Supplementation of Non-Starch Polysaccharide Enzymes Cocktail in a Corn-Miscellaneous Meal Diet Improves Nutrient Digestibility and Reduces Carbon Dioxide Emissions in Finishing Pigs

Simple Summary The objective of the present study was to investigate the effect of the non-starch polysaccharide enzymes cocktail (NSPEC) on growth performance, nutrient digestion and gas emissions on finishing pigs. The addition of the NSPEC into a corn-miscellaneous meal diet improved feed conversion ratio and increased the apparent total tract digestibility of dry matter, neutral detergent fiber, acid detergent fiber, crude protein and gross energy of the finishing pigs. Furthermore, the digestible energy of the diet was also increased by the NSPEC supplementation in the diet. In addition, the inclusion of NSPEC in the corn-miscellaneous meal diet reduced carbon dioxide emissions of a finishing pig house. The accumulation of manure could increase the release of ammonia in a pig house. Abstract This study was carried out to evaluate the effect of the addition of the non-starch polysaccharide enzymes cocktail (NSPEC) on growth performance, nutrient digestibility and gas emissions in a corn-miscellaneous meal-based diet for finishing pigs. The NSPEC is a combination of cellulase, xylanase, β-glucanase, β-mannanase, α-galactosidase and pectinase optimized by assessing the in vitro dry matter digestibility (IVDMD) of corn-miscellaneous meal diet using an in vitro method of simulating digestion in the stomach and intestine of growing pigs. Growth performance and apparent total tract digestibility (ATTD) of nutrients and energy were measured. The gas concentration of ammonia, carbon dioxide, nitrous oxide and methane in the environmental assessment chambers were determined. The gas detecting period was divided into three frequencies of manure removal of every 1d, 2d and 3d. The addition of NSPEC into the corn-miscellaneous meal diet decreased feed conversation rate (FCR) and increased the ATTD of dry matter, crude protein, gross energy, neutral detergent fiber and acid detergent fiber of pigs (p < 0.05). The digestible energy was also improved (p < 0.05) significantly by NSPEC supplementation in the diet. Furthermore, the supplementation of the NSPEC reduced (p < 0.05) carbon dioxide concentration in the chambers. The ammonia emissions were significantly increased according to average 1d, 2d and 3d manure removal procedures (p < 0.01). These results indicated that the inclusion of optimal NSPEC in a corn-miscellaneous meal diet improved growth performance, nutrient digestibility and reduced carbon dioxide emissions on finishing pigs. The accumulated manure could increase the release of ammonia in a pig house.

test diets are summarized in Table 1. The feed was manufactured in dry mash form and formulated to meet or slightly exceed the nutritional requirements of finishing pig as recommended by the National Research Council [22]. The methodology for screening the NSPEC in a diet using an in vitro method of simulating digestion in the stomach and intestine of pigs was developed in our lab [5]. The optimal NSP enzyme cocktail in the corn-miscellaneous meal diet was 1002 U/kg cellulase, 18,076 U/kg xylanase, 1376 U/kg β-glucanase, 14,765 U/kg β-mannanase, 337 U/kg α-galactosidase and 138 U/kg pectinase in the corn-miscellaneous meal diet. The NSPEC was screened by assessing the IVDMD of the corn-miscellaneous meal diet using an in vitro method of simulating digestion in the stomach and intestine of growing pigs.
Sixteen crossbred barrows (Duroc × (Landrace × Large White)); initial body weight of 117.89 ± 0.85 kg; Beijing Breeding Swine Center, Beijing, China) were randomly allotted into 2 dietary treatments and each treatment had 8 replicates which were randomly divided into 2 environmental control chambers and each chamber had 4 pigs. Each environmental control chamber fed 4 pigs which were housed in stainless steel metabolic cages (1.2 m × 1.5 m). The daily feed allowance was calculated at 3.5% of the initial weight of pigs. Pigs were fed one-half of the daily feed allowance each at 8:00 and 15:00 per day and provided ad libitum access to water during the entire experimental period. After a 5d adaption period and 3 d feces collection period, feces were collected via grab sampling and stored at −20 • C immediately after collection. To avoid differences between the environmental control chambers, the pigs of the CT group and NSPEC group were exchanged between the two experimental periods, and each period had a 3d gas detecting period. The 3d gas detecting period was divided into 3 frequencies of manure removal of every 1d, 2d and 3d.

