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مقاله علمی – پژوهشی:‌ تفاوت‌‌های ریختی و الگوی رشد بین جمعیت‌‌های مختلف کپور معمولی در ایران Phenotype and growth trend differences in varied populations of common carp in Iran 1 1 از مهم‌ترین اقدامات آغازین در تشکیل یک بانک ژن زنده، تشکیل جمعیت مرجع با تنوع ژنتیکی و ریختی گسترده است. در این مطالعه، تفاوت‌‌های ریختی بین چهار جمعیت مختلف کپور معمولی (شمال ایران، جنوب ایران، چینی و کاسپی) در اولین بانک ژن زنده این ماهی درکشور بررسی شد که در ابتدا تمایز ژنتیکی آنها با نشانگر SNP بر پایه توالی‌‌یابی کل ژنوم از یکدیگر مشخص شد. رشد طولی و وزنی و ارتفاع و قطر بدن در طول دو سال در جمعیت‌‌ها بررسی شد. نتایج نشان‌دهنده آن بود که به دلیل فاصله ژنتیکی کم بین جمعیت‌‌های شمال و جنوب ایران و جمعیت تاتا، لایه‌‌بندی جمعیتی بین این جمعیت‌‌های این مناطق وجود نداشت و نمونه‌‌ها از نظر ژنتیکی یکنواخت بودند. جمعیت‌‌های چینی و کاسپی، تفاوت ژنتیکی را با سه جمعیت دیگر نشان دادند. نتایج آزمون‌های آنالیز واریانس یک‌طرفه و چندطرفه نشان داد که نوع جمعیت، جنسیت، زمان و اثرات تعاملی آنها بر تغییرات تمام اندازه‌گیری‌های بدنی اثرات معنی‌‌دار دارد (05/0> p). بیشترین ارتفاع بدن در همه فصول متعلق به کپور جنوب ایران بود که با شکل بشقابی آن مطابقت دارد که نتیجه انتخاب چندین ساله تکثیرکنندگان این جمعیت‌‌ها در خوزستان با توجه به بازارپسندی آن است. پس از کپور جنوب ایران، کپور تاتا نیز دارای ارتفاع و قطر بیشتر بدن نسبت به دو جمعیت دیگر (شمال ایران و چینی) است. همچنین کمترین تغییرات در مقدار ارتفاع بدن متعلق به کپور جنوب ایران و پس از آن تاتا بود که با یکدست بودن ریخت این جمعیت‌‌ها مطابقت داشت. در همه جمعیت‌‌ها، اندازه‌‌های بدنی در جنس ماده بزرگتر از جنس نر بود به‌‌ویژه بیشترین تفاوت در وزن و طول بدن مشاهده گردید. ارتباط بین طول کل و وزن در سه جمعیت تاتا مجارستان، چینی و شمال ایران کاملاً مشهود بود، اما در جمعیت جنوب ایران، داده‌‌ها بیانگر پراکندگی بودند و کمترین ارتباط بین طول و وزن در این جمعیت مشاهده شد. روند رشد در تاتا بهتر از شمال ایران و چینی است. با توجه به وجود تفاوت‌های ریختی و رشدی بین جمعیت‌ها، باید برنامه‌های انتخاب ژنتیکی با رویکرد بهینه‌سازی و استفاده از تنوع ژنتیکی موجود صورت پذیرد. 2 Introduction Common carp is one of the most widely cultured freshwater fish species worldwide, having been introduced to all continents except Antarctica over the past two centuries. There are different populations of common carp, which can be morphologically distinguished, including Cyprinus carpio carpio, C. c. haematopterus, C. c. viridiviolaceus, and C. c. aralensis, each native to various regions such as Ponto-Caspian, the far East, northern Vietnam, and central Asia. These populations inhabit diverse environments due to their genetic diversity and adaptation capabilities. These genetic diversities lead to distinct phenotypic modifications, including variations in growth rate, body color, scale pattern, body shape, temperature, and hypoxia. One crucial initial step in establishing a living gene bank is to form a reference population that exhibits wide genetic and morphological diversity. Genetic diversity enables species to adapt to changing environments, and it provides a valuable source of individuals and genes for selective breeding programs in aquaculture, which can help meet production and consumer demands for fish strains. Knowledge of the population structure and genetic variation of existing strains/ populations is necessary for fish management and conservation, and many molecular markers and genetic tools have been used to evaluate the genetic diversity of common carp populations. In this study, we investigated the morphological differences between various common carp populations collected for the country's first live gene bank. These populations included Hungarian TATA (Cyprinus carpio carpio), China-originated (C. c. haematopterus), North Iran, and South Iran populations, all of which were initially genetically differentiated from one another using whole-genome single-nucleotide polymorphism (SNP) markers. Methodology The populations (South Iran, North Iran, TATA, and China-originated carp) were cultured under the same conditions in earthen ponds with the flow-through system at the South Iran Aquaculture Research Institute (SIARI). Each individual was tagged with a Passive Integrated Transponder (PIT) tag, and the phenotypic indices, including the length and weight growth, body height, and body width, were investigated in the populations over two years. Samples for genomic analysis were taken from the caudal fin and preserved in ethanol. Genomic DNA was isolated with a Denazist Kit (S-1033 GBS, Iran) with some modifications to the protocol. The purity of DNA was quantified and assessed with a NanoDrop Microvolume Spectrophotometer (Hercuvan Lab System Nano.300, Malaysia), while DNA integrity was evaluated through 1% agarose gel electrophoresis. After ensuring the quality of the extracted DNA, the samples were sent to BGI China for sequencing. The 2100 Bioanalyzer system was used for quality control of DNA samples. FastQC was employed to check the quality of the raw sequence data from high-throughput sequencing pipelines, and data editing was performed using Trimmomatic software version 0.35. The BWA-MEM algorithm was used to align the sequence reads against a large reference genome for alignment. The pairwise population genetic differentiation index (FST) was used to differentiate the populations. It is important to note that the Caspian-sea common carp population was collected for the gene bank but not included in the breeding stage, and no morphological data was recorded for it. This population was included in this study solely to assess the differentiation and genetic identity of the breeding populations from North and South Iran and to understand its relationship with the breeding populations. Results The results indicated no population stratification among the North Iran, South Iran, and TATA populations, as evidenced by the low genetic distance between them. The samples from these regions were relatively genetically uniform. However, the populations from China and the Caspian Sea showed genetic differences compared to the other three populations (Fig 1). Figureure 1: Tagging and recording the phenotypic and biometric indices of selected individuals After the south Iran carp population, the TATA population exhibited the highest average body weight, length, and width at the end of the period. The distribution of individuals based on weight growth in all three populations is skewed, resulting from the selection of superior individuals after one year of breeding. However, at the end of the breeding period, the distribution in the Hungarian and North Iran populations is normal, displaying no skewness or kurtosis. In contrast, the China-originated and south Iran show skewness and kurtosis in one direction (Fig. 2). Figure 2: Cluster analysis of Common carp population originated from five different region Wild refers to Caspian carp Results from one-way ANOVA and Multivariate analysis of variance tests revealed that factors such as population, sex, time, and their interaction influenced all body measurements. The highest body height across all seasons belonged to the South Iran carp, which is consistent with their plate shape. This is likely a result of several years of selective breeding by farmers in Khuzestan, emphasizing marketability. The TATA carp also exhibited greater body height and width than the northern and Chinese populations. Additionally, the South Iran population had the lowest variance in body height, followed by the TATA population, which aligns with the morphological uniformity observed in these groups. In all populations, males exhibited larger body sizes than females, with the most significant differences noted in terms of body weight and length. The TATA, China-originated, and North Iran populations showed a clear correlation between total length and weight. In contrast, the South Iran population demonstrated more dispersion, showing the lowest correlation between length and weight. Notably, the growth trend in the TATA population was superior to that of the Northern Iran and China-originated carp. Discussion and conclusion The common carp populations in Iran originated from Hungary approximately 70 years ago. Since genetic diversity is the origin of morphological differences between populations, the morphological characteristics of these populations closely resemble those of the Hungarian TATA population. This similarity in body shape (characterized by high body height and a plate-like shape) in these populations can be attributed to their genetic background. The