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Introduction
In modern formulations of birds food additives are used that significantly influence on the yield and health of animals. These supplements, among which are probiotics, prebiotics, symbiotics, enzymes and phytobiotics (Angelakis 2017), are used in small concentrations. Specifically, the probiotic term refers to live microorganisms that, when are administered in adequate amounts, confer benefits to the health of the host (FAO/WHO 2001).
Currently, probiotics are postulated as a potential alternative for the replacement of antibiotics that are used as subtherapeutics, as growth promoters. The advantage of probiotics is that they do not leave residues in the eggs or in the meat of birds, and do not generate risks of antibiotic resistance in the human microbiota. In birds feeding, the use of these zootechnical additives, mainly bacteria that produce lactic acid and Bacillus, helps maintain the integrity and stability of the intestinal microbiota, reducing the proliferation of harmful microorganisms. Thus, the appearance of diseases is prevented, and the productive yield of animals improves (Milián et al. 2013 and Díaz-López et al. 2017).
Generally, it is proposed that biopreparations with more than one isolate have higher chances of success, when complementing their effects synergistically, since they favor the colonization of a system as complex as the gastrointestinal tract (GIT) (Estrada 2015).
The objective of this study was to evaluate the probiotic effect of a mixture of Bacillus subtilis 20Bp and Lactobacillus brevis 40Lp on productive and health indicators of broilers.
Materials and Methods
Treatments and experimental conditions. The experiment was conducted between September-October 2016, at the Mr. Ariosto Mora poultry farm, located in Calceta parish, Bolívar canton, Manabí province, Ecuador. As an annual average, this area records temperatures of 26°C, and rainfalls of 1100 mm. Its height is 800 m o.s.l.
A total of 400 chicks of Cobb500® line were used, with 42 ± 2 g of weight, provided by the incubator from the Escuela Superior Agropecuaria de Manabí. A chicken coop with eight pens (1.25 × 3.75 m) was used. 50 animals were distributed per pen, with a density of 11 birds per square meter. The intake of water and food was ad libitum and cylinder feeders and nipple drinkers were used. The food was made in the school's concentrate factory (table 1). The temperature of the chicken coop was controlled with gas heaters using thermostats, fans and curtains. The management of animals was carried out in accordance as described by Cobb-Vantress (2012) for broilers.
The duration of the experiment was 1-42 d in a completely randomized design. Two treatments with four replications (pens) were evaluated: 1) corn-soybean diet as a control and 2) corn and soybean diet + mixture of the biopreparations Bacillus subtilis 20Bp and Lactobacillus brevis 40Lp. The strains, selected for their in vitro probiotic potential (Arteaga et al. 2017 and Arteaga et al. 2018), were isolated from the digestive tract of backyard chickens.
The food supply was carried out twice a day and the drinking water was treated with 0.1% calcium hypochlorite. The composition of the diet varied for starter, growing and finishing (table 1). Per kilogram of food (109 cfu.g-1) 1 mL of each biopreparation was supplied, according to Mutus et al. (2006) criteria. The food was weekly mixed with the biopreparation.
Table 1 Composition of diets used during the experiment
| Ingredients (%) | Starter (1-14 d) | Growing (15-28 d) | Finishing (29-42 d) |
|---|---|---|---|
| National yellow corn | 59.90 | 63.89 | 67.71 |
| Soybean paste (46%) | 30.94 | 27.13 | 23.96 |
| Fish meal (58%) | 5.00 | 4.00 | 2.50 |
| Palm oil | 0.76 | 1.56 | 2.44 |
| Calcium carbonate | 1.15 | 1.16 | 1.12 |
| Monocalcium phosphate (21% granulated) | 0.62 | 0.65 | 0.61 |
| Common salt | 0.21 | 0.22 | 0.24 |
| Vitamin and mineral premixture * | 0.25 | 0.20 | 0.20 |
| DL-methionine | 0.28 | 0.24 | 0.23 |
| L-lysine HCl | 0.15 | 0.16 | 0.16 |
| L-threonine | 0.03 | 0.03 | 0.04 |
| Rovabio MAX-AP | 0.05 | 0.05 | 0.05 |
| Milbond TX ( micotoxin trap) | 0.25 | 0.25 | 0.25 |
| Abiquim/Oxistop (antioxidant) | 0.02 | 0.02 | 0.02 |
| Cycostat (robenidine 6,6%) | 0.05 | 0.05 | 0.05 |
| Choline chloride | 0.08 | 0.07 | 0.12 |
| Sodium bicarbonate | 0.26 | 0.32 | 0.30 |
| Chemical composition calculated | |||
| ME, MJ/kg | 12.47 | 12.80 | 13.18 |
| Dry matter (%) | 89.36 | 89.39 | 89.41 |
| Crude fiber (%) | 2.46 | 2.40 | 2.36 |
| Ether extract (%) | 3.47 | 4.26 | 5.11 |
| Crude protein (%) | 22.74 | 20.75 | 18.75 |
| Calcium (%) | 0.83 | 0.79 | 0.70 |
| Sodium (%) | 0.21 | 0.22 | 0.21 |
| Available phosphorus (%) | 0.37 | 0.34 | 0.29 |
| Methionine + cystine (%) | 1.05 | 0.94 | 0.58 |
| Lysine (%) | 1.47 | 1.32 | 1.15 |
| Arginine (%) | 1.55 | 1.40 | 1.24 |
| Threonine (%) | 0.96 | 0.88 | 0.80 |
| Tryptophan (%) | 0.31 | 0.28 | 0.25 |
ME metabolizable energy)
*1kg of food contains viatamin and mineral supplemnts.
