3.1. In vitro temperature–growth, antibacterial activity and hydrophobicity test of bacteria
In Table 1, the amount of optical density (OD) measured from the culture of bacteria with probiotic power at temperatures of 10, 20, 30, and 37°C for 48 hours is given. During the 48 hours, there was a significant difference between the growth of bacteria at different temperatures (p < 0.05). All bacterial isolates were able to grow from 10 to 37°C based on the measured optical density. At a temperature of 10°C, all the isolates could grow at a very low rate. The highest values of optical density at a temperature of 20°C correspond to L1, at a temperature of 30°C and 37°C respectively to L2 and L1. L1 and L2 had good growth ability at the investigated temperatures (p < 0.05). Based on the bacterial growth test at different temperatures, it was found that by increasing the temperature of the greenhouse for 48 hours, the growth rate of bacterial isolates increases based on optical density. During the 48 hours of the experiment, the amount of optical density resulting from the growth of bacteria at a temperature of 10°C was very low, but at temperatures of 20, 30, and 37°C, the optical density increased and the growth of bacteria increased significantly. It arrived. It can be said that the results of the present study show that the obtained bacterial isolates can grow at the tested temperatures and the best temperature range for the growth of bacteria is the temperature range for the culture of Asian sea bass. Riaz et al., (2010) investigated the growth rate of Lactobacillus acidophilus and Lactobacillus fermentum at temperatures of 15, 20, 25, and 35°C. The optimal growth temperature of Lactobacillus acidophilus was 35°C and 25°C was introduced for Lactobacillus fermentum. The optimal temperature for the growth of bacteria in the current study is 37°C, which indicates that these isolates are thermophilic. The antibacterial characteristic of each probiont, including L1, and L2 was examined against pathogenic bacteria of trout (i.e., L. garvieae, A. hydrophila, Y. ruckeri, V. harvei), by using an in vitro test in which the inhibition zone of the cross culture medium was measured. Table (2) shows the higher inhibition zone for L2 rather than L1 in most cases (P < 0.001). Antimicrobial tests are one of the common tests for choosing a suitable probiotic. The probiotic bacteria must have antimicrobial properties and the ability to produce antimicrobial compounds (Zokaeifar et al.,2012). This effect is applied in several ways, some of which are as follows: The secretion of extracellular metabolites with bactericidal power by probiotic cells is the competition in occupying the place to stick to the mucus. In the present study, noteworthy antimicrobial activity against pathogenic bacteria was observed in lactic acid bacteria. Within probiotic strains, lactic acid bacteria exhibit the production of various compounds, including organic acids, lactic acid, bacteriocin, and hydrogen peroxide. These compounds contribute to the activation of non-specific immunity in the host. The subjects under investigation in this research are likely to possess a heightened capacity for producing growth-inhibitory substances. Vieira et al., (2016) demonstrated that Lactobacillus plantarum strains, isolated from shrimp intestines, exert an inhibitory effect on a broad spectrum of both Gram-positive and Gram-negative pathogenic bacteria, including V. harveyi and A. hydrophila. Additionally, Zapata and Lara-Flores, (2013) reported that Leuconostoc mesenteroides, a lactic acid bacterium isolated from tilapia fish intestines, exhibited the ability to inhibit V. harveyi and Mycobacterium marinum, both recognized as pathogenic bacteria. The percentage of hydrophobicity of the strains ranged from 6.6 ± 0.45 to9.37 ± 0.95% (L1 and L2 respectively).
