The effect of using mannan oligosaccharides extracted from fermented palm kernel cake and cassava by-product mixture in ration on broiler gut profile

Nurhayati^1,2, Hartutik³, Osfar Sjofjan³ and Eko Widodo³

¹ Faculty of Animal Science, University of Brawijaya Malang, East Java, Indonesia
hartutik@ub.ac.id
² Animal Science Department, Lampung State Polytechnic (Politeknik Negeri Lampung), Indonesia
³ Lecturer of Nutrition and Feed Department, Faculty of Animal Science, University of Brawijaya Malang, East Java, Indonesia

Abstract

Mannan oligosaccharides (MOS) are prebiotics that function to improve the balance of microflora, which is beneficial for the health of the broiler gut. This research evaluated the effect of MOS (extracted from fermented palm kernel cake (PKC) and cassava by-product (CB) mixture) and commercial MOS used in the diet and the effect of MOS feeding time nested to MOS use on broiler gut profile. Two hundred and eighty eight day old chick (DOC) broilers were reared until five weeks and applied with nine treatments that consisted of CTL without using MOS, MOS-F using extracted MOS and MOS-C using commercial MOS) and three systems: treatments in starter phase -S, finished phase -F and both starter and finisher phases -SF. Each treatment was repeated with 4 replications.

The use of MOS (both MOS-F and MOS-C) improved the gut profile of broilers the number of Lactobacillus sp., and of Escherichia coli, the pH, and viscosity of gut digesta, the villi surface area and intestinal mucosal surface area of the broilers. MOS supplementation from starter to finisher gave better results than in starter or finisher separately.

Keywords: Escherichia coli, Lactobacillus sp, prebiotic, villi surface area

Introduction

Mannan oligosaccharide (MOS) is a prebiotic which functions in improving microflora balance in the poultry gut by preventing pathogenic bacteria colonisation, so that non-pathogenic bacteria can develop well with resultant increase in digestion and absorption of nutrients and the growth of broilers.

MOS is useful for probiotic bacteria in the gut. Probiotic bacteria digest MOS by fermentation which results in volatile fatty acids (VFA) such as acetate, butyrate and propionate. In addition, probiotic bacteria also produce lactic acid. The acetate and lactate acid content produced by probiotic impact on the decrease of gut pH such that acid conditions is the gut will hamper the growth and development of pathogenic bacteria. Butyric acid as one of the MOS fermentation products that play a role in epithelium cell growth and differentiation and increase the intestine cell proliferation index. This is useful in the healing process after trauma, infection, and intestine inflammation. This also has positive effect in repairing the intestine such as increasing height and width of villi. The increase of villi height and width impacts on increasing of villi surface area and intestine mucosa area and finally increases the absorption efficiency of nutrients. The use of MOS has been investigated in broilers (Hooge 2004a), turkeys (Hooge 2004b), pigs (Miguel et al 2004) and rabbits (Bovera et al 2012).

Research on MOS increasing villi height of the gut meaning increasing fermentation enzyme activities of non-pathogenic bacteria have been reported (Yang et al 2007; Baurhoo et al 2009; Sinovec et al 2005). Research on the effect of Bio-MOS (commercial MOS) in improving microbiology and pH of broiler gut has been investigated by Yang et al (2008).

Mannan oligosaccharides can be produced from several raw materials such as extraction from the outer cell walls of Saccharomyces cerevisiae and physical or chemical extraction from palm kernel cake (PKC). Our previous research had succeeded in extracting MOS from products of fermentation of PKC and cassava by-product (CB) mixtures. This research (Nurhayati et al 2018) showed that using MOS extracted from fermented palm kernel cake (PKC) and cassava by-product (CB) mixture could decrease the number of pathogenic bacteria (Salmonella sp. and Escherichia coli) and increase non-pathogenic bacteria (Lactobacillus sp.). Against this background, the present study aimed to apply the use of MOS extracted from PKC-CB mixed fermentation products as prebiotics in the diet of broilers and specifically to study the effect on the profile of the broiler gut.

