Immunostimulation and increase of intestinal lactic acid bacteria with dietary mannan-oligosaccharide in Nile tilapia juveniles

In this experiment, the beneficial effects of mannan-oligosaccharide (MOS) on immunology and intestinal microbiology of Nile tilapia juveniles was demonstrated. Prior to this, three levels of MOS in Nile tilapia diets (1, 8, and 15 g.kg−1) were tested, and hematological parameters, serum lysozyme, and intestinal microbiology were analyzed. The fish blood was sampled at day zero (basal sample) and after 45 days of trial, and the intestinal microbiota was evaluated at the end of the experiment. After 45 days of trial, fish fed 8 and 15 g.kg−1 of MOS presented an increase in both aerobic and lactic acid bacteria numbers in their guts. The MOS feeding also increased the counts of total leukocytes, monocytes, and lymphocytes of fish, but a decrease in neutrophils was also observed. Additionally, the serum lysozyme was higher in all fish fed MOS. The dietary MOS is able to modulate the intestinal microbiota, increasing the number of beneficial bacteria, and immunostimulates the Nile tilapia juvenile, giving rise to white blood cells and serum lysozyme.


Introduction
Aquaculture has shown a consistent growth around the world over the last two decades, and one of the major contributors is the tilapia culture. Tilapia species are produced in large scale in more than 130 countries because they are recognized for their toughness, high meat quality, and low protein requirement (Fitzsimmons et al., 2011). Together with the rapid expansion of the tilapia aquaculture and subsequent intensification of production systems, the stress caused by the increase of stock densities, handling, and the use of artificial feeds gave rise to several disease outbreaks (Mauel et al., 2007;Mian et al., 2009;Iwama et al., 2011).
Previously, several strategies were employed to avoid the use of antibiotics, most of them as preventive approaches including non-antibiotics dietary additives for fish (Pohlenz and Gatlin, 2014). One possible alternative to avoid fish diseases and subsequent use of treatment procedures is the use of prebiotics such as the mannanoligosaccharide (MOS).
Mannan-oligosaccharide is a glycoprotein rich in mannose, usually isolated from the cell wall of Saccharomyces cerevisiae. The addition of MOS to fish feeding has been reported to modulate the intestinal microbiota of the host by promoting the colonization of the intestinal tract by benefic bacteria and eliminating some pathogenic microorganisms by adsorption, resulting in enhanced resistance against pathogens by the host (Gómez and Balcázar, 2008). Moreover, the mannose present in MOS particles can be recognized by innate immune receptors, immunomodulating the host (Torrecillas et al., 2014). Positive effects of dietary MOS on immune parameters have been observed in Oncorhynchus mykiss (Staykov et al., 2007), Labeo rohita (Andrews et al., 2009), Sciaenops ocellatus (Zhou et al., 2010), and Dicentrarchus labrax (Torrecillas et al., 2015).
Thus, the objective of the present study was to evaluate the effects of MOS on Nile tilapia (Oreochromis niloticus) intestinal microbiota, hematological parameters, and serum lysozyme.

