Effects of dietary administration of mannan oligosaccharides on growth performance, intestinal Vibrio, non-specific immunity and tolerance to low salinity in golden pompano, Trachinotus ovatus (Carangidae)

doi:10.21203/rs.3.rs-3102340/v1

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Effects of dietary administration of mannan oligosaccharides on growth performance, intestinal Vibrio, non-specific immunity and tolerance to low salinity in golden pompano, Trachinotus ovatus (Carangidae)

https://doi.org/10.21203/rs.3.rs-3102340/v1

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The effects of dietary supplementation with mannan oligosaccharide (MOS) on growth performance, haematological parameters, intestinal Vibrio, immune response, and stress tolerance to low salinity were evaluated in pompano (Trachinotus ovatus). Pompano (9.24 ± 0.15 g) were fed with 0 (control), 0.1, 0.2, 0.4, 0.6%, or 0.8% dietary mannan oligosaccharides for 8 weeks. The results showed that pompano fed 0.1–0.4% MOS had significantly improved (P < 0.039) specific growth rates. Dietary mannan oligosaccharides did not impact the survival rates of pompano (P = 0.192). The WBC was significantly higher in fish fed diets containing 0.1–0.6% MOS. Dietary MOS supplementation increased lymphocytes in pompano (P ≤ 0.042), while it reduced monocytes and basophils (P ≤ 0.019), but MOS did not show any effects on neutrophils and eosinophils (P ≥ 0.064). Intestinal Vibrio counts were reduced in pompano fed diets containing 0.1 to 0.4% MOS supplementation (P ≤ 0.035). The phagocytic ratio (PR) significantly increased in pompano fed diets containing 0.1 to 0.6% MOS (P ≤ 0.015), while the phagocytic index (PIs) and lysozyme in fish fed 0.1 to 0.4% MOS were significantly higher than fish fed control (P ≤ 0.035). There were significant positive correlations between MOS levels and fish growth at days 14, 28, 42, and 56 (P ≤ 0.049). In addition, dietary mannan oligosaccharide levels were highly correlated with blood parameters, intestinal Vibrio, and the immune index. Optimal dietary mannan oligosaccharides requirements for maximal growth of pompano were predicted to be 0.440% at day 21 (R² = 0.861), 0.385% at day 28 (R² = 0.877), 0.371% at day 42 (R² = 0.891) and 0.365% at day 56 (R² = 0.750), showing a reduction in MOS requirement when fish size increased. Furthermore, optimal MOS concentrations for maximal immunity based on lysozyme were predicted to be 0.431% (R² = 0.817) at day 56. Also, fish fed 0.1–0.4% MOS showed better resistance against low salinity stress (P ≤ 0.014). In conclusion, the 0.1–0.4% MOS supplementation could effectively enhance the growth, intestinal microbes, immune system, and tolerance capacity to the low salinity of the cultured pompano, T.ovatus.

Biological sciences/Immunology

Biological sciences/Physiology

Biological sciences/Zoology

Mannan oligosaccharide

pompano

nutrition

growth

immune system

stress resistance

Aquaculture has played a crucial role in meeting the increasing demand for animal protein by humans over the past decades. In 2020, total global aquaculture production reached 122.6 million metric tonnes, with a value of USD 281.5 billion. This production accounted for almost 56% of the world's seafood in 2020 and is expected to rise to 59% in 2030¹.

Pompano, Trachinotus ovatus (Carangidae), is a very important commercial species and is distributed in China, Japan, Australia, Viet Nam, and other countries². Pompano is a highly valued aquaculture species due to its rapid growth rate, high quality meat, and delicious flesh. Aquaculture of this species has recently grown quickly in Asia, and it is now one of the most prevalent marine species in Viet Nam^3,4.

In fact, the spread of intensive aquaculture, which has resulted in the release of more harmful organic and inorganic wastes, has caused outbreaks of infectious diseases, damage to the natural environment, particularly eutrophication, and the loss of biodiversity⁵. Also, antibiotics are commonly used to treat bacterial infections in aquaculture⁶. The use of antibiotics in aquaculture has; however, been related to a number of major issues, including the presence of residual antibiotics in commercially cultured products, the appearance of drug-resistant bacteria, concerns about food safety, and allergic and toxic reactions in humans⁷. Antibiotic resistance is one of the humanity's greatest challenges⁸. Research estimates that at least 10 million cases will be at risk annually by the year 2050 due to the rise of infections caused by antimicrobial-resistant strains⁹. There has also been an increase in evidence about antibiotic resistance in aquaculture^10–13. Also, more and more research has proven the risk of antibiotics for the environment and human health¹⁴, therefore, more research is needed to find antibiotic replacement¹⁵. Research has approved that a potential solution is to apply dietary supplements like pre- and pro-biotics^16,17.

Prebiotics are non-digestible substrates that nourish beneficial microorganisms, including probiotic bacteria and resident microbes¹⁸. Some of the effects of prebiotics include protecting the host against pathogens, modulation of the immune system, mineral intake, bowel function, metabolic effects, and safety¹⁹. Mannan oligosaccharide (MOS) is a kind of prebiotic that helps the host by selectively stimulating the growth and/or activating the metabolism of beneficial bacteria in the gut²⁰. In contrast to antibiotics, functional oligosaccharides are able to reach the colon without being inactivated and are free from drug residues and bacterial resistance²¹. Consequently, functional oligosaccharides are considered environmentally friendly substances for enhancing animal growth and resistance to infection²².

