Introduction

The excessive and indiscriminate use of antibiotics in poultry production industry poses a major threat to human health due to the high incidence of antibiotic-resistant bacteria. The growing global concerns about food security had led to the ban of antibiotics use as growth promoter in European Union countries. Probiotic, as a kind of green feed additive, has the ability to improve poultry production, intestinal health, morphology and microflora, and immune response (Abd El-Moneim and Sabic 2019, Abd El-Moneim et al. 2019, Ebeid et al. 2019, Fathi et al. 2018, Fathi et al. 2017). However, the probiotic sensitivity to environmental changes, particularly during the formulation and granulation of the diet, would reduce the amount of viable bacteria capable of reaching to the digestive tract of birds which in turn will reduce its beneficial effect (Abd El-Moneim et al. 2019, Leser et al. 2008). In adverse environmental condition, Bacillus subtilis can form spores that can grow fast with highly resistance to acid, alkali, heat, UV radiation, vacuum pressure, and chemicals (Hooge 2003). Thus, these spores can still grow and multiply in the intestinal tract upon arrival even after exposure to diets processing conditions and the acute acidic environment of proventriculus.

It is well known that B. subtilis is an aerobic bacterium that consumes large amounts of free oxygen in the digestive tract while proliferating. Therefore, it can inhibit aerobic pathogens growth and promote the growth of anaerobic probiotic such as bifidobacterium, yeasts, and Lactobacillus (Ebeid et al. 2019, Wang et al. 2006). Moreover, B. subtilis plays a vital role in the development of gastrointestinal tract and gut-associated lymphoid tissue as well as enhancing the innate and adaptive immunity of the host (Fathi et al. 2017, Huang et al. 2008). In brief, B. subtilis spores may exert their favorable effects in poultry diets and bodies by one or more of these mechanisms: (1) competitive exclusion in lowering the count of pathogenic bacteria (Abd El-Moneim et al. 2019, Ebeid et al. 2019); (2) oxygen consumption as mentioned above (La Ragione and Woodward 2003); (3) producing exogenous digestive enzymes (Abd El-Moneim and Sabic 2019, Li et al. 2014); (4) enhance immune response (Abd El-Moneim 2017, Huang et al. 2008, Yurong et al. 2005); and (5) promotion of intestinal function and development (Abd El-Moneim 2017, Abd El-Moneim et al. 2019, Yurong et al. 2005). These features make B. subtilis of particular interest for poultry producers to be used as an alternative supplement to antibiotics via various supplementation routes, especially in the diet. However, the optimal concentration for probiotic administration may be strain dependent and increasing the inclusion rate did not always perform better (Huang et al. 2004, Saleh 2014). Therefore, the present study was performed to evaluate the effect of dietary inclusion of serial doses of B. subtilis on growth performance, blood biochemical indices, oxidative status, and digestive enzyme activities in growing Japanese quail birds.

Materials and methods

Birds housing and experimental design

The present trial was conducted at the experimental poultry farm of the Poultry Research Unit, Biological Application Department, Radioisotopes Applications Division, Nuclear Research Center, Egyptian Atomic Energy Authority at Inshas, Egypt. All the procedures used in this trial were approved by Local Experimental Animals Care Committee, and approved by the Institutional Ethics Committee. The birds were cared using husbandry guidelines derived from Egyptian Atomic Energy Authority standard operating procedures. A total of 750 1-day-old Japanese quail chicks were randomly allotted equally into five experimental groups with five replicates (30 chicks each), to evaluate the effects of different concentrations of B. subtilis spores on growth performance, blood biochemical indices, antioxidant status, and digestive enzyme activities of fattening Japanese quails. Birds were caged in wire battery brooder cages equipped with stainless steel nipple drinker, which was supplying water ad libitum, and were kept under the same managerial, hygienic, and environmental conditions. House temperature was controlled during the first 14 days of the experiment ranged from 33 to 36 °C and then quails were reared until the end of the experimental period under the natural ambient temperature of summer condition. During the last period, ambient temperature was recorded from 1.00 a.m. to 9.00 p.m. (Fig. 1) and was ranged from 27 to 38 °C. Artificial light schedule was similar to the commercial condition (24 h light until the 4th day of age then followed by 23 h of light throughout the experimental period). The control group (BS0) received a maize-soybean mash basal diet without any additives. The basal diet was formulated to meet the nutrient requirements for growing Japanese quail from 1 to 6 weeks of age according to NRC (1994). The remaining experimental groups were fed the basal diet supplemented with B. subtilis spores with the levels of 1 × 103 (BS3), 1 × 105 (BS5), 1 × 107 (BS7), and 1 × 109 (BS9)/kg diet. The B. subtilis ATCC 35854 spores were obtained from Shandong Baolai-leelai Biotech Co., Ltd., Tai’an, China. The composition and the calculated analysis of the experimental diets are shown in Table 1.

