The administration of Ambon banana stem extract (EBPA) through feed in this study provided good growth performance with daily administration frequency for 30 days of maintenance before the challenge test. In Table 1, it can be observed that the highest survival rate was found in treatment P2 (0.75 g.kg− 1) at 95 ± 5.03% before the challenge test and 80 ± 4.08% after the challenge test. These values indicate that treatment P2 had significantly higher survival rates (P < 0.05) compared to all treatments before the challenge test, significantly lower rates (P < 0.05) than the negative control but significantly higher rates (P < 0.05) than the positive control, P1, and P3 after the challenge test. The specific growth rate and weight values in this study also showed that the administration of EBPA at a dose of 0.75 g.kg− 1 had significantly higher values (P < 0.05), namely 4.11 ± 0.02% and 4.85 ± 0.02 g, compared to treatment P1 (0.50 g.kg− 1) at 3.74 ± 0.12% and 4.50 ± 0.11 g, and treatment P3 (1.0
g.kg− 1) at 3.65 ± 0.07% and 4.23 ± 0.06 g. Additionally, the feed conversion ratio value in treatment P2 was lower at 1.55 ± 0.05 compared to other treatments.
The high growth and growth rate in treatment P2 (0.75 g.kg− 1) compared to other treatments are suspected to be due to the appropriate dosage of EBPA administration, which can stimulate faster and better growth synthesis. Additionally, it is influenced by the active compounds present in EBPA, which can function as immunostimulators. The high survival rate in the negative control treatment is due to the shrimp not being infected with V. parahaemolyticus bacteria, the cause of shrimp death. This is also consistent with Awaludin et al. (2020), explaining that the lower the feed conversion ratio value, the more effective the feed given will be for shrimp growth, and the feed will be utilized effectively during maintenance.
Qualitatively, EBPA is known to contain several phytochemical compounds such as flavonoids, hydroquinone, saponins, steroids, triterpenoids, alkaloids, and tannins, while quantitatively, it has been shown that EBPA contains 19.52% flavonoids, 8.38% saponins, and 4.94% tannins (Asuquo and Udobi 2016; Simanjuntak et al. 2016; Ramadhan et al. 2017). Compounds found in EBPA such as flavonoids, saponins, alkaloids, and tannins are known to play a role in maintaining the health of shrimp, enhancing immune function and growth, and inhibiting bacterial growth, which ultimately affects survival, growth, and immune response of shrimp through protection from the immune system acquired after feeding containing EBPA (Malangngi et al. 2012; Astria 2017). The growth performance results are also consistent with increased immune response such as total haemocyte count (THC), phagocytosis activity (AF), respiratory burst (RB), and phenoloxidase (PO).
The values of cellular (THC and AF) and humoral (PO and RB) immune responses of shrimp fed with EBPA-containing feed were higher than the control (Fig. 1) and directly contributed to combating V. parahaemolyticus bacteria. These results indicate that feeding EBPA with daily frequency for 30 days before challenge testing can enhance shrimp resistance to pathogen attacks, as evidenced by treatments (P1, P2, and P3) fed with EBPA containing feed having higher survival rates compared to the control treatment. This condition is caused by continuous administration of immunostimulants, which can regulate and maintain the immune system in optimal condition until their administration is stopped (Jane et al. 2015). Shrimp immune response consisting of THC, AF, RB, and PO indicates that shrimp fed with EBPA at a dose of 0.75 g.kg− 1 had the highest values compared to other treatments. The high values of shrimp immune response obtained in treatment P2 occurred not only because of the active compounds present in EBPA but also because the dose of Ambon banana stem extract greatly influences the use of EBPA. Higher doses of EBPA feed cause a decrease in immune response in the test shrimp, and low doses of EBPA feed yield good results, but better results are obtained when used at the optimal dose in this study, namely treatment P2 (0.75 g.kg− 1). These results are in line with the statement by Putri et al. (2013) that high doses of immunostimulants can suppress defense mechanisms, while low doses affect immune responses less effectively. Additionally, Giri et al. (2023) stated that administration of immunostimulants above the optimal dose can cause immunosuppression in test animals, but can protect fish or shrimp from disease attacks.
Observations of THC in test shrimp after 30 days of EBPA feeding before and after the challenge test indicate that treatment P2 is the best among all treatments. The THC bar graph shows a significant difference between treatment P2 and the control treatment. This significant difference is presumed to be due to the increased production of hemocyte cells in the shrimp's body as a result of EBPA feeding. The increase in hemocyte cells in the shrimp's body plays a crucial role in inhibiting or destroying pathogens that enter the shrimp's body (Ding et al. 2020). THC values were seen to decrease in treatments fed with EBPA containing feed and positive control on day 1 post-challenge test due to the migration of hemocyte cells from the shrimp's body circulation system to tissues where many cells are infected (Simanjuntak et al. 2016; Xian et al. 2016). Then, an increase in THC occurred on day 7 and then decreased again on days 14 to 21, followed by an increase on day 28. The increase in THC values on day 7 post-challenge test is due to the infection of V. parahaemolyticus bacteria in a critical phase, causing hemocyte cells to react to the body's defense mechanism consisting of pathogen recognition, phagocytosis, melanization, cytotoxicity, and cell communication in fighting pathogens (Azhar and Yudiati 2023; Dhewang et al. 2023; Kemal et al. 2023), and the increase on day 28 is presumably because the shrimp's body has returned to normal, thus it is able to produce hemocyte cells again. There are also parameters of shrimp's immune defense system that can be observed, namely phagocytosis activity (AF), which is the ability of non-specific immune response cells to phagocytose disease agents entering the body. Observations of AF indicate an increase in treatment P2 compared to other treatments. The increase in AF can be interpreted as the administration of EBPA in feed as an immunostimulant has been proven to stimulate or enhance the shrimp's immune system. This is in line with Kumar et al. statement (2023) that the mechanism of action of immunostimulants in stimulating the immune system is by increasing the activity of phagocytic cells. The increase in THC and AF values becomes the main defense system in fighting pathogens entering the shrimp's body (Panigrahi et al. 2020).
