Effect of capsaicin and capsaicin plus DL-methionine hydroxy analog in diet on growth performance and gastrointestinal conditions of nursery pigs 

After-weaning period is a critical point in swine production due to the changes in feed, management, behavior and environmental conditions. Antibiotic have been used for the protection and prevention of these negative effects. Because of the spread of antibiotic resistance, however, many countries have banned the use of these drugs. Therefore, varieties of alternative feed additives to replace antibiotic have been subjected to intensive focus.
        
As an alternative feed additive, capsaicin (CS; 8-methyl-N-vanillyl-6-nonenamide) from various species of Capsicum has been investigated (Lokaewmanee et al., 2012). Sads and Bilkei (2003) found that weaned pigs fed CS had greater growth performance and lower disease incidence, although Nofrarias et al., (2006) could not find any beneficial effects on growth performance. Several investigators reported that CS might improve gut health by increasing the stomach contents (Nofrarias et al., 2006), reducing the number of intraepithelial lymphocytes and increasing the villus height/crypt depth in the distal small intestine (Manzanilla et al., 2006).
        
LMA has been indicated as a precursor of antioxidants (Yodseranee and Bunchasak, 2012). Yodseranee and Bunchasak (2012) found that supplementing LMA enhanced plasma Met, cystine, taurine and the uric acid concentration in plasma. Under different pro-oxidant conditions (e.g. protein level and heat stress), LMA appears to sustain the antioxidant status of the cell, mainly by maintaining a higher GSH/GSSG ratio (Zou et al., 2015). In addition, Kaewtapee et al., (2009) found that LMA positively affected small intestinal morphology.       
        
The synergistic effects caused by the combination of phytogenic (CS) and Met precursor (LMA) may improve the productive performance and gut health of piglets. Recently, under tropical conditions, Arparjirasakul et al., (2018) found that adding CS+LMA in diet improve body weight of broiler chickens. Furthermore, the improvement of small intestinal morphology and FCR of piglets caused by CS+LMA supplementation was also reported (Jarupan et al., 2018). Therefore, this study was conducted to confirm the effect of CS and CS+LMA supplementations in diets on the growth performance and the gastrointestinal conditions in nursery pigs.

Animals and management
 
Piglets were kept, maintained and treated in adherence to accepted standards for the humane treatment of animals. Seventy-two castrated crossbred piglets (Landrace x Large White x Duroc) were used (7.79 ± 0.02 kg). The piglets were divided into 3 treatments and each treatment consisted of 6 replicates of 4 pigs each. They were kept in slatted pens with an evaporative cooling system. The house temperature and humidity were recorded daily.
 
Experimental diet
 
A completely randomized design was used. The treatment groups were: 1) control diet, 2) control diet with capsaicin 2.5 ppm (CS) and 3) control diet with capsaicin 2.5 ppm and LMA 0.05% (CS+LMA). All nutrients were formulated to meet or exceed the recommendation of National Research Council (NRC, 2012). The experimental periods were divided into 2 phases as follows: 1) Pre-Starter (21 to 35 days of age) and 2) Starter (36 to 64 days of age) (Table 1). Feed and water were provided ad libitum.
 

 
Measurements
Growth performance
 
The body weight of each piglet were recorded to calculate the weight gain and average daily gain (ADG). Feed intake was recorded weekly and the average daily feed intake (ADFI) and feed conversion ratio (FCR) were calculated.
 
Blood collection and pH in gastrointestinal track
 
At the end of the trial, 6 pigs from each treatment were randomly selected across all pens and exsanguinated after withdrawing the feed for 5 h. The blood was analyzed for the blood urea nitrogen (BUN) concentration using a commercial test kit (Urea liquicolor No.10505, Human). Subsequently, the pigs were euthanized, then the pH levels in each part of the gastro- intestinal tract were immediately measured using a pH meter.
 
Bacterial counts
 
The bacterial count in the caecum was conducted according to the procedure described by Brown (1974). The bacterial population was determined by using serial 10 fold dilutions with 1% peptone solution onto De Man, Rogosa and Sharpe agar (DifcoTM; Becton, Dickinson and Co., Sparks, USA) for determination of Lactobacillus spp. and onto MacConkey agar (Oxoid Laboratories) for Escherichia coli determination.
 
Analysis of lactic acid and SCFAs concentrations in the caecum
 
The samples of caecum digesta were centrifuged for 10 min at 14,000xg at 4°C and the supernatants were sampled to determine the lactic acid and short-chain fatty acids (SCFAs) concentrations. The concentrations of lactic acid and SCFAs were determined under the following conditions using a HPLC (Krutthai et al., 2015).
 
Morphology of small intestine
 
Tissue samples were collected from the duodenum, jejunum and ileum and were immediately fixed in 10% neutral buffer formalin. Then, the tissues were carefully embedded in paraffin. For each specimen, at least 10 sections of 7 µm thickness were prepared. Tissues were then stained with haematoxylin-eosin for histological evaluation. The morphology of the small intestines in this study including the villous height, crypt depth and villous height to crypt depth ratio was studied using a computer-assisted image-analysis system (Nunez et al., 1996).
 
Statistical analysis
 
All data were statistically analyzed using ANOVA. Statements of statistical significance were tested at P<0.05. The difference between the means of the groups were separated using Duncan’s Multiple Range Test.

Growth performance
 
The effects of CS and CS+LMA on the growth performance of piglets are shown in Table 2. Supplemental CS or CS+LMA in the diets (21 to 64 days of age) did not influence the body weight, feed intake and FCR of piglets (P>0.05).
 