genetic differences observed between different common carp populations in Iran highlight the importance of maintaining these diverse populations in live gene banks. Except for the South Iran population, the TATA population showed satisfactory growth compared to other populations. The variations in growth between these populations point to genetic influences, suggesting potential for improvement through selective breeding. Additionally, sex has been identified as a factor that affects body measurements, which should be taken into account in selection and breeding programs. Weight and length are traits that exhibit both genetic and phenotypic correlations. Nielsen et al. (2010) observed a relatively high heritability for weight, length, and survival during the six months leading up to harvest in common carp, indicating that selective breeding for growth and survival is likely to be successful. Given the substantially estimated within-population additive genetic variance for weight, there is considerable potential for creating a synthetic population with a strong genetic foundation. This could be utilized to establish a selective breeding program for common carp at SIARI. The findings of this study are valuable for developing genetic selection programs for common carp and enhancing the potential for utilizing this species' genetic resources. This research can serve as a foundation for future studies and practical applications in various aspects of common carp aquaculture. In aquaculture programs, it is essential to prioritize the maintenance and increase of genetic diversity within different common carp populations. Conflict of Interest The authors declare that there are no conflicts of interest. Acknowledgment We would like to express our gratitude to the Khuzestan Governorate, Iran's Vice-Presidency for Science, Technology, and Knowledge-Based Economy Affairs, and the Iranian Fisheries Research Institute (IFSRI) for their financial support of common carp breeding project. This study is part of project No. 124-74-12-033-98043-981235. 1 17 2025/04/27 1404/2/7 2025/09/1 1404/6/10 آیه سادات صدر Ayeh Sadat Sadr سازمان تحقیقات، آموزش و ترویج کشاورزی Iran AyehSadr@gmail.com سمیرا ناظم رعایا Samira Nazemroaya سازمان تحقیقات، آموزش و ترویج کشاورزی Iran samira.nazemroaya@gmail.com محمد یونس زاده فشالمی Mohammad Youneszadeh Feshalami سازمان تحقیقات، آموزش و ترویج کشاورزی Iran m_yooneszadeh@yahoo.com فاطمه حکمت پور Fatemeh Hekmatpour سازمان تحقیقات، آموزش و ترویج کشاورزی Iran hekmatpourf@gmail.com حسین هوشمند Hossein Houshmand سازمان تحقیقات، آموزش و ترویج کشاورزی Iran houshmand.hossein@gmail.com مینا آهنگرزاده Mina Ahahangarzadeh سازمان تحقیقات، آموزش و ترویج کشاورزی Iran Minaahangarzadeh@gmail.com مهدی گلشن Mahdi Golshan سازمان تحقیقات، آموزش و ترویج کشاورزی Iran mahdigolshan@gmail.com محمود حافظیه Mahmoud Hafezieh مؤسسه تحقیقات علوم شیلاتی کشور، سازمان تحقیقات، آموزش و ترویج کشاورزی، تهران، Iran منصور شریفیان Mansour Sharifian سازمان تحقیقات، آموزش و ترویج کشاورزی Iran sharif_23@yahoo.com الهام جرفی Elham Jorfi سازمان تحقیقات، آموزش و ترویج کشاورزی Iran ejorfi@gmail.com فرحناز کیان ارثی ننادگانی Farahnaz Kianersi پژوهشکده آبزی‌پروری جنوب کشور، مؤسسه تحقیقات علوم شیلاتی کشور، سازمان تحقیقات، آموزش و ترویج کشاورزی، اهواز، ایران Iran Cyprinus carpio Genetic diversity growth Selection Cyprinus carpio تنوع ژنتیکی رشد به‌‌گزینی 1. 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مقاله علمی – پژوهشی:‌ تأثیر پودر قارچ دکمه‌ای سفید بر ظرفیت آنتی اکسیدانی، سیستم ایمنی و بیان سایتوکاین‌های ایمنی در ماهی تیلاپیای نیل (Oreochromis niloticus) Effect of white button mushroom meal on antioxidant capacity, immune systems, and expression of immune-related cytokine genes in Nile Tilapia (Oreochromis niloticus) 1 1 این مطالعه برای بررسی اثرات مکمل غذایی با پودر قارچ دکمه‌ای سفید (Agaricus bisporus) بر سیستم ایمنی و آنتی اکسیدانی ماهی تیلاپیا نیل، انجام شد. در یک آزمایش کنترل شده، ماهی‌ها با جیره‌های 0، 5/0، 1 و 2 درصد پودر قارچ طی 56 روز تغذیه شدند. فعالیت آنزیم‌های آنتی اکسیدانی شامل کاتالاز، سوپراکسید دیسموتاز، گلوتاتیون پراکسیداز، سطوح آنتی اکسیدان کل و پراکسیداسیون لیپیدی با استفاده از تعیین سطح مالون دی آلدهید (MDA) سرم به همراه با پاسخ‌های ایمنی از جمله فعالیت باکتری‌کش سرم، غلظت ایمونوگلوبولین M (IgM) و سطوح لیزوزیم مورد ارزیابی قرار گرفت. همچنین بیان ژن سایتوکاین‌‌های اینترلوکین-6، شاخص نکروز توموری آلفا و اینترلوکین-1 بتا در بافت سر کلیه و طحال نیز اندازه‌گیری شد. نتایج نشان داد که ماهی‌های تغذیه شده با جیره‌های غنی‌شده با قارچ به‌ویژه در سطوح ۱ و ۲ درصد، افزایش قابل‌توجهی در فعالیت آنزیم‌های آنتی‌اکسیدانی و کاهش MDA نشان دادند. شاخص‌های ایمنی ((IgM، فعالیت لیزوزیم و بیان سایتوکاین‌ها به طور قابل‌توجهی افزایش یافته  که نشان‌دهنده بهبود سیستم ایمنی است. با توجه به نتایج مطالعه حاضر، پودر قارچ دکمه‌ای سفید یک مکمل غذایی امیدوارکننده برای تقویت سلامت ماهی و عملکرد ایمنی است و به ‌طور بالقوه نیاز به آنتی‌بیوتیک را در آبزی‌پروری کاهش می‌دهد. همچنین یافته‌های تحقیق حاضر، نشان‌دهنده نقش افزودنی‌های غذایی در پرورش پایدار ماهی است، اگرچه تحقیقات بیشتری در مورد اجزاء زیست‌فعال موجود در قارچ دکمه‌ای سفید و اثرات آن بر آبزیان نیاز است. 2 Introduction Nile tilapia (Oreochromis niloticus), a globally significant aquaculture species, plays a critical role in addressing food security due to its high nutritional value and adaptability to diverse farming environments (Shafiujjaman et al., 2024). However, the intensification of aquaculture practices has introduced stressors that adversely affect fish health and increase vulnerability to diseases (Li et al., 2022). Conventional solutions like antibiotics and chemical therapeutics, while effective, raise concerns about environmental degradation, drug resistance, and food safety. Functional feed additives, particularly those derived from natural sources, have emerged as promising alternatives for enhancing fish health and immune function. White button mushrooms (Agaricus bisporus), recognized for their rich bioactive compound profile—including polysaccharides, phenolics, and antioxidants—have shown potential in modulating immune responses and oxidative stress (Habib et al., 2021). Despite the demonstrated benefits of mushrooms in aquaculture, the molecular mechanisms underlying their effects, particularly on cytokine-mediated immune responses, remain underexplored. This study evaluates the impact of dietary supplementation with A. bisporus powder on antioxidant activity, immune parameters, and cytokine gene expression in Nile tilapia, providing valuable insights into its potential as a sustainable aquafeed additive (Habib et al., 2021; Harikrishnan et al., 2018). Methodology White button mushrooms were sourced locally, cleaned, sliced, dried at 50°C for 24 hours, and ground into powder. Experimental diets were formulated to include 0%, 0.5%, 1%, and 2% mushroom powder (Amiri et al., 2018; Hoseinifar et al., 2019). Diets were adjusted to maintain iso-nitrogenous and iso-caloric properties and pelletized into uniform 2 mm pellets for feeding. A total of 240 juvenile Nile tilapia, weighing approximately 3.1±0.3 g, were distributed randomly into 12 aquaria (250 liters) in a completely randomized design. Each treatment was replicated thrice, with 20 fish per aquarium. The fish were fed their respective diets to apparent satiation four times daily over 56 days. Water quality parameters, including temperature (28.01±0.10°C), pH (7.13±0.20), and dissolved oxygen (5.23±0.18 mg/L), were maintained within optimal ranges. At the conclusion of the feeding trial, six fish per treatment group were anesthetized using 0.3 mL/L of 2-phenoxyethanol. Blood samples were collected via caudal vein puncture for serum isolation. Immune tissues, specifically the head kidney and spleen, were aseptically excised and stored in RNA later solution for subsequent molecular analysis. Antioxidant enzyme activities, including catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx), as well as total antioxidant capacity (T-AOC), were quantified using commercial kits. Lipid peroxidation was assessed by measuring serum malondialdehyde (MDA) levels. Immune parameters, including serum bactericidal activity, immunoglobulin M (IgM) concentrations, and lysozyme levels, were evaluated using standard methodologies. RNA was extracted from head kidney and spleen tissues and reverse-transcribed into cDNA. Quantitative PCR (qPCR) was used to measure the expression of immune-related cytokines, including interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and interleukin-1 beta (IL-1β). Relative expression levels were normalized