Methionine 2.00 2.00 2.00 2.00 Fish oil h 25.00 25.00 25.00 25.00 Viligen® NE 0.00 0.50 1.00 2.00 Proximate Composition (g 100 g − 1 dry matter) Dry matter 96.08 93.60 90.65 94.35 Crude protein 38.65 37.86 36.77 38.70 Crude Lipid 9.83 7.10 8.39 7.49 Minerals 23.83 24.46 22.03 22.82 Gross Energy (kcal/g) 4173.0 3994.05 3878.92 3934.40 * All results are expressed on a natural product basis. ** Physical-chemical characterization analyses were conducted according to AOAC ( 1999 ) methodologies: Dry Matter by method 950.01; Mineral Matter by method 942.05; Protein by LECO Dumas method 990.03, conversion factor 6.25; Ether Extract by Soxhlet by method 920.39C. Gross energy was determined in a calorimetric bomb. a Rosa Branca, Type 1: 13 mg kg − 1 crude protein and 3440 kcal kg − 1 crude energy. b Soybean meal distributed by BRF Ingredients (Itajaí, Santa Catarina, Brasil): 56.61 mg kg − 1 crude protein and 4500 kcal kg − 1 crude energy. c Fish meal distributed by Agroforte Ind (Biguaçu, Santa Catarina, Brasil): 59.35 mg kg − 1 crude protein and 4,056.13 kcal kg − 1 crude energy. d Poultry viscera meal distributed by BRF Ingredients (Itajaí, Santa Catarina, Brasil): 62 mg kg − 1 crude protein and 4661 kcal kg − 1 crude energy. e Soybean oil: Soya: 7,909 kcal kg − 1 crude energy. f Composition of Vitamin: vit. A − 900 mg kg − 1 ; vit. D3–25 mg kg − 1 ; vit. E – 46.900 mg kg − 1 ; vit. K3–1.400 mg kg − 1 ; cobalamina (B12) – 50 mg kg − 1 ; piridoxina (B6) – 33.000 mg kg − 1 ; riboflavina – 20.000 mg kg − 1 ; ácido nicotinico – 70.000 mg kg − 1 ; ácido pantotênico – 40.000 mg kg − 1 ; biotina – 750 mg kg − 1 ; ácido fólico – 3.000 mg kg − 1 ; g Mineral Premix: cobre – 2.330 mg kg − 1 ; zinco – 10.000 mg kg − 1 ; manganês – 6.500 mg kg − 1 ; selênio − 125 mg kg − 1 ; iodo – 1.000 mg kg − 1 ; cobalto – 50 mg kg − 1 ; magnésio – 20 g kg − 1 ; potassio – 6,1 g kg − 1 . h Fish oil distributed by BFP Bio Food Products (Itajaí, Santa Catarina, Brasil): 8445 kcal kg − 1 crude energy. 2.3 EXPERIMENTAL DESIGN The experimental diets were administered over a period of six weeks to a total of 384 shrimp, distributed into four treatment groups (0, 0.5, 1, and 2 g Viligen® NE kg⁻¹), each with four 48 L replicate tanks. Each tank was stocked with 96 shrimp, corresponding to a stocking density of 2000 post-larvae m⁻³. Each tank was equipped with a water heating system (temperature maintained at 28.1 ± 0.9°C) and aeration provided via perforated tubing (dissolved oxygen > 5 mg L⁻¹). Water was sourced from an inoculum consisting of 30% mature biofloc water from a shrimp-rearing tank with established nitrification, presenting the following parameters: ammonia 0.15 ± 0.0 mg mL⁻¹; nitrite 0.08 ± 0.0 mg mL⁻¹; alkalinity 170 ± 0.0 mg CaCO₃ L⁻¹; total suspended solids (TSS) 484 ± 0.0 mg L⁻¹. Dissolved oxygen and temperature were monitored daily, while concentrations of total ammonia, nitrite, salinity, pH, and TSS were measured weekly. During the six-week rearing period, shrimp were fed six times daily (8:00, 9:30, 11:30, 13:30, 15:00, and 17:00) following the feeding table proposed by Van Wyk and Scarpa (1999). Weekly, 20 shrimp per tank were individually weighed to monitor growth and adjust feed quantities accordingly. At the end of the trial, growth performance was assessed based on survival, final weight, specific growth rate, yield, and feed conversion ratio. 2.4 INTESTINAL MICROBIOLOGY ANALYSES On the day of sampling, shrimp were not fed. At the end of the six-week feeding period, intestines from 10 shrimp per tank were collected, totaling 40 shrimp per treatment. The intestinal tracts were homogenized and serially diluted (1:10) in sterile 3% saline solution, then plated on Marine Agar and TCBS agar to enumerate total heterotrophic bacteria and Vibrio spp., respectively. No prior filtration of the intestines was performed before dilution. The plated samples were incubated at 30°C for 24 hours, after which total colony-forming units (CFU g⁻¹ of intestine) were counted. 2.5 THERMAL SHOCK RESISTANCE To assess thermal shock resistance, at the end of the six-week period, 15 shrimp from each experimental unit (60 shrimp per treatment) were transferred from the rearing tanks (water at 28.1 ± 0.9°C) to 60 L aquaria containing saltwater at 13.5 ± 0.1°C and maintained for one hour. This procedure followed a previously established 50% lethal temperature protocol (Pontinha, Vieira, and Hayashi 2018 ). During the exposure period, water temperature in the aquaria was continuously monitored. After one hour, the shrimp were simultaneously transferred to containers with water at 28°C. Survival was assessed at 12, 24, 36, and 48 hours post-transfer. Shrimp were not fed during the thermal shock test or the recovery period. 2.6 STATISTICAL ANALYSIS Water quality parameters and microbiological data were subjected to Shapiro–Wilk and Levene’s tests to verify normality and homoscedasticity, respectively. Once assumptions were met, one-way ANOVA was applied, followed by Tukey’s post hoc test when appropriate. Growth performance data were also analyzed using second-degree polynomial regression, with the optimal inclusion level estimated from the vertex of the quadratic regression equations. Thermal shock resistance data were analyzed using two-way ANOVA (treatment × time). A significance level of 5% (p < 0.05) was adopted for all statistical tests. 3. RESULTS 3.1 WATER QUALITY PARAMETERS Ammonia and nitrite levels differed significantly among treatments; however, these variations did not influence shrimp growth performance, as the values remained within acceptable limits for Penaeus vannamei reared in biofloc systems. Temperature and dissolved oxygen were maintained at 28.12 ± 0.97°C and 6.27 ± 0.26 mg L⁻¹, respectively, and remained stable throughout the experimental period. No significant differences were observed among treatments for alkalinity, pH, total suspended solids, or nitrate concentrations (Table 2 ). Table 2 Water quality parameters in Penaeus vannamei nursery tanks in a biofloc system, fed for six weeks with diets containing 0.5, 1, and 2 g kg − 1 of Viligen® NE and a control (0 g kg − 1 ), without the product addition. Treatments Ammonia (mg L − 1 ) Nitrite N-NO 2 (mg L − 1 ) Nitrate N-NO 3 (mg L − 1 ) Alkalinity (mg CaCO 3 L − 1 ) pH TSS (mg L − 1 ) Control 0.68 ± 0.88a 0.09 ± 0.03b 70.43 ± 24.89 163.67 ± 19.31 7.97 ± 0.10 613.26 ± 255.54 0.5g kg − 1 0.66 ± 0.95ª 0.12 ± 0.08b 64.25 ± 47.52 168.10 ± 19.05 7.96 ± 0.09 601.60 ± 266.09 1g kg − 1 0.29 ± 0.17b 0.14 ± 0.07b 72.44 ± 54.25 165.25 ± 14.83 7.95 ± 0.10 584.90 ± 264.54 2g kg − 1 0.30 ± 0.19b 0.20 ± 0.18a 79.42 ± 67.24 164.83 ± 16.11 7.95 ± 0.10 567.08 ± 285.75 p-value** 0.0001 0.0001 0.961 0.642 0.573 0.812 *Data presented as means ± standard deviation. ** One-way ANOVA. TSS – Total Suspended Solids. 3.2 GROWTH PERFORMANCE After the six-week period, the inclusion of Viligen® NE resulted in a quadratic effect on final weight, and specific growth rate (Table 3 ). Through polynomial regression, the optimal inclusion level was estimated at 1.17g kg − 1 of Viligen® NE for the highest response in terms of specific growth rate (Fig. 1 A) and 1.174 g kg − 1 of Viligen® NE for weight gain (Fig. 1 B). Table 3 Growth performance in the nursery of Penaeus vannamei in a biofloc system, fed for six weeks with diets containing 0.5, 1, and 2 g kg-1 of Viligen® NE and a control (0 g kg-1), without the product addition. Variables Viligen® NE supplementation g kg − 1 Values for Regression* 0 0.5 1 2 R² p Final Weight (g) 0.87 ± 0.17 1.02 ± 0.12 1.11 ± 0.06 0.99 ± 0.13 0.995 0.045 Weight gain (g) 0.83 ± 0.16 0.98 ± 0.12 1.07 ± 0.06 0.95 ± 0.13 0.995 0.007 Yield (kg m³) 1.75 ± 0.28 1.99 ± 0.21 2.08 ± 0.10 1.81 ± 0.32 0.9997 0.001 Specific Growth Rate (%) 1.98 ± 0.39 2.34 ± 0.29 2.55 ± 0.15 2.26 ± 0.31 0.995 0.045 Feed Conversion Ratio 1.58 ± 0.15 1.48 ± 0.16 1.55 ± 0.12 1.59 ± 0.19 - NS*** Survival (%) 97 ± 0.03 95 ± 0.03 95 ± 0.03 94 ± 0.05 - NS 3.3 INTESTINAL MICROBIOLOGY ANALYSES No significant differences were observed among the experimental groups in the total counts of heterotrophic bacteria (p = 0.182) or Vibrio spp. (p = 0.549) in shrimp intestines after six weeks of feeding with either non-supplemented or Viligen® NE-supplemented diets at different inclusion levels (Figs. 2 A and 2 B). 3.4 THERMAL SHOCK RESISTANCE A significant difference (p < 0.001) was observed in Penaeus vannamei mortality during the 48-hour period following thermal shock exposure among the experimental groups. Shrimp fed diets containing 1 g kg⁻¹ of Viligen® NE exhibited significantly lower mortality compared to those in the 0 g kg⁻¹ (control), 0.5 g kg⁻¹, and 2 g kg⁻¹ treatment groups (Fig. 3 ). 4. DISCUSSION This study represents the first evaluation of Viligen® NE as a dietary additive for Pacific white shrimp. Several benefits were observed, including improvements in final weight, weight gain, and specific growth rate following dietary supplementation with Viligen® NE. The optimal inclusion level, based on weight gain and specific growth rate, was estimated at 1.17 g kg⁻¹. While the effects observed in this study are attributed to Viligen® NE as a compound product, the existing literature on similar feed additives provides useful comparisons. In agreement with the present results, Lin et al. ( 2013 ) reported that among three organic zinc sources (zinc-lysine, zinc-glycine, and zinc-methionine), zinc-methionine supplementation led to the highest weight gain and improved survival following a Vibrio harveyi challenge in P. vannamei . Regarding yeast-based additives, Jin et al. ( 2018 ) observed improved weight gain, specific growth rate, and feed conversion ratio in juvenile P. vannamei fed diets supplemented with 1% hydrolyzed yeast compared to brewer’s yeast and a control group. Therefore, in the present study, the inclusion of Viligen® NE, which contains components similar to those tested individually in other studies, may have produced synergistic effects, contributing to enhanced growth performance. Similarly, Silva et al. ( 2016 ) observed significant improvements in growth and feed efficiency in P. vannamei when diets were supplemented with 20 g kg⁻¹ of sodium butyrate. However, optimal dietary levels for a given additive may vary depending on species, developmental stage, production system, and duration of supplementation. For instance, Pessini et al. ( 2021 ) tested Viligen™ (0.60, 1.20, 2.40, and 4.80 g kg⁻¹) in Nile tilapia diets over 60 days and estimated an optimal inclusion level of 2.80 g kg⁻¹ for maximum daily weight gain. It is important to note that the Viligen™ used in that study is related to but distinct from the product evaluated here. Although