Source of OA blend

The MOA mixture used in the experiment was a commercial product procured from Morning Bio Co., Ltd. (Cheonan, South Korea). The active ingredients were 17% fumaric acid, 13% citric acid, and 10% malic acid.

Experimental design, animals, and housing

In the 6-week trial, a total of 80 crossbred (Landrace × Yorkshire) × Duroc) growing pigs with average initial body weight (BW) of 22.66 ± 2.45kg and sex were allotted to one of two dietary treatments with 8 replications and 5 pigs (3 gilts and 2 castrated barrows) per pen in a randomized complete block design. The dietary treatment included a basal diet based on corn-soybean meal and a basal diet supplemented with a 0.2% OA blends. The composition of the basal diets is presented in Table 1. The basal diets contained 3300kcal of metabolized energy/kg and 15.50% crude protein and they were formulated to meet or exceed the nutritional requirements of swine (NRC 2012). All pigs were kept in an environmentally controlled room with slatted plastic floors. Each pen was equipped with a single-sided self-feeder and a nipple drinker, allowing ad libitum access to feed and water throughout the experiment.

Table 1.

Composition of growing diet (as-fed basis).

Sampling and measurements

Individual BW of growing pigs was measured at the beginning and the end of weeks 2, 4, and 6. Feed consumption and residues were weighed and recorded on a pen basis to monitor average daily gain (ADG), average daily feed intake (ADFI), and gain to feed ratio (G:F).

On day 36, 2g/kg of chromic oxide (Cr₂O₃) as an indigestible marker was mixed in growing pigs’ diets to calculate ATTD of DM, nitrogen, and gross energy (GE). At the end of week 6, fresh fecal samples were collected from 2 pigs per pen (1 gilt and 1 barrow) by rectal massage, placed on ice box transported to the laboratory, and stored at −20°C until analyzed. All feed samples and fresh fecal samples were dried at 70°C in the forced air oven for 72h and then finely ground to pass through a 1mm screen sieve. DM was analyzed following the methods outlined by the AOAC (2007). Chromium concentration was determined through UV absorption spectrophotometry (Shimadzu, UV-1201, Shimadzu, Kyoto, Japan). The GE was determined by measuring the heat of combustion in the samples, using a bomb calorimeter (Parr 6100; Parr Instrument Co., Moline, IL, USA). Nitrogen (protein) content was determined by using a Kjeltec 8600 analyzer (Foss Tecator AB, Hoeganaes, Sweden). The following formula was used to calculate the ATTD: N digestibility = 1 − [(Nf × Cd)/(Nd × Cf)], where: Nf is the nutrient concentration in feces, Nd is the nutrient concentration in diet, Cd is the chromium concentration in diet, and Cf is the chromium concentration in feces.

During second, fourth, and sixth weeks of the experiment, the rectal massage technique was used to collect fresh fecal samples from two pigs per pen for fecal microbial count analysis. One gram of feces sample was diluted with 9mL of 1% peptone broth (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) and mixed evenly. The total viable bacterial count in the fecal sample was found by plating a MacConkey agar plate (Difco Laboratories, Detroit, MI, USA) and Lactobacilli medium III agar plates (Medium 638, DSMZ, Braunschweig, Germany) with a 10-fold serial dilution to isolate both Escherichia coli and Lactobacillus, respectively. Lactobacilli medium III agar plates were kept in an incubator at 39°C for 48h under anaerobic conditions and MacConkey agar plates were kept in an incubator at 37°C for 24h. Immediately after removing the plate from the incubator, the number of E. coli and Lactobacillus colonies was counted. The microflora concentration was finally expressed as log₁₀ CFU/g of feces.

At the end of weeks 2, 4, and 6 of the trial, fresh fecal samples were collected from randomly selected 2 pigs per treatment (1 gilt and 1 barrow per pen per treatment) to analyze fecal NH₃, H₂S, methyl mercaptan, CO₂, and acetic acid. Then, samples were packed in 2.6L box with a small hole in the middle of one side that was sealed with adhesive plaster and filled with a total of 300g of fecal samples. Samples were fermented for 24h at room temperature (25°C), and 100mL samples were taken from the headspace from approximately 2.0cm above the fecal sample. After that, the box was re-sealed with adhesive plaster to measure the fecal noxious content. The fecal samples were shaken manually for about 30s before the measurement to disrupt any crust formation on the surface of the fecal sample and to homogenize the samples. Concentrations of NH₃, H₂S, methyl mercaptan, CO₂, and acetic acid were measured within the scopes of 5.0–100.0ppm (No. 3La, detector tube; Gastec Corp., Kanagawa, Japan) and 2.0–20.0ppm (4LK, detector tube; Gastec Corp.).

Statistical analysis

SAS's GLM technique was used to analyze all of the data as a completely randomized block design (version 9.2; SAS Institute, Cary, NC, USA). When significant differences between treatment means were found, they were separated using the T test. The pen was utilized as a testing unit. The standard errors of mean (SEM) were used to represent data variability, and values of P < 0.05 were considered statistically significant. Trends were defined as P < 0.1.

Growth performance

The effects of MOA mixture supplementation on growth performance of the growing pigs are shown in Table 2. Dietary MOA supplementation showed trends or significant effects to improve the daily gain of growing pigs at weeks 2 (P = 0.074) and 6 (P < 0.05), respectively. Also, pigs fed a diet containing MOA supplement significantly increased the G:F (P < 0.05) compared with those fed CON. Over the entire trial, except ADG (P = 0.019) and G:F (P = 0.101), there were no differences observed in the BW and ADFI of pigs.

Table 2.

The effect of dietary microencapsulated organic acid mixture supplementation on the growth performance of growing pigs.

