Effects of Mannan-Oligosaccharide Supplementation On Gut Health, Immunity, and Production Performance of Broilers

The study was designed to investigate the effect of mannan-oligosaccharide (MOS) supplementation on intestinal histomorphology, immunity against Newcastle disease virus (NDV) and productive parameters of broilers. A total of 1800 day old broiler chicks of Cobb-500 strain were selected and randomly assorted into 6 treatment groups: T1 (basal diet without antibiotics as negative control); T2 (basal diet plus antibiotics as positive control group); T3 (basal diet plus 200g/ton MOS); T4 (basal diet plus 400g/ton MOS); T5 (basal diet plus 600g/ton MOS) and T6 (basal diet plus 800g/ton MOS). Each treatment was having 6 replicates and the feed intake, body weight gain and feed conversion ratio (FCR) were recorded on weekly basis. Results showed that, MOS supplemented birds have signicantly higher feed intake, weight gain and FCR (P < 0.05). Similarly, supplementation of MOS showed positive effect on villus height and crypt depth both in jejunum and ilium. Goblet cell density was unaffected by MOS addition (P < 0.05). Furthermore, birds fed with diets containing MOS, exhibited better productive performance in comparison to positive and negative control groups. In conclusion, MOS can replace antibiotic growth promoters (AGPs) as non-microbial performance-enhancing feed advocates. effect of MOS supplementation on average weekly feed intake in broilers at 5 consecutive weeks. Results showed that supplementation of MOS signicantly (P < 0.05) affected the feed intake. At 1st week, highest average feed intake (FI) was observed in T5 followed by T3, T2 and T1 groups. At 2nd week of age, higher FI was seen in T6 group followed by T5 and T1. However, at 3rd week, elevated FI was observed in T6 and T3 followed by T2 and T1. At 4th week, highest FI was seen in T6 followed by T2. At the end of trial, highest feed intake was calculated in T4 followed by T6 and lowest FI was observed in T1. Birds fed on T6 and T5 showed highest antibody titer against NDV. Lowest values for titer were found in negative control group. Similar results were also published by Shahir et al. (2014) as they observed that MOS supplemented group gained higher antibody titer against NDV as compared to control group but lower than probiotic group. Results of Muhammad et al. (2020) were also similar to our study they recorded that MOS supplemented group has signicantly higher antibody titer against NDV as than control group but lower than probiotic group. Similar results were also found by Waqas et al. (2019) as MOS supplemented group showed higher antibody titer against NDV as compared to control group.


Introduction
Over past several years, there has been an enormous increase in consumption of poultry products due to enriched nutrients present in it. Poultry sector is playing a pivotal role in minimizing the gap between the requirement and availability of proteins for human. In Pakistan, poultry is one of the well-organized sectors producing 1.39 million tons of meat and contributes 32.7% of total meat production. The pro tability of poultry sector depends on e cient manufacturing of feed, proper utilization of nutrients, growth rate, improved feed conversion ratio (FCR) and better gastrointestinal tract (GIT) health of birds. Poultry production is facing several problems, including climatic changes, microbial load and stress during rearing which leads to disturbance of gastrointestinal tract (GIT) that lead to poor performance of birds (Grashorn 2010). Gut micro ora which is a key to the proper utilization of nutrients, can affect the immune status of birds as it in uences the intestinal wall (Klasing 2007). It is well documented that for good performance and healthy GIT showed good effect on overall poultry production (Chen et al. 2009). Moore et al. (1946) was rst who claimed that there is an improvement in performance, when birds fed with streptomycin.
The use of antibiotics in poultry feed is banned due to problem of antimicrobial resistance and appearance of antibiotic residuals in poultry products (eggs and meat). Consequently, it has encouraged the researchers to nd out the antibiotics-alternatives to be used in poultry feed. Therefore, use of probiotics, prebiotics, synbiotics, phytobiotics, enzymes, organic acids antimicrobial peptides, hyperimmune egg yolk antibodies, bacteriophages, clay and metals have been extensively studied as AGPs replacer in poultry feed (Gadde et al. 2017). Probiotics as stated by Reid (2016) are live strains of strictly selected microorganisms which, when fed to animals in adequate amounts, causes an improvement in health and performance of the host. Phytobiotics are plant derived compounds which are being added to animals feed and improves the productivity and quality of meat (Windisch and Kroismayr 2006). A prebiotic is a non-nutritive ingredient that may be digestible by intestinal micro ora and brings bene cial changes in health by changing the proportion of bene cial bacteria to pathogenic bacteria (De Vrese and Schrezenmeir 2008). Many prebiotics including fructo-oligosaccharides (FOS), lactulose, inulin, galactooligosaccharides (GOS), and polydextrose are already used as source of prebiotics in poultry feed.
Mannan-oligosaccharide (MOS) is one of the main prebiotics used in poultry feed that can improve the average daily feed intake, feed conversion ratio and overall performance of broiler chicks when fed in feed as they increase (Kocher et al. 2005). Many studies have revealed that MOS can improve the gut health of the birds by inhibiting the adhesion of harmful bacteria such as Escherichia coli and Salmonella pullorum to coco-2 cells and by promoting the Bi dobacterium in gut (Kocher et al. 2005;Xu et al. 2017). Therefore, this study was designed to check the effect of MOS supplementation on gut health, immunity against Newcastle disease virus (NDV), and production performance of broilers.

