Ethical statement

All animal management and experimental procedures were approved by the Institutional Animal Care and Use Committee of Dankook University, Republic of Korea (Approval No. DK-1-1937).

Source of probiotics

The probiotic compound used in the current experiment was acquired from a commercial company (Synerbig Co. Ltd., Seoul, South Korea), named SynerZymeF10. This product is assured to comprise at least 1 × 10¹² CFU/kg B. coagulans, 5 × 10¹¹ CFU/kg B. licheniformis, 1 × 10¹² CFU/kg B. subtilis, and 1 × 10¹¹ CFU/kg C. butyricum, which is a combination of B. coagulans, B. licheniformis, B. subtilis, and C. butyricum spray-dried spores.

Experimental design and animal management

In a 42-day trial, 180 crossbred weanling piglets ([Yorkshire × Landrace] × Duroc; 6.61 ± 1.29 kg; 28 days of age) were used. According to starting BW and sex, total pigs were arbitrarily allocated to one of four nutritional treatments (nine duplicate pens, each including two gilts and three barrows). The nutritional treatment groups were TRT1, positive control: basal diet added with 3000 ppm ZnO (HZ, high ZnO); TRT2, HZ + 0.1% probiotic complex (HZP); TRT3, negative control (NC): basal diet added with 300 ppm ZnO (LZ); and TRT4, LZ + 0.1% probiotic complex (LZP). The trial period was allocated into three phases: days 0–7 of age (phase 1), days 8–21 of age (phase 2), and days 22–42 of age (phase 3). The diets (Tables 1–3) were designed to meet or surpass the National Research Council (NRC)'s nutritional requirements (NRC 2012). All pigs were kept in a biologically maintained room with a mechanical aeration system that provided a 0.26 m × 0.53 m area for each pig. All over the experiment, each pen was supplied with a self-feeder along with a nipple drinker made of stainless steel to permit the piglets ad libitum access to feed and water. Twelve hours of artificial light for each day was automatically adjusted. The room's ambient temperature was maintained at around 30 °C during the first week, and afterward the temperature was reduced by 1 °C per week.

Table 1.

Experimental diet composition on a fed basis.

Table 2.

Experimental diet composition on a fed basis.

Table 3.

Experimental diet composition on a fed basis.

Growth performance and nutrient absorption

On initial, 7th, 21th, and 42nd days of the trial, BWs of individual pigs were measured and feed intake was documented on a pen basis during the research to examine ADG, average daily feed intake (ADFI), and gain-to-feed ratio (G:F). Chromium oxide (Cr₂O₃, 0.20%; Samchun Pure Chemical Co., Ltd., Gyeonggi, South Korea) was included in the diet as an indigestible indicator 1 week prior to fecal assembly. On day 42, fecal specimens were collected via rectal massage from two pigs for each pen (one barrow and one gilt). On a pen basis, they were combined and pooled, and a random specimen was kept in a freezer at −20 °C until analyzed. For chemical study, fecal specimens were dried out at 60 °C for 72 h, and then finely crushed to a size that could go through a 1 mm screen. By utilizing the method developed by AOAC (2010), diet and fecal samples were examined to regulate dry matter (DM) and nitrogen (N). To determine energy (E) and excreta samples, specimens were taken and put in a calorimeter (Parr Instruments, Moline, IL, USA) to assess thermal combustion in the samples as well as chromium, which was evaluated by atomic absorption spectrophotometry (Shimadzu UV-1201; Shimadzu, Kyoto, Japan). Apparent total tract digestibility (ATTD) was then measured by the following formula:

Digestibility (%) = [1 − (N_f × C_d)/(N_d × C_f) ] × 100, where N_f represents the nutrient concentration in feces (% DM), N_d represents the nutrient concentration in diet (% DM), C_d represents the chromium concentration in diet (% DM), and C_f represents the chromium concentration in feces (% DM).

Fecal noxious gas emissions

At the end of the study, fresh fecal specimens (300 g) were amassed randomly from each pen (two pigs), mixed evenly, kept in a 2.6 L sealed plastic box with small holes on any side, fixed firmly with tape, and fermented for 7 days to determine ammonia (NH₃) at 25 °C, hydrogen sulfide (H₂S), methyl mercaptans, CO₂, and acetic acid, utilizing a GV-100 gas sampling pump (model GV100S; Gastec Corp., Tokyo, Japan). Various measurement tubes (No. 3L, No. 4LT, and No. 70L; Gastec) were used to detect total mercaptans. On the 8th day, a 100 mL sample was grabbed from the headspace (2 cm) for air passage, and it was sealed again. To understand the creation of a hard crust on the top, the specimen box was shaken manually for 30 s. Finally, the concentrations of NH₃, H₂S, methyl mercaptan, CO₂, and acetic acid were evaluated.

