مجله علمی شیلات ایران
سال سی و سوم شماره 4 (پیاپی 144، مهر و آبان 1403)

Emerging anthropogenic pollutants in wastewater treatment plant (WWTP) effluents have been shown to pose a particular risk to aquatics and potentially affect the aquatic environment from the molecular to the ecosystem level (Santos et al., 2010). Micropollutants are emerging pollutants that include pharmaceuticals and personal care products (PPCPs). Due to the widespread consumption of a wide range of micropollutants and their discharge into wastewater worldwide, their occurrence and fate in aquatic environments has become a research topic in recent decades (Ryu et al., 2014). The common wastewater treatment process is not able to completely remove these compounds from the wastewater, and as a result, a significant amount of these compounds enters the aquatic water environment (Rehman et al., 2015). Dexamethasone is a potent synthetic glucocorticoid with anti-inflammatory and immunosuppressive effects that is used to treat many diseases including allergies, asthma, covid-19, rheumatic problems, and skin diseases (Sanders et al., 2020). Today, the increasing use of dexamethasone from sources such as hospitals is a global concern. High levels of this drug, which is a cortisone derivative, have been detected in wastewater (Herrero et al., 2013). As one of the important components of the innate immune system in ectothermic vertebrates, including fishes, the complement system has been less studied. Evaluation of serum complement activity is considered as a valuable tool to diagnose the health status of fish. Complement in the non-specific immune response to a compound can have a direct effect such as killing the pathogen by lysing it (Ellis, 1999).

Fifteen mature Arabian sea bream, A. arabicus were collected from the Bahrakan Port in Abadan. Fish were transported to the 300 L tanks. After seven days of adaptation, the fish were anesthetized using 2-phenoxy ethanol (0.35 ml/L). Then, the fish were dissected under aseptic conditions and their spleens and kidney heads were separated (Wen et al., 2008). The separated tissues were immediately washed three times with 100 ml antibiotic medium (Leibovitz's L-15 (L-15) with 400 IU/ml penicillin, 400 µg/ml streptomycin and 200 µg/ml amphotericin B) each time for 30 minutes. The spleen cell culture was performed according to Huang et al. (2009). The head kidney cell culture followed the protocols described by Ribera et al. (2020). Dexamethasone cytotoxicity was assessed by MTT method according to Momeni et al. (2010). Cultivated spleen and head kidney cells were plated in 24-well microplates (105 cells / ml L-15 medium / well) and incubated at 28°C for 24h. The cell viability was assessed using trypan blue exclusion test. Then, 200 μL of fresh L-15 culture medium with different experimental concentrations of dexamethasone was added to each well and microplates were then incubated at 28°C for 48 h. Each treatment runs in five replicates. For this purpose, the cell suspensions were collected at 0, 12, 24 and 48 hours of experiment and transferred to microtubes for further analysis. The level of C3 and C4 was measured using immunoturbidimetry method by a double–antibody sandwich ELISA kits. ACH50 was measured by hemolytic method according to Sunyer and Tort (1995).

Measurement of dexamethasone in hospital wastewater: To measure dexamethasone in hospital wastewater, samples were taken from the wastewater of Taleghani Hospital in Abadan, and after scanning the dexamethasone samples and determining the maximum absorption wavelength at 213 nm for it, the concentration of dexamethasone was measured using an HPLC device. The obtained concentration was 86 ng/l. Primary culture of head kidney and spleen cells: Using the trypan blue test, the survival rate of spleen and head kidney cells was determined to be 95%. Then, cells derived from the spleen and head kidney were incubated in L-15 culture medium for two weeks at 28°C. After 48 hours, cells started to grow and colony formation started 5 days after incubation. The cells grew well and completely occupied the culture dish within 12 days. All the time, the cells were floating in the culture medium as round cells and did not stick to the bottom of the culture container. The trypan blue test showed that more than 90% of the cells were alive and healthy during the test. Twelve days after incubation, the cells were passaged. The second passage was performed fifteen days after incubation and then the cells were used for cytotoxicity assay. The C3 contents notably decreased in spleen cells exposed to all concentrations of dexamethasone except for the lowest concentration (3 μM; P < 0.05). In the head kidney, the C3 contents only notably decreased in cells treated with the higher concentrations of dexamethasone (3×10 and 3×102 μM; P < 0.05). The complement C4 has no obvious change following exposure of spleen and head kidney cells to all concentrations of dexamethasone except for the highest concentration. The C4 content of spleen and head kidney cells significantly decreased after 24 h exposure to highest concentration of dexamethasone (P < 0.05).  The concentrations of dexamethasone (except the highest concentration of dexamethasone) caused a significant change in the ACH50 content of spleen cells compared to the controls (P > 0.05). The exposure of spleen cells to 3 mM dexamethasone led to an 11% decrease in alternative complement activity after 48 hours of exposure (P < 0.05). ACH50 activity in head kidney cells treated with 3×102 nM and 3 μM of dexamethasone showed no difference compared to control (P > 0.05). A significant decrease in ACH50 was observed after exposure of head kidney cells to 3×10 and 3×102 μM of dexamethasone (P < 0.05). The amount of ACH50 activity in head kidney cells treated with 3 mM dexamethasone decreased by 27% after 48 hours of exposure (P < 0.05)

This study is the first study that reports the suppressive effects of dexamethasone on the activity of complement components of head kidney cells and spleen of Arabian yellow fin sea bream (Acanthopagrus arabicus) in cell culture medium. Dexamethasone is commonly known as an immunosuppressant drug that is toxic to immune cells and inhibits the function of the immune system (Ribas et al., 2016). The complement system plays an important role in innate and acquired immunity by facilitating the function of phagocytizing cells and cell lysis. The components of this system, which are activated in a cascade manner, lead to the destruction of pathogens by targeting the membrane components of invading agents (Boshra et al., 2006). C3 is an important component of the complement system, whose stimulation activates other components. This component belongs to the acute phase cellular proteins and is the first component of the complement system to be activated (Bayne et al., 2001). In the present study, dexamethasone led to a significant decrease in C3 levels in spleen and head kidney cells. On the other hand, the level of C4 in spleen and head kidney cells was suppressed only by the highest concentration of dexamethasone. In the study of Zach et al. (1993), corticosteroids can suppress the expression level of genes related to the synthesis and activity of C3 through the glucocorticoid (GR) and mineralocorticoid (MR) receptors. Most Osteichthyes have three corticosteroid receptors, including two glucocorticoid receptors (GR1 and GR2) and one mineralocorticoid receptor (MR), which are encoded by three different genes (Zach et al., 1993). This can explain the decrease in complement levels under the influence of dexamethasone (corticosteroid) in the present study. In the present study, dexamethasone did not affect the activity of alternative complement component (ACH50) of spleen and head kidney cells, and only its highest concentration decreased ACH50 levels after 48 hours. Milla et al. (2018) did not observe any changes in gene expression, protein quantity or activity of proteins related to innate immunity (such as ACH50 level) up to two weeks after exposure of Perca fluviatilis to 11-deoxycorticosterone (corticosteroid). While after two weeks, a significant increase in the ACH50 level of the spleen was observed. They stated that acute exposure to corticosteroids (as immunosuppressants) does not seem to have a significant effect on some innate immune proteins (Milla et al., 2018).

