Iranian Scientific Fisheries Journal

fa مقاله علمی – پژوهشی:‌ تأثیر عصاره هیدروالکلی ریزپوشانی‌شده برگ گیاه آقطی سفید (Sambucus ebulus) بر شاخص‌های خونی، بیوشیمیایی، ایمنی و مقاومت قزل‌آلای رنگین‌کمان (Oncorhynchus mykiss) در برابر باکتری Yersinia ruckeri Effect of microencapsulated hydroalcoholic extract of dwarf elder (Sambucus ebulus) leaf on hematological, biochemical, immunological parameters and disease resistance of rainbow trout (Oncorhynchus mykiss) against Yersinia ruckeri بهداشت و بيماريهاي آبزيان بهداشت بيماريها كاربردي Applicable با توجه به نقش کلیدی قزل‌آلا در صنعت آبزی‌پروری ایران و کاهش اثربخشی آنتی‌بیوتیک‌ها در کنترل بیماری‌هایی مانند Yersiniosis، استفاده از گیاهان دارویی به ‌عنوان راهکاری نوین برای تقویت ایمنی و افزایش مقاومت ماهیان، ضروری به‌نظر می‌رسد. هدف از مطالعه حاضر، ارزیابی اثرات عصاره هیدروالکلی کپسوله شده گیاه آقطی بر شاخص‌‌های ایمنی، خون، بیوشیمیایی و مقاومت در برابر باکتری Yersinia ruckeri بوده است. بدین‌منظور، عصاره هیدروالکلی برگ گیاه آقطی به ‌صورت ریزپوشانی‌شده با ژلاتین در غلظت‌های 0 (شاهد)، 1/0، 25/0 و 5/0 درصد به جیره غذایی ماهیان قزل‌آلای رنگین‌کمان (میانگین وزنی 55/2±11/59 گرم) افزوده شده و ماهیان به مدت 8 هفته تغذیه شدند. در پایان هفته‌های 4 و 8 آزمایش، از 8 عدد برای سنجش شاخص‌های خونی، بیوشیمیایی و ایمنی خون‌گیری انجام گرفت. در پایان هفته 8 نیز به 30 عدد ماهی از هر تیمار، 1/0 میلی‌لیتر از باکتری Y. ruckeri به ‌صورت داخل ‌صفاقی تزریق شد. نتایج این بررسی نشان داد که تعداد گلبول‌های قرمز، هماتوکریت، هموگلوبین، حجم متوسط گلبولی، میزان هموگلوبین گلبولی و غلظت متوسط هموگلوبین گلبولی ، آلبومین، آسپارتات آمینوترانسفراز تفاوت معنی داری در پایان هفته‌های 4 و 8 آزمایش بین شاهد و تیمارها نشان ندادند. تعداد گلبول‌های سفید، کلسترول، تری‌‌گلیسرید، پروتئین تام سرم، آلانین آمینوترانسفراز، IgM تام سرم، لیزوزیم، انفجار تنفسی، در گروه‌‌های تیمار در مقایسه با شاهد بهبود معنی‌‌داری داشتند. در مواجهه با باکتری، درصد بازماندگی در تیمارهای 0 (شاهد)، 1/0، 25/0 و 5/0 درصد به‌ترتیب 3/23، 7/56، 7/66 و 3/73 درصد بودند. نتایج این بررسی نشان داد که عصاره گیاه آقطی از توانائی بسیار خوبی در بهبود شاخص‌های بیوشیمیائی، ایمنی و مقاومت ماهیان قزل آلا دارد در برابر باکتری بیماریزا برخوردار بوده و بهترین دوز مصرفی 25/0 درصد است. <jarvis-extension style="position: relative; z-index: 2147483647;"></jarvis-extension><jarvis-injection></jarvis-injection><jarvis-injection style="position: absolute; left: 635px; top: 465.969px; z-index: 2147483647;"></jarvis-injection> Introduction Rainbow trout (Oncorhynchus mykiss) is one of the most widely farmed fish species in Iran and globally, valued for its economic and nutritional significance. However, intensive aquaculture practices often lead to physiological stress, liver dysfunction, and increased susceptibility to bacterial infections such as yersiniosis, caused by Yersinia ruckeri. The emergence of biotype 2 strains with reduced sensitivity to conventional vaccines has raised concerns about vaccine failure and disease outbreaks in trout farms (Austin et al., 2003; Kumar et al., 2017). In recent years, medicinal plants have gained attention as natural immunostimulants and hepatoprotective agents in aquaculture. Elderberry (Sambucus ebulus) is one such plant, known for its flavonoid compounds with antimicrobial, antioxidant, anti-inflammatory, and immune-boosting properties (Ivanova et al., 2014; Ngugi et al., 2015). Previous studies have demonstrated the positive effects of dietary elderberry leaf supplementation on blood parameters, serum biochemistry, immune response, and disease resistance in rainbow trout (Ghiasi et al., 2023). Despite promising findings, previous research suggests that this plant has strong potential to be introduced as a