Effect of dietary supplementation of β-mannanase on growth performance, carcass characteristics, excreta microflora, blood constituents, and nutrient ileal digestibility in broiler chickens

Objective The aim of the present study was to investigate the effects of dietary supplementation of β-mannanase on growth performance, carcass characteristics, excreta microflora, blood constituents, and nutrient digestibility in broiler chickens. Methods A total of 680 one-d-old Ross 308 (as hatched) broiler chickens were used in a 35-d growth assay. Chicks were sorted into pens with 17 birds/pen and 10 pens/treatment. Treatment diets were contained either 44% or 48% crude protein (CP) soybean meal (SBM) with or without β-mannanase. Results Using SBM containing 48% CP led to an improvement (p<0.05) in feed conversion ratio (FCR) from d 1 to 14. Addition of β-mannanase to the diets significantly improved body weight gain (BWG) and FCR from d 1 to 14. During overall experimental period, BWG was affected (p<0.05) by CP level of SBM and inclusion of β-mannanase, but FCR and feed intake were not affected. Carcass characteristics were not influenced by treatment diets. The results showed that digestibility of dry matter (DM), nitrogen (N), and energy was not affected by CP level of SBM and/or inclusion of β-mannanase. Among essential amino acids (EAA) apparent digestibility of valine, methionine, and leucine improved (p<0.05) by the addition of β-mannanase to the diets. The results demonstrated that ileal digestibility of DM, N, and energy was not affected by treatment diets. Among EAA, the ileal digestibility of valine and arginine was higher (p<0.05) in the diets containing 48% CP SBM and/or β-mannanase. Excreta Lactobacillus count increased (p<0.05) by the addition of β-mannanase to the diets. Blood urea nitrogen, creatinine, and total protein level were not affected by treatments. Conclusion Feeding chickens with diets containing 44% CP SBM resulted in detrimental effects on growth performance and digestibility of nutrients, but addition of β-mannanase to the 44% CP diet improved the growth performance of chickens without any effects on carcass characteristics.


INTRODUCTION
Soybean meal (SBM) is one of the most used protein sources in poultry and swine nutri tion around the globe. Relatively high levels of crude protein (CP), an excellent profile of amino acids (AA), and high AA digestibility are some reasons for the universal accept ability of SBM [1]. Similar to most plantderived feedstuff, SBM has some antinutritional factors such as trypsin inhibitor (TI) and nonstarch polysaccharides (NSP) that can limit the bioavailability of SBM protein. Heat processing is one effective approach to eliminate or reduce the level of TI [1]. It has been reported that higher content of fiber in processed SBM leads to lower growth performance of broiler chickens [2]. Researchers stated that there is a relationship between watersoluble xylose content of SBMs and improvement in weight gain when sunflower meal is used to replace some SBM [3]. It has been reported that SBM contains 1.3% βmannans [4]. βMannan, classified as a soluble NSP, is composed of sequential units of mannose complexed with galactose or glucose linked to βmannan backbone [47]. Due to the lack of endogenous NSP degrading enzymes in poultry and swine, NSP cannot be hydrolyzed and digested in their gut [8]. The only known way to digest them is through bacterial fermentation. It has been well documented that the addi tion of exogenous enzymes such as βmannanase, xylanase, αamylase, and βglucanase can alleviate the antinutritional effects of NSP [9,10]. Previously, other researchers reported the antinutritional effect of soybean oligosaccharides and soluble NSP in weaning piglets [11,12]. Reducing the fiber content of feedstuff and consequently reducing the concen tration of NSP can also be beneficial in improving the performance of poultry and swine. It has been suggested that removing soluble NSP and oligosaccharide by ethanol/ water extraction improved intestinal health and growth performance in weaning piglets [13]. Dehulling is another possible approach to reduce the NSP content of SBM. Re searches have shown that NSP can increase the viscosity of digesta, modify the physiology of gastrointestinal tract, and change the ecosystem in the gut [14]. It has been stated that a commonly found NSP in feedstuff is βmannan that can negatively affect the performance of animals [6]. How ever, there is some evidence showing that βmannan can stimulate the innate immune system and is potentially ca pable of stimulating nonproductive energy draining innate immune responses [1517]. Some researchers have sug gested that dehulled SBM contains a higher concentration of protein and a lower level of fiber compared to hulled SBM [18]. Therefore, the objective of this study was to in vestigate the effect of using dehulled SBM and supplementing the diets with βmannanase on growth performance, car cass characteristics, excreta microflora, blood constituents, and nutrient ileal digestibility in broiler chickens.

Animal care
The experimental procedures used in this trial were approved by the Animal Care and Use Committee of Dankook Uni versity (approval number DK11516).