Fig. 1
figure 1

Average daily ambient temperature during summer season from 1:00 am to 9:00 pm for 42 days of experiment

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Table 1 Composition and calculated analysis of basal diet of Japanese quail
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Growth performance

Individual live body weight (BW) per replicate was recorded in early morning at 2, 4, and 6 weeks of age. Daily body weight gain (DBWG) was calculated during the experimental periods. Daily feed intake (DFI) was recorded weekly on replication basis to estimate feed conversion ratio (FCR) as g feed/g gain. Birds were monitored twice a day for mortality.

Blood sampling and biochemical analysis

At the end of the experimental period, five quails from each group were randomly chosen and blood samples were collected during slaughtering in a serum-separating tube. Blood samples were immediately centrifuged at 4500 r.p.m. for 15 min., and the serum was frozen at − 80 °C until analysis. Serum total protein (TP), albumin (ALB), globulin (GLO), glucose (GLU), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP), uric acid (UA), urea-N, creatinine (CR), total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL), very low-density lipoprotein (VLDL), and high-density lipoprotein (HDL) concentrations were analyzed using spectrophotometer (Spectronic 1201, Milton Roy, Ivyland, PA, USA) using commercial kits (Spinreact Co., Girona, Spain) according to the manufacturer’s instructions. Serum concentrations of triiodothyronine (T3) and thyroxine (T4) were measured in all blood samples using radioimmunoassay (RIA) kits. Serum contents of malondialdehyde (MDA) and reduced glutathione (GSH), and catalase (CAT) activity were analyzed using commercial kits (Cell Biolabs Inc., San Diego, CA, USA).

Enzyme activity assay

At the end of the experiment, five quails were randomly collected from each experimental group and slaughtered by severing the jugular vein, and then duodenum samples were immediately collected after birds’ evisceration. A crude mixture of homogenous duodenum content was obtained by massaging the tract from both ends following the operating on ice method of Jin et al. (2000). Based on the sample weight, samples were diluted 10× with ice-cold phosphate-buffered saline (PBS, pH 7.0), subsequently homogenized using a hand-held glass homogenizer and centrifuged at 5000g for 20 min at 4 °C. The supernatants were separated and stored at 4 °C until analysis. All enzymatic assays were performed within 24 h after extraction. Amylase, lipase, and protease activities were determined using the methods of Coles (1986), Boutwell (1962), and Lowry et al. (1951), respectively.

Nutrients digestibility coefficients

For each experimental group, eight male quails were weighed before and after the collection period and housed in metabolic cages individually. During the collection period (4 days), all birds had free access to food and water and a 24-h period had elapsed, as adaptation period, before the commencement of the excreta collection period. The proximate analysis of diets and dried excreta was carried out according to AOAC (2003) for determination of dry matter (DM) (#930.15), organic matter (OM) (#942.05), crude protein (CP) (#954.01), crude fiber (CF) (#978.10), ether extract (EE) (#920.29), and ash (#923.03). Fecal nitrogen and urinary OM were estimated using trichloroacetic acid procedure (Jacobsen et al. 1960) and the equation of Abou-Raya and Galal (1971), respectively.

Statistical analysis

Data were analyzed by one-way analysis of variance using the general linear models (GLM) using SPSS software, Version 18.0. The experimental unit for growth and productive performance traits was a cage, whereas for the rest of the parameters it was the individuals’ data. Homogeneity of variance and normality of distribution were tested using Levene and Shapiro-Wilk tests, respectively. Tukey’s multiple range tests were performed to detect differences among dietary treatments at a significance level of P < 0.05.

Results

Growth performance

As presented in Table 2, live body weight showed a linear (P < 0.01) increase to B. subtilis supplementation at all studied ages. Moreover, daily body weight gain also observed a linear (P < 0.01) increase during the starter period (1–14 days of age) and the overall one (1–42 days of age). Daily feed intake was not affected by the feeding treatments for all periods considered. No mortality was recorded in all experimental groups throughout the trail period. Feed conversion ratio was linearly (P < 0.05) decreased with increasing dietary B. subtilis concentration during the starter and overall fattening periods. The highest levels of B. subtilis (BS7 and BS9 groups) recorded the best values of body weight, body weight gain, and feed conversion ratio.