Birds and treatments
The experiment has been conducted at research and development farm of Sultan Feed Mills, Sargodha, Pakistan. Before the arrival of chicks, oor brooding area and equipments were cleaned and disinfected. Five days prior to arrival of chicks, the whole shed was fumigated with formaldehyde. Two days before arrival of chicks, brooder have been switched on to maintain inside shed feeling temperature at 32°C and humidity was set to 65±5%. Feed and water were supplied adlib, while light duration was set at 22-24 hours for entire duration.
The research trial was conducted using 1800 Cobb-500 day old chicks. All the birds were randomly divided into 6 treatment groups (T1, T2, T3, T4, T5 and T6) having 300 birds in each group. Each group was consisted of 6 replicates containing 50 birds per replicate. T1 served as negative control, T2 as a positive control supplemented with meduramycine and avomycine, T3 was supplied with Actigen at level of 0.2gm/kg of feed, T4 was supplied with Actigen at level of 0.4gm/kg of feed, T5 was supplied with Actigen at level of 0.6gm/kg of feed and T6 was supplied with Actigen at level of 0.8gm/kg of feed.

Weekly body weight gain
Live weight (g) of each bird was recorded at the beginning of trial. Birds were wing banded and live body weight (g) of each bird was recorded at the start and end of experimental period, 35 days of age in the morning before accesses to feed.

Feed intake and feed conversion ratio
Feed was weighed at the start and after end of each week. Feed residues were collected and weighed every week to calculate the amount of feed consumed per each bird per day for each treatment (g/bird/day). The FCR was calculated as (FCR = kg feed consumed/kg weight gain of birds).

Gut histomorphology
At the end of trial, six birds per treatment group were slaughtered by Halal method (Farouk et al. 2014). Their small intestines were removed and washed with normal saline and its segments; duodenum (pancreatic loop), jejunum and ilium were measured in centimeter, and then 2 cm segments were xed in 10% formalin solution for further processing. Villus height and crypt depth were recorded in jejunum and ilium and goblet cells per villus were counted using microscope. To measure villus height and crypt depth, 2 cm segments of jejunum and ilium were cut down and washed with physiological saline solution, and then xed in 10% buffered formalin. Histological sections were examined microscopically. Villi were photographed with Nikon spot camera and PixelPro software was used for all measurements (Brümmer et al. 2010).

Antibody titer against Newcastle disease virus
Antibody titer was tested against the Newcastle disease virus (NDV) using hemagglutination inhibition test (HI). After 7 days of vaccination, 2 ml fresh blood was collected from the wing vein of the birds in a sterile way and transferred to the vacutainer. The (Newcastle Virus) suspension was prepared with a known HA titer. 0.025 ml of phosphate saline (PBS) solution was distributed in each well of the microtiter plate. 0.025 ml of serum was placed in the rst well. Then the dual serial dilution was made through this suspension across the plate. After that, 0.025 ml of 4HAU of virus/antigen was added to each well and the plate is left for 30 minutes at room temperature. Prepare 1% (v\v) of the chicken RBCs by adding 100ml of PBS into 1 ml of suspended RBCs. Then add 0.025 ml of 1% (v/v) of the chicken RBCs, to each well and mix gently. Red blood cells (RBCs) were allowed to settle for 40 minutes at room temperature. HI titer was the highest serum dilution causing complete inhibition of 4HAU (Shahir et al. 2014).

