Effect of dietary supplementation with essential oils and a Bacillus probiotic on growth performance, diarrhoea and blood metabolites in weaned pigs

Additional keywords: classical swine fever virus, gas emission, immunity.

Weaned pigs commonly suffer from post-weaning diarrhoea (PWD), which has an economic impact on pig production worldwide. PWD is caused by many factors, such as the weaning period, removal from the sow, gastrointestinal and dietary changes, adaption to new environments, and social stress (Lallès et al. 2004; Lallès et al. 2007). It has been demonstrated to increase the mortality rate, and contribute to dehydration, gut dysfunction and growth retardation in surviving piglets (Amezcua et al. 2002; Rhouma et al. 2017). Colistin is an effective antibiotic used to mitigate PWD induced by Gram-negative bacteria, particularly Escherichia coli (Nguyen et al. 2016; Rhouma et al. 2017), whereas amoxicillin is a moderate-spectrum antibiotic, which effects both Gram-positive and Gram-negative bacteria. Despite the effectiveness of colistin in controlling PWD, several studies have reported high rates of colistin-resistant E. coli in swine (Amezcua et al. 2002; Nguyen et al. 2016). In response to this, some farmers have resorted to using a combination of antibiotics. Although co-treatment using colistin and amoxicillin is effective against PWD, the overuse of these antibiotics has raised concerns regarding the spread of multidrug resistant bacteria. Therefore, the European Medicines Agency has recommended reducing the use of colistin and amoxicillin in animal production, and restricting their use to the treatment of unhealthy animals (European Medicines Agency 2016).

To curb the escalating problem of antimicrobial resistance exacerbated by the livestock industry, probiotics and phytogenic compounds, such as essential oils, have been proposed as alternatives to the in-feed antibiotics. Numerous studies have confirmed the positive roles of Bacillus probiotic on growth performance and feed efficiency of piglets (Kyriakis et al. 1999), growing-to-finishing pigs (Davis et al. 2008; Upadhaya et al. 2015), and weaning-to-finishing pigs (Giang et al. 2012; Jørgensen et al. 2016). Lee et al. (2014) found that weaned pigs fed a 4500-g/tonne diet of Bacillus subtilis had lower faecal Clostridium spp. and coliforms counts. Bacillus species have also been observed to have positive effects on pig productivity when combined with other substances, such as organic acids and essential oils (Giang et al. 2012; Jiang et al. 2015). Ahmed et al. (2013) demonstrated that oregano oil successfully mitigated PWD caused by E. coli. in weaned pigs. Many reports have observed that the active substances present in essential oils, such as carvacrol and thymol, exhibit antimicrobial (Michiels et al. 2010) and immunostimulant (Li et al. 2012ab) properties, anti-oxidant activity, and safe-guard intestinal morphology (Xu et al. 2018). Our hypothesis is that combinations of essential oils and probiotics are more likely to modulate growth performance and immunity than in-feed antibiotics. Consequently, the primary goal of this research was to evaluate the combined or sole effect of essential oils (thymol and carvacrol) and a probiotic as a replacement for colistin and amoxicillin on the growth performance and incidence of diarrhoea in weaned pigs.

The Institutional Animal Care and Use Committee at King Mongkut’s Institute of Technology Ladkrabang reviewed and approved the animal protocol for the current trial.

The minimum inhibitory concentration of essential oils (mixture of 1.1% thymol and 2.2% carvacrol) against Gram-negative bacteria (E. coli) was determined using a broth microdilution method. Briefly, E. coli ATCC 25922 culture was first prepared by streaking a loopful of stock culture (kept at –80°C) onto Tryptic Soy Agar (Oxoid, Richmond, VA, USA) + 0.6% Yeast Extract (Lab M, Heywood, Lancashire, UK) agar plate and incubated overnight at 37°C. After incubation, several colonies were aseptically picked with a sterile loop, suspended in Tryptone Soy Broth (Oxoid) + 0.6% Yeast Extract (Lab M) broth and vortexed to ensure homogeneity. The bacterial suspension was subsequently diluted with Tryptone Soy Broth Yeast Extract broth until a final working concentration of ~6.7 × 10⁵ cells/mL. A total of 150 μL of essential oil mixture in varying final concentrations (0.10–0.50% with an increase concentration of 0.10%; diluted in culture broth) were first added to individual wells of a 96-well microplate. The bacterial culture was inoculated into each well to yield a density of ~1.0 × 10⁵ cells/well. After 48 h of incubation at 37°C, the minimum inhibitory concentration of Bacilli-based probiotic against Gram-negative bacteria was determined. The minimum inhibitory concentration is defined as the minimum concentration of tested product required to inhibit bacterial growth.

