Supplemental Cap-Met in diet to promote growth performance and immune response of nursery pigs

The stress from environmental change normally causes disorders in the physiology, microbiology and immunology in the gastrointestinal tract of weaning pigs (Pluske et al., 1997). The use of antibiotics such as Colistin has been recognized to control gut pathogens and improve growth performance. Since antibiotic resistance genes regarding bacteria have spread worldwide, the European Union has totally banned the use of antibiotics in farm animals as growth and health promoters (Samanta et al., 2018), including Colistin (Mohamed et al., 2016).

In order to replace the use of such antibiotics, alternative feed additives such as probiotic, prebiotic, antioxidant, acidifier and herbal extract have been investigated (Kaewtapee et al., 2009). It is generally known that antioxidants are essential compounds for host defense against oxidative stress and improving immunity (Miladi and Mohamed, 2008). Capsaicin and its related compounds are herbal extracts from chili and pepper and are known as capsaicinoids (Ramandeep et al., 2018). The beneficial properties of capsaicinoids such as a source of vitamin C and E and antioxidant, antibacterial, anti-inflammatory and perhaps immunoregulatory effects have been reported (Krinsky, 1994). Since derivatives of DL-Methionine hydroxy analogue free acid such as taurine, cystathionin or glutathione, also play a key role in intestinal epithelial antioxidant function (Shoveller et al., 2005), the combination of Capsiacin with DL-Methionine hydroxy analogue free acid (Cap-Met) seems to have greater potential as an alternative feed additive rather than by using Capsaicin alone. Recently, Wararat et al., (2018) showed that adding Cap-Met improved the growth performance of broilers under open-housed conditions.

Since the removal of antibiotics as growth promoters has led to reduced growth performance and feed efficiency as well as increased incidence of certain animal diseases (Dibner and Richards, 2005), Cap-Met may be able to be used as an alternative feed additive. Therefore, the objectives of this study were to compare the effects of supplementing Colistin and Cap-Met in diets of nursery pigs on growth performance, fecal score, short chain fatty acids in the caecum and small intestinal immune response.

The present study was conducted at the animal research farm of the Department of Animal Science, Faculty of Agriculture of the Kasetsart University, Bangkok, Thailand. Nursery pigs were housed under similar managerial, hygienic and environmental conditions during the total experimental period of 66 days of age. Throughout the trial, the nursery pigs were handled according to the principles for the care of experimental animals and the experiment was approved by the committee of Animal Nutrition Care of the Animal Science Department of the Kasetsart University.

In total, 72 crossbred barrows (Duroc x Large White x Landrace) were used. The piglets were weaned at 24 days of age. There were randomly allotted into 3 treatments with 6 replicates (4 piglets in each replicate). The average body weight of each replication was homogenized and balanced. Experimental periods were divided into two periods as a pre-starter period (24-38 days of age) and a starter period (38-66 days of age) and an evaporative cooling system was used to control air ventilation and temperature. The pigs were kept, maintained and treated in accordance with accepted standards for the animals’ welfare. Feed were offered ad libitum and water was provided via water nipples. During the feeding trial, the house was cleaned weekly, while the fecal of piglets were removed every day.

Experimental diets were divided into two phase: pre-starter (24-38 days of age) and starter (38-66 days of age). The experimental diets offered were: 1) basal diet, 2) basal diet+ 40 ppm Colistin sulfate and 3) basal diet + Cap-Met 0.2% of diet. The feed ingredients and nutrients composition of experimental diets in the pre-starter and starter diets are shown in Table 1. The experimental diets were formulated to provide the same amounts of nutrients and met the requirement of NRC (2012).



At 38 and 66 days of age, the body weight of each pig was recorded and the body weight gain was determined. The feed consumption was recorded weekly to calculate for the average daily feed intake per day (ADFI), average daily gain (ADG) and feed conversion ratio (FCR) for each period (Seidu et al., 2018).

Fecal scores of all pigs were determined at 6 time points during this experiment on d 31, 38, 45, 52, 59 and 66. To determine the severity of postweaning diarrhea, feces were scored by determining the moisture content as described by Pedersen and Toft (2011). Scores were 0 = normal (firm and shaped); 1 = soft fecal (soft and shaped); 2 = mild diarrhea (loose) and 3 = severe diarrhea (watery). An overall diarrhea score per diet and sampling day was then calculated.

Each sample of digesta from the caecum was centrifuged (TOMY model MX-301, TOMY Kogyo Co., Ltd., Tokyo, Japan) in a microfuge tube at 14,000 rpm and 4°C for 10 min and 1.5 mL of the supernatant was transferred to a clean microfuge tube. The concentrations of volatile fatty acids were analyzed using high-performance liquid chromatography (HPLC). The HPLC system consisted of a Water Alliance model e2695 Serarations Module (Waters Corporation, Milford, USA), an Aminex HPX-87H Ion Exclusion Column (7.8 mm i.d. x 330 mm) (BioRad, Richmond, USA), a Micro-Guard Cation-H guard column (4.6 mm i.d. x 30 mm) (Bio-Rad) and a 2998 Photodiode Array Detector (Waters Corporation).