previous studies with dietary additives have reported positive modulation of shrimp intestinal microbiota, no significant differences were found in this study in the total counts of heterotrophic bacteria or Vibrio spp. among treatments. This could be due to the relatively low inclusion levels used here, as studies showing microbial modulation typically employed higher doses. For example, Silva et al. ( 2016 ) demonstrated that sodium butyrate at 5, 10, and 20 g kg⁻¹ significantly reduced Vibrio spp. abundance and increased serum agglutination activity in P. vannamei . Thus, further research is needed to determine the optimal inclusion level of Viligen® NE to influence the intestinal microbiota of Pacific white shrimp effectively. Temperature plays a key role in the metabolism of crustaceans, influencing both growth and survival in penaeid shrimp. Cold stress can lead to immunosuppression, increasing susceptibility to pathogens (Gomez-Jimenez et al. 2000 ; Kautsky et al. 2000 ; Wang and Chen 2006 ). Certain organic acids are known to enhance membrane fluidity in intestinal cells, which can improve survival under cold stress due to the importance of homeoviscous membrane adaptation as a cold-resistance mechanism (Hayward, Manso, and Cossins 2014 ). Insects, crustaceans, microorganisms, and cold-adapted plants typically exhibit greater membrane fluidity through increased unsaturation, a shared trait among organisms resistant to temperature fluctuations (Corcoles-Sáez et al. 2016; Takahashi et al. 2016 ). In this study, cumulative mortality after thermal shock differed significantly among treatments. Shrimp fed 1 g kg⁻¹ of Viligen® NE exhibited the lowest mortality after 48 hours compared to other treatments and the control. This enhanced resistance may be linked to improved membrane fluidity in shrimp hemocytes, allowing better thermal adaptation. For ectothermic animals like shrimp, cold stress can induce physiological dysfunctions with serious implications for production (Hayward et al. 2014 ). Therefore, dietary supplementation with Viligen® NE appears to be a promising strategy for improving resilience in environments with sudden temperature fluctuations. Further studies are needed to elucidate the underlying mechanisms that enhance shrimp resistance to thermal stress and to determine the optimal supplementation levels to promote benefits in growth performance, intestinal microbiota, and survival under challenging environmental conditions. 5. CONCLUSION Benefits in growth performance were observed for Penaeus vannamei reared in a biofloc system during the nursery phase, with an optimal inclusion level of 1.17 g kg⁻¹ of Viligen® NE for improved weight gain and specific growth rate. Additionally, significant differences were found among treatments in shrimp mortality following thermal shock, with the 1 g kg⁻¹ supplementation level enhancing thermal resistance. However, no significant differences were observed in the total counts of heterotrophic bacteria or Vibrio spp. in shrimp intestines. Therefore, it can be concluded that dietary supplementation with 1 g kg⁻¹ of Viligen® NE is the most effective among the tested doses for P. vannamei during the nursery phase in a biofloc system. Declarations Funding: This project has received funding from Alltech, CNPq (403469/2023-6) and the Coordination for the Improvement of Higher Level Personnel (CAPES; finance code: 001). F.B.V and W.Q.S are research fellows from CNPq. Conflict of Interest: The authors declare no conflict of interest. Ethical approval: Not applicable (work with invertebrate - Law No. 11,794, of October 8, 2008 ( https://www.planalto.gov.br/ccivil_03/_ato2007-2010/2008/lei/l11794.htm ) . Conceptualization, A.K., C.S.M., D.M.F., F.B.V., P.L.; Methodology, C.S.M., F.B.V., I.C.P., I.L.S., N.C.B., R.F.S., S.A.P.D.; Validation, C.S.M., I.L.S., N.C.B., R.F.S.; Formal analysis, C.S.M., I.C.P., S.A.P.D.; Investigation, C.S.M., F.B.V., I.C.P., I.L.S., N.C.B., R.F.S.; Resources, A.K., P.L.; Writing—original draft preparation, C.S.M.; Writing—review and editing, A.K., C.S.M., D.M.F., F.B.V., P.L.; Supervision, F.B.V., S.A.P.D.; Project administration, F.B.V.; Funding acquisition, A.K., C.S.M., D.M.F., P.L. All authors have read and agreed to the published version of the manuscript. Author Contribution Conceptualization, A.K., C.S.M., D.M.F., F.B.V., P.L.; Methodology, C.S.M., F.B.V., I.C.P., I.L.S., N.C.B., R.F.S., S.A.P.D.; Validation, C.S.M., I.L.S., N.C.B., R.F.S.; Formal analysis, C.S.M., I.C.P., S.A.P.D.; Investigation, C.S.M., F.B.V., I.C.P., I.L.S., N.C.B., R.F.S.;Resources, A.K., P.L.; Writing—original draft preparation, C.S.M.; Writing—review and editing, A.K., C.S.M., D.M.F., F.B.V., P.L.; Supervision, F.B.V., S.A.P.D.; Project administration, F.B.V.; Funding acquisition, A.K., C.S.M., D.M.F., P.L. All authors have read and agreed to the published version of the manuscript. Acknowledgement We thank the companies Alltech Inc. (Nicholasville, KY, USA) for their financial support. Data Availability Data supporting the findings of this study are available from the corresponding author upon request. References AOAC (1999) Official Methods of Analysis of AOAC International. AOAC International, Gaithersburg, MD Avnimelech Y (2015) Biofloc Technology: A Practical Guidebook (3rd ed.). Baton Rouge, LA: The World Aquaculture Society. ISBN 978-1880443036 Browdy CL, Bratvold D, Stokes AD, McIntosh RP (2001) Perspectives on the application of closed shrimp culture systems. In C. L. Browdy & D. E. Jory (Eds.), New Wave: Proceedings of the Special Session on Sustainable Shrimp Farming (pp. 20–34). Baton Rouge, LA: World Aquaculture Society Córcoles-Sáez I, Hernández ML, Martínez-Rivas JM, Prieto JA, Randez-Gil F (2016) Characterization of the S. cerevisiae inp51 mutant links phosphatidylinositol 4,5-bisphosphate levels with lipid content, membrane fluidity and cold growth. Biochim et Biophys Acta (BBA) - Mol Cell Biology Lipids 1861:213–226. https://doi.org/10.1016/j.bbalip.2015.12.014 De Schryver P, Crab R, Defoirdt T, Boon N, Verstraete W (2008) The basics of bio-flocs technology: The added value for aquaculture. Aquaculture 277:125–137. https://doi.org/10.1016/j.aquaculture.2008.02.019 Ferreira NL, Vieira FN, Mouriño JLP, Seiffert WQ, Fracalossi DM (2023) Effect of microbial inoculants and fertilization strategies in super-intensive biofloc shrimp culture system. Aquac Res 54(6):2394–2406. https://doi.org/10.1111/are.15746 Gomez-Jimenez S, Uglow RF, Gollas-Galvan T (2000) The effects of cooling and emersion on total haemocyte count and phenoloxidase activity of the spiny lobster Panulirus interruptus . Fish Shellfish Immunol 10:631–635. https://doi.org/10.1006/fsim.2000.0277 Gong H, Lawrence AL, Jiang DH, Castille FL, Gatlin III, D. M (2000) Lipid nutrition of juvenile Litopenaeus vannamei I. Dietary cholesterol and de-oiled soy lecithin requirements and their interaction. Aquaculture 190(3–4):305–324. https://doi.org/10.1016/S0044-8486(00)00414-2 Hargreaves JA (2006) Photosynthetic suspended-growth systems in aquaculture. Aquacult Eng 34(3):344–363. https://doi.org/10.1016/j.aquaeng.2005.08.009 Hayward SAL, Manso B, Cossins AR (2014) Molecular basis of chill resistance adaptations in poikilothermic animals. J Exp Biol 217:6–15. https://doi.org/10.1242/jeb.096537 Jin M, Xiong J, Zhou QC, Yuan Y, Wang XX, Sun P (2018) Dietary yeast hydrolysate and brewer’s yeast supplementation could enhance growth performance, innate immunity capacity and ammonia nitrogen stress resistance ability of Pacific white shrimp ( Litopenaeus vannamei ). Fish Shellfish Immunol 82:121–129. https://doi.org/10.1016/j.fsi.2018.08.020 Kautsky N, Rönnbäck P, Tedengren M, Troell M (2000) Ecosystem perspectives on management of disease in shrimp pond farming. Aquaculture 191(1–3):145–161. https://doi.org/10.1016/S0044-8486(00)00424-5 Krummenauer D, Cavalli R, Ballester E, Wasielesky W (2010) Feasibility of Pacific white shrimp Litopenaeus vannamei culture in southern Brazil: Effects of stocking density and a single or a double crop management strategy in earthen ponds. Aquac Res 41:240–248. https://doi.org/10.1111/j.1365-2109.2009.02326.x Lightner DV (1996) A handbook of shrimp pathology and diagnostic procedures for diseases of cultured penaeid shrimp. World Aquaculture Society, Baton Rouge, LA Lin S, Mai K, Tan B, Liu Y, Zhang W (2013) Comparison of chelated zinc and zinc sulfate as zinc sources for growth and immune response of shrimp ( Litopenaeus vannamei ). Aquaculture , 406–407, 79–84. https://doi.org/10.1016/j.aquaculture.2013.04.026 Liu H, Wang X, Wang G, Gao Q, Yan H, Li L (2011) The intestinal microbial diversity of Chinese shrimp ( Fenneropenaeus chinensis ) as determined by PCR-DGGE and clone library analyses. Aquaculture 317(1–4):32–36. https://doi.org/10.1016/j.aquaculture.2011.04.008 Lorenzo MA, Schveitzer R, Nuñer APO, Vinatea L (2016) Intensive hatchery performance of Pacific white shrimp in the biofloc system under three different fertilization levels. Aquacult Eng 72:40–44. https://doi.org/10.1016/j.aquaeng.2016.04.001 Lückstädt C (2008) The use of acidifiers in fish nutrition. CAB Reviews: Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources , 3(044), 1–8. https://doi.org/10.1079/pavsnnr20083044 NRC (2011) Nutrient requirements of fish and shrimp. National Academies, Washington, DC Ng W, Koh C (2016) The utilization and mode of action of organic acids in the feeds of cultured aquatic animals. Reviews Aquaculture 9(4):342–368. https://doi.org/10.1111/raq.12141 Pessini J, Daitx de Oliveira V, Souza de Sá L, Mourio P, Pettigrew JL, J., Fracalossi DM (2021) Dietary supplementation of Viligen® to Nile tilapia improves growth and gut morphology. Aquacult Nutr 27:1065–1076. https://doi.org/10.1111/anu.13247 Pontinha VdeA, Vieira F do N., Hayashi L (2018) Mortality of Pacific white shrimp submitted to hypothermic and hyposalinic stress. Boletim do Instituto de Pesca , 44(2), [páginas] Silva BC, Vieira FN, Mouriño JLP, Ferreira GS, Seiffert WQ (2013) Salts of organic acids selection by multiple characteristics for marine shrimp nutrition. Aquaculture 384:104–110. https://doi.org/10.1016/j.aquaculture.2012.12.017 Silva BC, Vieira FN, Mouriño JLP, Ramírez N, Seiffert WQ (2016) Butyrate and propionate improve the growth performance of Litopenaeus vannamei . Aquac Res 47:612–623. https://doi.org/10.1111/are.12520 Takahashi D, Imai H, Kawamura Y, Uemura M (2016) Lipid profiles of detergent resistant fractions of the plasma membrane in oat and rye in association with cold acclimation and freezing tolerance. Cryobiology 72:123–134. https://doi.org/10.1016/j.cryobiol.2016.02.003 Van Wyk P (1999) Nutrition and feeding of Litopenaeus