3.2. Growth performances
In this study, the results of the tests indicate that the percentage of weight gain in fish fed with L1 probiotics was significantly higher than that of L2 probiotic treatments and the control group. Both probiotic treatments led to an improvement in the food conversion ratio and specific growth rate compared to the control group. Moreover, the protein efficiency ratio was also significantly improved in the probiotic treatments compared to the control group; however, this improvement was not seen to be significant between the two probiotic treatments. There was no significant effect of probiotic feeding on the condition factor. Additionally, no cases of mortality were observed during the test period in any of the groups. (Table 3). Bacterial pathogens are a significant contributor to disease damage in aquaculture, with vibriosis standing out as a major cause of extensive losses and damages in the marine aquaculture industry. This phenomenon is closely linked to the invasive capabilities of these bacteria, particularly under stressful conditions (Ina-Salwany et al.,2019). In this investigation, a notable observation was the 100% survival rate (SR) across all examined groups throughout the entire culture period, implying optimal maintenance conditions in each treatment. The study revealed that fish utilizing feed enriched with L1 (to a greater extent) and L2 (to a lesser extent, compared to another probiotic group) over a 45-day culture period outperformed the control group, which had no added probiotics. Additionally, these treated groups exhibited superior growth indices, particularly in terms of final weight factors. Our results are those of Reda et al., (2018) (Clarias gariepinus), Yang et al., (2019) (Carassius auratus), Gisbert et al., (2013) (Oncorhynchus mykiss), Yang et al., (2015) (Apostichopus japonicas) fed with host-associated probiotic compared to fish fed with commercial probiotics. Similar results have been reported in Asian seabass fed with Bacillus subtilis E20 and a mixture of Bacillus licheniformis and Bacillus subtilis (Lin et al., 2017; Adorian et al.,2019). In previous investigations, probiotic bacteria in aquaculture have been linked to enhanced growth by upregulating the insulin-like growth factor system. This is often associated with a concurrent decrease in myostatin gene expression and lower cortisol levels, contributing to improved overall health and growth in aquatic species (Carnevali et al.,2006; Hauville et al.,2016). It sounds like the researchers are proposing a hypothesis that suggests L1, in addition to its high probiotic potential, may enhance growth performance through the extracellular metabolites of protease, carbohydrase, and lipase. These metabolites could potentially break down complex dietary proteins, lipids, and carbohydrates into more usable forms, such as amino acids, peptides, fatty acids, and monosaccharides, ultimately improving digestibility and promoting growth.
Table 3
Growth performance of Asian seabass fed either regular feed or feed supplemented with probiotics for 45 Day
Parameters | Treatments |
Control | L1 | L2 |
IBW (g) | 75.55 ± 0.09a | 74.36 ± 1.2a | 74.91 ± 0.43a |
FBW (g) | 115.78 ± 2.05b | 123.67 ± 2a | 118.33 ± 1.5ab |
FTL (cm) | 19.06 ± 0.11b | 19.4 ± 0.2a | 18.9 ± 0.17b |
RGR (% / 45 day) | 53.19 ± 1.99b | 65.4 ± 2.5a | 57.95 ± 2.35b |
FCR (g / g) | 1.14 ± 0.12a | 1.002 ± 0.04b | 1.01 ± 0.04b |
SGR (% / d) | 1.41 ± 0.14b | 1.67 ± 0.05a | 1.52 ± 0.04ab |
PER (g gain / g pro) | 2.03 ± 0.16b | 2.32 ± 0.06a | 2.29 ± 0.09a |
FER (%) | 87.55 ± 6.2b | 99.77 ± 2.8a | 98.89 ± 4.07a |
Survival (%) | 100a | 100a | 100a |
The data represent the Mean ± SD of three tanks per treatment (n = 3). Values with various lowercase letters in each row indicate significant differences (p < 0.05). |
3.3. Digestive enzyme activities