Fishmeal price were increasing every year and predicted that fish diet will have a wider range of ingredients replaced by fishmeal, such as vegetable sources, microbial protein sources, waste from fishing and animal protein waste (Glencross et al., 2007). Vegetable proteins can replace fish meal to some extent and have a negative effect on the growth of aquatic animals at high levels. But many studies have shown that a mixture of vegetable proteins can improve the amino acid profile compared to single protein source replaced by fish meal (Amaya et al., 2007).  By this method, high replacement with a blend of protein Vegetables have been reported in aquatic animal (Hansen et al., 2007), but increasing fiber, increasing carbohydrates and inappropriate amino acids in diets based on animal and vegetable proteins can led to a negative effect on fish digestive system that showed by growth reduction, immune system depress , low quality meat and even fish fatality in a long-term feeding period (Raskovic et al., 2011). therefore, in this experiment, the effect of a mixture of animal and vegetable proteins replaced by fish meal (levels of 20, 40, 60 and 80%) on the growth indices and digestive system tissue Huso huso were studied.

In order to reduce the share of fishmeal in the diet of beluga, a basal diet based on 54% of fishmeal was made. A mixture of animal and vegetable proteins (corn gluten: 35%, wheat gluten: 19.34%, processed soybean flour: 3%, chicken by-products: 20%, meat and bone meal: 8%, blood meal: 3.4%) at 20, 40, 60 and 80% levels replaced with fishmeal (FM) (CGM20, CGM40, CGM60, and CGM80, respectively) and finally 5 isonitrogenous (44% crude protein) and isoenergetic (18 MJ/kg) diets were produced. The fish (Average weight: 167±6.5 gr) were fed to saturation for 12 weeks.  Biometry were carried out on month interval and by carrying out information obtained from fish length, weight and food consumption, growth indices and feed efficiency ratio were calculated. At the end of the feeding period, 30% of the fish population was collected from each tank for histological studies. Three parts of the intestine (hindgut, midgut, and foregut) were removed. It was fixed and stained. Histological studies were carried out by an optical microscope equipped with a monitor.

There was no significant difference in the final weight and length of fish of different treatments (P>0.05). condition factor and percentage of weight gain of fish fed with FM, CGM20, CGM40, CGM60 and CGM80 had no no significant difference and percentage of weight gain was close to each other (243 to 298%) (P>0.05). No significant difference was observed in specific growth rate and daily growth rate and food conversion ratio in fish fed with all diets (P>0.05). Fish fed FM had the highest protein efficiency ratio (1.75±0.23), but protein efficiency ratio in fish fed with CGM20, CGM40, CGM60 and CGM80 was in the range of 1.53 to 1.73%. But no significant difference was observed (P>0.05). Histological studies were carried out by an optical microscope equipped with a monitor. The intestinal tissue of fish fed with CGM80 diet had necrosis and biliary stagnation. However, histological studies in fish fed with FM, CGM20, CGM40, and CGM60 diets showed that the lamina propria space is adequate, the necrosis of enterocytes is negligible, and the number of goblet cells in the anterior, middle, and distal intestine is balanced. The final results confirmed that the CGM80 did not a negative effect on the growth fish, but it caused damage to the digestive system.  In the present study, there is no significant difference in growth indices and food conversion ratio of fish fed with diets based on fish meal and diets based on corn gluten flour (a mixture of corn gluten: 35%, wheat gluten: 19.34%, soybean meal processed: 3%, poultry by product: 20%, meat and bone meal: 8%, blood powder: 4.3%). In this order, it seems be that alternative diets formulation based on a mixture of Animal and vegetable protein sources  were led to enriching of profile and biochemical composition of diet, In addition beluga Compared to white and Siberian sturheon is a species that can be fed with high carbohydrates percentages without growth reduction (Mohseni et al., 2011). In the present experiment, by increasing mixtures based on corn gluten-based in fish diet,  goblet cells were increased and reached to highest value in CGM80. goblet cells are related to immune system and mucus secretion in order to fluidity of digestive tract and create a protective barrier against mechanical and chemical damage (Marchetti et al., 2006). it seems that the increase of goblet cells in beluga fed with a mixture of vegetable proteins (CGM60 and CGM80) is a reaction against to mucous layer necrosis and reduction of intestinal villi in order to further damage of folds by secreting more mucins (Li et al., 2007), but in general, digestive tract epithelium damage in fish fed CGM20, CGM40 diets and CGM60 was negligible compared to fish fed with FM diets and did not lead to enteritis (intestinal inflammation). Therefore, it is suggested to replace fishmeal with a mixture of animal and vegetable proteins at 20 and 40% in Huso huso diet during the fattening stage.

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.

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.

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.Figure 1: Comparative graphs of mean and standard deviation of steroid hormones in blood serum of Oscar fish in LHRHa2 hormone injection treatment.Figure 2: Comparative graphs of mean and standard deviation of steroid hormones  in the blood serum of Oscar fish in LHRHa2 hormone implant treatment

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.