A three-phase feeding program was carried out: the diets for Phases I (0–2 weeks), II (3–5 weeks) and III (6–8 weeks) contained 22.7%, 21.26% and 19.71% crude protein, and 1.38%, 1.18% and 1.07% total lysine, respectively. Experimental diets were provided by a local company (Top Feed Mills, Pathum Thani, Thailand), and both feed additives were provided by Kemin Industries, Singapore, and supplemented to the basal diets according to each treatment. The formulae and nutrient compositions of the basal diets are presented in Table 1. All experimental diets were fed to the pigs in a mash form.

Table 1.  Nutrient composition of the basal diets (%, as fed basis)
CP, crude protein; Met, methionine; Cys, cysteine

A total of 96 crossbred ([Yorkshire × Landrace] × Duroc) pigs averaging 7.25 ± 0.24 kg in initial bodyweight (BW) were randomly assigned to each treatment based on sex and initial BW according to a randomised complete block design. Each treatment consisted of four replicates with four pigs per pen. The treatments were: (i) positive control (PC), basal diet supplemented with colistin 150 g/tonne and amoxicillin 200 g/tonne; (ii) negative control (NC), basal diet without supplementation; (iii) T3, basal diet supplemented with essential oils at 300 g/tonne diet; (iv) T4, basal diet supplemented with essential oils at 600 g/tonne diet; (v) T5, basal diet supplemented with essential oils at 1000 g/tonne diet; and (vi) T6, basal diet supplemented with essential oils at 300 g/tonne diet and Bacillus probiotics at 1000 g/tonne. The essentials oils consisted of a mixture of 2.2% carvacrol and 1.1% thymol (marketed as ORSENTIAL), whereas the probiotic contained 4 × 10¹¹ cfu/kg Bacillus subtilis PB6 (marketed as CLOSTAT).

All experimental pigs were raised in an evaporative house with half-slatted concrete floors (0.96 × 2.16 m²). The pigs were provided with water, and feed ad libitum from a self-feeder and a nipple drinker throughout the experimental period. The lighting program was artificial 20 h daily. The temperature was maintained at 31°C during the first 7 days and gradually decreased by 1°C on a weekly basis up to the 6th week, and subsequently maintained at 20°C for the 6th to 8th week. BW and feed intake were recorded on Week 0, 2, 5 and 8 to determine average daily gain, average daily feed intake, gain : feed ratio and feed efficiency.

The occurrence of diarrhoea for each piglet was recorded from Day 1 to 21 during the feeding period. Diarrhoea was assessed visually based on the faecal appearance collected at 0800 hours. A faecal consistency score was assessed visually in a range from 1 to 5 (1 = hard faeces; 2 = slightly soft faeces; 3 = soft, partially formed faeces; 4 = loose, semiliquid faeces; 5 = watery, mucus-like faeces; Gahan et al. 2009). Faecal consistency was determined per pen by recording the number of pigs within the category of faecal consistency scores. These data were used to calculate the diarrhoeal rate using the following equation: [total number of diarrhoeal pigs / (total number of pigs × days of experiment)] × 100.