Chemical Analysis
At the completion of the experiment, feces samples were thawed, mixed and oven-dried at 65 • C for 96 h. The feed and feces samples were ground through a 0.5 mm sieve in a centrifugal grinder before analysis. All samples of diets and feces were analyzed for dry matter (DM, method 930.15) and crude protein (CP, method 990.03) following the procedures outlined by the Association of Official Analytical Chemists [23]. Neutral detergent fiber (NDF) and acid detergent fiber (ADF) were determined using filter bags and fiber analyzer equipment following a modification of the procedures [24]. Samples of the feed were analyzed for extract ether (EE; method 954.02) and ash (method 942.05) [23]. The gross energy (GE) in the diets and feces were determined using an adiabatic bomb calorimeter (Model 6400; Parr Instrument, Moline, IL). All the analyses were performed in duplicate.

Calculations
The ATTD of DM, CP, NDF, ADF and GE were calculated in the diets according to former reported equations [25]: where ATTD is the apparent total tract digestibility of gross energy (%), N i is the total intake of DM, CP, NDF, ADF and GE in the feed, and N o is the total fecal output of DM, CP, NDF, ADF and GE.

Measurements of Gas Concentrations
The concentration of gas in all the chambers was measured with a Photoacoustic Field Gas-Monitor INNOVA 1412 (LumaSense Technology, Santa Clara, CA, USA). Simultaneous measurement of NH 3 , N 2 O, CH 4 and CO 2 was carried out throughout the entire experiment. The gas emissions expressed per day and per livestock unit were corrected to 500 kg body weight. This system was designed to continuously monitor incoming and exhaust concentrations of gases, including control chamber ventilation volume, temperature and humidity to ensure the stability of the cabin environment [26].
A TH100 thermal gas mass flow meter was placed at the ventilator vent to monitor the ventilation environmental controlled chamber in real time. All gas samples were collected in the middle of the intake and exhaust ducts. At the time of gas collection, the gas sampling system continuously delivered the collected gas to the gas detector. Each cabin gas sample was measured 5 times in a sequential manner (every time for 1 min) and it was continuously measured for 24 h.
Egas: daily gas emissions of per pig (g/pig/d); D: ventilation rate at house temperature and pressure, (L/min); Co: concentration of exhaust house ventilation air (ppm); Ci: gas concentration of incoming house ventilation air (ppm); N: the number of pigs in the house; T: ventilation time (24 h); M: molecular weight of the gas; t: the temperature in the house; Thereafter, the daily emissions were calculated for each series of measurements and expressed per pig and per livestock unit (LU) that equals 500 kg body weight.

Statistical Analysis
The UNIVARIATE procedure (SAS Version 9.2, SAS institute Inc., Cary, NC, USA) was used to confirm the homogeneity of variance and also analyze for outliers, but no outliers were identified. The normality of the data distribution was also tested prior to the final comparison by SAS. Growth performance and nutrient digestibility data were analyzed by Student's t-test. Diet was a categorical variable. Frequency of manure removal was treated as an ordinal variable. According to a completely randomized design for gas emissions, the diet, frequency of manure removal and diet × frequency of manure removal interaction were treated as fixed effects, whereas animals, chambers and periods were treated as random effects by using the MIXED procedure of SAS. The differences were considered significant if p < 0.05 and were considered a trend if the p-value was between 0.05 and 0.10.

