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Cuban Journal of Agricultural Science

versión impresa ISSN 0864-0408versión On-line ISSN 2079-3480

Cuban J. Agric. Sci. vol.55 no.1 Mayabeque ene.-mar. 2021  Epub 01-Mar-2021


Animal Science

Evaluation of the zootechnical additive SUBTILPROBIO® E-44 in productive and health indicators in heavy pure breeds birds under production conditions

1Universidad de Matanzas. Autopista Varadero km 3 ½. Matanzas, Cuba

2Universidad Estatal Amazónica. km. 2 ½. Vía a Tena (Paso Lateral). Puyo, Pastaza. Departamento de Ciencias de la Tierra. Ecuador

3Unidad Empresarial de Base Líneas Puras Pesadas de la Empresa Genética Avícola y Pie de Cría. Matanzas


In order to evaluate the zootechnical additive SUBTILPROBIO® E-44 in productive and health indicators in E1 heavy pure breeds birds under production conditions, an experiment for a period of six weeks was developed. A completely randomized design was applied, with two treatments: control group corn-soybean basal diet and group I basal diet + SUBTILPROBIO® E-44 zootechnical additive. A total of 1800 birds with an average weight of 43 g were used, distributed in 900 birds per treatment. They were located on the floor, in similar conditions of management and feeding. To determine the probiotic effect of this zootechnical additive, live weight, conversion, uniformity, viability and mortality were evaluated. The results showed that the birds from the fourth week to the sixth expressed a probiotic response in the productive indicator live weight (4:1472g/1406), (5:1897g/1826g) and (6:2387g/2336g) for P <0.01. At the end of the experiment, the productive indicators live weight (2387g/ 326g) and conversion showed significant values (1.8/1.9) for P <0.01, as well as uniformity (88/84 %) for P <0.001, with respect to the control group and to the standard for the breed under study. The mortality (3.8/8) and viability (96.2/92 %) indicators showed differences with respect to the control for P <0.01. The results of the research show the feasibility of using this zootechnical additive as an improver of the productive and health indicators for this poultry category.

Key words: probiotics; endospores of Bacillus subtilis; heavy birds

With the triumph of the Revolution, the poultry industry in Cuba was remarkably developed. The Combinado Avícola Nacional (CAN) was created on May 22, 1964, and with it a group of production units, whose premise was to guarantee the genetic line and its main descendants, the foundation stock, as well as the obtaining of reproducers and their hybrids for the production of egg and birds meat. This freed the country has to invest to import replacement regularly. In addition, it allowed maintaining and improving heavy pure breeds, and guaranteeing the poultry genetic basis with the existing breeds to promote the increase in meat and egg yields and their commercialization in poultry enterprises in the country (Ramírez 2014).

In order to achieve the mentioned goals, work is carried out with great productive intensity, which favors the incidence of different factors that lead to constant stress situations in animals, and this gives rise to imbalances in the intestinal microbiota, with its consequent negative impact on the health and productivity of animals (García-Hernández et al. 2016 and Beruvides et al. 2018). To mitigate these difficulties, growth-promoting antibiotics (GPA) have been used for decades. However, its prolonged and indiscriminate use caused undesirable side effects and the rejection of consumers towards animal origin products (Díaz et al. 2017). In this context, zootechnical additives with a probiotic effect based on Lactobacillus spp., yeasts and Bacillus spp. spores were introduced into the feeding and management systems (Gao et al. 2017a and Medina-Saavedra et al. 2017), in order that they contribute to avoid the negative effects of the use of growth-promoting antibiotics. These additives, which have the advantages of being natural and economic products, that do not leave residues in the final products, stimulate the responses of the immune system and are enhancers of animal productivity, which allows obtaining flocks more productive, healthy and resistant to diseases (Blanch 2017 and Arteaga et al. 2018).

The world knows about the use of zootechnical additives. However, Cuba does not use them systematically, despite having products obtained in the country from national resources and the vast experience that the University of Matanzas (UM) and the Institute of Animal Science (ICA) have in this matter (Pérez 2000 and Rondón 2009). SUBTILPROBIO® E-44 is among the group of additives obtained by researchers from these institutions, which is the result of a simple biotechnological process, obtained under laboratory conditions. This zootechnical additive was made with endospores of Bacillus subtilis sub species subtilis, which gives it durability over time (Milián et al. 2014 and 2019a). It was applied in different livestock categories with favorable results in the physiological, productive and health indicators (Milián et al. 2017a and 2019b). Based on the potential of this product, the objective of this study was to evaluate the zootechnical additive SUBTILPROBIO® E-44 in the feeding of E1 heavy pure breeds birds in a commercial production unit, taking into account productive and health indicators.