to β-actin, and fold changes were calculated using the 2−ΔΔCt method. Data were analyzed using one-way ANOVA followed by Tukey's post hoc test for pairwise comparisons. Results were presented as mean ± standard deviation, with statistical significance set at p<0.05. Results Dietary supplementation with mushroom powder significantly enhanced antioxidant enzyme activities, with the 1% and 2% treatments showing the greatest increases in CAT, SOD, and GPX activities compared to the control group (p<0.05). Total antioxidant capacity (T-AOC) was similarly elevated in these groups, while serum MDA levels, indicative of lipid peroxidation, were markedly reduced. These results underscore the potent antioxidant properties of A. bisporus, attributed to its phenolic and flavonoid compounds, which act as radical scavengers and reduce oxidative stress. Comparable studies have demonstrated the role of mushroom-derived antioxidants in enhancing oxidative resilience in fish, including common carp and rainbow trout. Mushroom supplementation significantly increased serum bactericidal activity, IgM concentrations, and lysozyme levels. The 1% and 2% inclusion levels elicited the strongest responses (p<0.05), reflecting improved non-specific immune defenses. Polysaccharides such as beta-glucans, abundant in mushrooms, are known to activate macrophages and stimulate the release of immune mediators, enhancing humoral immunity. The observed immune improvements align with previous findings in aquaculture species, where mushroom-based diets augmented pathogen resistance and overall health. The expression of pro-inflammatory cytokines IL-6, TNF-α, and IL-1β in head kidney and spleen tissues was significantly upregulated in fish fed mushroom-enriched diets, particularly at the 1% and 2% inclusion levels (p<0.05). Cytokines play a pivotal role in coordinating immune responses, mediating inflammation, and facilitating pathogen clearance. The enhanced expression observed in this study likely results from mushroom-derived polysaccharides interacting with toll-like receptors on immune cells, triggering downstream signaling pathways. These findings corroborate previous studies that reported similar cytokine upregulation in fish fed functional diets containing prebiotics or plant-derived compounds. The dual antioxidant and immunomodulatory effects of A. bisporus are attributed to its diverse bioactive compounds, including phenolics, flavonoids, and beta-glucans. These molecules neutralize reactive oxygen species (ROS), enhance the activity of antioxidant enzymes, and stimulate immune responses. Additionally, mushrooms may influence gut microbiota composition, indirectly bolstering immune function. While this study provides compelling evidence of the benefits of A. bisporus, further research is warranted to isolate specific bioactive compounds and elucidate their precise mechanisms of action. The findings of this study highlight the potential of white button mushroom powder as a sustainable alternative to antibiotics and chemical treatments in aquaculture. By enhancing both antioxidant defenses and immune responses, mushroom supplementation can improve fish health, reduce disease incidence, and mitigate environmental risks associated with conventional therapeutics. Functional diets incorporating natural additives like A. bisporus align with the goals of sustainable aquaculture, promoting health and productivity without compromising ecological integrity. Discussion and conclusion This study demonstrates the efficacy of dietary supplementation with white button mushroom powder in enhancing the antioxidant and immune systems of Nile tilapia. The 1% and 2% inclusion levels yielded the most significant improvements in antioxidant enzyme activities (CAT, SOD, GPX), total antioxidant capacity (T-AOC), and reductions in serum MDA levels. Immune parameters, including bactericidal activity, IgM concentrations, and lysozyme levels, were also significantly enhanced. Furthermore, mushroom supplementation upregulated the expression of cytokines IL-6, TNF-α, and IL-1β, indicating robust immunomodulatory effects. These findings underscore the potential of A. bisporus as a functional feed additive, offering a natural and sustainable approach to improving fish health and reducing reliance on chemical therapeutics in aquaculture. Future research should focus on identifying and characterizing the specific bioactive compounds responsible for these effects and optimizing their application in aquafeeds. Conflict of interest We wish to confirm that there are no known conflicts of interest associated with this study. Acknowledgment The authors are grateful to the Fisheries Lab at Science and Research Branch Institute and the Aquatics Production & Trade Union of Iran for their kind cooperation. 19 31 2025/04/272024/11/29 1403/9/9 2025/09/12025/09/1 1404/6/10 کسری لطفی Kasra Lotfi گروه شیلات، واحد علوم و تحقیقات، دانشگاه آزاد اسلامی Iran kasra.lotfi1995@yahoo.com مهدی شمسایی مهرجان Mehdi Shamsaie Mehrgan گروه شیلات، واحد علوم و تحقیقات، دانشگاه آزاد اسلامی Iran drshamsaie@gmail.com فرهاد فرودی Farhad Forodi گروه شیلات، واحد علوم و تحقیقات، دانشگاه آزاد اسلامی Iran f.foroudi@gmail.com هومن رجبی اسلامی Houman Rajabi Islami گروه شیلات، واحد علوم و تحقیقات، دانشگاه آزاد اسلامی Iran rajabi.h@srbiau.ac.ir سید پژمان حسینی شکرابی Seyed Pezhman Hosseini Shekarabi مؤسسه تحقیقات علوم شیلاتی کشور Iran hosseini.pezhman@yahoo.com Nile tilapia White button mushroom Antioxidant enzymes Immune system modulation Cytokine gene expression تیلاپیا نیل قارچ دکمه‌ای سفید آنزیم‌های آنتی‌اکسیدان تعدیل سیستم ایمنی بیان ژن سایتوکاین Adineh, H., Naderi, M., Nazer, A., Yousefi, M., Ahmadifar, E., 2021. 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مقاله علمی – پژوهشی:‌ ریزپوشانی باکتری پروبیوتیک Lactobacillus plantarum با ژلاتین- پروتئین هیدرولیز شده ماهی با استفاده از روش خشک‌کن انجمادی و ارزیابی خواص آنتی‌اکسیدانی Microencapsulation of Lactobacillus plantarum with gelatin-hydrolyzed fish protein using the freeze-drying method and evaluation of antioxidant properties 1 1 استفاده از پروتئین‌ها در اسکلت ریزپوشانی باکتری پروبیوتیک Lactobacillus plantarum می‌تواند گزینه مناسبی برای توسعه محصولات غذایی پروبیوتیک باشد. در این پژوهش، تأثیر ترکیب برابر ژلاتین و پروتئین هیدرولیز شده ماهی (نسبت ۵۰: ۵۰) بر پایداری L. plantarum بررسی شده و اثر شرایط فرآوری بر خصوصیات فیزیکوشیمیایی پودر حاصل از ریزپوشانی این باکتری به روش خشک‌کردن انجمادی مورد ارزیابی قرار گرفته است. در بررسی ساختار سطحی ذرات ریزپوشانی‌شده با استفاده از میکروسکوپ الکترونی روبشی (SEM) مشاهده شد، ذرات دارای ساختاری نسبتاً یکنواخت و کروی بودند و سطح آنها بافتی متراکم و بدون ترک را نشان داد. میزان رطوبت 5/7 درصد و فعالیت آبی مطلوب 02/0±32/0 بود. علاوه‌براین، ظرفیت آنتی‌اکسیدانی‌ لایه‌های محافظ پس از ریزپوشانی کردن بررسی شد. نتایج نشان داد فعالیت مهار رادیکال‌ (DPPH) تا 33/25٪ و پتانسیل آنتی‌اکسیدانی احیاءکننده آهن (FRAP) نیز 187/5 بوده است (05/0p<) .همچنین همبستگی مثبت قوی بین غلظت و فعالیت آنتی‌اکسیدانی بر اساس آزمون  DPPH و FRAP به‌ترتیب 8347/0= R2 و 9265/0= R2 وجود دارد (05/0p<). پوشش به عنوان یک مانع محافظ برای مهار مؤثر رادیکال‌های فعال اکسیژن عمل می‌کند. این ویژگی، امکان استفاده از این ترکیب را در صنایع غذایی و دارویی به ‌عنوان جایگزینی طبیعی و ایمن برای آنتی‌اکسیدان‌های مصنوعی نظیر BHA فراهم می‌‌آورد. 2 Introduction Among various probiotic strains, lactic acid bacteria (LAB) have been the most extensively studied (Anal and Singh, 2007). Lactobacillus plantarum, a prominent LAB species, exhibits a wide range of bioactivities, including inhibition of pathogenic bacteria, improvement of gut microbiota, modulation of the immune system, and regulation of glucose and lipid metabolism. These features make L. plantarum a promising candidate for promoting gastrointestinal, metabolic, and cardiovascular health (Collantes et al., 2023). Nevertheless, like many other probiotic species, L. plantarum is susceptible to environmental stresses such as high temperature, low pH, salt, oxygen, and bile salts during processing and digestion, which may limit its functionality and efficacy (Ranadheera et al., 2010). One promising approach to improve probiotic viability is microencapsulation, a technique that involves embedding microorganisms within protective coatings or matrices. This not only shields probiotics from adverse conditions but also allows for targeted and controlled release at the desired site in the gastrointestinal tract. Additionally, microencapsulation can mask undesirable flavors in probiotic-containing food products (Fraj et al., 