Blood samples were collected from the jugular vein of piglets in control and treatment groups at the beginning (5 samples) and the end (24 samples; 4 samples per treatment) of the experiment. Five millilitres of blood was transferred immediately into non-heparinised tubes, and incubated at room temperature for 2 h before centrifugation at 3000g for 15 min. The serum was collected for blood urea nitrogen (BUN) and immunoglobulin analyses. The BUN was measured using a commercial test kit (Boehringer Mannheim, Mannheim, Germany), immunoglobulin A (IgA) and immunoglobulin G (IgG) were measured using an enzyme-linked immunosorbent assay (ELISA) kit (ELISA Stater Accessory Package, Pig IgG ELISA Quantitation Kit, Pig IgA ELISA Quantitation Kit, Bethyl Laboratories, Montgomery, TX, USA). For immunoglobulin measurement, each sample was analysed in triplicate with 1 : 10 000 (IgA) and 1 : 100 000 (IgG) fold dilution under the same conditions. The rest of the blood samples were sent directly to a commercial laboratory (Betagro Science Centre, Pathum Thani, Thailand) for field strain classical swine fever virus antibody analysis using a serum neutralisation test (neutralising peroxidase-linked assay against ALD strain).

Fresh faecal samples were collected in Weeks 0, 5 and 8 by direct rectal massage to determine the effect of dietary supplementation with essential oils and B. subtilis PB6 on ammonia reduction in pig excreta. Faecal samples were collected from four pigs (two gilts and two barrows, n = 24) in each treatment. A total of 50 g of fresh faecal samples were collected in duplicate and kept in a 2.6-L sealed plastic box. The samples were incubated at room temperature for 7 days to allow for fermentation using the method described by Cho et al. (2008). A total of 100 μL of headspace air was sampled for ammonia quantification using a gas detector. The ammonia concentration was detected within the ranges of 5–100 mg/kg (model GV-100S; Gastec Corp., Kanagawa, Japan) and 2–20 mg/kg (4 LK Detector tube; Gastec Corp.).

Data were analysed in a randomised complete block design using the general linear models procedure from statistical software package SAS (SAS Institute, Cary, NC, USA). Each pen was defined as an experimental unit for detecting growth performance and faecal consistency score, whereas selected individual pig was the experimental unit for BUN, immunological assay and ammonia emission. Duncan’s new multiple range test declared statistically significant differences between the dietary treatments at the probabilities of P < 0.05 and P < 0.01. A split-plot in time was used to show significant differences in collection periods for faeces and blood ammonia, BUN, and immunoglobulin concentrations among treatments.

The irresponsible use of antibiotics in the livestock industry has contributed to the emergence and widespread of antibiotic-resistant bacteria (Economou and Gousia 2015; Brown et al. 2017; Ronquillo and Hernandez 2017). Dietary supplements, such as probiotics and phytogenic compounds, have been proposed as the next-generation antibiotic alternatives (Ri et al. 2017; Poulsen et al. 2018). The current study reported the impact of dietary supplementation with essential oils and Bacillus probiotics on the incidence of diarrhoea and growth performance of weaned pigs.

The pigs receiving diet supplements (antibiotics, essential oils or combination of essential oils and probiotics) had better growth performance than the NC, in terms of BW (P = 0.019) and average daily gain (P = 0.001; Table 2). In addition, piglets treated with essential oils with and without probiotics gained, on average, 25–45 g per day more than the PC. It is important to note that average daily feed intake and feed efficiency were comparable among all groups, suggesting feed intake by piglets was not compromised, even at the highest essential oil concentration (1000 g/tonne). These findings are consistent with previous reports showing the beneficial effects of oregano essential oils on pig growth performance and productivity (Hong et al. 2004; Cho et al. 2006; Li et al. 2012a, 2012b; Xu et al. 2018). Overall, our findings and others support the positive role of essential oils (Michiels et al. 2010; Zou et al. 2016; Wei et al. 2017) and this may be further enhanced by the addition of probiotics to diets.