Materials and Methods

Treatments and experimental conditions. The study was carried out at Unidad de Genética Avícola y Pie de Cría, Matanzas, in the E1 heavy pure breeds category. The evaluation was carried out in May and June during the first six weeks of life of birds. During this period, the mean temperature was 29 ºC ± 2; the maximum of 30 ºC ± 1, and the minimum of 28º C ± 3. The average relative humidity was 78 % ± 3. The experiment was carried out according to a completely randomized design, with two treatments: control group (CG): basal diet (corn-soybean) and group I (G-I): basal diet + SUBTILPROBIO® E-44 zootechnical additive. A total of 1800 one-day-old birds were used, with an average weight of 43 g. A total of 900 birds were distributed per treatment.

Elaboration of the zootechnical additive SUBTILPROBIO®. From the Bacillus subtilis strain sub species subtilis C-31 (Milián et al. 2014), 30L of the product were made, according to the methodology proposed by Milián et al. (2017b).

Diet. The composition of the supplied diet is shown in table 1. The food was offered twice a day in the form of corn-soybean meal. The zootechnical additive was supplied in the G-I ration (basal diet + SUBTILPROBIO® E-44 zootechnical additive). It was offered every day and the additive was manually mixed with the diet. The dose that was used was 109 cfu.g-1 concentrate.

Table 1 Basal diet composition 

Raw matter, % Starting (0- 14 d) Growing (15- 28 d) Finishing (29-42 d)
Corn meal 42.43 54.32 60.27
Soybean meal 43.88 33.68 28.58
Sunflower oil 8.80 7.28 6.52
Dicalcium phosphate 2.57 2.45 2.39
Calcium carbonate 0.74 0.72 0.71
Common salt 0.25 0.25 0.25
DL- methionine 0.33 0.30 0.29
Vitamin -mineral pre-mixture* 1.00 1.00 1.00
Calculated analysis
Metabolizable energy (MJ/ kg) 13.38 13.38 13.38
Crude protein (%) 23.00 20.00 18.80
Calcium (%) 0.95 0.95 0.95
Asimilable phosphorus (%) 0.42 0.42 0.42
Methionine + cystine (%) 0.92 0.87 0.82

* 1 kg of food contains vitamins: A (10000 UI), D3 (2000 UI), E (10 mg), K3 (2 mg),Thiamine (1 mg) - B1, Riboflavin (5 mg) - B2, Pyridoxine (2 mg) - B6, B12 (15.4 mg), nicotinic acid (125 mg) calcium pantothenate (10 mg), folic acid (0.25 mg) and biotin (0.02 mg), as well as minerals: 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).

Animal management. The buildings were subjected to a health rating before the birds arrived at them. The animals were distributed by treatment for a total of 900 birds, distributed in three paddocks (300 each). The paddock dimensions were 6 m long x 7 m wide (42m2). The rearing was on the floor and the water was ad libitum.

Experimental procedure for sample analysis. To determine the probiotic effect of the in vivo zootechnical additive, all the birds under study were selected. The average live weight of birds was determined weekly for each treatment. For this, 10 % of the mass was taken, and never less than 50 birds. They were weighed in early hours. All the weights of each sampled bird were added and divided by the number of birds to obtain the average weight. The food intake and the weight conversion per kilogram of intake food were determined. The percentage of uniformity and viability was also calculated, as well as the mortality during the six weeks that the experiment lasted, according to the technical instructions for the category.

Statistical processing. For data analysis, the statistical program INFOSTAT, version 2012 (Di Rienzo et al. 2012) was used. A total of 900 birds per treatment were selected. Each bird constituted an experimental unit. The results were compared with the standards for the breed under study. For means comparison, the LSD Fisher (1935) test was used. For the original variables mortality and viability, the theoretical assumptions of the analysis of variance were verified. For the normality of errors, the Shapiro and Wilk (1965) test was applied and for the homogeneity of variance was proceeded with Levene (1960. Both fulfill the assumptions, so a completely randomized design was used.

