Available energy content, nutrients digestibility of chili meal and effects on performance of growing pigs

Highlights • Chili meal is classified as a roughage due to its high dietary fiber content.• Chili meal has moderate DE, ME and nutrients digestibility for growing pigs.• Addition of chili meal have adverse effect on ADG and ATTD of nutrients.• The combination of 50 g/kg chili meal and proper soybean has no significant negative effects for growing pigs.

chemical composition of chili has been reasonably well documented. Abundant nutrients in chili include vitamins (C, E), β-carotene and carotenoid pigments (Palevitch and Craker, 1996), which appear to be critically important in preventing chronic and age-related diseases (Minguez-Mosquera and Hornero-Mendez, 1994). In recent years, a growing number of chili was processed deeply to extract capsaicinoids, because they are important in the pharmaceutical industry for treatment of neurological disorders. Chili meal (CM), as the by-product of capsicum oleoresin extraction, is a potential source of feed material for abundant nutrients and increasing output.
In 2014, the world production of chili was 462955 ton and as the biggest producer, consumer and exporting country, the overall output in China was 32891 ton (FAO). When capsaicin is extracted form dried chili peel, more than 80% CM was left. However, being lack of research, most of the CM was ignored and discarded which leads to severe wasting of resources and environmental pollution. It is very important and necessary to develop CM as a new source of feed, because it can promote the utilization of CM and reduce the feeding cost. The research about CM in swine production seem empty in the world and vast research is needed to exploit potentialities of CM. Firstly, a digestion and metabolism experiment is wanted to provide an essential data about utilizability of CM in swine diet. Furthermore, Goncalves et al. (2012) reported that Brazilian red pepper meal (BRPM) contains tannins thus it needs to be evaluated through liver function and animal performance. When 78.9 g/kg CM was fed to broilers reared under high stocking density, greater growth performance and lower malondialdehyde (MDA) in serum were observed (Thiamhirunsopit et al., 2014). It suggested that CM can enhance antioxidant capacity for broilers. In order to study the effect of CM in swine production, a feeding experiment was conducted to evaluate the growth performance, nutrients digestibility, antioxidant index and conventional physiological and biochemical indexes in serum.
Therefore, the objective of this study was to evaluate the feeding value of CM for growing pigs and played a guidance role when it was applied in production.

Materials and methods
All procedures used in these experiments were approved by the Institutional Animal Care and Use Committee of China Agricultural University (Beijing, China). These experiments were conducted in the Metabolism Laboratory and Finishing Facility of the Fengning Pig Experimental Base (Hebei Province, China).
The processing methods of CM in China are similar and the main difference is the organic solvent when capsicum oleoresin was extracted. CM in present experiment was purchased from Chenguang Biotech Group Co., Ltd (Handan, China) and the processing flow was as follows: fresh chili was air dried, ground, and the husk of the chili was pelleted after seed-and-husk separation. Acetone was used with the pelleted chili husks to extract the capsaicin and the residue was dried. The dried residue is referred to as CM. The chemical composition of CM is shown in Table 1.

Animals, diets and experimental designs
Exp. 1 was conducted to evaluate the digestible energy (DE), metabolizable energy (ME) content and apparent total tract digestibility (ATTD) of nutrients in CM. Twelve barrows (Duroc x Landrace x Yorkshire) with an initial average body weight (BW) of 50.9 ± 1.8 kg were housed individually in stainless steel metabolism crates (1.4 × 0.7 × 0.6 m 3 ) and randomly allocated to one of two treatments. The basal diet contained 971.0 g/kg corn-soybean meal, and the treatment diet was formulated to contain 194.2 g/kg CM which replaced corn and soybean meal in the basal diet (Table 2). All the pigs were fed at 4% of their initial BW determined 1 day before the trial, and the daily feed allotment was divided into two equal portions which were fed at 08:30 and 15:30 h. All the pigs were allowed ad libitum access to water through a nipple waterer located at the front of the crate. The room temperature was maintained at 20 ± 1°C and this experiment lasted for 12 days.
In Exp. 2, 150 crossbred barrows and gilts (Duroc x Landrace x Yorkshire) weighing 58.4 ± 1.2 kg BW were used in a 28-d experiment. Pigs were allocated to 1 of 5 treatments on the basis of weight and gender in a completely randomized design with 6 replicates (pens) per treatment and 5 pigs per pen. The pigs were housed in pens of 2.6 × 1.8 × 0.9 m 3 with half of the floor cement and the other half woven mesh. All pigs had free access to water and feed throughout the 28-d experiment period. The temperature of the barn was set at 25°C. Diets composition and nutrients concentration in Exp. 2 are presented in Table 3. Treatment 1 was corn-  (Li et al., 2016). Pigs were weighed at the beginning and the conclusion of the trial. The amount of feed offered to each pen was recorded, and at the end of the experiment (d 28), the amount of feed left in the feeder was weighted and used to calculate feed disappearance. The average daily gain (ADG), average daily feed intake (ADFI), feed: gain ratio (F: G) were calculated. As an exogenous indicator, acid-insoluble ash (AIA) in feed  and feces were analyzed to calculate the digestibility coefficients of nutrients. Antioxidant indexes, physiological and biochemical indexes of serum were detected.

