EFFECTS OF LOW LEVELS OF MANNANOLIGOSACCHARIDES IN FEED ON GROWTH AND INTESTINAL SALMONELLA REDUCTION IN JUVENILE RAINBOW TROUT ( Oncorhynchus mykiss )

Low levels of added mannanoligosaccharides (MOS) in the feed for juvenile rainbow trout significantly promote growth rate, food conversion and overall culture results. Three levels of added MOS: 0.05%, 0.10% and 0.15% were incorporated in commercial pelleted feed and fed three and later two times per day. The experiment started with juveniles of 48±2.1 grams and lasted 90 days. The best growth result was obtained with the smallest (0.05%) MOS level, if compared to the control group and 0.1% and 0.15% MOS supplemented feed. The growth was improvedby11.5% with 0.05% MOS. The best feed conversion ratio (FCR) of 1.29 was found with 0.05% MOS supplement compared to 1.54 in the control group. FCR in 0.1% and 0.15% MOS treatments was 1.46, also significantly better than in the control group. The experiment shows that 0.05% MOS in trout feed is not fully successful in total elimination of salmonella colonies from fish intestine. But this is achieved with 0.1% MOS after only 30 days of experiment. The results of experiment point that smaller levels of MOS, if compared to previous research, are beneficial for rainbow trout juvenile culture.


Introduction
Improving growth performances and fish health with novel food agents in commercial production is an important factor in the aquaculture industry (Wache et al., 2006;Helland et al., 2008;Sado et al., 2008;Gultepe et al., 2012).Available products such as probiotics (Burr et al., 2005;Balcázar et al., 2006), and prebiotics (Ringø et al., 2010) have become popular in recent years.The application of probiotics and prebiotics in fish food may result in elevated health status, improved disease resistance, growth performance, body composition, reduced malformations and improved gut morphology and microbial balance, increasing villi height, uniformity, and integrity resulting in greater absorptive efficiency of digestive tract (Ringø et al., 2010;Merrifield et al., 2010).
One such example is mannanoligosaccharides (MOS), which improve gut function by increasing microvilli height, uniformity, and integrity resulting in greater absorptive efficiency of digestive tract (Merrifield et al., 2010).
This article presents the effect of food enriched with low levels of mannan oligosaccharides on growth, food conversion, survival and salmonella intestine infestation of rainbow trout juveniles cultured in cages.

Materials and Methods
This research was realized in Ramsko Lake (Bosnia and Herzegovina) which is an artificial reservoir of a hydropower station (43°48'14'' N and 17°31' 52'' E).The experiment was performed in eight net cages (dimensions: 1 m x 1 m and depth of 1.4 m) with 1m 3 of water volume and it lasted for 90 days.Cages were mounted in two lines with 4 cages each, with a wooden walking platform between.Average water temperatures during experiment varied from 9.5 o C in April, 13.4 o C in May, 16.5 o C in June to 21.2 o C in July.The temperature during this period fluctuated from 8.5 o C at the beginning of the experiment to 22.3 o C at the end of the experiment.Oxygen levels varied from 8.3 mg/l to 10.8 mg/l through the whole experiment.The pH values varied from 7.33 to 7.86.
Each cage was stocked with 105 specimens of juvenile rainbow trout.The average weight of fish was 48±2.1 g.Four treatments were performed in duplicate.The control group was fed commercial pelleted food without added mannan oligosaccharides (Bio-Mos ® , Alltech, Kentucky, USA).Three treatments with different levels (E1−0.05%,E2−0.1%,E3−0.15%) of added MOS were implemented.
The fish were fed daily: three times (at 8, 13 and 19 hours) in the first 50 days and later two times (at 8 and 19 hours), following feeding tables of the producer.Daily ratios were determined based on water temperature and oxygen level.
Pelleted feed was produced by "Poljoprerada", Zagreb, Croatia.The crude protein content in feed was 45%, crude oil content was 12%, ash content was 7% and crude fibre content was 2%.
The fish were measured every 30 days after anaesthesia with benzocaine.The fish were weighed using a digital balance and total length was measured with a digital calliper.Every 30 days, 10 fish from each cage were sacrificed for bacterial analysis.
The effects of MOS on intestine were investigated using measurement of Salmonella colonization of intestinal tract.Intestine was dissected into sterile petri dishes and 1 g was mixed with 9 ml of sterile solution and homogenized.After dissolution to 10 -9 , a sample was cultured using buffered peptone water (Merck, Germany) at 37 o C and Salmonella enrichment broth (Merck, Germany) incubated for 24 hours, and later it was incubated for 24 hours on XLD agar at 37 o C. Bacterial number is expressed as colony-forming units per gram of intestine tissue (cfu/g).
Statistical analyses were performed using Statistica software (StatSoft, Inc., 2005).Differences among treatments were analysed using Duncan post-hoc test.

