Effects of multi-enzyme supplementation in a corn and soybean meal-based diet on growth performance, apparent digestibility, blood characteristics, fecal microbes and noxious gas emission in growing pigs

Abstract

The objective of this study was to determine the effect of multi-enzyme supplementation in a corn and soybean meal-based diet on the growth performance, apparent nutrient digestibility, blood profile, fecal microbes and noxious gas emission in growing pigs. A total of 80 crossbred [(Landrace ${\times}$ Yorkshire) ${\times}$ Duroc] growing pigs with an average body weight (BW) of $25.04{\pm}1.44kg$ were used in a 6-week experiment. The experimental treatments were as follows: CON, basal diet and; T1, basal diet + 100 mg/kg multi-enzyme. During the experiment, the pigs fed the diet with multi-enzyme supplementation had a higher gain to feed ratio (G/F) (p < 0.05) than the pigs fed the diet without multi-enzyme supplementation. On day 42, the pigs fed the diet with multi-enzyme supplementation had decreased $H_2S$ and $NH_3$ emissions (p < 0.05) than the pigs fed the diet without multi-enzyme supplementation. However, no effect was observed on nutrient digestibility, blood profiles and fecal microbes among the treatments (p > 0.05). In conclusion, it is suggested that multi-enzyme supplementation in a corn and soybean meal based diet can partly improve the growth performance and noxious gas emission of growing pigs.

Introduction

Enzyme products have been extensively evaluated in poultry, cattle and swine diets focusing on enzyme preparations, the physiologicalstatus of the animal and feed ingredient. Beneficial effects of addition of single or multiple enzyme preparations, such as cellulase, β-glucanase, α-amylase, β-mannanase, xylanase, and protease to diets fed to swine have been reported (Ngoc et al., 2011; O’Shea et al., 2014; Passos et al., 2015; Upadhaya et al., 2016a; Upadhaya et al., 2016b). Corn has been considered as the best quality grain especially for pig. In addition, the response and its degree due to dietary enzyme supplementation have been negligible or relatively less to other grains. Since the primary non-starch polysaccharides (NSP) in corn is cellulose, a structural insoluble fiber, enzymes hydrolyzing soluble fiber did not respond well upon supplementation. Therefore, cellulase and protease supplementation to swine diet was proven more effective compared to other enzymes without cellulase (de Souza et al., 2007). In addition, Willamil et al.(2012) showed that multi-enzyme (xylanase and β-glucanase as main activities) supplementation to wheat-based diet improved nutrient utilization and growth performance in growing pigs. However, mannanase-only supplementation to corn based diet was failed to improve feed to gain ratio (F/G), but the F/G was improved by multi-enzyme supplementation including cellulase as well as mannanase in nursery pig (Ragland et al., 2008). On the other hand, mannanase supplementation to distillers dried grains with solubles (DDGS) included pig diet was effective to improve average daily gain (ADG) (Yoon et al., 2010), probably due to relatively higher mannan in DDGS than corn grain. Owing to decreased fiber content and high nutrient digestibility, it has beenmore challenging to obtain beneficial effectsfor corn and soybeanmeal (SBM) dietsthrough the exogenous enzymes supplementation.

Most of the available information on the application of multi-enzyme has been generated from younger pig studies. In addition, older pigs appear less effective because older pigs are more able to digest fiber than younger pigs (Ao et al., 2010). The objective of the currentstudy wasto examine the effects of multi-enzyme (protase, cellulase, β-glucanase, β-mannanase, xylanase, and amylase) supplementation on growth performance, apparent nutrient digestibility, blood characteristics, fecal microbial, and noxious gas emission in growing pigsfed corn and SBM diet.

Materials and Methods

The experimental protocol used in thisstudywas approved by theAnimalCare andUseCommittee ofDankookUniversity. The multi-enzyme was provided by commercial company (Da-Sion Product Co., Ltd., Busan, Korean). The multi-enzyme contained 180 units/g protase, 5,826 units/g cellulase, 2,677 units/g β-glucanase, 518 units/g β-mannanase, 6,299 units/g xylanase, and 1,624 units/g amylase.