Seafood has been an important source of nutrients and has attracted human attention from a health perspective (Dehghani et al., 2018; Khanlar et al., 2018). Important marine sources include shrimp, which are highly perishable and are associated with microbial, chemical, and physical changes during storage (Na et al., 2018). The growth of decay-causing bacteria in shrimp leads to protein degradation, resulting in the production of ammonia, indole, methanethiol, putrescine, trimethylamine, and other odorless compounds. Hence, it is vital to maintain the quality of white shrimp using effective methods (Liu et al., 2020). In general, artificial or chemical preservatives pose potential toxicity and carcinogenicity risks; therefore, consumers tend to prefer natural additives. Many studies have focused on natural food coatings for food preservation, such as alginate/nanocomposite active films containing essential oils, which have been produced and evaluated for their antimicrobial properties. These active films exhibited antimicrobial effects on a wide range of microorganisms, and it was also found that, in the presence of nano-receptors, they maintained their antimicrobial properties for a longer duration (Albofetileh et al., 2014). Additionally, due to the microbial changes in shrimp during cold storage and the issues associated with artificial preservatives, the use of biodegradable natural materials that can simultaneously maintain the quality and safety of food products is necessary. The present study examined the comparative effects of a nanocomposite coating (alginate/nano-clay) combined with Chuchagh plant extract on the quality of western white shrimp (Litopenaeus vannamei). Chuchagh plant extract is known for its phenolic and flavonoid compounds, as well as its antioxidant and antimicrobial properties. Alginate film is recognized for its gelling properties, ability to enhance tissue strength, stabilize structures, and form films (Liu et al., 2020). The research objectives include: 1) studying the phenolic composition of chuchagh plant extract; 2) determining the physical and mechanical characteristics of alginate-based films containing various concentrations of Chuchagh essential oil; and 3) assessing the physicochemical and sensory changes in fried shrimp coated with alginate-nanoclay films containing different concentrations of Chuchagh essential oil. Overall, the use of Chuchagh extract at a concentration of 1500 ppm as a natural preservative, along with clay nanoparticles in the active edible coating structure based on alginate, delays chemical and microbial decay and increases the shelf life of western white shrimp.

Preparation and extract of the essence of Chuchagh was done and total phenolic compounds were measured (Hasanzadehaidenlu et al., 2017). The alginate-nanoclay film containing the essence of the Chuchagh plant was prepared (Ojagh et al., 2010; Eslamian, 2021). Identification of films containing a better concentration of the essence was conducted by performing physical and mechanical tests across five treatments. The thickness of the films was measured according to ASTM (1996). The humidity of the films was measured (Ojagh et al., 2010). The rate of water vapor permeability through the films and also opacity of the films were measured (ASTM, 1996; Pang and Li, 2014). The mechanical testing of the films was based on the modified method ASTM D0882-02 (1996). After examining the results of indicators related to the physical and mechanical properties of the films, better concentrations of the essence (1000 and 1500 parts per million) were identified. To evaluate the efficacy of the nanocomposite activated by the essence produced in a real environment, and to compare it with the nanocomposite without essence and the alginate coating, five treatments were selected: alginate+nanoclay+essence oil (1500 ppm), alginate+nanoclay+essence oil (1000 ppm), alginate+nanoclay, and control. Preparation and processing of shrimp were carried out (Basiri et al., 2015). Physicochemical tests conducted after frying the shrimp included measuring the moisture percentage (Avak, 2005), frying efficiency (Daraei Garmkhanei et al., 2012), measuring peroxide levels (Ronald and Ronald, 1991), and measuring the sum of volatile nitrogen bases (Avak, 2005). Chemical, microbial, and sensory tests were performed to determine the total viable count (TVC) and psychrotrophic bacteria (PTC) (Arancibia et al., 2014). The sensory test was evaluated using a five-point hedonic scale (Dehghannasiri et al., 2012).

This study investigates the effects of alginate and nanocomposite coatings containing Chuchagh essential oil on the microbial load, chemical properties, sensory characteristics, and oxidation properties of white shrimp during their shelf life at refrigeration and frying temperatures. First, the phenolic compounds in the Chuchagh essential oil were determined. The results indicated that the Chuchagh oil contained high levels of phenolic compounds. Five edible films were then prepared: alginate; alginate combined with nano-clay; alginate combined with nano-clay+Chuchagh essential oil at 500 parts per million (ppm); alginate combined with nano-clay+Chuchagh essential oil at 1000 ppm; and alginate combined with nano-clay+ Chuchagh essential oil at 1500 ppm. The physical and mechanical characteristics of the films were examined according to ASTM standards. Based on the results of the physical and mechanical tests, the optimal concentrations of Chuchagh essential oil (1000 and 1500 ppm) were identified. The mechanical tests demonstrated that increasing the concentration of Chuchagh essential oil decreased the tensile strength of the alginate film until the point of tearing, while the physical tests indicated that the addition of Chuchagh essential oil improved the film's physical properties. Overall, the best characteristics of the film were observed in the formulations containing nano-clay+Chuchagh essential oil at 1000 ppm and at 1500 ppm (Ojagh et al., 2017). To further explore the effects of the alginate-nanocomposite coating and Chuchagh essential oil on extending the shelf life of shrimp during storage, five treatments were produced: a control group, alginate, alginate+nano-clay, alginate+nano-clay+ Chuchagh essential oil at 1000 ppm, and alginate+ nano-clay+Chuchagh essential oil at 1500 ppm. These treatments were periodically evaluated for microbial and chemical properties (Arancibia et al., 2014). The results of the present study indicated that the alginate-nanocomposite coating possesses antioxidant and antimicrobial properties, and the addition of Chuchagh essential oil enhanced these characteristics. Specifically, shrimp treated with alginate+nano-clay+Chuchagh essential oil at 1500 ppm exhibited a significant delay in the processes of microbial growth and oxidation, thereby increasing their shelf life (Dehghannasiri et al., 2012). In conclusion, the addition of Chuchagh essential oil helps maintain the quality of shrimp in terms of chemical and microbial indicators and extends their shelf life in refrigeration compared to the other samples.

The present study showed that the alginate- nanoclay coating possesses antioxidant and antimicrobial properties. Compared to previous research on the addition of essential oils, this study demonstrated that shrimp containing the alginate-nanoclay-essence not only exhibited antioxidant properties (similar to those of (Thymus vulgaris and Froriepia subpinnata plant) but also significantly delayed the microbial spoilage and oxidation processes, thereby increasing the shelf life of the shrimp. According to the results, incorporating essence into shrimp preserves its quality in terms of chemical and microbial indicators and extends the shelf life in refrigeration compared to other samples. Recommendations for the use of natural preservatives include: 1) investigating the feasibility of replacing artificial preservatives with alginate film combined with nano-clay and clay essence at a concentration of 1500 ppm in meat products such as sausages and hamburgers, 2) using native nano-capsule essences with alginate film and nano-clay to produce active packaging to extend the shelf life of meat products and reduce microbial contamination, and 3) studying the simultaneous application of essential oil in both free-form and micro-encapsulated forms, as well as packaging under vacuum conditions, to enhance the shelf life of various meat products.