vannamei in intensive culture systems. In: Van Wyk P et al (eds) Farming marine shrimp in recirculating freshwater systems. Harbor Branch Oceanographic Institution, Tallahassee, pp 125–140 Wang FI, Chen JC (2006) The immune response of tiger shrimp Penaeus monodon and its susceptibility to Photobacterium damselae subsp. damselae under temperature stress. Aquaculture , 258, 34–41. https://doi.org/10.1016/j.aquaculture.2006.03.043 Wasielesky W, Atwood H, Stokes A, Browdy C (2013) Nursery of Litopenaeus vannamei reared in a biofloc system: The effect of stocking densities and compensatory growth. J Shellfish Res 32(2):799–806. https://doi.org/10.2983/035.032.0323 Weston DP (1996) Environmental considerations on the use of antibacterial drugs in aquaculture. In: Baird DJ, Beveridge MCM, Kelly LA, Muir JF (eds) Aquaculture and water resource management. Institute of Aquaculture, Blackwell Science, pp 140–165 Xu WJ, Pan LQ, Zhao DH, Huang J (2012) Preliminary investigation into the contribution of bioflocs on protein nutrition of Litopenaeus vannamei fed with different dietary protein levels in zero-water exchange culture tanks. Aquaculture 350–353. https://doi.org/10.1016/j.aquaculture.2012.04.003 Zhou QC, Tan BP, Mai KS, Liu YJ, Ceng C (2012) Dietary arginine requirement of juvenile Pacific white shrimp, Litopenaeus vannamei . Aquaculture , 364–365, 252–258. https://doi.org/10.1016/j.aquaculture.2012.08.020 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 03 Sep, 2025 Reviews received at journal 01 Sep, 2025 Reviews received at journal 29 Aug, 2025 Reviewers agreed at journal 29 Aug, 2025 Reviews received at journal 12 Aug, 2025 Reviewers agreed at journal 02 Aug, 2025 Reviewers agreed at journal 31 Jul, 2025 Reviewers agreed at journal 13 Jul, 2025 Reviewers invited by journal 11 Jul, 2025 Editor assigned by journal 29 Jun, 2025 Submission checks completed at journal 27 Jun, 2025 First submitted to journal 25 Jun, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6974226","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":484692558,"identity":"71096a92-dc5f-4575-bbf0-245f6f42e86a","order_by":0,"name":"Camilla Souza Miranda","email":"","orcid":"","institution":"Universidade Federal de Santa Catarina, Barra da Lagoa","correspondingAuthor":false,"prefix":"","firstName":"Camilla","middleName":"Souza","lastName":"Miranda","suffix":""},{"id":484692559,"identity":"c56291ba-598b-471f-95c2-08e7735e3a0c","order_by":1,"name":"Ivanilson de Lima Santos","email":"","orcid":"","institution":"Universidade Federal de Santa Catarina, Barra da Lagoa","correspondingAuthor":false,"prefix":"","firstName":"Ivanilson","middleName":"de Lima","lastName":"Santos","suffix":""},{"id":484692560,"identity":"dd7e2643-f006-4bb9-b325-226d3e34e91c","order_by":2,"name":"Ramon Felipe Siqueira Carneiro","email":"","orcid":"","institution":"Universidade Federal de Santa Catarina, Barra da Lagoa","correspondingAuthor":false,"prefix":"","firstName":"Ramon","middleName":"Felipe Siqueira","lastName":"Carneiro","suffix":""},{"id":484692561,"identity":"f3c0e3f6-c8dc-4d86-ba6b-bac4402b68f5","order_by":3,"name":"Isabela Claudiana Pinheiro","email":"","orcid":"","institution":"Alfred Wegener Institute Helmoholtz Centre for Polar and Marine Research","correspondingAuthor":false,"prefix":"","firstName":"Isabela","middleName":"Claudiana","lastName":"Pinheiro","suffix":""},{"id":484692562,"identity":"a98cb770-2e25-4508-bec3-921be05f8c25","order_by":4,"name":"Norha Constanza Bolívar","email":"","orcid":"","institution":"Universidade Federal de Santa Catarina, Barra da Lagoa","correspondingAuthor":false,"prefix":"","firstName":"Norha","middleName":"Constanza","lastName":"Bolívar","suffix":""},{"id":484692563,"identity":"4a4a1e39-cf9f-4ae3-992b-8e42f11d8992","order_by":5,"name":"Débora Machado Fracalossi","email":"","orcid":"","institution":"Universidade Federal de Santa Catarina","correspondingAuthor":false,"prefix":"","firstName":"Débora","middleName":"Machado","lastName":"Fracalossi","suffix":""},{"id":484692568,"identity":"1cfac069-c329-431b-83a9-e64806a7066e","order_by":6,"name":"Philip Lyons","email":"","orcid":"","institution":"Alltech Biotechnology Inc","correspondingAuthor":false,"prefix":"","firstName":"Philip","middleName":"","lastName":"Lyons","suffix":""},{"id":484692573,"identity":"f2cbc099-5221-4a5b-a60a-fe5de7def008","order_by":7,"name":"Anne Koontz","email":"","orcid":"","institution":"Alltech Inc","correspondingAuthor":false,"prefix":"","firstName":"Anne","middleName":"","lastName":"Koontz","suffix":""},{"id":484692574,"identity":"36a690a7-f119-452f-a8d1-ca605fb3ad83","order_by":8,"name":"Scheila Anelise Pereira Dutra","email":"","orcid":"","institution":"Universidade Federal de Santa Catarina, Barra da Lagoa","correspondingAuthor":false,"prefix":"","firstName":"Scheila","middleName":"Anelise Pereira","lastName":"Dutra","suffix":""},{"id":484692576,"identity":"559c3fcb-ae53-4961-a2c2-e0940b057892","order_by":9,"name":"Felipe Boéchat Vieira","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA60lEQVRIie3RsWrDMBCA4RMGaZHJeoZgv0JCxxbyKtGUNaMHUy4I7CmdXQh9jMw2B87iB8hqsmYIdOrQUqVLvVTJ2EH/dBo+OEkAodA/TFFEzXWQIAggh8nP7Es3Ykx6SOge8nsS5T1EbTa8zsG8KFtePt4Ys6qRp9xHdEtc92BK3drX7Z5x1i/VvPeQBRriuIQHicZCvOfnGYJMyEN0Njjy5Ug2WPG5c4vVtwgKRwhSicJGMTHC8RbRbrG6w1RqY6Npt3J3MdXcSxTz+7p40ll1OIlz8ehejLvBR65FADg+j3/qbxIKhUIhT99J3EhniPJ/egAAAABJRU5ErkJggg==","orcid":"","institution":"Universidade Federal de Santa Catarina, Barra da Lagoa","correspondingAuthor":true,"prefix":"","firstName":"Felipe","middleName":"Boéchat","lastName":"Vieira","suffix":""}],"badges":[],"createdAt":"2025-06-25 11:38:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6974226/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6974226/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":86776562,"identity":"fad018bd-5f46-4012-adc4-751029edda50","added_by":"auto","created_at":"2025-07-15 12:46:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":54553,"visible":true,"origin":"","legend":"\u003cp\u003ePolynomial regression of growth performance of \u003cem\u003ePenaeus vannamei\u003c/em\u003e shrimp raised in a biofloc system, fed for six weeks with diets containing 0.5, 1, and 2 g kg\u003csup\u003e-1\u003c/sup\u003e of Viligen\u003csup\u003e®\u003c/sup\u003e NE and a control (0 g kg\u003csup\u003e-1\u003c/sup\u003e), without the product addition. A) Weight gain (g) and B) Specific growth rate (SGR%).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6974226/v1/7f570090eb1208f4475e717b.png"},{"id":86776564,"identity":"7ca337ef-cf77-40f2-842d-b1874f95f9b8","added_by":"auto","created_at":"2025-07-15 12:46:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":24482,"visible":true,"origin":"","legend":"\u003cp\u003eTotal count of A) Heterotrophic bacteria and B) Count of \u003cem\u003eVibrio\u003c/em\u003e spp. in the intestine of \u003cem\u003ePenaeus vannamei\u003c/em\u003e fed for six weeks with diets containing 0.5, 1, and 2 g kg\u003csup\u003e-1\u003c/sup\u003e of Viligen\u003csup\u003e®\u003c/sup\u003e NE and a control (0 g kg\u003csup\u003e-1\u003c/sup\u003e), without the product addition. Data represent colony-forming units (CFU mL\u003csup\u003e-1\u003c/sup\u003e g\u003csup\u003e-1\u003c/sup\u003e in Log 10).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6974226/v1/c22460f3a7967081e3635049.png"},{"id":86776566,"identity":"5a91102b-dcbb-4426-ac83-65fc6451aab1","added_by":"auto","created_at":"2025-07-15 12:46:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":115538,"visible":true,"origin":"","legend":"\u003cp\u003eCumulative mortality of \u003cem\u003ePenaeus vannamei\u003c/em\u003e post-larvae fed for six weeks with diets containing 0.5, 1, and 2 g kg\u003csup\u003e-1\u003c/sup\u003e of Viligen\u003csup\u003e®\u003c/sup\u003e NE and a control (0 g kg\u003csup\u003e-1\u003c/sup\u003e), without the product addition for 48 hours after being subjected to thermal shock challenge.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6974226/v1/754ea9f1f91b54ffe3423868.png"},{"id":86780276,"identity":"6fc4fd19-bd75-4be7-9517-240a893fbb1d","added_by":"auto","created_at":"2025-07-15 13:18:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":994136,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6974226/v1/ec7e75ec-c114-4561-9141-333083ab9a61.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Evaluation of Viligen® NE as a feed additive for pacific white shrimp reared in a biofloc system during the nursery phase","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eThe biofloc system is a sustainable technology developed for shrimp farming, offering high productivity with low water exchange. In this system, excess nitrogen compounds such as ammonia and nitrite can be controlled through a microbial community and the formation of aggregates composed of microalgae, protozoa, bacteria, yeast, and organic and inorganic matter, known as bioflocs (Avnimelech 2014; de Schryver et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Benefits of this system include the ability to rear shrimp in smaller areas away from coastal regions, using lower water volumes, with high biosecurity (Hargreaves \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), and allowing high stocking densities and organic matter input maintained through intense aeration (Browdy et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2001\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe nursery phase is an intermediate stage between larviculture and grow-out, designed to acclimate post-larvae to grow-out conditions. Several studies have demonstrated the importance of the biofloc system during early rearing phases, resulting in a more controlled environment that supports optimal shrimp growth and health, ultimately improving performance during grow-out (Krummenauer et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Wasielesky et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Lorenzo et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Moreover, combining the biofloc system with a nursery phase can help maintain suitable water quality parameters, even without water exchange (Xu et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eDespite the many benefits of the biofloc system, certain challenges remain. Due to the high organic matter and stocking densities, proper system balance and dimensioning are essential to avoid issues such as pathogenic bacterial outbreaks. \u003cem\u003eVibrio\u003c/em\u003e species are common in marine and estuarine environments and are part of the shrimp\u0026rsquo;s natural microbiota (Lightner \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). However, they can become harmful when shrimp experience stress or immunosuppression, often due to poor water quality, suboptimal nutrition, or temperature fluctuations.