Based on the results of the enzyme activity analysis presented in Fig. 1. On the 45th day of sampling, a significant difference in the activity of α-amylase, alkaline phosphatase, trypsin, and chymotrypsin was observed between group 2 and the other experimental groups. The study conducted on sea bass over 45 days revealed that the administration of both types of endogenous probiotics led to a significant increase in various enzymes, including trypsin, chymotrypsin, amylase, lipase, alkaline phosphatase, and protease. The observed enhancement in enzyme levels could be attributed to two possible mechanisms. Firstly, probiotic bacteria may secrete a diverse range of enzymes (exoenzymes). Alternatively, the activity of the fish's digestive enzymes (endoenzymes) might increase. Furthermore, certain probiotic bacteria can produce foreign enzymes like cellulase, amylase, protease, lipase, and phytase. These foreign enzymes, in addition to the host's digestive enzymes, contribute to the digestion of organic matter and enhance the overall activity of the fish's digestive enzymes (Del’Duca et al.,2013). Our observations are in agreement with those of Ghanei-Motlagh et al., (2021), Adorian et al., (2019), and Reda et al., (2018), who reported increased activities of protease, lipase, and amylase in L. calcarifer and C. gariepinus nourished by diets containing Bacillus spp. for 60 days, respectively. The improvement of the activities of digestive protease, lipase, and α-amylase can be partly attributed to the potential of probiotic strains for the production of extracellular enzymes including protease, lipase, and amylase as confirmed in our previous study (Mohammadian et al.,2018). Marlida et al., (2014) found that probiotic bacteria from the humpback grouper's (Chromileptes altivelis) digestive tract positively influenced the growth factors and digestive enzymes in their study. This aligns with the idea that probiotics can enhance the digestive system, leading to better digestion of food particles and ultimately promoting growth in the fish. The findings from studies employing probiotics have demonstrated an augmentation in the breakdown of dietary proteins, fats, and starch (Wang and Xu,2006). Hence, it is plausible that the probiotic bacteria investigated in the current research have enhanced the utilization efficiency of proteins, fats, and carbohydrates within the sea bass diet. Comparable instances exist where gastrointestinal bacteria have the ability to produce extracellular digestive enzymes (Suzer et al.,2011; Son et al.,2009). These instances substantiate the outcomes of the present experiment, indicating an elevation in digestive enzymes specifically α-amylase, trypsin, and lipase in rainbow trout. Lara-Flores et al., (2003) explored the impact of administering a blend of Enterococcus faecium and Lactobacillus acidophilus alongside the yeast Saccharomyces cerevisiae on Nile tilapia, focusing on growth rate and changes in alkaline phosphatase enzyme. Their findings indicated superior growth factors in treatments involving either bacteria or yeast compared to the control treatment. Regarding the alkaline phosphatase enzyme, the yeast treatment exhibited the highest activity level. The authors suggested that the heightened enzyme activity likely signifies enhanced development of brush membranes in fish enterocytes. It is plausible that the probiotic bacteria in the current study exerted a substantial influence on the development of sea bass enterocyte brush-like appendages, contributing to increased fish growth through various mechanisms. In the current investigation, a significant disparity emerged between the groups administered probiotics and the control group to the study's conclusion. Given the optimal rearing conditions and minimal stress experienced by all fish groups during the cultivation period, noteworthy augmentation in the activity of digestive enzymes α-amylase, lipase, trypsin, and chymotrypsin was observed in the probiotic treatments during the final sampling. This effect is likely attributed to the enhanced retention of probiotic bacteria within the digestive system of the fed fish. L2, and to a lesser extent L1, seemingly heightened digestive enzyme activity and secretion within the sea bass digestive system, thereby enhancing the capacity for food digestion and absorption. Consequently, this led to an increased specific growth rate. Notably, despite its comparatively lower impact on digestive enzymes, the L1 probiotic exhibited superior growth indicators among the experimental treatments. The outcomes suggest that the L2 treatment potentially established a dominant intestinal flora in sea bass, with a consequential elevation in lactic acid bacteria influencing growth indicators more profoundly than other bacteria. Another potential factor contributing to the superior performance of L1 may involve the influence of unidentified growth factors inherent in the growth process of sea bass within this specific probiotic treatment.
