Iranian Scientific Fisheries Journal

fa مقاله علمی – پژوهشی:‌ تأثیر افزودن پروبیوتیک تک‌سل (Bacillus subtilis) بر شاخص‌های رشد، بقاء و کیفیت آب میگوی, Boone, 1931 Litopenaeus vannamei پرورش‌یافته در سیستم بیوفلاک Effect of supplementing single cell probiotic (Bacillus subtilis) on growth performance, survival, and water quality of Litopenaeus vannamei, Boone, 1931 cultured in a biofloc system تكثير و پرورش تكثير و پرورش كاربردي Applicable <a name="_Hlk204975112"></a><a name="_Hlk204975112">پرورش میگوی پا سفید غربی (</a>Litopenaeus vannamei, Boone, 1931) نیازمند راهکارهایی برای حفظ کیفیت آب و بهبود شاخص‌های رشد است. یکی از رویکردهای نوین در این زمینه، استفاده هم‌زمان از فناوری بیوفلاک و مکمل زیستی مانند پروبیوتیک است. پژوهش حاضر، با هدف ارزیابی تأثیر کاربرد بیوفلاک و پروبیوتیک تک‌سل بر کیفیت آب و عملکرد رشد میگوی پا سفید غربی در مزارع پرورش میگو واقع در سایت پرورش میگوی گمیشان، انجام شد. پست لاروهای مرحله 12 (PL12) با میانگین وزنی 05/0±52/0 گرم با تراکم 200 هزار قطعه در هکتار در مزارع پرورش میگو ذخیره‌‌سازی شدند. در طول دوره پرورش، شاخص‌‌های فیزیکوشیمیایی (شوری، هدایت الکتریکی، دما، (pH به‌ صورت منظم پایش گردید. تغذیه میگوها با استفاده از خوراک تجاری کنسانتره، به میزان 4-2 وعده در روز و در محدوده 10-5/2 درصد زیست توده کل، از خرداد لغایت شهریور 1402 انجام شد. طرح آزمایشی به‌ صورت کاملاً تصادفی با یک گروه شاهد و سه تیمار آزمایشی، هر کدام با سه تکرار، اجرا گردید: گروه شاهد: (بدون افزودنی)، تیمار 1: (افزودن بیوفلاک و پروبیوتیک تک‌‌سل با غلظت 300 گرم در هکتار)؛ تیمار 2 (افزودن بیوفلاک و پروبیوتیک تک‌‌سل با غلظت 400 گرم در هکتار)؛  تیمار 3 (افزودن بیوفلاک و پروبیوتیک تک‌‌سل با غلظت 500 گرم در هکتار). نتایج حاصل از تجزیه‌وتحلیل آماری نشان داد که تفاوت معنی‌‌داری در درصد بازماندگی بین تیمارها وجود نداشت. با این‌حال‌، تیمار حاوی 300 گرم پروبیوتیک در هکتار، بالاترین میانگین وزن نهایی، بیشترین نرخ رشد روزانه و کمترین ضریب تبدیل غذایی را نسبت به سایر تیمارها به‌ویژه تیمار شاهد و تیمار 500 گرم در هکتار (غلظت پیشنهادی کارخانه سازنده)، نشان داد. بر این اساس، به‌نظر می‌‌رسد که استفاده از غلظت 300 گرم پروبیوتیک تک‌سل در هکتار، ضمن بهبود عملکرد رشد، از نظر اقتصادی نیز مقرون به‌صرفه‌تر است و می‌تواند به‌ عنوان دوز بهینه در سیستم‌های پرورش میگوی پا سفید غربی، پیشنهاد شود. <a name="_Hlk205631899"></a><a name="_Hlk205632165">Introduction </a> Aquaculture now represents a primary sector in global food production, having surpassed capture fisheries in aquatic animal production as of 2022 (Abdel-Rahim et al., 2023; Caputo et al., 2023). Among the farmed species, Litopenaeus vannamei (Boone, 1931) has become the dominant species due to its rapid growth, omnivorous diet, high metabolic rate, and adaptability to tropical marine environments (De Silva et al., 2021; Huang et al., 2025; Fadel et al., 2025). Although this species is native to the eastern Pacific, more than 85% of its global aquaculture production now occurs in Asian countries (FAO, 2020; Amiin et al., 2023). Specifically, in Iran, more than 180,000 hectares are suitable for shrimp farming, including 4,000 hectares in Golestan Province. Nevertheless, intensive shrimp farming is confronted with ecological challenges, particularly the accumulation of nutrients and deterioration of water quality due to animal feed (Chaikaew et al., 2019). Maintaining optimal water parameters including temperature, dissolved oxygen, salinity, pH, and conductivity is critical for preventing stress and disease in shrimp culture (Kautsky et al., 2000; Kuncha et al., 2025). Biofloc technology (BFT), which stimulates the formation of beneficial microbial flocs through the addition of carbon sources, has been shown to improve productivity and promote environmental sustainability (Kumar et al., 2014; Mansour et al., 2022; Iber et al., 2025). The incorporation of probiotics into recirculating aquaculture systems further enhances water quality, gut health, and immune function (Menaga et al., 2023; Kaya, 2025). Probiotics exert their effects by competitively excluding pathogens, producing antimicrobial substances, and stimulating host immune responses (Angahar, 2016; Hoseinifar et al., 2018); certain strains also facilitate the reduction of nitrogenous waste (Dalmin et al., 2001; Mang et al., 2024). Moreover, probiotics are recognized as cost-effective, readily isolatable, and ecologically sustainable interventions (Gullian et al., 2004; Vidhya and Thomas, 2023). In larval stages, probiotics enhance digestion through enzyme secretion (Bairagi et al., 2002; Lara-Flores, 2011; Qiu et al., 2023; Vulla et al., 2024). When combined with biofloc technology (BFT), they improve growth performance, feed efficiency, disease resistance, and water quality (Dash