Introduction

Poultry is one of the most important sources of protein (meat and eggs) for humans. Due to the growing demand for food products over the past few years, poultry production has increased significantly, both quantitatively and qualitatively, resulting in the broilers’ weight reaching 3 kg at just 40 days and in the egg laying capacity of 330 eggs per 52 weeks [1].

Internationally, antibiotics such as tetracycline, amoxicillin, penicillin, bacitracin, and more are used routinely as a chicken growth promoter and as a preventive antimicrobial measure [2]. However, the use of antibiotics in poultry farming leads to the spread of antibiotic resistance and the development of microbiota disturbances in birds [2, 3]. For these purposes, probiotics should be considered as an alternative to antibiotics [4]. The World Health Organization defines probiotics as “live microorganisms which when administered in adequate amounts confer a health benefit on the host” [5]. Similar to antibiotics, some probiotics inhibit the growth of microbial pathogens in the intestines of birds, thus reducing morbidity. Moreover, probiotics do not trigger antibiotic resistance in the gut bacteria and their use does not lead to the accumulation of toxic antibiotics in bird tissues [6, 7].

Most of the probiotic microorganisms used in poultry farming belong to Lactobacillus spp., Bifidobacterium spp., and Enterococcus spp. They are utilized either as monocultures or in multispecies formulations. Additionally, there is a noticeable increase in the use of bacilli-based probiotic formulations in poultry farming. Bacilli species are technologically suitable feed additives because of their spores’ stability in the presence of numerous stresses and ability to produce a variety of enzymes such as protease, amylase, and lipase. Bacilli probiotics supplementation was reported as improving egg mass, production, and quality (e.g., increase of the shell’s strength and thickness). The observed positive effects were proportional to the number of the probiotic cells administered to the birds [8].

Materials and Methods

The research was carried out according to the approved conditions at JV “Svetly,” which is a structural unit of CJSC “Agrofirma” Vostok (Volgograd region, Russia), the sow farm of the second order for poultry breeding “Highsex brown.”

Probiotics

Two strains of probiotic bacteria were used: B. subtilis KATMIRA1933, the fermented milk product isolate [9], and B. amyloliquefaciens B-1895, the soil-derived microorganism.

The protocol for solid-phase fermentation of probiotic bacilli was described in detail in our study [10]. Briefly, bacterial strains were inoculated on plates with solid LB medium (Difco, MI) and incubated for 1 day at 37 °C. Soybeans (1 kg) were washed with running water, soaked for 12 h at room temperature, sterilized at 115 °C for 40 min, placed in an incubator, and cooled to 60 °C. The soybean preparation was inoculated with the biomass of bacteria from one plate, mixed thoroughly and incubated for 24 h at 42 °C aerobically. The fermented substrate was milled with a meat grinder, distributed in a thin layer on metal trays, and dried at 50 °C to a humidity of 8–10%. Viable cells were enumerated at each step of the process by seeding on the appropriate solid medium.

In Vivo Experimental Procedures

Parent herd of the “High-sex brown” cross (hatched on August 25, 2016) was obtained from the Sverdlovsk PPR Ltd. (Sverdlovsk Region). Eight groups of 1-day-old chicks were formed: four groups of female chickens with 70 animals per group and four groups of male chickens with 7 animals each. These groups consisted of a control and experimental (I, II, and III) subgroups. The control group received a standard diet, while experimental animals received the diet with probiotic strains (group I received a probiotic preparation based on the B. subtilis strain KATMIRA1933, group II received a probiotic preparation based on the strain B. amyloliquefaciens B-1895, and group III received a probiotic preparation based on the mixture of the two bacillus strains).

These preparations were introduced into the diet as additives. Additive No. 1 included a probiotic preparation based on the B. subtilis strain KATMIRA1933 (107–109 CFU viable spores per gram of the probiotic supplement) and extruded pumpkin press cake (included in the main diet) as a filler; additive no. 2 included a probiotic preparation based on the strain B. amyloliquefaciens B-1895 (107–109 CFU viable spores per gram of the probiotic supplement) and extruded pumpkin press cake as a filler; additive No. 3 included probiotic preparation based on B. subtilis KATMIRA1933 and B. amyloliquefaciens B-1895 (equal amounts, 107–109 CFU viable spores per gram of the probiotic supplement) and extruded pumpkin press cake as filler.

Doses of the preparations’ administration were 1% in the overall structure of the poultry diet, and the dose of probiotic supplements was 0.1%.

Each experimental bird was contained in the cell battery Big Dutchman (Germany). The microclimate parameters were set according to the recommendations of the manufacturer of cross-country “High-sex brown” company “ISA Hendrix Genetics” (Holland).

The birds were fed with the standard mixed fodder manufactured at the feed mill of the company. Feeding of the experimental birds was carried out according to NRC [11]. Weighing of the experimental young animals was carried out on the weekly basis. The conversion of the feed was calculated as the ratio of the weight of the expended feed to the weight gain of the bird.