Vitamin supplement: vitamin A (10000 UI), D3 (2000 UI), E (10 mg), K3 (2 mg), thiamine - B1 (1 mg), riboflavin - B2 (5 mg), pyridoxine- B6 (2 mg), vitamin B12 (15.4 mg), nicotinic acid(125 mg), calcium pantothenate (10 mg), folic acid (0.25 mg), biotin (0.02 mg).
Mineral supplement: selenium (0.1 mg), iron (40 mg), copper (12 mg), zinc (120 mg), magnesium (100 mg), iodine (2.5 mg) and cobalt (0.75 mg).
Determination of productive and health indicators. For determining in vivo the effect of biopreparations on productive and health indicators, the live weight (LW) was recorded and the average daily gain (ADG), food intake and food conversion were calculated, which were controlled at 7, 14, 21, 28, 35 and 42 d. The mortality and viability were observed throughout the experimental period. The relative weight of the carcass was only determined at 42 d. A total of 20 animals were selected and slaughtered (by puncturing the jugular vein) per treatment at the end of the experiment. The selection was performed based on the average weight of each group of birds, in a range of ± 10 %. All these indicators were calculated according to Pronaca (2017) criteria.
Statistical methods. The INFOSTAT program, version 2012 (Di Rienzo et al. 2012) was used to analyze the results. For the statistical treatment of data, analysis of variance was performed, according to a completely randomized design. The Duncan (1955) comparison test was used to verify differences. In addition, the CompaPro-version 1 (2007) Test was used, with 95 % confidence, with the objective of determining statistical differences for viability and mortality.
Results and Discussion
The performance of the live weight of animals is showed in figure 1. There are differences (P≤ 0.001) from 14 to 42 d, when weight gain is observed in animals that intake the mixture with probiotic potential.
7 d: SE± 1.72 (P=0.067); 14 d: SE± 2.54 (P=0.001); 21 days: SE± 11.26 (P=0.001); 28 d: SE± 11.38 (P=0.001); 35 d: SE± 18.64 (P=0.001) and 42 d: SE± 16.71 (P=0.001).
Figure 1 Effect of the mixture of Bacillus subtilis 20BP and Lactobacillus brevis 40LP on the live weight performance of broilers.
In this experiment it was found that the effect of the mixture began to be observed from the 14 d of its application. It is explained that the use of probiotics, formulated specifically for the initial development of the GIT of newborn chicks, is a promising topic, which can have a positive effect on birds yield (Wolfenden and Hargis 2014). The colonization of the digestive tract with probiotic microorganisms guarantees from the first hours of life the stability of the beneficial microbiota, without the development of pathogenic microorganisms, as well as the neutralization of toxins that are produced and the stimulation of the immune system, as reported by Cisek and Binek (2014) and Milián et al. 2017). These authors also suggest that colonization favors the activation of the mucin gene for the increase of this protective layer, as well as the production of lactic acid and short-chain fatty acids. They also report that the histomorphology of the intestinal epithelium that increases the surface of assimilation of nutrients is modified, as another factor that influences on birds life and, consequently, improves their weight.
Other authors, who also used Lactobacillus in their biopreparations, achieved improvements in the live weight of animals with respect to the control, without observing differences regarding to the weight of birds treated with antibiotics as animal growth promoters (AGP).
Olnood et al. (2015) reported that when they evaluated a culture of Lactobacillus johnsonii, as a candidate for probiotic in broilers, they observed differences in live weight with respect to the control group. However, they were not found in relation to the treatment that included the antibiotic Zinc bacitracin (50 mg.kg-1 of food). These results show that when biopreparations with Lactobacillus spp. are used, results similar to those achieved with antimicrobials, used as AGP, can be obtained.