Materials and methods

This research was done in a farm in Malang Regency, East Java, Laboratory and in the Microbiology Department of the Central Laboratory of Life Sciences and the Laboratory of Histology, Faculty of Medicine, University of Brawijaya, Malang-Indonesia. The materials used were the extracted MOS from fermented PKC-CB product (Nurhayati et al 2018) and commercial MOS both a at 4000 ppm of the diets (Table 1 and Table 2).

Broiler chicks (n= 280) were raised in two stages: 1-3 weeks (starter period) and 4-5 weeks (finisher period). ND vaccine was given at 4-day-old by eye drops and at 21 days of age in the drinking water.

The treatments in a 3*3 nested factorial consisted of CTL without using MOS, MOS-F using extracted MOS and MOS-C and the three systems when the treatments were applied: in starter phase -S, finisher phase -F and both starter and finisher phases -SF. Each treatment was repeated with 4 replications. The chicks ere raised in cages (1x1 m) on the floor with litter.

The variables observed included:

The number of pathogenic bacteria (Salmonella sp. and Escherichia coli) and non-pathogenic bacteria ( Lactobacillus sp.) in gut digesta, based on Sari et al (2013) method.

Viscosity and pH of gut digesta according to McDonald et al (2001) and Mirzaie et al (2012), respectively.

Intestinal villi surface area (µm²/villi) and intestinal mucosal surface area (µm²/µm²). Villi surface area was based on the method of Iji et al (2001) and the intestinal mucosal surface area according to Drozdowski et al (2005).

Data were tabulated with the Microsoft Excel program and analyzed based on the Completely Randomized Nested Design. If there was a treatment effect, further testing was done by Duncan's Multiple Range Test according to Steel and Torrie (1989). The data were analyzed using the R program with the Agricolae package (de Mendiburu 2017).

Results and discussion

Lactobacillus sp.

The numbers of Lactobacillus sp. of broilers fed with MOS either commercial MOS or extraction MOS were higher compared to those fed with control ration. This is caused by the MOS action to suppress harmful bacteria by binding to the fimbriae of pathogenic bacteria and being excreted with feces. The decrease in the population of pathogenic bacteria resulted in reduced competition to obtain nutrients compared with non-pathogenic bacteria (Lactobacillus sp.). This condition has a positive impact enabling Lactobacillus sp. to grow and develop better, so that the amount of Lactobacillus sp. increased in the digestive tract of the chicken fed MOS higher than in birds fed the control diet. These results are in accordance with the findings of Baurhoo et al (2007) who reported that the amount of Lactobacillus sp. of broilers fed with MOS is higher than those fed with control diet. Furthermore, Koc et al (2010) reported that the number of Lactobacillus sp. in broiler gut of birds given Saccharomyces cerevisiae and MOS in the diet was higher than in those fed with control diet.

As expected, the continuous feeding of MOS from starter to finisher gave highest counts of Lactobacillus sp. MOS is useful for lactic acid bacteria (Lactobacillus sp.). Lactic acid bacteria ( Lactobacillus sp.) are able to ferment MOS and produce nutrients that further stimulate the growth and development of Lactobacillus sp. bacteria. Baurhoo et al (2007) stated that the high Lactobacillus sp. numbers and other beneficial bacteria in the digestive tract contribute significantly to the increase in height of intestinal villi height and cause the digestive tract to become healthier. This condition can increase the absorption of nutrients in the feed consumed by chickens which in turn can increase the growth rate.