Table 2.  Growth performance of weaned pigs fed diets with essential oils and a Bacillus probiotic
PC, basal diet with colistin 150 g/tonne + amoxicillin 200 g/tonne; NC, basal diet without supplementation; T3, basal diet + essential oils 300 g/tonne; T4, basal diet + essential oils 600 g/tonne diet; T5, basal diet + essential oils 1000 g/tonne diet; T6 = basal diet + essential oils 300 g/tonne diet + Bacillus probiotics 1000 g/tonne diet; BW, bodyweight; ADG, average daily gain; ADFI, average daily feed intake; G : F, gain : feed ratio; FE, feed efficiency. Means within a row not sharing common lowercase letters differ significantly (P < 0.01)

The occurrence of PWD is common among piglets, and mainly results in E. coli infection (Kyriakis et al. 1999) and poor digestion (Li et al. 2012a). High rates of diarrhoea have been associated with piglets fed a diet without antibiotics or antibiotic replacement (Fairbrother et al. 2005). In this study, we showed that diarrhoea was significantly lower when piglets were given diets supplemented with antibiotics or essential oils alone, or in combination with Bacillus probiotics (P = 0.001; Table 2). These findings are consistent with previous reports indicating the anti-diarrhoeal effects of many plant extracts (with essential oils as the main ingredients; Jiang et al. 2015). For example, thymol and carvacrol, the active components in oregano essential oils, were reported to reduce faecal coliform (E. coli; Varel 2002; Jiang et al. 2015; Zou et al. 2016), suppress biofilm formation by pig faecal isolates (Oh et al. 2017) and increase Lactobacilli counts (Wei et al. 2017; Xu et al. 2018) in pigs. Jiang et al. (2015) demonstrated that essential oils primarily reduced the pathogen load in pigs’ gastrointestinal tracts through anti-microbial activity and by reducing the viscosity of the intestinal digesta. Similarly, our in vitro study demonstrated the antimicrobial effect of thymol and carvacrol mixture against E. coli (Fig. 1). ORSENTIAL at 0.3% slightly decreased the growth of E. coli, and from 0.4%, the growth was totally inhibited. In contrast, previous studies showed that Bacillus supplementation could improve nutrient digestion efficiency (Hermes et al. 2009), and reduce the occurrence and severity of PWD in piglets (Kyriakis et al. 1999). It is likely that probiotics play a role in digesting complex feed substrates, such as fibre and insoluble protein, reducing the production of branched chain fatty acids and ammonia (toxic metabolite), which may lead to the increased occurrence of diarrhoea. These observations suggest interactions between essential oils and probiotics, creating a favourable gastrointestinal tract environment for reducing the diarrhoea score of piglets.

Fig. 1.  Growth curve of Escherichia coli in response to different concentrations of essential oil mixture in liquid culture broth.

Piglets fed with essential oils or a combination of essential oils and probiotics had lower faecal ammonia emission compared with the NC and PC (P = 0.027; Table 3). Particularly, treatment with >600 g/tonne of essential oils or lower essential oils (i.e. 300 g/tonne) combined with probiotics showed significantly lower ammonia emission compared with the PC (P = 0.027). Chen et al. (2006) observed ammonia concentration reduced in growing pigs treated with the 0.2% complex probiotics (Lactobacillus acidophilus 1.0 × 10¹⁰ cfu/kg; Saccharomyces cerevisiae 4.3 × 10⁹ cfu/kg; B. subtilis 2.0 × 10⁹ cfu/kg). Zhao and Kim (2015) also observed a reduction in faecal noxious emissions when probiotics were fed directly to weaned pigs. Ammonia emission was probably lowered due to the increased total volatile fatty acids in the caecum following feeding of essential oils (Xu et al. 2018). In contrast, Zhao and Kim (2015) observed that direct-fed probiotic could enhance intestinal morphology, leading to better nutrient digestibility. This means fewer substrates are available for microbial fermentation in the large intestine, resulting in lower emissions of faecal noxious gas to the environment (Yan et al. 2010). This finding supports the result of decreased BUN in the weaned pigs. Therefore, it appears that both feed additives reduce odour release from pig manure.

