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Under production conditions, pigs can be affected by several environmental factors that stress them. In particular, neonatal and weaned animals are susceptible to physiological changes (feeding practices, farm management and nutritional needs), which can lead to the invasion of pathogenic bacteria that obstruct the microbial composition of the gastrointestinal tract of hosts (Yang et al. 2015).
To counteract this situation, antibiotics were used for a long time. However, different authors report that the indiscriminate use of these antimicrobials, in humans and animals, causes microbial resistance. Hence the need to replace these substances as growth promoters in animal production with other alternative additives, such as probiotics, which should be compatible with the environment and avoid negative effects on human health (Yeo et al. 2016 and Foko et al. 2018).
Lactobacillus spp. and Bacillus spp. cultures, from the digestive tract of animals, are among the products that show probiotic activity, which stand out for their effectiveness in improving production and health indicators (Rondón et al. 2018 and Milián et al. 2019).
The University of Matanzas and the Institute of Animal Science (ICA, initials in Spanish) carried out different research projects for the development of economically viable products that would improve productive yield and animal health (Pérez 2000, Milián 2009 and Rondón 2009). Among the obtained probiotics were SUBTILPROBIO® (culture of Bacillus subtilis C-31) and PROBIOLACTIL® (culture of Lactobacillus salivarius C-65), evaluated in broilers, layers (Rondón et al. 2018 and Milián et al. 2019), calves (del Valle 2017 and González 2019) and pigs (Ayala et al. 2014) with excellent results in weight gain, improvements in food conversion and decrease of diseases. However, the effect of these additives has not been evaluated yet, when they are applied mixed in the animal diet.
It is stated that when probiotics are supplied in the form of multi-strain cultures, or several microbial additives are mixed, the probiotic action tends to be enhanced in the digestive tract (Timmerman et al. 2004 and Zhang et al. 2017). Therefore, the objective of this study was to evaluate the probiotic effect of SUBTILPROBIO®, PROBIOLACTIL® biopreparations and their mixture, on productive and health indicators of growing pigs.
Materials and Methods
Preparation of PROBIOLACTIL® and SUBTILPROBIO® zootechnical additives. From the Lactobacillus salivarius C-65 and Bacillus subtilis C-31 strains respectively, 20 L of each bioproduct were produced. For the preparation of additives, the methodology described by Rondón (2009) and Milián et al. (2017) was followed.
Treatments and experimental conditions. The experiment was carried out in the Unidad Porcina Gelpis, in Matanzas province. An amount of 200 crossbred pigs was used, weaned at 33 d, from York-Land (YL), Large White-Landrace (LWxL), and Duroc Jersey and L-3534 sires, from 34 to 76 d (42 d). The study took place from November 19 to December 30, 2018. During this period, a mean temperature of 26 ºC ± 2, minimum of 20 ºC ± 3 and maximum of 27 ºC ± 1 was recorded. The mean relative humidity was 79% ± 3. A completely randomized design was applied, in which four treatments were included, 50 animals each: 1 control) basal diet without additives, 2) basal diet + PROBIOLACTlL® additive (10 mL/animal/d), 3) basal diet + SUBTILPROBIO® additive (10 mL / animal / d) and 4) basal diet + mixture of PROBIOLACTlL® + SUBTILPROBIO® additives (5mL/animal/d of each biopreparation).
Doses of additives was adjusted to 109 CFU.mL-1, which were mixed with water for homogenization. Samplings were carried out at 34, 45, 60 and 76 d.
Feeding and management conditions. Table 1 shows the composition of the provided diet. Food was provided in the form of corn-soy-based meal, according to NRC (2012).