dietary immunostimulant and growth enhancer in aquaculture. The aim of this study was to evaluate the effects of encapsulated elderberry leaf extract as a dietary supplement to promote growth and immunity. As a complementary strategy alongside conventional vaccines, it may play a significant role in reducing mortality and enhancing trout production in Iran. <div aria-label="Page Break" class="cke_pagebreak" contenteditable="false" data-cke-display-name="pagebreak" data-cke-pagebreak="1" style="page-break-after:always" title="Page Break"></div>Methodology  To prepare the herbal extract, S. ebulus leaves were collected from Sefrabad (Sari, Iran) and authenticated by the Semnan Agricultural Research Center. After shade-drying, the leaves were extracted using a modified Soxhlet method with 70% ethanol (Păvăloiu et al., 2020). The concentrated extract was freeze-dried and microencapsulated with gelatin through high-speed homogenization. Encapsulation efficiency and extract yield were assessed spectrophotometrically (Shu et al., 2006). Experimental diets were formulated weekly by spraying the dissolved microcapsules onto commercial trout feed (GF1, Beyza Co., Iran), followed by air-drying and cold storage. A total of 280 rainbow trout (O. mykiss, 59.11 ± 2.55 g) were randomly assigned to 12 fiberglass tanks. After a 2-week acclimation, fish were fed for 8 weeks with diets containing 0, 0.1, 0.25, and 0.5% extract, adjusted to 2–2.5% of body weight daily. At weeks 4 and 8, blood samples were collected from anesthetized fish to evaluate hematological and immunological responses. Standard methods were used to measure RBC, WBC, Hct, Hb, MCV, MCH, and MCHC (Blaxhall and Daisley, 1973; Seiverd, 1964), while differential leukocyte counts were performed on Giemsa-stained smears (Lee et al., 1998). Respiratory burst activity was assessed using luminol-based chemiluminescence (Binaii et al., 2022). Serum levels of TP, Alb, Chol, Tri, ALT, AST, total IgM, and lysozyme activity were measured using commercial kits and turbidimetric assays (Ellis, 1990; Binaii et al., 2014). At the end of the trial, fish were challenged with Y. ruckeri strain MT968739 via intraperitoneal injection (Ghiasi et al., 2023). Mortality was monitored for 14 days, and survival rates were calculated. All data were statistically analyzed using one-way ANOVA followed by Duncan’s multiple range tests at a 95% confidence level (Zar, 1994). Results Microencapsulation showed strong formulation performance, yielding 92% with an encapsulation efficiency of 78%. Hematological profiles at weeks 4 and 8 revealed no significant differences in RBC count, hemoglobin, hematocrit, MCV, MCH, or MCHC between treated and control groups, though treated fish tended to display slightly higher values. In the 0.25% group, WBC count and neutrophil percentage increased, accompanied by a reduction in lymphocytes, while other treatments followed similar but non-significant trends. Serum biochemistry remained stable for AST and albumin across all groups, yet treated fish generally exhibited improved profiles. Cholesterol declined slightly at week 4 and significantly by week 8 in all treatments. Triglycerides consistently decreased, while total serum protein rose in the 0.25% and 0.5% groups. ALT levels were lower in all treatments, suggesting enhanced liver function. Immunological responses were strengthened, with IgM levels significantly elevated in all treated groups at both time points. Lysozyme activity increased in the 0.25% group at week 4 and further in the 0.25% and 0.5% groups by week 8, while the 0.1% group showed a non-significant rise. Respiratory burst