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مقاله علمی – پژوهشی:‌ تأثیر انواع روش‌های پخت در میزان چربی و اسیدهای چرب چندغیراشباع ضروری (امگا-3) فیله ماهی کپور سرگنده (Hypophthalmichthys nobilis) Influence of different cooking methods on the lipid and essential polyunsaturated fatty acids (omega-3) contents of bighead carp (Hypophthalmichthys nobilis) fillet 1 1 ماهی بیگ هد پرورشی (Hypophthalmichthys nobilis) با وزن متوسط 100±700 گرم از یک مزرعه پرورش ماهی تهیه و به آزمایشگاه فرآوری پژوهشکده اکولوژی دریای خزر انتقال داده شد. نمونه‌ها پس از فیله شدن، با روش‌، سرخ کردن (با روغن آفتابگردان) و کبابی کردن در آون پخته شدند. سپس از لحاظ شاخص‌‌های شیمیایی (پروتئین خام، چربی خام، رطوبت و خاکستر) و پروفایل اسیدهای چرب با استفاده از کروماتوگرافی گازی و شاخص‌‌های اکسیداسیون (عدد پراکسید، تیوباربیوتیک اسید و بازهای نیتروژنی فرار)، فیله ماهی خام و پخته شده اندازه‌گیری گردید. نتایج نشان داد که مقدار رطوبت، چربی خام، پروتئین خام و خاکستر در نمونه شاهد ماهی بیگ هد به‌ترتیب، 86/76، 49/1، 06/19 و 91/1 درصد بود. اعمال روش‌های متفاوت پخت سبب کاهش رطوبت حدود 10-6 درصد، افزایش پروتئین حدود 7-5 درصد شد و مقادیر چربی در ماهی سرخ شده به میزان 2 درصد افزایش یافت. تمامی روش‌های پخت موجب افزایش شاخص‌های اکسایشی چربی شدند (05/0>P). سرخ کردن موجب افزایش مقادیر پراکسید (86/3 میلی اکی والان/ کیلوگرم چربی)، تیوباربیتوریک اسید (04/2میلی گرم آلدهید اسید) و بازهای نیتروژنی فرار (20/19 میلی گرم/ صد گرم) گردید. مقادیر اسید چرب در روش کبابی در اکثر موارد اختلاف معنی‌داری با فیله خام نداشت. کبابی کردن نیز تأثیری بر نسبت امگا ۳ به امگا 6 نداشت (05/0<P). دربین روش‌های مختلف پخت، سرخ کردن موجب کاهش شدید اسید های چرب غیراشباع از 63/22 (ماهی خام) به 72/16 درصد و برعکس افزایش قابل‌توجه اسید چرب تک غیر اشباع از 62/40 (ماهی خام) به 53/43 درصد شد که می‌تواند ناشی از نفوذ نوع روغن مایع مصرفی به درون بافت ماهی باشد. کاهش نسبت امگا 3 به امگا 6 و افزایش نسبت اسید چرب اشباع به اسید چرب تک غیر اشباع و اسید چرب اشباع به اسید چرب چند غیر اشباع در ماهی سرخ شده مشاهده شد. کاهش معنی‌داری در میزان EPA و DHA نمونه‌های سرخ شده مشاهده گردید (05/0>p). بنابراین، پخت به روش کباب کردن روش مناسب‏تری جهت حفظ ارزش تغذیه‌ای فیله ماهی کپور سرگنده ماهی نسبت به سایر روش‌های پخت است. 2 Introduction Seafood is an excellent source of high-quality protein, essential fatty acids, vitamins, and minerals. In terms of fish quality, the most basic element is to measure the approximate composition of the flesh along with its fatty acid composition (Hedayati Fard and Miri, 2017). The bighead carp (Hypophthalmichthys nobilis) is one of the most important warm-water fish species in the world, which has a special place in multi-species culture systems worldwide due to its high adaptability (Dadras et al., 2011). Fish oil contains high amounts of essential unsaturated fatty acids such as eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and 5 to 6 double bonds, essential minerals and vitamins (El-Lahamy et al., 2019). Humans obtain most of their essential fatty acids, especially EPA and DHA, from the consumption of fish, aquatic invertebrates and macroalgae (Hedayati Fard and Miri, 2017). In modern societies, meat is almost always cooked before consumption. Heating is a common method for food processing. Heat (boiling, steaming, frying, microwave, etc.) in foods inactivates pathogenic microorganisms and lipolytic enzymes, improves sensory quality (smell and taste), increases digestibility, and increases shelf life. Most changes in quality issues in cooked fish products are directly related to the quality of the initial raw material. Heating is one of the common methods in food processing. In research conducted on different cooking methods, the fatty acid composition of Biyah fish oil treated with fennel alcoholic extract was studied. The results showed that the oven-baked sample had the lowest moisture (51.97%) and the highest protein (39.69%) during cooking (Ramzani et al., 2016). Different cooking methods affect the quality characteristics of fish, including texture, taste, smell, color, and overall acceptance (Momenzadeh et al., 2017). The purpose of the research is to measure different cooking methods (grilling and frying) that cause significant changes in the quality indicators and fatty acid compositions of bighead carp fillets. Considering the above, the nutritional value of bighead carp was determined by applying different cooking methods and the effect of these cooking methods on the fatty acid analysis of the fillets, in order to introduce the best cooking method. Methodology Frying method: Fish fillets were fried in oil at 180°C for 8 minutes (Zakipur Rahimabadi and Bakr, 2011). Oven roasting method: Oven roasting (UN55, Memmert, GR) was performed at 180°C for 30 minutes (Hedayatifard and Miri, 2017). Approximate compounds measurement: Moisture weighing was performed by oven drying at 105°C (Avak and Glaser, 2005). The protein samples were analyzed using the Kjeldahl method (Avak and Glaser, 2005). Fat weighing was performed using the Soxhlet method (Bilgin et al., 2010). Ash measurement was determined by the 190°C oven method (Avak and Glaser, 2005). The measurement of (PV) peroxide values was performed by iodine titration method (Ronald and Ronald, 1991). Thiobarbituric acid (TBA) was measured colorimetrically (Hedayatifard and Miri, 2017). Total volatile bases-nitrogen (TVB-N) was measured by the Kjeldahl method (Hedayatifard and Miri, 2017). Identification of fatty acid profiles and composition was performed using a gas chromatography (Agilent Technol 7890-GC) equipped with a flame ionization detector (FID) with a capillary tube and a column of 50 m × 0.25 mm and 0.2 μm (Hedayatifard and Miri, 2017). Study The ratios of PUFA/SFA and MUFA/SFA fatty acids were investigated (Brimani et al.,2021). The nutritional quality index of EPA+DHA and the n-3/n-6 ratio were examined as an important index for comparing the nutritional value of fish oil in the present study (Bayir et al.,2006). Index (PI), index (HH), atherogenic index (AI) and thrombogenic index (TI) were investigated in raw, fried and oven-baked fillets. (Brimani et al.,2021). The data analysis and variance homogeneity were performed by the (ON-WAY ANOVA) method. Results The results of the moisture content study showed that it was significantly higher in raw fish than in other cooking methods (76.86%), and the lowest moisture content was observed in fried fish (66.62%) (p<0.05). The results showed that the percentage of protein in fried fish was significantly higher than other cooking methods (26.26%), and the lowest protein levels were observed in the raw fish treatment (19.60%) (p<0.05). The results showed that the percentage of fat in fried fish was significantly higher than other cooking methods (70.3%). The lowest fat levels were observed in the oven-baked fish treatment (17.1%) (p<0.05). The results showed that the ash content in oven-cooked fish (kebab) was significantly higher than other cooking methods (2.77%), and the lowest ash content was observed in raw fish treatment (1.90%) (p<0.05). The results of the peroxide value PV study showed that it was significantly higher in fried fish than in other cooking methods (386.3 milliequivalents per kilogram of fat) (p<0.05), and the lowest peroxide value was observed in the raw fish treatment (0.94 milliequivalents per kilogram of fat) (Figure 5) (p<0.05). The results of the study showed that the levels of thiobarbituric acid in fried fish were significantly higher than those in other cooking methods (2.04 mg malondialdehyde/kg acid) (p<0.05), and the lowest levels of thiobarbituric acid were observed in the raw fish treatment (0.54) (Figure 6) (p<0.05). The results of the study showed that the total volatile nitrogen bases TVB-N in fried fish were significantly higher than other cooking methods (17.10 mg/100 g) (p<0.05), and the lowest values ​​of volatile nitrogen bases were observed in the raw fish treatment (10.35 mg/100 g) (Figure 7) (p<0.05). Results related to the amounts of saturated fatty acids (SFA) in raw and cooked fish by different cooking methods identified five types of saturated fatty acids (C14:0, C16:0, C18:0, C20:0 and C22:0) (Table 1). Results of the study of polyunsaturated fatty acids (PUFA) identified 6 types of acids (C18:2n-6, C18:3n-3, C20:3n-6, C20:3n-3, C20:5n-3 (EPA), C22:6 n-3 (DHA) (Table 1). In the study of monounsaturated fatty acids (MUFA), 3 types of monounsaturated fatty acids were identified (C16:1, C18:1, and C20:1) (Table 1). The results of the composition of PUFA/SFA fatty acids showed that the minimum recommended value, i.e. 0.4, was higher (Brimani et al., 2021). The MUFA/SFA ratio for all 3 treatments showed that the highest amount was related to fried fish and the lowest amount was related to grilled fish (Table 2) (Hosseini et al., 2014). The DHA index was higher in raw fish than in cooked fish, and the lowest values ​​were observed in fried fish (Table 2). The n-3/n-6 index was shown in 3 treatments. The lowest n-3/n-6 ratio was observed in fried samples and the highest n-3/n-6 ratio was observed in raw fillets (Table 2) (Bayir et al., 2006). The lowest values ​​of pi were observed in fried fish (Table 2) (Brimani et al., 2021). The HH index in all 3 treatments was within the appropriate range (Table 2) (Brimani et al., 2021). The atherogenic index (AI) and thrombogenic index (TI) were very low in 3 samples (Table 2) (Brimani et al., 2021). As a result of the present study, grilling is a more suitable method for cooking fish and the fish has an acceptable nutritional value. Discussion and conclusion Maintaining the nutritional value of fish can be achieved by using appropriate cooking methods (Uran and Gokoglu, 2014). The decrease in moisture is due to denaturation of the protein structure and evaporation of water (Delfieh et al., 2013). The increase in protein in the fried treatment (kebab) is due to the decrease in moisture content of the samples compared to the relative increase in other components and the dissolution of some nitrogen (De Castro et al., 2007). The increase in fat in the fried sample is due to the decrease in meat juice due to evaporation and penetration of oil into the tissue (Qayumi et al., 2011). The increase in ash in the cooked sample is due to the decrease in moisture and increase in dry matter and the absence of minerals in the thermal process (Abroomand and Masoudi, 2012). The increase in peroxide in the fried sample is due to the increase in temperature. In fact, in the frying method, the production of free radicals is greater and they attack fatty acids more quickly and cause the production of hydroperoxides (Ozogul et al., 2009). The high content of thiobarbituric acid in fried samples and the low content in raw samples is due to the high temperature, malonaldehyde level and fat oxidation increase (Campo et al., 2006). An increase in (TVB-N) was observed in fried fish and its decrease in raw treatment. This is due to the thermal processing of the samples, which causes the decomposition of proteins, amino acids and other nitrogenous compounds (Mohan et al., 2006). Suggestions: 1- The effect of using natural preservatives on the fat content of fish cooked in different ways. 2- The effect of frying oils from different sources of oil plants on the quality of fish fillets. Conflict of Interest The authors declare that they have no conflict of interest Acknowledgment The authors wish to convey their heartfelt appreciation to all colleagues who supported and facilitated the experiment's necessary infrastructure. 1 15 2025/01/1 1403/10/12 2025/07/1 1404/4/10 غلامرضا رازقیان رستمی Gholamreza Razeghian Rostami گروه شیلات، دانشکده کشاورزی و منابع طبیعی، واحد قائم‌‌شهر، دانشگاه آزاد اسلامی، قائم شهر، ایران Iran rostamireza5@yahoo.com مسعود هدایتی فر Masoud Hedayatifard گروه شیلات، دانشکده کشاورزی و منابع طبیعی، واحد قائم‌‌شهر، دانشگاه آزاد اسلامی، قائم شهر، ایران Iran persiafish@gmail.com حسن نصراله زاده ساروی Hassan Nasrollahzadeh Saravi پژوهشکده اکولوژی دریای خزر، مؤسسه تحقیقات علوم شیلاتی کشور، سازمان تحقیقات، آموزش و ترویج کشاورزی، ساری، ایران Iran hnsaravi@gmail.com رضا صفری Reza Safari پژوهشکده اکولوژی دریای خزر، مؤسسه تحقیقات علوم شیلاتی کشور، سازمان تحقیقات، آموزش و ترویج کشاورزی، ساری، ایران Iran safari1351@gmail.com شراره فیروزکندیان Sharareh Firouzkandian پژوهشکده اکولوژی دریای خزر، مؤسسه تحقیقات علوم شیلاتی کشور، سازمان تحقیقات، آموزش و ترویج کشاورزی، ساری، ایران Iran sh50@yahoo.com وحید رنجبر Vahid Ranjbar پژوهشکده اکولوژی دریای خزر، مؤسسه تحقیقات علوم شیلاتی کشور، سازمان تحقیقات، آموزش و ترویج کشاورزی، ساری، ایران Iran vahid.ranjbar.k123@gmail.com Oxidation Fatty acid Chemical parameters Cooking methods Hypophthalmichthys nobilis اکسیداسیون اسید چرب شاخص‌‌های شیمیایی روش‌های پخت Hypophthalmichthys nobilis Avak,R.,and Glaser,R., 2005. ##Aberoumand. 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DOI: 10.22092/ijfs.2018.114140## ##

مقاله علمی – پژوهشی:‌ بررسی زی‌‌توده و گستره جوامع گیاهی غالب در تالاب انزلی Study of biomass and distribution of dominant plant communities in Anzali Wetland 1 1 گیاهان آبزی از اجزاء اصلی اکوسیستم تالابی به‌شمار می‌روند و نقش مهمی در جریان مواد مغذی دارند و زیستگاه بسیاری از جانوران محسوب می‌‌شوند. بنابراین، پایش تراکم و پراکنش گیاهان از ارکان اصلی در مدیریت تالاب‌ها به‌شمار می‌‌رود. در این بررسی تراکم گیاهان شناور، غوطه‌‌ور و حاشیه‌‌ای غالب در تالاب انزلی تعیین گردید. زی‌‌توده گیاهان به صورت فصلی در 10 ایستگاه از پیکره آبی تالاب برآورد گردید. گستره گیاهان حاشیه‌‌ای با استفاده از تصاویر ماهواره‌‌ای و شاخص NDVI)) و تفسیر بصری تصاویر Google earth، تعیین گردید. میانگین زی‌‌توده گیاهان آبزی غوطه‌‌ور، در حد 9/313±7/547 گرم در مترمربع بود که گونه‌‌ Ceratophyllum demersum با 8/53 درصد، بیشترین زی‌‌توده و گونه Hydrilla verticillata با 9/18 درصد، در رتبه بعدی قرار داشت. بیشترین و کمترین میانگین زی‌‌توده گیاهان در تیر و اردیبهشت ماه به‌ترتیب به میزان 730 و 388 گرم در مترمربع اندازه‌گیری شد. در بررسی گیاهان شناور دو گونه لاله مردابی Nelumbo nucifera و سنبل آبیPontederia crassipes به لحاظ گستره پراکنش و زی‌‌توده گیاهی غالب بوده و به‌‌ترتیب گستره‌‌ای درحد 350 و 970 هکتار از تالاب غرب را پوشش داده‌‌اند. در یک فصل رویشی میزان سنبل آبی 150 تن در هکتار برآورد شده است. گیاه نی (Phragmites australis) از گونه‌های غالب در نواحی حاشیه‌‌ای تالاب بوده و گستره آن در حد 10290 هکتار برآورد شده است. با توجه به حجم بالای انتقال مواد آلی به بستر تالاب و اثرات منفی این گیاهان، برداشت بهینه گیاهان آبزی یکی از ضروریات اصلی مدیریت تالاب انزلی به‌شمار می‌‌رود. 2 Introduction Anzali Wetland, located in the southwest of the Caspian Sea, is notable for its unique characteristics. It was one of the first wetlands registered on the Ramsar Convention's list. However, it is rapidly deteriorating and has been included in the Montreux Record, highlighting the need for conservation and restoration efforts (Ramsar Convention Bureau, 2014). Aquatic plants are a key component of the wetland ecosystem, playing a crucial role in nutrient cycling and providing habitat for many animals (Kurniawan et al., 2021; Dissanayaka et al., 2023). In recent years, the increase in nutrient influx has led to significant growth of aquatic plants in various parts of Anzali Wetland. This growth, including both floating and submerged species, has negatively impacted the oxygen levels, temperature, and pH of the wetland's water (Filizadeh and Khodaparast, 2005). Numerous studies have focused on the identification of aquatic plant species in different areas of Anzali Wetland. The most important studies include the investigation of plant species biodiversity in the 1960s, which identified 194 genera and 291 species (Ghahraman and Atar, 2003), as well as a more recent document that provides a checklist detailing 69 families and 362 species in Anzali Wetland (Zehzad, 2017). However, research on the growth and density of plant communities remains limited. Notably, only one study has addressed the distribution and abundance of aquatic plants in the western part of Anzali Wetland (Hosseinjani et al., 2017; Mirzajani et al., 2020a). Monitoring the distribution and biomass of dominant plant species from different ecological types is essential for effective wetland management. Therefore, this study investigates the biomass of dominant floating and submerged plants in the water body of Anzali Wetland and examines the distribution of prominent marginal plants along the wetland's boundaries. 