Results and Discussion

Table 2 shows the live weight results of the birds studied during the first six weeks of life with respect to the standards established for this production line. In both treatments, there were not differences in the first three weeks of rearing. However, from week 4, an improvement of this indicator is seen in the group that intake the zootechnical additive SUBTILPROBIO® E-44 (P <0.01) with respect to the control and the standards described for this rearing. This result can be associated with the characteristics of the microbial genus (Bacillus), among which it is a transit microorganism. Thus the need to offer it daily to achieve required levels, which allow demonstrating its probiotic effect on the animals that intake it.

Table 2 Performance of the live weight indicator with respect to the standard with the addition of the zootechnical additive SUBTILPROBIO® E-44 for six weeks 

Weeks Treatments Live weight, g SE ± Sign
1 Breed standard 185 -
G-I: SUBTILPROBIO® E-44 zootechnical additive 186 0.15 P=0.0096
CG: control 193
2 Breed standard 400 -
G-I: SUBTILPROBIO® E-44 zootechnical additive 443 0.70 P=.0042
CG: control 448
3 Breed standard 820 -
G-I: SUBTILPROBIO® E-44 zootechnical additive 926 0.17 P=0.0051
CG: control 894
4 Breed standard 1300 -
G-I: SUBTILPROBIO® E-44 zootechnical additive 1472 0.14 P=0.0026
CG: control 1406
5 Breed standard 1820 -
G-I: SUBTILPROBIO® E-44 zootechnical additive 1897 0.21 P=0.00036
CG: control 1823
6 Breed standard 2340 -
G-I: SUBTILPROBIO® E-44 zootechnical additive 2387 0.26 P=0.0010
CG: control 2336

The means between the rows differ to P <0.05 (LSD Fisher)

Diverse are the researches that show the positive response of the inclusion of zootechnical additives with probiotic effect in the food. These provide a state of eubiosis, and improve the physiological effect on the body, beyond its nutritional value, which is reflected in the birds in productive and health indicators (Sosa et al. 2018). Studies obtained by Wang et al. (2016) show this, when they included endospores of Bacillus subtilis in broilers feeding. These authors verified that with this inclusion, growth is stimulated and the adequate weight is reached in the animals before time.

According to Barros (2018) and Mozombite (2018), probiotics can replace antibiotic therapies and provide a new, less aggressive alternative; in addition, they allow reducing the economic losses that originate from the presence of pathogens in poultry farms. The cited authors evaluated a probiotic at two concentrations and obtained differences between the group that received the probiotic with respect to the control, for the indicator live weight, conversion and mortality, results that are similar to those obtained in this research.

Nuñez et al. (2017), when they assessed the effect of the commercial product Enterogermina (Bacillus clausii spores) on the productive performance of male broilers from the Cobb Line, supplied in the drinking water, obtained an increase in the weight of treated birds with respect to those of the control. This agrees with the results obtained in this study with the use of Bacillus subtilis spores, which allows us to infer that the use of this probiotic is a viable alternative in Cuban poultry farming.

Rodríguez et al. (2015), when evaluating a probiotic mixture of two zootechnical additives (PROBIOLACTIL® C65 and SUBTILPROBIO® E-44) with respect to the standard in Heavy Pure Breeds B4 birds for five weeks, obtained positive results in terms of weight increase from the third week of inclusion of the biopreparations (793, 1249 and 1587g). This result is reaffirmed in the Rendón et al. (2015) and Valdés (2018) reports, when they refer to the use of mixtures of microorganisms in biopreparations for animal production.

The obtained results allow inferring that the effect of probiotics on poultry farming resides, regardless of the experimental designs that are used, on the positive action they exert on the productive response of the animals that intake them.

Table 3 shows the results achieved in the productive indicators, when the first six weeks of life of the birds under study ended. There was difference (P <0.01) for live weight and conversion. However, for the intake indicator, there were not differences between the GI and the control group. The uniformity indicator showed differences (P <0.001) in the group treated with the nutritional additive SUBTILPROBIO® E-44 with respect to the control.

Table 3 Performance of the productive indicators up to the sixth week of the rearing cycle of heavy pure breeds E1birds, fed ad libitum with SUBTILPROBIO® E-44 

Indicators Breed standard Treatments SE ± Sign
Live weight, g 2340 2387 2336 0.89 P=0.0016
Intake, g 4.97 4.51 4.60 0.12
Conversion 2.12 1.88 1.96 0.15 P=0.0025
Uniformity, % 80.0 88.0 84.6 0.11 P<0.0001

The means between the rows differ to P < 0.05 (Ducan 1955)

Respect to the use of probiotics in poultry farming, there are infinite studies that report and show the effectiveness of biopreparations based on Bacillus spp. endospores. The results obtained in this research, as well as the reports available in the literature, together with Gao et al. (2017b) results show this. These authors, when using a B. subtilis strain as a probiotic microorganism, observed an improvement in the conversion and increased growth of the treated animals, with respect to the control group.