The supernatants were filtered using a 0.22 µm nylon syringe and 20 µl of each sample was injected into the HPLC system using an auto sampler, with 0.005M H₂SO₄ as the mobile phase. The running conditions provided for column heat at 60°C and a flow rate of 0.6 mL/min, with the absorbance detector operating at a wavelength of 210 nm. A mixture of acetic, propionic butyric and lactic acids was included as a standard in all analyses. The acid peaks were detected using the Empower 2TM software at a wavelength of 210 nm. Qualitative acid analysis was determined using the retention time of the acid peaks, while quantitative analysis was carried out using a standard curve composed of the various acid concentrations compared with the peak area ratio of the acid peaks and the internal standard (Akira et al., 2009).

At the end of the experiment, tissue was collected from the duodenum (D), jejunum (J) and ileum (I). The tissue samples were cut into small pieces and stored in RNA later at -80°C. Total RNA was extracted from 20 to 30 mg of tissue using the RNeasy Fibrous Tissue Mini Kit (Qiagen, Mississauga, Canada) according to the manufacturer’s protocol. Total RNA quantity and purity were determined based on the optical density (OD) at 260 and 280 nm wavelengths using a spectrophotometer.

One microgram of total RNA was used for a reverse transcription reaction for converse to cDNA. The resulting single-stranded cDNA was then used in the CFX Connect Real-Time System (BIO-RAD, USA) for evaluation of relative expression. The total quantity of relative expression for Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), Interleukin-1 beta (IL-1beta) and Immunoglobulin A (IgA), the different primers used were: F- 5' GTT TGT GAT GGG CGT GAA C3' (forward) and R-5¢ATG GAC CTG GGT CAT GAG T 3' (reverse) for GAPDH; and for expression of IL-1 beta were F-5' GTG ATG GCT AAC TAC GGT GACAA 3' (forward) and R- 5' CTC CCA TTT CTC AGA GAACCA AG 3' (reverse) (adapted from Shirkey et al., 2006). The primers for IgA were F- 5' CCG TGA ACG TGC CCT GCA AAG3' (forward) and R-5' GAG CCC AGG AGC AGG TCT3' (reverse) (adapted from Navarro et al., 2000). Thermal cycling was performed in the CFX Connect Real-Time System (BIO-RAD, USA) according to the manufacturer’s instructions. Real-time PCR dilution was performed in 20.0 µl reaction mixture containing 10 µl of iTaq universal SYBR Green super mix (BIO-RAD, USA), 300 nmol of each primer and 100 ng of cDNA. The real-time PCR conditions were 95°C for 5 min with denaturation, annealing at 60°C for 30 sec for GAPDH, 68°C for 30 sec for IL-1 beta and 65°C for 30 sec for IgA with 40 cycles and elongation at 72°C for 1 min. The qPCR reactions were run on a Bio-Rad iCycler iQ (Bio-Rad, Hercules, USA). Relative expression levels of IL-1 beta and IgA were calculated using the 2-DDÄCT method (Livak and Schmittgen, 2001).

The effects of diet were assessed by ANOVA using the GLM procedure of SAS software. A significance level of P<0.05 was criteria for all cases. The differences between the means of groups were separated using Duncan’s new multiple range test. The polynomial contrasts for linear and quadratic effect were used to evaluate time effect on fecal scores.

The effects of Colistin and Cap-Met in the diets of pigs on growth performance are presented in Table 2. The body weight, average daily gain (ADG) and average daily feed intake (ADFI) of piglets did not significantly differ between Colistin and Cap-Met supplementations. Supplemental Cap-Met significantly (P<0.01) improved the FCR of piglets during 24-38 days of age, although neither Colistin nor Cap-Met influenced any of these parameters during 24-66 days of age.

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Torrallardona et al., (2007) indicated that supplementing Colistin in diets improved the growth rate and FCR in pigs during the second week of weaning (8-14 days of experiment). In the current study, there was no positive effect from adding Colistin (40 ppm) on growth performance, while 0.2% Cap-Met significantly improved FCR during the 24-38 days of age (pre-starter period). Alter weaning pigs is the one most stressful period that can contribute to intestinal and immune system dysfunctions that in turn cause reduced feed intake, growth performance and immunity (Campbell et al., 2013).  However, there were no incidents of outbreaks or diarrhea; which implied that if the piglets are kept under good management, antibiotics would have less positive effect on feed utilization. Interestingly, supplementation of capsaicin in combination with DL-methionine hydroxy analog (Cap-Met) improved the FCR of the piglets during the pre-starter period. Capsaicin or some herbs enhance the synthesis of bile acids in the liver and stimulate the function of pancreatic enzymes (lipases, amylases, proteases), with a consequent increase in the digestibility of nutrients (Suresh and Srinivasan, 2007). Moreover, a derivative of the methionine hydroxy analog can play a role in intestinal epithelial antioxidant function as a precursor for taurine and glutathione (Shoveller et al., 2005) and/or its function as an acidifier. Therefore, during the pre-starter period Cap-Met may have greater potential to improve gut function than Colistin.