Animals, diets, and management
In this experiment, 680 onedold Ross 308 (as hatched) broiler chicks with an average initial body weight (BW) of 43±0.54 g were used in a 35d experimental period. The chicks were sorted into pens (17 birds per pen and 10 pens per treat ment) and housed in battery cages (1.55×0.75×0.55 m/cage), in an environmentally controlled room (32°C to 24°C and 65% relative humidity). They were allowed free access to feed and water during the experiment. Each cage was equipped with two feeders (one feeder in each side) and two nipple drinkers.
The treatment diets were: i) T1, 44% CP SBM; ii) T2, T1 +0.05% βmannanase; iii) T3, 48% CP SBM, and iv) T4, T3+0.05% βmannanase. Four isocaloric diets were formu lated to meet Ross 308 nutrient recommendations (Table 1) [19]. Enzyme preparation used in this study was provided by a local manufacturer (CTC Bio Inc., Seoul, Korea). It was produced by using Bacillus subtilis (WL1) grown on Luria broth at guaranteed activity of 800 international unit/g (re ducing sugar assay). One IU is defined as the quantity of enzyme required to generate 1 μmol of reducing sugar/min, at pH 6.0 and 50°C [19]. According to the manufacturer's information, βmannanase was purified from crude solution produced by Bacilli optimized to produce only βmannanase.
The BW and remained feed were measured on d 0, 10, and 35 to calculate body weight gain (BWG), feed intake (FI), and feed conversion ratio (FCR). Mortality was recorded daily to allow the further correction of FCR. The finisher diet contained 0.2% of chromium oxide (Cr 2 O 3 ) as marker to allow determination of apparent retention (AR) and di gestibility of components.

Sampling
Excreta samples for AR assay; 10 samples per treatment) were collected in 3 consecutive days from d 32 to 35 and stored at -20°C until further analysis. On d 35, 160 birds (40 birds per treatment) were randomly selected (10 birds per treatment were bleed before euthanasia, samples were col lected from a wing vein into vacuum tubes) and euthanized by cervical dislocation. Breast muscle, liver, abdominal fat, and empty gizzard were weighed. Ileal digesta (portion of the small intestine from Meckel's diverticulum to approxi mately 1cm proximal to the ileocecal junction) were collected into sample bags and placed on ice and stored at -20°C.
Ileal digesta samples were weighed and then freezedried at -53°C for 72 h by freezedryer (FD5510, Freeze Dryer, Ilshin Lab, Dongducheon, Korea) after which they were finely ground to the size that could pass through a 1 mm screen. Chromium was analyzed by UV absorption spectro photometry (Shimadzu, UV1201, Shimadzu, Kyoto, Japan) [24]. The excreta samples were pooled and homogenized, and the moisture content determined by placing in an oven at 80°C for 48 h. Diet samples and airdried excreta samples were finely ground. All the samples were analyzed for dry matter (DM, method 930.15) [21], N (Leco N analyzer; FP 528; Leco, Saint Joseph, MI, USA), gross energy (GE; IKA bomb calorimeter, C5000; IKA Works, Wilmington, NC, USA), and Cr 2 O 3 . The AR of components was calculated [25] as followed: Where (NT/Cr) diet is ratio of component and chromium oxide in the diet, and (NT/Cr) excreta is ratio of component and chromium oxide in excreta. Component can be DM, N, or GE. The relative weights of breast meat, abdominal fat, and organs were expressed as percentage of live BW.
Serum samples were obtained by spinning blood samples at 3,000×g for 15 min at 4°C. Blood urea nitrogen (BUN), creatinine, and total protein concentrations were measured using an automatic biochemistry blood analyzer (Hitachi  747, Hitachi, Tokyo, Japan).

Statistical analysis
All data were subjected to statistical analysis in a completely randomized design, with a 2×2 factorial arrangement using general linear model procedures of SAS [26]. Each pen was used as an experimental unit. The main effects included dif ferent source of SBM and enzyme inclusion. The mean values and standard errors were reported. Probability values of less than 0.05 were considered as statistically significant.

Growth performance
Growth performance results are summarized in Table 3.
Using different SBM containing 44% CP or 48% CP in the diets did not significantly affect BWG from d 1 to 10. But supplementing the diets with βmannanase improved (p< 0.05) BWG during the starter phase. The results showed that feeding chickens with the diet containing SBM 48% CP significantly improved (p<0.05) FCR. It was also ob served that the addition of βmannanase to the diets improved (p = 0.05) FCR from d 1 to 10. The data showed that feed ing the chickens with the diets containing SBM with 44% CP resulted in lower (p = 0.02) BWG but addition of βman nanase to the diets improved (p = 0.02) BWG during the overall experimental period. Both FI and FCR were not significantly affected by treatment diets.

Relative organ weights
Results of the relative organ weights and breast meat yield are summarized in Table 4. The results showed that using  different SBM containing 44% or 48% CP or adding βman nanase to diets did not affect the relative weights of abdominal fat, liver, gizzard, and breast meat yield.