2021). Several encapsulation techniques are available, including extrusion, emulsification, spray drying, freeze drying, and cold spraying, each with its specific advantages and limitations (Luca and Oroian, 2021). Among these, freeze drying has attracted considerable attention due to its capacity to preserve the structure and bioactivity of probiotics. The selection of appropriate wall materials during freeze drying is critical for minimizing cellular damage and enhancing bacterial viability. Moreover, controlling parameters such as temperature and residual moisture is essential to maintain probiotic stability. L. plantarum thrives in slightly acidic conditions (pH 5.0–6.2), a factor that should be considered when designing preservation and storage conditions (Pramono et al., 2025). The choice of wall material plays a vital role in determining the efficiency, release profile, and functional properties of the microcapsules (Ozdemir et al., 2021). Commonly used wall materials include polysaccharides, proteins, and lipids. Among them, proteins are desirable due to their biocompatibility, low cost, availability, and desirable physicochemical characteristics (Vaziri et al., 2018). Gelatin, a widely used protein-based material, is biodegradable, biocompatible, and cost-effective, exhibiting a high molecular interaction capacity, making it suitable for encapsulation applications (Bastos et al., 2021). On the other hand, fish protein hydrolysate (FPH), derived from aquatic by-products such as skin, scales, and bones, is rich in bioactive peptides with antioxidant, antimicrobial, anti-inflammatory, immunomodulatory, and antihypertensive properties, and is widely used in the food and pharmaceutical industries (Nemati et al., 2021). These peptides, produced through enzymatic hydrolysis of fish proteins, possess low molecular weight, high solubility, and desirable bioactivity, making them effective protective agents in the microencapsulation of probiotics such as L. plantarum (González-Serrano et al., 2022). Due to its antioxidant capacity, FPH protects probiotic cells against oxidative stress during freeze-drying and storage. Furthermore, its antibacterial peptides contribute to enhanced product safety. When combined with gelatin, FPH forms a protective matrix that shields bacteria from harsh gastric conditions (Nemati et al., 2024). Additionally, the peptides and amino acids present in FPH can serve as a nutrient source for probiotics, thereby enhancing their viability (Shori, 2017). From an environmental perspective, the utilization of fish waste for FPH production represents a sustainable and cost-effective approach (Kristinsson and Rasco, 2000). Given these advantages, the present study employed a combination of commercial bovine gelatin and fish protein hydrolysate as a wall material for the microencapsulation of L. plantarum using freeze-drying technology. This research aimed to evaluate the potential of this biopolymeric blend in enhancing the viability and stability of L. plantarum under adverse environmental conditions. Methodology A 50g sample was mixed with 100 ml of distilled water and heated at 85°C for 20 min to inactivate endogenous enzymes. After cooling, pepsin was added (1% of total protein) and hydrolysis occurred at pH 5.8 and 58°C for 90 min. The mixture was then heated at 90°C for 10 min to inactivate the enzyme, cooled, centrifuged, and the supernatant was collected and freeze-dried (Ojagh et al., 2012). To prepare L. plantarum cultures, 1g of lyophilized powder was dissolved in 9 ml of sterile phosphate buffer, and 0.1 ml of the suspension was spread on an MRS agar plate. After anaerobic incubation at 37°C for 24 h, the cells were scraped off the plate with 2 ml of sterile phosphate buffer, and the resulting slurry was centrifuged at 13,000×g for 1 min. Gelatin (5g) and fish protein hydrolysate (5g) were dissolved in 1 liter of distilled water, maintained at 45°C for 30 min, and cooled to 25°C. A L. plantarum suspension (1×10⁸ CFU/g) was added, and the mixture was homogenized at 350 rpm for 15 min (Heinzelmann et al., 2000). The suspension was frozen at −80°C for 24 h and then freeze-dried for 3-4 days at 30 Pa with a condenser temperature of -60°C (Machado et al., 2022). The DPPH radical scavenging activity was assessed according to the method described by Dadmehr et al. (2024). In brief, 100 µl of each sample solution was mixed with 3.9 ml of DPPH solution. Absorbance was measured at 518 nm using a spectrophotometer at 5-min intervals. Calibration was performed in the range of 5–30 min. The FRAP assay was performed as previously described by Dadmehr et al. (2024). The FRAP reagent was prepared by mixing acetate buffer (300 mM), TPTZ solution (10 nM in 40 mM HCl), and FeCl₃ (20 mM) in a 10:1:1 (v/v/v) ratio and incubated at 37°C. Then, 100 µl of the sample was added to 3 ml of the FRAP reagent, and absorbance was recorded at 593 nm. A calibration curve was constructed within 5–30 min. BHA was also analyzed under the same conditions as a reference antioxidant. Results Moisture content was 7.5% and water activity was 0.32 ± 0.02. Particle size analyzer output showed that the average diameter of microcapsules in the final freeze-dried product was 1.62 ± 0.47 μm (mean ± ST). Phase contrast microscopy observations showed relatively uniform and round capsules in the final product. SEM observation showed that L. plantarum was successfully encapsulated with gelatin-hydrolyzed fish protein and the capsules produced were mostly spherical and elliptical. The results of this study showed that the DPPH free radical activity increased with increasing concentration. (p<0.05) Microencapsulated Lactobacillus had the highest antioxidant activity of 25.33% at a concentration of 250 mg/ml, and the DPPH free radical activity at this concentration was not significantly different from the synthetic antioxidant BHA. (p>0.05). The results of this study showed that the reducing power (FRAP) increased with increasing concentration. Microencapsulated L. plantarum had the highest antioxidant activity at a concentration of 250 mg/ml, and the FRAP at this concentration was significantly higher than the synthetic antioxidant (BHA) (187.5 μmol/g) (p< 0.05). Discussion and conclusion The results showed that the microencapsulated wall containing Lactobacillus, combined with gelatin and hydrolyzed protein, had a moisture content of 7.5% and a water activity of 0.32 ± 0.02, indicating the physical stability and good shelf life of the system. The low water activity, which prevents the growth of unwanted microorganisms and chemical degradation, plays a crucial role in protecting probiotics. Low moisture also prevents the fragility of the structure (Hasniah, 2022). The use of gelatin and hydrolyzed protein has created a resistant and controlled matrix in water retention, increased mechanical stability and resistance to environmental conditions (Mushtaq et al., 2022). Examination of the surface structure of the microencapsulated particles using scanning electron microscopy (SEM) indicated the success of the microencapsulation process with a combination of gelatin and hydrolyzed fish protein. The particles had a relatively uniform and spherical structure and their surface showed a dense texture without cracks. It has been confirmed that the stability of probiotics decreases under oxidative conditions. Therefore, the use of agents with antioxidant activity can help increase the survival of microencapsulated probiotics (Dadmehr et al., 2024). The compounds resulting from protein hydrolysis, depending on the type of initial protein and the hydrolysis method, can include free amino acids, small peptides, and in some cases, larger protein molecules remaining (Ramezani et al., 2018). Gelatin and bioactive peptides have been reported as natural antioxidants, which were used as a protective layer in this study (Yaghoubzadeh et al., 2019; Nurilmala et al., 2020). The antioxidant activity of gelatin is due to its peptides, which contain high amounts of arginine, tyrosine, and phenylalanine and are thought to have higher antioxidant activity (Shiao et al., 2021). These coatings may enhance the antioxidant activity of microencapsulated L. plantarum. The results obtained from the antioxidant tests showed that the radical scavenging activity and reducing power of the microencapsulated samples increased significantly with increasing concentration. In the DPPH test, the percentage of free radical scavenging increased from 10.25% at a concentration