Sample collection
In Exp. 1, after a 7-D-adaptation period, a 5-d-total collection of feces and urine was conducted. Feed refusals and feed spillage were collected, dried and weighed, and feed intake was calculated. Feces were collected immediately as they appeared in the metabolism crates, placed in plastic bags and stored at −20°C. Urine was collected in a bucket placed under the metabolism crates. The bucket contained 10 mL of 6 N HCl for every 1000 mL of urine to avoid the loss of nitrogen. Each day, the total urine volume was measured, then a 10% aliquot of urine was filtered through gauze and pooled together per pig, and 50 mL of the mixed urine sample was transferred into a screw-capped tube and immediately stored at −20°C. At the end of the collection period, the sampled feces and urine were pooled for each pig and subsamples were collected for chemical analysis. The subsamples of feces were dried for 72 h at 65°C and ground through a 1-mm screen.
In Exp. 2, Feces was sampled on d 27-28 for all the pigs and mixed by each replicate, then dried by the same method as Exp. 1. Blood samples (1 pig/replicate) were collected from pigs after a 12 h fast at 8:00 am via the superior vena cava into vacuum blood collection tubes on d 29. The blood samples were centrifuged at 3500 × g for 15 min at 4°C (Ciji 800 Model Centrifuge; Surgical Instrument Factory, Changzhou, China) and the serum was stored at −20°C for analysis.

Chemical analysis and calculation
All chemical analyses were conducted in duplicate. Samples of CM, diets and feces in both two experiments were analyzed for dry matter ( Organic matter (OM) was computed as 100 minus the content of ash and water. Neutral detergent fiber (aNDF) and acid detergent fiber (ADF) were determined using fiber filter bags and fiber analyzer equipment (Fiber Analyzer, Ankom Technology, Macedon, NY) following an adaptation of the procedure described by Van Soest et al. (1991). The concentration of aNDF was analyzed using heat stable α-amylase and sodium sulfite without correction for insoluble ash (inclusive of residual ash). The ADF fraction was analyzed in a separate sample (inclusive of residual ash). Samples of CM, diets, feces and urine were analyzed for gross energy (GE) with an Isoperibol Oxygen Bomb Calorimeter (Parr 6400 Calorimeter, Moline, IL). The diets and feces in Exp. 2 were analyzed for AIA by the methods described by McCarthy et al. (1974).
In Exp. 1, DE and ME content of the CM were calculated using the difference method (Adeola, 2001). The ATTD of nutrients in diets was determined by following equation (Kong and Adeola, 2014): In this equation, D bd , D td and D ti are the digestibility (%) of the component in the basal diet, test diet, and test ingredient (CM), respectively, and P ti are the proportional contribution of the component by the test ingredient to the test diet, respectively.
In Exp. 2, the ATTD of nutrients were calculated as follows (Kong and Adeola, 2014): In this equation, CI input and CI output are the concentration of index compound (AIA) in feed and feces, respectively; CC input and CC output are the concentration of component in feed and feces, respectively. In these two experiments, digestibility coefficients were then determined by dividing grams of component digested by the grams of component consumed.