Escherichia coli

Birds fed the control ration had the highest number of E. coli (Table 4) This is due to the function of MOS which can suppress pathogenic bacteria (Escherichia coli) in the broiler gut. The MOS acts as a high binding power molecule to attach to fimbriae type-1 pathogenic bacteria. The attachment of sugar mannose to fimbriae type-1 of pathogenic bacteria inhibits the attachment of the pathogenic bacteria to intestinal epithelial cells and so they are removed from the digestive tract along with the feces. Therefore, the presence of MOS can prevent the colonization of pathogenic bacteria in the chicken's digestive tract (Bozkurt et al 2008). These results agree with those of Baurhoo et al (2007) who stated that the number of Escherichia coli in chickens fed with MOS is less than in chickens fed with control diet. Koc et al (2010) also reported that the number of Escherichia coli in intestine of chicken supplemented with Saccharomyces cerevisiae and MOS was lower compared with those fed the control diet.

Broilers given MOS from starter period until finisher in both extraction and commercial MOS use gave effect to the lowest number of Ecoli (8.03x10³ cfu/ml for M₁ and 8.21x10³ cfu/ml for M₂) and were different (p<0.05) from those given MOS during finisher period (9.56x10³ cfu/ml for M₁ and 10.52x10³ cfu/ml for M₂) and those given MOS at starter (10.18x10³ cfu/ml for M₁ and 8.86x10³ cfu/ml for M₂). This was due to the presence of MOS in the digestive tract which can continuously lead to a stable balance of microflora population in the broiler digestive tract. The stability of broiler microflora balance gave a positive effect on broiler gastrointestinal health and improvement of broiler performance. Meanwhile, the number of Escherichia coli of gut of broiler given MOS during the finisher phase on the use of extraction and commercial MOS were the highest compared to those given MOS from starter to finisher and at starter phase only. This is maybe caused by the provision of MOS at finisher phase is not sufficient to reduce pathogenic bacteria that have already developed in the digestive tract.

pH

The effect of MOS use in the ration and MOS feeding time period in each MOS type use on broiler intestine pH are presented in Table 5. The result of variance analysis showed that the treatment of MOS use in ration influenced (p<0.05) the pH of broiler gut, while the treatment of MOS feeding time period in both extraction and commercial MOS use did not affect (p> 0.05) on the pH of broiler gut. Based on Table 5 it can be seen that control ration gave effect to the highest pH of gut digesta of broiler (6.72) and differed (p<0.05) from that of extraction MOS (6.57) and commercial MOS (6.52). Meanwhile, pH of gut digesta of broiler fed with extraction MOS was not different (p >0.05) from those fed with commercial MOS. The results of this study are in accordance with the results of Markovic et al (2009) who reported that pH of gut digesta of chicken fed with diet containing MOS was lower than those fed with control diet.

Chickens fed with commercial and extracted MOS resulted in lower pH of gut digesta compared to those fed with control diet. MOS is a material that can be fermented by beneficial bacteria, especially lactic acid bacteria in the gastrointestinal tract of broiler. MOS fermentation products include organic acids such as acetate, propionate, butyrate and lactic acid. Acetate, propionate, butyrate and lactic acid affected on decreasing pH of gastrointestinal tract (Rachmawati et al 2005; Samal and Behura, 2015). Acid conditions in the gastrointestinal tract resulted in inhibition of growth and development of pathogenic bacteria, thereby reducing the colonization of pathogenic bacteria in the gastrointestinal tract of chicken (Macfarlane et al 2008; Bozkurt et al 2008).

Viscosity

The effect of the use of MOS in rations and the period when giving MOS for each type of MOS on the viscosity of broilers is presented in Table 6. The result of variance analysis showed that the treatment of MOS use affected (p<0.01) viscosity of gut digesta of broiler, so did the treatment of the MOS feeding time period affected (p<0.05) viscosity of gut digesta of broiler. Based on Table 6 it can be seen that the control ration gave an effect on the highest viscosity of broiler gut digesta (3.54) and differed (p<0.05) from that of extraction MOS (2.29) and commercial MOS (1.86). Meanwhile, the viscosity of gut digesta of broiler fed ration containing extraction MOS was not different (p>0.05) from that of broiler fed ration containing commercial MOS. MOS feeding time period in the use of either extraction or commercial MOS affected (p<0.05) on viscosity of broiler gut digesta. Viscosity of gut digesta of broiler given MOS at finisher both in extraction and commercial MOS use was the highest although its effect was not different from those given MOS at starter, but different (p<0.05) from those given MOS from starter to finisher.