Aquatic oligochaetes are members of a main group of macroinvertebrates and include about 1,700 species (Caramelo and Martinez-Ansemil, 2012) of 13 families with worldwide distribution (Martin et al., 2008). These species commonly inhabit within sediments of rivers, streams, lakes, marshes, ponds, springs and ground-waters (Collado and Schmelz, 2001), showing that these species have been adapted to a wide variety of habitats and environments, such as freshwater, brackish or seawater.The freshwater oligochaete genus Stylodrilus is a large and heterogeneous. Although the genus has a Holarctic distribution, approximately 28, are exclusive to the Palaearctic region (Martin et al., 2007), except Stylodrilus heringianus a peregrine species recorded in the Australasian region and assumed to have been introduced by shipping (Brinkhurst & Jamieson, 1971).From the Lumbriculidae family, only two species, L. variegatus and S. heringianus, have cosmopolitan, asexual reproduction (fragmentation) and use as live food in fish farming are the most important factors that have contributed to the distribution of these two species in the world (Pinder and Brinkhurst, 1994).Cheshmehkileh of Tonekabon and Sardabroud of Chalus Rivers are the most important, mountainous and permanent rivers running from high elevation to the sea (southern waters of Caspian Sea). These rivers are important for reproductive migration of two indigenous valuable fisheries species Salmo caspius (Caspian trout) and Rutilus frisii (Caspian Kutum) as well as other migratory fish (Khara, 2016). For these reasons, estuary of these rivers is regarded as a ‘Protected Area’ and is conserved by the Department of Environment of Iran (DOE, 1996). 26 species of aquatic oligochaetes have been reported from inland waters of Iran so far, of which only 1 species belongs to the Lumbriculidae family and from Lumbriculus genus: L. variegatus (Ahmadi et al., 2012; Basim et al., 2012).  The aim of this study was to introduce Stylodrilus heringianus from Cheshmehkileh of Tonekabon and Sardabroud of Chalus Rivers in the Southern Caspian Sea basin for the first time and investigation of its distribution pattern in the three ecosystems of river, estuary and marine combined with some environmental factors in water (temperature and salinity) and sediment (grain size and total organic matter).

Sampling of oligochaetes and environmental factors in both Sardabroud and Cheshmekile rivers, was carried out between November 2014 and September 2015 and random sampling was done at six stations with three replicates for each bimonthly sampling along each river. Sampling was done using 0.03 m2 Van Veen grab for soft sediments at the estuary area and for sampling at inner parts of the river with pebbles a 0.1 m2 and 0.2 mm-mesh size Surber sampler was applied. Biotic samples were fixed in situ using a 5% formalin solution. In the laboratory, sediments were sieved through mesh sizes of 1, 0.5 and 0.25 mm and specimens were preserved in 70% ethanol and then sorted and counted under a stereomicroscope (Nikon SMZ800, Japan) and eventually the wet weight of worms was measured using a digital balance (0.0001 g). For identification at species level, worm specimens were mounted on glass slides in Amman’s lactophenol clearing agent (Smith, 2001) and covered by a coverslip and left for several hours to a day or two, and then for observation of setae and other details, a microscope was used (Nikon E200). The main identification keys used were: Brinkhurst (1971a and b, Brinkhurst and Wetzel (1984), Brinkhurst (1986), Pinder and Brinkhurst (1994), Smith (2001), Arslan and Sahin (2003), Krieger and Stearns (2010), and Pinder (2010). In this study environmental variables, such as temperature and salinity in water were measured in situ by multimeter portable HACH-HQ40d model, and organic matter content was measured by the weight lost during ashing (Wildsmith et al., 2011), and grain size was analyzed by dry mechanical separation through a column of standard sieves of different mesh sizes, corresponding to classes described by Wentworth.

In this study, the annual average water temperature was 18.2±0.2ºC. The average water temperature showed a significant difference between the sampling months (P<0.05), so that the lowest average water temperature were recorded in March as 10.1±0.08ºC and the highest in July as 30±0.8ºC. In both studied rivers, station (S1) has the lowest annual average salinity (0 g/l), station (S2) has average salinity in moderate level (6.7±1.2 g/l), and station (S3) has the highest annual average salinity (10.6±0.05 g/l). In both studied rivers, the average water salinity between sampling stations and months showed a significantly different (P<0.05), so that in station (S2), the highest average salinity was in July (8.3±1.5 g/l) and its lowest in January (5±0.8 g/l) and in station (S3) the highest average salinity in July (11.6±0.06 g/l) and its lowest in March (9.2±0.04 g/l) was recorded (Fig. 1).The systematic account and description for the described species is as follows:Kingdom: Animalia ; Phylum: Annelida Linnaeus, 1758 ; Class: Clitellata Linnaeus, 1740 ;  Subclass: Oligochaeta Grube, 1850 ; Order: Lumbriculida Claus, 1872 ; Family: Lumbriculidae Claus, 1872; Genus: Stylodrilus Claparède, 1862 ; Species: heringianus Claparède, 1862.Figure 1: Locality of sampling stations in studied transects (Google Earth, 2014-2015).  (S1: River, S2: Estuary, S3:Marine) Worms are olive gray or dark yellow with brown spots (Fig. 2A). Prostomium is without proboscis and most specimens have no eyes (Fig. 2B). Worms's body length was 23 to 69 mm and 0.5 to 1.3 mm in diameter with 28-71 segments. Dorsal and Ventral chaetae beginning in segment II and have 2 chaetae per bundle and 8 chaetae per segments (Fig. 2C). Chaetae in the anterior segments were simple pointed (Fig. 2D) and the posterior segments, bifid with upper tooth very short and reduced (Fig. 2E, F), with 35-55 μm long. A pair of penes on ventral side of X segment were visible in mature individuals (Fig. 2G).Figure 2: Comparison of average (± standard deviation) of water temperature in sampling stations and months in studied transects, (S1: River, S2: Estuary, S3: Marine). Different letters indicate significant differences among averages (p<0.05).In total, 40 individuals of S. heringianus were examined. During the present study, this species occurred in stations 1 (river) but was absent in station 2 (estuary) and 3 (marine) in both sampling areas. Density and biomass of this species among sampling months andstations were significantly different (p<0.05), as the highest average density and biomass were observed in Cheshmehkileh (station 1) in January (78±32.6 ind m-2 and 197±70 mg m-2), and the lowest of those were observed in Sardabroud (station 1) in September (9.7±4.3 ind m-2 and 18±7 mg m-2) respectively. Spatial distribution of S. heringianus among sampling stations (S1, S2 and S3) showed significant differences (p<0.05). In freshwater stations (S1) within the river average density and biomass were 42.6±17.3 ind m-2 and 113.3±40.3 mg m-2 respectively, and no specimens were observed in the estuary and marine stations (Fig. 3). In other words, no specimens were observed in the estuary (S2) and marine (S3) stations of both studied transects in all the sampling months (Table 1).Temporal distribution of this worm in both studied transects showed significantly different among sampling months (p<0.05), as the highest average density and biomass of this species were observed in January as 58.6±23.7 ind m-2 and 175±55 mg m-2, respectively. While, the lowest of those values were in September as 9.7±4.3 ind m-2 and 18±7 mg m-2 (Fig. 4).Figure 4: Comparison of average (± standard deviation) of sediment TOM percentage in sampling stations and months in studied transects, (S1: River, S2: Estuary, S3: Marine). Different letters indicate significant differences among averages (p<0.05).A significant correlation (p<0.05) between density and biomass of S. heringianus with environmental variables was found (Table 2). A positive correlation was found between this species and gravel, silt and clay, while its correlations with temperature, salinity, TOM and sand were negative.