Quality of Sperm

Semen from the birds was collected by abdominal massage [12] and evaluated for the selected gross semen variables such as semen volume, sperm concentration, and live and abnormal sperm.

Sperm viability and abnormality were evaluated using a portion of ejaculate stained with an eosin-nigrosin solution. The stained seminal smears were prepared in duplicates and 200 sperm per slide were evaluated for viability, where unstained spermatozoa were considered as live. Spermatozoa with detached heads, abaxial heads, malformed heads, bent tails, coiled tails, double tails, and protoplasmic droplets were considered as abnormal, as described [13, 14].

Sperm concentration was determined in duplicate, using a Neubauer hemocytometer [14].

Egg Production and Quality of Eggs

Egg production was calculated using the following formula:

$$ \mathrm{Hen}-\mathrm{Day}\ \mathrm{Egg}\ \mathrm{Production}\ \left(\mathrm{HDEP}\right)=\frac{\mathrm{Total}\ \mathrm{number}\ \mathrm{of}\ \mathrm{eggs}\ \mathrm{produced}\ \mathrm{during}\ \mathrm{the}\ \mathrm{period}}{\mathrm{Total}\ \mathrm{number}\ \mathrm{of}\ \mathrm{hen}-\mathrm{days}\ \mathrm{in}\ \mathrm{the}\ \mathrm{same}\ \mathrm{period}}\times 100\% $$

Haugh unit (H.U.) was calculated using the formula:

$$ \mathrm{H}.\mathrm{U}.=100\times \log \left(h-1.7\ {\mathrm{w}}^{0.37}+7.6\right) $$

Where, h is albumen height in millimeters, measured by a spherometer, and w is the observed weight of the egg in grams [15].

The eggs’ length and breadth were measured with a digital caliper and the shape index was calculated as the ratio of breadth to length times 100.

Albumen weight was calculated as egg weight − (yolk weight + shell weight). Albumen and yolk ratios were calculated taking their individual weights as the percentage of the total egg weight. Albumen and yolk indices were estimated as a percentage, taking the ratio of their respective heights to the average of breadth and length as suggested in previously published reports. Yolk albumen ratio was calculated as the weight of yolk/weight of albumen [16, 17].

Hatchability was calculated as the percentages of all the egg sets that hatched.

Statistical Processing of Experimental Data

The statistical significance of the differences was determined by Student’s t test for independent samples at p < 0.05.

Ethics of Biological Experiments

Experiments on animals were conducted in accordance with the principles of the European Convention for the Protection of Vertebrate Animals, used for experiments or for other scientific purposes.

Results

Quality of Rooster Sperm Production

In pedigree roosters, the males of the experimental groups exceeded the control volume of the ejaculate, the spermatozoa concentration, and the total number of spermatozoa in the ejaculate. The number of morphologically abnormal cells in the ejaculate of the roosters of the experimental groups decreased (Table 1).

Table 1 Quality of the rooster sperm production (n = 5)
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Egg Production

The age of the first egg laying was found to be dependent on the reproductive organ development which was followed during the pullet production. In the second and third experimental groups, the first egg was laid at the age of 126 days, in the control group at 127 days, and in the first test group at 128 days. The poultry productivity in all experimental groups during the first 5 months of oviposition (39 weeks) was higher than that in the control group (Table 2, Fig. 1).

Table 2 The number of eggs laid by the control and test groups up to the age of 39 weeks
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Fig. 1
figure 1

Egg production of control group birds (a) and the difference in egg production of the experimental groups from the control group (b), in percent

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At the age of 39 weeks, the birds of all the groups reached the peak of productivity. However, during the entire period of observations, the number of laid eggs in the first experimental group was higher than that in the test groups II and III by 69 and 56 more eggs, respectively, and it measured 119 eggs more than that of the control group.

Hatching Egg Quality

For the study’s purposes, the eggs were incubated from the 28-week-old birds. Prior to the incubation, morphological and chemical analyses of the eggs were conducted (Table 3).

Table 3 Morphological indices of the hatched eggs (n = 10)
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Morphological analysis of incubation eggs showed that the weight of eggs in all experimental groups exceeded the control. The increase of the eggs’ mass was due to the mass of the yolk.

The protein index and the number of Haugh unit in the experimental groups were significantly higher than those of the control. The thickness of the eggshell in experimental groups exceeded that of the control, too. The chemical composition of the experimental laying hens’ eggs was within the physiological norm and did not differ significantly from the eggs in the control group.

Egg Hatchability

Poultry is characterized by high reproductive qualities, which are determined by a number of factors such as the intensity of laying, high fertilization, and hatchability of eggs. Egg hatchability characterizes the biological fullness of fertilized eggs and the viability of embryos and hatched young animals. Our results indicate that, in all the experimental groups, the output of the chickens was high and corresponded to the standard characteristic to the cross (Table 4).

