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Cuban Journal of Agricultural Science

Print version ISSN 0864-0408On-line version ISSN 2079-3480

Cuban J. Agric. Sci. vol.53 no.2 Mayabeque Apr.-June 2019  Epub Apr 01, 2019


Animal Science

Evaluation of the zootechnical additive SUBTILPROBIO® C-31 on feeding of laying hens in a commercial production unit

Grethel Milián1  * 

Marlen Rodríguez1 

Damarys Días2 

Ana J. Rondón1 

M. L. Pérez4 

R. Boucourt3 

Yadileiny Portilla5 

A. Beruvides1 

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

2Combinado Avícola de Matanzas, Cuba

3Instituto de Ciencia Animal, Apartado Postal 24. San José de las Lajas, Mayabeque, Cuba

4Universidad Estatal Amazónica. Km. 2 ½. Vía a Tena (Paso Lateral), Puyo, Pastaza. Departamento de Ciencias de la Tierra. Ecuador

5Universidad Autónoma de España, Madrid, España


This study was conducted in a farm belonging to the Combinado Avícola de Matanzas, Cuba, using Leghorn L33 line laying hens, and lasted three months. The objetive of this study was to evaluate the zootechnical additive SUBTILPROBIO® for feeding laying hens. This additive was produced with strains of Bacillus subtilis, subtilis C-31 subspecies, in a concentration of 109 endospores/g. The experiment was conducted according to a completely randomized design, with two treatments: group I, base diet (maize-soybean) and group II: base diet + SUBTILPROBIO® C-31. An amount of 600 hens, with a mean weight of 1,192 grams and 113 days old was used, divided into 300 hens/treatment. Hens were located in cages under similar conditions of management and feeding. In order to determine the probiotic effect of this zootechnical additive, liveweight, intake, conversion, total production of eggs and cracked eggs, and total disqualified eggs, as well as mortality, death by pecking, cannibalism and viability were evaluated. Results showed that hens consumed the zootechnical additive had a higher increase of productive indicators like liveweight (1640.0g/1585.0g), intake (10780 kg-1 DM), conversion (1.92/2.10), egg production (15,540/15,397), cracked eggs (1,092/1,114) and disqualified eggs (69/76) for P<0.01 regarding control group. In other indicators like mortality (1/4), viability (99.6/98.6 %) and death by pecking/cannibalism (1/2), there are no differences among treatments. Results of this research demonstrate feasibility of the use of this zootechnical additive as improver of productive and health indicators for this category.

Key words: zootechnical additives; endospores of Bacillus subtilis; laying hens; eggs


Poultry production is currently one of the most important branches of animal production worldwide, which contributes at present to the satisfaction of more than 35 % of protein needs of the world population. This is achieved through the exploitation of two basic aspects that make up this branch: meat production and egg production (Peinado 2015 and FAO 2017).

The laying hen is one of the most efficient domestic animals since it is capable of producing more than 10 times its weight in eggs. This allows to convert food unfit for human intake into a proper source of protein for humans. For developing countries, poultry farming is an invaluable source that must be taken into account so that its people aspire to gradually satisfy their needs for protein intake in such a way that the economic balance is profitable enough to maintain an adequate and competitive production level (Lamazares 2000).

During the 90s, a set of products was developed that do not create the problems of microbial resistance or residual effect produced by antibiotics for poultry farming. They are grouped, generically, under the name of zootechnical additives, which can be microorganisms, mainly lactic bacteria, Bacillus endospores, fungi, yeasts or substances, which contribute to maintain a favorable ecological balance in the intestine and a good functioning of the immune system (Pérez et al. 2015 and Zhang et al. 2017). Among these additives, probiotics currently play an important role because they have the advantage of being natural and economic products that leave no residue in the final products, stimulate the immune system response and are animal productivity enhancers, which allows to obtain more productive, healthy and disease- resistant flocks (Carvalho et al. 2016). From the exposed elements, the objective of the following research was to evaluate the zootechnical additive SUBTILPROBIO® C-31, in the feeding of laying hens in a commercial production unit.


Treatments and experimental conditions. This study was carried out in the Unidad Productiva- Económica de Base “Güira” belonging to the Combinado Avícola de Matanzas, Cuba, in the category of laying hens of Leghorn Line L33 breed. The evaluation was carried out in the months between March 5th and June 5th (14 weeks). During this period, mean temperature was 29 ºC ± 2; the maximum was 30 ºC ± 1, and the minimum, 28 ºC ± 3. Mean relative humidity was 78 % ± 3. The experiment was carried out according to a completely randomized design, with two treatments: Group I: Basal diet (maize-soybean) and Group II: Basal diet + zootechnical additive SUBTILPROBIO® C-31. An amount of 600 hens, with a mean weight of 1,192 grams and 113 days old was used, divided into 300 hens/treatment.