Statistical analysis
Data were checked for normality and outliers were detected using the UNIVARIATE procedure of SAS (SAS Institute, Cary, NC). No outliers were identified. In Exp. 1: Data were analyzed using the PROC GLM procedure of SAS (SAS Institute). Pig was treated as the experimental unit. One-way ANOVA was used for data analysis. Treatment means were calculated using the LSMEANS statement and statistical differences among the treatments were separated by SNK test. In Exp. 2: Pen was treated as the experimental unit and two methods were used to data analysis. Orthogonal contrast was used to determine the effect of 50 g/kg CM (-SBO) vs. 50 g/kg CM (+SBO), 100 g/kg CM (-SBO) vs. 100 g/kg CM (+SBO) and basal diet vs. 50 g/kg CM (+SBO) vs. 100 g/kg CM (+SBO). Polynomial contrasts were conducted to determine linear and quadratic effects of basal diet vs. 50 g/kg CM (-SBO) vs. 100 g/kg CM (-SBO).
Y.F. Fan et al. Animal Feed Science and Technology 229 (2017) 97-105 Significant differences were declared at P < 0.05. Differences at 0.05 ≤ P < 0.10 were considered as a trend toward significance.

Experiment 1. Nutrients concentration, digestibility, digestible and metabolizable energy content in chili meal
The GE in CM was 18.91 MJ/kg (DM basis; Table 1). The concentration of CP, ash, and crude fiber (CF) were 177.7, 110.3 and 246.8 g/kg respectively. There was no significant difference in the GE intake between the basal diet and CM diet ( Table 4). The CM diet fed group had greater (P < 0.01) dry feces output, GE in dry feces, and fecal GE output than the basal diet group. As a consequence, values for DE, ME, and ME/DE, DE/GE and ME/GE ratio were less (P < 0.05) in the CM diet, and pigs fed the CM diet had lower (P < 0.01) ATTD of DM, CP, aNDF, ADF, Ca, GE, and OM compared with pigs fed the basal diet (Table 5). However, there was a trend that CM diet had higher (0.05 < P < 0.10) ATTD of EE than basal diet for growing pigs. The DE and ME content of CM were 9.08 and 8.48 MJ/kg (as-fed basis), and the ME/DE ratio was 0.93. The ATTD of DM, aNDF, ADF, GE, and OM were 0.60, 0.38, 0.33, 0.54 and 0.66, respectively.

Experiment 2. Effects of chili meal on growth performance and nutrients digestibility for growing pigs
No significant differences were observed in ADFI and F:G among treatments (Table 6). With the inclusion of CM, the ADG and ATTD of DM, GE, and OM linearly (P < 0.05) decreased with a trend for quadratic decrease (0.05 < P < 0.10; 0.53 vs. 0.33 vs. 0.38) for ADF, and the ATTD of CP changed quadratically (P < 0.05; 0.87 vs. 0.85 vs. 0.86; Table 7). When SBO was fed with 50 g/ kg CM, there was a trend that ME intake and ADG were improved (0.05 < P < 0.10), and the ATTD of DM, CP, EE, aNDF, ADF, GE and OM were greater (P < 0.05) in the diet formulated with SBO. In 100 g/kg CM treatments, the ATTD of DM, EE, GE, and OM were greater (P < 0.05) when SBO was added. Compared with control treatment, the ATTD of EE were greater (P < 0.01) in two SBO treatments, but the ATTD of CP in 100 g/kg CM treatment with SBO was lower than control or 50/kg CM treatments with SBO (P < 0.01).

Experiment 2. Effects of chili meal on antioxidant indexes, physiological and biochemical indexes of serum for growing pigs
Neither CM nor SBO affected the levels of MDA and SOD in serum (P > 0.10; Table 8). With increasing levels of CM in diets, the  value for ALB/GLB (A/G) changed quadratically (P < 0.05; 0.86 vs. 0.66 vs. 0.75), and the level of LDL-C increased linearly (P < 0.05). There was also a trend that CM linearly increased (0.05 < P < 0.10) the level of TC and changed the value of AST/ ALT ratio quadratically (0.05 < P < 0.10; 0.68 vs. 1.09 vs. 0.84). When SBO was fed with 50 g/kg CM, a reduction (P < 0.05) of T-AOC and a trend of reduction (0.05 < P < 0.10) for CREA were found. In 100 g/kg CM treatments, the TC was lower (P < 0.05) in pigs fed with SBO compared to those without SBO, and there was a trend that SBO decreased (0.05 < P < 0.10) the LDL-C and Ca levels. Pigs fed with two SBO diets had a trend (0.05 < P < 0.10) to increase AST/ALT ratio than control diet.