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 consisted 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., 2020b). Today, the water bodies in most areas, including Shijan, Sorkhankul, and much of Siah Keshim, have dried up or become very limited and shallow. Sampling of submerged plants was carried out using a rake at 10 stations during four seasons in 2023. The aquatic plants were extracted from the water by rotating the rake with a circular motion, harvesting diameter of 30 cm. The samples were then washed with water to remove mud before being identified using reference materials (Abbasi, 1377; Riazi, 1996). Afterward, the samples were air-dried for 8 to 24 hours, and their wet weight was measured using a scale with an accuracy of 0.1 grams. The samples were then oven-dried at 70°C for 24 hours and weighed again. To measure the organic matter and ash content of each species, several subsamples were weighed on a balance with an accuracy of 0.0001 grams, burned in a furnace at 450 degrees Celsius for 4 hours, and weighed again (ASTM, 2000). The extent of marginal plants was determined using satellite images and the Normalized Difference Vegetation Index (NDVI), while the distribution of floating plants within the water body was assessed through visual interpretation of Google Earth images. Results In the western part of the Anzali Wetland, the submerged species Ceratophyllum demersum, Myriophyllum spicatum, Stuckenia pectinata, and Hydrilla verticillata exhibited high densities, while Najas marina and Potamogeton crispus were abundant in some areas. The average biomass of submerged aquatic plants was 547.7±313.9 g/m², varying from 400 to 983 g/m² across different stations. C. demersum showed the highest biomass at 53.8%, followed by H. verticillata at 18.9% and M. spicatum at 10.6%. The biomass of C. demersum ranged from 146 to 595 g/m² at various stations. The highest average plant biomass was measured in July (730 g/m²), while the lowest was recorded in May (388 g/m²) (Fig 1). The organic matter content of submerged plants was found to be 1.72% of their dry weight, and the contribution of organic matter to the sediment of Anzali Wetland due to winter die-off was estimated at 246-560 grams per square meter. Among the floating plants, Nelumbo nucifera and Pontederia crassipes were the dominant species, covering areas of 350 and 970 hectares, respectively. The final weight of water hyacinth was estimated at 150 tons per hectare. Other floating plants, including Hydrocotyle spp., Nymphoides cristata, Lemna spp., Spirodela spp., Salvinia natans, and Trapa natans, were observed in limited and patchy distributions across some stations. The leaf dry weight of N. nucifera was measured at 219.5 g/m², while the total dry biomass of P. crassipes was measured at 1620 g/m². Their total organic matter values were approximately 88.5% and 78.2% of their dry weight, respectively. P. crassipes occupied marginal areas and shallow zones of the western wetland, along all canals, drains, and entrances that have slow water flow, with no area completely devoid of this plant. When this plant dies back in winter, it contributes about 1267 grams per square meter of organic matter to the wetland substrate. The common reed, Phragmites australis, is also a dominant species in the marginal areas of the wetland. Its distribution was estimated to cover 10,290 hectares, as determined through visual interpretation of Google Earth images. Figure 1: Average dry weight of submerged plants in different areas of the western part of Anzali Wetland (a) during the months of 1402 Discussion and conclusion  In recent years, the shrinking of the Anzali Wetland and the conversion of water bodies to other land uses have intensified. Comparing the extent of land uses within the wetland bed in 2022 to that in 2001 showed that the water body had decreased by about 40%, while urban areas increased by 114%, paddy fields by 4.7%, forests by 54%, and common reeds by 3.6%. Comparing the results of this research with wetland plant surveys from previous years (Hosseinjani et al., 2017; Mirzajani et al., 2020a) indicates rapid changes in the aquatic plant communities of the wetland. The highest distribution and density of Azolla filiculoides was recorded in the summer of 2017, while it was not observed in the main areas of the wetland in subsequent years. The biomass of submerged plants in this survey was almost similar to that of the 2017 survey, but the composition of dominant species changed slightly. In 2017, the submerged species C. demersum and M. spicatum had high distribution and density (Mirzajani et al., 2020a), while in this study, the dominant species were C. demersum and H. verticillata. In 2017, two floating species, Nelumbo nucifera and Hydrocotyle spp., were dominant, especially in marginal areas (Mirzajani et al., 2020a). However, after five years, N. nucifera significantly occupied more areas of the wetland, and the invasive water hyacinth P. crassipes has grown explosively, occupying a large area of Anzali Wetland at high densities. This indicates that the high density of aquatic plants in the water body and marginal areas has become a serious problem. The excessive growth of both floating and submerged species impacts the growth and survival of many aquatic organisms, especially fish and epiphytic macroinvertebrates, sometimes leading to their mortality due to decreased water oxygen levels and changes in water temperature and pH. Therefore, optimal harvesting of aquatic plants is essential for the management of Anzali Wetland and should not be overlooked. 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 14-73-12-036-01051-011054. We also appreciate the help of our colleagues at Inland Waters Aquaculture Research Center. 17 26 2025/01/12025/04/1 1404/1/12 2025/07/12025/07/1 1404/4/10 علیرضا میرزاجانی Alireza Mirzajani پژوهشکده آبزی پروری آبهای داخلی Iran armirzajani@gmail.com وحید غلامی Vahid Gholami گروه مهندسی مرتع و آبخیزداری، دانشکده منابع طبیعی Iran gholami.vahid@gmail.com سعید نادری Saeed Naderi گروه محیط زیست، دانشکده منابع طبیعی Iran ssnadery@gmail.com رضا محمدی دوست Reza Mohammadidost پژوهشکده آبزی پروری آبهای داخلی Iran rezamohamadidoust@gmail.com Aquatic plants floating and submerge water hyacinth lotus Common Coontail common Reed گیاهان آبزی غوطه ور شناور سنبل آبی لاله مردابی چنگال آبی نی Abbasi, H., 1998. Aquatic plants. Natural Sciences of Padidae, Gorgan. 228 P. (In Persian).##Arshad, A., 1994. Study the effects of Azolla on Anzali wetland. Bandar Anzali. 81 P. (In Persian).##Asri, Y. and Eftekhari, T., 2002. An introduction to the flora vegetation of Siah-keshim and wetland. 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مقاله علمی – پژوهشی:‌ تاثیر برخی غذاهای وارداتی مراکز تکثیر میگو با آزمایش واکنش زنجیره‌ای پلیمراز (Real time PCR) مثبت ویروس لکه سفید و توانایی آنها در آلودگی پست‌لاروها The effect of some imported foods from shrimp breeding centers on positive real-time PCR tests for white spot virus and their ability to infect post-larvae 1 1 پرورش میگو به عنوان یکی از ارکان آبزی‌پروری به دلیل ارزش غذایی بالا، مورد استقبال آبزی‌پروران، تجار و مردم قرار گرفته است. طی سال‌های اخیر توسعه صنعت میگو در ایران، به دلیل عدم دسترسی کافی نهاده‌های اولیه تولید غذا از خارج از کشور، با مشکل جدی مواجه شده است. بررسی‌های اخیر نشان داده است که عمده غذاهای تکثیر میگو وارداتی است. در بررسی‌های اولیه کارشناسان که مبتنی بر آزمایش‌های واکنش زنجیره‌ای پلیمراز کمی (Real time PCR) بوده، مشخص گردیده است که برخی از غذاهای وارداتی مراکز تکثیر میگو آلوده به ویروس لکه سفید میگو هستند. برخی دیگر از کارشناسان معتقدند که وجود قطعاتی از ژنوم ویروس موجب می‌گردد تا آزمایش‌های واکنش زنجیره‌ای پلیمراز مربوطه مثبت گردد. بنابراین، بر اساس همین ابهام که آیا مثبت بودن آزمایش واکنش زنجیره‌ای پلیمراز غذا، می‌تواند دال بر وجود ویروس فعال باشد و این‌که این غذا کانونی برای ایجاد و انتقال ویروس گردد، تحقیقی تدوین گردید که مشتمل بر 4 گروه تحقیقاتی شامل یک گروه کنترل و 3 گروه تیمار در 3 تکرار با 90 قطعه میگو در هر گروه (30 قطعه در هر تکرار) در آکواریوم‌های شیشه‌ای 150×30×50 بوده است. گروه شاهد با غذای عاری از ویروس لکه سفید (Real time منفی) و گروه‌های بعدی با نتایج Real time  مثبت به‌ترتیب گروه‌های 2، 3 و 4 با غذاهای تجاری  