Discussion and conclusion :

This study aims was done to identify and determine the distribution and density of Stylodrilus heringianus in Cheshmehkileh and Sardabroud river and estuary, alongside Iranian coasts and has not been previously reported from Iran’s freshwater fauna and Southern Caspian Sea basin. According to the taxonomic and morphological studies of the samples and the use of valid identification keys, and also sending photos of the samples to experts to confirm the identified species, it was determined that the samples belong to S. heringianus species. This species was recorded for the first time from Iran. This paper updated a short checklist of Iranian aquatic oligochaetes to 27 species (for Lumbriculidae to 2 species and for Stylodrilus genus to 1 species). In Table 3, the identified species of Lumbriculidae from Iran until now are listed and S. heringianus was not on the list.Different letters indicate significant differences among averages (p<0.05). Capital letters indicate variation among stations (horizontal), small letters indicate variation among months (vertical).According to the results of this study, the highest average density and biomass of this species was observed in the cold months of the year (January and March) and the lowest in the warm months of the year (July and September), whereas density and biomass of this species show a significant negative correlation with water temperature, that this increase in density can be due to the asexual reproduction of Architomy and Parathomy (Smith, 2001) of this species in the cold months of the year, and its reduction in the warm months of the year (July and September) can probably be related to the feeding of fish with this worm. In the present study, S. heringianus was observed only in the river's freshwater ecosystem (salinity 0 ppt) and it was not observed in both estuarine brackish ecosystems with a salinity range of 5 to 8 g/l and marine brackish ecosystem with a salinity range of 9 to 11.5 g/l, which indicating that this species is oligohaline (does not tolerate salinity more than 1 g/l). Considering that the sediments grain size and the percentage of total organic matter were almost the same in the both ecosystems of river freshwater and estuarine brackish, it can be concluded that the limiting factor for distribution of this species in the estuarine and marine environments, maybe it is because of salinity intolerance, even to a very low level.Substrates in both rivers at Freshwater sampling stations (S1) were covered with vegetation in littoral and some central zone, but substrates in estuary and marine brackish stations (S2 and S3) vegetation cover was absent, According to the results of other researchers' studies about dependency of this species to vegetation habitats, probably, the main reason of density and biomass increase of this species in freshwater sampling stations was presence of vegetation habitats and its decrease in brackish water stations was due to absence of this habitat. Possibly, for this is reason, S. heringianus showed significant negative correlation with salinity. Among the investigated environmental factors, temperature and salinity of water and sand have the most influence on the distribution of this species. In addition to its important role in the food chain of water resources, this species is also considered as a bioindicator of water quality and is used as live feed in the aquaculture industry.

Dissolved oxygen is one of the most important indicators of water quality in marine environments and a critical element for the survival of various aquatic species (Breitburg et al., 2018). Lack of oxygen in marine environments can have irreparable environmental consequences, such as changes in biogeochemical cycles, damage to ecosystem functioning, disruptions in the trophic network, and reductions in biodiversity. With the emergence of climate change effects and global warming in recent decades, the Oman Sea, similar to many marine environments, has experienced deoxygenation and the expansion of hypoxic (dissolved oxygen less than 2 mg/L) areas (Queste et al., 2018; Lachkar et al., 2021). Based on measurements made in the 1960s, permanent hypoxic and anoxic conditions existed in the Oman Sea at depths ranging from about 150 to 1000 m (Bopp et al., 2013; Long et al., 2016; Stramma et al., 2010; Paulmier and Ruiz-Pino, 2009). However, the hypoxic conditions prevailed at depths greater than 50 m based on measurements taken in the summer of 2018 (Saleh et al., 2021). Therefore, studying the effective factors influencing changes in dissolved oxygen in this sea is crucial to protect and manage aquatic resources. Since the Oman Sea has water exchanges with the Persian Gulf (Garcia-Soto et al., 2021; Reynolds, 1993), this research investigated how dense outflow from the Persian Gulf (penetrating to a depth range of 150–300 m in the Oman Sea through the Strait of Hormuz) affected dissolved oxygen levels in this sea and their spatial variations, using available global measurement data. Furthermore, this study examined the trends of dissolved oxygen changes in the Persian Gulf over recent decades and their effects on decreasing dissolved oxygen levels in the Oman Sea.

To investigate the influence of high salinity and dense outflow from the Persian Gulf on dissolved oxygen levels in the Oman Sea, we utilized available global measurements of dissolved oxygen in both water bodies. We specifically analyzed long-term trends in dissolved oxygen in the Persian Gulf and their impact on the reduction of dissolved oxygen levels in the Oman Sea. Data on dissolved oxygen profiles at 25 points in the Oman Sea in 2010 and all available dissolved oxygen profiles in the Persian Gulf from 1980 to 2010 were downloaded from the World Ocean Database. At each study point in the Oman Sea, vertical profiles of dissolved oxygen at each depth were averaged, and this result was used as the annual mean profile for each point. The profiles obtained from the 25 study points were then analyzed using Ocean Data View software as four cross-sections to evaluate areas influenced by dissolved oxygen from the Persian Gulf outflow. To evaluate long-term trends in dissolved oxygen in the Persian Gulf, measurement data from 1980 to 2010 were studied as an annual average time series.