Table 1 Composition and contribution, under dry basis, of the diet consumed by pigs
| Ingredients | Inclusion, % |
|---|---|
| Corn meal | 70.36 |
| Soy bean meal | 27.00 |
| Sodium chloride | 0.50 |
| Calcium carbonate | 0.50 |
| Dicalcium phosphate | 1.00 |
| Premix1 | 0.50 |
| Choline | 0.14 |
| DM, % | 90.81 |
| CP, % | 19.00 |
| ME, MJ kg-1 | 18.97 |
| Ca, % | 0.61 |
| P, % | 0.49 |
| C, % | 4.55 |
1Premix of vitamins and minerals per kg of concentrate: vitamin A, 12,000 IU; vitamin D, 2,600 IU; vitamin E, 30 IU; vitamin B12.12 ug; vitamin K, 3 mg; calcium pantothenate, 15 mg; nicotinic acid, 40 mg; choline, 400 mg; Mn, 40 mg; Zn, 40 mg; Fe, 40 mg; Cu, 8.8 mg; I, 0.35 mg and Se, 0.3 mg.
DM: Dry matter; CP: Crude protein; ME: metabolizable energy; Ca: calcium; P: phosphorus; C: ashes
Facilities, in which pigs were housed (Flat Decks system), underwent a sanitary authorization, as established for these animals (IIP 2008). Ten pigs were located per pen, for a total of 20 pens. Water was provided ad libitum and food intake was restricted according to consumption regulations established for this category (GRUPOR 2017).
Determination of the effect of biopreparations and the mixture on microbiological, productive and health indicators. For determining microbiological indicators, samples of fecal content (1 g) were taken from 10 piglets per treatment at 34, 45, 60 and 76 d. They were homogenized in 9 mL of diluent medium (Caldwell and Bryant 1966) and immediately processed under anaerobic conditions (5% CO2 atmosphere). To perform the count of lactic acid bacteria (LAB) and coliforms, serial dilutions of samples (1:10, w/v) were carried out in a diluent medium until 10-11. Out of these dilutions, 10-9, 10-10 and 10-11 were used for LAB, and 10-6,10-7 and 10-8 for coliforms. Each was replicated three times (0.5 mL) on plates containing selective culture medium. For coliforms, violet red bile agar (OXOID) was used, and, for LAB, MRS agar (CONDO, Spain). After incubating at 37 ºC (for 72 h for LAB and 24 h for coliforms), microbial count was performed. The CFU number was determined under magnifying glass by visual colony count. In productive and health indicators, live weight of pigs was evaluated every week and daily mean gain (DMG), food conversion (FC) and weight gain (WG) were calculated. The incidence of diarrhea and mortality in animals was also observed every day.
Statistical processing. For data analysis, the statistical program INFOSTAT, version 2012 (Di Rienzo et al. 2012) was used. For productive variables, a one-way analysis of variance model was applied, after checking the normal distribution of data and the variance homogeneity using Shapiro and Wilk (1965) and Levene (1960) tests, respectively. Microbial counts were transformed to LN and subsequently to √X, and normality was checked again. Differences between groups were verified using Duncan (1955) multiple range comparison test. Results of diarrhea incidence were analyzed using ComparPro version 1 program (Font et al. 2007).
Results and Discussion
Figure 1 shows the effect of PROBIOLACTIL®, SUBTILPROBIO® and its mixture, on the performance of coliform and LAB populations in feces of growing pigs. At 34 d, there were no differences in the count of these microbial groups in the different treatments. However, after 45, there was an increase (P <0.05) of LAB and a decrease of coliforms in the groups in which biopreparations were applied, with respect to control. Furthermore, it was found that, out of the additives used, PROBIOLACTIL® was the one that caused the greatest increase (P <0.05) of LAB population.
a,b,c Columns with different letters differ at P ˂ 0.05 (Duncan 1955)Coliforms: 34 d SE ± 0.39, P=0.3307; 45 d, SE ± 0.83, P=0.001; 60 d, SE ± 0.70, P=0.001 and 76 d, SE ± 0.88, P=0.001, LAB: 34 d, SE ±0.089, P=0.2206; 45 d, SE ± 0.42, P=0.001; 60 d SE ± 0.56, P=0.001 and 76 d, SE±0.31, P=0.001.SE: Standard errorFigure 1 Effect of PROBIOLACTIL®, SUBTILPROBIO® and their mixture on the performance of coliform and LAB populations in feces of growing pigs
The addition of this biopreparation, containing Lactobacillus salivarius, should have increased LAB population in the GIT, which are part of the native microbiota of this ecosystem. According to Pieper et al. (2006), Lactobacillus salivarius, L. fermentum and L. acidophilus species are the most abundant lactobacilli of the microbial community that inhabits the pig ileum during weaning period.