MPZ،MEF و Flak تغذیه شدند. پس از گذشت 20 روز از غذادهی، ردیابی ویروس لکه سفید در نمونه میگوها در آزمون‌های Real time ، Nested-PCR (PCR آشیانه‌ای) و در نهایت از نظر هیستوپاتولوژی منفی گردید. نتایج این تحقیق نشان داد، انتقال عفونت لکه سفید میگو از طریق غذاهای پلت وارداتی مراکز تکثیر محتمل نیست. 2 Introduction The growing global population and increasing demand for healthy protein have made aquaculture, particularly shrimp farming, a crucial solution. However, rapid industry expansion has led to disease outbreaks like White Spot Syndrome Virus (WSSV), causing $20 billion in Asian losses over two decades (Davies, 2016). Global farmed shrimp production surged from 50,000 tons (1975) to 4.5 million tons (2018) (FAO, 2018), with Iran's production reaching 60,000 tons in 2022 and targeting 150,000 tons. Intensive farming practices (up to 1.5 million PLs/0.8 ha) and excessive feeding (20-25% unused protein) exacerbate disease risks. WSSV already affects 10-14% of Iranian farms (Madani, 2021), causing annual losses of 5,000 tons (~$17 million) - potentially doubling by 2023. This study evaluates WSSV transmission through contaminated imported feed using real-time PCR, comparing results with nested PCR, histopathology, and control groups to address this critical biosecurity gap. Methodology This experimental study evaluated the potential transmission of White Spot Syndrome Virus (WSSV) through imported shrimp feeds using four test groups: one control group fed WSSV-free feed (RT-PCR negative) and three treatment groups fed RT-PCR positive commercial feeds (MPZ, MEF, and Flak). Each group consisted of 90 PL5 shrimp (30 per replicate, 3 replicates) maintained in 150×30×50 cm glass aquariums containing 60% sterilized coastal water with continuous aeration. The trial lasted 20 days with four daily feedings, alternate-day siphoning, and 20% water exchange. Survival rates were calculated and compared statistically. For molecular analysis, samples from moribund shrimp underwent DNA extraction using commercial kits, followed by TaqMan RT-qPCR with specific WSSV primers (50°C for 2 min, 95°C for 10 min, then 40 cycles of 95°C/15 sec and 60°C/1 min) and nested-PCR using the IQ2000 WSV kit. Histopathological examination involved fixing six PLs per group in Davidson's solution, followed by standard ethanol-xylene processing, paraffin embedding, 4μm sectioning, and H&E staining. Data were analyzed using one-way ANOVA with Tukey's post-hoc test (α=0.05). Results The experimental feeds for groups 2-4 tested positive for WSSV by RT-PCR at an accredited veterinary laboratory, with Ct values consistently above 30, indicating low viral genome loads (Figures 2-4). Survival rates after 20 days showed no significant differences (p>0.05) between groups: control (95.55%), MPZ (95.55%), MEF (97.77%), and Flak (94.44%) (Figure 5). Although the Flak group had slightly lower survival, statistical analysis revealed no significant differences among groups. All shrimp samples tested negative for WSSV infection through both RT-PCR (Table 2) and nested-PCR confirmation (Figure 6) using the IQ2000 kit. Histopathological examination of hepatopancreas, intestine, muscle and gill tissues showed no viral inclusion bodies in any experimental group (Figures 7-10). The study results clearly demonstrate that while traces of WSSV genetic material were detected in the imported shrimp feeds, these findings do not indicate any actual disease risk. The high Ct values (above 30) from PCR testing show the viral material present was minimal and likely non-infectious fragments rather than live virus. Most importantly, after 20 days of feeding, comprehensive testing revealed no evidence of WSSV infection in any shrimp across all experimental groups. All shrimp samples tested negative through both molecular methods (real-time PCR and nested PCR) and histological examination, with no viral inclusion bodies found in any tissues. The survival rates across all groups remained consistently high (94.44-97.77%) with no statistically significant differences, confirming that the PCR-positive feeds performed just as well as the control feed. This strongly suggests that the presence of viral genetic material in these imported feeds does not translate to actual disease transmission or negative impacts on shrimp health and survival. The consistent negative results across all diagnostic methods provide robust evidence that these feeds do not pose a WSSV transmission risk under normal aquaculture conditions. These findings have important implications for the shrimp farming industry, indicating that proper PCR testing can effectively distinguish between non-infectious viral fragments and genuine disease threats in imported feeds. The findings conclusively show that the presence of WSSV genetic material in imported feeds did not lead to actual infection or affect shrimp survival under these experimental conditions. Discussion and conclusion This study aimed to assess the potential pathogenicity of shrimp hatchery feed that tested positive in real-time PCR for white spot syndrome virus (WSSV). The results confirmed trace amounts of WSSV genetic material in imported larval-stage feed (Figures 2-4), though high Ct values indicated low viral genome levels, suggesting contamination rather than active infection. The detected genetic material likely originated from natural shrimp-based pigments or antioxidants added during feed production. While some researchers claim WSSV is inactivated by heat or freezing, inconsistent global measures (sometimes conflicting with OIE standards) have perpetuated uncertainty (Durand et al., 2000). Deadly viruses can spread via: Transport of infected live stocks (Schnurrenberger et al., 1987), Bird vectors (Garza et al., 1997), Import/reprocessing of frozen food (Humphrey, 1995). Durand et al. (2000) found WSSV survives freezing/cold storage, prompting this study to evaluate processing methods beyond freezing. WSSV has been detected in post-larvae (PL) of various Penaeus species across different regions (Withyachumnarnkul et al., 2003), with multiple potential transmission routes proposed. Vertical transmission was confirmed by Mohan et al. (1997), who observed WSSV inclusions in reproductive organs and eggs of P. monodon. Momoyama et al. (1998) reported WSSV survival after freezing, while Durand et al. (2000) suggested that block freezing could degrade WSSV DNA, attributing viral inactivation to ice crystal damage or repeated freeze-thaw cycles during processing. Contradictory findings exist: Hasson et al. (2006) and Sritunyalucksana et al. (2010) noted WSSV viability in frozen shrimp. However, Aranguren Caro et al. (2020) demonstrated that boiling WSSV-infected shrimp for 1–30 minutes eliminated infectivity, despite qPCR detecting viral DNA (with no significant Ct differences). Nested PCR and histopathology confirmed the loss of infectious WSSV in boiled samples, with no pathological lesions observed—aligning with this study’s results (Figures 7–10), which suggest heat-treated feed poses no infection risk to broodstock. Experimental WSSV infection via feeding contaminated shrimp tissue has been demonstrated (Chou et al., 1998; Hameed et al., 2002; Momoyama et al., 1998). However, wild shrimp/crabs of unknown WSSV status are still used as feed in some systems (Corsin et al., 2001). Few studies evaluate feeding practices' impact on WSD outbreaks. While MPEDA/NACA found no link (Corsin et al., 2005), Vietnam associated higher feed amounts with WSD incidence—possibly due to water quality or stocking density (Corsin et al., 2001). Contrary to this study’s conclusion (no transmission via feed), Maeda et al. (1998) suggested commercial feed could influence WSSV positivity. Similarly, Corsin et al. (2001) linked specific feed brands to WSSV presence in harvested shrimp, though without disease correlation. In India, 43% of ponds used WSSV-positive feed, with some brands showing higher contamination—likely due to raw materials or milder processing (Corsin et al., 2002). However, no direct feed-WSD relationship was found, implying low-quality feed may weaken immunity, exacerbating natural infections. Pongmaneerat et al. (2001) observed no WSSV in hemolymph after feeding WSSV-positive feed, despite oral transmission being highly effective (Soto and Lotz, 2001). This study concludes that hatchery feed cannot transmit WSSV to post-larvae; real-time PCR likely detects non-infective viral fragments from raw materials. Conflict of interest The authors declare no conflict of interest. 