Other studies revealed that MOS can increase the health of fish by boosting their immunity, stress tolerance, and resistance to infections²³. The reduced rate of disease in animals fed a diet supplemented with MOS is likely due to their enhanced gastrointestinal health and cell membrane integrity^24–26. Also, studies have shown that prebiotics can help fish by raising their overall resilience to infections caused by bacteria, improving their non-specific immune responses^27–29, and enhancing both the health of the digestive tract and its performance by modifying the ultrastructure of the intestinal mucosa^30,31. Conversely, dietary MOS limited growth in other species^32–34. According to Xue, et al.³⁵, the effect of MOS on fish growth may be dependent on the species and its developmental stage.

Although the efficiency of MOS on other species has been reported, the impact of MOS on juvenile pompano is still not entirely understood. To the authors’ knowledge, this is the first study to examine the effects of dietary MOS on pompano juveniles. Also, very few studies have examined the optimal levels of MOS requirements for growth and immunity in fish. This is crucial because providing the fish with an improper dose of feed additives could cause negative effects^36–38. Thus, the purpose of this study was to determine the impact of mannan oligosaccharide on growth performance, haematological parameters, intestinal Vibrio, immunity, MOS requirement, and salinity stress tolerance of juvenile pompano fish, Trachinotus ovatus. Due to the importance of dietary mannan oligosaccharides and pompano fish, this topic is worthy of investigation.

Ethical statement

This study was conducted under the ethical principles and guidelines according to the animal use protocol approved by Institute of Oceanography, Vietnam Academy of Science and Technology (VAST). In addition, the search carried out in accordance with ARRIVE's instructions³⁹.

Experimental fish and culture systems

Pompano, Trachinotus ovatus (9.24 g ± 0.15, mean weight ± SD) obtained from a local hatchery. Healthy fish were selected and transported to a laboratory at the Institute of Oceanography, Nha Trang and acclimated for 3 weeks in a 2 m³ tank prior to the experiment. During acclimation, fish were given a commercial diet at a rate of 5% body weight per day. An independent recirculation system was installed in each of the experimental tanks with a volume of 300 L (50 × 80 × 80 centimetres), for a total of 36 tanks. Water chemistry was measured every second day, while temperature was measured daily. The mean water temperature was 28.5 °C; salinity: 33 – 34 ppt; dissolved oxygen: 6.1 ± 0.5 mg L^-1; pH: 8.1 - 8.3, NH₄⁺: 0.05 mg L^-1, NO₂^-: < 0.01 mg L^-1. Throughout the experiment, fish were maintained in a natural photoperiod environment.

Experimental diets and feeding

Mannan oligosaccharide, MOS (Active-Mos^® provided by Biorigin), was used to add to a base diet (Control), as shown on Table 1. This diet formulation has been used previously and has demonstrated favourable growth and survival for pompano³⁸. In order to prepare the diets, the ingredients listed in Table 1 were minced and extruded twice to create noodles with a diameter of 2 mm and supplemented with different levels of Active-MOS^®. Six diet treatments were used: a control basal diet without MOS (MOS0.0) and five concentrations of Active-MOS^®, including 0.10% (MOS0.1), 0.20% (MOS0.2), 0.4% (MOS0.4), 0.6% (MOS0.6), and 0.8% (MOS0.8) were supplemented to the basal diet, and the filler was corn starch. During the feeding trial, the fish were fed twice a day (7:00 and 17:00). Pompanos were provided their feed in a slight excess of their satiety. The amount of uneaten food was monitored daily, and the feed was adjusted daily to attain satiety. Fish condition was monitored in the morning, and then uneaten pellets and waste were removed before the morning feed.

Table 1. The basal diet composition (crude protein 48.6%, crude lipid 6.7%, gross energy 19.78 MJ.kg^-1, dry matter 92.1% and ash 11.31%).

Ingredients	Percentage
Fish meal (62% - Vietnam)	44.60
Gluten (wheat)	21.10
Soybean	10.40
Fish oil	3.60
Binder	1.10
Mineral premix	1.50
Vitamin premix	1.50
Corn starch	16.20
TOTAL	100

Experimental design

The design of the experiment was to compare pompano growth, mortality, haematological parameters, immune response, intestinal Vibrio and the low salinity stress tolerance of pompano when given a base diet supplemented with various MOS concentrations. There were six replicate tanks for each dietary treatment, and 20 fish had been stocked randomly in each tank. After the acclimation period, fish were weighed, and treatment diets were fed since the experiment started (day 0).

Sampling and data collection

At initial (day 0) and then every two weeks, the weight and length of all pompano fish in each tank were measured. Fish were starved for 24 hours before weighing or sampling. The fish was weighed using a balance with an accuracy of 0.01 g. At day 56 (end), 12 fish in each treatment were randomly sampled and sacrificed for haemolymph samples, and tissues (head-kidney, liver, and intestine). Prior to dissecting, the fish were anesthetized with 100 mg L^-1 tricaine methanesulfonate (MS-222, Sigma, USA) for 2 - 5 minutes. The pompano was washed with 2% sterile saline water prior to dissection.

Heamatological assays

A hemocytometer was used to count white blood cells (WBC) and red blood cells (RBC)⁴⁰. In order to obtain differential leukocyte counts, including monocytes, lymphocytes, neutrophils, and eosinophils, fish blood was smeared, then air-dried before fixing in methanol, and then stained with Diff-Quick^®. Leucocytes were classified based on morphology and counted at × 1000 magnification using a microscope.

