Iranian Scientific Fisheries Journal

fa مقاله علمی – پژوهشی:‌ مطالعه مقایسه‌ای ایمپلنت‌‌های آهسته‌رهش و تزریق داخل صفاقی هورمون LHRHa2 بر شاخص‌های رسیدگی جنسی و میزان هورمون‌‌ها‌‌ی استروئیدی جنسی در ماهی اسکار Investigating the comparative effect of slow releasing hormone implants and intraperitoneal injection of LHRHa2 hormone on sexual maturity indicators in oscar fish بهداشت و بيماريهاي آبزيان بهداشت بيماريها پژوهشي Research ماهی اسکار از جمله معروف‌ترین ماهیان زینتی از خانواده سیکلیده با اهمیت تجاری بالاست. تخمدان‌‌ها در ماهی اسکار در دسته تخمدان‌‌های ناهمزمان تقسیم‌‌بندی می‌‌شود و استفاده از هورمون‌های اگزوژن برای کنترل چرخه‌‌های تولید‌‌مثلی آنها کاربرد زیادی دارد. هدف از این مطالعه، مقایسه‌‌ ایمپلنت‌‌های آهسته رهش و تزریق داخل صفاقی هورمون LHRHa2 در ماهی اسکار است. در این مطالعه 18 عدد ماهی اسکار پرتغالی‌‌ که به‌‌ صورت تصادفی به 3 گروه که هر گروه شامل 6 عدد ماهی (3 جفت) بودند، تقسیم شده و هر جفت ماهی در آکواریوم‌‌های 150 لیتری با دمای آب 27-25 درجه سانتی‌‌گراد نگهداری شدند. هر 3 گروه در شرایط نوری یکسانی بودند و به‌‌ صورت دو نوبت در روز غذا‌‌دهی شدند. گروه اول تحت القاء هورمون LHRHa2 به‌‌ صورت تزریقی (2 مرحله برای جنس ماده: 10% مرحله اول و 90% مرحله دوم) و گروه دوم تحت القاء هورمون به ‌‌صورت ایمپلنت و به گروه سوم تنها سرم فیزیولوژی تزریق شد. خونگیری از ماهیان ماده پس از بیهوشی، قبل از تزریق یا کاشت ایمپلنت و در فاصله‌‌های زمانی معین پس از تزریق تا 24 ساعت و پس از کارگذاری ایمپلنت تا 20 روز انجام گرفت. در بررسی هورمون‌‌های استروئیدی ماهیان تحت القاء هورمون LHRHa2، به دو روش تزریقی و ایمپلنت، سطح هورمون پروژسترون و 17-بتا استرادیول در هر دو گروه افزایش یافته و سطح هورمون تستوسترون کاهش یافت اما میزان هورمون‌‌های استروئیدی جنسی در گروه ایمپلنت در قیاس با گروه تزریق به صورت مدت‌‌دار در سطح بالایی قرار داشت و تلفاتی مشاهده نشد، اما در گروه تزریقی تلفاتی مشاهده گردید. پس از گذشت مدت زمان القاء تولید مثلی در گروه ایمپلنت در مقایسه با گروه تزریقی، کاهش استرس و به ‌‌دنبال آن رهاسازی بهتر تخمک مشاهده گردید که می‌توان آن را دلیلی بر موفقیت‌‌آمیز بودن روش ایمپلنت‌‌گذاری برای القاء تولیدمثلی در ماهی اسکار پرتغالی قلمداد نمود. Introduction In this study, LHRHa2 hormone was induced in Portuguese Oscar fish by two methods: intraperitoneal injection and slow-release implant implantation (made with innovative PLGA_b_PEG copolymer), and the levels of steroid hormones in the blood serum of the fish and sexual maturity indices were investigated. Oscar fish are among the most famous ornamental fish of the Cichlid family with high commercial importance, which accounts for 95% of the world's ornamental fish. The ovaries of these fish are classified as asynchronous (multiple spawning) and problems in the absence of natural spawning in certain seasons of the year have been reported in this family (Mylonas and Zohar, 2000). For this purpose, the use of exogenous hormones to control their reproductive cycles is of great use. According to studies, the use of hormones by injection is ineffective in some fish due to problems such as rapid hormone clearance, excessive stress, gonadal atresia, and pre-spawning mortality. Also, in studies, slow-release hormone implants have been successfully used in fish such as European sea bass (Dicentrarchus labrax) and northern bluefin tuna (Thunnus thynnus) to gradually release the hormone over days to weeks in the fish's circulatory system, and good results were obtained (Rainis et al., 2003; Mylonas et al., 2007). Therefore, this study attempts to compare these two methods with each other and investigate the effectiveness of the hormone LHRHa2, which has generally been limited in this field. Methodology For this purpose, 18 adult male and female Oscar fish were randomly selected into 3 groups (3 pairs of adult fish in each group) and kept in 150 liter aquariums with water temperature of 25-27 degrees Celsius and identical light conditions (each pair in one aquarium) and fed twice a day. The first group was induced with LHRHa2 hormone by injection (2 stages for the female: 10% of the first stage and 90% of the second stage), the second group was induced with hormone by implant, and the third group was injected with only physiological serum. LHRHa2 hormone was calculated at a dose of 80 micrograms per kilogram according to the weight of each fish and injected intramuscularly under the pectoral fin. The