27 39 2025/01/12025/04/12023/09/16 1402/6/25 2025/07/12025/07/12025/07/1 1404/4/10 شاپور کاکولکی S. Kakoolaki مؤسسه تحقیقات علوم شیلاتی کشور، سازمان تحقیقات، آموزش و ترویج کشاورزی، تهران، ایران Iran bsh443@gmail.com بابک قائدنیا B. Ghaednia مؤسسه تحقیقات علوم شیلاتی کشور، سازمان تحقیقات، آموزش و ترویج کشاورزی، تهران، ایران Iran babak.ghaednia@gmail.com سید امیر مختار بهاری میمندی S.A.M. Bahari Meymandi سازمان دامپزشکی ایران، اداره کل هرمزگان، هرمزگان، ایران Iran amir.bahari@yahoo.com ابوالفضل سپهداری A. Sepahdari مؤسسه تحقیقات علوم شیلاتی کشور، سازمان تحقیقات، آموزش و ترویج کشاورزی، تهران، ایران Iran asepahdari@yahoo.com عیسی شریف پور I. Sharifpour مؤسسه تحقیقات علوم شیلاتی کشور، سازمان تحقیقات، آموزش و ترویج کشاورزی، تهران، ایران Iran isharifpour@yahoo.com مینا آهنگرزاده M. Ahangarzade پژوهشکده آبزی پروری جنوب کشور، مؤسسه تحقیقات علوم شیلاتی کشور، سازمان تحقیقات، آموزش و ترویج کشاورزی، اهواز، ایران Iran rkbs_kh@yahoo.com عاطفه همتی A. Hemati مؤسسه تحقیقات علوم شیلاتی کشور، سازمان تحقیقات، آموزش و ترویج کشاورزی، تهران، ایران Iran atefeh.hemati2543@gmail.com محمد خلیل پذیر M.K. Pazir پژوهشکده میگوی کشور، مؤسسه تحقیقات علوم شیلاتی کشور، سازمان تحقیقات، آموزش و ترویج کشاورزی، بوشهر، ایران Iran dr.pazir@gmail.com Shrimp Hatchery Larval feed WSSV Diagnosis پست لارو میگو غذای لاروی بیماری لکه سفید میگو مراکز تکثیر میگو تشخیص Aranguren Caro, L.F., Mai, H.N., Nunan, L., Lin, J., Noble, B. and Dhar, A.K., 2020. Assessment of transmission risk in WSSV‐infected shrimp Litopenaeus vannamei upon cooking. Journal of Fish Diseases, 43(4):403-411. DOI: 10.1111/jfd.13134##Chou, H.Y., Huang, C.Y., Lo, C.F. and Kou, G.H., 1998. Studies on transmision of white spot syndrome associated baculovirus (WSBV) in Penaeus monodon and P.japonicus via water borne contact and oral ingestion. Aquaculture, 164:263-276. DOI:10.1016/S0044-8486(98)00192-8##Corsin, F., Turnbull, J., Hao, N., Mohan, C., Phi, T., Phuoc, L., Tinh, N. and Morgan, K., 2001. Risk factors associated with white spot syndrome virus infection in a Vietnamese rice-shrimp farming system. Diseases of Aquatic Organisms, 47(1):1-12. DOI:10.3354/dao047001##Corsin, F., Thakur, P., Padiyar, P., Madhusudhan, M., Turnbull, J., Mohan, C., Hao, N. and Morgan, K., 2002. Feeding farmed shrimp with shrimp waste—the lessons for aquaculture from BSE. Research in Veterinary Science, 72(8). DOI:10.1053/rvsc.2001.0531##Corsin, F., Turnbull, J., Mohan, C., Hao, N. and Morgan, K., 2005. Pond-level risk factors for white spot disease outbreaks. Diseases in Asian aquaculture V, 75-92.##Davies, R., 2016. Disease has cost asia shrimp sector over $20bn. In: Undercurrent News. Avaialabe at: https://www.undercurrentnews.com/2016/09/09/diseasehas-cost-asia-shrimp-sector-over-20bn/ accessed on 9.11.2021.##Durand, S., Tang, K. and Lightner, D., 2000. Frozen commodity shrimp: potential avenue for introduction of white spot syndrome virus and yellow head virus. Journal of Aquatic Animal Health, 12(2):128-135. DOI:10.1577/1548-8667(2000)012<0128:FCSAPF>2.0.CO;2##FAO, 2018. The state of world fisheries and aquaculture 2018. Meeting the sustainable development goals.##Garza, J., Hasson, K., Poulos, B., Redman, R., White, B. and Lightner, D., 1997. Demonstration of infectious Taura syndrome virus in the feces of seagulls collected during an epizootic in Texas. Journal of Aquatic Animal Health, 9(2):156-159. DOI:10.1577/1548-8667(1997)009<0156:DOITSV>2.3.CO;2##Hameed, A.S., Murthi, B., Rasheed, M., Sathish, S., Yoganandhan, K., Murugan, V. and Jayaraman, K., 2002. An investigation of Artemia as a possible vector for white spot syndrome virus (WSSV) transmission to Penaeus indicus. Aquaculture, 204(1-2):1-10. DOI:10.1016/S0044-8486(01)00640-9##Hasson, K., Fan, Y., Reisinger, T., Venuti, J. and Varner, P., 2006. White-spot syndrome virus (WSSV) introduction into the Gulf of ## Mexico and Texas freshwater systems through imported, frozen bait-shrimp. Diseases of Aquatic Organisms, 71(2): 91-100.##Humphrey, J., 1995. Quarantine policies and practices for aquatic animals and their products: A review for the Australian quarantine and inspection service. Bureau of Resource Sciences, Canberra.##Iber, B.T. and Kasan, N.A., 2021. Recent advances in shrimp aquaculture wastewater management. Heliyon, 7(11). DOI:10.1016/j.heliyon.2021.e08283##Jang, I.K., Meng, X.H., Seo, H.C., Cho, Y.R., Kim, B.R., Ayyaru, G. and Kim, J.S., 2009. A TaqMan real-time PCR assay for quantifying white spot syndrome virus (WSSV) infections in wild broodstock and hatchery-reared postlarvae of fleshy shrimp, Fenneropenaeus chinensis. Aquaculture, 287(1-2):40-45.##Kakoolaki, S., Soltani, M., Ebrahimzadeh Mousavi, H.A., Sharifpour, I., Mirzargar, S., Afsharnasab, M. and Motalebi, A., 2011. The effect of different salinities on mortality and histopathological changes of SPF imported Litopenaeus vannamei, experimentally exposed to white spot virus and a new defferential hemocyte staining method. Iranian Journal of Fisheries Sciences, 10(3): 447-460.##Lightner, D. 1996. A handbook of pathology and diagnostic procedures for diseases of penaeid shrimp (1 ed.). LA, Baton Rouge: World Aquaculture Society.##Madani, V., 2021. Fourty-nine thousands of shrimp was produced in Iran. Bushehr Fishery Office Report, [in Persian].##Maeda, M., Kasornchandra, J., Itami, T., Suzuki, N., Hennig, O., Kondo, M., Albaladejo, J.D. and Takahashi, Y., 1998. 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مقاله علمی – پژوهشی:‌ بهینه‌‌سازی استخراج آلژینات‌‌سدیم با استفاده از طرح ‌‌مرکب ‌‌مرکزی (CCD) از جلبک دریایی قهوه‌‌ای Sargassum angustifolium Optimization of sodium alginate extraction conditions using a central composite design from the brown seaweed Sargassum angustifolium 1 1 جلبک‌های دریایی قهوه‌ای منبع ارزشمندی از بیوپلیمرهای مختلف هستند که آلژینات به ‌عنوان یک فیکوکلوئید از اهمیت صنعتی بالایی برخوردار است. این تحقیق به منظور بهینه‌‌‌‌سازی استخراج آلژینات‌‌سدیم از جلبک دریایی قهوه‌‌ایSargassum angustifolium با استفاده از طرح‌‌ مرکب ‌‌مرکزی (CCD) روش سطح پاسخ (RSM)[1] با در نظر گرفتن متغیرهای مستقل زمان، دما و pH انجام شد. بازده استخراج آلژینات‌‌سدیم و میزان اسیدیورونیک پاسخ‌‌های مدل بودند. نتایج نشان داد که شرایط بهینه 4 ساعت، 90 درجه سانتی‌‌گراد و pH 10 منجر به 64/34 درصد بازده آلژینات‌‌سدیم شد درحالی‌که شرایط بهینه برای بیشترین میزان اسیدیورونیک با 12/27 درصد در pH 10، زمان 4 ساعت و دمای 40 درجه سانتی‌‌گراد به‌‌دست آمد. نتایج نشان داد که pH دارای بیشترین تاثیر در بین سایر متغیرهای مستقل بر پاسخ‌‌ها بود و به دنبال آن دما و زمان قرار داشتند. نتایج نشان داد که مدل‌‌درجه‌‌دوم قادر است، بازده آلژینات‌‌سدیم و میزان اسیدیورونیک را با R2، R2Adj و R2Pre به‌ترتیب با مقادیر 9873/0، 9746/0، 9275/0 و 9744/0، 9489/0، 923/0پیش‌‌بینی کند. افزایش pH منجر به آلژینات‌‌های با رنگ روشن‌‌تر شد. نتایج این مطالعه نشان داد، استخراج آلژینات‌‌سدیم با استفاده از طرح ‌‌مرکب ‌‌مرکزی برای بهینه‌‌سازی آن و بهبود شرایط استخراج، رضایت‌‌بخش بود.   [1] Response Surface Methodology (RSM) 2 Introduction Marine algae, with large size and wide variety of species, are marine ecosystems. They are cultivated both naturally and commercially in the coastal areas of the world. 