Gut bacterial content and culture microbiology

Fish were sampled and cleaned in 2% sterilised saline water. Midgut was taken and hand-ground using a ceramic mortar and pestle. After that, the ground tissue was mixed with 0.85% saltwater (1:10), and an examination for the presence of Vibrio spp. was carried out using Thiosulfate Citrate Bile Salt Sucrose (TCBS Agar) as a conventional medium recommended to isolate pathogenic Vibrio on fish following the APHA standard method⁴¹. After incubation at 37 ºC for 24 h, the total colony-forming units (cfu) were counted and then calculated per unit of fish tissue (g).

Head-kidney macrophage isolation

Macrophage isolation was done using a technique developed by Secombes ⁴². The head-kidneys of 12 fish per treatment were sampled. The collected head-kidney was then cut into small pieces, and 10 mL of RPMI-1640 culture medium was added, subsequently supplemented with 100 IU mL^-1 streptomycin, 100 IU mL^-1 penicillin, 10 IU mL^-1 heparin and 2% neonatal calf serum (FCS). To obtain leukocytes (HKLs), the head-kidney pieces were ground through a 100-µm mesh. Then 5 mL of 51% Percoll, 5 mL of 34% Percoll were added to a 15-mL Falcon tube to make a 34%/51% Percoll solution. Next, the head-kidney cell suspension was mixed with 34%/51% Percoll and centrifuged at 400 g for 35 minutes at 4 °C. Subsequently, the leukocyte cell interphase was collected and centrifuged at 200 g for 10 minutes after being washed with phosphate-buffered saline (PBS) (2:1) for 10 minutes. Afterwards, the cell pellet was resuspended in 5 mL of L-15 medium. The viable cell was stained with 1% trypan blue and counted using a haemocytometer.

Non-specific immune response of pompano fed different levels of MOS

Phagocyte activity in pompano fed different concentrations of MOS

The leukocyte phagocytosis index assay was conducted following the method described by Siwicki, et al. ⁴³. Leukocytes were adjusted to a density of 1 × 10⁸ cells mL^-1 by using 0.1 M phosphate citrate buffer (PBS) at pH 5.8 before incubating for 5 min. Then, zymosan (Sigma) at a concentration of 1×10⁷ cells mL^-1 was added to the mixture, followed by incubation at a temperature of 22 °C for 2 h. The cells were then stained with 1 mL of Congo red and incubated again for 5 minutes, after which they were washed three times with PBS. Then, about 50 µL of the mixture were taken and smeared on a microscope slide, and the slides were left to dry for 2 hours at room temperature. On each slide, approximately one hundred phagocytes were counted using a microscope with a 1000 × magnification. To determine phagocytic activity, both the phagocytic index (PI) and the phagocyte ratio (PR, %) were calculated as follows: PR = no. of phagocyte cells with engulfed zymosan/no. of phagocyte cells; PI = no. of engulfed zymosan/no. of phagocyte cells⁴⁴.

Lysozyme activity in pompano fed different MOS concentrations.

For lysozyme analysis, the collected blood of pompano was left at room temperature for 30 minutes to clot and then centrifuged at 1500 g for 15 minutes at 4 °C to get serum. Afterwards, 25 µL of serum (three replicates) and 25 µL of hen egg white lysozyme (Sigma) with a serial dilution of 0 to 20 L mL1 in PBS were added to a 96-well microplate. The blank wells, which served as controls, were filled with the same amount of PBS alone. After adding 175 µL of Micrococcus lysodeikticus suspension (0.75 mg mL^-1 in PBS) to each well, the microplate was thoroughly mixed. Afterwards, a microplate reader was used to measure the turbidity at 440 nm and 20 degrees Celsius after 15 and 30 minutes²⁸^,⁴⁵.

Predicting MOS requirements for maximal growth and maximal immune response in pompano

Using Pearson’s correlation (r) to evaluate the relationships between the MOS concentrations in the diets, growth rate of fish, intestinal microbiota, and haematological parameters. The present study indicated that specific growth rate (SGR) and serum lysozyme activity in pompano had the strongest significant correlations with dietary MOS levels, so they were used to estimate the MOS level necessary to gain maximum growth and health in pompano. MOS level requirements were analysed using six regression models in Microsoft Excel 2007, including exponential, linear, logarithmic, quadratic polynomial, power and moving average. Input parameters in each case included the concentrations of MOS as independent variables and growth rate data and serum lysozyme activity as dependent variables. The model chosen for the prediction analysis was based on a higher value of the correlation coefficient (R²). The results indicated that the quadratic polynomial model had a higher R² value; thus, this model was used for the analysis of growth response as well as immune response. The quadratic polynomial model is shown as the following equation: y = ax² + bx + c; where y is the specific growth rate over time or serum lysozyme of pompano at day 56; x is dietary MOS concentrations, a, b are coefficients, and c is a constant of the equation.

Salinity stress test

At the end of the feeding trial (Day 56), fish were exposed to low salinity stress. Eighteen fish from each diet treatment were randomly captured and moved into tanks containing water with a salinity of 1 ppt. The low salinity level and the exposed time were based on prior research on pompano³⁸. Deaths were recorded every 30 minutes for a total of four hours. When the fish's gill operculum stopped moving, the fish was recorded as dead.