implants were made of PLGA-b-PEG copolymer, which coated 75 μg of LHRHa2 hormone by double emulsion method (Avgoustakis., 2004; Locatelli and Franchini., 2012), and were made into oval tablets, sterilized with an ultraviolet device, and implanted under the pectoral fin by observing the principles of surgery and anesthesia. The drug release rate from slow-release implants was also evaluated by making a drug model and using the Franz chamber diffusion method. Blood samples were taken from female fish after anesthesia, before injection or implant placement, and at specific intervals after injection for up to 24 hours and after implant placement for up to 20 days. Then, the levels of steroid hormones in the fish's blood serum, including 17-beta estradiol, progesterone, testosterone, and cortisol, were determined by ELISA using commercial monobind kits. Result Results of serum steroid hormones in Oscar fish in injection treatment are shown in Figure 1. As can be seen, the amount of cortisol hormone in the blood serum of fish after injection of LHRHa2 hormone had a significant difference at the level of p≤0.05 compared to before injection, and the level of cortisol hormone in the blood serum after injection had an increasing trend. The amount of testosterone hormone in the blood serum of fish 6 hours after injection of the hormone increased compared to the level of hormone in the blood serum before injection, and the level of testosterone hormone in the serum, 18 hours after injection of the hormone, compared to 6 hours after injection, decreased, and there was a significant difference at the level of p≤0.05. The amount of progesterone hormone in the blood serum of fish 6 hours after injection increased compared to the level of hormone in the serum before injection, and there was a significant difference at the level of p≤0.05, but 18 hours after injection, the level of hormone decreased. The level of beta-estradiol hormone in the blood serum of fish injected with LHRHa2 was at its lowest level before injection and the level of E2 hormone increased 6 hours after injection but there was no significant difference with the level before injection. 18 hours after injection, the level of E2 hormone in the blood serum continued to increase and there was a significant difference with the level before hormone injection (p≤0.05). Steroid hormones in Oscar fish serum after slow-release implant induction are shown in Figure 2. As can be seen, the level of cortisol hormone in fish serum increased significantly after implant implantation up to 216 hours after implantation and then decreased. However, the hormone level was still higher on the last day of blood collection (480 hours after implantation) compared to before implant implantation, and there was a significant difference at the p≤0.05 level. The level of testosterone in the serum of fish induced with LHRHa2 implants increased up to 24 hours after implant placement, and then decreased until 336 hours after implantation. The hormone level was lower at 336 hours after implant placement compared to before implant placement, and there was a significant difference at the p≤0.05 level. However, at 480 hours after implantation, the level of testosterone increased slightly, but was still lower than the level of testosterone before implant placement. The level of progesterone hormone in the blood serum of fish induced with LHRHa2 implants showed an overall increase 216 hours after implantation and was significantly higher compared to before implantation, and there was a significant difference (p≤0.05). It decreased at 336 hours after implantation and then increased. At 480 hours after implantation, the level of progesterone hormone was higher compared to before implantation, and there was a significant difference (p≤0.05). The level of beta-estradiol hormone in the blood serum of fish induced with LHRHa2 implants showed an increase 144 hours after implantation and there was a significant difference (p≤0.05) compared to before implantation, and after that, the level of E2 hormone in the blood serum of fish decreased somewhat, but the level of E2 hormone was still higher 480 hours after implantation compared to before implantation, but there was no significant difference. Finally, two of