25,000 tons of algae are produced worldwide each year and its economic value is about 6.5 billion, which appears to reach $ 1.07 billion by 2028 (Yang et al., 2025). Marine algae are mainly used as food in most Asian countries such as China, Japan, South Korea and North Korea, Indonesia, Philippines, Vietnam and Thailand, and for the first time in Japan in 1670 they were raised (Khan et al., 2024). Due to their high consumption in Asia, brown algae make up 47.3 percent of the world's cultivation level with 16.4 million tonnes (Nam et al., 2024; Radulovich et al., 2015). Marine macroalgae are a sustainable raw material with antioxidant, antimicrobial, non-condensed, biodegradable and biological properties. The most important polysaccharide in algae cell is alginate (40 % dry weight), laminarin and fokoidan (Faidi et al., 2025). In algae, the percentage of alginate can reach up to 60 % dry weight. Alginates contain sodium, calcium, magnesium, barium and monomeric acid units D-mannuronic (M) and L-guluronic (G). Uronic acids are often organized in the forms of heteropolymer block (MG) or hemopolymer (MM or GG) (Fawzy et al., 2017). Methodology From the shallow and lower parts of the tide of the rocky beds of the Persian Gulf and the Oman Sea (Chabahar area), the S. angustifolium algae was carried out. The Persian Gulf and Oman Sea Atlas and Algae base platform data were used to confirm the species. The algae were washed with distilled water several times to remove impurities and algae sand. Then in the dry sun, they were powdered with a mill. Then, in polyethylene bags, they were transferred to the laboratory under 10°C (Fig. 1). Figure 1: Sargassum angustifolium Central Composite Design (CCD) using Response Surface Methodology (RSM) to optimize the process of extraction of sodium alginate in brown algae S. angustifolium by considering 3 factors or independent variables temperature (A), time (b) and pH (c) consisting of 19 points and 5 repeats at point the central was used with 19 treatments. In RSM it is selected for each domain variable. The third level as the central level or (0) is minimal and maximum (Table 1). Table 1: Experiment design levels for independent variables using CCD Control factors Surface -1 0 +1 A Temperature (°C) 40 65 90 B Time (h) 1 2.5 4 C pH 8 9 10 Results The results of the CCD test plan with 19 repetitions are presented in Table 2 to evaluate the optimum parameters of sodium alginate extraction from S. angustifolium. As Table 2 shows, the actual and coded values ​​of each factor and 19 experimental points include three repetitions of central points. Independent variables were the study of temperature (A), time (b) and pH (c). The second -degree polynomial equation shows the correlation between the independent variable and the responses. Multiple regression analysis and analysis of variance (ANOVA) were performed on experimental data and two multi -order multi -order equations were created in terms of encoded factors (Table 3). Second -degree equations for alginate efficiency and acidic amount were obtained as follows: Alginate yield: 14.6029 + 2.0921 A + 1.0935 B + 9.3055 C - 0.41 AB + 1.6825 AC + 0.7995 BC - 1.15968 A2 + 0.0553247 B2 + 6.60732 C2 Uronic acid content: 14.0044 - 0.8655 A + 0.1275 B + 8.9599 C - 1.247 AB - 1.8205 AC - 0.413 BC - 4.59434 A2 + 1.19566 B2 + 4.29266 C2 Table 2: CCD design from RSM and the observed responses; Sodium alginate yield and uronic acids (%) Std Run Factor 1 Factor 2 Factor 3 Response 1 Response 2 A: Temperature B: Time C: pH Sodium alginate yield Uronic acid content °C h % % 10 1 90 2.5 9 15.501 8.981 18 2 65 2.5 9 13.407 17.208 9 3 40 2.5 9 12.104 10.070 2 4 90 1 8 12.184 7.167 11 5 65 1 9 12.778 14.435 13 6 65 2.5 8 11.652 9.278 7 7 40 4 10 26.902 27.881 14 8 65 2.5 10 31.487 27.547 4 9 90 4 8 10.559 6.489 3 10 40 4 8 11.183 6.122 15 11 65 2.5 9 16.318 12.608 17 12 65 2.5 9 14.277 13.027 8 13 90 4 10 34.648 18.743 12 14 65 4 9 17.257 16.196 1 15 40 1 8 9.528 4.035 19 16 65 2.5 9 12.825 11.413 16 17 65 2.5 9 14.75 15.304 5 18 40 1 10 23.689 25.223 6 19 90 1 10 31.435 23.296  Table 3: Analysis of variance (ANOVA) for CCD model of sodium alginate extraction yield and uronic acid content Source Sum of Squares df Mean Square F-value p-value Model of yield 1123.64 9 124.85 77.75 < 0.0001 significant A-Temperature 43.77 1 43.77 27.26 0.0005 B-Time 11.96 1 11.96 7.45 0.0233 C-pH 865.92 1 865.92 539.28 < 0.0001 AB 1.34 1 1.34 0.8375 0.3840 AC 22.65 1 22.65 14.10 0.0045 BC 5.11 1 5.11 3.18 0.1080 A² 3.67 1 3.67 2.29 0.1646 B² 0.0084 1 0.0084 0.0052 0.9440 C² 119.29 1 119.29 74.29 < 0.0001 Residual 14.45 9 1.61 Lack of Fit 7.20 5 1.44 0.7952 0.6046 not significant Pure Error 7.25 4 1.81 Cor Total 1138.09 18 Model of uronic content 934.89 9 103.88 36.12 < 0.0001 significant A-Temperature 7.49 1 7.49 2.60 0.1410 B-Time 0.1626 1 0.1626 0.0565 0.8174 C-pH 802.80 1 802.80 279.16 < 0.0001 AB 12.44 1 12.44 4.33 0.0673 AC 26.51 1 26.51 9.22 0.0141 BC 1.36 1 1.36 0.4745 0.5083 A² 57.68 1 57.68 20.06 0.0015 B² 3.91 1 3.91 1.36 0.2738 C² 50.35 1 50.35 17.51 0.0024 Residual 25.88 9 2.88 Lack of Fit 4.35 5 0.8703 0.1617 0.9643 not significant Pure Error 21.53 4 5.38 Cor Total 960.77 18 Discussion and conclusion Optimizing the process conditions to achieve maximum efficiency and uronic acid amount can be obtained by analyzing multiplication using utility function. Under the experimental conditions, the projected extraction efficiency of sodium alginates was 33.54 % and 0.956, respectively. High desirability (0 ≤d ≤ 1) indicates better accuracy of independent variables in optimization. Both responses were empirically approved under optimal parameters, indicating that the model is sufficient to predict these responses and are comparable with other researchers: Sargassum natans 23 %, Sargassum vulgare 17 %, Padina gymnospora 16 %, Padina antillarum 22 %, Laminaria digita 29 %, Macrocystis pyriffra 26 % (Rhein-Knudsen et al., 2017), Sargassum vulgare 40 % (Sari-chmaysme et al., 2016), 19% Sargassum natans (Mohammed et al., 2018). Kanagesan et al. (2022) gained the efficiency of 13.55 % alginate by alkaline with 3 % carbonate extraction. Changes in alginate extraction efficiency among various studies, including the current study, may be due to differences in sargassum species, growth conditions, or extraction conditions (Khan et al., 2024). The efficiency of the extracted alginate is different among the brown marine algae, as these algae have physiological and metabolic processes that allow it to produce different metabolites. The concentration of nutrients in the environment directly affects the amount of polysaccharide in different metabolites. The production of polysaccharides is influenced by several factors such as species, age, sampling time and extraction method (El-Sheekh et al., 2024). Conflict of interest The authors declare no conflict of interest. Acknowledgment  The authors would like to thank the Islamic Azad University, Ahvaz Branch for arranging the experimental samples. 41 56 2025/01/12025/04/12023/09/162025/02/25 1403/12/7 2025/07/12025/07/12025/07/12025/07/1 1404/4/10 اکبر نوروزی akbar norozi گروه شیلات، واحد علوم و تحقیقات تهران، دانشگاه آزاد اسلامی، تهران، ایران Iran Aidanorouzi203@gmail.com مهدی شمسایی mehdi shamsaie گروه شیلات، واحد علوم و تحقیقات تهران، دانشگاه آزاد اسلامی، تهران، ایران Iran drshamsaie@gmail.com لاله رومیانی laleh roomiani گروه شیلات، واحد اهواز، دانشگاه آزاد اسلامی، اهواز، ایران Iran l.roomiani@yahoo.com هومن رجبی اسلامی Houman rajabi islami گروه شیلات، واحد علوم و تحقیقات تهران، دانشگاه آزاد اسلامی، تهران، ایران Iran rajabi.h@srbiau.ir مهدی رییسی mehdi Raissy گروه بهداشت و بیماری‌های آبزیان، واحد شهرکرد، دانشگاه آزاد اسلامی، شهرکرد، ایران Iran mreissy@yahoo.com Sodium Alginate Uronic Acid Algae Sargassum angustifolium آلژینات‌‌سدیم اسیدیورونیک جلبک Sargassum angustifolium Abraham, R.E., Su, P., Puri, M., Raston, C.L. and Zhang, W., 2019. 