3.4. Innate immune parameters
The data related to the serum innate immune response are summarized in Fig. 4. The highest and lowest serum lysozyme, anti-trypsin, myeloperoxidase, Respiratory burst activity (NBT reduction), and bactericidal activity levels were in L2, L1, and Control, respectively (P < 0.05, Fig. 2). However, total immunoglobulin was significantly higher in the L2 compared to the L1 and control groups at day 45, respectively (Fig. 2) (P > 0.05). Fish in the L1 group had higher serum total complement than the other treatments. The study demonstrated a significant enhancement in immune responses, including bactericidal, antiprotease, and respiratory burst activities in fish subjected to probiotic-supplemented diets compared to those on basal diets. Specifically, serum bactericidal activity, indicative of the serum's ability to resist bacterial infections, showed elevated levels in probiotic-fed fish over 45 days. Probiotics were found to enhance the host's immune competence by activating various protective mechanisms, as elucidated by Lazado et al., (2010). Furthermore, serum respiratory burst, a phagocyte-mediated killing mechanism, exhibited a significant increase in probiotic-fed fish. Antiprotease activity, impeding pathogen infiltration with protein-degrading enzymes, was significantly elevated in all probiotic-treated groups. Consistent with these findings, prior research by Newaj-Fyzul et al., (2007) indicated that incorporating Bacillus subtilis AB1 in rainbow trout's diet substantially heightened antiprotease activity. Similarly, Kim et al., (2012) reported increased serum antiprotease activity in olive flounder following dietary supplementation with E. fascium. Myeloperoxidase (MPO) plays a pivotal role in pathogen eradication, and in this investigation, there was a notable augmentation in MPO activity across L1 and L2 groups at this study. Mohammadian et al., (2018) demonstrated that endogenous probiotic supplementation resulted in a significant increase in lysozyme and MPO activities. Lysozyme, possessing antibacterial properties, exhibited heightened activity in fish receiving L1 and L2 in their diet. Lactic acid bacteria have been shown to stimulate immunoglobulin synthesis (Mohammadian et al.,2022; Gopalakannan and Arul,2011 Mohammadian et al.,2021). Similar enhancements in immune responses have been documented in various fish species, including Nile tilapia, pirarucu, catla, red seabream, striped catfish, snakehead, Mrigal, and Japanese eel (Makled et al.,2019; do Vale Pereira et al.,2019; Bhatnagar and Saluja,2019; Dawood et al.,2019; Thy et al.,2017; Talpur et al.,2014; Bhatnagar and Lamba,2017; Lee et al.,2013)
3.5. Intestinal histopathology and histomorphology
In the present experiment, the height of the villus, average width of the villus, and epithelium thickness were evaluated across different treatment groups. Statistical analyses were performed to examine the potential differences in these parameters between the experimental groups at the end of the testing period. Concerning the height of the villus, width of the villus, number of goblet cells, thickness of the submucosal layer, and epithelium thickness by the end of the experiment, the L2 group exhibited a statistically higher height compared to other treatment groups. In the current investigation, pathological examinations revealed an absence of damage in the prepared sections. (Fig. 4) Groups receiving probiotics with food exhibited reduced damage in microvilli presence compared to the control group, along with decreased dead tissue in the lumen. Noteworthy is the absence of histological changes in intestinal epithelial tissue post probiotic exposure, with no indications of basement membrane connection rupture in intestinal cells. By the findings, Kristiansen et al., (2011) observed that the administration of food containing Carnobacterium divergens did not inflict damage on intestinal structure. Martinsen et al., (2011) demonstrate that C. maltaromaticum did not change and had adverse effects on the intestinal structure of cod. Probiotics can bind to the intestine and pass through it without damaging the structure of the intestinal wall, which indicates that their entry method is not destructive. Kristiansen et al., (2011) electron microscope observations indicate no visible difference between the groups in terms of the presence of cell debris in the lumen, increased amount of mucus, the number of bacteria-like particles in the lumen and between microvilli, disruption of microvilli order. and did not show a loss of integrity of strong joints. Normal and intact cell connections, along with a healthy zona adherence area, were observed in all groups, emphasizing the non-destructive impact of probiotics on intercellular junctions. This is crucial, as compromised junctions could serve as an entry point for potential pathogens. The study underscores the significance of intestinal morphology and histometry in assessing probiotic performance in fish, aligning with Ringo et al., (2007) emphasis on intestinal morphology in selecting lactic acid bacteria as fish probiotics. While Lactobacillus species from non-fish sources are generally considered beneficial for fish ( Bagheri et al.,2008) Salma et al., (2011) cautioned against the use of bacteria isolated from Iranian cheese due to severe destructive effects on Huso huso's intestine. Evaluating endogenous bacteria's impact on intestinal morphology is deemed crucial in probiotic selection. Macroscopic