Experimental design, animal, and housing

A total of 80 crossbred [(Landrace × Yorkshire) × Duroc] growing pigs with an average body weight (BW) of 25.04 ± 1.44 kg were used in a 6-week experiment. Pigs were randomly assigned into one of the two experimental diets according to BW. Pigs were housed in groups of five barrows per pen with eight replicates per treatment. Pigs were housed in an environmentally controlled facility and room temperature was maintained at approximately 24°C. Each pen (1.2 × 1.6) was equipped with a self-feeder and nipple waterer to allow ad libitum access to feed and water throughout the experimental period. The experimental treatments included: CON, basal diet; T1, basal diet + 100 mg/kg multi-enzyme. All diets were provided in mash form and formulated to meet or exceed the NRC (2012) recommendation for all nutrients and regardless of treatments (Table 1).

Table 1. Composition of the basal growing diets (as-fed basis; g kg^-1).

Sampling and measurements

BW and feed intake were recorded initially and week 6 of the experiment period. Feed consumption was recorded on a pen basis during the experiment to calculate the ADG, average daily feed intake (ADFI) and gain to feed ratio (G/F). Chromic oxide (Cr₂O₃) was added to the diet as an indigestible marker at 2 g/kg of the diet for 7 days prior to fecal collection at 6th week to calculate dry matter (DM) and nitrogen (N) digestibility. Fecalsamples were collected randomly from 2 pigsin each pen, mixed and pooled. All fecalsamples, as well asfeed samples, were stored at - 20°C until analysis.

Before chemical analysis, the fecalsamples were thawed and dried at 60°C for 72 h, after which they were ground to pass through a 1-mm screen. The procedures used for determination of DM and N digestibility were in accordance with the methods established by AOAC (2005). N was determined using a Kjeltec 2300 Nitrogen Analyzer (Foss Tecator AB, Hoeganaes, Sweden). Crude protein was calculated as N × 6.25. Dietary DM (method 930.15), crude protein (method 968.06), calcium (method 984.01), phosphorus (method 965.17), crude ash (method 942.05), ether extract (method 920.39), and crude fiber (method 962.09) were analyzed according to the procedures described by AOAC (2005). Individual amino acid composition was measured using an Amino Acid Analyzer (Beckman 6300, Beckman Coulter Inc., Fullerton, USA) after a 24 h for 6 N-HCl hydrolysis at 110°C. Chromium was analyzed by ultraviolet absorption spectrophotometry (UV1201; Shimadzu, Tokyo, Japan) according to the methods of Williams et al. (1962). The digestibility was then calculated using the following formula: Digestibility (%) = [1 – {(N_f × C_d )/ (N_d × C_f )}] × 100, where Nf = nutrient concentration in feces (%DM), C_d = chromiumconcentration in diets (%DM), N_d = nutrient concentration in diets (%DM), and C_f = chromium concentration in feces (%DM).

For the blood profile, four pigs were randomly selected from each treatment and bled via jugular venipuncture and added to heparinized tubes for blood urea nitrogen (BUN) analysis, and non-heparinized tubes for serum creatinine analysis at the 6th week ofthe experiment, respectively. After collection, BUN concentration was analyzed using the Abbott Spectrum urea nitrogen test (Series II, Abbot Laboratories, Dallas, USA). Creatinine concentrations were determined using an Astra-8 Analyzer (Beckman Instruments, Inc., Brea, USA).

For fecal microbial, fecalsamples were collected directly by massaging the rectum of 2 pigsrandomly selected from each pen on day 42, and transported to the laboratory. The obtained fecalsample (1 g) from each pen was diluted with 9 mL of 10 g L^-1 peptone broth (Becton, Dickinson and Co., Franklin Lakes, USA) and homogenized. Viable counts of bacteria in the fecal samples were then conducted by plating serial 10-fold dilutions (in 10 g L^-1 peptone solution) onto MacConkey agar plates (Difco Laboratories, Detroit, USA) and lactobacilli medium III agar plates (Medium 638, DSMZ, Braunschweig, Germany) to isolate the E. coli and Lactobacillus, respectively. The MacConkey agar plates and lactobacilli medium III agar plates were then incubated for 24 h at 37°C and 48 h at 39°C, respectively. The E. coli and Lactobacillus colonies were counted immediately after removal from the incubator.