activity was consistently higher, with the 0.25% group showing the strongest response at week 4 and sustained elevations across treatments by week 8. Upon challenge with Y. ruckeri, survival improved markedly in all supplemented groups. Fish receiving 0.1%, 0.25%, and 0.5% supplementation achieved survival rates of 56.67%, 73.33%, and 66.67%, respectively, compared to only 23.33% in controls. Discussion and conclusion  This study shows that dietary supplementation with microencapsulated herbal extract can enhance both physiological and immunological status in rainbow trout. While hematological indices did not differ significantly, treated fish generally displayed improved profiles. Biochemical changes, including reduced ALT and triglycerides together with higher total protein, point to better liver function and metabolic stability. Immunological outcomes such as elevated IgM, lysozyme activity, and respiratory burst highlight strengthened innate immunity, especially in the 0.25% and 0.5% groups. Most importantly, survival after Y.ruckeri challenge was markedly higher in all supplemented groups, confirming the protective effect of the extract. Overall, the formulation proved effective in supporting fish health and disease resistance, with the 0.25% dose delivering the most consistent benefits Conflict of interest The authors declare that they have no conflict of interest. Acknowledgment This article is derived from a dedicated research project entitled “Evaluation of the effects of free and microencapsulated hydroalcoholic extract of elderberry (S. ebulus) leaves on growth, immunity, and disease resistance of  rainbow trout against Y. ruckeri”, approved under project number 001292-101-12-76-2. We sincerely thank all colleagues at the Iranian Fisheries Science Research Institute and the Caspian Sea Ecology Research Center for their valuable scientific and laboratory support throughout this study. <jarvis-extension style="position: relative; z-index: 2147483647;"></jarvis-extension><jarvis-injection></jarvis-injection><jarvis-injection style="position: absolute; left: 20px; top: 278.766px; z-index: 2147483647;"></jarvis-injection> آقطی, قزل آلای رنگین کمان, Yersinia ruckeri, پروتئین تام سرم, ایمنی, لیزوزیم Sambucus ebulus, Oncorhynchus mykiss, Yersinia ruckeri, total serum protein, immunity, lysozyme 55 72 http://isfj.ir/browse.php?a_code=A-10-647-6&slc_lang=fa&sid=1 Maryam Ghiasi مریم قیاسی ghiasimaryam4@gmail.com 100319475328460041571 100319475328460041571 Yes پژوهشکده اکولوژی دریای خزر، موسسه تحقیقات علوم شیلاتی کشور، سازمان تحقیقات، آموزش و ترویج کشاورزی، ساری، ایران Farzaneh Bahadori فرزانه بهادری farbahadori@gmail.com 100319475328460041572 100319475328460041572 No مرکز تحقیقات و آموزش کشاورزی و منابع طبیعی استان سمنان، سازمان تحقیقات، آموزش و ترویج کشاورزی، سمنان، ایران Razieh Azimi Atargoleh راضیه عظیمی اترگله razimi@rifr-ac.ir 100319475328460041573 100319475328460041573 No موسسه تحقیقات جنگل‌ها و مراتع کشور، سازمان تحقیقات، آموزش و ترویج کشاورزی، تهران، ایران Mohamad Binaii محمد بینائی bbinaii@gmail.com 100319475328460041574 100319475328460041574 No پژوهشکده اکولوژی دریای خزر، موسسه تحقیقات علوم شیلاتی کشور، سازمان تحقیقات، آموزش و ترویج کشاورزی، ساری، ایران Farshideh Habibi فرشیده حبیبی farshidehhabibi@gmail.com 100319475328460041575 100319475328460041575 No پژوهشکده اکولوژی دریای خزر، موسسه تحقیقات علوم شیلاتی کشور، سازمان تحقیقات، آموزش و ترویج کشاورزی، ساری، ایران Mohammad Javad Taghavi محمد جواد تقوی j_taghavi2001@yahoo.com 100319475328460041576 100319475328460041576 No پژوهشکده اکولوژی دریای خزر، موسسه تحقیقات علوم شیلاتی کشور، سازمان تحقیقات، آموزش و ترویج کشاورزی، ساری، ایران