of 15.625 mg/ml to 25.33% at a concentration of 250 mg/ml. Similarly, in the FRAP test, the reducing power value increased from 0.297 µmol Fe²⁺/g at a concentration of 625.15 mg/ml to 187.5 µmol Fe²⁺/g at a concentration of 250 mg/ml. This dose-dependent increase indicates the presence of bioactive compounds with antioxidant properties in the coating formulation. Hydrolyzed fish proteins are rich in peptides with functional groups (such as -NH₂ and -COOH) that can react with free radicals. (Harnedy and FitzGerald, 2012) In addition, the gelatinous structure of the matrix may also play a role in stabilizing the antioxidant compounds (Shahidi and Ambigaipalan, 2015). The test results are in line with similar studies. For example, Wang et al. (2017) in a similar study showed that microcapsules containing peptide-coated probiotics showed higher DPPH and FRAP activity at higher concentrations. Also, the study by Mehdipour Biregani and Ahari (2021) reported that hydrolysates derived from fish protein have a high ability to absorb free radicals. The results obtained indicate that the developed microencapsulation system not only increased the stability of L. plantarum, but also provided significant antioxidant properties, which can be used as a bioactive carrier in functional products. Wang et al. reported that microencapsulation of L. plantarum with gelatin increased its stability and antioxidant properties under different conditions (Wang et al., 2019). In another study, Cui et al. (2021) showed that the use of marine-derived peptides in the coating of microcapsules led to a significant improvement in antioxidant activity in DPPH and ABTS assays (Cui et al., 2021). They attributed this effect to the presence of amine and hydrophobic active groups in the coating composition. Razavi et al. (2020) reported that coating L. plantarum with gum arabic and alginate increased antioxidant activity, but the extent of this increase was less than that of protein coatings such as gelatin and FPH. Also, Hebert et al. (2017) pointed out the synergistic effects between probiotic bacteria and marine bioactive peptides, which led to the enhancement of biological functions, including antioxidant properties (Hebert et al., 2017). The results of this study are consistent with the study of Wang et al. (2019) who showed that gelatin microencapsulation can significantly increase the antioxidant activity of L. plantarum. Also, Cui et al. (2021) reported that the combination of marine peptides with probiotic bacteria resulted in high iron (Fe³⁺) reduction potential and enhanced antioxidant function, which is consistent with the results of the FRAP assay. In addition, the study conducted by Razavi et al. (2020) who investigated the antioxidant activity of L. plantarum microencapsulated with gum arabic and alginate confirmed a dose-dependent increase in FRAP; however, the reducing power was reported to be lower compared to protein functional groups in peptides derived from protein sources, which play an important role in electron transfer in the FRAP assay (Hebert et al., 2017). In the present study, the reduction in water activity due to the use of a combination of gelatin and hydrolyzed rainbow trout proteins can help create a limiting environment for destructive reactions (such as oxidation and enzymatic degradation), which is consistent with the results of previous studies. Microencapsulation of L. plantarum with a combination of gelatin and hydrolyzed fish protein not only increases bacterial survival under oxidative conditions, but also plays an effective role in enhancing antioxidant properties through chemical interactions with radicals and metal ions. This feature allows the use of this compound in the food and pharmaceutical industries as a natural and safe alternative to synthetic antioxidants such as BHA. Conflict of Interest The authors declare that they have no conflict of interest Acknowledgment This research was supported by Iranian Fisheries Science Research Institute (IFSRI). 33 45 2025/04/272024/11/292025/06/25 1404/4/4 2025/09/12025/09/12025/09/1 1404/6/10 زهرا یعقوب زاده zahra yaghoubzadeh موسسه تحقیقات علوم شیلاتی کشور، ، سازمان تحقیقات، آموزش و ترویج کشاورزی Iran za_yaghoub@yahoo.com فائزه ترک پهنابی Faezeh Turk Pahnabi موسسه تحقیقات علوم شیلاتی کشور، ، سازمان تحقیقات، آموزش و ترویج کشاورزی Iran pahnabifaezeh@gmail.com Hydrolyzed fish protein Lactobacillus plantarum microencapsulation antioxidant activity پروتئین هیدرولیز شده ماهی Lactobacillus plantarum ریزپوشانی فعالیت آنتی اکسیدانی Albadran, H.A., Chatzifragkou, A., Khutoryanskiy, V.V. and Charalampopoulos, D., 2015. Stability of probiotic Lactobacillus plantarum in dry microcapsules under accelerated storage conditions. Food Research International, 74: 208-216.##Arepally, D. and Goswami, T.K., 2019. 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مقاله علمی – پژوهشی:‌ تأثیر سطوح مختلف لیزین و هیستیدین در جیره فیل‌ماهی (Huso huso) بر شاخص‌های آنتی‌اکسیدانی کبد Effect of different lysine and histidine levels in the diet of beluga sturgeon (Huso huso) on liver antioxidant indices 1 1 هدف از این پژوهش، بررسی اثرات سطوح مختلف لیزین و هیستیدین در جیره غذایی فیل‌ماهی(Huso huso) بر عملکرد شاخص‌های آنتی‌اکسیدانی و میزان استرس اکسیداتیو در بافت کبد بود. بدین منظور، تعداد 315 عدد فیل‌ماهی با میانگین وزنی اولیه 2 ± 20 گرم به صورت تصادفی و پس از دو هفته سازگاری، در  7 تیمار با سه تکرار در 21 مخزن فایبرگلاس 2000 لیتری با تراکم 15 عدد در هر مخزن توزیع شدند. سطوح لیزین شامل 4/0و 8/0 درصد و سطوح هیستیدین شامل2/0 ، 5/0و 8/0 درصد در جیره پایه بود. پس از 8 هفته تغذیه، نمونه‌برداری از بافت کبد جهت اندازه‌گیری شاخص‏های آنتی اکسیدانی شامل فعالیت آنزیم‌های سوپراکسید دیسموتاز (SOD)،کاتالاز (CAT)،گلوتاتیون‌پراکسیداز (GPx) و شاخص‏های پراکسیداسیون لیپیدی: مالون‌دی‌آلدئید (MDA) صورت گرفت. نتایج نشان داد که بالاترین سطوح ترکیبی (8/0 درصد لیزین و 8/0 درصد هیستیدین) باعث بهبود معنی‌دار فعالیت سوپراکسید دیسموتاز، کاتالاز، گلوتاتیون پراکسیداز و کاهش سطح مالون دی آلدئید درکبد فیل‌ماهی گردید (05/0p<). یافته‌های این مطالعه نشان‌دهنده نقش کلیدی لیزین و هیستیدین در سطوح بهینه در تعدیل استرس اکسیداتیو و ارتقاء سلامت کبد در فیل‌ماهی است. 2 Introduction The beluga sturgeon (Huso huso) is one of the most economically important fish species in aquaculture, particularly for caviar production (Mohseni et al., 2016). Oxidative stress is a major challenge in aquaculture, leading to cellular damage, impaired growth, and increased susceptibility to diseases. It occurs when the balance between the production of reactive oxygen species (ROS) and the antioxidant defense system is disrupted. Fish, like other vertebrates, rely on endogenous antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR) to neutralize oxidative stress (Naji et al., 2023). Nutritional strategies play a fundamental role in modulating oxidative stress (Feng et al., 2013: Huang et al., 2021; Huang et al., 2022). Among various nutrients, amino acids have gained increasing attention due to their involvement in antioxidant defense mechanisms. Lysine is a key essential amino acid required for protein synthesis, enzyme function, and immune response (Ahmed and Ahmed, 2021; Dou et al., 2023), while histidine serves as a precursor for carnosine, a potent antioxidant that protects tissues against oxidative stress (Ramos-Pinto et al., 2021; Hussein et al., 2023). Studies suggest that histidine supplementation can enhance antioxidant capacity by scavenging free radicals and reducing lipid peroxidation. Despite their importance, the optimal levels of lysine and histidine required to mitigate oxidative stress in beluga sturgeon remain unclear. This study aimed to investigate the effects of different dietary levels of lysine and histidine on antioxidant enzyme activity and oxidative stress markers in the liver of beluga sturgeon. The findings will contribute to developing optimized dietary formulations to improve fish health and performance in aquaculture. Methodology At International Sturgeon Research Institute, a total of 315 juvenile beluga sturgeons (Huso huso) with an initial mean weight of 20±2 g housed in recirculating aquaculture systems (RAS) equipped with biological filtration, aeration, and temperature control to ensure optimal conditions. The experimental diets were formulated based on a fishmeal-based basal diet with varying levels of lysine (0.4% and 0.8%) and histidine (0.2%, 0.5%, and 0.8%) including control and six Treatments (Control: 0% Lysine and Histidine, T1: 0.4% Lysin -0% Histidine; T2: 0.8% Lysin -0% Histidine; T3:0% Lysin -0.2% Histidine; T4:0% Lysin -0.5% Histidine;T5: 0.4% Lysin -0.2% Histidine; T6:0.8% Lysin -0.8% Histidine), each consisting of three replicates (15 fish per replicate). The ingredients were finely ground, mixed, pelleted, and dried before storage. The fish were fed four times daily (08:00, 14:00, 20:00 and 2:00) at a rate of 3% of body weight for a period of eight weeks. Feed intake was monitored daily to ensure uniform consumption across treatments. At the end of the feeding trial, fish were euthanized with clove powder and liver samples were collected and stored at -80°C for biochemical analysis (Bahrami et al.,2022). In the present study liver antioxidant enzymes activity including Catalase, Superoxide dismutase, Glutathione peroxidase, Glutathione reductase, as well as the lipid peroxidation index, malondialdehyde were evaluated in Huso huso fed with different levels of lysine and histidine. Enzymes activity was determined using spectrophotometric assays, following standard protocols. Results Catalase activity in samples that received the combination of lysine and histidine showed a significant increase. The highest catalase activity was observed in the treatment with levels of 0.8% lysine and 0.8% histidine (P<0.05).  