\u003c/p\u003e\u003cp\u003eTraditionally, harmful bacteria have been controlled with antibiotics. However, excessive or improper use of antibiotics can lead to the emergence of resistant strains and result in residues in both water and shrimp tissue (Weston \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). Therefore, various alternative and preventive approaches are under investigation to control pathogens and enhance shrimp health. Among these alternatives, feed additives such as sodium butyrate, hydrolyzed yeast, and zinc proteinate have shown promising results.\u003c/p\u003e\u003cp\u003eRegarding commercial additives, Viligen\u0026reg; NE (Alltech\u0026reg;, USA) is a zootechnical acidifier used as an alternative to antibiotics to promote growth in aquaculture (L\u0026uuml;ckst\u0026auml;dt \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Acidifiers may improve productivity, general health, immune function, and intestinal integrity, while also supporting a balanced gut microbiota. In feed, acidifiers inhibit microbial growth, enhance quality, and reduce nutrient loss during storage (Ng and Koh \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In the digestive tract, they improve digestion by increasing nutrient availability and suppress the growth of pathogenic bacteria (Ng and Koh \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Additionally, acidifiers provide a source of energy due to their high gross energy values (L\u0026uuml;ckst\u0026auml;dt \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn \u003cem\u003ePenaeus vannamei\u003c/em\u003e, the use of sodium butyrate has been shown to improve growth, palatability, feed intake, nutrient digestibility, and survival (Silva et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In another study with \u003cem\u003eP. vannamei\u003c/em\u003e post-larvae, supplementation with the marine yeast \u003cem\u003eMeyerozyma guilliermondii\u003c/em\u003e in a super-intensive biofloc system reduced solid accumulation and increased survival (Ferreira et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Zinc proteinate has also been reported to enhance shrimp growth and immunity (Lin et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eBased on this evidence, individual feed additives can support the growth performance of \u003cem\u003eP. vannamei\u003c/em\u003e shrimp reared in both clear-water and biofloc systems. Therefore, the present study aimed to evaluate the effects of Viligen\u0026reg; NE at three concentrations (0.5, 1, and 2 g kg⁻\u0026sup1;) and a control treatment (without Viligen\u0026reg; NE) on growth performance, intestinal microbiota, and thermal shock resistance in Pacific white shrimp reared in a biofloc system during the nursery phase.\u003c/p\u003e"},{"header":"2. METHODOLOGY","content":"\u003cp\u003eThe experiment was conducted at the Marine Shrimp Laboratory (LCM), and the experimental diets were produced at the Laboratory of Aquatic Species Nutrition (LABNUTRI), both located at the Federal University of Santa Catarina (UFSC), in Florian\u0026oacute;polis, SC, Brazil.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 ANIMALS\u003c/h2\u003e\u003cp\u003ePost-larval \u003cem\u003ePenaeus vannamei\u003c/em\u003e with 20 days (PL20), obtained from the commercial hatchery Aquatec, were acclimated in a biofloc system at the Marine Shrimp Laboratory (LCM/UFSC) until reaching an average weight of approximately 41 mg.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 PREPARATION OF EXPERIMENTAL DIETS\u003c/h2\u003e\u003cp\u003eFour experimental diets were formulated to provide Viligen\u0026reg; NE at 0.5, 1, or 2 g kg⁻\u0026sup1;, along with a non-supplemented control diet (0 g kg⁻\u0026sup1;). The formulations were developed using Optimal Formula 2000\u0026reg; software, based on the nutritional requirements for \u003cem\u003ePenaeus vannamei\u003c/em\u003e (Gong et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; NRC \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Zhou et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Viligen\u0026reg; NE was included by replacing kaolin in the formulation. Dry ingredients were pre-ground and sieved through a 600 \u0026micro;m mesh. Micro-ingredients were homogenized and then mixed with the macro-ingredients. Oils, soy lecithin, and water were subsequently added. The resulting mixture was pelletized using a microextruder (Inbramaq MX-40), dried in an oven at 40\u0026deg;C, and stored under refrigeration at 4\u0026deg;C until use. The ingredients and proximate composition, analyzed following AOAC (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1999\u003c/span\u003e) guidelines, are presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eFormulation of the Experimental Diet containing 0.5, 1, and 2 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of Viligen\u0026reg; NE and the Control Diet 0 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, without the product addition.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eIngredients (g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Dry Weight)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e\u003cp\u003eAmount in g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of Viligen\u0026reg; NE\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.0\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.0\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWheat meal \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e100.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e100.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e100.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e100.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSoybean meal \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e350.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e350.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e350.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e350.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFish byproduct meal \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e200.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e200.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e200.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e200.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePoultry viscera meal \u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e80.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e80.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e80.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e80.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCarboxymethyl cellulose\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e5.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e5.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e5.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e5.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSoy lecithin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e20.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e20.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e20.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e20.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMonocalcium phosphate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e25.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e25.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e25.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e25.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSoybean oil \u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e5.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e5.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e5.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e5.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVitamin C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.70\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVitamin premix \u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e4.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e4.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e4.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e4.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMineral premix \u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e17.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e17.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e17.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e17.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMagnesium sulfate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e15.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e15.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e15.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e15.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eKaolin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e80.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e75.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e70.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e60.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSodium Chloride\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e10.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e10.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e10.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSalmon byproduct meal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e51.30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e51.30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e51.30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e51.30\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePotassium Chloride\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e10.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e10.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e10.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMethionine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFish oil \u003csup\u003eh\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e25.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e25.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e25.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e25.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eViligen\u0026reg; NE\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eProximate Composition (g 100 g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e dry matter)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDry matter\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e96.