Aquafeeds containing plant or herbal extracts have improved aquatic species' growth, immunological response, and feed consumption [4, 43]. It has been demonstrated that adding powdered or extracted medicinal plants to fish diets enhances appetite, growth, immunity, and stress tolerance [44, 45]. In the current study, the inclusion of grass carp diets has beneficial effects on feed utilization and the growth of the fish. Although there may be a few differences based on the plant species from which the active substance is extracted and the species of fish that are being cultivated, the favorable results are generally consistent with the trend of the use of phytogenics as reported in the literature [46]. Mocanu et al. [47] reported a reduced FCR of grass carp dietary phytogenics supplementation. Since feed accounts for more than half of the overall cost of grass carp production, the FCR is a critical economic measurement. Feed manufacturers often optimize their feed formulas to minimize FCR; however, there may be variances based on aquaculture type, water quality, temperature, and other factors [48]. In the current study, MS supplementation lowers the FCR, whereas other research has shown that the use of phytogenics decreases the FCR [49]. There was an increase in crude lipid and moisture contents of the body composition among the MS group compared with the control. This is comparable to a previously published study that found that the nutritional profile of whole fish is improved by the inclusion of phytogenic feed additives [7]. The largemouth bass increased crude lipid content, which could be partially responsible for the higher HSI.
Blood is widely utilized as a promising indicator of pathological and physiological changes in toxicological research and environmental monitoring [50]. The hematological parameters are commonly studied to assess toxic effects triggered by aquatic pollutants. Fish in aquatic environments are often subjected to a wide range of physical and chemical stresses, which could weaken their immune systems and increase their susceptibility to infections by pathogenic microorganisms [51, 52]. The hematocrit value is an important tool in aquaculture to determine the health status of fish [53]. In the current study, dietary MSE increased the levels of HB, RBC, WBC, HCT, and MCV and reduced the levels of MCH and MCHC in grass carp in all the MS extract treatments compared with the control. Parallel studies have also been recorded in other fish species, including Huso huso [54] and common carp, C. carpio [55]. Burducea et al. [46] reported an increase in the levels of HB, RBC, and MCV and a reduction in the levels of MCH in common carp-fed phytogenic feed additives. It is believed that fish with higher serum albumin, globulin, and total protein levels have stronger innate immune responses [53]. Transaminases, including GPT and GOT, are essential for proteins and amino acids; while oxicants cause tissue damage or dysfunction, they can release these enzymes into the plasma [56]. Globulin and albumin are the major plasma proteins in fish. The current study showed an improvement in the levels of TG, TC, TP, GOT, GPT, glucose, albumin, and globulin in grass carp in all the MSE groups compared to the control. This is consistent with earlier research employing quercetin [57], garlic [58], Avena sativa [53], and herbal seed [52], all of which elevated serum TG, TC, TP, glucose, albumin, and globulin levels in various fish species. Many studies on herbal and medicinal plants [4, 53] have shown that including herbal seeds in diets has erythropoietic and immune-boosting properties. According to reports, these effects improve cellular resistance to infections, oxidative stress adaptation, oxygen transport capability, and eventually lower death rates [59].
Overall, the results of the feeding experiment showed that as the content of MS extracts increased, it also increased the activities of the antioxidant enzymes. Although the role of the role of antioxidant enzymes in grass carp growth has not been fully understood, the assessment of antioxidant enzyme activities is frequently done to evaluate an organism's ability to defend against oxidative stress [60]. The present study's results are consistent with earlier research findings that demonstrated enhanced antioxidative mechanisms in fish with the supplementation of plant extract [61]. SOD and AOC are vital antioxidant enzymes that can be found in almost every organism [60, 62]. Improvements in superoxide anion and hydrogen peroxide, along with increases in the activities of CAT, GSH-Px, SOD, and AOC, may be indications of enhanced NADPH-oxidase activity and mass production of reactive oxygen species [63, 64]. Reactive oxygen species are a defense mechanism against microbial infections, but high concentrations can harm an organism's immune system by affecting essential proteins, unsaturated fatty acids, and nucleic acids [60]. Lipid peroxidation produces MDA, which is a byproduct that puts cells under toxic stress and is used as a biomarker for measuring an organism's amount of oxidative stress. Consequently, antioxidant activity is typically associated with a living system defense mechanism. The improved levels of SOD, T-AOC, CAT, and GSH-Px activities indicated that the grass carp may have benefited from the MS extract during inclusion. Our results are in agreement with Hoseinifar et al. [65], who reported an improvement in the activity of SOD, GSH-Px, and AOC and a decrease the activity of MDA in the liver of common carp through dietary supplementation with phytogenic feed additives. Salomón et al. [66] reported that medical plant extract can increase SOD and GSH-Px and decrease MDA in the gut gut of juvenile gilthead seabream. However, Das et al. [67] established that dietary inclusion of coriander oil has beneficial effects on the antioxidant activity of Nile tilapia. Regulation of these selective antioxidant enzymes by MS was detected by the nuclear factor E2-related factor 2/Kelch-like ECH-related protein 1 signaling pathway, which plays an important role in protecting the body from oxidative damage [68].