Data calculation and statistical analysis

Specific growth rate (SGR % d^-1), survival rate, and relative percent survival (RPS) was calculated as follows: SGR = 100 × [Ln(W_t) – Ln(W_o)]/days; S = 100 × (N_t/N_o); RPS = 100 × (1 - M_d/M_o)⁴⁶, where W_t and W_o were the weight of the fish at time t; and at the commencement; S: Survival rate (%), N_t and N_o were the number of the fish at time t and at the commencement, M_d and M_o were the percentage mortalities of pompano fish on MOS diets and control diet treatments, respectively.

Data are displayed as treatment means and standard error (S.E.). ANOVA F-tests were used to compare data among treatments. Before the data were analysed, they were checked to see if they were normal distribution, and if not, they were then transformed. If the results of the F-test were significant, then differences between treatment means were compared using the least significant difference (LSD) method. To analyse survival data, the non-parametric Kruskal-Wallis ANOVA test was used. Pearson correlation (r) was used to examine the correlations between dietary MOS concentrations, growth performance, haematological index, intestinal microbes, and immune responses. When comparing data, if P < 0.05, then the difference is significant. SPSS 18 (IBM, Chicago, IL) was used for all statistical analyses.

Growth performance of pompano fed different levels of MOS

Fish were similar in weight for all treatments at the beginning of the trial without significant differences (ANOVA, P = 0.512). By day 56, the weight of pompano among the treatments ranged from 10.84 to 15.69 g. At the end of the experiment (day 56), the group of fish given 0.2% MOS showed the largest weight increment (4.83 times higher compared to the weight of fish fed control), followed by fish fed diets adding 0.1% and 0.4% MOS (4.61 and 4.41 times higher than the weight of fish fed control, respectively). The lowest weight gain was observed in the group fed the control diet (3.44 times) (Fig. 1).

Significant variations in the SGR of pompano were seen between diet treatments at days 14, 28, 42 and 56 (Fig. 2). After 14 days, SGRs of pompanos fed diets adding 0.1% to 0.6% MOS were significantly higher than the SGR of pompanos fed the control diet (P ≤ 0.030). However, the SGR of pompano fed diets with 0.8% MOS did not significantly differ from the SGRs of fish fed control or other MOS levels (P ≥ 0.062). At days 28 and 42, there were significant differences among SGRs of pompanos fed a diet containing 0.1% to 0.4% MOS (P ≤ 0.032) compared to SGR of pompano fish fed the control (MOS0.0). Nevertheless, there were no significant distinctions between the SGRs of pompanos fed basal, MOS0.6, and MOS0.8 (P ≥ 0.064). Also, no statistical significance was found among the SGR of groups of pompanos fed diets containing 0.6% and 0.8% MOS compared to the SGR of groups of pompanos fed diets containing 0.1 - 0.4% MOS (P ≥ 0.071). After 52 days of feeding (end), SGRs of pompanos fed diets with 0.1 - 0.2% MOS were significantly higher than SGR of pompanos fed the basal diet (P ≤ 0.020), while feeding 0.4 - 0.8% MOS inclusion in the diet did not significantly impact SGRs of pompanos (P ≥ 0.124). SGR of pompano fed diets added any levels of MOS were not significantly different from each other (P ≥ 0.412). (Fig. 2).

Survival rate and relative survival rate

At the end of the investigation (day 56), survival rates of pompano fish fed various MOS diets ranged between 92% and 98% (Fig. 3). The fish that were given diets with 0.1 - 0.6% MOS had the greatest survival rate, which ranged from 97.5% to 98%. The survival rates of pompano fed at all levels of MOS were not significantly different (P = 0.192). Relative percentage survival (RPS) was highest in the group of pompanos fed diets that added 0.2% MOS, followed by the RPS of pompanos fed diets that added 0.1% and 0.4% MOS (both RPS were 72.5%), and pompanos fed diets that added 0.6% and 0.8% MOS had RPS of 68.75% and 50%, respectively (Fig. 3).

Haematological parameters of pompano fed dietary MOS supplementation

The haematological parameters showed statistically significant variations among the treatments. Red blood cells (RBC) of pompano fed all levels of MOS were higher than those of the fish fed the control diet, but only the fish fed diets MOS0.1, MOS0.2 and MOS0.4 had higher RBC than that in fish fed the control (P ≤ 0.039), while RBC in fish fed diets MOS0.6 and MOS 0.8 from RBC in fish fed control diet (P ≥ 0.076); and there was no significant difference in RBC among the fish fed any levels of MOS-supplemented diets (P ≥ 0.104). Pompano fed 0.1 to 0.6% MOS had a higher WBC compared to the WBC of pompano fed control (P < 0.041), but when adding higher levels of MOS (0.6 and 0.8%) the WBC did not differ from the WBC of control (P ≥ 0.073) (Table 2).

When compared to fish given the control diet, pompano fed a diet containing any levels of MOS had higher proportions of lymphocytes (P ≤ 0.042), while monocytes and basophils were significantly decreased in fish fed MOS inclusion in the diet (P ≤ 0.019). In addition, dietary MOS showed no significant influence on the quantity of neutrophils and eosinophils (P ≥ 0.064) (Table 2).

Table 2. Haematological parameters of pompano fish (day 56 MOS0.0: control; MOS0.1: 0.1%; MOS0.2: 0.2%; MOS0.4: 0.4%; MOS0.6: 0.6%; MOS0.8: 0.8% mannan oligosaccharide added to the diets. The data is presented with the mean and standard error of the mean. The use of distinct letters to indicate significant differences between treatments.