Fecal microbiota

Fecal specimens were taken from two pigs from each pen by massaging the rectums and kept on ice for transfer to the research laboratory, where they were instantly tested to determine the gastrointestinal microbial flora. Fecal samples were obtained to study the microbiota in the feces. Every sample had 1 g of fecal droppings combined with 9 mL of 1% peptone broth mixed using a vortex stirrer for 1 min. Samples were serially blended from 10⁻¹ to 10⁻⁶, and were injected by 50 µL in two selective agar media, MacConkey agar plates (Difco Laboratories, Detroit, MI, USA) and Lactobacillus medium III agar plates (Medium 638, DSMZ, Braunschweig, Germany), for the purpose of isolating Escherichia coli and Lactobacillus. For the Lactobacillus bacterial count, the agar plates in the media were incubated in anaerobic environments for 48 h at 39 °C, and incubated anaerobically for 24 h at 37 °C for the E. coli counts. Colony counts were then totaled and the outcomes were displayed as log₁₀ transformed data.

Fecal score

Fecal scores were estimated and documented on a pen source at 08:00 and 20:00 h on days 7, 14, 21, 28, 35, and 42. Using the above fecal scoring scheme, the fecal score was calculated as the average of the five pigs in each pen: 1, hard, dry pellet; 2, firm, formed stools; 3, soft, moist stools that maintain shape; 4, soft, unformed stools that adapt to the form of the vessel; and 5, liquids that are watery and pourable.

Statistical analysis

The data were analyzed with the generalized model with a 2 × 2 factorial arrangement of SAS (SAS Institute Inc., Cary, NC, USA), using the pen as a trial unit. Data were log transformed prior to statistical analysis of microbial counts. The data variability was expressed as pooled SE, with P < 0.05 being significant and P < 0.10 indicating a trend.

Growth performance

Table 4 summarizes the outcomes of the growth performance. When HZ was added to the piglets' diets, BW increased significantly (P < 0.05) at week 6 compared with when LZ was added to the diet. Similarly, in week 3, week 6, and overall period, the ADG in pigs fed HZ diets increased (P < 0.05) compared with those fed LZ diets. Pigs fed HZP diets had a trend of higher G:F (P < 0.05) than pigs fed LZP diets. Furthermore, G:F was greater (P < 0.05) for the HZ diets at week 3 compared with the LZ diets. Throughout the experiment, the addition of ZnO (HZ vs. LZ) and the probiotic to the diet had no effect on ADFI (P > 0.05). In the growth performance measures, no interacting effects between ZnO and probiotic were found.

Table 4.

The effect of dietary supplementation of probiotic complex additive on growth performance in weaning pigs.

Nutrient digestibility

The ATTD values for DM, E, and N are summarized in Table 5. When HZ was added to the diets of weaning pigs, there was no significant change in nutritional digestibility (P > 0.05) when compared with the LZ diet. Furthermore, there was no significant improvement in nutritional digestibility when probiotics were added to the diet. In terms of nutrient digestibility, no interaction between ZnO and probiotic was discovered.

Table 5.

The effect of dietary supplementation of probiotic complex additive on nutrient digestibility in weaning pigs.

Fecal noxious gas emissions

As shown in Table 6, the supplementation of HZ into diet had no significant effect (P > 0.05) on fecal noxious gas emissions (NH₃, H₂S, and total mercaptans) when compared with LZ diet. Furthermore, the piglet administered a probiotic complex in its diet showed no influence on gas emission (P > 0.05). No interaction between ZnO and the probiotic was observed.

Table 6.

The effect of dietary supplementation of probiotic complex additive on gas emission in weaning pigs.

Fecal microbiota

Table 7 shows the findings of the fecal microbiota analysis. HZ-supplemented diets had significantly lower (P < 0.05) E. coli counts as compared with LZ, while failed (P > 0.05) to affect Lactobacillus enumeration. However, piglets fed the HZP diet showed decreased E. coli (P < 0.05) and increased (P < 0.05) Lactobacillus counts compared with the LZP diet. For fecal microbiota, no interactions between probiotic and ZnO were observed.

Table 7.

The effect of dietary supplementation of probiotic complex additive on fecal microbiota in weaning pigs.