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e93.60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e90.65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e94.35\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCrude protein\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e38.65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e37.86\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e36.77\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e38.70\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCrude Lipid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e9.83\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e7.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e8.39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e7.49\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMinerals\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e23.83\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e24.46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e22.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e22.82\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGross Energy (kcal/g)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e4173.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3994.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3878.92\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e3934.40\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e* All results are expressed on a natural product basis.\u003c/p\u003e\u003cp\u003e** Physical-chemical characterization analyses were conducted according to AOAC (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1999\u003c/span\u003e) methodologies: Dry Matter by method 950.01; Mineral Matter by method 942.05; Protein by LECO Dumas method 990.03, conversion factor 6.25; Ether Extract by Soxhlet by method 920.39C. Gross energy was determined in a calorimetric bomb. \u003csup\u003ea\u003c/sup\u003e Rosa Branca, Type 1: 13 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e crude protein and 3440 kcal kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e crude energy. \u003csup\u003eb\u003c/sup\u003e Soybean meal distributed by BRF Ingredients (Itaja\u0026iacute;, Santa Catarina, Brasil): 56.61 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e crude\u003c/p\u003e\u003cp\u003eprotein and 4500 kcal kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e crude energy. \u003csup\u003ec\u003c/sup\u003e Fish meal distributed by Agroforte Ind (Bigua\u0026ccedil;u, Santa Catarina, Brasil): 59.35 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e crude protein and 4,056.13 kcal kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e crude energy. \u003csup\u003ed\u003c/sup\u003e Poultry viscera meal distributed by BRF Ingredients (Itaja\u0026iacute;, Santa Catarina, Brasil): 62 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e crude protein and 4661 kcal kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e crude energy. \u003csup\u003ee\u003c/sup\u003e Soybean oil: Soya: 7,909 kcal kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e crude energy. \u003csup\u003ef\u003c/sup\u003e Composition of Vitamin: vit. A \u0026minus;\u0026thinsp;900 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; vit. D3\u0026ndash;25 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; vit. E \u0026ndash; 46.900 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; vit. K3\u0026ndash;1.400 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; cobalamina (B12) \u0026ndash; 50 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; piridoxina (B6) \u0026ndash; 33.000 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; riboflavina \u0026ndash; 20.000 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; \u0026aacute;cido nicotinico \u0026ndash; 70.000 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; \u0026aacute;cido pantot\u0026ecirc;nico \u0026ndash; 40.000 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; biotina \u0026ndash; 750 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; \u0026aacute;cido f\u0026oacute;lico \u0026ndash; 3.000 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; \u003csup\u003eg\u003c/sup\u003e Mineral Premix: cobre \u0026ndash; 2.330 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; zinco \u0026ndash; 10.000 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; mangan\u0026ecirc;s \u0026ndash; 6.500 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; sel\u0026ecirc;nio \u0026minus;\u0026thinsp;125 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; iodo \u0026ndash; 1.000 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; cobalto \u0026ndash; 50 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; magn\u0026eacute;sio \u0026ndash; 20 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; potassio \u0026ndash; 6,1 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. \u003csup\u003eh\u003c/sup\u003e Fish oil distributed by BFP Bio Food Products (Itaja\u0026iacute;, Santa Catarina, Brasil): 8445 kcal kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e crude energy.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 EXPERIMENTAL DESIGN\u003c/h2\u003e\u003cp\u003eThe experimental diets were administered over a period of six weeks to a total of 384 shrimp, distributed into four treatment groups (0, 0.5, 1, and 2 g Viligen\u0026reg; NE kg⁻\u0026sup1;), each with four 48 L replicate tanks. Each tank was stocked with 96 shrimp, corresponding to a stocking density of 2000 post-larvae m⁻\u0026sup3;.\u003c/p\u003e\u003cp\u003eEach tank was equipped with a water heating system (temperature maintained at 28.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u0026deg;C) and aeration provided via perforated tubing (dissolved oxygen\u0026thinsp;\u0026gt;\u0026thinsp;5 mg L⁻\u0026sup1;). Water was sourced from an inoculum consisting of 30% mature biofloc water from a shrimp-rearing tank with established nitrification, presenting the following parameters: ammonia 0.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0 mg mL⁻\u0026sup1;; nitrite 0.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0 mg mL⁻\u0026sup1;; alkalinity 170\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0 mg CaCO₃ L⁻\u0026sup1;; total suspended solids (TSS) 484\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0 mg L⁻\u0026sup1;.\u003c/p\u003e\u003cp\u003eDissolved oxygen and temperature were monitored daily, while concentrations of total ammonia, nitrite, salinity, pH, and TSS were measured weekly. During the six-week rearing period, shrimp were fed six times daily (8:00, 9:30, 11:30, 13:30, 15:00, and 17:00) following the feeding table proposed by Van Wyk and Scarpa (1999). Weekly, 20 shrimp per tank were individually weighed to monitor growth and adjust feed quantities accordingly. At the end of the trial, growth performance was assessed based on survival, final weight, specific growth rate, yield, and feed conversion ratio.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 INTESTINAL MICROBIOLOGY ANALYSES\u003c/h2\u003e\u003cp\u003eOn the day of sampling, shrimp were not fed. At the end of the six-week feeding period, intestines from 10 shrimp per tank were collected, totaling 40 shrimp per treatment. The intestinal tracts were homogenized and serially diluted (1:10) in sterile 3% saline solution, then plated on Marine Agar and TCBS agar to enumerate total heterotrophic bacteria and \u003cem\u003eVibrio\u003c/em\u003e spp., respectively. No prior filtration of the intestines was performed before dilution. The plated samples were incubated at 30\u0026deg;C for 24 hours, after which total colony-forming units (CFU g⁻\u0026sup1; of intestine) were counted.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 THERMAL SHOCK RESISTANCE\u003c/h2\u003e\u003cp\u003eTo assess thermal shock resistance, at the end of the six-week period, 15 shrimp from each experimental unit (60 shrimp per treatment) were transferred from the rearing tanks (water at 28.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u0026deg;C) to 60 L aquaria containing saltwater at 13.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u0026deg;C and maintained for one hour. This procedure followed a previously established 50% lethal temperature protocol (Pontinha, Vieira, and Hayashi \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). During the exposure period, water temperature in the aquaria was continuously monitored. After one hour, the shrimp were simultaneously transferred to containers with water at 28\u0026deg;C. Survival was assessed at 12, 24, 36, and 48 hours post-transfer. Shrimp were not fed during the thermal shock test or the recovery period.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 STATISTICAL ANALYSIS\u003c/h2\u003e\u003cp\u003eWater quality parameters and microbiological data were subjected to Shapiro\u0026ndash;Wilk and Levene\u0026rsquo;s tests to verify normality and homoscedasticity, respectively. Once assumptions were met, one-way ANOVA was applied, followed by Tukey\u0026rsquo;s post hoc test when appropriate. Growth performance data were also analyzed using second-degree polynomial regression, with the optimal inclusion level estimated from the vertex of the quadratic regression equations. Thermal shock resistance data were analyzed using two-way ANOVA (treatment \u0026times; time). A significance level of 5% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) was adopted for all statistical tests.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. RESULTS","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.1 WATER QUALITY PARAMETERS\u003c/h2\u003e\u003cp\u003eAmmonia and nitrite levels differed significantly among treatments; however, these variations did not influence shrimp growth performance, as the values remained within acceptable limits for \u003cem\u003ePenaeus vannamei\u003c/em\u003e reared in biofloc systems. Temperature and dissolved oxygen were maintained at 28.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.97\u0026deg;C and 6.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26 mg L⁻\u0026sup1;, respectively, and remained stable throughout the experimental period. No significant differences were observed among treatments for alkalinity, pH, total suspended solids, or nitrate concentrations (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eWater quality parameters in \u003cem\u003ePenaeus vannamei\u003c/em\u003e nursery tanks in a biofloc system, fed for six weeks with diets containing 0.5, 1, and 2 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of Viligen\u0026reg; NE and a control (0 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), without the product addition.