Nutrients content, DE, ME, and nutrients digestibility of chili meal
The concentration of CP and ash in CM were 177.7 and 110.3 g/kg in this experiment, which were greater than the values (150.0 and 80.3 g/kg, respectively) reported by Thiamhirunsopit et al. (2014), but the concentrations of CF, Ca, P and EE (246.8, 3.92, 3.70 and 4.03 g/kg, respectively) in this study were lower than those in their report (253.0, 20.3, 6.9, 115 g/kg, respectively). This discrepancy might result from factors, such as different varieties and environments under which the chili was grown, stored and processed into CM. In their study, CM was the by-product of ethanol extraction of whole dried chili which included the chili seeds.  Y.F. Fan et al. Animal Feed Science and Technology 229 (2017) 97-105 However, the CM used in our experiment was the residue of chili husk after acetone extraction. This might imply that chili husk had greater CP but lesser Ca, P and oil content than chili seed. Chili meal is classified as a roughage due to its high dietary fiber content. Total dietary fiber primarily consists of non-starch polysaccharides, which are well known to encapsulate nutrients, and monogastric animals do not have proper enzymatic ability to digest such complex structures (Bedford and Schulze, 1998). Therefore, an increasing fiber content is negatively correlated with digestibility and the energy content of feedstuffs ). In the present study, the inclusion of CM with high fiber content in diets increased the concentration of aNDF and ADF compared with the corn-soybean meal basal diet, which resulted to the increase of fecal output. This is consistent with previous reports (Wilfart et al., 2007) and related to the water holding capacity of soluble dietary fiber and increase in fecal bulk of insoluble dietary fiber (Serena et al., 2008). Therefore, higher fecal gross energy out resulted in lower DE and ME content and nutrients digestibility. Y.F. Fan et al. Animal Feed Science and Technology 229 (2017) 97-105 4.2. Effect of chili meal on growth performance and nutrients digestibility for growing pigs In this research, the DE of diets in treatments 2 and 3 formulated with 50 g/kg and 100 g/kg CM were lower than the energy requirements due to lower energy content in CM. No significant difference in ADFI was observed among the treatments and lower DE intake was observed for pigs fed the CM diets. In growing pigs, it has been previously reported that 1% increase in dietary NDF reduced the energy digestibility by 0.9% (Le , because the dietary increase of NDF may stimulate bowel movement and reduce the transit time of digesta (Bindelle et al., 2008;Bastianelli et al., 1996). Consistent with those reports, lesser ATTD of DM, CP, GE and OM was evident in pigs fed CM diets. Therefore, the ADG of pigs linearly decreased with the dietary inclusion of CM. The concentrations of CP, aNDF and ADF in alfalfa meal (162.5, 420.0 and 321.5 g/kg, respectively) are similar to those of CM (NRC, 2012). In research with alfalfa meal, Chen et al. (2014) reported that ADG and the ATTD of DM, OM, CP, NDF, ADF and GE reduced linearly as the level of alfalfa meal in the diet increased (50, 100 and 200 g/kg).