Nutrient digestibility
Results of AR of DM, N, and gross energy are shown in Table  5. The AR of DM, N, and GE were not significantly affected by treatment diets. Apparent ileal digestibility analysis also showed that DM, N, GE, and total essential amino acids (EAA) were not significantly influenced by dietary treat ments. Among EAA valine and arginine showed a tendency (p<0.10) to be affected by treatments. Feeding the chickens with the diets composed of dehulled SBM and adding βman nanase to the diets improved (p<0.05) the apparent ileal digestibility of valine and arginine (Table 5).

Excreta microbiota and blood constituents
The results of excreta microbiota assay presented in Table 4 showed that regardless of the CP level of SBM in the diet, addition of βmannanase significantly increased (p = 0.002) the count of excreta Lactobacillus; these results also indicated that there was a trend (p = 0.06) in reducing the count of ex creta Escherichia coli (E. coli).
Blood sample analysis showed that using different sources of SBM did not significantly affect the concentration of blood urea nitrogen, creatinine, and total protein. Supplementing diets with βmannanase tended to increase (p = 0.06) the level of serum total protein. The levels of BUN and creatinine were not significantly affected by the addition of βmannanase to the diets.

DISCUSSION
In general enzymes are added to feeds in order to augment low levels of natural endogenous enzymes or to add novel enzymatic systems not naturally produced by the bird [27]. Several researchers have reported that supplementing enzymes in the diets of broiler chickens improved overall performance [28 30]. It was also reported that replacing hulled SBM (44% CP) with dehulled SBM (48% CP) led to an improve ment in the performance of pigs [18]. Our findings in the current study showed that feeding broiler chickens with di ets containing hulled or dehulled SBM supplemented with exogenous enzyme improved BWG at the starter phase.
The results of the current study agreed with the results of previous studies showing beneficial effects of adding NSP degrading enzymes to the diet of broiler chickens [31]. Pre vious researchers reported that broiler chickens fed the diet supplemented with βmannanase showed 3.5% higher BW compared to the birds fed diets without βmannanase [32]. It has been reported that the content of βmannan in broiler chickens starter diet containing 35% dehulled SBM is approxi mately 0.44% [6]. The results of previous studies suggested that simple sugars released from this amount (0.44% βman nan) would not be enough to improve the performance of chickens [32]. They concluded that βmannanase supple mentation improved the apparent metabolizable energy (AME) of diets. The results of the current study were con  [4,6,33]. It has been reported that highly viscous nature of βman nans is the main reason for their adverse effect on the function of digestive system. The high viscous nature of βmannans results in lowering gastric emptying rate, restricting the contact of nutrients with absorptive epithelium [34].
Our findings showed that the addition of βmannanase to the diets composed of different sources of SBM did not affect the retention of DM, N, and GE. The content of βmannans in hulled and dehulled SBM was 1.7% and 1.6%, respectively, indicating that there was not a considerable difference be tween the content of substrate for βmannanase. The low amount of βmannans as the substrate of βmannanase might be the reason why the retention of nutrients was not signifi cantly improved. Previous researchers reported that mannans content of guar gum in broiler feed significantly increased intestinal supernatant viscosity [6]. It has been suggested that the effect of βmannanase in reducing intestinal viscosity was the solution to overcome the antinutritional effect of mannans [35]. Supplementing the diets with βmannanase resulted in preparing a suitable environment for the growth of Lactobacillus. It has been observed that supplementing broiler chicken diets with βmannanase improved AR and apparent ileal digestibility of DM during the starter phase [32]. It can be speculated that the addition of βmannanase to the diet of broiler chickens led to the generation of man nanoligosaccharides, mannotriose, and mannobiose, as well as a small amount of mannose [32] which might have sup ported the improvement in BWG during starter phase.
Other researchers supplemented the diet of broiler chickens with βmannanase and reported that addition of βman nanase did not influence the blood proteins (albumin α1, albumin α2, albumin β, and γglobulins) [36]. The results of current study agreed with previously published data. We did not observe any significant effect on BUN showing that source of SBM and/or addition of βmannanase to the diets did not influence the efficiency of nitrogen which is consistent with the results of previous studies [31,37]. In a trial with growing pigs fed with diets containing hulled or dehulled SBM with or without βmannanase the results showed that the count of excreta microbiota was not influenced by the source of SBM. They reported that addition of βmannanase reduced the count of excreta E. coli [38].

CONCLUSION
In conclusion, the results of the current study showed that supplementing the diet of broiler chickens with βmannanase improved their growth performance. Our findings indicated that dietary βmannanase could improve the gut environ ment in favor of beneficial bacteria such as Lactobacillus while reducing the activity of E. coli which ultimately resulted in improving feed efficiency and BWG especially during the starter phase which is the critical stage of commercial broiler chickens.