	Diets
	MOS0.0	MOS0.1	MOS0.2	MOS0.4	MOS0.6	MOS0.8
RBC (× 10⁶ mm^-3)	3.56 ± 0.24^a	4.61 ± 0.28^b	4.91 ± 0.23^b	4.75 ± 0.43^b	4.07 ± 0.41^ab	3.77 ± 0.41^ab
WBC (× 10⁵ mm^-3)	0.50 ± 0.06^a	0.87 ± 0.09^b	0.75 ± 0.07^b	0.73 ± 0.07^b	0.68 ± 0.06^b	0.59 ± 0.08^ab
Monocyte (%)	33.76 ± 3.80^a	26.85 ± 2.0^b	20.68 ± 1.88^b	23.09 ± 3.73^b	25.75 ± 4.25^b	22.75 ± 4.25^b
Lymphocyte (%)	48.72 ± 3.76^a	59.09 ± 2.38^b	66.20 ± 2.30^b	61.90 ± 4.61^b	60.94 ±4.30^b	62.94 ±2.20^b
Neutrophil (%)	13.70 ± 2.54	11.16 ± 2.92	10.44 ± 1.53	12.34 ± 2.88	10.56 ± 2.48	11.56 ± 2.32
Eosinophil (%)	1.07 ± 0.42	1.05 ± 0.34	1.02 ± 0.35	0.94 ± 0.24	1.11 ± 0.21	1.51 ± 0.12
Basophil (%)	2.76 ± 0.48^a	1.84 ± 0.35^b	1.65 ± 0.44^b	1.72 ± 0.51^b	1.64 ± 0.51^b	1.24 ± 0.31^b

Intestinal microorganisms of pompano fed dietary MOS supplementation

The Vibrio counts in the gut of pompano fish fed a 0.1 to 0.4% MOS diet were significantly lower than Vibrio in pompano fed the control diet (P ≤ 0.035), while pompano fed diets containing 0.6-0.8% MOS did not significantly influence Vibrio in pompano. Vibrio counts, however, did not significantly differ among pompano fed different levels of the MOS diet (P = 0.165) (Fig. 4).

Immune response of pompano fish fed different levels of MOS

Phagocytic ratio (PR) and phagocytic index (PI) of fish fed MOS diets

At the end of the feeding trial (day 56), the pompano fed the diets added with 0.1 to 0.6% MOS had significantly greater phagocytic ratios (PR) compared to the PR of the pompano fed the control diet (P ≤ 0.015). However, the PR value of the pompano fed diet added with 0.8% MOS did not significantly differ from the PR of the pompano fed control diet (P = 0.210). Also, there were no statistically significant differences in the PR values of pompano fed any levels of MOS inclusion in the diets (P ≥ 0.162) (Fig. 5).

There were significant increases in the phagocytic index (PI) of pompano fish fed MOS diets. The PI values in pompano fed 0.1 to 0.4% MOS increased significantly compared to the PI value in fish fed a control diet (MOS0.0) (P ≤ 0.035), while no significant differences were observed in the PI of pompano fed a diet supplemented with 0.6% and 0.8% MOS compared to PI in fish fed the control (P = 0.068). Also, the PR values were not significantly different between fish fed any levels of MOS added (P ≥ 0.267) (Fig. 5).

Serum lysozyme of pompano fed different MOS concentrations

The lysozyme activity of pompano increased significantly (P ≤ 0.001) after 56 days of feeding them diets with MOS supplementation. The highest lysozyme levels were in the pompano fed diet containing 0.2% and 0.4% MOS (1.25 times and 1.24 times higher than the lysozyme of the pompano fed control), followed by the pompano fed diets containing 0.1 and 0.6% MOS, which were both 1.18 times greater than the lysozyme of the pompano fed control diet. The lowest lysozyme activity was in the pompano fed diet, which added 0.8% MOS (1.17 times higher than the lysozyme of the pompano fed control diet) (Fig. 6). The lysozyme activity in fish fed 0.1 to 0.6% MOS was considerably greater than the lysozyme activity in fish fed control (P ≤ 0.005). However, no significant difference was observed in lysozyme activities of pompano fed diet added 0.8% MOS compared to lysozyme of pompano fed control (P = 0.061). Also, no significance was found in lysozyme activity among pompano fed any levels of MOS after 56 days (P = 0.352) (Fig. 6).

Relationship between MOS concentrations and the growth of pompano over time

Results of Pearson correlation analyses showed significantly positive relationships between MOS levels and the fish's growth rate at days 14, 28, 42 and 56 (P ≤ 0.049). Among growth performance parameters, MOS levels had the strongest correlations with specific growth rate (SGR) across all time points (Table 3). On day 14, it was found that the concentrations of MOS in the diet had a significantly positive correlation with SGR (r = 0.471, P = 0.004). The correlation values (r) between SGR and MOS levels were 0.494 and 0.483 at days 28, and 42, respectively, with the significance level (P) less than 0.001. On day 56, there was a slight downward trend in this correlation, but it was still highly significant (r = 0.452, P = 0.008). At all-time points, the SGR had the highest values of correlation (r) (Table 3); thus, the SGR was used as an independent variable in the regression model to predict the optimal MOS requirement for the maximal growth rate of pompano. (Table 3).

Table 3. Pearson correlation (r) among MOS concentrations in the diet and the growth rate of pompano over time.