The viscosity of gut digesta of broiler fed either with extraction or commercial MOS was lower than those fed with control ration. This is due to the benefits of MOS in maintaining the stability of the balance of microflora in the digestive tract of broiler. The decrease in pathogenic bacteria as a result of the presence of MOS can increase the dominance of beneficial bacteria. Beneficial bacteria in the digestive tract can produce enzymes that help improve the digestive process of nutrition. Feed digestion process converts feed molecules from large sizes into small sizes. This decrease in feed molecules results in a decrease in viscosity of the gut digesta (Moftakharzadeh et al 2017). Decreased viscosity of the gut digesta makes it easier to absorb feed nutrients, which in turn increases the ADG of chicken (Barros et al 2015). Likewise, the viscosity of the gut digesta of broiler fed with MOS continuously from the starter to the finisher resulted in a lower viscosity compared to the viscosity of gut digesta of broiler given the MOS at the starter or finisher only.

Villi Surface Area

The average effect of MOS use in ration and MOS feeding time period on villi surface area of broiler intestine are presented in Table 7. The result of variance analysis showed that treatment of MOS use in ration affected (p<0.05) villi surface area of broiler intestine, while feeding time period of ration containing MOS did not influence ( p>0.05) villi surface area in each of MOS use. Table 7 showed that birds fed with ration containing commercial MOS had the highest villi surface area (1754.99 µm²), although the effect did not differ ( p>0.05) from those fed ration containing extraction MOS (1669.73 µm²). Villi surface area of birds fed ration containing either commercial MOS or extraction MOS were higher (p<0.05) than those fed with control ration (1401.10 µm²). This result agree with the result of Baurhoo et al (2007) who reported that intestine villi height which related to villi surface area of intestine of birds fed ration containing MOS was higher than those fed with control ration. Broilers that were given MOS from starter to finisher in the use of extraction MOS or commercial MOS tended to have the highest effect (1786.93 µm² on M₁ and 1948.36 µm² in M₂ ) on the area of the intestinal villi, although the effect was not statistically different (p>0.05) from broilers given MOS during the starter period (1709.89 µm² at M₁ and 1561.15 µm² in M₂) and from broilers given MOS at finisher time (1512.38 µm² at M₁ and 1755.45 µm² in M₂).

The area of intestinal villi of broiler fed either with commercial or extraction MOS was higher than those fed with control ration. This is caused by the function of MOS which can reduce the damage of cells of intestinal wall and renew cells in the digestive tract. In addition, the energy obtained from nutrients of feed consumed is used for cell growth, thereby increase the area of intestinal villi (Zikic et al 2011). MOS also functions to protect the intestinal mucosa from the incidence of attaching pathogenic bacteria and carrying pathogenic bacteria to get out of the digestive tract with feces, so that beneficial bacteria (probiotics) can develop better.

Broilers that were given MOS starting from the starter to the finisher period on the use of extraction and commercial MOS, although statistically not different, tend to produce the highest villi surface area compared to those given MOS only during the starter or finisher period. Giving MOS starting from the starter period to the finisher has an effect on maintaining the balance of the microflora in the digestive tract. Domination of beneficial bacterial populations in the digestive tract can improve the integrity of the small intestine such as increasing villi height and villi width. The dominance of beneficial bacteria in this study was proven by the amount of Lactobacillus sp. which was higher than Escherichia coli (Table 3 and Table 4), especially in the gut of chicken that received MOS treatment from starter to finisher, namely 13.45x10¹⁰ cfu / ml at M₁ and 15.73x10¹⁰ cfu / ml at M₂ (Lactobacillus sp.) and 8.03x10³ cfu / ml at M₁ and 8.21x10³ cfu / ml at M₂ (Escherichia coli). Increased height and width of villi resulted in increased villi surface area, and could have an effect on the more effective absorption of feed nutrients and the higher performance of chickens.