Other studies, such as that of Ortiz et al. (2013), show results similar to those of this research, when they added the probiotic ECOBIOL (Bacillus amyloliquefaciens CECT 5940) to the diet of 480 broilers (Arbor Acres Plus), and obtained better yield when reaching the final weight (2 575 g) in 2.5 d less than in the control group. These authors stated that probiotics can improve conversion (1.98 vs. 2.06) and the efficiency index (259 vs. 242), as well as decrease mortality (6.28 vs. 6.77 %).

Bai et al. (2016) reported improvements in weight gain and feed conversion rate in one-day-old Arbor Acres males, when they supplemented basal diets with Bacillus subtilis mbJ (BSfmbJ) at doses of 2, 3 and 4 x 1010 cfu/kg , without using antibiotics. The reports by Zhang et al. (2013) refer the benefits of including B. subtilis in the diets, by achieving better weight gain and feed conversion with the use of the probiotic, which exceeded the results obtained with the diets in which an antibiotic was added. Nuñez et al. (2017), when evaluating the productive performance of broilers supplemented with Eterogermina® in drinking water, recorded differences for the weight and conversion indicators, and not for intake, which coincides with what was obtained in this study. These results, which support those of this study, confirm the importance of zootechnical additives with a probiotic-type effect on birds. Specifically, SUBTILPROBIO® E-44 showed that the treated animals made more efficient use of the nutrients provided by the intake food, when obtaining a higher live weight with a similar food intake.

Table 4 shows the results obtained in the mortality and viability indicators. For both there is a difference of the G-I with respect to the control group (P <0.01). One of the marked effects of additives made with Bacillus spp. strains is to have a positive effect on the intestinal microbiota, in favor of reducing the presence of E. coli, staphylococci and clostridia, while increasing the presence of beneficial bacteria, such as Lactobacillus spp. and Bifidobacterium spp. (Forte et al. 2016, Li et al. 2016 and Medina et al. 2017). This makes the viability indicator favorable and therefore reduces the number of deaths.

Table 4 Health indicators up to the sixth week of the rearing cycle of heavy pure breeds E1birds, fed ad libitum with SUBTILPROBIO® E-44 

Indicators Treatments SE ± Sign
Breed standard GI GC
Mortality, % 7 3.8 8 0.89 P=0.0031
Viability, % 93 96.2 92 0.08 P=0.0018

The means between the rows differ to P<0.05 (Ducan 1955). The values were transformed to arcsen%+0.375 and correspond to the original means

Rodríguez et al. (2015), who evaluated the effect of the inclusion of a probiotic mixture composed of Lactobacillus salivarius C65 and Bacillus subtilis E44 in birds of heavy pure breeds B4, obtained results very similar to those of this research. These authors observed improvements in the percentage of mortality and viability, in favor of treatment with the probiotic mixture. The value of this result lies in the application of the zootechnical additive SUBTILPROBIO® E-44 under production conditions, and the consequent improvement in mortality and viability indicators, regardless of the action of biological factors present in the animals and exogenous ones.

From the results obtained in this study, it could be inferred that the zootechnical additive SUBTILPROBIO® E-44, under the studied experimental conditions, shows real possibilities for its use in the heavy breeder category in the first six weeks of life. At present, this new zootechnical additive with probiotic activity constitutes a promising alternative with respect to the use of antibiotics as growth promoters.


Thanks to the UEB Lines Puras Pesadas, from the Empresa Genética Avícola and Pie de Cría, for the support for the development of this study.


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Recebido: 18 de Junho de 2020; Aceito: 03 de Fevereiro de 2021


Conflict of interest: The authors declare that there are no conflicts of interests among them

Author´s contribution: Grethel Milián Florido: Original idea, design the experiment, data analysis, manuscript writing. Marlen Rodríguez Oliva: Elaboration of the zootechnical additive, data analysis, manuscript writing. Onel González Espino: Conducting the experiment, data analysis. Julia Rondón Castillo: Elaboration of the zootechnical additive, data analysis, manuscript writing. Manuel Lázaro Pérez Quintana: Original idea, adviser the experimental design, manuscript review. Agustín Beruvides Rodríguez: Conducting the experiment, adviser the experimental design. Iraní Placeres Espinosa: Statistical analysis, data review.

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