	Day 14		Day 28		Day 42		Day 56
	r	*Sig.*	r	*Sig.*	r	*Sig.*	r	*Sig.*
Mean weight (g)	0.454^**	0.005	0.442^**	0.007	0.435^**	0.008	0.335^*	0.049
Weight gain (g d^-1)	0.441^**	0.007	0.442^**	0.011	0.447_**	0.006	0.435^**	0.010
SGR (% d^-1)	0.471^**	0.004	0.494^**	0.000	0.483_**	0.001	0.452^**	0.008

* Correlation is significant at the 0.05 level (2-tailed).

** Correlation is significant at the 0.01 level (2-tailed).

Relationship between MOS concentrations and haematological index, intestinal microbes, and non-specific immunity (day 56)

Table 4. Pearson correlation (r) between immune response, haematological parameters, intestinal microbiota and MOS levels (day 56). Mono: Monocyte, Lym: Lymphocyte, Neu: Neutrophil Eos: Eosinophil, Bas: Basophil, TAC: total aerobic bacteria, PR: Phagocytic index, PR: Phagocytic ratio, Lys: Lysozyme.

	RBC	WBC	Mon.	Lym.	Neu.	Eos.
r	0.386^**	0.362^*	-0.383^*	0.481^*	-0.104	0.048
Sig.	0.033	0.035	0.000	0.033	0.531	0.695
	Baso	*Vibrio*	PR	PI	Lys.
r	-0.434^**	-0.330^*	0.341^*	0.421^**	0.559^**
Sig.	0.015	0.045	0.039	0.008	0.000

* Correlation is significant at the 0.05 level (2-tailed).

** Correlation is significant at the 0.01 level (2-tailed).

Pearson correlation analyses at day 56 showed that the concentrations of MOS in the diet were significantly positively correlated with RBC, WBC, and lymphocytes (P ≤ 0.035) and negatively correlated with monocyte, basophil, and Vibrio counts (P ≤ 0.045). However, there were no significant correlations between concentrations of MOS in the diet and other haematological parameters of pompano (P ≥ 0.531). At the end of the experiment (day 56), the immune parameters of pompano fish fed graded levels of MOS were significantly and positively correlated with MOS concentrations supplemented in the diet (P ≤ 0.039) (Table 4). Among the haematological indices, intestinal microbes, and immune parameters, lysozyme gained the highest values of correlation (r) (Table 4); thus, lysozyme activity was used to predict the optimal MOS requirement for pompano to reach maximal immune response.

Prediction of the optimal level of MOS requirements for maximal growth of pompano fish, T. ovatus

At day 56, there was a positive correlation between MOS inclusion and growth according to the quadratic polynomial models (R² ≥ 0.750). As fish weight rose, the predicted optimal concentrations of dietary MOS supplementation decreased. To obtain the maximal growth of pompano in the experiment, the optimum levels of MOS requirement were predicted to be 0.440%, 0.385%, 0.371% and 0.365% at days 14, 28, 42 and 56, respectively (Fig. 7, Table 5).

Table 5. Nutritional requirement of dietary mannan oligosaccharide based on specific growth rate, SGR, % d^-1(determined by the quadratic polynomial model, y = ax² + bx + c) of pompano, T. ovatus, fed various MOS supplements in the diets.

	a	b	c	R²
*Day 14*	-2.346	1.934	2.045	0.861
*Day 28*	-3.431	2.566	1.617	0.877
*Day 42*	-2.259	1.718	1.545	0.891
*Day 56*	-1.735	1.254	1.367	0.750

Predicting MOS level to gain maximal immune response in pompano (day 56)

At the end of the trial (day 56), in relation to haematological indices, intestinal Vibrio, and immune parameters, the lysozyme activity of pompano fish showed the highest significant correlations with MOS concentrations in the diet (Table 4). Therefore, lysozyme activity values were used as independent variables, and MOS concentrations were used as dependent variables in the regression model for prediction for maximal immune response. The procedure to find an appropriate prediction model was the same as for growth data. In this experiment, the quadratic polynomial model gained the highest (R²). Hence, MOS supplemented levels were predicted using the quadratic polynomial regression model, and the result showed that the requirement for MOS was predicted to be 0.431% for pompano to gain maximal lysozyme level (R²= 0.817) (Fig. 8).

Cumulative mortalities, relative risk, and hazard ratio of pompano fish challenged against salinity

We used Kaplan-Meier curves and log-rank tests to compare the number of pompano that died after being challenged to a low salinity of 1 ppt. After 4 h of challenge in a 1 ppt salinity condition, it was observed that there were significant differences in the survival rates of pompano fish among the treatments (P = 0.021). Pompano given diets added with 0.1 to 0.4% MOS had significantly higher survival rates compared to the survival rate of pompano fed a control diet (P ≤ 0.014). However, the survival rates of fish fed 0.6% and 0.8% MOS inclusion in the diets did not differ significantly from the survival rates of pompano fed control (P ≥ 0.189). Also, there was no significant difference among the survival rates of pompano fed any levels of the diets (P ≥ 0.086). (Fig. 9).

In addition, the relative risk index (RRI) was lower in pompano fed the MOS diet when exposed to low salinity than in those fed the control diet. RRI values in pompano fed a MOS-supplemented diet were less than one when compared to pompano fed a control diet, showing that fish fed a MOS-supplemented diet had a higher capacity to tolerate salinity stress.

When compared to the relative risk indices (RRI) of pompano in control diets, the RRI values and confident intervals (CI) of those fish fed MOS inclusion diet were computed as follows: MOS0.1: 0.256 (CI: 0.0791 to 0.830); MOS0.2: 0.182 (CI: 0.057 to 0.583); MOS0.4: 0.359 (CI: 0.108 to 1.195); MOS0.6: 0.547 (CI: 0.161 to 1.864); and MOS0.8: 0.598 (CI: 0.187 to 1.908). The results showed that when exposed to low salinity, the risks were significantly reduced in pompano when fed MOS inclusion diets at 0.1 - 0.4% MOS (P ≤ 0.032) (Fig. 9).