and microscopic images indicate that endogenous probiotics enhance epithelium development in the intestine, confirmed by changes in epithelium surface and the growth of intestinal villi under the probiotic influence. Assessing intestinal tissue, particularly microvilli diameter and size, serves as a valuable indicator of the intestine's physiological state and digestive system health. Increased villi diameter and height signify enhanced absorption levels, indirectly improving food ration utilization efficiency. This information is pivotal for understanding fish's nutritional needs. The measured lengths of intestinal villi (Fig. 3,4) align with macroscopic observations of the intestine in this study. Histological examinations reveal variations in villi length, with the L2 group exhibiting the most significant increase in villi length after the sampling period. The use of L1 also positively influences villi growth, potentially contributing to the microscopic structural integrity of the intestine and fostering expansion of intestinal epithelial tissue. Over the probiotic supplementation period, villi length increased across all groups, with a more pronounced increase compared to the control group in the treatments receiving a ration containing probiotics. Similar results were noted for other factors such as villus width, thickness of the submucosal layer, epithelium thickness, muscle layer thickness, submucosa thickness, and the number of goblet cells. Consistent with findings from Nakandakare et al., (2013) in Nile tilapia fish, as well as Burrells et al., (2001) in salmon, treatments incorporating probiotics demonstrated a significant difference in villi height in the middle intestine compared to the control group. The augmentation in villi width and the amplification of intestinal goblet cell numbers are conspicuously evident in both probiotic treatments upon course completion. Additionally, parameters such as the thickness of the submucosal layer, epithelium thickness, and muscle layer thickness manifest an increment in both treatments by day 45. Numerous anatomical factors influence absorption levels within the digestive system. Notably, elongated, narrower, and more regular villi, coupled with an increased villus count per area, signify heightened activation of intestinal villi (Heidarieh et al.,2012). In this investigation, the observation of elongated villi in the group administered with a combination of L2 (to a greater extent) and L1 (to a lesser extent) appears promising. However, further scrutiny is warranted, particularly concerning intestinal villi width and epithelium layer thickness, necessitating additional studies to elucidate effects on fish subjected to probiotics. Comparing the effects of the shape of the intestine with the activity of digestive enzymes shows that the most changes in the administration of bacteria occur at the end of the period. The two treatment groups of L1 and L2 had better results compared to the control group, and it should be noted that each of the probiotic bacteria may have different morphological effects in each of the fish species (Ferguson et al.,2010). The increase in the height of villi in treatments containing probiotics is probably due to the improvement of growth conditions and proliferation of lactic acid bacteria under the positive effects of probiotics. In the present study, an increase in the thickness of different tissue layers was observed in the groups treated with probiotics, especially L2 treatment at the end of the period. The anterior and middle intestines, which are the main places of digestion and absorption of food in the intestine, probably indicate the adaptive response of fish to increase the ability to absorb nutrients inside the intestine, and this increase in the diameter of the covering muscle layer is caused by the production of chain fatty acids. Zahran et al., (2014) showed the effect of probiotic use on increasing the length of intestinal villi in tilapia fish. On the other hand, there was no significant effect on the thickness of the muscle layer. Contrary to the present results, Enferadi et al. (2018) investigated the effect of Lactobacillus plantarum on the intestinal morphology of salmon and did not observe a significant change in the height of the villus, the thickness of the epithelium layer, the width of the villus and the thickness of the muscle layer. In this study, after feeding with food containing a probiotic supplement, the number of goblet cells increased at the end of the period in the L2 group. Although there are studies on the increase of goblet cells in the skin and digestive tract after being challenged with infectious diseases (Buchmann and Bresciani,1998), there are few studies on increasing the density of mucous cells in fish. Ramos et al., (2015) investigated the effect of commercial probiotics including Bacillus, Pedicoccus, Enterococcus, and Lactobacillus in different amounts on the intestine of rainbow trout. They found an increase in the area of the anterior intestine and the number of goblet cells in the group containing 3 grams of probiotics per kilogram of food ration and also an increase in hair length in the group containing 1.5 grams of probiotics per kilogram of food ration. Goblet cells in the digestive tract act as the first sensitive layer of fish immune defense factors by forming a viscous watery coating on the surface of the mucus and providing protection against the damage caused (Ringø et al.,2003).