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مقاله علمی – پژوهشی:‌ شاخص‌‌های جمعیتی و وضعیت برداشت عروس دریایی موزی (Catostylus perezi, Ranson, 1945) در آبهای شمالی دریای عمان Population parameters and exploitation status of banana jellyfish (Catostylus perezi, Ranson, 1945) in the northern waters of the Oman Sea 1 1 یکی از ظرفیت‌های آبزیان غیر معمول خوراکی آبهای شمالی دریای عمان (استان سیستان و بلوچستان)، عروس دریایی موزی بوده که در سالیان گذشته مورد بهره‌برداری قرار گرفته است. طی سال 1403 تعداد 503 عدد عروس دریایی موزی زیست‌سنجی گردید و میانگین طولی (دامنه) میزان 6±20 (40-8) سانتی‌متر و میانگین وزنی (دامنه) میزان 326±899 (1700-180) گرم به‌دست آمد. رابطه طول کل و وزن بدن (88/0=R2، 503=N)06/1L 28/27=W به‌دست آمد و میزان b رابطه طول و وزن نشان‌دهنده رشد ناهمسان بود. معادله وان‌‌برتالنفی برای این گونه به‌ترتیب به صورت: Lt =46 (1- exp (-0.6 (t +0.24)) محاسبه شد. حداکثر محصول پایدار (MSY) و نسبت بیوماس فعلی به بیوماس بهینه (B/BMSY) به‌ترتیب 9969 تن، 09/1 برآورد شد. حداکثر محصول ثابت (MCY) و صید مجازکل (TAC) عروس دریایی موزی به‌ترتیب 6580 و 4984 تن تخمین زده شد. نسبت پتانسیل مولدین براساس طول (LBSPR) در سال 1403 این نسبت (01/1- 74/0) 81/0 به‌دست آمد. ضریب بهره‌‌برداری، شاخص LBSPR، شاخص مرگ‌ومیر صیادی موجود به مرگ‌ومیر صیادی حداکثر محصول پایدار (1F/FMSY<) و شاخص بیوماس موجود به بیوماس پایدار بیش از یک (1B/BMSY>) می‌تواند دلیلی بر وجود بهره‌‌برداری کمتر از بهینه (پایین‌تر از صید بهینه) در آبهای شمالی دریای عمان بوده و این اطلاعات در زمینه مدیریت بهره‌‌برداری و صید از این گونه با ارزش می‌تواند مفید و ضروری باشد. 2 Introduction More than 1,400 jellyfish species have been identified worldwide (Brotz et al., 2017). However, only 23 edible jellyfish species have been studied for their nutritional properties, with a growing commercial market primarily observed in Asia (Edelist et al., 2021). Various studies and field reports indicate that large-scale and frequent jellyfish blooms have occurred globally since the 1950s. Jellyfish have long been consumed as food in many Southeast Asian countries, particularly China, largely due to their high collagen content (Subhan et al., 2021). Jellyfish blooms have been shown to significantly impact human activities worldwide, including tourism, aquaculture, fishing operations, power plant cooling systems, and seawater desalination plants. Despite these challenges, jellyfish also contribute positively to marine ecosystems by providing habitat for various fish species and playing a role in carbon sequestration (Edelist et al., 2021). Jellyfish are polypless members of the phylum Cnidaria. These gelatinous marine animals possess a bell-shaped body and trailing tentacles. They use the pulsation of their umbrella-like bell for locomotion, while their tentacles are primarily used for capturing prey (Sealifebase, 2023). Jellyfish inhabit all of the world’s oceans, occurring from the surface to great depths. Fossil evidence suggests that jellyfish have existed in marine environments for over 500 million years—possibly up to 700 million years—and they represent some of the oldest known multicellular organisms with differentiated tissues (Gueroun et al., 2021). In the southern waters of Iran, at least six jellyfish species belonging to three families have been documented. Among these, Cassiopea andromeda is endemic to the Persian Gulf and Nayband Bay, whereas the remaining five species exhibit broader distributions across the region (Owfi, 2020). In the Tis (Chabahar) and Pozm areas, the dominant jellyfish species is the banana jellyfish (Catostylus perezi), which is also the predominant species in the northern Indian Ocean and is currently subject to commercial harvesting (Gueroun et al., 2021). C. perezi, commonly known as the banana jellyfish, is distributed across the western Indian Ocean from Kuwait to India. It belongs to the subphylum Metazoa, phylum Cnidaria, class Scyphozoa, order Rhizostomeae, family Catostylidae (Riyas et al., 2019). Methodology The study area is situated in the northern part of the Oman Sea, within Sistan and Baluchistan Province, Iran, covering an east longitude range of 28°60′ to 20°61′ and a north latitude range of 14°25′ to 60°25′. Two landing sites—Pozm Port (28°60′ E, 14°25′ N) and Tis Port (62°60′ E, 35°25′ N)—were selected as sampling stations for the banana jellyfish. At each station, several specimens were collected, counted, and rinsed before being transported to the laboratory for subsequent biochemical analysis. The primary method of jellyfish capture in this region involves the use of a scoop net/cast net. Result Sampling of banana jellyfish  was conducted in the Tis and Pozm regions from June to December 2024 (corresponding to the Persian year 1403). The highest frequency of occurrence was recorded in August (48%), while the lowest was observed in June (1%). A total of 503 individuals were subjected to biometric measurements during the study period. The mean total length of the sampled jellyfish was 20±6 cm, with a range of 8–40 cm (Fig. 1). The mean wet weight was 326 ± 899 g, with individual weights ranging from 180 g to 1,700 g. The highest length frequency was observed in 17–20 cm size class (approximately 20%), whereas the lowest frequency was recorded in the 38–41 cm size class (1%). Figure 1: Location of jellyfish data collection areas in the northern waters of the Sea of ​​Oman (Sistan and Baluchestan Province) Discussion and conclusion The estimated values of maximum sustainable yield (MSY), maximum constant yield (MCY), and total allowable catch (TAC) were all higher than the actual catch recorded for banana jellyfish in 2024. This suggests that the current level of harvesting in the northern Oman Sea (Sistan and Baluchistan Province) remains below the optimal exploitation level. The exploitation coefficient (E) was less than 0.5, and fishing mortality (F) was lower than natural mortality (M), further indicating the absence of significant fishing pressure on this species. According to Sparre and Venema (1998) and King (2007), the exploitation rate should not exceed 0.5, and fishing mortality should remain below natural mortality; otherwise, it signals overfishing. Two primary factors influence stock pressure:1) The intensity of fishing and harvesting from the population, and 2) Environmental variables affecting the survival and accessibility of the stock (Mateus and Estupiñán, 2002). The length-based spawning potential ratio (LBSPR) index for the study period exceeded 0.8, indicating no signs of overfishing and a healthy stock status (Cousido-Rocha et al., 2022; Deng et al., 2024). The LBSPR serves as an estimator of the spawning potential ratio (SPR), where values below approximately 0.2 (B/B₀ ≈ 0.2) suggest severe depletion, while values above 0.6 (B/B₀ ≈ 0.6) reflect a favorable stock condition (Zhai et al., 2020; Froese et al., 2018). The ratio of available fishing mortality to maximum sustainable yield fishing mortality was less than one (F/FMSY<1) and the ratio of available biomass to sustainable biomass was greater than one (B/BMSY>1), indicating no overfishing. Froese and Pauly (2020) consider the fishing mortality at maximum sustainable yield (FMSY) and natural mortality (M) to be equivalent (FMSY ≈ M). F/FMSY values ​​above one mean overfishing and less than one means underfishing, and B/BMSY values ​​above one mean underfishing and less than one mean overfishing (Ji et al., 2019). Overall, the results indicate that the banana jellyfish population in the northern Oman Sea is currently underexploited and shows no signs of overfishing. Continued monitoring and adaptive management strategies are recommended to ensure the long-term sustainability of this resource. Conflict of Interest There are no known conflicts of interest associated with this study. 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مقاله علمی – پژوهشی:‌ مطالعه کمی و کیفی رشد ریز جلبک Chlorella vulgarisدر آب چاه ژرف سیستان Quantitative and qualitative study of the microalgae growth, Chlorella vulgaris in deep aquifer well water of Sistan 1 1 در این مطالعه به بررسی کمی و کیفی رشد ریز جلبک Chlorella vulgaris در در محیط کشت گیلارد و آب ژرف منطقه سیستان پرداخته شده است. این آزمایش با 5 تیمار (تیمار 1: 100 درصد محیط کشت، تیمار 2: 25 درصد آب ژرف، تیمار 3: 50 درصد آب ژرف، تیمار 4: 75 درصد آب ژرف، تیمار 5: 100 درصد آب ژرف) و 3 تکرار طی مدت زمان 10 روز در دمای 27 درجه سانتی‌گراد و روشنایی 3500 لوکس و هوادهی به صورت مداوم در ظرف‌های 500 میلی لیتر انجام شد. نتایج این آزمایش نشان داد، pH در تیمارهای آب ژرف به میزان 63/10-89/8 بوده است. به‌علاوه، همه تیمارها نسبت به روز اول آزمایش (تراکم 104×15 سلول در میلی لیتر) تا روز 5 آزمایش از رشد برخوردار بودند و بعد از آن روند رشد آنها نزولی شد. نتایج نشان داد که در بین تیمارها، تیمار 2(25 درصد آب ژرف) از کمترین رشد 104× 24/50 سلول در میلی لیتر و تیمار 5 (100 درصد آب ژرف) با تراکم 104×33/129 سلول در میلی لیتر نسبت به سایر تیمارها دارای بیشترین رشد بود. بیشترین و کمترین نرخ رشد ویژه (SGR) به‌ترتیب برای تیمار یک (100 درصد محیط کشت) و 4 (75 درصد آب ژرف) بود. همچنین بیشترین و کمترین زمان دو برابر شدن (DT) به‌ترتیب مربوط تیمار 4 (75 درصد آب ژرف) و یک (100 درصد محیط کشت) بود. همچنین بیشترین و کمترین بیومس به‌ترتیب برای