et al., 2018; Pratiwi et al., 2020; Qiu et al., 2023). This study aims to evaluate the synergistic effects of probiotics and BFT on L. vannamei with a focus on water quality improvement, enhanced survival, optimized growth, and increased pathogen resistance. The integrated approach offers a sustainable framework for efficient and environmentally friendly shrimp aquaculture. Methodology This study was conducted at the shrimp aquaculture site in Gomishan County, located in northern Golestan Province, Iran. Field experiments were carried out in 12 shrimp ponds to evaluate the effects of biofloc and the probiotic Tak-Cell (containing Bacillus subtilis) on the growth performance and survival of Pacific white shrimp (L. vannamei). A total of 30 kg of the probiotic was applied over a 120-day period across nine ponds, with an additional 500 g administered during the pond preparation phase. Biofloc was prepared using carbon sources such as sugarcane molasses, rice flour, and wheat flour. These substrates were incubated in warm water (40°C) and subsequently diluted at a specific ratio before being introduced into the culture system. The carbon and nitrogen ratio (C: N) was optimized based on the assumption that 50% of nitrogen from feed is excreted into the aquatic environment. Post-larvae with an initial mean weight of 0.52±0.12 g were stocked at a density of 200,000 individuals per hectare in aerated ponds. Throughout the culture period (June to September), water quality parameters including salinity, temperature, pH, ammonia, nitrate, and phosphate were regularly monitored. Shrimp were fed commercial formulated diets 2–4 times daily at feeding rates ranging from 2.5% to 10% of biomass. The experimental design followed a completely randomized structure with four treatments: (1) biofloc without probiotic (control), and (2–4) biofloc combined with probiotic at concentrations of 300, 400, and 500 g/ha, respectively. Each treatment was replicated three times. Growth parameters including mean body weight, average daily gain, specific growth rate, feed conversion ratio, and survival rate were calculated using standard formulas. Data normality was assessed via the Shapiro–Wilk test. Upon confirmation of normal distribution, one-way ANOVA and LSD post hoc tests were conducted at a 5% significance level using SPSS version 23. <a name="_Hlk205632204">Result</a> During the period of shrimp farming in the test ponds, considerable fluctuations in air temperature were observed between the morning and evening hours, occasionally reaching up to 15°C. However, the water temperature, salinity and pH remained relatively stable throughout the sampling period and were consistently within the optimum range for shrimp farming. Statistical analysis revealed no significant differences in survival rates between the different treatments (p>0.05). Nevertheless, the 500 g/ha single-cell probiotic treatment showed the least variation in survival rate, indicating more stable culture conditions. In terms of growth performance, the highest mean final weight was observed in the 300 g/ha of probiotic treatment. Although this difference was not statistically significant compared to the 400 g/ha treatment, it was significantly higher than that of 500 g/ha and control groups. The highest specific growth rate (SGR) was recorded in the 400 g/ha treatment, which was not significantly different from other treatments, including the control. The highest average daily growth rate (ADGR) was also recorded in the 300 g/ha group, but no statistically significant differences were observed between treatments. For live biomass, the 300 g/ha treatment yielded the highest values and showed a statistically significant difference compared to the 500 g/ha group. Feed consumption was highest in the control group and was significantly higher than in the other treatments. The lowest feed conversion ratio (FCR) was found in the 300 g/ha treatment, which differed significantly only from the control group. Discussion and conclusion Probiotics play a crucial role in improving growth performance in aquaculture by optimising metabolic processes and creating favourable ecological conditions (Liu et al., 2009; Todorov et al., 2024; Tao et al., 2025). Their enzymatic activity improves digestion and nutrient uptake, leading to better growth results (Ghosh et al., 2023; Calcagnile et al., 2025). In the present study, water quality parameters such as temperature, salinity, and pH remained within the optimal range, which is consistent with the findings of Naik and Srinivasulu Reddy (2020b) and Van Wyk and Scarpa (1999), who emphasized