Material and Methods
Research on animals was conducted according to the institutional committee on animal use (case number 22.517/10).
The present experiment was conducted in Jaboticabal (São Paulo State, Brazil -21º15'17" S, 48º19'20" W). Masculinized Nile tilapia (~1 g), GIFT strain, were obtained from a commercial farm. Initially, the fish were maintained in 310-L fiberglass tanks with continuous aeration and water flow. During this grow-out period, fish fed the control diet (Table 1) twice a day until apparent satiation.
After eight weeks, treatments were started by including MOS in the feed. Fish (~101.42±2.71 g) were randomly distributed into 12 fiberglass tanks of 310 L (30 fish per tank) and maintained in the same conditions as described above. The experimental diets were prepared according to NRC (1993) for Nile tilapias (Table 1). The experimental diets were prepared according to the formulation expressed in Table 1, using the basal formulation as in the-grow out stage. ActiveMOS (Biorigin ® , Lençois Paulistas, Brazil) was added to the diets at zero (control), 1, 8, and 15 g.kg −1 , and inert Kaolin was added to balance the diets, assuring similar levels of crude energy and crude protein. All the ingredients were ground and then mixed three times. The resultant mixture was extruded in a single screw extruder machine (Ex Micro, Exteec, Brazil) into 1.0 mm pellets and then dried by forced ventilation at room temperature. Fish were fed using the experimental diets twice a day for 45 days. Growth and survival rates were measured during this period. Water parameters including pH, dissolved oxygen, and temperature were measured weekly.
Before starting the experiment, 15 fish were randomly caught, anesthetized in clove oil solution (0.1 mL of clove oil per liter of water), and had the blood sampled, corresponding to the basal sampling. After 45 days of MOS feeding, five fish from each tank (15 per treatment) also had the blood sampled following the same procedures. Blood was collected by puncture in the caudal vein, using 3-mL syringes with a 22 gauge needle and without anticoagulant. Each blood sample was divided into three aliquots for subsequent analysis. For hematological parameters, a 100-µL aliquot was transferred to a 2-mL microtube containing 15 μL of anticoagulant (0.65% NaCl, sodium heparin 100 IU.mL −1 ). Then, hematocrit was determined by the microhematocrit method (Goldenfarb et al., 1971), hemoglobin by the cyanmethemoglobin method (Collier, 1944), and red blood cells (RBC) were counted in a Neubauer chamber after diluting 10 µL of blood in 2 mL of a citrate-formaldehyde solution. Blood smears were prepared with drops of blood directly from the syringe without anticoagulant to be air-dried and stained with May Grünwald-Gyemsa-Writh (Tavares-Dias and Moraes, 2006). This was done in preparation for total counts of thrombocytes and leukocytes and differential counts of leukocytes (Hrubec and Smith, 2000). Hematimetric equations (Wintrobe, 1934) were used to determine mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC).
The remaining blood sample was transferred to 5-mL glass tubes and incubated for 45 min at room temperature to obtain the serum, which was collected using a micropipette and maintained at −20 °C for later analysis. The lysozyme concentration was then determined using a modification of Abreu et al. (2009). An aliquot of 60 µL of serum sample was diluted in 40 µL of phosphate buffer solution in 96-well plates. Then, 100 µL of PBS containing 1 µg µL −1 of Micrococcus lysodeikticus were added to the mixture. The optical density was measured at 5 and 10 min at 540 nm in spectrophotometer to estimate lysozyme concentration. After 45 days of MOS feeding, one fish from each tank was euthanized with highly concentrated clove oil solution (0.2 mL.L −1 ) and sampled for microbiological analysis. Fish was rinsed with 70% ethanol, and the gut was removed. The lumen was opened and rinsed twice with 0.9% saline solution for the removal of intestinal content. Then, the whole intestinal lumen was gently scraped with a swab, and the material removed was placed into 15-mL tubes containing 10 mL of 0.85% NaCl solution to be gently homogenized for 3 min. Each sample was diluted to 10 −7 in 0.85% NaCl solution. For the determination of aerobic bacteria, 100 µL of each suspension were spread onto triplicate nutrient agar plates (Himedia, China) and incubated at 36 °C for 72 h. For the determination of anaerobic bacteria, the same procedure was conducted with the plates placed inside anaerobic jars with a Gas Pak system and incubated under the same conditions. For the lactic acid bacteria (LAB) culture, 100 µL of each dilution were spread onto triplicate MRS agar plates (DeMan, Rogosa and Sharpe agar, Sigma, USA) and also incubated at 36 °C for 72 h. Each bacterial population (given in units per mL) was calculated from plates containing 30 to 300 cfu.
Results were expressed as mean ± standard error. The statistical analyses were performed with the software R V3.0.3. All data were checked for homoscedasticity and normality with Levene's test and Cramer-Von Mises' test, respectively, and were transformed to fit normal distribution using log(x + 1.5). A one-way ANOVA was performed following the model below: Y ij = μ + τ i + ε ij , in which Y ij is the response of animal j of treatment group i; μ is the overall mean; τ i is the fixed effect of treatment i (dietary levels of MOS); and ε ij is the random error of the response of animal j of treatment group i. The resultant means were compared using Tukey's multiple range test (α<0.05).