The activity of hemocytes in shrimp bodies also produces additional antibacterial compounds, such as hydrogen peroxide (H2O2) and superoxide anion (O2−), which can enhance shrimp's resilience to pathogen infection. Superoxide anion (O2−) is the first reaction generated in the process of respiratory burst (RB) activity. RB activity is detected when foreign objects are expelled through phagocytosis; when phagosomes engulf foreign objects, it involves the release of degradative enzymes that produce reactive oxygen intermediates (ROIs) (Risjani et al. 2021). The results of this study indicate that shrimp fed with EBPA for 30 days before the challenge test have higher RB activity compared to the control. Based on the results of the study, RB activity is directly proportional to THC. The decrease in RB activity on day 1 post-challenge test is related to the decrease in the number of hemocytes in the shrimp's body due to the migration of hemocytes to infection sites to perform phagocytosis processes and initiate defense responses (Muharrama et al. 2021).
Phenoloxidase (PO) activity is one of the humoral immune responses. In hemolymph, PO is present in an inactive form called prophenoloxidase (proPO). The ability of shrimp to recognize foreign objects entering its body indicates the high value of PO activity and the presence of shrimp's immune defense activity (Mahasri et al. 2017; Barrios et al. 2023). PO will later oxidize phenols to form quinones and produce the end product melanin, a dark brown pigment that inactivates and prevents the spread of pathogens (Amparyup et al. 2013). Measurement results show that the PO value in treatment P2 is higher than in other treatments, both before and after the challenge test. These results indicate that feeding EBPA-containing feed induces the activation of non-specific immune mechanisms in shrimp, thereby triggering the activation of intracellular genes that can increase the production of antimicrobial molecules (Santhosh et al. 2023). With increased immune response, it can also reduce and suppress the growth of bacteria infecting shrimp as the primary defense against pathogen attacks.
The administration of Ambon banana stem extract (EBPA) through feed in this study also suppressed the growth of bacteria in the intestines of vannamei shrimp, including total Vibrio count and V. parahaemolyticus count (Fig. 2). Total Vibrio count and V. parahaemolyticus count in each treatment P1, P2, and P3 significantly suppressed bacterial populations (P < 0.05) compared to treatment K+. Treatment P2 was able to significantly suppress bacterial populations (P < 0.05) compared to other dosage treatments, including total Vibrio count and V. parahaemolyticus count up to day 28 after the challenge test. This is because EBPA contains flavonoids as antibacterial compounds, which work by breaking down the bacterial cell membrane, causing a cessation of ion and compound transport into bacterial cells. This transport constraint will result in reduced bacterial nutrition needed for growth (Kusdarwati et al. 2010; Krisnata et al. 2014). Additionally, according to Munaeni et al. (2020), the use of herbal materials can suppress bacterial growth and enhance immune responses in shrimp infected with V. parahaemolyticus. This is also emphasized by Rozik et al. (2022), stating that the use of herbal products in fish farming can be the initial step in preventing disease spread and inhibiting the growth of pathogenic bacteria. The reduction in bacterial populations in this study also correlates with the ability to reduce damage to the hepatopancreas organ (Fig. 3). This is evident from the reduction in vacuolization, normal tubules, lysis compared to the positive control. According to Raja et al. (2017), shrimp infected with V. parahaemolyticus can cause damage to the hepatopancreas organ. In this study, the use of EBPA was able to enhance immune responses in defense against V. parahaemolyticus infection, both through direct and indirect defense mechanisms, thus minimizing damage to the shrimp's hepatopancreas.
Furthermore, this study also observed immunity genes including Lipopolysaccharide- and β-1,3-glucan-binding protein (LGBP) and peroxinextine (PE) genes. Lipopolysaccharide- and β-1,3-glucan-binding protein (LGBP) gene plays a major role in recognizing and responding to entering bacteria (Chen et al. 2016) and plays a crucial role in activating the proPO system (Du et al. 2019). proPO is a key gene in activating the PO enzyme and then induces melanization and phagocytosis (Rowley 2016), while the PE gene plays a vital role in degranulation and phagocytosis, so when pathogens attack, hemocyte cells will undergo degranulation. During the degranulation process, the peroxinectin (PE) enzyme is released and stimulates the phagocytosis reaction. This is also related where peroxinectin (PE) is an essential amino acid in an inactive form and will be activated when the LGBP gene is present, so these two genes are interrelated in influencing the shrimp's immune response (Muharrama et al. 2021). In this study, it can be seen that the use of EBPA provided higher results compared to the control for LGBP and PE gene expression parameters (Fig. 4). The increased expression of the LGBP gene in EBPA usage after treatment and challenge tests indicates that the process providing resistance to pathogens by the shrimp's immune system is more effective in treatment compared to the positive control. Treatment P2 (0.75 g.kg− 1) in this study provided higher values compared to other dosages that could enhance the expression of immunity related genes. This is also emphasized by Hsieh et al. (2013), indicating that the use of herbal plants can enhance immune responses, immunity gene expression such as LGBP and PE, thereby increasing the survival of vannamei shrimp from bacterial infections.