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTreatments\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAmmonia (mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNitrite N-NO\u003csub\u003e2\u003c/sub\u003e (mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNitrate N-NO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\u003cp\u003e(mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eAlkalinity (mg CaCO\u003csub\u003e3\u003c/sub\u003e L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003epH\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eTSS (mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.88a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e70.43\u0026thinsp;\u0026plusmn;\u0026thinsp;24.89\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e163.67\u0026thinsp;\u0026plusmn;\u0026thinsp;19.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e7.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e613.26\u0026thinsp;\u0026plusmn;\u0026thinsp;255.54\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0.5g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.95\u0026ordf;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e64.25\u0026thinsp;\u0026plusmn;\u0026thinsp;47.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e168.10\u0026thinsp;\u0026plusmn;\u0026thinsp;19.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e7.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e601.60\u0026thinsp;\u0026plusmn;\u0026thinsp;266.09\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e72.44\u0026thinsp;\u0026plusmn;\u0026thinsp;54.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e165.25\u0026thinsp;\u0026plusmn;\u0026thinsp;14.83\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e7.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e584.90\u0026thinsp;\u0026plusmn;\u0026thinsp;264.54\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e79.42\u0026thinsp;\u0026plusmn;\u0026thinsp;67.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e164.83\u0026thinsp;\u0026plusmn;\u0026thinsp;16.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e7.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e567.08\u0026thinsp;\u0026plusmn;\u0026thinsp;285.75\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ep-value**\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.0001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.0001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.961\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.642\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.573\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.812\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e*Data presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. **\u003cem\u003eOne-way ANOVA. TSS \u0026ndash; Total Suspended Solids.\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.2 GROWTH PERFORMANCE\u003c/h2\u003e\u003cp\u003eAfter the six-week period, the inclusion of Viligen\u0026reg; NE resulted in a quadratic effect on final weight, and specific growth rate (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Through polynomial regression, the optimal inclusion level was estimated at 1.17g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of Viligen\u0026reg; NE for the highest response in terms of specific growth rate (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) and 1.174 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of Viligen\u0026reg; NE for weight gain (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eGrowth performance in the nursery of \u003cem\u003ePenaeus vannamei\u003c/em\u003e in a biofloc system, fed for six weeks with diets containing 0.5, 1, and 2 g kg-1 of Viligen\u0026reg; NE and a control (0 g kg-1), without the product addition.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eVariables\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e\u003cp\u003eViligen\u0026reg; NE supplementation\u0026nbsp;g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003eValues for Regression*\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eR\u0026sup2;\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cem\u003ep\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFinal Weight (g)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e0.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e1.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e1.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e0.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.995\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.045\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWeight gain (g)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e0.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e0.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e1.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e0.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.995\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.007\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eYield (kg m\u0026sup3;)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e1.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e1.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e2.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e1.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.9997\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSpecific Growth Rate (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e1.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e2.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e2.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e2.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.995\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.045\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFeed Conversion Ratio\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e1.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e1.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e1.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e1.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNS***\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSurvival (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.3 INTESTINAL MICROBIOLOGY ANALYSES\u003c/h2\u003e\u003cp\u003eNo significant differences were observed among the experimental groups in the total counts of heterotrophic bacteria (p\u0026thinsp;=\u0026thinsp;0.182) or \u003cem\u003eVibrio\u003c/em\u003e spp. (p\u0026thinsp;=\u0026thinsp;0.549) in shrimp intestines after six weeks of feeding with either non-supplemented or Viligen\u0026reg; NE-supplemented diets at different inclusion levels (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.4 THERMAL SHOCK RESISTANCE\u003c/h2\u003e\u003cp\u003eA significant difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) was observed in \u003cem\u003ePenaeus vannamei\u003c/em\u003e mortality during the 48-hour period following thermal shock exposure among the experimental groups. Shrimp fed diets containing 1 g kg⁻\u0026sup1; of Viligen\u0026reg; NE exhibited significantly lower mortality compared to those in the 0 g kg⁻\u0026sup1; (control), 0.5 g kg⁻\u0026sup1;, and 2 g kg⁻\u0026sup1; treatment groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. DISCUSSION","content":"\u003cp\u003eThis study represents the first evaluation of Viligen\u0026reg; NE as a dietary additive for Pacific white shrimp. Several benefits were observed, including improvements in final weight, weight gain, and specific growth rate following dietary supplementation with Viligen\u0026reg; NE. The optimal inclusion level, based on weight gain and specific growth rate, was estimated at 1.17 g kg⁻\u0026sup1;. While the effects observed in this study are attributed to Viligen\u0026reg; NE as a compound product, the existing literature on similar feed additives provides useful comparisons.\u003c/p\u003e\u003cp\u003eIn agreement with the present results, Lin et al. (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) reported that among three organic zinc sources (zinc-lysine, zinc-glycine, and zinc-methionine), zinc-methionine supplementation led to the highest weight gain and improved survival following a \u003cem\u003eVibrio harveyi\u003c/em\u003e challenge in \u003cem\u003eP. vannamei\u003c/em\u003e. Regarding yeast-based additives, Jin et al. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) observed improved weight gain, specific growth rate, and feed conversion ratio in juvenile \u003cem\u003eP. vannamei\u003c/em\u003e fed diets supplemented with 1% hydrolyzed yeast compared to brewer\u0026rsquo;s yeast and a control group. Therefore, in the present study, the inclusion of Viligen\u0026reg; NE, which contains components similar to those tested individually in other studies, may have produced synergistic effects, contributing to enhanced growth performance.\u003c/p\u003e\u003cp\u003eSimilarly, Silva et al. (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) observed significant improvements in growth and feed efficiency in \u003cem\u003eP. vannamei\u003c/em\u003e when diets were supplemented with 20 g kg⁻\u0026sup1; of sodium butyrate. However, optimal dietary levels for a given additive may vary depending on species, developmental stage, production system, and duration of supplementation. For instance, Pessini et al. (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) tested Viligen\u0026trade; (0.60, 1.20, 2.40, and 4.80 g kg⁻\u0026sup1;) in Nile tilapia diets over 60 days and estimated an optimal inclusion level of 2.80 g kg⁻\u0026sup1; for maximum daily weight gain. It is important to note that the Viligen\u0026trade; used in that study is related to but distinct from the product evaluated here.\u003c/p\u003e\u003cp\u003eAlthough previous studies with dietary additives have reported positive modulation of shrimp intestinal microbiota, no significant differences were found in this study in the total counts of heterotrophic bacteria or \u003cem\u003eVibrio\u003c/em\u003e spp. among treatments. This could be due to the relatively low inclusion levels used here, as studies showing microbial modulation typically employed higher doses. For example, Silva et al. (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) demonstrated that sodium butyrate at 5, 10, and 20 g kg⁻\u0026sup1; significantly reduced \u003cem\u003eVibrio\u003c/em\u003e spp. abundance and increased serum agglutination activity in \u003cem\u003eP. vannamei\u003c/em\u003e. Thus, further research is needed to determine the optimal inclusion level of Viligen\u0026reg; NE to influence the intestinal microbiota of Pacific white shrimp effectively.