the importance of a stable environment for shrimp health. Survival rates showed no significant differences between treatments, which is consistent with previous Biofloc studies (Naik and Srinivasulu Reddy, 2020b), although the 500 g/ha probiotic treatment showed greater stability. In particular, the 300 g/ha treatment gave better results for final weight, biomass, and feed conversion ratio (FCR), outperforming the manufacturer‑recommended dose. This result contrasts with Llario et al. (2020), who reported limited growth benefits from Bacillus amyloliquefaciens alone, but supports studies emphasizing the improved efficacy of probiotics when used in combination (Souza et al., 2012; Qiu et al., 2023; Preena et al., 2025). The addition of carbon sources such as molasses and rice bran contributed to improved water quality and plankton density, which is consistent with the results of Naik and Srinivasulu Reddy (2020a), Menaga et al. (2023), and Aparna et al. (2024). These improvements probably supported the observed growth and feed efficiency. The synergistic effects of probiotics and biofloces reduction of microbial load, immune stimulation and supplementary nutrition; are well documented (Serra et al., 2015; Santos et al., 2024; Marimuthu et al., 2024) and were reflected in the result of the present study, with FCR values around 2 and final weights between 13.21 and 20.80g. Overall, the use of 300 g/ha of single-cell probiotic proved to be more effective than the recommended dose and provided both biological and economic benefits. This supports the view that optimised probiotic dosing in conjunction with carbon supplementation and biofloc management can significantly improve the sustainability and productivity of shrimp aquaculture (Khanjani et al., 2024; Ziaei-Nejad et al., 2006; Megahed et al., 2019; Kaya et al., 2022). Based on the results of this study, it is recommended that future research focus on the synergistic application of multi-strain probiotic formulations in combination with optimized carbon sources. This approach can further improve growth performance, feed efficiency, and environmental stability in intensive shrimp aquaculture systems. In addition, adjusting probiotic dosing based on empirical evidence rather than manufacturer guidelines may improve both biological efficacy and economic sustainability. Conflict of interest The authors declare that there are no known financial conflicts of interest or personal relationships that could have appeared to influence the work presented in this article. Acknowledgment The authors sincerely express their appreciation to all colleagues and experts who provided assistance at various stages of this research. This study was financially supported by Gorgan University of Agricultural Sciences and Natural Resources, under the research project entitled “Improving the growth performance and survival of Pacific white shrimp (L. vannamei) through the addition of probiotics and biofloc to pond water” (Project ID: 21‑484‑02). بیوفلاک, پروبیوتیک تک‌سل, میگوی پاسفید غربی, پرورش میگو, شاخص رشد Biofloc, Single cell probiotic, Pacific white shrimp, Shrimp culture, Growth performance 75 91 http://isfj.ir/browse.php?a_code=A-10-709-4&slc_lang=fa&sid=1 Rasoul Ghorbani رسول قربانی rasoulghorbani@gmail.com 100319475328460041521 100319475328460041521 Yes Gorgan Unviversity of Agricultural Sciences and Natural Resources دانشگاه علوم کشاورزی و منابع طبیعی گرگان، استان گلستان، گرگان، ایران Seyed Abbas Hosseini سید عباس حسینی seyedabbas_hosseini@yahoo.com 100319475328460041522 100319475328460041522 No Gorgan Unviversity of Agricultural Sciences and Natural Resources دانشگاه علوم کشاورزی و منابع طبیعی گرگان، استان گلستان، گرگان، ایران Fatemeh Abbasi فاطمه عباسی f.abbasi59@yahoo.com 100319475328460041523 100319475328460041523 No Gorgan Unviversity of Agricultural Sciences and Natural Resources دانشگاه علوم کشاورزی و منابع طبیعی گرگان، استان گلستان، گرگان، ایران Meysam Sabzeh میثم سبزه meysam.sabzeh2002@gmail.com 100319475328460041524 100319475328460041524 No Gorgan Unviversity of Agricultural Sciences and Natural Resources دانشگاه علوم کشاورزی و منابع طبیعی گرگان، استان گلستان، گرگان، ایران Hadiseh Kashiri حدیثه کشیری hadiskashiri@gmail.com 100319475328460041525 100319475328460041525 No Gorgan Unviversity of Agricultural Sciences and Natural Resources دانشگاه علوم کشاورزی و منابع طبیعی گرگان، استان گلستان، گرگان، ایران Abdol Azim Fazel عبدالعظیم فاضل f.fazel58@gmail.com 100319475328460041526 100319475328460041526 No Iranian Fisheries Science Research Institute مرکز تحقیقات ذخایر آبزیان آبهای داخلی، استان گلستان، گرگان، ایران