According to the results of this study, the birds reached practically, at 35 d, the average weight that the control animals achieved at 42 d. Therefore, if this mixture were used systematically in broilers feeding, the breeding days could be reduced. Wang et al. (2016) also included Bacillus subtilis endospores in chicken feeding and found that growth was stimulated and the target weight of the animals was early reached.
Table 2 shows the effect of the probiotic mixture on productive indicators of broilers, from seven to 42 d. It was found that the mixture had effects on weight gain, food conversion and relative weight of the carcass. There were not differences in food intake.
Table 2 Effect of the addition of the probiotic mixture on the productive indicators of broilers at 7, 14, 21, 28, 35 and 42 d of age
| Indicators | Age (d) | Control | Probiotic mixture | SE±Sign |
|---|---|---|---|---|
| Average daily gain (g) | 7 | 18.37 | 20.38 | 0.25 P=0.072 |
| 14 | 24.06 | 28.95 | 0.18 P=0.001 | |
| 21 | 34.04 | 42.77 | 0.53 P=0.001 | |
| 28 | 41.90 | 51.30 | 0.41 P=0.001 | |
| 35 | 47.47 | 58.34 | 0.53 P=0.001 | |
| 42 | 56.96 | 65.61 | 0.40 P=0.001 | |
| Food intake (g) | 7 | 156.25 | 161.0 | 3.72 P=0.401 |
| 14 | 498.50 | 528.75 | 19.04 P=0.304 | |
| 21 | 1169.25 | 1183.75 | 15.60 P= 0.737 | |
| 28 | 2100.00 | 2117.75 | 12.05 P= 0.221 | |
| 35 | 3280.25 | 3320.50 | 20.29 P=0.210 | |
| 42 | 4644.50 | 4694.00 | 22.18 P=0.165 | |
| Food conversion | 7 | 0.92 | 0.88 | 0.04 P=0.390 |
| 14 | 1.32 | 1.18 | 0.05 P=0.147 | |
| 21 | 1.55 | 1.27 | 0.027 P=0.001 | |
| 28 | 1.75 | 1.43 | 0.027 P=0.001 | |
| 35 | 1.93 | 1.59 | 0.017 P=0.001 | |
| 42 | 1.93 | 1.70 | 0.018 P=0.001 | |
| Relative carcass weight (%) | 42 | 71.55 | 75.26 | 0.58 P=0.004 |
SE Standard error. Sign. Significance
Núñez et al. (2017) evaluated the effect of enterogermin (Bacillus clausii endospores) on the productive performance of broilers. As reported, the treatment with the probiotic showed better weight gain (2972.65 g) with respect to the control (2626.90 g). Food intake was not statistically affected. However, these authors observed better food conversion, with a value of 2.02 in the group treated with the biopreparation. The highest mortality was recorded in the control treatment (17.14 %). The T2 showed better European efficiency index (292), as well as higher carcass yield (75.25 %). A similar performance was observed in this experiment.
The application of the probiotic mixture in this study caused higher increase of the animals weight, results that coincide with what was reported by Aliakbarpour et al. (2012). These authors report that the additives made with Bacillus subtilis and lactic acid bacteria (LAB), which are used in chicken feeding, affect the increase of this indicator. They also show that this effect can be related to the extension of the expression of the MUC2 gene in goblet cells and the morphological changes that occur in the intestinal tract. They suggest that after probiotic intake, mucin gene activation occurs, and its synthesis is higher. This can positively influence on the bacterial interactions that take place in the GIT, as well as in the proliferation of mucosal cells. Consequently, nutrient absorption becomes more efficient.
Other studies show results that also agree with this research. In a study by Li et al. (2016) a culture of Bacillus subtilis CGMCC 1.1086 was administered in the diet, and its effect on live weight yield was evaluated, as well as its relation with the manipulation of the caecal microbiota of broilers. The probiotic improved the daily weight gain in 27.7 %, and the food conversion rate in 10.3 % with respect to control. It was also found that, in the caecum of chickens supplemented with this additive, the bacteria of the genera Alistipes, Odoribacter, Ruminococcus, Blautia and Desulfovibrio were increased, while the populations of Staphylococcus, Escherichia and Shigella were lower than in the control birds. These references show that these microorganisms increased the population of beneficial microorganisms, and decreased the potentially pathogenic.