Fecal score

The results of the piglets' fecal scores are summarized in Table 8. In comparison to LZ, adding HZ to the diet had no significant influence on the fecal score of weaned pigs. Similarly, the addition of the probiotic complex to the piglets' diet had no effect on the piglets' fecal score (P > 0.05), and no interacting effects were detected.

Table 8.

The effect of dietary supplementation of probiotic complex additive on fecal score in weaning pigs.

Effect of high- versus low-dose ZnO diet

In the present research, pigs provided therapeutic levels of ZnO had significantly greater BW, ADG, and G:F than those provided 300 ppm ZnO. Weaning pigs fed therapeutic doses of ZnO increased ADG, ADFI, and G:F (Hill et al. 2001; Sales 2013). Moreover, pigs fed ZnO as a positive control (ZH) diet increased ADG and BW compared with ZnO as an NC diet (ZL) (Shi et al. 2019). Supplementing with 3000 ppm standard ZnO and 200 ppm customized ZnO enhanced BW (Cho et al. 2015), and 2500 mg/kg of Zn from ZnO increased ADG (Hollis et al. 2005), but had no effect on ADFI or G:F. In agreement with our study, the addition of protected ZnO resulted in significantly greater BW, ADG, and ADFI in weaning pigs (Upadhaya et al. 2018) compared with pigs fed a basal diet with the nutritional content of zinc. In contrast, research found no change in growth performance (BW, ADG, ADFI, and G:F) in weanling pigs fed HZ compared with LZ (Shen et al. 2014). The enhancements in BWG, ADG, and G:F found in this trial may be due to the antibacterial properties of ZnO administration and improved gut health.

Several studies have indicated enhanced growth rate and nutrient digestibility using a therapeutic level (3000–4000 ppm) of ZnO as a substitute for in-feed antibiotics (Cho et al. 2015; Milani et al. 2017). In line with present research, nutrient digestibility was not influenced by supplementing ZH into diet (Shi et al. 2019) compared with ZL. In contrast, adding protected ZnO resulted in higher ATTD of DM and E when compared with the NC diet in piglets (Upadhaya et al. 2018). As the concentration of ZnO reduces, the nutritional digestibility also reduces quantitatively, where the ATTD of DM and N were found to be identical (Wang et al. 2021). Fecal noxious gas emissions such as NH₃ and H₂S have become one of the leading air pollutants in recent intensive pig production. There are few reports on the effects of ZnO feeding on noxious gas emissions in weaned piglets. In line with our finding, incorporating a probiotic complex into a diet with LZ and HZ did not affect fecal gas emissions in weaned piglets (Shi et al. 2019; Wang et al. 2021). Our findings found no differences in nutrient consumption; therefore, it was reasonable to presume that there would be no differences in noxious gas emissions as well, consistent with Hoque et al. (2021).

Escherichia coli concentrations in the feces of weaned pigs fed diets containing HZ were found to be significantly lower, and Lactobacillus was higher (Kaevska et al. 2016; Upadhaya et al. 2018), coherent with current research. In contradiction to this study, consuming ZnO at a level of 2500 mg/kg enhances the level of E. coli (Bednorz et al. 2013) in the intestine. With high dosages of ZnO, Hojberg et al. (2005) found that Lactobacillus was reduced, but there was no impact on E. coli (Broom et al. 2006). Carlson et al. (2008) and Shen et al. (2014) revealed that a greater level of ZnO lowered the diarrhea incidence of weaning pigs. The fecal score, which could indicate diarrhea status, was lower in piglets fed the customized ZnO (Cho et al. 2015) and ZnO nanoparticles (Milani et al. 2017) compared with control diet. The fecal count, which can indicate diarrhea severity, was similar across all treatments, confirming no diarrhea (Shen et al. 2014; Upadhaya et al. 2018), coherent with current research. Higher ZnO doses were thought to minimize the incidence of diarrhea by reducing harmful bacteria counts (Sales 2013). These findings indicated that the impact of ZnO in decreasing diarrhea could be unrelated to the direct inhibition of pathogenic E. coli, but rather is linked to an enhancement in gut microbiome, which promotes competition among enterobacteria (Amezcua et al. 2008; Wang et al. 2021). In the current investigation, diarrhea was not observed in pigs given ZnO diet, which could be related to the decreased E. coli count and pigs being raised in a hygienic method.