3.6. Intestinal microflora
It has been evidenced that probiotics, particularly those derived from the host, can modulate the gut microbiota. However, functional probiotics require to be colonized in the host intestinal tract before exerting their beneficial effects. In this study, no significant alteration in populations of Bacillus spp., total cultivable bacterial counts, and Vibrio spp. was observed between the treatments (p > 0.05). Moreover, LAB was significantly higher in the probiotic groups than in the other groups (p < 0.05) (Table 4). This could be associated with the high survival of these strains within the gastrointestinal tract from which they isolated and their appropriate abilities to adhere to mucus as described in our previous study (Ghanei-Motlagh et al.,2021).
Table 4
The intestinal populations of total cultivable bacteria, Bacillus spp. and Vibrio spp. in Asian seabass fed or un-fed with probiotics at different time intervals
Parameters | Treatments | Initial (day 0) | Day 45 |
Total aerobic heterotrophic bacteria (Log CFU/ml) | Control | 25.7 ± 4.05a,A | 27.13 ± 3.5a,A |
L1 | 27.6 ± 6.75a,A | 26 ± 3.86a,A |
L2 | 25.1 ± 6.006a,A | 26.7 ± 4.57a,A |
Lactic Acid Bacteria (Log CFU/ml) | Control | ND | ND |
L1 | ND | 40.3 ± 14.7a,A |
L2 | ND | 45.3 ± 20.31a,A |
Bacillus spp. (Log CFU/ml) | Control | 1.5 ± 0.57a,A | 1.16 ± 0.81a,A |
L1 | 1.4 ± 0.54a,A | 1.66 ± 0.43a,A |
L2 | 1.16 ± 0.4a,A | 1.28 ± 0.46a,A |
Vibrio spp. (Log CFU/ml) | Control | 15.03 ± 2.53a,A | 14.8 ± 2.58a,A |
L1 | 13.4 ± 2.45a,A | 12.5 ± 2.83a,A |
L2 | 14.4 ± 2.4a,A | 12.8 ± 2.57a,A |
ND: No detected. For each parameter, values (Mean ± SD, n = 9) bearing different lowercase letters or different uppercase letters represent significant differences within each row or each column, respectively (p < 0.05). |
3.7. Expression gens
On the 45th day of sampling, fish fed with the L2 probiotics had higher GMCFC and IL-10 in the gut than those fed the L1 probiotic and control group (Fig. 5). Fish in L1 and L2 groups had higher gut EGF gene expression values than the control group. The highest TGFβ gene expression value in the gut was in L2, meanwhile, the lowest values were in the control group (P < 0.05). Results of the present study showed that all probiotics, applied here, can up-regulate the immune-related gene expression of the L. calcarifer intestine. probiotic bacteria can influence gene expression, including genes related to the immune system and growth factors. Some studies in aquatic animals, including fish, have explored the impact of probiotics on genes such as interleukin-10 (IL-10), granulocyte-macrophage colony-forming cells (GMCFC), epidermal growth factor, and Transforming Growth Factor Beta (TGF-β). In the present study, feeding with L1 and L2 probiotics induced higher transcription levels of EGF, TGFβ, GMCFC, and IL-10 genes in the gut, which may correlate with better immune and hematological parameters in these groups. The increase in IL-10 mRNA expression may have led to the enhanced expression of pro-inflammatory cytokines such as GMCFC in the L. calcarifer fed L1 probiotic to balance the immune responses for better immunocompetence. The mechanism of action of probiotics in the expression of interleukin 10 genes is related to their effect on internal signaling pathways in cells. These processes usually involve molecular interactions between probiotics and the surface of host cells. One possible mechanism is that probiotics activate signaling communications by combining with various cells of the gastrointestinal tract. This interaction can lead to the activation of specific signaling pathways, which ultimately leads to increased expression of TGF and interleukin 10 genes. Additionally, probiotics may help improve the gut microbiome. A better microbiome balance can lead to different production of chemical compounds and molecular signals that regulate gene expression. Although the exact mechanism is not