Results and Discussion
The best growth was found in fish fed 0.05% MOS.Fish weight after 90 days of experiment was 109.52±2.461g in the control group and 122.15±1.23 g in E1, which is 11.5% more than in the control.A higher weight was also measured in E2 group (114.25±0.364g) and E3 group (113.47±1.864g).A significant difference was found among the control group and all experimental groups (p<0.001).Furthermore, a significant difference was also found between E1 treatment and both E2 and E3 treatments (p<0.001).There was no significant difference between E2 and E3 experimental groups (p<0.001),Table 1.Daily growth rate (DGR) varied from 0.69 grams in control groups, to the highest rate of 0.83 grams in E1 treatment groups.Significantly lower DGRs of 0.75 g and 0.74 g were found in E2 and E3 treatments.E1 treatment was significantly better (p<0.001)than other three treatments.E2 and E3 treatments were significantly better (p<0.001)than the control.No difference was found between E2 and E3.The improvement in daily growth rate of fish fed 0.05% MOS was 16.8% if compared to the control group, 9.6% if compared to 0.1% MOS and 10.75% if compared to 0.15% MOS.
The best feed conversion ratio (FCR) of 1.29 was found in E1 groups and the lowest ratio of 1.54 was found in the control group.FCR ratio in E2 and E3 groups was 1.46.A significant difference (p<0.001) was found among E1 treatments and all other treatments, as well as between the control and E2 and E3 treatments.
The best protein efficiency ratio (PER) of 1.75 was found in E1 treatment group and was significantly higher (p<0.001)than in other treatments.In the control group, PER was 1.53, and in E1 and E2, it was 1.61.PER in E2 and E3 treatments was significantly higher than in the control.
Fish mortality was low and only 14 fish died during the experiment.In control groups, six fish died.Other fish were recorded in treatment groups: in E1 group mortality was two fish, in E2 group three fish died and in E3 group three fish died.The difference was significant between the control and all three experimental treatments, while there was no difference among E treatments (p<0.001).
At the start of the experiment, the initial number of Salmonella in rainbow trout intestine (CFU/g) was 0.92x10 1 .
In the control group, this number showed the trend of a constant increase to 1.5x10 1 after 90 days of the experiment.In E1 treatment, the number of Salmonella decreased after 30 days to 0.3x10 1 and to 0.1x10 1 after 60 and 90 days of the experiment.During the experiment, Salmonella was not found in E2 and E3 treatments.The experiment showed that 0.05% MOS in trout feed was not fully successful in total elimination of Salmonella colonies from fish intestine.But this was achieved with 0.1% MOS supplemented in feed (Table 2).
This experiment showed that low levels of MOS produced good results in culture of juvenile rainbow trout in cage conditions.The best results were obtained with the lowest (0.05%) used MOS level.The growth rate, daily growth rate, feed conversion ratio and protein efficiency ratio were significantly better with 0.05% added MOS than in treatments with 0.1%, 0.15% MOS and no MOS supplemented feed.Previous research on MOS in rainbow trout dealt with a higher MOS content.
A significantly improved growth performance and immune status of rainbow trout were also observed when applying 0.2% MOS in the net cage trial−improved weight gain of 13.7%, reduced feed conversion ratio and mortality and improved indicators of immune status (Staykov et al., 2007).An enhanced growth performance was observed in rainbow trout fed the diet supplemented with 0.15% MOS, but not with 0.3% or 0.45% MOS.Those higher percentages of MOS supplement also produced no significant differences in feed conversion ratio, protein efficiency ratio, or hepatosomatic index (Yilmaz et al., 2007), as we observed with lower levels of MOS in the present study.The level of 0.2% MOS supplemented feed did not produce a significant growth benefit for juvenile rainbow trout, whereas increased gut absorptive surface area and an increasein microvilli length and density in the subadults (weight around 120 grams) fed MOS supplemented diet were reported (Dimitroglou et al., 2009).But, in a study on rainbow trout fingerlings fed 0.4% MOS for 12 weeks, Rodriguez-Estrada et al. (2009) reported that inclusion of MOS to the diet stimulated growth, haemolyticand phagocytic activity, mucosa weight and improved survival when the fish were challenged with Vibrio anguillarum.Although it seems that higher MOS supplement (above 0.2%) does not produce significant growth benefits in rainbow trout juvenile culture in most studies, some studies confirmed beneficial effects.The reasons for these differences are difficult to elaborate and more research is needed to introduce commercial practice with MOS supplement in rainbow trout culture, although all studies reported some benefits for the fish and culture practice.

Conclusion
This study showed that the low level (0.05%) of MOS supplemented trout feed produced better overall results on culture performance of juvenile rainbow trout when compared with higher MOS percentages.This should enable the production of MOS supplemented trout feed at lower prices and subsequently better overall economic results of rainbow trout culture in cage farms.

Table 1 .
The effect of mannan oligosaccharides on the growth of rainbow trout, Oncorhynchus mykiss juveniles after feeding different levels of mannan oligosaccharides (MOS) for 90 days.

Table 2 .
The effect of mannan oligosaccharides (MOS) on the number of Salmonella in rainbow trout, Oncorhynchus mykiss intestine (CFU/g) after feeding on MOS supplemented feed for 90 days.