Effect of probiotics

Probiotics have a favorable influence on the host's health and improve the functioning of the intestinal tract. Decades of research has indicated that the use of probiotics in farm animals is beneficial as it improves feed efficiency, weight gain, and immune response (Al-Shawi et al. 2020). In this study, probiotic inclusion showed a tendency to decrease G:F. Previous research from our laboratory revealed that including multistrain probiotics (0.1%, B. licheniformis and B. subtilis) in the diets had a positive impact on ADG, ADFI, and G:F all through the experiment (Lan et al. 2016; Zhang et al. 2020). Indeed, adding a probiotic complex into diet increased growth performance in weaned pigs and broilers in our prior study (Balamuralikrishnan et al. 2017; Nguyen et al. 2019). Moreover, the diet inclusion of ZnO with probiotic complex decreased ADG, but had no effect on ADFI and G:F (Wang et al. 2021). The administration of Bacillus-based probiotics in the diet of finishing pigs had no effect on ADFI and G:F (Chen et al. 2006), ADG, or feed efficiency (Munoz et al. 2007), which is slightly in line with our research. Some recent studies found that feeding B. subtilis and B. licheniformis to basal diet improved BW and FCR for grower–finisher pigs (Alexopoulos et al. 2004), and enhanced ADG and ADFI for fattening goats (Lu et al. 2021). Probiotics would support stabilizing the microbiota by encouraging the beneficial bacteria, increasing gut health, decreasing the occurrence of diarrhea, and boosting growth performance (Hu et al. 2014).

Weaning pigs' feeds enriched with multimicrobe probiotic-containing products such as B. subtilis enhanced ATTD of DM, N, and E, according to Nguyen et al. (2019), which correlates with Lee et al. (2014). Multistrain probiotic (0.1%) administration can improve ATTD of DM, N, and E (Upadhaya et al. 2015; Lan et al. 2016), which is supported by Hu et al. (2021). However, diverse effects were accompanied by Chen et al. (2006), who showed that addition of Bacillus-based probiotics into diet had no effect on DM and N digestibility, in agreement with current study. In contrary, probiotics (B. subtilis and C. butyricum endospore) administration in growing–finishing pig diet enhanced ATTD of N and E, whereas interactive effects of probiotics were detected on the digestibility of N at week 10 and E at week 5 (Meng et al. 2010). These variable probiotic actions were linked to different types of probiotics and the level of feeding and management (Yu et al. 2020). Nutritive probiotic addition is assumed to have optimistic influence on the gut microbiome ecosystem, causing a shift in the intestinal flora that leads to increased nitrogen consumption and, in turn, reduced noxious gas emissions from the feces (Jeong et al. 2015). In comparison to control diet, Chen et al. (2006) and Hu et al. (2021) discovered that dietary addition with a Bacillus-based complex (0.2%) lowered NH₃ and amounts of total mercaptan. As observed, probiotic treatment with B. subtilis and B. licheniformis considerably reduces slurry noxious NH₃ emission, but had no effect on H₂S and mercaptan discharge in growing pigs (Wang et al. 2009). In the present investigation, dietary inclusion of probiotic had no effect on noxious gas emission. If there is no change in digestibility, there will be no change in noxious gas discharges.

While the beneficial bacterium Lactobacillus is responsible for maintaining intestinal health and can help to prevent diarrhea, E. coli is one of the most common intestinal pathogens, and some strains are serious enough to cause diarrhea (Hu et al. 2014). In contrast with current research, E. coli increased, but Lactobacillus decreased in the intestinal flora of weaned piglets (Konstantinov et al. 2006). Lan et al. (2016) and Upadhaya et al. (2017) found that probiotic treatment resulted in higher intestinal Lactobacillus counts and decreased E. coli counts in weanling piglets, which is in agreement with our findings. Probiotics benefit the host animal by maintaining intestinal barrier and producing gut microecological environments that suppress infectious organisms while increasing helpful bacteria (Chen et al. 2013). In weaned piglets, Giang et al. (2012) discovered that Bacillus-based microbiota lowered diarrhea incidence and reduced fecal score. Furthermore, Alexopoulos et al. (2004) discovered that feeding probiotic spores significantly reduced occurrence of diarrhea in postweaning period. In this study, addition of dietary probiotic to the diet increased fecal Lactobacillus and reduced E. coli counts in piglets, resulting in no diarrhea incidence. Probiotics in the feed had a significant impact on intestinal bacterial populations, thus decreasing diarrhea scores (Dowarah et al. 2017).

Interactive effects between ZnO and probiotics

In all of the analyzed parameters (growth performance, nutrient utilization, fecal microbiome, noxious gas production, and fecal index), there were no interaction effects between ZnO and probiotics.