This study emphasised the effectiveness of dietary mannan oligosaccharide in boosting the growth, survival, haematological parameters, intestinal Vibrio, immune system, and stress resistance of the pompano fish, T. ovatus. The results in this study are in line with previous research that revealed MOS diet enhanced growth in common carp, Cyprinus carpio³⁵, Nile tilapia, Oreochromis niloticus²², Atlantic salmon, Salmo salar⁴⁷, red sea bream, Pagrus major⁴⁸, hybrid grouper, Epinephelus lanceolatus ♂ × Epinephelus fuscoguttatus ♀⁴⁹. Conversely, dietary mannan oligosaccharides limited the growth of Mexican sturgeon, Acipenser oxyrinchus desotoi³³. According to Xue, et al. ³⁵ varied outcomes regarding the effect of MOS on fish growth may be dependent on the fish species or its developmental stage.

Haematological indices are useful indicators for estimating the health, nutrition, and environmental factors affecting fish⁵⁰. It was recommended that leukocyte profiles be used to determine stress in vertebrates⁵¹. Also, lymphocytes, specifically B cells, play a crucial role in the immune system⁵² by participating in the antibody-production processes of teleost fish⁵³. In previous research, Khosravi-Katuli, et al. ⁵⁴ reported that the addition of MOS to the diet of gilthead sea bream, Sparus aurata significantly boosted their haematocrit, haemoglobin, lymphocytes, white blood cells (WBC), and monocytes.

In addition, gut microbes play a crucial role in fish metabolism, nutritional balance, immunity, and physiological processes⁵⁵^,⁵⁶. In line with our research, other studies indicated that MOS-supplemented diets decreased Vibrio and improved intestinal bacteria³⁰^,⁵⁷. Many studies have proved that the application of probiotics, prebiotics, and their combinations has been used to control vibriosis in aquaculture⁵⁸. Thus, the reduction in Vibrio in pompano fed MOS in the present investigation may indicate they are healthier and reduce the chance of Vibrio infection. In aquaculture, many Vibrio species have caused disease in farmed animals, resulting in massive economic losses⁵⁹. However, to fulfil this knowledge on vibriosis, further research needs to be done on the composition of intestinal microbes.

Lysozyme and phagocytosis are non-specific immune responses involved in the protection of the fish body against pathogenic invaders⁶⁰. According to Luo, et al.⁶¹ lysozyme has the ability to deactivate the peptidoglycan layer in the cell walls of harmful microbes, resulting in significant resistance to infection. In the present investigation, increased lysozyme activity, phagocytic activity, and white blood cell counts were observed in pompano fed diets containing 0.1-0.6% MOS compared to fish fed control diet. This is an implication of an improvement in the bactericidal activity of phagocytic activity and immunity⁶². Similar to our findings, MOS has been shown to boost the immune response of common carp³⁵, Nile tilapia⁶³, and rainbow trout⁶⁴. Lu, et al.⁶⁵ stated that the action of MOS begins with the activation of the local intestinal immunity, which is linked to the immune system of the entire fish body. Furthermore, the role of the mannose receptor, which is the key molecule involved in antigen detection and phagocytosis, is another explanation for the immunomodulatory effects of mannan oligosaccharide⁶⁶. Also, mannose binding lectin activation via liver secretion is another way that MOS may influence the immune system⁶⁷.

Determining optimal nutrient levels is one of the most important parts of aquaculture, as supplying an excess or deficiency of a nutrient can cause adverse effects on cultured animals³⁷^,³⁸^,⁶⁸. In fact, the majority of publications on dietary mannan oligosaccharide in aquaculture have utilised one or two 'recommended' administration levels^69-73, rather than determining the requirements of nutrition concentrations. Although the effects of MOS levels on growth performance and feed utilisation have been reported previously³¹^,⁷⁴^,⁷⁵. For instance, Torrecillas, et al.⁷⁶ reported that adding 0.4% dietary MOS increased growth performance in European Sea Bass, Dicentrarchus (Morone) labrax compared with both fish fed 0.2% MOS and controls, which is an indication for dose impact on fish. However, limited research has determined the optimal MOS dose to obtain optimal growth³¹.

In the present study, we measured growth over time using dose-response models to determine the effect of MOS on growth. The determination method applied in this study is similar to previous studies that determined the nucleotide requirements for prawn growth³⁷, MOS for lobster³¹, and β-glucan for pompano²⁸^,³⁸. In this study, six regression models were used. The model with the highest correlation coefficient (R²) was chosen to predict the suitable MOS concentration for maximal growth of pompano. According to Ryan ⁷⁷, R²value is the most important indicator in determining the quality of the relationship between the models.

Similar to previous studies²⁸^,³⁸, the current finding demonstrated that either excessive or deficient amounts of MOS supplementation adversely affected pompano’s growth. The current study also revealed that the optimal level of MOS requirement in the diets of pompano decreases as fish size increases. This is possibly because of the higher growth rate in smaller fish compared to larger ones. On days 28 and 42, the highest correlation coefficients between pompano growth and diet MOS supplementation were observed. Therefore, it is recommended that studies on the effects of MOS on pompano should be conducted for two to four weeks. However, since size affects the MOS requirement, further examination of different sizes is required to support this statement.