The results showed that, in general, the surface mixed layer of the Oman Sea has the maximum dissolved oxygen concentration. With increasing depth, this concentration decreases until it reaches 2 mg/L at a depth of about 80 m. In the layer between depths of 80 and 1000 m, hypoxic and anoxic conditions prevail in the sea. Between depths of 150 and 300 m, a relative increase occurs in dissolved oxygen concentration. The results demonstrated that the dense and salty outflow from the Persian Gulf, which has higher oxygen levels than the low-oxygen waters of the Oman Sea, increases dissolved oxygen levels by 1–4 mg/L in the depth range of 150–300 m in this sea. These effects are greatest along the southern coast; moving toward the Iranian coast, this influence diminishes. Additionally, along the southern coast of the Oman Sea, moving from the Strait of Hormuz to the Arabian Sea, these effects decrease. Considering these analysis results, a criterion of at least a 0.5 mg/L local increase in dissolved oxygen concentration at depths ranging from 150 to 350 m indicated that approximately 76,000 km2 of the Oman Sea is affected by the dense currents coming from the Persian Gulf. Examining dissolved oxygen concentration data from the Persian Gulf during 1980–2010 revealed that deoxygenation occurred in its deep waters at a rate of 0.13 mg/L per decade, with concentrations decreasing from about 4.5 mg/L in 1980 to 4.1 mg/L in 2010. Correspondingly, the influence of the Persian Gulf outflow on dissolved oxygen levels in the Oman Sea has decreased from an average of 1.2 mg/L to 0.7 mg/L.

This study assessed the effects of outflow from the Persian Gulf on dissolved oxygen in the Oman Sea. The results indicated a relative increment in dissolved oxygen concentration between 150 and 300 m in the Oman Sea. Considering that this zone is affected by the Persian Gulf outflow (Ghazi et al., 2021; Pous et al., 2015), this increase in oxygen levels can be attributed to these currents. Shenoi et al. (1993), Prasad et al. (2001), Acharya and Panigrahi (2016), and Queste et al. (2018) also demonstrated the penetration of dense Persian Gulf outflow below the thermocline of both the Oman and Arabian Seas, along with its effects on their temperature and salinity. The results showed that outflows from the Persian Gulf increase dissolved oxygen levels over a wide area of the Oman Sea, extending more than 160 km from the southern coast and up to 550 km from the Strait of Hormuz. These effects are greatest along the southern coast; moving toward the Iranian coast, these effects decrease and reach zero near the shores. The results also demonstrated that along the southern coast of the Oman Sea, moving from the Strait of Hormuz to the Arabian Sea, these effects on dissolved oxygen levels of the Oman Sea decrease. Examining dissolved oxygen concentration data from the Persian Gulf during 1980-2010 showed that dissolved oxygen in its deep waters decreased at a rate of 0.13 mg/L per decade; as a result, the ventilation effect of its outflows in the affected area of the Oman Sea has diminished.

Introduction  :

Stickwater is a solution obtained by squeezing cooked fish meat in the process of producing fishmeal. It contains soluble proteins, fat, insoluble solids, vitamins, and minerals, constituting more than 60% of the weight of the raw material. In advanced fishmeal factories, after separating the fish oil, the stickwater is concentrated and added to the cooked and compressed fish meat to increase the amount of protein and other nutrients in the fishmeal. In the fishmeal production factories in Iran, the stickwater concentration process is not carried out, and it is discharged into the environment after incomplete oil separation. This not only causes economic loss but also leads to various environmental problems. However, research has shown that stickwater obtained from the fish meal production process can serve as an excellent adhesive agent in animal feed formulation as well as a potent absorbent and appetite enhancer for farmed aquatic animals. Therefore, a co-drying process of stickwater with drying aids was developed to address these issues and create a new product for livestock, poultry, and aquatic animals feed. Rice bran, a main byproduct of rice milling, is commonly used in animal feed due to its nutritional value. It can also serve as a processing aid or drying aid for drying stickwater. The affordability and accessibility of rice bran, coupled with its nutritional benefits, make it an ideal material for this purpose. Despite a lack of scientific reports on the drying of stickwater using drying aids for animal and aquatic feed, production of this innovative approach shows promise in increasing the nutritional value of rice bran while economically drying stickwater. The primary objective of this study was to determine the drying condition and nutritional value of the resulting prototypes for use in animal, poultry, and aquatic feed.  However, the obtained technical knowledge can solve the environmental problems related to the disposal of stickwater.

Methodology   :

The production process was conducted using a pilot-scale hot air dryer. After prototyping on a laboratory scale, by keeping the temperature of the dryer constant, different ratios of stickwater rice to bran flour (1:0.42, 1:0.65, and 1:1) were tested. The inlet air temperature was considered to be 65±5°C, and the heating continued until the moisture content of the product was below 8%. The yield percentage, proximate analysis including dry matter, crude protein, crude fiber, ether extract, ash, and pH, calcium, phosphorus, fatty acid profile, amino acid profile, total volatile nitrogen (TVB-N), and peroxide index were measured. The amount of nitrogen-free extract was also obtained by reducing the total percentage of moisture, fat, protein, fiber, and ash from 100. The insoluble fibers in acid detergent (ADF), non-protein nitrogen (NPN), insoluble nitrogen in acid detergent (ADIN), and neutral detergent insoluble fibers (NDF) were also assessed. Measurement of these indexes is important for ruminant nutritionists and was performed to evaluate the fiber components of the product and its nitrogen partitioning. The metabolizable energy of the product was also tested.