the three female producers in the injection treatment succeeded in spawning, but one of the two producers died due to ovarian rupture and ascites. In the implant treatment, two of the three female producers succeeded in spawning after 40 days, and one of the two producers spawned repeatedly 3 times at 3-day intervals. <img alt="" id="_x0000_i1025" o:ole="" src="file:///C:/Users/falahi/AppData/Local/Temp/msohtmlclip1/01/clip_image001.emz" style="width:229.5pt; height:258.75pt" > Figure 1: Comparative graphs of mean and standard deviation of steroid hormones                 in blood serum of Oscar fish in LHRHa2 hormone injection treatment <img alt="" id="_x0000_i1026" o:ole="" src="file:///C:/Users/falahi/AppData/Local/Temp/msohtmlclip1/01/clip_image002.emz" style="width:223.5pt; height:258pt" > Figure 2: Comparative graphs of mean and standard deviation of steroid hormones  in the blood serum of Oscar fish in LHRHa2 hormone implant treatment Discussion and Conclusion In the present study, the increase in cortisol levels in the injection and implant groups indicated the effect of the blood collection and manipulation stages of the fish and the indirect effect of the LHRHa2 hormone on cortisol levels. In line with the present study, in the investigation of steroid hormone levels in Chilasoma dimerus fish, cortisol levels naturally increase before spawning (Varela et al., 2017). In this study, the level of E2 hormone increased in both groups because during the internal yolk formation process, the concentration of E2 increases and reaches its maximum level and remains at the same level during the external yolk formation process. Then, during the maturation and fluidization stage of the eggs, the hormone concentration decreases rapidly (King and Pankhurst, 2003). The level of testosterone in both groups initially increased and then decreased, which was due to the effect of LHRHa2 on the pituitary gland and the effect on the cells of the ovarian theca layer, followed by the secretion of progesterone and the conversion of this hormone to testosterone in the cells of the granulosa layer, and the decrease was due to the conversion of testosterone by the aromatase enzyme to 17-beta estradiol. The level of progesterone in both groups initially increased and then decreased, which was due to the effect of LHRHa2, and the level of progesterone decreased following the conversion of progesterone to 17-alpha hydroxyprogesterone. The results of this study show that the implanted fish survived the postoperative period normally and no deaths were observed, and the secretion of steroid hormones in this group of fish was at a high level compared to the long-term injection group. After a period of induction with the implant method, stress was reduced and egg release was improved, which can be considered evidence of the success of implantation for reproductive induction in Portuguese Oscar fish. Acknowledgment Thanks to Dr. Mohammad Vali Mohammadpour, Resident of Aquatic Health and Diseases. تولید‌‌مثل, سیکلید, القاء هورمون, کارگذاری ایمپلنت, کوپلیمر PLGA-b-PEG Oscar fish, LHRHa2 Hormone, Implant, Slow release 39 51 http://isfj.ir/browse.php?a_code=A-10-2925-1&slc_lang=fa&sid=1 Fatemeh Tobaee فاطمه طوبایی fati.toobaee@gmail.com 100319475328460039518 100319475328460039518 No Department of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran دانشکده دامپزشکی، دانشگاه شهید چمران اهواز، اهواز، ایران Takavar Mohammadian تکاور محمدیان t.mohammadian@scu.ac.ir 100319475328460039519 100319475328460039519 No Department of Animal, Poultry and Aquatic Health, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran گروه بهداشت دام، طیور، آبزیان، دانشکده دامپزشکی، دانشگاه شهید چمران اهواز، اهواز، ایران Soroush Sabiza سروش سابیزا s.sabiza@scu.ac.ir 100319475328460039520 100319475328460039520 Yes Department of Clinical Sciences, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran گروه علوم درمانگاهی، دانشکده دامپزشکی، دانشگاه شهید چمران اهواز، اهواز، ایران Eskandar Moghimipour اسکندر مقیمی پور moghimipour@yahoo.com 100319475328460039521 100319475328460039521 No Department of Pharmaceutics, Faculty of Pahrmacy, Jondi Shapour University of medical sciences, Ahvaz, Iran گروه فارماسیوتیکس، دانشکده داروسازی، دانشگاه علوم پزشکی جندی شاپور، اهواز، ایران