Table 4 Results of the egg incubation
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However, in experimental group I, the hatching rate exceeded the control by 2.14%, with 84.64 against 82.50 in the control. In group II, the observed excess in hatching was 1.43%, and it reached just 0.71% in experimental group III (almost equivalent to control). The higher yield of chicks in the experimental groups was obtained by increasing the egg fertilization and reducing the number of embryo deaths during the first 7 days of incubation. This indicates a biological incorporation of the bacilli from the feed that stems from the hen to their young.

Discussion

Our study was conducted in the industrial technological environment, aimed at the results’ implementation in the poultry production. That is why the hen to rooster ratio correlated with that commonly accepted for optimal insemination [18]. Our preliminary (data not shown) and reported studies here showed that the number of roosters (seven) was appropriate for the study’s objective. Moreover, the low variability in the analyzed sperm quality parameters allowed for identification of statistically significant differences between control and experimental groups of animals.

According to the literature, probiotics affect numerous parameters in hens and eggs. These factors include biochemical blood indices showing the intensity of carbohydrate and protein metabolism (protein, glucose, urea content); hematological composition of blood (number of blood corpuscles); dynamics of live weight (weight gain); conversion rate of feed (apparently, it is increased by improving digestion and absorption of nutrients, leading to better performance); quantitative and qualitative compositions of the microbiota; the level of oxidative stress (mRNA expression of antioxidant genes, oxidative damage index, etc.); meat quality (pH, drip loss, cooking loss, shear force, color); laying performance; egg quality (yolk cholesterol level, improved shell thickness, egg weight); intestinal barrier function of laying hens [8, 19,20,21].

In our study, the introduction of probiotic bacteria into the diet of birds led to the increase in sperm production, egg production, egg quality, and hatchability. We speculate that these qualities resulted from the production of a large number of lytic enzymes and metabolites exhibiting antioxidant and DNA-protective properties by the studied strains [22]. The observed effects can also be due to the bacilli-produced proteases, amylases, and cellulases which contribute to the better digestion of the feed.

Probiotics strains of Lactobacillus, Streptococcus, Bacillus, Bifidobacterium, Enterococcus, Aspergillus, Candida, and Saccharomyces species have been shown to increase resistance of chickens to Salmonella, Escherichia coli, and Clostridium perfringens infections. In addition, oral inoculation of Bacillus subtilis spores reduced intestinal colonization of pathogenic E. coli in chickens [19, 23].

The use of bacilli-based probiotic formulations also seems to be a promising health-promoting approach. Bacillus spp. are widely used in the poultry industry [24, 25, 36]. They demonstrate adaptability to diverse conditions and long shelf life. Bacillus spp., including B. amyloliquefaciens, can be found in the normal intestinal microbiota and are capable of germinating and resporulating in the gastrointestinal tract [25,26,27,28,29,30]. Moreover, their ability to form biofilms is important for functionality as a medical and veterinary probiotic [31].

Noticeably, probiotics affect the characteristics of the laid eggs. Enterococcus faecium supplementation was shown to result in a significant increase in egg production, eggshell thickness, and nutrient digestibility in laying hens, and a decrease in fecal coliform counts [32].

Data on the impact of probiotic on the egg production are somewhat contradictory. For instance, hens fed with 0.01 and 0.06% of B. licheniformis had improved egg production over the control group (98.4 and 94.0%, respectively) [8]. Kurtoglu et al. [33] showed that the hens fed with up to 750 mg of probiotic (3.2 × 109 CFU/g)/kg of diet had improved egg production, whereas Li et al. [34] and Yalcin et al. [35] demonstrated no statistically significant effect of probiotics on hen egg production. These effects seem to be strain and animal specific.

In the present study, we observed a similar situation: the number of laid eggs significantly increased, as well as their quality [8, 33]. In addition, the quality of the sperm of roosters improved.

Probiotic supplementation may be even more effective in stress than in normal conditions. Thus, Jia et al. showed that B. subtilis reduced the adverse effects of mycotoxins on laying performance, effectively improving egg quality and reducing the accumulation of aflatoxin residues in the egg [36].

Based on the data presented here, it can be concluded that the use of probiotic preparations based on the Bacillus subtilis KATMIRA1933 and Bacillus amyloliquefaciens B-1895 positively affects the rate of growth and condition of the birds, both the rearing flocks and the laying hens. The weight, egg production, egg quality, and hatchery increase. Considering the simplicity and economical effectiveness of the studied fermented soybean-based probiotic preparations, the use of these formulations can present some benefits for modern livestock production.

The ongoing investigation is dedicated to the observation of the birds’ conditions, productivity, and incubatory qualities of eggs with the duration of the study extended up to 45–50 weeks.