ATTD of nutrients

The ATTD results are summarized in Table 3. There were no significant differences observed in ATTD of DM, N, and GE between CON and MOA groups during the end of the trial (P > 0.05).

Table 3.

The effect of dietary microencapsulated organic acid mixture supplementation on the apparent total tract digestibility (ATTD) of nutrients of growing pigs.

Fecal gas emission

The effects of MOA supplementation on the fecal gas emission of growing pigs are presented in Table 4. The dietary MOA supplementation had no influence on fecal gas emission in growing pigs during the entire experiment (P > 0.05).

Table 4.

The effect of dietary microencapsulated organic acid mixture supplementation on gas emission in growing pigs.

Fecal microbial composition

Fecal microbial composition test results are represented in Table 5. Compared with the CON diet, pigs fed a diet supplemented with MOA significantly reduced (P = 0.0143) E. coli counts at week 4. However, the Lactobacillus population (P > 0.05) remained unaffected throughout the experiment.

Table 5.

The effect of dietary microencapsulated organic acid mixture supplementation on fecal microbial composition in growing pigs.

Growth performance

Previous researchers demonstrated that feeding a protected blend of OAs led to improved growth performance of piglets and growing–finishing pigs (Cho et al. 2014; Upadhaya et al. 2014a, 2014b; Lei et al. 2017; Xu et al. 2018). In this study, we observed that when growing pigs were fed a diet supplemented with an MOA mixture, the ADG and G:F ratio improved compared with the diet without MOA supplementation. This is consistent with Walsh et al. (2007), who found better results in G:F when 0.4% OAs were included in piglets’ diets. Similarly, Kuang et al. (2015) found that weaning pigs fed the diet supplemented with an OA blend showed improved ADG. This is also in agreement with Upadhaya et al. (2018) who found an increase in ADG when 0.1% and 0.2% of the OA mixture was supplemented to the diets of weaning pigs. The possible reasons for improvements are the mechanism of action for organic acids that includes lowering the pH of the digesta in the GIT (Ravindran and Kornegay 1993), which might have helped in regulating the balance of microbial populations and promoting the growth and restoration of intestinal morphology (Papatsiros et al. 2012).

In contrast, Zentek et al. (2013) also reported that 0.416% fumaric acid or 0.328% lactic acid in the feed had no influence on the growth performance of weaned piglets. In addition, Manzanilla et al. (2004) observed that there are no effects with individual OAs, such as citric, formic, or fumaric acids on early weaned pigs. The inconsistent results among different studies could be due to age differences of animals, the types of OAs used, and OA dosage used.

ATTD of nutrients

Lowering the pH in the upper region of the GIT may improve the digestibility of nutrients. OAs were commonly used as an acidifying agent in livestock feed and are considered a promising alternative to antibiotics as they can improve the digestibility of nutrients (Nguyen et al. 2020). In the present study, we noticed that inclusion of MOA to the diet had no significant effect on the digestibility of DM, N, and GE, which is consistent with results of Upadhaya et al. (2016) and Muniyappan et al. (2021) who observed similar results when OA supplementation was introduced to the diets of pigs. However, some studies have reported a positive effect on ATTD when organic acid was used in pig diets (Upadhaya et al. 2014a; Kuang et al. 2015; Hossain et al. 2018). ATTD has not improved maybe due to the lack of influence of MOA on Lactobacillus counts in the current study as it helps in the breakdown of feeds and facilitates absorption.

Fecal gas emission

Intensive pig farming is responsible for significant air pollutant emissions (Costantini et al. 2020). The significant air pollutants from pig farming include NH₃, H₂S, and total mercaptans (Lesschen et al. 2011). Therefore, it is important to find some useful methods to reduce the noxious gas emission either by proper management or by dietary modification. Many studies explained that supplementing OAs to pigs’ diets significantly reduced noxious gases (Eriksen et al. 2010; Upadhaya et al. 2014a, 2014b; Devi et al. 2016; Hossain et al. 2018). However, in the current study, the supplementation of 0.20% MOA in the diet of growing pigs did not influence the noxious gas emission of NH₃, H₂S, methyl mercaptans, CO₂, and acetic acid. The obtained findings are in agreement with Nguyen et al. (2018) who did not find any influence on NH₃, H₂S, and acetic acid in finishing pigs. Also, Upadhaya et al. (2018) pointed out that the dietary supplement of OA mixture had no effect on NH₃, H₂S, and total mercaptans in weaning pigs. The reduced fecal pH inhibits the invasion and proliferation of pathogenic bacteria in the GIT, which further limits the production of toxic bacterial metabolites and ammonia (Kil et al. 2011; Upadhaya et al. 2014a). Therefore, in this study, the pH of the fecal might have not been reduced by the supplementation of MOA in growing pigs, which led to the insignificant effects in gas emission.

Fecal microbial composition

The GIT is the interface at which digestion, secretion, and absorption take place (Ramani et al. 2021). The gastrointestinal microbiota plays a crucial role in the host's gut-associated immune system. In addition, the intestinal microbiota affects physiological development, health, and productivity (Upadhaya et al. 2021), leading to the hypothesis that the use of feed additives such as organic acids can be useful to control the microbial community. The recent results agree with the published evidence that indicate that the dietary supplementation of OAs had reduced E. coli and increased Lactobacillus counts in weaned piglets and weaning pigs (Long et al. 2018; Upadhaya et al. 2018; Yang et al. 2019). However, other researchers have shown no significant difference in fecal microbial composition (E. coli and Lactobacillus) by the addition of OAs. (Oh et al. 2018; Lee et al. 2021) and Cho et al. 2018 found no significant difference in Lactobacillus population in weanling pigs. Inconsistency in results may be related to the age of the animals, the composition of the diet, or the amount and type of OAs mixed.