Feces and urine were collected on d 42 from 4 pigs per treatment. The urine was collected in a bucket via a funnel below the cage. Samples were kept in sealed containers and were immediately stored at - 4°C for the duration of the period. After the collection period, feces and urine samples were pooled and each mixed well for each pen. The subsamples ofslurry (150 g feces and 150 g of urine were mixed well; 1 : 1 on the wet weight basis) were taken and stored in 2.61 plastic boxes in duplicate as described by Cho et al.(2008). Each box had a small hole in the middle of one side wall, which wassealed with adhesive plaster so as to maintain anaerobic condition. The samples were permitted to ferment for 7 d at room temperature (25°C). After the fermentation period, a Gastec (model GV-100) gas-sampling pump was utilized for gas detection (Gastec Corp., Gastec detector tube No. 3M and 3La for NH₃ and H₂S; No. 70 and 70L for R.SH (total mercaptan), Gastec Corp., detector tube, Japan). The adhesive plasters were punctured, and 100 mL of headspace air was sampled approximately 2.0 cm above the fecessurface.

Statistical analysis

All data were subjected to the general linear model (GLM) procedures of SAS as a randomized complete block design (SAS institute, 2001). Pen was used as experimental unit for growth performance, digestibility and fecal microbial, whereas pig was used as experimental unit for blood profile and noxious gas emission. Variability in data was expressed as standard error of means (SEM). Differences among all treatments were separated by using the Tukey’stest. A probability level of p < 0.05 was considered to be statistically significant.

Results

The effects of dietary multi-enzyme on growth performance, nutrient digestibility, blood profile, fecal microbial, and noxious gas emission were summarized in Table 2 - Table 6. However, there were no differences (p > 0.05) in ADFI, ADG, DM and N digestibility, blood profile and fecal microbial among all the treatments. During the experiment, pigs fed the diet containing multi-enzyme had higher G/F (p < 0.05) compared with pigs fed the diet without multi-enzyme supplementation. On day 42, pigs fed the diet containing multi-enzyme had decreased H2S and NH3 emission (p < 0.05) compared with pigs fed the diet without multi-enzyme supplementation.

Table 2. Efect of dietary multi-enzyme on growth performance in growing pigs.

Table 3. Efect of dietary multi-enzyme on apparent digestibility in growing pigs.

Table 4. Efect of dietary multi-enzyme on blood characteristics in growing pigs.

Table 5. Efect of dietary multi-enzyme on fecal microbial in growing pigs.

Table 6. Efect of dietary multi-enzyme on noxious gas emission in growing pigs.

Discussion

Growth performance

The results of the current study indicated that multi-enzyme supplementation affected on G/F but had no influence on ADFI and ADG among all treatment groups. Our findings are similarly with a study conducted by Omogbenigun et al.(2004), who reported that an improvement in ADG and G/F had been observed in piglets fed diets based on corn and wheat supplemented with an enzyme cocktail containing cellulase, galactanase, mannanase and pectinase. Besides, Ao et al.(2010) observed that ADG and G/F was increased with 100 mg/kg enzyme complex (α-1,6-β-galactosidase, β-1,4-mannanase, and β-1,4-mannosidase) supplementation to corn and SBM based diets for growing pigs. However, the results are not always consistent. In a study with rape seed meal and DDGS-based dietssupplemented with xylanase and β-glucanase enzymes, Mc Alpine et al. (2012) found no improvement in ADFI, ADG and feed conversion efficiency of grower-finisher pigs. The contradictions in the impact of multi-enzyme supplementation on growth performance may be attributed to the differences in the diets composition, and the age of pigs used. In addition, the enzyme source, the situations under which the specific ingredient was grown, the storage and process of feed, the interactions among dietary compositions and health status may also exert a significant effect on growth performance (Kim et al., 2003; Willamil et al., 2012).

Nutrient digestibility

Previous reports indicate that different exogenous enzyme supplementation in swine diet can enhance the digestibility, leading to improved growth performance and nutrient digestibility (Nyachoti et al., 2006; Kiarie et al., 2013). A study by Omogbenigun et al.(2004) demonstrated that multi-enzyme (cellulase, galactase, mananase, and pectinase) having activity when supplemented to corn and SBM diet on the digestibility of nutrients in both ileum and total tract in piglets. In contrast, Wubben et al. (2000) found that multiple enzymes (cellulase, hemicullulase, amylase, xylanase, alpha-galactosidase, and protease) supplementation in corn and SBM diets did not exert benefits on nutrient digestibility of growing pigs. In addition, the ileal digestibility of DM and N were not affected in growing pigs fed corn and SBM diet supplemented or not with α-galactosidase (Smiricky et al., 2002). In the current experiment, there were no differencesin DM and N digestibility among all the treatments.