Elaboration of SUBTILPROBIO® zootechnical additive. From the Bacillus subtilis sub subtilis C-31 strain (Milián et al. 2014), 20L of the product were elaborated according to the Milián et al. (2017).

Diet. Composition of the supplied diet met the requirements of the layer, shown in table 1. Food was offered twice a day in the form of maize-soybean meal, with a composition, according to NRP (1994), at a rate of 110 g per animal per day. The zootechnical additive was supplied in the G-II ration, in the morning hours, every day. It was manually mixed with the diet, at a concentration of 109 CFU.g-1.

Table 1 Nutritional requirements for L33 laying hen 

Animal management. Before the arrival of hens, the farm was subjected to a health rating, as established by the Technical Instructions for the handling of layer (UCAN-IIA 2003). Hens were housed in metal cages with capacity for four hens, at a rate of 400-533 cm2/bird; they received ad libitum water through automatic nipples.

Experimental procedure for sample analysis. To determine the probiotic effect of the zootechnical additive, the birds under study were weighed at the beginning and at the end, intake (kg-1 DM), and conversion (kg of food / kg of total eggs) were recorded, and all the eggs per each treatment were collected, in order to determine the number of postures, the total number of eggs that are broken and disqualified. Total of dead hens was also determined: (initial existence-final existence / initial existence) x 100 %, death by pecking and cannibalism: (total death by pecking and cannibalism / initial existence) x 100 % and viability percentage.

Statistical processing. For data analysis, INFOSTAT statistical software, version 2012 (Di Rienzo et al. 2012) was used. An amount of 300 hens were selected per treatment, where each hen constituted an experimental unit. Differences among groups were verified through Duncan (1955) multiple range comparison test.


Table 2 shows the results of the evaluation of SUBTILPROBIO® C-31 zootechnical additive in productive indicators: liveweight, intake and conversion. Hens that consumed the zootechnical additive showed a better performance in the indicator liveweight and conversion (P <0.01) at the end of the experiment, a result that reflects the veracity of the use of zootechnical additives with a marked probiotic effect. Egg conversion per each kilogram of consumed food by kilograms of total eggs is an indicator of productive efficiency of the unit, hence the results correlate with the above and it can be said that the G-II made an efficient use of the nutrients provided in the feed from the inclusion of the zootechnical additive. These results are in the ranges reported in the Guía de manejo de ponedoras (2016) of the layer for these indicators.

Table 2 Performance of productive indicators at the beginning and at the end of the experiment with the addition of the zootechnical additive SUBTILPROBIO® C-31 for 14 weeks 

Means between lines differ at P<0.05 (Duncan 1955) **P<0.01

According to Pérez et al. (2012), the use of probiotic biopreparations in laying hens is a routine practice in modern poultry production, which provides favorable results. These authors evaluated a probiotic mixture of Lactobacillus salivarius and the Bacillus subtilis strain E-44 in the commercial farm of layers "Baro Chiquito" and obtained differences between the group that received the probiotic and the control for the conversion indicator, similar to those obtained in this research.

One of the important elements that is taken into account in layer rearing is the balance in diets which will improve nutrient balance, timely use of synthetic amino acids, and some other aspects (Bermúdez 2012). Hence, the option of including zootechnical additives that are currently on the market, is a real possibility.

Research carried out by Pérez et al. (2012) and Rodríguez et al. (2016) demonstrated the effect of zootechnical additives in activating a group of mechanisms in animals of livestock interest, specifically in the layer category. In their research they showed how the nutritional value of diets is favored with the inclusion of these additives.

Studies carried out in other categories of birds were those obtained by Gamboa (2014) when the addition of a homemade microbial culture in broiler chickens was evaluated. This author obtained favorable results in productive and health indicators as weight gain (2,902.1 g vs. 2,564 g) and mortality (3.3% vs 8.3%).

Other inclusions of probiotics based on spores of Bacillus spp. in birds were reported by Boaro (2015) and Lie et al. (2015), who had similar results to those of this research for the productive indicator liveweight. Likewise, Cerón (2016) obtained similar results to this study when adjustments to diets were performed and reached body weights and optimal uniformity in the experimental birds.

The use of the zootechnical SUBTILPROBIO® C-31 additive in the diet of laying hens showed a positive effect in terms of the number of postures, decrease in the number of broken and disqualified eggs with respect to GI group (control), which did not receive the additive, when comparing both groups at the end of the experiment, for a significance of (P <0.01). Table 3 shows the results.

Although the results in the group treated with the zootechnical additive were superior to the control, when evaluating the zootechnical response, it should be integrally assessed, and if their positive effect is true, it is important to point out that the environmental effect, adequate management and high potential of the layer favored this result. Herrera (2014) advocated taking these factors into account as this allows to obtain more eggs, which is translated into an improvement in the laying plateau.