Besides influencing growth performance, it has been documented that nutrition plays a vital role in boosting fish immunity⁷⁸ and diet may have an impact on fish disease resistance⁷⁹. However, nutritional requirements are often determined based on growth data rather than immunological responses and disease resistance⁷⁹^,⁸⁰. In this study, among the investigated immune parameters, lysozyme activity showed the highest correlation with levels of MOS in the diet; hence, lysozyme from fish fed different MOS levels was employed to predict the optimal MOS requirement for the highest level of immune response in juvenile pompano. To the authors’ knowledge, this is the first attempt to investigate the dietary mannan oligosaccharide dosage requirement for such an immune response in pompano. On day 56, the predicted MOS requirement for optimum growth in pompano was 0.365%; however, a slightly higher concentration of MOS (0.431%) is required for the fish to achieve its maximum lysozyme activity value. Our results are consistent with earlier research that reported MOS stimulates the immune system in aquatic species, but that the dosage of MOS is crucial in achieving the desired outcomes⁸¹. Similar to this, a study on rainbow trout, Oncorhynchus mykiss found that higher levels of MOS produced a more effective stimulant outcome compared with a lower level⁸². Additionally, it was discovered that when the amount of MOS in the diet increased, the phagocytic and lysozyme activities milkfish, Chanos chanos continually increased⁸³.

In this study, the MOS-supplemented diet could enhance the pompano’s capacity to suffer from stress. Pompano fed 0.1% - 0.4% MOS had higher survival rates when challenged with low salinity conditions. In line with our results, other studies also revealed that dietary MOS supplementation improved the tolerance against low salinity stress in red sea bream⁴⁸, white sea bream (Diplodus sargus L.) larvae⁸⁴, cobia larvae Rachycentron canadum⁷². In aquaculture practise, in Vietnam, pompano cage farms are typically situated near the coast and are highly influenced by freshwater from the land during the rainy season. Thus, the findings in this study showed the effectiveness of the MOS diet to improve salinity tolerance in pompano, which has a meaningful role for aquaculture practise. Research revealed that when exposed to stress, fish secrete more stress hormone, which affects immune function by suppressing lymphocyte and immunoglobulin synthesis⁵³. In this study, we have not examined the hormone of pompano; however, the increase in WBC, lymphocytes, and non-specific immune responses in pompano fed MOS diets may indicate a better response to growth, health, and stress tolerance.

In the current study, pompano fish fed 0.1-0.6% MOS had significantly higher WBC, lymphocyte counts, and immune indices. Increased WBC, lymphocyte, and immunological parameters; reduction in gut Vibrio in pompano fed a MOS diet may indicate that the fish's innate immune system is being stimulated, resulting in boosted defences and salinity stress resistance. It is known that the use of antibiotics in aquaculture may cause some risks, such as an increase in the transfer of antibiotic resistance genes to terrestrial animals and human pathogens⁸⁵. Our research reveals that the haematological parameters, immune system, and stress are enhanced, particularly the decrease in Vibrio count in the gut of pompano fed diets with MOS inclusion, so MOS can be proposed as an antibiotic alternative for pompano aquaculture. Thus, the findings in this study have significant implications for the future of pompano aquaculture, the improvement of sustainable aquaculture practises, and the reinforcement of food safety and security. Also, as Vibrio causes illness in human⁸⁶, the decrease in Vibrio in pompano fed MOS diets may indicate an improvement in food safety and an increase in the value of commercial products. Since MOS administration has not been reported to have any adverse effects on the environment, it is thought to be an environmentally friendly alternative to antibiotics for the treatment of disease. Hence, the use of MOS is recommended to develop sustainable pompano aquaculture⁵⁹.

This report concludes that the addition of mannan oligosaccharide to the diet of juvenile pompano, Trachinotus ovatus, improves growth performance, haematological indices, intestinal Vibrio reduction, immune parameters, and salinity stress resistance. It was also determined that including MOS at a level of 4.0 g kg^-1 is ideal for promoting juvenile pompano’s maximum growth and for gaining a maximal immune response. However, other potential benefits of MOS supplementation should be examined, such as increased resistance to other environmental stressors or pathogens at various life stages and physiological conditions in fish.

CRediT authorship contribution statement

Do Huu Hoang: conceptualization, study design, supervision, project administration, methodology, data analysis, writing – original draft, review and editing.

Nguyen Thi Nguyet Hue: study design, methodology, drafting, fish husbandry

Vo Hai Thi: study design, methodology, drafting, fish husbandry, writing review and editing.

Acknowledgements

This research was funded by the Vietnam National Foundation for Science and
Technology Development (NAFOSTED) under grant number: 106-NN.05-2019.04. We particularly would like to thank Dr. Michael Wang, Nguyen Gia Tuong, and Biorigin for generously providing Active-MOS^®. The authors would like to express special thanks to Dr. Nguyen T.T.T, Nguyen T.K.B, Cao V.N, Hua T.A and Tran L.K.H for their great help.

Data availability

The original data supporting the findings of this study are available upon reasonable request from the corresponding author.

Competing interests

The authors declare no competing interest.

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No competing interests reported.

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Effects of dietary administration of mannan oligosaccharides on growth performance, intestinal Vibrio, non-specific immunity and tolerance to low salinity in golden pompano, Trachinotus ovatus (Carangidae)

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Abstract

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Introduction

Materials and Methods

Results

Discussion

Declarations

References

Additional Declarations

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