Growth Performance and Nutrient Digestibility
The supplementation of the NSPEC in corn-miscellaneous meal diet had no effect on the average daily gain (ADG) and feed intake (FI) in finishing pigs (Table 2). However, the pigs fed the NSPEC diet had lower feed conversion rate (FCR) than the CT diet (p < 0.05). Compared with the CT group, the inclusion of the NSPEC improved ATTD of DM by 2.4%, CP by 2.84%, NDF by 4.9% and ADF by 5.93% during the overall period (p < 0.05). In addition, the ATTD of the GE and DE were also improved by the NSPEC supplementation (p < 0.05).  according to different frequency of manure removal, respectively. The NH 3 emissions were significantly increased according to average 1d, 2d and 3d manure removal procedures (p < 0.01). The evolution of N 2 O and CH 4 emissions showed no particular trends throughout the experimental period. No differences were observed for the N 2 O and CH 4 emissions compared the NSPEC group with the CT group. However, the ADC (average daily gas concentration) of CO 2 was significantly lower (p < 0.05) in the NSPEC group than in the CT group. Furthermore, the ADEU of CO 2 was remarkably decreased or showed a downward trend (p = 0.06) in pigs fed a diet supplement with NSPEC.

Discussion
In the present study, corn-miscellaneous meal diet with high fiber content was mainly composed of corn, soybean meal, wheat bran, cottonseed meal and sugar beet pulp. The xylan content accounts for the main part of the NSP composition of these feed ingredients. In addition, the mannan content also occupies a large proportion. Therefore, the enzyme cocktail mainly included xylanase and β-mannanase in our study. The pigs fed the NSPEC diet had a relatively lower FCR than the pigs fed the CT diet. The reduced FCR might result from the supplementation of enzyme cocktail in the diet improving the relative NSP digestibility. Our results are also in agreement with a previous study which reported that non-starch polysaccharide-degrading enzymes supplementation improved the FCR of growing pigs fed diets with multi-enzyme [27]. In addition, some studies observed that a complex of non-starch polysaccharide-degrading enzymes could improve the growth performance of the weaned piglets and growing-finishing pigs [28][29][30][31]. However, some studies failed to observe a positive effect of enzyme cocktail supplementation on growth performance of pigs [32]. The apparent contradictions in the effectiveness of multi-enzyme supplementation on growth performance among studies may be mainly attributed to the differences in age of the pigs and the composition of diets used. In addition, the enzyme source and the combination of various NSP enzymes may also exert a different effect on growth performance.
The NSPEC supplementation increased the ATTD digestibility of DM, NDF and ADF by 2.4%, 4.9% and 5.93%, respectively in growing pigs. The ATTD of CP, GE and DE were also improved when the NSPEC was added in the diet. These results were consistent with a previous study which reported that multi-enzyme supplementation increased nutrient digestibility in pigs [33,34]. For example, Li et al. reported an increase in DM, GE and CP in growing pigs fed a diet supplemented with an amylase, protease and xylanase blend compared to a corn-soybean meal-based diet [35]. The improvement in nutrient digestibility in our study indicated that the NSPEC exerted its beneficial effects on nutrient digestibility of the finishing pigs, probably through first breaking down the plant cell wall structure and then releasing the nutrients for use by the pig [36].
The level of gas concentrations is the balance between the production by the animals' respiration and/or the manure and the evacuation by the exhaust fans. All the NH 3 , N 2 O, CH 4 and CO 2 emission patterns reflect the increase of feed intakes, the higher metabolism of animals and the accumulation of manure during the entire experiment. We found that the NH 3 emissions were significantly increased according to average 1d, 2d and 3d manure removal procedures. The N 2 O, CH 4 and CO 2 emissions showed an upward trend with the accumulation of manure. A previous study has reported that accumulated manure could increase the release of gases by a pig house [37]. Furthermore, manure removal frequency has been proposed to serve as an efficient means to reduce the emissions of harmful gases from pig buildings. Some researchers found that cumulative CH 4 emissions were shown to be lower