Results   :

The study found that increasing the ratio of rice bran led to a decrease in the percentage of crude protein in the final product and significantly reduced the drying time. The prototype with a ratio of 1 to 1 (rice bran to stickwater) exhibited the highest production efficiency (67.70%) and the lowest amount of moisture content (highest amount of dry matter), indicating its potential as an optimal feed supplementation. The highest amount of crude fat and the lowest amount of ash were observed in combination with a ratio of 0.42 to 1 (rice bran to stickwater), and the lowest amount of crude fat and the highest amount of ash were observed in the product with a ratio of 1 to 1 (rice bran to stickwater), which showed a significant difference with other samples (Table 1).a-c Different letters show statistical significance in each column (p<0.05).The amount of nitrogen-free extract in the optimal prototype (ratio of 1 to 1 rice bran to stickwater) was 23.96%. The lowest moisture content and the highest peroxide index and TVB-N were observed in the optimal product. The lowest amount of ash and the highest pH value was observed in stickwater, and the highest amounts of insoluble fiber in acid and acid detergent (ADF and NDF) were observed in common rice bran. In general, the results showed that the nutritional value of the optimal prototype was higher compared to stickwater and common rice bran. The levels of myristic, palmitoleic, heptadecanoic, arachidic, gadolic, margaric, stearic, vaccinic, eicosadienoic, eicosatrianoic, eicosapentaenoic (EPA), arachidonic, urosic, docosa-tetraanoic, docosapentaenoic, and docosahexaenoic (DHA) fatty acids in stickwater were significantly higher than rice bran and co-dried stickwater. The levels of all amino acids including aspartic acid, glutamic acid, serine, histidine, glycine, threonine, arginine, alanine, tyrosine, methionine, valine, phenylalanine, isoleucine, leucine and lysine in stickwater dried with rice bran were significantly higher than common rice bran and lower than stickwater. Apparent metabolizable energy (AME) and true metabolizable energy (TME) in the optimal prototype were 4012 and 4447 respectively.

One of the most important issues in developing livestock, poultry, and aqua feed is the amount of consumption energy in the production phase, which will directly affect the cost of the product. Increasing the drying time will lead to more energy consumption and consequently increase the cost of the product. On the other hand, this issue may also affect the quality of the product and can also show its negative effect on animal performance. Therefore, due to the importance of drying time and production efficiency, the product with a ratio of 1 to 1 (rice bran to stickwater) with the lowest drying time and the highest production efficiency was selected as the optimal prototype. The use of drying aids is one of the methods of drying liquids or materials with high humidity in the food industry and animal feed production. These materials have moisture absorption properties and are used to reduce the moisture of the material intended for drying and thus reduce the drying time. The use of these materials is very important from a technical and economic point of view because it saves energy while minimizing the quality loss of the product. Rice bran contains significant amounts of protein, fat, carbohydrates, and minerals, and as a result, it has desirable physicochemical properties for formulation and feed production. The use of this technology can improve the nutritional value of rice bran. The presence of proteins and lipids of marine origin in co-dried stickwater with rice brans has positive effects on feeding livestock, poultry, and aquatic animals. The innovative approach not only addresses environmental concerns related to stick water disposal but also offers a cost-effective solution for fishmeal production units in the country.

Aquaculture development is challenged with limitation and high costs of supplying protein diets, so searching for alternative protein sources in fish diets is inevitable (Tacon, 2020). Insect meals are considered as a promising substitution protein ingredients last decades (Alfiko et al., 2022). Among candidates’ insect meals, Silkworm is well-balanced in protein as well as lipid and can be served as feedstuff (Mahanta et al., 2023). A study conducted to investigate if low level of silk worm pupa inclusion in fingerling Beluga sturgeon (Huso huso) diet could provide a protein source replacement accompanying with high benefit for the species.

Fingerling Beluga sturgeons (5±0.2 g, mean±SE) fed with experimental diets for a period of 8 weeks. Four experimental diets includes: no (SWP) inclusion as a control treatment (T0), 5, 10 and 15 percent (SWP) substitution referred as (T5, T10 and T15) treatments. At the end of the experiment, Weight Gain (WG), Specific Growth Rate (SGR) and Feed Conversion Ratio (FCR) calculated to assess the diets quality on fish production. Hepatic enzyme (ALP, AST, and ALT) and serum biochemical (IgM, total protein and lysozyme) analyzed for revealing the health status of fish.

(SWP) treatments, in particular (T15) had a significant increase in (WG) compared to the control group (p<0.05). (SGR) as well as (CF) in (SWP) treatments showed no significant differences compare to the (T0) (p<0.05). (FCR) was significantly improved among (SWP) treatments, although the difference was not significant (p<0.05). Serum lysozyme, Serum immunoglobin and total protein in (T10) and (T15) were significantly increased compared to the control group, (T0) (p<0.05).

Our findings indicate the protein level adjusts and fixes with the (SWP) was efficiently enough to be replaced with fish meal in fingerling Beluga sturgeon (H. huso). The results of (SGR) as well as (CF) suggest that the replacement level could be changes to more to get a differently improved result. Hepatic enzymes showed that (SWP) did not affect impairment in digestive tract.  What’s more, blood biochemical analysis showed that (SWP) can have immunomodulatory effects on fingerling Beluga sturgeons (Zhou et al., 2022)

The emergence of diseases poses significant challenges for the aquaculture sector. While diseases are natural components of aquatic ecosystems, they often lead to economic repercussions for both aquaculture and fisheries. The restrictions on antibiotic use in aquaculture have prompted the development of environmentally friendly alternatives, such as probiotics and organic acids, which serve as immune stimulants. Short-chain fatty acids and organic acids, along with their corresponding salts are emerging as potential alternatives to antibiotic growth promoters in aquaculture, garnering increasing interest. Probiotics, on the other hand, are live microbial feed supplements that help maintain a healthy microbial balance in the host's gut.

This study was carried out to evaluate individual and combined effects of the dietary Lactobacillus casei and sodium propionate supplementation on the growth performance and immunity of zebrafish (Danio rerio). A total of 120 zebra fish (335.22 ± 0.23 mg) were placed in 12 tanks with 10 fish per tank and fed with diets containing 0 % Lactobacillus casei and sodium propionate (control, T1) 0.1% Lactobacillus casei (5 x 106 CFU, T2), 1% sodium propionate (T3), and 0.1% Lactobacillus casei bacteria and 1% sodium propionate (T4) for 8 weeks. At the end of the experiment, feeding was halted for 24 hours. Eight fish from each tank were anesthetized with clove oil at a dose of 50 μl/l. After removing the head and tail, the fish were frozen in liquid nitrogen and homogenized using a manual homogenizer. The resulting homogenate was then prepared in 25 mM Tris-HCl buffer (pH 7.2) for the measurement of immune factors.