The difference between treatments 5 and 6 was not significant (P>0.05), while a significant difference was observed with other groups (P<0.05). Analysis of superoxide dismutase activity showed that adding high amounts of lysine and histidine (0.8 lysine and 0.8 histidine) increased the highest level of activity of this enzyme. Unlike catalase, the difference between treatments 5 and 6 was significant (P<0.05). GPx activity increased significantly with increasing histidine and lysine. Treatment 6 showed the highest level of activity (P<0.05). GR activity gradually increased with increasing histidine and lysine levels in the diet. The highest level of activity of this enzyme was also related to treatment 6. Discussion and conclusion The observed increase in antioxidant enzyme activity (SOD, CAT, and GPx) in response to higher lysine and histidine supplementation suggests that these amino acids play a crucial role in enhancing the antioxidant defense system of beluga sturgeon. Histidine, as a precursor of carnosine, likely contributed to the neutralization of reactive oxygen species, thereby protecting liver tissue from oxidative damage (Holeček, 2020; Sui et al., 2023). Additionally, the reduction in MDA levels supports the hypothesis that optimal dietary amino acid levels mitigate lipid peroxidation and oxidative stress. Similar findings have been reported in other fish species, indicating that adequate lysine and histidine supplementation improves oxidative stability and overall health. This study highlights the importance of lysine and histidine in regulating oxidative stress and enhancing hepatic antioxidant defense mechanisms in beluga sturgeon. The optimal dietary levels (0.8% lysine and 0.8% histidine) significantly improved antioxidant enzyme activity while reducing lipid peroxidation markers. These findings provide valuable insights into nutritional strategies for improving fish health in aquaculture. Future studies should investigate long-term effects of amino acid supplementation on growth performance, immune response, and metabolic functions in sturgeon. Conflict of Interest The authors declare no conflict of interest Acknowledgment The authors wish to extend their heartfelt gratitude to the International Sturgeon Research Institute at the Agricultural Research Education and Extension Organization (AREEO). We also sincerely appreciate the support from the Beluga Sturgeon Farm for providing fish and diets essential for our research. It is important to note that this study did not receive any specific funding from public or non-profit sector agencies. 47 58 2025/04/272024/11/292025/06/252025/01/27 1403/11/8 2025/09/12025/09/12025/09/12025/09/1 1404/6/10 محمود محسنی Mahmoud Mohseni انستیتو تحقیقات بین المللی ماهیان خاویاری، موسسه تحقیقات علوم شیلاتی کشور، سازمان تحقیقات، آموزش و ترویج کشاورزی، رشت، گیلان Iran mahmoudmohseni73@gmail.com سارا مهدی زاده Sara Mehdizadeh بخش بهداشت مواد غذایی و آبزیان ، دانشکده دامپزشکی، دانشگاه سمنان، سمنان، ایران Iran smehdizadeh@semnan.ac.ir علی مهدوی mahmoud Mahdavi بخش بهداشت مواد غذایی و آبزیان ، دانشکده دامپزشکی، دانشگاه سمنان، سمنان، ایران Iran mahmoudmohseni@yahoo.com سمیه حسن پور لسکوکلایه Somayeh Hassanpour مزرعه پرورش ماهی خاویاری بلوگا، رشت، گیلان، سنگر Iran somayehhasanpoor1982@yahoo.com Beluga sturgeon (Huso huso) lysine histidine liver antioxidant indices فیل‌ماهی (Huso huso) لیزین هیستیدین شاخص‌های آنتی‌اکسیدانی کبد Aebi, H., 1984. Catalase in vitro. 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مقاله علمی – پژوهشی:‌ مطالعه فراوانی و زی‌‌توده درشت بی‌مهر‌‌گان تالاب انزلی و تخمین پتانسیل تولید ماهیان کفزی‌خوار Study of the abundant and biomass of macroinvertebrates in Anzali Wetland and estimation of the potential production of benthophagous fish 1 1 تالاب انزلی در جنوب غربی دریای کاسپین دارای ارزش‌‌های شیلاتی و اقتصادی بالایی است، اما امروزه برداشت ماهی از آن در حد ناچیزی قرار دارد. درشت بی‌‌مهر‌‌گان آبزی نقش ویژ‌‌ه‌‌ای در تولیدات شیلاتی به‌عهده دارند که در این بررسی مورد مطالعه قرار گرفتند. درشت بی‌‌مهر‌‌گان کفزی و موجودات مستقر در گیاهان غوطه‌‌ور (اپی‌‌فیت) از بخش‌‌های مختلف تالاب انزلی طی 7 مرحله از اسفند 1401 لغایت دی 1402 نمونه برداری شدند. زی‌‌توده این موجودات با هدف برآورد توان تولید ماهی اندازه‌‌گیری شد. در این بررسی 21 خانواده متعلق به 12 راسته از درشت بی‌‌مهرگان شناسایی گردید. تنوع و تراکم گروه‌های جانوری در ایستگاه‌های واقع در پهنه‌‌های آبی و ایستگاه‌‌های رودخانه‌‌ای دارای پوشش گیاهی بیشتر بود. خانواده‌‌های Naididae و Chironomidae در بین موجودات کفزی و خانواده‌‌های Gammaridae، Lymnaeidae و Physidae در بین درشت بی‌‌مهرگان اپی‌‌فیتی دارای بالاترین درصد مشاهده بودند. زی‌‌توده موجودات طی زمستان و بهار بیشتر از تابستان و پائیز بود. میانگین زی‌‌توده درشت بی‌‌مهرگان کفزی 9/0±4/4 و اپی‌‌فیتی 6/0±1/3 گرم در مترمربع بود. تولید طبیعی ماهی کفزی خوار براساس زی‌‌توده درشت بی‌‌مهرگان 1/15 کیلوگرم در هکتار در سال برآورد گردید. با توجه به گستره آبی تالاب که 3700  هکتار گزارش شده است، این میزان زی‌‌توده از درشت بی‌‌مهر‌‌گان، تولیدی معادل 9/55 تن از ماهیان را در سطح تالاب انزلی سبب خواهد شد. بررسی میزان برداشت ماهی از تالاب انزلی در سال 1403 تا حد زیادی با تخمین مذکور همخوانی دارد. کاهش توان شیلاتی تالاب انزلی نسبت به سالیان گذشته محسوس بوده که با خشک شدن اکثر بخش‌‌ها و تغییر سیمای اکولوژی آن در ارتباط است. بهبود عرصه‌‌های زیستگاهی شامل گسترش پهنه‌های آبی، ایجاد عمق مناسب و اجرای برنامه‌های احیاء تالاب انزلی کمک خواهد نمود تا وضعیت این زیست بوم و صید ماهی در آن بهبود یابد. 2 Introduction Wetlands occupy only about 6% of the land on Earth, yet they provide habitat for 40% of all species and offer countless benefits (Mitsch et al., 2015; Kingsford et al., 2016; Khatun et al., 2021). In Iran, large wetlands cover approximately 1.7% of the country’s surface area; however, their destruction is occurring rapidly, with a loss of about 14% of this area between 2011 and 2021 (Mahdian et al., 2024). Anzali Wetland, located southwest of the Caspian Sea, is notable for its unique characteristics and was one of the first wetlands registered on the Ramsar Convention list in June 1975 (RIS, 2023). Like other aquatic ecosystems in Iran, Anzali Wetland has undergone rapid destruction. Despite being geologically young (Leroy et al., 2011), it has aged quickly and is approaching its final stage of succession. Long-term monitoring shows that the wetland's water surface area decreased by approximately 80%, from about 258 km² in 1930 to about 52 km² in 1989. This trend continued from 1989 to 2020, with a decrease of approximately 0.75 km² per year, resulting in a shrinkage of the wetland to 25.9 km². This pattern of degradation raises concerns about the wetland's potential complete disappearance in the near future (Aghsaei et al., 2020; Mahdian et al., 2023). Currently, the Caspian Sea level is decreasing by 8 cm per year (Chen et al., 2017; Prange et al., 2020; Lahijani et al., 2023), contributing to the wetland's shrinkage, as it is hydraulically connected to the sea. Finally, the Anzali Wetland will be desiccated between 2058 and 2062 in non-conservative approach (Mahdian et al., 2024). Due to these factors and other anthropogenic influences, Anzali Wetland was included in the Montreux Record, highlighting the urgent need for conservation and restoration efforts (JICA et al., 2005; RIS, 2023). The connection between the Caspian Sea and Anzali Wetland