These results indicate that the application of the additive PROBIOLACTIL® in the established dose caused the colonization of the gastrointestinal tract of the pigs under study. Many probiotics, especially lactic acid bacteria, ferment carbohydrates to produce short-chain fatty acids, such as lactic acid and acetic acid, which lowers luminal pH to levels in which potentially pathogenic bacteria do not develop (Segura and de Bloss 2000).
It is also known that these bacteria produce bacteriocins and hydrogen peroxide (Price and Lee 1970 and Nazef et al. 2008). These substances generally destroy the integrity of the cytoplasmic membrane by forming transmembrane pores, causing the release of amino acids and ATP. Therefore, the electrochemical gradient is not generated nor the proton-motive force is reached, necessary for ATP synthesis (Bajagai et al. 2016).
Rondón et al. (2012) determined in vitro that Lactobacillus salivarius C-65 species inhibited Escherichia coli 0157: H7 from the production of acids and bacteriocins. It is known that L. salivarius is a very prolific microorganism in the intestine, making it capable of competing with many others, including some pathogenic bacteria (Riboulet-Bisson et al. 2012). Among them, L. salivarius is characterized by being a very effective probiotic bacteria, since it performs an essential function in maintaining a healthy digestive system.
Figure 2 shows the performance of live weight in the evaluated animals, with the use of PROBIOLACTIL®, SUBTILPROBIO® and its mixture during pre-fattening. An increase of live weight (P <0.05) was observed in pigs treated with the biopreparations with respect to control group. This increase was observed after 11 d of application, which corresponds to 45 d of birth. Regarding control, it was found that from that age, up to 76 d, weight gain occurred in all the treatments in which biopreparations were applied. However, the best results were achieved with the administration of PROBIOLACTIL®, followed by SUBTILPROBIO® and their mixture.
a,b,c Columns with different letters differ at P ˂ 0.05 (Duncan 1955)34 d, SE ±0.019, P=0.3307, 45 d, SE ± 0.093, P=0.001, 60 d, SE ± 0.070, P=0.001 and 76 d, SE ± 0.098, P=0.001Figure 2 Live weight performance during the experiment
The best results were obtained in pigs treated with PROBIOLACTIL®. This biopreparation contains Lactobacillus salivarius C-65, a microorganism found in the digestive tract of different animals of zootechnical interest.
Iñiguez-Palomares et al. (2007) isolated eight strains of Lactobacillus, with probiotic potential, from the GIT of pigs due to their resistance to gastrointestinal transit barriers, to the property of adhering to the intestinal mucosa and to antibacterial activity against pathogenic microorganisms. However, L. salivarius was the species that fulfilled all the criteria to be selected as a probiotic.
The results of this study are in correspondence with criteria of authors who state that, with the use of zootechnical additives, apparent retention of nutrients included in the diet is increased (amount of consumed nutrients minus the amount of excreted nutrients) and the N, P and Ca (Ángel et al. 2005) retention is favored. Lactobacillus supplementation increases the activity of digestive enzymes, such as β-galactosidase, which stimulates gastrointestinal peristalsis and promotes nutrient digestibility (Zhao and Kim 2015).
Table 2 shows the performance of productive indicators in pigs that consumed the different biopreparations.
Tabla 2 Performance of productive indicators in growing pigs, supplemented with PROBIOLACTIL®, SUBTILPROBIO® and their mixture
| Indicators | Treatments | SE±Sign | |||
|---|---|---|---|---|---|
| Control | PROBIOLACTIL® | SUBTILPROBIO® | Mixture | ||
| DMG, g | 408.65c | 445.27a | 431.58b | 431.89b | 0.40 P=0.001 |
| Weight gain, kg | 16.36c | 19.42a | 18.22b | 18.28b | 0.10 P=0.001 |
| DM intake, kg-1LW | 47.43 | 47.43 | 47.43 | 47.43 | - |
| Food conversion | 2.90a | 2.44c | 2.60b | 2.59b | 0.024 P=0.001 |
a, b, cMedias con letras distintas difieren para P ˂ 0.05 (Duncan 1955).