Table 3.  Ammonia concentration of weaned pigs fed diets with essential oils and a Bacillus probiotic
PC, basal diet with colistin 150 g/tonne + amoxicillin 200 g/tonne; NC, basal diet without supplementation; T3, basal diet + essential oils 300 g/tonne; T4, basal diet + essential oils 600 g/tonne diet; T5, basal diet + essential oils 1000 g/tonne diet; T6, basal diet + essential oils 300 g/tonne diet + Bacillus probiotics 1000 g/tonne diet. Means within a row not sharing common lowercase letters differ significantly (P < 0.05)

The serological profiles of weaned piglets, including BUN and immunoglobin (i.e. IgG, IgA and classical swine fever virus-specific antibody), were determined (Tables 4 and 5). BUN is widely used to quantify nitrogen utilisation and excretion rates (Kohn et al. 2005); a lower BUN may indicate better utilisation of nitrogen for protein synthesis (Lan et al. 2016) and muscle generation (Kohn et al. 2005). Piglets fed with a diet containing essential oils alone or mixed with probiotics had lower BUN concentrations than the piglets in the NC (P = 0.027; Table 4), which is consistent with observations in broilers and nursery pigs (Zhu et al. 2014; Cai et al. 2015). However, it remains unknown, mechanistically, how essential oils or probiotics may lower the circulating concentration of BUN.

Table 4.  Blood metabolites of weaned pigs fed diets with essential oils and a Bacillus probiotic
PC, basal diet with colistin 150 g/tonne + amoxicillin 200 g/tonne; NC, basal diet without supplementation; T3, basal diet + essential oils 300 g/tonne; T4, basal diet + essential oils 600 g/tonne diet; T5, basal diet + essential oils 1000 g/tonne diet; T6, basal diet + essential oils 300 g/tonne diet + Bacillus probiotics 1000 g/tonne diet. CSFV, classical swine fever virus. Means within a row not sharing common lowercase letters differ significantly (P < 0.01)

Table 5.  Immunological parameters of weaned pigs fed diets with essential oils and a Bacillus probiotic
PC, basal diet with colistin 150 g/tonne diet + amoxicillin 200 g/tonne diet; NC, basal diet without supplementation; T3, basal diet + essential oils 300 g/tonne diet; T4, basal diet + essential oils 600 g/tonne diet; T5, basal diet + essential oils 1000 g/tonne diet; T6, basal diet + essential oils 300 g/tonne diet + Bacillus probiotics 1000 g/tonne diet; IgA, immunoglobulin A; IgG, immunoglobulin G. Means within a row not sharing common lowercase letters differ significantly (P < 0.01)

Serum immunoglobulins are generally considered an indicator of humoral immunity in animals. Dietary supplementation with antibiotics, essential oils or Bacillus probiotics did not elicit the classical swine fever virus antibody titre or IgA concentration (Tables 4 and 5, respectively). However, the serum IgG concentration was significantly increased by dietary supplementation with the 600 g/tonne essential oils or combination of 300 g/tonne essentials with probiotics compared with the NC (P = 0.014). This finding is consistent with that of Trevisi et al. (2007), who demonstrated that thymol supplementation could increase IgG concentration in the sera of weaning pigs and in non-challenged pigs’ intestine (Li et al. 2012b). Thymol supplementations may increase goblet cells in animals’ ileum (Zhang et al. 2017) and suppress the growth of pathogenic bacteria (Hedegaard et al. 2016). Together, these results clearly suggest that dietary supplementation with essential oils and probiotics can enhance pig immunity by modulating the humoral immune system. This may provide additional protection against microbial infection and thus reduce diarrhoea among piglets at weaning.

Dietary supplementation with essential oils can improve growth performance and reduce diarrhoea of weaned pigs in a dosage-dependent manner (i.e. 300 g/tonne to 1000 g/tonne). The positive effects of essential oils on pig growth and diarrhoea control can be further enhanced with the inclusion of Bacillus probiotics. These findings provide a basis for the application of phytogenic compounds and probiotics as antibiotic growth promoter alternatives in feed.

The authors declare that they have no conflicts of interest with any financial organisation regarding the material discussed. Tan Boon Fei and Lim Tricia are employees of Kemin Industries (Singapore). Waewaree Boontiam conducted the experiment, collected and analysed trial data, and did not receive any personal benefits from the funder. All authors have read and approved this research article.