تیمار 5 (100 درصد آب ژرف) و 3 (50 درصد آب ژرف) بود. بیشترین و کمترین میزان کلروفیل a نیز به‌ترتیب برای تیمار 5 (100 درصد آب ژرف) در روز سوم و تیمار 3 (50 درصد آب ژرف) در روز آخر بود. بنابراین، طبق نتایج این مطالعه، پرورش ریزجلبک C. vulgaris در آب چاه ژرف امکان‌پذیر است. 2 Introduction Microalgae can produce valuable compounds, such as pigments, minerals, amino acids and fatty acids, proteins, and vitamins that can be used in pharmaceutical, health, and food fields, with three economically important genera including Chlorella, Spirulina, and Dunaliella (Priyadarshani and Rath, 2012). Green algae include various forms: filamentous, membranous, unicellular, plate-like, or tubular. Chlorella is spherical in shape and have relatively thin walls. Reproduction in Chlorella is also very simple and occurs through asexual reproduction (Kerem et al., 2008). C. vulgaris can stimulate plant growth in agriculture and suppress the growth of pathogenic microorganisms (Allaguvatova et al., 2019). Chlorella is an important species of green algae that is fed to rotifers, various aquatic larvae and some phytophagous fish in fish farming ponds and aquatic ecosystems (Salavatian and Fallahi, 2005). Deep aquifer well water is a source of groundwater that is considered a strategic asset in any country. Deep water, which is located at a depth of 300 to 1200 meters, is not suitable for drinking due to high salinity and dissolved solids, but it is suitable for agricultural activities. These water resources are valuable in countries with arid and desert climates. The first deep water well in Iran was commissioned in 2018 in the Sistan region with the capacity to produce 1,500 m3 of water per day (Khosravanizadeh et al., 2021). This research was conducted considering the closeness of the salinity level of deep water in Well No. 1 of Sistan to the requirements of C. vulgaris algae, and can be an introduction to the possibility of further studies on various algae in these waters. Methodology The experiment was conducted in the Microalgae Research Laboratory of Zabol Research Institute. The microalgae stock C. vulgaris was obtained from the Fars Algae Biological Reserves Development Company. To achieve the appropriate concentration of microalgae for cultivation, the prepared stock was cultured in 500 ml Erlenmeyer flasks for one week at a salinity of 24.5 parts per thousand (distilled water and Lake Urmia salt) and 1.5 ml of Gaillard culture medium per liter. This experiment was conducted with 5 treatments (treatment 1: 100% culture medium. Treatment 2: 25% deep aquifer well water plus 75% culture medium. Treatment 3: 50% deep aquifer well water plus 50% culture medium. Treatment 4: 75% deep aquifer well water plus 25% culture medium. Treatment 5: 100% deep aquifer well water, and 3 repetitions during the duration of 10 days. It was done at a temperature of 27˚c and 3500 lux lighting and continuous aeration in 500 ml containers. pH was measured using a pH meter. To measure the density in different treatments, the number of cells was counted every other day using a Neobar slide and a light microscope (magnification ×40). Algae growth rate in different treatments including specific growth rate (SGR) (percentage per day) and: doubling time (DT) (per day) were calculated using the relevant formulas (Hibbered, 1981). Biomass dry weight was calculated using the method of Colusse et al. )2020(, and chlorophyll a content was calculated using the method of Lim et al. in 1991 and the corresponding formula. The collected data were statistically analyzed using SPSS version 22 software. The normality of the data and the homogeneity of variances were checked with Kolmogorov-Smirnov and Levene's tests. One-way ANOVA was used to determine the difference in means. Also, Duncan's test with a level of (p˂0.05) was used to compare means. Results An examination of the pH changes in the experimental treatments shows that the highest fluctuation of these changes occurred on the first day of the experiment, from pH 8.98 to 10.63 on the fifth day. The initial density in all treatments on the first day of the experimental period was 15×104 cells ml-1. Chlorella algae showed increasing growth in all treatments until day 5 of the experiment, with the highest density in the fifth treatment being 129.33×104 cells ml-1. However, no significant difference was observed between treatments (p > 0.05). Also, on the third day of the experiment, the highest specific growth rate (SGR) of C. vulgaris algae was related to treatment 1 (100% Gaillard culture medium) and the lowest was on the last day for treatment 4 (75% deep aquifer well water). In addition, the results of this study show that the highest doubling time (DT) is observed in treatment 4 (25% deep aquifer well water) on the last day and the lowest time for treatment 1 (100% Gaillard medium) on the third day. The results of measuring the concentration of chlorophyll a show that the highest and lowest levels of this pigment were observed on the third day of the experiment in treatment 5 (100% deep aquifer well water) and treatment 3 (50% deep aquifer well water), respectively. Also, the results related to the dry weight of this alga indicate that the highest average was recorded on the tenth day (treatment 5) and the lowest for treatment 3 on the third day of the experiment. Also, a significant difference was observed between the treatments only on the third day of the experiment (p˂0.05). Discussion and conclusion Algae have the ability to utilize waste and can produce valuable materials such as pharmaceuticals, chemicals, and human and animal food through light energy and inexpensive natural materials such as carbon dioxide (De la noue and Pauw, 1988). Therefore, the necessity of cultivating this type of algae in saline water environments is felt, and considering the new water source in the Sistan region, the necessity of faster and cheaper growth in its mass production seems necessary. For this reason, Chlorella vulgaris is used in this study to investigate its growth process in the deep aquifer well water of the Sistan region and the Gaillard culture medium. Light, temperature, pH, water quality, aeration rate and the presence of nutrients are considered as factors affecting the production of microalgae (Ayala, 1998). In the present study, the pH fluctuation in Table 2 for treatments containing deep water was in the range of 10-9. pH is one of the most important parameters for algae growth because it can determine the availability of carbon dioxide and nutrients. A study by Habibi et al. in 2011 showed that the highest growth of Chlorella was recorded at pH 9.5, although it maintained its growth and survival at higher pH (12). The results of this study showed that the highest growth was in the pH range of 7 to 8, which could be explained by the difference in environmental conditions and the type of algae species. The results of this study showed that the highest density of C. vulgaris species was observed in treatment 5 (100% deep aquifer well water) on the fifth day with an average density of 129.33×104 cells ml-1. A study by Rahdari et al. (2023) investigating the ability to cultivate the microalgae Dunaliella tertiolecta in the deep aquifer well waters of Sistan also showed that the highest number of this microalga was 24 ×106 cells ml-1. It seems that factors such as the type of species and the conditions of the microalgae cultivation environment, such as the type of water, the salinity of the water, the culture medium, etc., are effective in the cell density of this alga in various studies. According to the results of this research, it seems that the microalga C. vulgaris has the ability to be cultivated in deep water, but further studies are needed to achieve the appropriate combination of this water (deep water) with municipal or distilled water to achieve the desired salinity. Conflict of Interest The authors declare that they have no conflict of interest. Acknowledgment This work was financially supported by a grant (UOZ-8746) from the Vice Chancellor for Research Affairs of University of Zabol. 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