Intestine Mucosa Surface Area

Treatment effect of MOS use in ration and its feeding time in each MOS use on mucosa surface area of broiler intestine are presented in Table 8. The result showed that MOS use in ration and its feeding time period in each MOS use affected (p<0.01) intestine mucosa surface area of broiler. Birds fed with ration containing commercial MOS had the highest intestine mucosa surface area (26946.33 µm²) although they were not different from those fed with ration containing extraction MOS (24642.41 µm²), but both of them were different from those fed with control ration (17455.74 µm²).

Meanwhile, the effect of MOS feeding time period in the use of extraction and commercial MOS were different. Broilers that were given MOS from the start to the finisher on the use of MOS extraction (M₁) gave the highest effect on the surface area of the intestinal mucosa (27786.60 µm²) although the effect was not statistically different (p>0.05) from those given MOS at starter period (27212.62 µm ²), but different (p<0.05) from those given MOS at finisher phase (18927.99 µm²). Furthermore, the intestinal mucosal surface area of chickens given MOS at starter on the use of commercial MOS use was not different (p>0.05) from those given MOS at finisher, but both were lower (p<0.05) than those given MOS from starter until finisher (31712.45 µm²).

Intestinal mucosal surface area of chicken given ration containing either extraction or commercial MOS was higher than those given control ration. This is due to the function of MOS which can provide a residential stability for beneficial microflora in the digestive tract. This situation causes cells in the intestinal lining (enterocytes) to live or last longer before cell turnover and ultimately increases villi height and broadens the intestinal mucosa and increases absorption of feed nutrients (Markovic et al 2009; Zikic et al 2011).

The results also showed that chickens given MOS from the start to the finisher in the use of extraction or commercial MOS had a higher intestinal mucosal surface area compared to those fed ration containing MOS at starter or finisher only. This means that the continuous administration of MOS from the starter to the finisher gives the effect of expanding the surface of the chicken intestine. MOS can be fermented by probiotic bacteria (beneficial bacteria). MOS fermentation results by beneficial bacteria include organic acid acids such as butyric acid, acetic acid and propionic acid (Pan et al 2009). Butyric acid is useful as an energy source to help the growth and differentiation of epithelial cells and increase cell proliferation index in the intestine (Kinoshita et al 2002). This process benefits the healing of trauma, infection, and inflammation of the digestive tract and improves intestinal morphology such as increasing the height and width of the intestinal villi.

Conclusion

• The use of extraction Mannan oligosaccharides (MOS) and commercial MOS differed in influencing the amount of Lactobacillus sp. but not differed in influencing the number of Escherichia coli, pH, viscosity, villi surface area and intestinal mucosal surface area of broilers, and the effects of both extraction and commercial MOS were better than controls.
  
  
• MOS feeding time period from starter to the finisher in the use of either extraction or commercial MOS did not affect the pH and villi surface area, but the effect was better when compared to the administration of MOS during starter or finisher only on the amount of Lactobacillus sp. and Escherichia coli, the viscosity of gut digesta and the surface area of intestinal mucosa.

Implications

Implication of the results of this research is that extraction MOS could be an alternative apart from commercial MOS to improve gut profile of poultry. Research on the dosage of extraction MOS in ration is still needed to investigate the role of MOS in improving performance and the health of gut of poultry.

Acknowledgment

The authors gratefully acknowledge to Directorate General of Human Resource for Science, Technology and Higher Education of The Republic of Indonesia for funding PhD study at University of Brawijaya Malang, East Java, Indonesia.

Conflict of Interest

The authors declare that they have no conflict of interest.

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