Results
Throughout the experiment, the water parameters were maintained at pH of 7.53±0.15; dissolved oxygen at 4.15±1.04 mg L −1 ; and temperature at 28.12±0.89 °C with no disease outbreaks or natural mortality being detected.
Regarding the erythrocytic parameters (Table 2), no statistical differences were observed for Ht, Hb, VCM, MCH, MCHC, and RBC (P>0.05). In relation to the white   (Table 2), no differences were observed for thrombocytes and basophils (P>0.05). The fish fed 8 and 15 g.kg −1 of MOS presented more leukocytes than the basal and control groups (P = 0.0016). The basal fish presented a lower number of monocytes than fish sampled after 45 days of experiment, and all fish fed MOS presented more monocytes than the control group (P<0.0001). A similar pattern was observed for lymphocytes, but only the groups fed 8 and 15 g.kg −1 MOS presented more lymphocytes than the control (P = 0.0022). On the other hand, all fish fed MOS presented a lower number of neutrophil than the control and basal groups (P = 0.0017) (Figure 1). The serum lysozyme (Figure 2) for fish sampled after 45 days of trial was significantly higher than that of basal fish, and the fish fed MOS presented a higher concentration of serum lysozyme than the control group (P<0.0001).

Discussion
Several researchers have described positive effects in gut health and innate immunity of fish feeding MOS (Lara-Flores et al., 2003;Pirarat et al., 2006;Aly et al., 2008a;Aly et al., 2008b;Ngamkala et al., 2010;Pirarat et al., 2011), but it is the first time that this is described in Nile tilapia. Mannan-oligosaccharide is a polymer rich in mannose, a complex carbohydrate that can be used as a substrate to beneficial microorganisms, especially LAB (Katakura et al., 2010). Lactic acid bacteria are present in invertebrate and vertebrate animals and have an important role in the maintenance of health in mucous environments by producing antimicrobial substances that act against pathogens or competing for cell-surface and mucin-binding sites (Liu et al., 2013). In the present study, a significant increase of aerobic bacteria and LAB in the intestine of fish fed 8 and 15 g.kg −1 MOS was observed but not for fish fed the 1 g.kg −1 MOS and control diets, suggesting the effectiveness of dietary MOS as prebiotic for Nile tilapia. Previous studies using MOS as feed additive also reported alterations in the intestinal microbiota of Oncorhynchus mykiss (Dimitroglou et al., 2009), as well as showed an increase in LAB number in Sparus aurata guts (Dimitroglou et al., 2010). However, in review of the related literature, this is the first study showing beneficial changes to the intestinal microflora of Nile tilapia fed MOS.
In the present study, no significant effects were observed on erythrocytic parameters from dietary MOS, as also observed in Huso huso (Mansour et al., 2012), O. niloticus (Sado et al., 2008), Piaractus mesopotamicus (Sado et al., 2014), and Channa striata (Talpur et al., 2014 On the other hand, after 45 days of MOS feeding, an increase in leukocyte, monocyte, and lymphocyte numbers was observed, which is concomitant to a decrease in the number of neutrophils. A higher content of leukocytes in peripheral blood usually indicates organic response to infections and parasitical infestations (Grant, 2015), but some substances or microorganisms are able to modulate, directly or indirectly, some of the innate immune response, both cellular and humoral, resulting in higher protection of fish against diseases (Anderson, 1992;Bricknell and Dalmo, 2005;Galina et al., 2009;Dimitroglou et al., 2011). Different results found by Mansour et al. (2012) presented a decrease in Huso huso lymphocytes after 46 days of feeding 2 g.kg −1 of MOS with no changes in the other white cell counts. Also in contrast with our data, Sado et al. (2014) observed no differences in white cell profile when giving MOS to Piaractus mesopotamis with.
The lysozyme is an important protein in the innate immune system of fish. It acts as an enzyme, hydrolyzing the peptidoglycan present on the cell wall of Grampositive bacteria, and also as an opsonin, activating the complementary system and promoting the phagocytosis (Ellis, 1999;Magnadóttir, 2006;Saurabh and Sahoo, 2008). In the present study, MOS feeding during 45 days successfully increased the serum lysozyme levels in Nile tilapia juveniles, which can also be considered an immunostimulation. Akrami et al. (2012) also observed an increase in lysozyme levels of Carassus aurata gibelio. However, their supplementary doses were considerably higher than ours (1.5, 3, and 4.5 g.kg −1 MOS), and only the 4.5 g.kg −1 MOS was statistically different from the control.

Conclusions
The presented data shows that mannan-oligosaccharide, as a dietary supplement, exerts a prebiotic effect and is able to immunostimulate Nile tilapia juveniles, presenting potential as a prophylactic additive for Nile tilapia in the substitution of antibiotics. The authors recommend the enrichment of Nile tilapia diets with 8 g.kg −1 of mannanoligosaccharide due to the better results on monocytes, lysozyme, and intestinal lactic acid bacteria.