The experimental diets significantly affected the final weight and weight gain of the fish. Fish fed with Lactobacillus casei and sodium propionate had a higher weight compared to the control group (Table 1, p<0.05). The specific growth rate also varied among the diets, with the lowest growth rate observed in the control group and the highest in the groups receiving sodium propionate and Lactobacillus casei, which differed significantly from the control group (p<0.05). No significant differences were found among the treatments in triglyceride, cholesterol, and glucose levels (Table 2, p<0.05). Serum albumin analysis indicated no significant difference between treatments 1 and 3, nor between treatments 2 and 4 (Table 2, p<0.05). However, significant differences were noted between treatments 1 and 3 and treatments 2 and 4, with the highest levels found in treatments 2 and 4. Regarding the globulin index, there was no significant difference between the control group and treatment 3, but significant differences were observed with the other two treatments (Table 2, p<0.05). The highest activities of superoxide dismutase, catalase, and glutathione peroxidase were recorded in treatment 4, which significantly differed from the control group (p<0.05) but showed no significant difference from treatment 3 (Table 3, p<0.05). Significant differences were also observed in total serum protein and immunoglobulin levels, with the highest levels in treatments containing Lactobacillus casei and sodium propionate (treatments 2, 3, and 4), which differed significantly from the control group (Table 4, p<0.05). Notably, significant variations were found in lysozyme and complement levels among the treatments, with the highest levels in treatments 3 and 4 (Table 4, p<0.05).

Fish growth and immunity improved in fish fed supplemented diets compare to the control group. Improving fish performance through organic acids and probiotics is possible in several ways, including reducing gastric pH, which increases pepsin activation, reducing intestinal pH, which leads to increased mineral dissolution and subsequent absorption by organic and probiotics. It can be concluded that the use of a diet containing Lactobacillus casei and sodium propionate in zebrafish has numerous positive effects. Overall, the findings suggest that these supplements can be effectively utilized both separately and in combination, specifically at levels of 1% sodium propionate and 0.1% Lactobacillus casei (5 × 106 CFU) in aquaculture.

Ensuring food and nutritional security is a top issue for global food-producing sectors among the projections of a 60% increase in food consumption by 2050 due to population growth. To address this challenge sustainably, the food and feed industry must conserve resources while feeding a growing population (Saiprasad et al., 2023a). This rapid expansion of aquaculture has led to increased demand for protein sources for feed production (Wang et al., 2023) and straining the supply of natural protein ingredients, such as fishmeal and plant protein sources. As a necessary way to reduce protein inclusion levels in aquaculture feed, lipids and carbohydrates are extensively used. Lipids can serve as an energy source with high energy density, and fish has good lipid absorption and utilization ability. Carbohydrate is a cheaper energy source than lipids and protein, which also serves as a swelling and stabilizer agent in fish feed (Kamalam et al., 2017). However, numerous studies have demonstrated that long-time ingestion of a high lipid/carbohydrate diet causes disturbance of lipid metabolism, excessive hepatic lipid accumulation, and mitochondria stress, thus inducing fatty liver disease and affecting the physiological condition and growth of fish (Ding et al., 2020; Jin et al., 2019a; Jin et al., 2020; Lin et al., 2018; Wang et al., 2021; Wang et al., 2020). Using functional feed additives is an effective way to relieve hepatic metabolic stress and improve fish health which has drawn considerable attention in the aquaculture nutrition field. Up to now, L-carnitine (Jin et al., 2019a), choline (Jin et al., 2019b), herb extract (He et al., 2021; Liu et al., 2021; Wang et al., 2021), and other feed additives showed positive effects on fish metabolic disorder. Bile acids (BAs) play important roles in lipid metabolism. They are synthesized from cholesterol exclusively by the liver. In the intestine, BAs act as detergents to emulsify and facilitate the absorption of dietary fats and lipid-soluble vitamins. Therefore, the objective of this study was to evaluate the effects of dietary BA supplementations on growth performance, carcass biochemical composition and Biochemical Factors of rainbow trout (Oncorhynchus mykiss).

A 75- day feeding experiment was conducted to investigate the effects of dietary bile acids (BAs, Roneon 30%) on nutritional index, growth, carcass biochemical composition, Biochemical Factors and economic return coefficient of rainbow trout (Oncorhynchus mykiss). An experiment was designed with four treatments each with three replicates. Experimental diets were made by supplementing a control diet (BA0) with 300, 600 and 900 mg bile acid (BA) kg− 1 diet, respectively. 600 juvenile rainbow trout average initial weight of 52±5 g (±SD) were assigned to twelve 2000 L experimental tanks. Fish were sampled on day 75 and growth performance indices including Food conversion ratio (FCR), Protein efficiency ratio (PER), Specific growth rate (SGR), Condition factor (CF), and Weight gain percentage (WGP) were compared among the treatments. Also carcass composition (protein, fat, ash, moisture) and bichemical factors of serum including Aspartate transaminase enzyme (AST), Alanine transaminase enzyme (ALT), Alkaline phosphatase enzyme (ALP), Lactate Dehydrogenase, Glucose, Triglyceride, Albumin and total protein were analyzed.

At the end of the experiment food conversion ratio, (<1) in the treatment of 900 mg/kg showed the best performance among the experimental treatments. The final weight (187.33±7.28 g) had a significant difference compared to other treatments(P<0.05). Specific growth rate, Condition factor, Protein efficiency ratio and Weight gain percentage were improved in fish fed with diets 600 and 900 mg/kg bile acid supplementation compered with other treatments. Also Treatments receiving bile acid supplementation had more crude protein and less ash than the control treatment and the 300 and 600 mg treatments had higher moisture content than other treatments (P<0.05). Glucose, Triglyceride, Albumin and Lactate Dehydrogenase did not change by the addition of bile acid supplementation (P>0.05). Cholesterol content was significantly higher in fish fed diet containing 900 mg/kg bile acid supplementation compared with control group (P< 0.05). Aspartate transaminase enzyme in the treatment of 900 mg/kg compared to the control group and 300 mg/kg, Alanine transaminase enzyme in the treatment of 900 mg/kg and Alkaline phosphatase enzyme in the treatment of 300 mg/kg compared to other treatments showed significant differences (P<0.05) (Tables 1- 3).Different lowercase letters in the same row indicate statistically significant differences (P<0.05).

Discussion and conclusion :

The changes of serum biochemical factors as well as the relative improvement of growth and nutrition factors indicated the positive performance of using 900 mg/kg bile acid supplementation in the diet of juvenile rainbow trout, while the effects of increasing Liver enzymes, the Serum Biochemical Factors in the mentioned treatment should be studied and investigated. Bile acid supplementation could also enhance crude protein and decrease of ash and moisture in treatment of 900 mg/kg while bile type, level and fish species must be considered. This study discusses these aspects in regard to fish nutrition to help increase the inclusion of dietary fishmeal alternatives and thus enhance aquaculture sustainability.