The apparent contradictions in the effectiveness of enzyme supplementation among studies are mainly attributable to differences in age of the pigs and the composition of diets used (Omogbenigun et al., 2004). In general, the impact of enzymes supplementation on nutrient digestion declines with age of the pig particularly, because digestive capacity in pigs improves with age as the enzyme system matures and gut microbial population increases (Lindemann et al., 1986). Older pigs have a more mature gastrointestinal system, increasing the ability of the gut to digest cereal components of the ration through the effects of both pancreatic enzyme section and bacterial fermentation. Also, the extent to which enzymes supplementation improve nutrient digestibility tend to be low when using diets containing highly digestible ingredients (Johnson et al., 1993). From a practicalswine nutrition perspective, it would appear thatsupplementing enzymesto pig diets will be more beneficial when using diets based on ingredientsthat are of lower quality and poorly digested.

Blood profile

The BUN and creatinine values generally help to assess renal damage in animals and humans. Creatinine and urea both are metabolic wastesthat enter into the bloodstream and are discharged out by kidneys. When kidney filtration rate declines, creatinine and urea levels in the blood increase spontaneously (Kaneko et al., 2008). Previous studies showed that addition of multi-enzymes to the diets had no significant effect on blood constituents (Wang et al., 2009; Ao et al., 2010), which is in agreement with the result of the current study. Moreover, Wang et al. (2009) reported that BUN was not affected by the addition of the enzyme cocktail (α-1,6-β-galactosidse, β-1,4-mannanase and β-1,4-mannosidase) to corn-SBM diets. Considering the above results, in our study, it indicates that the addition of multi-enzyme to corn-SBM diet results in a diet with the same protein quality as the control diet. To our best knowledge, a few experiments have been conducted to compare the BUN or creatinine coefficients ofmulti-enzyme containing corn-SBMdietsin pigs. However, the results ofthisstudy are not adequate to conclude that corn-SBM diets supplemented with multi-enzyme have inferior nitrogen balances when compared to nonmulti-enzyme diets. Therefore, furtherstudies are necessary to investigate the carcasstrait amino acid profile of pigs provided with the experimental diets used in this study.

Noxious gas emission

When dietary protein is not fully utilized by growing pigs can result in a build-up of N-rich compounds in the resulting manure stimulating NH₃ and odor emissions (Nahm, 2003; Dourmad and Jondreville, 2007). In the current study, the fecal NH₃ and H₂S concentration was significantly decreased by multi-enzyme supplemented diet. Similarly, Mc Alpine et al. (2012) reported that finisher pigs offered protease-xylanase supplemented diet had reduced NH₃ emissions. In contrast, Atakora et al.(2011) founded that growing finishing pigs fed phytase-xylanase supplementation of wheat grain based diets had no effect on gas emission. The large differencesin age and diet might be the reasonsfor the differencesin results across these inconsistent reports. Ferket et al. (2002) indicated that nitrogen gaseous emissions are related to intestinal microbial ecosystem and nutrient utilization. From ourstudy, reduction of NH₃ and H₂S emission could possibly be due to a reduction of the pathogenic bacterial population in the gastrointestinal tract or due to enhancement of beneficial microbial activity (Dibner and Buttin, 2002; Upadhaya et al., 2016a). Microbial fermentation of undigested proteins and amino acids in the hindgut produces NH3 and contributesto the NH3 output in the manure (Gaskins, 2000). Therefore, the lower fecal NH₃ and H₂S concentration in the multi-enzyme supplementation on pigs may indicate better digestion of dietary proteins and amino acids. This is through limiting the availability of non-digested proteins, which then serve as substrate for NH₃ production in the large intestine (Tactacan et al., 2016). However, no comparisons could be made with other studies because there was a scarcity of information on the effects of multi-enzyme supplementation on fecal noxious gas emission in pigs. Further research is necessary to elucidate the important factors involved.

Conclusion

Multi-enzyme supplementation was effective in enhancing G/F. Besides, fecal NH₃ and H₂S emission were reduced by enzymes supplementation. This suggested that multi-enzyme be utilized in corn-SBM diets had positive effects on growing pig performance.