yet fully understood, these effects may improve cellular activity and balance the immune system. In this context, Siddik et al., (2022); Choi et al., (2022); Panigrahi et al., (2011) and Abarike et al., (2020) reported administration of a diet with probiotic significantly increased IL-10 gene expression in the head kidney of olive flounder and Nile tilapia. In the present study, the growth performance of sea bass fed with L1 probiotic was proportional to TGFβ gene expression. Probiotics may modulate the immune response and contribute to the regulation of growth factors, potentially promoting a more balanced and beneficial gene expression profile. However, the effectiveness can vary depending on factors like the specific probiotic strains used, the host species, and environmental conditions.
3.8. Relative survival rate after challenge with V. alginolytichus
Seven days after the injection challenge of fish with V. alginolyticus bacteria, the percentage of survival in unchallenged control groups (100%), challenged control (64.95%), L1 treatment (76.2%), treatment L2 (80.95%) was obtained (Table 5). Statistically, the relative survival percentage of fish fed with both Lactobacillus plantarum bacteria was significantly higher than the control group (p < 0.05). Probiotics increase survival and natural resistance against unfavorable factors. In the current investigation, a notable enhancement in the survival rates of sea bass fish exposed to V. alginolyticus bacterial challenge was evident in probiotic interventions, notably with L2. This finding aligns with prior research conducted on European migratory eel and Indian carp, where increased survival rates were observed against A. hydrophila infection after dietary supplementation with Bacillus probiotics (Das et al.,2011). The heightened resistance observed in probiotic treatments is likely attributable to an augmentation in non-specific immune responses influenced by the administration of endogenous probiotics. The augmentation in non-specific immune responses in probiotic-treated groups is evident from the results presented in Table 5. Notably, the introduction of L2 and L1 into the diet of sea bass fish beyond day 45, followed by intraperitoneal injection of V. alginolyticus bacteria, resulted in enhanced resistance in both probiotic treatments compared to the control group. However, the reduction in losses observed in the group receiving L2 was particularly noteworthy (p < 0.05). Clinical signs and bacteriological examination demonstrated the infection of V. alginolytichus in the mortalities. Son et al. [83] investigated the effect of oral administration of Lactobacillus plantarum on common grouper fish for four weeks and then challenged with Streptococcus species. Probiotic treatment showed 108 CFU/gr compared to the control group. Balcazar et al.,( 2006) investigated the ability of three species of lactic acid bacteria (Lactobacillus lactis, Lactobacillus plantarum, and Lactobacillus fermentum) in inhibiting the adhesion of several fish pathogens (Aeromonas hydrophila, Aeromonas salmonicida, Yersinia ruckeri, and Vibrio anguillarum), and the results showed that Lactobacillus plantarum reduces the adhesion of A. hydrophila and A. salmonicida and Yersinia ruckeri, but it had no effect on V. anguillarum. Increasing the level of non-specific immune defense, reducing the penetration power of pathogenic agents, reducing access to essential nutritional factors, and producing antibacterial substances can be considered among the reasons for increasing resistance to the challenge of pathogenic agents.
Table 5
Relative Survival percentage of fish of different treatments after challenge with Vibrio alginolyticus
Parameters | Treatments |
Control | L1 | L2 |
Survival (%) | 64.95b | 76.2a | 80.95a |
The data represent the Mean ± SD of three tanks per treatment. Values with various lowercase letters in each row indicate significant differences (p < 0.05). |