Effect of soybean oil on growth performance and nutrients digestibility for growing pigs
Increased dietary fiber level is associated with a reduced available energy content in feed, in most practical conditions, fiber-rich ingredients are combined with high energy ingredients such as animal fat or vegetable oil in order to maintain the dietary energy level (Bakker, 1996;Noblet and Shi, 1994). Therefore, in the present experiment, SBO were added in treatments 4 and 5 to improve DE content to required levels. Addition of soybean oil contributed to a reduced rate of gastric emptying (Gentilcore et al., 2006), and increased the retention time of digesta in intestinal tract (Cervantes-Pahm and Stein, 2008), which increased the fermentability of fiber due to a longer exposure of substrates to the intestinal microbiota (Wilfart et al., 2007;Morel et al., 2006). Therefore, addition of soybean oil can increase digestibility of energy. Our research results showed that addition of SBO in CM diets improved the ATTD of most nutrients compared to diets without SBO. Moreover, compared with the control treatment, the ATTD of EE were greater in the two SBO treatments. The increased ATTD of EE in diets by addition of SBO is a consequence of a higher digestibility of SBO compared to corn and soybean meal, because intact fat from corn and soybean meal is encased with fat cell membranes and thus is more resistant to the formation of emulsions and enzymatic digestion than extracted fat from SBO (Kil et al., 2010). Another reason for that is the addition of SBO increased the EE content in diets and apparently digested fat was linearly related to dietary fat intake (Kil et al., 2010). Consistent with results from this research, other reports have shown that, with the dietary inclusion of SBO, the ATTD of DM, GE, AEE and NDF increased (Gutierrez et al., 2016;Du et al., 2009). Dietary inclusion of SBO with 50 g/kg CM tended to increase the ADG of growing pigs, which was consistent with the DE intake and the improvement of nutrients digestibility induced by SBO. Pettey et al. (2002) also reported that, when feeding a corn-dehulled soybean meal basal diet to finishing pigs, the addition of 20 g/kg SBO tended to improve F: G compared with the control. Conversely, Kil et al. (2013) indicated that addition of SBO had no effect on growth performance but increased ATTD of acid hydrolyzed ether extract and GE. Thiamhirunsopit et al. (2014) found that CM in diets reduced the MDA level to the normal levels when broilers were under high stocking density. In their study, CM contained 0.43 g/kg capsaicin, an important alkaloid due to its neurological benefit, and potential lipid peroxidation properties (Kentaro et al., 2002;Conforti et al., 2007). In present study, with the dietary inclusion of CM, no significant difference was observed in MDA, SOD or T-AOC. It is possible that acetone extraction was sufficiently efficient so that there was little remaining capsaicin in CM in the current study. However, when 50 g/kg CM were added to the diets, significant reduction of T-AOC was found in diet formulated with SBO, which indicated that the inclusion of SBO might reduce antioxidant capacity. Total antioxidant capacity is considered to be the integrated action of all the antioxidants present in plasma and body fluids, thus providing an insight into a delicate balance in vivo between oxidants and antioxidants (Ghiselli et al., 2000). This is consistent with a report that high fat significantly elevated MDA levels and lowered T-AOC levels in the serum of rabbits (Sun et al., 2014).

Effect of chili meal and soybean oil on serum antioxidant indicators, physiological and biochemical indicators of growing pigs
Changes in serum biochemical indicators suggest the change in tissue cell permeability and body's metabolic function With the dietary inclusion of CM, no significant difference was observed in the serum concentrations of AST or ALT. The levels of the enzymes ALT and AST are tools in the diagnosis of liver function. Similar results was observed that the serum concentrations of AST and ALT in broiler chickens fed diet with 12 g/kg of Brazilian red pepper meal did not differ from the negative control.
Concentrations of TC, UREA and TP reflect the health and nutritional status of pigs (Etim et al., 2014). UREA concentration indicates protein catabolism. Increased TP concentration suggests that more protein is available for utilization and the decrease in UREA suggests sufficient protein consumption (Hlatini and Chimonyo, 2016). With the inclusion of CM, the level of A/G had a quadratic change, and the lowest numerical number was observed in the diet containing 50 g/kg CM. The decline of A/G ratio indicated that more globulin was synthesized to elevate the immune status of the pigs. With the inclusion of CM, the level of LDL-C increased linearly but the addition of SBO decreased the level of TC when 100 g/kg CM was used. Excessive cholesterol can contribute to arthrosclerosis and lead to coronary problems in humans. In pigs, the normal blood cholesterol range is 81-134 mg/dl (Radostits et al., 2000) and all the TC contents of serum in present study were lower than it. The decreased serum TC may be associated with a decline in lipid mobilization (Prvulovic et al., 2007).

Conclusion
The present study demonstrated that inclusion of chili meal in swine diets, as a novel fibrous feedstuff, results in moderate DE, ME Y.F. Fan et al. Animal Feed Science and Technology 229 (2017) 97-105 and nutrients digestibility for growing pigs. The inclusion of chili meal decreases the average daily gain and digestibility of most nutrients. Supplementation of CM diets with soybean oil increases the average daily gain and digestibility of nutrients. The combination of 50 g/kg chili meal and proper soybean has no significant negative effects for growing pigs.