On the other hand, during the starter period (24-38 days of age), neither Colistin nor Cap-Met had an effect on piglet growth performance. This indicated that piglets can regain their health or physiological functions lost due to stressors during the pre-starter period. This was consisted with Campbell et al., (2013) who stated that the first week after weaning seriously contributed to intestinal and immune system dysfunctions.

The comparative effect of Colistin and Cap-Met supplementations in the diets on the fecal score is shown in Table 3. There were no significant effects of Colistin and Cap-Met supplementations on the fecal score. By polynomial contrast in the effect of periods, the fecal scores were changed in a linear and quadratic trend for Colistin and Cap-Met supplementations in the diets respective (<0.01).

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Diarrhea is often life threatening to piglets and it can cause major financial losses in the pig industry. According to Pedersen and Toft (2011) the average fecal score of current study of 1.28-1.58 indicates no incidence of diarrhea (score of 2-3), although the score reached its maximum at 3 weeks into the experimental period (mild diarrhea in the control and Cap-Met groups). However, there were statistically no significant differences. This might be explained by the piglets being kept in a closed-house system, so that the environmental conditions and sanitation system were controlled according to the requirement of the pigs, resulting in fewer negative effects of the pathogenic bacteria that cause diarrhea. The high productive growth performance of piglets in this study also confirmed that the pigs were kept under good management throughout the experimental period and the effect of antibiotics or feed additive supplementation would have less effect on fecal score.

The effect of supplemental Colistin and Cap-Met in diets on short-chain fatty acids in the caecum is presented in Table 4. Neither Colistin nor Cap-Met affected the concentration of acetic acid, butyric acid and propionic acid in the caecum of the piglets. However, both Colistin and Cap-Met significantly increased the lactic acid level in the caecum of the piglets.

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The concentration of lactic acids clearly increased with Colistin or Cap-Met supplementations. Lactic acid is produced by many bacterial species, primarily those of the genera Lactobacillus, Bifidobacterium, Streptococcus and Leuconostoc (Mduduzi, 2017). This showed that supplementing with Colistin and Cap-Met may promote the activities or populations of these lactic acid bacteria. Since lactic acid primarily can metabolized bacteria and many molds or yeasts (Foegeding and Busta, 1991). The comparison of the effect of Colistin and Cap-Met in pig diets on the immune response of the small intestine is presented in Table 5. There were no significant differences between the treatments in the IL-1 beta and IgA levels in the duodenum and ileum. However, the IL-1 beta was significantly decreased in the segment of the jejunum with supplementation of Colistin or Cap-Met in diets (P<0.05).

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IL-1 beta is a cytokine known to mediate and function in the inflammatory response (Dinarello 1988) and has been linked to altered nutrient uptake and utilization (Spurlock, 1997). Our study indicated that adding Colistin and Cap-Met decreased the IL-1 beta level in the segment of the jejunum; it seems that Cap-Met increased the level of jejunal IgA (P=0.05). This indicated that both Colistin and Cap-Met significantly reduced the inflammatory response of the jejunum which is one of the most important absorptive site of nutrients in the small intestine. Again, Cap-Met showed high potential as an immune enhancer in this segment of the small intestine. The positive immune and inflammatory responses of Cap-Met were confirmed by the improvement in the FCR of piglets during the pre-starter period.

In addition to the direct antibacterial activity, the addition of Colistin reducing IL-1beta is not surprising as it also has potent anti-endotoxin activity via binding and neutralization of LPS (Senturk, 2005). A plant extract mixture including capsaicin showed the property of anti-inflammation in rats (Srinivasan, 2005). Cap-Met with multi-antioxidants also showed anti-inflammation by reducing the level of IL-1 beta as Colistin, although the mechanism of Cap-Met is unclear.

Under good management without any evidence of diarrhea, adding Colistin and Cap-Met did not promote growth performance. However, Cap-Met improved the FCR of piglets during the pre-starter period. Both Colistin and Cap-Met increased lactic acid in the caecum and decreased IL-1 beta in the jejunum.

This research was supported in part by a Graduate Program Scholarship from the Graduate School, Kasetsart University, Bangkok, Thailand.