DMG: daily mean gain. SE: standard error. Number of animals per treatment: 50
These results are similar to those observed by Dowarah et al. (2018), who described that probiotic supplementation to growing pigs improved DMG and FC during 180 d. These criteria demonstrate that these microorganisms improve the use of nutrients contained in the diet.
Table 3 shows results of diarrhea incidence during the experiment. These may be associated with different action mechanisms proposed for probiotics, including normalization of altered microbial population, improvement of the intestinal immune barrier, particularly through secretory IgA response, and decrease of intestinal inflammatory responses. (Zhang et al. 2011, Khare et al. 2018 and Plaza et al. 2019).
Table 3 Diarrhea incidence on pre-fattening pigs, supplemented with PROBIOLACTIL®, SUBTILPROBIO® and their mixture for 42 d
| Indicators | Treatments | Number of diarrheas | % | SE± Sign |
|---|---|---|---|---|
| Diarrhea incidences | Control | 94 | 67.14a | 0.56 P<0.001 |
| PROBIOLACTIL® | 12 | 8.57c | ||
| SUBTILPROBIO® | 22 | 15.71b | ||
| Mixture | 20 | 14.28b | ||
| Total of animals | 200 | 140 | 100 |
a.b.cPercentages with different letters differ at P ˂ 0.001 (Duncan 1955)
The incidence of diarrhea showed lower values in the animals that consumed the additives, a result that coincides with studies of Lu et al. (2018), who referred to the reduction of mortality and the presence of animals with diarrhea when probiotics are applied. Kim et al. (2018) studied the dynamics of intestinal microbial diversity during pig weaning, after providing food supplemented with probiotic bacteria. The bioassay results indicated that, in the presence of Lactobacillus acidophilus 30SC, the activity of enterohemorrhagic Escherichia coli (EHEC) O157: H7 was inhibited, while lactobacilli population increased in weaned pigs.
Other researchers, such as Deng et al. (2013), also applied a mixture of Bacillus RJGP16 and Lactobacillus salivarius B1 to assess its effect on the stimulation of the immune system of pigs. These authors observed an increase of the production of interleukin (IL)-6 and porcine beta-defensins (pBD)-2 in the duodenum and ileum (P <0.01). They also confirmed an increase in the number of immunoglobulin (Ig) A (P <0.01) in the intestinal lumen. All these actions contribute to the reduction of potentially pathogenic microorganisms, which cause the presence of diarrhea.
Regarding these results, it is stated that probiotic microorganisms create a complex with animal own bacteria to favor defense mechanisms, production of antimicrobial substances, intestinal pH decrease, bacterial antagonism and stimulation of the activity of macrophages and lymphocytes, which influence on better productive yields (Bajagai et al. 2016 and Markowiak and Śliżewska 2018).
Results similar to those of this study were obtained by Dowarah et al. (2017), who evaluated the efficacy of two probiotics (Lactobacillus acidophilus NCDC-15 and Pediococcus acidilactici FT28) in weight gain, diarrhea incidence, intestinal microbiota composition and pig health. These authors concluded that supplementation of these additives in basal diet improved growth performance, fecal microbial count and intestinal morphology in pigs. In addition, they considered that P. acidilactici FT28 strain was more effective in reducing diarrheas and maintaining the acidic environment of the digestive tract, indicating a synergic probiotic effect between these bacteria and the intestinal microbiota to promote animal health.
These results may be given because lactobacilli, unlike Bacillus, have the ability to colonize and adhere to intestinal mucosa, thus inhibiting potentially pathogenic microorganisms. They produce organic acids and maintain the integrity of epithelial cells. In addition, they can multiply under anaerobic conditions and remain viable for 28 d, after stopping treatment. They also activate the immune system and improve host health (Blajman et al. 2015, Pluske et al. 2018 and Hernández et al. 2019). Regarding the results of the mixture, it is evident the need to evaluate higher doses than those established for each additive, in order to analyze its effect on animals.