Statistical analysis
Data were analyzed using SPSS 21.0 software (SPSS Inc., Chicago, IL, USA). Effects of mannanoligosaccharide supplementation on gut health, immunity, and production performance of broilers were analyzed using one-way ANOVA. The signi cance level was set at 5% and calculated using Duncan's multiple range test. The data were presented as the means ± standard deviations.

Average weekly feed intake
Results shown in Table 3 reveals the effect of MOS supplementation on average weekly feed intake in broilers at 5 consecutive weeks. Results showed that supplementation of MOS signi cantly (P < 0.05) affected the feed intake. At 1st week, highest average feed intake (FI) was observed in T5 followed by T3, T2 and T1 groups. At 2nd week of age, higher FI was seen in T6 group followed by T5 and T1. However, at 3rd week, elevated FI was observed in T6 and T3 followed by T2 and T1. At 4th week, highest FI was seen in T6 followed by T2. At the end of trial, highest feed intake was calculated in T4 followed by T6 and lowest FI was observed in T1.

Average weekly weight gain
Results presented in Table 4 shows the outcome of MOS on average weekly body weight gain (BWG) in broilers at 5 consecutive week intervals. Supplementation of MOS signi cantly (P < 0.05) affected the BWG in broilers.
During 1st week, higher BWG was observed in T6 followed by T3 group. During 2nd week, higher BWG was seen in T6 followed by T3. During 3rd week of age, higher BWG was found in T3 and T6 followed by T1, T4 and T5 groups. At 4th week of trial, birds reared on T6 showed signi cantly (P < 0.05) highest body weight gain followed by T5, T4, T3 and T2. At last week, higher BWG was recorded in T6 and T4 group and no signi cant difference was observed between T1, T2, T3 and T5 groups (P > 0.05).

Feed conversion ratio
The Table 5 demonstrates the impact of MOS supplementation on weekly feed conversion ratio.
Supplementation of MOS signi cantly affected FCR in broilers (P < 0.05). 1st week data showed that birds of T6 group had the best FCR followed by T3, T2 and T1. Similarly, at the end of 2nd week best (P < 0.05) value of FCR was found in T6 followed by T4 and T3. At the end of 3rd week, best value for FCR was calculated in T6 and T3 followed by T5 and T4. At 4th week, birds of T6 group showed best FCR. However, at the end of trial, best FCR was calculated in T6 group followed by T4 and T2 (P < 0.05).

Length of different intestinal sections
Results presented in Table 6 reveals the impact of MOS addition on the length of duodenum, jejunum and ilium. Results showed that MOS signi cantly (P < 0.05) affected the length of intestinal sections. At the end of trial, birds supplemented with T2 group have signi cantly (P < 0.05) highest length of duodenum followed by T4. Smallest length of duodenum was found in negative control group T1. Highest jejunum length was found positive control group T2 followed by T5 and lowest values for jejunum length was found in T6. Similarly, signi cantly (P < 0.05) highest ilium length was found positive control group and T5 followed by T4 and T3.
Lowest values for ilium length were found in negative control group.

Histomorphological parameters
Results presented in Table 7

Antibody titer against NDV
Data presented in Table 8 shows the impact of MOS supplementation on antibody titer against NDV. Supplementation of MOS signi cantly (P < 0.05) affected the antibody titer against NDV. Birds fed on T6 and T5 showed signi cantly (P < 0.05) highest antibody titer against NDV. Lowest values of the titer were found in negative control group. Supplementation of MOS signi cantly affected the antibody titer against NDV. Birds fed on T6 and T5 showed highest antibody titer against NDV. Lowest values for titer were found in negative control group. Similar results were also published by Shahir et al. (2014) as they observed that MOS supplemented group gained higher antibody titer against NDV as compared to control group but lower than probiotic group. Results of Muhammad et al. (2020) were also similar to our study they recorded that MOS supplemented group has signi cantly higher antibody titer against NDV as than control group but lower than probiotic group. Similar results were also found by Waqas et al. (2019) as MOS supplemented group showed higher antibody titer against NDV as compared to control group.

Conclusion
The results indicated that birds fed with diets containing MOS, exhibited better productive performance in comparison to positive and negative control group. In conclusion, MOS can be used in place of AGPs as nonmicrobial performance-enhancing feed advocates and can play a part in minimizing the irrational use of antibiotics in poultry feed.

Funding
No funding was received.