enhances its role as a habitat for spawning and as a nursery ground for anadromous fish. Historically, more than 80% of the total fishing along the Iranian coast of the Caspian Sea occurred in Anzali Wetland (Holčík and Oláh, 1992). Historical data indicate that total fish catches in the wetland between 1932 and 1940 ranged from 4,000 to 7,500 tons (~218 kg/ha), primarily consisting of Caspian Sea fish such as kutum, bream, and pike-perch. The fishery value of Anzali Wetland has declined due to reductions in water depth and surface area, reaching only 17 kg/ha by 1990 (Hydropriject, 1965; Kimbal and Kimbal, 1974; Nezami, 1994). The severe decline in fish stocks prompted a limnological investigation of Anzali Wetland in collaboration with Iranian and World Fisheries Organization experts in 1980 (Holčík and Oláh, 1992). This investigation yielded a report on the status of fishing and proposed solutions for stock rehabilitation. Subsequent studies have periodically explored various topics, including the determination of fisheries potential, hydrology, hydrobiology, and the ecology of Anzali Wetland (Nezami, 1994; Khodaparast, 2003; Mirzajani, 2009; Fallahi, 2018). Aquatic macroinvertebrates are crucial organisms in all aquatic ecosystems. Their abundance and distribution respond significantly to nutrient levels and pollution, making them valuable indicators for ecological assessments ( Hilsenhoff, 1988; Barbour et al., 1996; Lenat, 1998; Overton, 2001; Bode et al., 2002). They also serve as food resources and contribute to energy transfer for fish and other aquatic organisms (López–López et al., 2015). Additionally, macroinvertebrates act as a link between primary producers, decomposers, and higher trophic levels, playing a major role in the detritus food chain (Neogi et al., 2016) and enhancing the productivity of aquatic environments (Sarkar et al., 2020; Karmakar et al., 2022). Their biomass positively affects fish density and growth rates (Richardson, 1993). Macroinvertebrates are among the most important organisms in Anzali Wetland, and they play a crucial role in fisheries production, yet only a few studies have been conducted on them. Notable research from the last two decades includes assessments of the inlet rivers to Anzali Wetland based on benthic communities (Mirzajani et al., 2008), surveys of macro-benthic organisms in the southwestern region of the wetland (Jalili et al., 2011), identification of Oligochaetes (Annelida, Clitellata) in Anzali Wetland (Nazarhaghighi et al., 2014), identification of Limnodrilus species in the wetland (Naeemi et al., 2015), studies on benthic macroinvertebrates during 2014 (Ghane et al., 2017), and health assessments of the Shanbeh-Bazar River using macroinvertebrates and water quality parameters (Foomani et al., 2020). None of the previous studies examined the relationship between fish production and macroinvertebrates. This study aims to investigate the abundance and biomass of benthic and epiphytic macroinvertebrates to assess the potential for benthophage fish production in Anzali Wetland. Methodology Anzali Wetland is located at a latitude of 37°28' North and a longitude of 49°25' East, with an average elevation of -23 meters below sea level. In the recent past, Anzali Wetland was comprised of four main sections: the eastern part (Shijan), the central part (Sorkhankul), the western part (Abkenar), and the southern part (Siah Keshim) (Mirzajani et al., 2020). Today, the water bodies in most areas, including Shijan, Sorkhankul, and much of Siah Keshim, have dried up or become very shallow or limited in extent. Benthic and epiphytic macroinvertebrates were sampled from various locations within the wetland, and their biomass was measured. For benthic sampling, a Van Veen grab with a surface area of 225 cm² was used at 11 stations, with three replicates taken at each location. The sampling periods occurred approximately every 45 days in March, April, June, August, October, November 2023, and December 2024. Epiphytic macroinvertebrates were seasonally sampled from submerged plants at 10 stations, specifically in the western part of the wetland in April, July and October 2023 and February 2024. Aquatic plants were collected with three replicates using a rake with a 30 cm diameter, rotated in a circular motion. The plants were washed several times, and the organisms were carefully separated. In the laboratory, the collected specimens were classified into taxonomic groups at the family or genus level, using established references (Macan, 1968; Merritt et al., 2008; Thorp and Covich, 2009). The biomass of the organisms was measured with a balance accurate to 0.0001 g. The prediction of benthophage fish productivity was based on macroinvertebrate biomass, calculated using the following equation (Li and Mathias, 1994): Fish productivity= where B is the biomass of macroinvertebrates, P/B is the ratio of production to standing biomass of food organisms, which was considered to be 4 according to Li and Mathias (1994). The food utilization coefficient (Uf) and the feed conversion ratio (FCR), were set at 25% and 5, respectively (Li and Mathias, 1994). Results Benthic macroinvertebrates were classified into 12 families across 8 orders, including insects, worms, mollusks, and crustaceans. Diversity and abundance were higher at stations located in the open water bodies, particularly in the Siah Keshim and western parts, as well as at stations 4 and 8. The families Naididae and Chironomidae were the most abundant among the benthic organisms, with densities of 128 and 61 individuals/m², respectively. The highest occurrence percentages were also observed in these families, while Coenagrionidae and Simuliidae had the lowest percentages. The epiphytic macroinvertebrates were identified across 18 families belonging to 11 orders. The highest occurrence percentages were found in the orders Amphipoda and Gastropoda. Chironomidae were the most abundant epiphytic macroinvertebrates, with 82 individuals/m², followed by Planorbidae, with 58 individuals/m². The total abundance of benthic organisms was higher at stations 9, 10, and 11 compared to other stations, ranging from 400 to 625 individuals/m², primarily due to the dominance of the Naididae, Chironomidae, and Lumbriculidae families. Benthic biomass was highest at station 4, followed by stations 11, 10, and 8, varying between 6.4 and 11.1 g/m², linked to the dominance of the Sphaeriidae and Lumbriculidae families. Total biomass of epiphytic macroinvertebrates varied from 1.9 to 6.4 g/m², dominated by the Gammaridae and Lymnaeidae families. The mean (±SE) total biomass of benthic and epiphytic macroinvertebrates was 4.4 ± 0.9 g/m² and 3.1 ± 0.6 g/m², respectively. Temporal changes in macroinvertebrate biomass showed that the biomass of organisms was greater in winter-spring compared to summer-autumn, decreasing to less than 1 g/m² in summer and early autumn. Overall, benthophage fish production was estimated at 15.1 kg/ha. Discussion and conclusion In this study, the greatest diversity of macroinvertebrates was observed at the Bahmbar station, which has been reported to have good water quality, with total nitrogen and phosphorus levels lower than in many other sites (Abedini et al., 2018). The least diversity and abundance of organisms were found in most regions such as Siahdarvishan, the Sorkhankul outlet, and the eastern stations. This decline is attributed to the degradation of these areas to a riverine state, minimal water availability, and high exposure to nutrients or pollutants for most of the year. Nutrient levels were also greater in the eastern and Sorkhankul areas compared to Siah Keshim and the western areas (Abedini et al., 2018). In this study, the diversity of epiphytic macroinvertebrates was greater than that of benthic macroinvertebrates (Table 2). Annual investigations on benthic organisms from 1992 to 2002 (Mirzajani, 2009) and in 2014 (Ghane et al., 2017) observed a similar number of macroinvertebrate groups, with Chironomidae and Tubificidae having the highest percentages in terms of occurrence, abundance, and biomass among benthic organisms. The total biomass of benthic organisms was reported to range from 1.15 to 7.76 g/m² during different years from 1992 to 2002 (Mirzajani, 2009) and with a maximum biomass of 5.4 g/m² in 2014 (Ghane et al., 2017). Temporal changes in organism abundance can be interpreted through their biological cycles; for instance, the low abundance of Chironomidae in summer has been attributed