Table 3 Performance of the amount and quality of the egg, with the addition of the zootechnical additive SUBTILPROBIO® C-31 during 14 weeks 

Means between lines differ at P<0.05 (Duncan 1955) **P<0.01

Similar researches were carried out by Pérez et al. (2012), when they evaluated probiotic additives (SUBTILPROBIO® E-44 and PROBIOLACTIL® C65) with positive effects in terms of the number of eggs, as well as in the conversion of eggs per each kilogram of food consumed.

Guo et al. (2017) in studies with cultures of B. subtilis as probiotic in laying hens (Hy-Line Brown) of 28 weeks old, obtained an improvement in the resistance of egg shell.

Results of health indicators evaluated in both groups of animals are shown in table 4. These were favored by two factors, one the effect exerted by the zootechnical additive SUBTILPROBIO® C-31 in the intestinal microbiota in favor of the reduction of enteropathogens, which favored the decrease of the indicator total death and death by pecking/cannibalism, as well as, the viability of G-II with respect to the control group.

Table 4 Response of health indicators in birds fed with the zootechnical additive SUBTILPROBIO® C-31 for 14 weeks. 

As a second, the study was performed with young hens at the start of laying. They were in a satisfactory health state, which helped no infectious outbreak occur in the experimental units during the entire experimental stage. Results are in correspondence with the exposed in the Guía de manejo de la ponedora (2016).

It is known that some the important effects of zootechnical additives are the nutrient digestibility modification, digestive mucosa development, reduction of colonization by pathogens, decrease of adverse effects of mycotoxins and modification of the immune response (Lourenço et al. 2016, Kizerwetter-Świda and Binek 2016 and Rodríguez 2017), so the results of this research are associated with these mechanisms.

According to Pérez et al. (2012), colibacillosis, erysipelas, coccidiosis, lymphatic leukosis and cannibalism are among the most common causes that influence on hen mortality, which were not detected during the development of the experiment.

Studies conducted by Milián et al. (2014) and Rodríguez et al. (2015) demonstrated the antibacterial effect of an enzymatic hydrolyzate of Saccharomyces cerevisiae yeast and endospores of Bacillus subtilis E-44 in the prevention of infectious diseases in animals of zootechnical interest.

Ricke et al. (2015), when they applied the commercial prebiotic Biolex® MB40 (composed of mannan oligosaccharides) in conventionally bred chickens, found beneficial effects in the reduction of Salmonella in the treated animals. In contrast, other researchers obtained no differences among treatments by including live strains of Saccharomyces cerevisiae, Pediococcus acidilactici, and yeast cell wall extract, individually or in combination (Purdum and Hahn 2016).

Regularly, literature reports that when yeast (S. cerevisiae) and derivatives of its wall are applied in the diet of birds, bacterial attacks decrease, intestinal bacterial populations are modified, improve the productive performance and nutrient digestibility, which decrease the number of deaths, which is component found in the provided additive (Jahanian and Ashnagar 2015 and Rodríguez 2017).

Mountzouris et al. (2015) evaluated the application of a zootechnical additive based on Saccharomyces cerevisiae var. boulardii in the diet of broilers treated with Salmonella enteritidis. In this study, the effect of yeasts on the growth of animals and on the prevalence of pathogenic bacteria in the caeca, cloaca and carcass skin was determined through culture procedures, while the performance of cecal microbiota was characterized by real-time polymerase chain reaction (PCR). These authors concluded that when zootechnical feed additives were supplied independently of the strain used for producing them, there is an improvement of growth of birds, increase of eggs, the improvement of their quality and their health status is improved, which shows a decrease of mortality indicator and favors the viability of treated animals.

Several experiences show that the use of strains of Bacillus spp. have an important function in reducing and even preventing intestinal colonization of Salmonella spp.González (2016) provided a suspension of endospores of B. subtilis specific pathogen-free chickens before treating them with S. enteritidis and C. perfringens. According to the authors, the treatment with B. subtilis suppresses completely the persistence and colonization of both germs and favors the increase of productive indicators with a low mortality, reason why it is inferred that the results of this research correlate with the previously mentioned facts.

In this sense, Corrigan et al. (2015) studied the effects of these probiotic compounds on the microbial community and on the physiology of the caeca of these birds, and determined that they can modify the microbiota with the increase of Bacteroides. It means that the native or beneficial bacteria proliferate, and mortality levels and cannibalism incidence in this birds are reduced.

As a conclusion, the results obtained reflect a multifactorial probiotic response, where there is an incidence in the productive and health indicators. Animals treated with the SUBTILPROBIO® C-31 zootechnical additive showed favorable results for both indicators. Therefore, it can be stated that this additive is a promising, viable, ecological and sustainable alternative in current Cuban poultry industry.


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Received: November 23, 2017; Accepted: February 17, 2019

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