\u003c/p\u003e\u003cp\u003eTemperature plays a key role in the metabolism of crustaceans, influencing both growth and survival in penaeid shrimp. Cold stress can lead to immunosuppression, increasing susceptibility to pathogens (Gomez-Jimenez et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Kautsky et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Wang and Chen \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Certain organic acids are known to enhance membrane fluidity in intestinal cells, which can improve survival under cold stress due to the importance of homeoviscous membrane adaptation as a cold-resistance mechanism (Hayward, Manso, and Cossins \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Insects, crustaceans, microorganisms, and cold-adapted plants typically exhibit greater membrane fluidity through increased unsaturation, a shared trait among organisms resistant to temperature fluctuations (Corcoles-S\u0026aacute;ez et al. 2016; Takahashi et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn this study, cumulative mortality after thermal shock differed significantly among treatments. Shrimp fed 1 g kg⁻\u0026sup1; of Viligen\u0026reg; NE exhibited the lowest mortality after 48 hours compared to other treatments and the control. This enhanced resistance may be linked to improved membrane fluidity in shrimp hemocytes, allowing better thermal adaptation. For ectothermic animals like shrimp, cold stress can induce physiological dysfunctions with serious implications for production (Hayward et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Therefore, dietary supplementation with Viligen\u0026reg; NE appears to be a promising strategy for improving resilience in environments with sudden temperature fluctuations.\u003c/p\u003e\u003cp\u003eFurther studies are needed to elucidate the underlying mechanisms that enhance shrimp resistance to thermal stress and to determine the optimal supplementation levels to promote benefits in growth performance, intestinal microbiota, and survival under challenging environmental conditions.\u003c/p\u003e"},{"header":"5. CONCLUSION","content":"\u003cp\u003eBenefits in growth performance were observed for \u003cem\u003ePenaeus vannamei\u003c/em\u003e reared in a biofloc system during the nursery phase, with an optimal inclusion level of 1.17 g kg⁻\u0026sup1; of Viligen\u0026reg; NE for improved weight gain and specific growth rate. Additionally, significant differences were found among treatments in shrimp mortality following thermal shock, with the 1 g kg⁻\u0026sup1; supplementation level enhancing thermal resistance. However, no significant differences were observed in the total counts of heterotrophic bacteria or \u003cem\u003eVibrio\u003c/em\u003e spp. in shrimp intestines. Therefore, it can be concluded that dietary supplementation with 1 g kg⁻\u0026sup1; of Viligen\u0026reg; NE is the most effective among the tested doses for \u003cem\u003eP. vannamei\u003c/em\u003e during the nursery phase in a biofloc system.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding:\u003c/h2\u003e\u003cp\u003eThis project has received funding from Alltech, CNPq (403469/2023-6) and the Coordination for the Improvement of Higher Level Personnel (CAPES; finance code: 001). F.B.V and W.Q.S are research fellows from CNPq.\u003c/p\u003e\u003cp\u003eConflict of Interest: The authors declare no conflict of interest.\u003c/p\u003e\u003cp\u003eEthical approval: Not applicable (work with invertebrate - Law No. 11,794, of October 8, 2008 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.planalto.gov.br/ccivil_03/_ato2007-2010/2008/lei/l11794.htm\u003c/span\u003e\u003cspan address=\"https://www.planalto.gov.br/ccivil_03/_ato2007-2010/2008/lei/l11794.htm\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) .\u003c/p\u003e\u003cp\u003eConceptualization, A.K., C.S.M., D.M.F., F.B.V., P.L.; Methodology, C.S.M., F.B.V., I.C.P., I.L.S., N.C.B., R.F.S., S.A.P.D.; Validation, C.S.M., I.L.S., N.C.B., R.F.S.; Formal analysis, C.S.M., I.C.P., S.A.P.D.; Investigation, C.S.M., F.B.V., I.C.P., I.L.S., N.C.B., R.F.S.; Resources, A.K., P.L.; Writing\u0026mdash;original draft preparation, C.S.M.; Writing\u0026mdash;review and editing, A.K., C.S.M., D.M.F., F.B.V., P.L.; Supervision, F.B.V., S.A.P.D.; Project administration, F.B.V.; Funding acquisition, A.K., C.S.M., D.M.F., P.L. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization, A.K., C.S.M., D.M.F., F.B.V., P.L.; Methodology, C.S.M., F.B.V., I.C.P., I.L.S., N.C.B., R.F.S., S.A.P.D.; Validation, C.S.M., I.L.S., N.C.B., R.F.S.; Formal analysis, C.S.M., I.C.P., S.A.P.D.; Investigation, C.S.M., F.B.V., I.C.P., I.L.S., N.C.B., R.F.S.;Resources, A.K., P.L.; Writing\u0026mdash;original draft preparation, C.S.M.; Writing\u0026mdash;review and editing, A.K., C.S.M., D.M.F., F.B.V., P.L.; Supervision, F.B.V., S.A.P.D.; Project administration, F.B.V.; Funding acquisition, A.K., C.S.M., D.M.F., P.L. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe thank the companies Alltech Inc. (Nicholasville, KY, USA) for their financial support.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData supporting the findings of this study are available from the corresponding author upon request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAOAC (1999) Official Methods of Analysis of AOAC International. AOAC International, Gaithersburg, MD\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAvnimelech Y (2015) \u003cem\u003eBiofloc Technology: A Practical Guidebook\u003c/em\u003e (3rd ed.). Baton Rouge, LA: The World Aquaculture Society. ISBN 978-1880443036\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBrowdy CL, Bratvold D, Stokes AD, McIntosh RP (2001) Perspectives on the application of closed shrimp culture systems. In C. L. Browdy \u0026amp; D. E. Jory (Eds.), \u003cem\u003eNew Wave: Proceedings of the Special Session on Sustainable Shrimp Farming\u003c/em\u003e (pp. 20\u0026ndash;34). Baton Rouge, LA: World Aquaculture Society\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eC\u0026oacute;rcoles-S\u0026aacute;ez I, Hern\u0026aacute;ndez ML, Mart\u0026iacute;nez-Rivas JM, Prieto JA, Randez-Gil F (2016) Characterization of the \u003cem\u003eS. cerevisiae inp51\u003c/em\u003e mutant links phosphatidylinositol 4,5-bisphosphate levels with lipid content, membrane fluidity and cold growth. Biochim et Biophys Acta (BBA) - Mol Cell Biology Lipids 1861:213\u0026ndash;226. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.bbalip.2015.12.014\u003c/span\u003e\u003cspan address=\"10.1016/j.bbalip.2015.12.014\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDe Schryver P, Crab R, Defoirdt T, Boon N, Verstraete W (2008) The basics of bio-flocs technology: The added value for aquaculture. Aquaculture 277:125\u0026ndash;137. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.aquaculture.2008.02.019\u003c/span\u003e\u003cspan address=\"10.1016/j.aquaculture.2008.02.019\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFerreira NL, Vieira FN, Mouri\u0026ntilde;o JLP, Seiffert WQ, Fracalossi DM (2023) Effect of microbial inoculants and fertilization strategies in super-intensive biofloc shrimp culture system. Aquac Res 54(6):2394\u0026ndash;2406. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/are.15746\u003c/span\u003e\u003cspan address=\"10.1111/are.15746\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGomez-Jimenez S, Uglow RF, Gollas-Galvan T (2000) The effects of cooling and emersion on total haemocyte count and phenoloxidase activity of the spiny lobster \u003cem\u003ePanulirus interruptus\u003c/em\u003e. Fish Shellfish Immunol 10:631\u0026ndash;635. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1006/fsim.2000.0277\u003c/span\u003e\u003cspan address=\"10.1006/fsim.2000.0277\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGong H, Lawrence AL, Jiang DH, Castille FL, Gatlin III, D. M (2000) Lipid nutrition of juvenile \u003cem\u003eLitopenaeus vannamei\u003c/em\u003e I. Dietary cholesterol and de-oiled soy lecithin requirements and their interaction. Aquaculture 190(3\u0026ndash;4):305\u0026ndash;324. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0044-8486(00)00414-2\u003c/span\u003e\u003cspan address=\"10.1016/S0044-8486(00)00414-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHargreaves JA (2006) Photosynthetic suspended-growth systems in aquaculture. Aquacult Eng 34(3):344\u0026ndash;363. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.aquaeng.2005.08.009\u003c/span\u003e\u003cspan address=\"10.1016/j.aquaeng.2005.08.009\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHayward SAL, Manso B, Cossins AR (2014) Molecular basis of chill resistance adaptations in poikilothermic animals. J Exp Biol 217:6\u0026ndash;15. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1242/jeb.096537\u003c/span\u003e\u003cspan address=\"10.1242/jeb.096537\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJin M, Xiong J, Zhou QC, Yuan Y, Wang XX, Sun P (2018) Dietary yeast hydrolysate and brewer\u0026rsquo;s yeast supplementation could enhance growth performance, innate immunity capacity and ammonia nitrogen stress resistance ability of Pacific white shrimp (\u003cem\u003eLitopenaeus vannamei\u003c/em\u003e). Fish Shellfish Immunol 82:121\u0026ndash;129. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.fsi.2018.08.020\u003c/span\u003e\u003cspan address=\"10.1016/j.fsi.2018.08.020\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKautsky N, R\u0026ouml;nnb\u0026auml;ck P, Tedengren M, Troell M (2000) Ecosystem perspectives on management of disease in shrimp pond farming. Aquaculture 191(1\u0026ndash;3):145\u0026ndash;161. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0044-8486(00)00424-5\u003c/span\u003e\u003cspan address=\"10.1016/S0044-8486(00)00424-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKrummenauer D, Cavalli R, Ballester E, Wasielesky W (2010) Feasibility of Pacific white shrimp \u003cem\u003eLitopenaeus vannamei\u003c/em\u003e culture in southern Brazil: Effects of stocking density and a single or a double crop management strategy in earthen ponds. Aquac Res 41:240\u0026ndash;248. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1365-2109.2009.02326.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1365-2109.2009.02326.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLightner DV (1996) A handbook of shrimp pathology and diagnostic procedures for diseases of cultured penaeid shrimp. World Aquaculture Society, Baton Rouge, LA\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLin S, Mai K, Tan B, Liu Y, Zhang W (2013) Comparison of chelated zinc and zinc sulfate as zinc sources for growth and immune response of shrimp (\u003cem\u003eLitopenaeus vannamei\u003c/em\u003e). \u003cem\u003eAquaculture\u003c/em\u003e, 406\u0026ndash;407, 79\u0026ndash;84. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.aquaculture.2013.04.026\u003c/span\u003e\u003cspan address=\"10.1016/j.aquaculture.2013.04.026\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiu H, Wang X, Wang G, Gao Q, Yan H, Li L (2011) The intestinal microbial diversity of Chinese shrimp (\u003cem\u003eFenneropenaeus chinensis\u003c/em\u003e) as determined by PCR-DGGE and clone library analyses. Aquaculture 317(1\u0026ndash;4):32\u0026ndash;36. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.aquaculture.2011.04.008\u003c/span\u003e\u003cspan address=\"10.1016/j.aquaculture.2011.04.008\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLorenzo MA, Schveitzer R, Nu\u0026ntilde;er APO, Vinatea L (2016) Intensive hatchery performance of Pacific white shrimp in the biofloc system under three different fertilization levels. Aquacult Eng 72:40\u0026ndash;44. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.aquaeng.2016.04.001\u003c/span\u003e\u003cspan address=\"10.1016/j.aquaeng.2016.04.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eL\u0026uuml;ckst\u0026auml;dt C (2008) The use of acidifiers in fish nutrition. \u003cem\u003eCAB Reviews: Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources\u003c/em\u003e, 3(044), 1\u0026ndash;8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1079/pavsnnr20083044\u003c/span\u003e\u003cspan address=\"10.1079/pavsnnr20083044\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNRC (2011) Nutrient requirements of fish and shrimp. National Academies, Washington, DC\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNg W, Koh C (2016) The utilization and mode of action of organic acids in the feeds of cultured aquatic animals. Reviews Aquaculture 9(4):342\u0026ndash;368. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/raq.12141\u003c/span\u003e\u003cspan address=\"10.1111/raq.12141\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePessini J, Daitx de Oliveira V, Souza de S\u0026aacute; L, Mourio P, Pettigrew JL, J., Fracalossi DM (2021) Dietary supplementation of Viligen\u0026reg; to Nile tilapia improves growth and gut morphology. Aquacult Nutr 27:1065\u0026ndash;1076. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/anu.13247\u003c/span\u003e\u003cspan address=\"10.1111/anu.13247\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePontinha VdeA, Vieira F do N., Hayashi L (2018) Mortality of Pacific white shrimp submitted to hypothermic and hyposalinic stress. \u003cem\u003eBoletim do Instituto de Pesca\u003c/em\u003e, 44(2), [p\u0026aacute;ginas]\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSilva BC, Vieira FN, Mouri\u0026ntilde;o JLP, Ferreira GS, Seiffert WQ (2013) Salts of organic acids selection by multiple characteristics for marine shrimp nutrition. Aquaculture 384:104\u0026ndash;110. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.aquaculture.2012.12.017\u003c/span\u003e\u003cspan address=\"10.1016/j.aquaculture.2012.12.017\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSilva BC, Vieira FN, Mouri\u0026ntilde;o JLP, Ram\u0026iacute;rez N, Seiffert WQ (2016) Butyrate and propionate improve the growth performance of \u003cem\u003eLitopenaeus vannamei\u003c/em\u003e. Aquac Res 47:612\u0026ndash;623. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/are.12520\u003c/span\u003e\u003cspan address=\"10.1111/are.12520\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTakahashi D, Imai H, Kawamura Y, Uemura M (2016) Lipid profiles of detergent resistant fractions of the plasma membrane in oat and rye in association with cold acclimation and freezing tolerance. Cryobiology 72:123\u0026ndash;134. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.cryobiol.2016.02.003\u003c/span\u003e\u003cspan address=\"10.1016/j.cryobiol.2016.02.003\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVan Wyk P (1999) Nutrition and feeding of \u003cem\u003eLitopenaeus vannamei\u003c/em\u003e in intensive culture systems. In: Van Wyk P et al (eds) Farming marine shrimp in recirculating freshwater systems. Harbor Branch Oceanographic Institution, Tallahassee, pp 125\u0026ndash;140\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang FI, Chen JC (2006) The immune response of tiger shrimp \u003cem\u003ePenaeus monodon\u003c/em\u003e and its susceptibility to \u003cem\u003ePhotobacterium damselae\u003c/em\u003e subsp. \u003cem\u003edamselae\u003c/em\u003e under temperature stress. \u003cem\u003eAquaculture\u003c/em\u003e, 258, 34\u0026ndash;41. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.aquaculture.2006.03.043\u003c/span\u003e\u003cspan address=\"10.1016/j.aquaculture.2006.03.043\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWasielesky W, Atwood H, Stokes A, Browdy C (2013) Nursery of \u003cem\u003eLitopenaeus vannamei\u003c/em\u003e reared in a biofloc system: The effect of stocking densities and compensatory growth. J Shellfish Res 32(2):799\u0026ndash;806. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2983/035.032.0323\u003c/span\u003e\u003cspan address=\"10.2983/035.032.0323\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWeston DP (1996) Environmental considerations on the use of antibacterial drugs in aquaculture. In: Baird DJ, Beveridge MCM, Kelly LA, Muir JF (eds) Aquaculture and water resource management. Institute of Aquaculture, Blackwell Science, pp 140\u0026ndash;165\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXu WJ, Pan LQ, Zhao DH, Huang J (2012) Preliminary investigation into the contribution of bioflocs on protein nutrition of \u003cem\u003eLitopenaeus vannamei\u003c/em\u003e fed with different dietary protein levels in zero-water exchange culture tanks. Aquaculture 350\u0026ndash;353. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.aquaculture.2012.04.003\u003c/span\u003e\u003cspan address=\"10.1016/j.aquaculture.2012.04.003\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhou QC, Tan BP, Mai KS, Liu YJ, Ceng C (2012) Dietary arginine requirement of juvenile Pacific white shrimp, \u003cem\u003eLitopenaeus vannamei\u003c/em\u003e. \u003cem\u003eAquaculture\u003c/em\u003e, 364\u0026ndash;365, 252\u0026ndash;258. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.aquaculture.2012.08.020\u003c/span\u003e\u003cspan address=\"10.1016/j.aquaculture.2012.08.020\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"","identity":"aquaculture-international","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"10499","submissionUrl":"https://submission.nature.com/new-submission/10499/3","title":"Aquaculture International","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"","reportingPortfolio":"VoR Journals","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Penaeus vannamei, growth performance, thermal shock, final weight","lastPublishedDoi":"10.21203/rs.3.rs-6974226/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6974226/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe aim of this study was to evaluate Viligen\u0026reg; NE (Alltech\u0026reg;, USA) as a feed additive for \u003cem\u003ePenaeus vannamei\u003c/em\u003e reared in a biofloc system during the nursery phase, focusing on growth performance, intestinal microbiological analysis, and resistance to thermal shock. Shrimp post-larvae (41 mg) were fed for six weeks with four experimental diets: (a) 0.5 g Viligen\u0026reg; NE kg⁻\u0026sup1;; (b) 1 g Viligen\u0026reg; NE kg⁻\u0026sup1;; (c) 2 g Viligen\u0026reg; NE kg⁻\u0026sup1;; and (d) a control diet (0 g Viligen\u0026reg; NE kg⁻\u0026sup1;). The experiment was conducted in quadruplicate, totaling 16 experimental tanks, with a stocking density of 2000 post-larvae m⁻\u0026sup3;. Shrimp growth performance parameters were positively influenced by the inclusion of Viligen\u0026reg; NE in the diet. Polynomial regression indicated an optimal inclusion level of 1.17 g kg⁻\u0026sup1; for maximizing specific growth rate and weight gain. Additionally, supplementation at 1 g kg⁻\u0026sup1; enhanced shrimp resistance to thermal shock. However, no significant differences were observed in intestinal counts of total heterotrophic bacteria or \u003cem\u003eVibrio\u003c/em\u003e spp. among treatments. In conclusion, dietary supplementation with Viligen\u0026reg; NE at an optimal level of 1.17 g kg⁻\u0026sup1; improved growth performance, and 1 g kg⁻\u0026sup1; enhanced thermal shock resistance of nursery-phase shrimp reared in a biofloc system.\u003c/p\u003e","manuscriptTitle":"Evaluation of Viligen® NE as a feed additive for pacific white shrimp reared in a biofloc system during the nursery phase","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-15 12:45:57","doi":"10.21203/rs.3.rs-6974226/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-03T13:48:40+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-02T01:02:04+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-29T22:47:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"168339787157394633253347354158545000495","date":"2025-08-29T22:12:06+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-12T10:34:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"322174647561773413256219711335077061574","date":"2025-08-02T06:09:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"64478591255971997082386881522656572292","date":"2025-07-31T14:33:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"220639136676316618760377262013793259460","date":"2025-07-13T15:02:11+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-11T14:51:12+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-29T18:44:21+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-27T04:22:55+00:00","index":"","fulltext":""},{"type":"submitted","content":"Aquaculture International","date":"2025-06-25T11:26:58+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"","identity":"aquaculture-international","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"10499","submissionUrl":"https://submission.nature.com/new-submission/10499/3","title":"Aquaculture International","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"","reportingPortfolio":"VoR Journals","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"0417a602-a326-424a-aaae-a65dc829fa40","owner":[],"postedDate":"July 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-01-05T15:54:04+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-15 12:45:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6974226","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6974226","identity":"rs-6974226","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}