to their departure from substrates and water for the final metamorphosis stage (Valipour, 1997). The decline of Anzali Wetland, coupled with the drying of large areas, excessive growth of invasive species like water hyacinth, and increasing pollutant concentrations, has severely impacted the diversity and abundance of macroinvertebrates. Even non-native species, such as Macrobrachium nipponense, which previously exhibited high abundance and biomass (Ghane et al., 2021), have seen a sharp decline in density and were not observed in the current survey's sampling units. On the other hand, high concentrations of pollutants in some regions have led to a notable decline in benthic communities. The Naididae family was the most abundant benthic group in this study (Table 2). This family can tolerate low oxygen levels (Aston, 1973; Nijboer et al., 2004); however, a mass mortality of Naididae was observed during field surveys in May 2024 in the eastern region, where there is significant discharge from the highly polluted city of Rasht. Field observations also indicated a lack of benthic organisms beneath the cover of water hyacinth, which has become extensively distributed in Anzali Wetland recently (Mirzajani, 2024). The recent negative degradation in Anzali Wetland has decreased its fisheries potential, estimated at 15.1 kg/ha for benthophage fish in this study. The highest average fish catch recorded was 218 kg/ha in 1941, followed by sharp decreases to 105 kg/ha in 1951 and 19 kg/ha in 1962 (Hydropriject, 1965). There was an increase to 75 kg/ha in 1993 due to rising Caspian Sea levels (Holčík and Oláh, 1992). Currently, fish production potential is lower than in many Iranian inland waters, such as Shovir, Taham, and Todebin, which average around 30 kg/ha (Mirzajani et al., 2020), and in various wetlands worldwide, including those in Italy (30–300 kg/ha) and countries in West Africa (~80 kg/ha) (Nezami, 1994). Considering the current water body area of 3,700 ha (Mirzajani, 2024), the total benthophage fish production is predicted to be 55.9 tons. In 2023, fish harvests in Anzali Wetland reached 93.1 tons, with cyprinid species comprising 56.6% of the total catch; common carp accounted for 24.8%, and Prussian carp for 22.1% (Daghigh Roohi et al., 2025). Anzali Wetland has undergone significant environmental changes and cannot revert to its previous status. Furthermore, predictions indicate a continued decline in Caspian Sea levels at a rate of 8 cm per year (Chen et al., 2017; Lahijani et al., 2023; Mahdian et al., 2024). Habitat rehabilitation could effectively enhance the growth and survival of many aquatic organisms, particularly macroinvertebrates and fish. Expanding water body areas, creating depth in various regions, controlling sediment in the watershed area, managing water hyacinth, and optimally harvesting aquatic plants could help restore some of the wetland’s lost capacity. Conflict of Interest The authors have no conflicts of interest to declare that are relevant to the content of this article. Acknowledgment The authors would like to thank the Department of Environment of Guilan Province for financially supporting this project, registered in AREEO under the code number 14-73-12-037-01051-011055. We also appreciate the help of our colleagues in Inland Waters Aquaculture Research Center. 59 73 2025/04/272024/11/292025/06/252025/01/272025/04/1 1404/1/12 2025/09/12025/09/12025/09/12025/09/12025/09/1 1404/6/10 علیرضا میرزاجانی Alireza Mirzajani پژوهشکده آبزی پروری آبهای داخلی Iran armirzajani@gmail.com سیامک باقری Siamak Bagheri پژوهشکده آبزی پروری آبهای داخلی Iran Siamakbp@gmail.com یعقوبعلی زحمتکش Yaghobali Zahmatkesh پژوهشکده آبزی پروری آبهای داخلی Iran younes_zahmatkesh@yahoo.com احمد قانع Ahmad Ghane پژوهشکده آبزی پروری آبهای داخلی Iran ahmad4566@yahoo.com Anzali Wetland Macro-benthic epiphytic fish production potential تالاب انزلی ماکروبنتوز اپی‌‌فیتی توان تولید ماهی Abedini, A., Mirzajani, A.R. and Fallahi, M., 2018. Physicochemical conditions and trophic levels of the Anzali Wetland. Iranian Scientific Fisheries Journal, 26: 113-123. (In Persian).##Aghsaei, H., Dinan, N.M., Moridi, A., Asadolahi, Z., Delavar, M., Fohrer, N. and Wagner, P.D., 2020. 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مقاله علمی – پژوهشی:‌ اثر نانو ذرات نقره(AgNPs) و دی‌اکسید سیلیس (SiO2NPs) بر سنجه‌‌‌های رشد و رنگدانه‌های فتوسنتزی ریز جلبک Nannochloropsis oculata Study of the effect of silver nanoparticles (AgNPs) and silica nanoparticles (SiO2NPs) on growth indices and photosynthetic pigments of the phytoplankton, Nannochloropsis oculata 1 1 چکیده به منظور بررسی تأثیر نانو ذرات نقره و نانو ذرات دی‌اکسید سیلیس بر Nannochloropsis oculata، این فیتوپلانکتون به مدت 72 ساعت طبق استاندارد شماره 201 سازمان توسعه و همکاری اقتصادی در معرض غلظت‌های 005/0، 010/0، 025/0، 050/0، 075/0 و 100/0 میلی‌گرم در لیتر نانو ذرات نقره و غلظت‌های 75، 100، 150 و 200 میلی‌گرم در لیتر نانو ذرات دی‌اکسید سیلیس قرار گرفتند و در نهایت شاخص‌های متوسط نرخ رشد ویژه، درصد بازدارنگی متوسط نرخ رشد ویژه و میزان رنگدانه‌های فتوسنتزی در هر غلظت محاسبه گردید. همچنین مقادیر غلظت‌های بازدارنده (ICs)، از جمله غلظت­های بازدارنده میانی (IC50)، بر اساس درصد بازدارندگی متوسط رشد، به­وسیله نرم­افزار پروبیت محاسبه گردید. طبق نتایج، نانو ذرات نقره در کمترین غلظت مورد بررسی سبب کاهش رشد و کاهش میزان رنگدانه‌های فتوسنتزی در فیتوپلانکتون مورد مطالعه گردیدند و با افزایش غلظت آن‌ها به 075/0 و 100/0 میلی‌گرم در لیتر، سبب توقف کامل رشد گردیدند. غلظت‌های مختلف نانو ذرات دی‌اکسید سیلیس سبب کاهش نرخ رشد و تراکم زی‌توده فیتوپلانکتون ‌گردیدند، اما سبب توقف کامل رشد نگردیدند. تاثیر سمیت نانو ذرات نقره و نانو ذرات دی‌اکسید سیلیس بر رنگدانه‌های فتوسنتزی در فیتوپلانکتون به غلظت نانو ذرات و مدت زمان رویارویی وابستگی نشان داد؛ به­‌طوری­که با افزایش غلظت و مدت زمان رویارویی، میزان کلروفیل  aو b و کلروفیل کل و کارتنوئیدها کاهش معنی‌داری را نشان دادند. نانو ذرات نقره و نانو ذرات دی‌اکسید سیلیس در تمام غلظت‌های مورد استفاده سبب ایجاد سمیت، افزایش درصد بازدارندگی متوسط رشد و کاهش میزان رنگدانه‌های فتوسنتزی گردیدند.   2 Abstract To investigate the effect of silver nanoparticles and silica dioxide nanoparticles on Nannochloropsis oculata, this microalgae was exposed for 72 hours to concentrations of 0.005, 0.010, 0.025, 0.050, 0.075, and 0.100 mg/L of silver nanoparticles and 75, 100, 150, and 200 mg/L of silica dioxide nanoparticles according to OECD Test Guideline No. 201,  and the growth inhibition percentage, specific growth rate, and photosynthetic pigment concentrations were subsequently examined for each treatment. The values of inhibitory concentrations (ICs), including the IC50, were calculated based on the average growth inhibition percentage using Probit software. According to the results, silver nanoparticles at the lowest concentration examined caused a reduction in growth and a decrease in the amount of photosynthetic pigments in the studied microalgae, and with an increase in their concentration to 0.075 and 0.1 mg per liter, they caused complete growth cessation. Different concentrations of SiO2NPs reduced the growth rate and biomass density of the microalgae but did not completely stop their growth. The toxicity effect of silver nanoparticles and silica dioxide nanoparticles on the photosynthetic pigments in microalgae showed a concentration-and time-dependent manner of the nanoparticles, such that with increasing concentration and duration of exposure, the levels of chlorophyll a, b, total chlorophyll, and carotenoids showed significant reductions. Silver nanoparticles and silica dioxide nanoparticles caused toxicity, increased the percentage of average growth inhibition, and reduced the amount of photosynthetic pigments across all tested concentrations 75 89 2025/04/272024/11/292025/06/252025/01/272025/04/12025/01/19 1403/10/30 2025/09/12025/09/12025/09/12025/09/12025/09/12025/09/1 1404/6/10 محمدجواد جامی Mohammad javad jami jami.mohammadjavad@yahoo.com علی جوهری Ali Johari sajohari@gmail.com محمد بهزادی طائمه Mohamad Behzadi tayemeh behzadimohamad72@gmail.com حسام الدین عبایی Hesamoddin Abaie hesam.abaie@gmail.com Nanotechnology Aquatic nanotoxicology Microalgae Acute toxicity Chlorophyll. نانوفناوری؛ نانوسم­شناسی آبزیان؛ ریزجلبک؛ سمیت حاد؛ سبزینه. جوهری، س. ع.، قادرسربازی، ژ.، سوری‌نژاد، ا. 1395. مطالعه اثر سمیت نانو ذرات نقره کلوئیدی بر ریزجلبک دریاییNannochloropsis oculata . بوم‌شناسی آبزیان، 6، 83 تا90.##Asghari, S., Johari, S. A., Lee, J. H., Kim, Y. S., Jeon, Y. B., Choi, H. J., Moon, M. C. & Yu, I. J. 2012. 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