In this study, in the group the mixture was applied, food conversion values were obtained very close to the standard (1.68) (Cobb-Vantress 2015). Similar results were obtained by Aguavil (2012), who stated that the inclusion of a probiotic mixture of Lactobacillus acidophilus and Bacillus subtilis positively influenced on weight gain and food conversion, and decreased the mortality rate. This author refers that these effects are related to the increase in the colonization of beneficial bacteria, and the pathogens or gastrointestinal parasites, such as Escherichia coli, Eimeria spp. and Salmonella spp., did not proliferate in the birds treated with the mixture.
Gao et al. (2017) studied the influence of different levels of Bacillus subtilis on growth performance, nutrition metabolism and intestinal microbiota of broilers. The use of this probiotic increased the ADG and the average higher food intake in treated chickens. The feeding-gain ratio of the experimental groups was lower than that of the control. These results coincide with those of this research, where B. subtilis was also used as a probiotic microorganism. Improvement in conversion and growth increase was observed in the treated groups in relation to the control.
Bai et al. (2013) used a mixture of Lactobacillus fermentum and Saccharomyces cerevisiae to research its effects on growth performance and intestinal immune status in broilers. Their reports show that the probiotic mixture stimulated the production of T lymphocytes at the intestinal level, without diminishing the growth performance of broilers, in a period of 1 to 21 d. These results coincide with what was obtained in this study, since the differences in weight were observed from the first 14 d of breeding.
According to Blajman et al. (2015), current tendencies in intensive production systems postulate probiotics as a good alternative for the replacement of growth-promoting antibiotics. The intake of these biopreparations in birds production increased considerably in recent years, due to the benefits it generates in the host. This practice is aimed at activating the microbial balance in the GIT, inhibiting the growth of pathogenic bacteria, stimulating the immune response and improving the productive performance of animals. Hence, the use of this probiotic mixture of Bacillus subtilis 20BP and Lactobacillus brevis 40LP in the production of broilers could constitute a strategy to improve sanitary conditions and the production of intensive farms, since it affects the increase in productivity and bird health.
Barrera et al. (2014) evaluated the supplementation of broilers with a commercial probiotic. These authors observed that the weight gain, on day 35 of age, showed a better productive performance with the addition of the probiotic compared to other groups under study. They pointed out that, possibly, this is because these microorganisms have the ability to ferment simple sugars, stimulate the production of enzymes and lactic acid, which translates into a better use of nutrients. They also stated that, when these microorganisms come into contact with the mucous membranes, they have anti-inflammatory properties, inhibit germs that cause infectious processes or putrefaction, and thus efficiently facilitate the absorption process.
Similar results to those of this study obtained Rodríguez et al. (2015), who evaluated the effect of the inclusion of a probiotic mixture, composed of Lactobacillus salivarius C65 and Bacillus subtilis E44. They used 600 started birds, heavy pure breeds, and determined the live weight, the intake, food conversion, mortality and viability of birds. The animals, except in the first two weeks, in which there were no differences between treatments, improved (P <0.05) the food conversion per bird and the percentage of mortality and viability, in favor of the treatment with the probiotic mixture.
Table 3 shows the effect caused by the probiotic mixture on the health indicators. It was found that in animals that intake the biopreparation, mortality decreased and viability increased. The highest percentage of viability corresponded to the birds treated with the probiotic mixture, while the control group failed to reach the standard percentage of the breed (96%) (Cobb-Vantres 2012).
Table 3 Effect of the addition of the probiotic mixture on the health indicators of broilers, from 0-42 d of age
| Indicator | Age (d) | Control | Probiotic mixture | SE± |
|---|---|---|---|---|
| Mortality (%) | 0-42 | 8.50 | 3.00 | 0.02 P=0.04 |
| Viability (%) | 0-42 | 91.50 | 97.00 | 0.02 P=0.04 |
These results are in correspondence with those of Rondón et al. (2018), who used biopreparations made with Lactobacillus salivarius C65. The cited authors observed improvements in the eubiosis status of the GIT. They also verified an increase in the beneficial population of Lactobacillus and total anaerobes, and a decrease in coliforms. As reported, the inclusion of these additives in the food increased the relative weight of the spleen and the bursa of Fabricius, as well as the HI titles for the Newcastle vaccine. These results show that these biopreparations improve the microbiological indicators and stimulate the immune system, which guarantees the protection of birds against pathogenic microorganisms, with the consequent increase in viability and decrease in mortality.
The mixture of Lactobacillus brevis 40Lp and Bacillus subtilis 20Bp improved the productive and health indicators in broilers at production scale, so that their application could be considered as probiotic additive in broilers.