by 16% and N 2 O emissions remained the same when manure was removed three times a week instead of only one time in growing pigs [38]. The NH 3 emissions data demonstrated that there were no significant differences observed between the NSPEC and CT group. The results of this experiment are consistent with a previous study in which exogenous enzyme supplementation in cereals increased ammonia emissions on finisher pigs [39]. However, it was observed that enzyme supplementation decreased ammonia emissions in wheat based diets, while in barley-based diets enzyme supplementation increased ammonia emissions in pigs [40,41]. These opposing results could be elucidated by the difference of the NSP composition of these cereals. For instance, the NSP fraction of barley mainly contains a mixture of β-glucans and arabinoxylans, while arabinoxylans are the main NSP component of the wheat [42]. In pig houses, the formation of nitrous oxide originates only from manure. N 2 O is an intermediate product and its formation mainly takes place during incomplete nitrification and denitrification processes [43]. The supplemental NSPEC in the diets had no significant effect on N 2 O emissions for fattening pigs. There were a few data on N 2 O emissions in the literature, which accounts for approximately 10% of the NH 3 emission mass [44]. Some authors suggested that reduced NH 3 emission strategies could also limit N 2 O emissions since NH 3 is the precursor of the formation of N 2 O [45]. Another greenhouse gas in pig houses is methane. CH 4 emissions mainly result from enteric fermentation in the gut [46]. In our study, the levels of CH 4 emissions were not altered after the NSPEC treatment. However, some authors observed a tendency for higher CH 4 emissions with xylanase supplementation [47]. Although the amount of CH 4 emissions from pig houses is thought to be very little, its emissions are closely related to the fiber content in the diet [48,49].
The carbon dioxide emissions from pig houses mainly originate from two sources including exhalation by pigs and release from manure. Several authors measured a 25% reduction in CO 2 emissions from pig breathing, as a consequence of reduced pig activity [50]. CO 2 release from manure was ignored for many decades [51,52]. However, some researches indicated that the levels of CO 2 emissions from manure have been evaluated to be 4-5% of the entire amount of CO 2 exhaled by livestock [53]. The CO 2 emissions of manure principally comes from three sources, namely: (1) the rapid hydrolysis of urea into NH 3 and CO 2 catalyzed by the urease; (2) the aerobic degradation of organic matter; (3) the anaerobic fermentation of organic matter into intermediate product such as volatile fatty acids (VFAs), CH 4 and CO 2 [54]. The third process is usually regarded as the principal source of CO 2 [55]. The present study indicated that the ADC of CO 2 in the supplemental NSPEC group was significantly lower than the CT group. These results may correspond to the increase of the ATTD of NDF and ADF in the NSPEC supplementation group. We have demonstrated for the first time that supplementation with NSPEC could reduce CO 2 emission from swine houses according different frequencies of manure removal. Nevertheless, further investigations have to be carried out to clarify detailed gas emission mechanisms under supplemental NSP enzyme cocktail. We only showed that the optimal NSPEC supplementation in corn-miscellaneous meal-based diet could reduce CO 2 emissions and increase NH 3 emissions with the manure accumulation for finishing pigs. Therefore, further studies need to be carried out in order to evaluate the effect of the optimal NSPEC on NH 3 and GHG emissions in fattening pigs fed other diets.

Conclusions
In conclusion, the supplementation of NSPEC in a corn-miscellaneous meal-based diet could improve pigs' growth performance. Furthermore, the beneficial effects of the NSPEC supplementation in pigs' diet on nutrient digestibility improvement and CO 2 emission reduction are more obvious compared with the CT diet. Therefore, supplemental NSPEC could promote swine production efficiency and improve the feeding environment of fattening pigs. In the meantime, the accumulated manure could increase the release of ammonia in pig houses. Funding: This work was financially supported by the National Natural Science Foundation (31702119) and the Agricultural Science and Technology Innovation Program (ASTIP-IAS07) in China.