        
Positive effects of supplementing CS, DL-LMA or CS+LMA on growth performance and health of piglets have been observed (Kaewtapee et al., 2009; Frankic et al., 2010; Jarupan et al., 2018). However, several reports showed that CS supplementation did not support the growth rate and feed utilization (Udompoka, 2006) and less positive response in growth performance from the supplementation of various feed additives has been also reported (Walsh et al., 2012). The CS+LMA group failed to enhance the productive performance of the pigs, although LMA can be converted to be Met that is an essential amino acid for the animals. Under the good management of current study, Met and other nutrients in diets were offered to meet the requirement for maximal growth performance, so the additional DL-LMA (CS+LMA) would not support more growth or protein synthesis.
 
Gastrointestinal pH and BUN concentration
 
The effect of CS or CS+LMA on pH throughout the gastrointestinal tract and BUN are presented in Table 3. The gastrointestinal pH levels and BUN concentration of pigs were not affected by CS or CS+LMA supplementation (P>0.05).
 

        
There have been indicated that the extracted CS from Capsicum spp. positively influences the functions of the gastrointestinal tract such as an increase in the stomach contents (Nofrarias et al., 2006) and an increase in the villus height/crypt depth in the distal small intestine (Manzanilla et al., 2006). However, this study shows that the CS dose not effects on the gastrointestinal acid-base value.
        
Although LMA can act as an acidifier in the gastrointestinal tract (Dibner and Buttin, 2002), supplemental CS+LMA could not induce acidity in the gastrointestinal tract (Jarupan et al., 2018). Accordingly, supplemental plant extracts (capsaicin, cinnamon and oregano) or formic acid in diets (Manzanilla et al., 2006) could not induce evidence of an acidity change in their gastrointestinal pH. This absence of acidifier potential may have been due to the buffering properties of the feed and the intestinal biostatics on the acid-base balance (Fuller and Perdigon, 2003).
        
Too high or low levels of BUN indicate the nutritional or health status of the animal (Kongkeaw et al., 2013). The BUN concentrations in the present study were around 7.86-9.37 mg/dL which were in accordance with normal range of BUN in pigs (2-22 mg/dL; average 10 mg/dL) (Kongkeaw et al., 2013). The lack of an effect of CS or CS+LMA on the BUN concentration indicated that at least the supplementations did not influence in the amino acids or protein requirements of the pigs.
 
Bacterial populations in caecum
 
The effects of CS or CS+LMA supplementation on bacterial populations in the caecum of piglets are shown in Table 4. Supplemental CS in the diet did not significantly affect populations of Escherichia coli or Lactobacillus spp. in the caecum. However, supplemental CS+LMA tended to increase the population of Lactobacillus spp. (P=0.09).             
 
  
 
In this study, a combination of DL-LMA with CS seemed to promote the growth of Lactobacillus spp. Kaewtapee et al., (2009) reported that using DL-LMA as the methionine source also increased the population of Lactobacillus spp. in the caecum of piglets. Likewise, acidifiers and/or plant extracts supplementation can increase the population of Lactobacillus spp. (Manzanilla et al., 2006). Although supplemental CS or CS+LMA in a diet with sufficient nutrients diet did not promote growth performance and influence the acid-base balance throughout the gastrointestinal tract, the supplementation may affect the ecology in the hind gut.
 
Lactic acid and SCFAs concentration in the caecum
 
The effects of supplementing CS or CS+LMA on the concentrations of lactic acid and SCFAs in the caecum of piglets are shown in Table 5. The concentration of lactic acid was increased by the CS+LMA supplementation (P<0.05). Nevertheless, CS or CS+LMA showed no significant effects on the SCFAs in the caecum.
 

        
The fermenting processes by microorganisms in the hind gut produce SCFAs, lactate, ammonia and various gases (Jensen and Jorgensen, 1994). It is known that lactic acid is effective against pathogenic bacteria and is generated from the fermentation of Lactobacillus spp. (Kristian et al., 2014). According to the tendency of the increasing population of Lactobacillus spp. by CS+LMA supplementation, the concentration of lactic acid was increased. Van Winsen et al., (2001) found that the total lactobacilli and Lactobacillus Plantarum increased the production of lactic acid in the gut. So, CS+LMA could influence the activities of some beneficial bacteria involving the production of lactic acid in the gut, but not SCFAs production. Manzanilla et al., (2006) also reported that supplemental plant extracts mixture (CS, oregano and cinnamon) did not affect the total VFA concentrations in the caecum and colon of piglets.
 
Morphology of small intestine
 
The villous height, crypt depth and villous height to crypt depth ratio of the small intestines are shown in Table 6 and Fig 1. Supplementing CS or CS+LMA increased the villous height (P<0.01), while CS+LAM increased the crypt depth compared to the control group (P<0.05) in duodenum.
 

 

        
Adding CS or CS+LMA promoted the morphology of the small intestine in the segment of the duodenum. Similarly, due to the CS+LMA supplementation, the improvement of small intestinal morphology (duodenum and ileum) of piglet was found (Jarupan et al., 2018). Bakir and Sari (2015) found that CS increased the absorption of substances in the small intestine by increasing the number of goblet cells and providing a protective effect on the digestive system. In combination with CS, it seems that LMA has a synergistic effect on the morphology. This may due to derivatives of DL-LMA such as taurine or glutathione (antioxidants) can protect the villous from damage caused by oxidative stress (Kaewtapee et al., 2009).

Although CS or CS + LMA could not provide any extra benefit to growth performance due to maximal genetic potential of piglets was achieved, there were ecological and morphological improvements in the gut system.

The authors gratefully acknowledge that the funding has come from Sumitomo Chemical Co., Ltd., Japan; Better Pharma Co., Ltd. Thailand and the Center for Advanced Studies for Agriculture and Food, Institute for Advanced Studies, Kasetsart University Under the Higher Education Research Promotion and National Research University Project of Thailand.