Fish have a more developed innate immune system than mammals, which is their primary line of defense against a wide range of infections [12]. The humoral immune components respiratory burst, myeloperoxidase, lysozyme, alternative complement, ceruloplasmin, antiprotease, haemagglutination, bacterial agglutination, and total immunoglobulin are closely linked to the adaptive and innate immune responses in fish and are tested to determine the health state of fish, its capacity to stimulate phagocytes and the complement system, as well as its activity towards both Gram-negative and Gram-positive bacteria [12, 40, 42]. Mohseni et al. [40] reported increased myeloperoxidase, lysozyme, and alternative complements due to Japanese eel-fed Eucommia ulmoides Oliver bark extracts supplemented. The dietary supplementation of phytogenics can increase lysozyme activity in turbot [69]. In the present study, the dietary supplementation of MS extracts could improve the activities of nitroblue tetrazolium, myeloperoxidase, lysozyme, alternative complement, ceruloplasmin, antiprotease, haemagglutination, bacterial agglutination, and total immunoglobulin in the serum of grass carp. The major organ of fish that synthesizes complement protein is the liver [40]. However, the enhanced liver health, boosted by increased serum glucose, albumin, and globulin activities, might be responsible for the improvement in the alternative complement activity of grass carp fed MS-supplemented diets. Myeloperoxidase, nitroblue tetrazolium activity, ceruloplasmin,, and antiprotease could activate neutrophils and activate macrophages [70]. Due to its opsonic properties, ability to activate phagocytes and complement systems, and ability to fight Gram-negative and Gram-positive bacteria, an essential component of the innate immune system is lysozyme [71]. Because of this, dietary supplements containing MS may offer a cost-effective and sustainable means of enhancing the innate immunity of grasscar and minimizing their need for chemotherapy while protecting them against various infections.
Nutrients are digested by digestive enzymes like cellulose, amylase, and lipase [40], and it has been reported that food handling can affect enzyme activity [72]. Since the activity of digestive enzymes impacts the effectiveness of nutrient absorption, their characterization offers significant insight into fish's capacity to hydrolyze fat, protein, and carbohydrates in diets. Digestive enzymes are important for fish utilization [73, 74]. A lack of MS in animals has been shown to cause digestive issues in various ways, even though the mechanisms by which MS impacts fish digestive action are unknown. In a study by Almeida-Bezerra et al. [9], the vagus nerve innervates the established enteric neural system of the gastrointestinal tract, which can function autonomously and promote digestive secretions. Thus, deficiencies in MS may impact these areas' metabolic processes, potentially leading to adverse consequences for the gastrointestinal system [68]. Additionally, cellulose conversion and protein digestibility are decreased while a stomach's pH increases [75, 76]. Accordingly, the decrease of lipase and increase of amylase and cellulose in this study could be related to a possible improvement in gastric pH. Additionally, it improves the amount of enzymes secreted into the midgut [7], which is responsible for the increase of midgut amylase and cellulose and the reduction of midgut lipase activity in MS fed the control diet. Improvement in digestive enzymes activated by dietary herbal extract inclusion has been reported in grass carp [77, 78], tilapia [79], and white leg shrimp [60]. It has high growth efficiency in fish. To address the association between the growth performances of grass carp fed varied doses of dietary MS extract and the digestive enzymes, more research is required.
In the current study, grass carp exhibited increased resistance to A. hydrophila in a dose-dependent manner in dietary MS extract, even after intraperitoneal injection of 1×107 cells/mL of the pathogen. Compared to 36% in the control diet, the survival rate increased to 47%, 63%, 88%, and 86% in MS1, MS2, MS3, and MS4. Das et al. [67] showed that dietary phytogenic supplementation improved the survival rate of Nile tilapia against A. hydrophila. MS mainly causes damage to bacterial membranes, which leads to the loss of membrane potential, respiratory activity, and efflux activity, and finally results in cell death [9]. Besides, MS extracts also increase the immunological state by controlling the innate immune indicators, including lysozyme and immunoglobulin expression, in grasscarp. Previously, Sattanathan et al. [80] documented that C. aerea in L. rohita exhibits anti-fungal and anti-bactericidal effects against fish infections. However, dietary supplementation with phytogenics could show an increased survival percentage after confronting A. hydrophila [81]. Therefore, an increased survival percentage after conflict with A. hydrophila has been shown in rainbow trout supplemented with Coriandrum sativum extract [82].
The immunomodulation mechanism in grass carp infected with A. hydrophila is still unclear. In response to immunization with A. hydrophila, we examined the expression levels of immune-related genes (NKEF-B, Lysozyme-C, Lysozyme-G, TNFα, TLR 22, β2M) in the grass carp head kidney and spleen in the present study. Because the head kidney is the leading main lymphoid tissue for the B-lymphocyte line and the spleen is a prominent peripheral lymphoid organ, these organs are appropriate for investigating the expression of such genes in vivo [42, 83]. Major antibacterial enzymes called lysozymes are crucial to fish's innate immunity. The study of fish lysozymes helps manage pathogen-induced fish illnesses. TLR22 and β2M, exclusive to antigen-presenting cells and activating CD4 + T cells [84], are helpful markers for comprehending the processes of epitope recognition and tracking the migration of immune-related cells. Following immunization, there was a significant rise in TCRα expression levels [85]. In the grass carp, the mRNA levels of expression NKEF-B, Lysozyme-C, Lysozyme-G, TNFα, TLR 22, and β2M improved after 14 days of A. hydrophila challenge supplemented with MS extract. After A. hydrophila infection, Lysozyme, TNFα, and β2M are thought to exacerbate the inflammatory response in grass carp. The explanation of species-specific immune reactions in A. hydrophila could be found in the expression patterns of immune-related genes.