Table 2 Effect of dietary supplementation of Bacillus subtilis on productive performance of Japanese quails birds
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Blood biochemical indices

Data presented in Table 3 indicate that dietary inclusion levels of B. subtilis significantly affect total serum protein, albumin, urea-N, and creatinine concentrations as well as liver enzymes activities. Increasing levels of B. subtilis led to a linear (P < 0.05) increase in serum concentrations of total protein and albumin, and a linear decrease in creatinine (P < 0.01), urea-N (P < 0.05 or P < 0.05 quadratically), ALT (P < 0.01), and AST (P < 0.01 or P < 0.01 quadratically) values. However, serum levels of globulin, uric acid, and ALP were not linearly or quadratically affected by dietary levels of B. subtilis.

Table 3 Effect of dietary supplementation of Bacillus subtilis on blood components of Japanese quails birds at 6 weeks of age
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Table 4 reports the concentrations of serum TC, TG, HDL, LDL, and VLDL as influenced by dietary inclusion of B. subtilis spores. Feeding treatments linearly reduced serum levels of TC and TG in groups BS7 and BS9, and BS9, respectively. Furthermore, serum concentration of VLDL was linearly decreased in groups BS5, BS7, and BS9 while LDL was insignificantly affected. HDL values were linearly elevated in experimental groups BS5, BS7, and BS9. The hypolipidemic impact of feeding B. subtilis spores was greatly observed and enhanced by increasing its inclusion level in quail diet.

Table 4 Effect of dietary supplementation of Bacillus subtilis on serum lipid constituents, glucose and thyroid hormones activity of Japanese quails birds at 6 weeks of age
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Thyroid hormones activities

All dietary supplemental doses of B. subtilis were linearly and quadratically (P < 0.01) lowered serum glucose level compared with the unsupplemented group (Table 4), while T3 and T4 activities showed linear (P < 0.01) and quadratic (P < 0.05, for T4 only) elevation in treated groups compared with the control one. Groups BS7 and BS9 recorded the highest levels of T3 and T4 and the lowest level of glucose.

Antioxidative status and lipid peroxidation

Serum MDA (as an index of lipid peroxidation in blood serum) and GSH levels and CAT activity as influenced by dietary levels of B. subtilis are depicted in Fig. 2. Antioxidative status of growing Japanese quails fed diets containing B. subtilis spores was greatly enhanced. All examined dietary levels of B. subtilis linearly and quadratically (P < 0.01) decreased serum content of MDA compared with the control group, while serum content of GSH and CAT activity were linearly (P < 0.01) increased in groups BS7, BS9, and BS5.

Fig. 2
figure 2

Effect of dietary supplementation of Bacillus subtilis on oxidative status (A) MDA, (B) GSH, and (C) CAT in the serum of Japanese quail birds. Treatment groups: BS0—corn-based diet, BS3—1 × 103B. subtilis spores/kg, BS5—1 × 105B. subtilis spores/kg, BS7—1 × 107B. subtilis spores/kg, and BS9—1 × 109B. subtilis spores/kg. Data presented as mean values with their standard errors. Values with different superscript letters are statistically different (P < 0.05)

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Digestive enzyme activities

Data illustrated in Fig. 3 revealed a linear (P < 0.01) increase in the activities of protease, lipase, and amylase due to dietary supplementation of B. subtilis. The highest activity of the aforementioned enzymes was observed in BS9 group followed by BS7.

Fig. 3.
figure 3

Effect of dietary supplementation of Bacillus subtilis on digestive enzyme activities (A) proteases, (B) lipase, and (C) amylase of Japanese quail birds. Treatment groups: BS0—corn-based diet, BS3—1 × 103B. subtilis spores/kg, BS5—1 × 105B. subtilis spores/kg, BS7—1 × 107B. subtilis spores/kg, and BS9—1 × 109B. subtilis spores/kg. Data presented as mean values with their standard errors. Values with different superscript letters are statistically different (P < 0.05)

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Nutrients digestibility coefficients

As presented in Table 5, digestion coefficients of DM, OM, CP, and EE were linearly improved by the supplementation levels of B. subtilis spores. The abovementioned parameters were significantly increased in groups BS7 and BS9 and numerically increased in the rest of the treatment groups compared with the control group. However, digestibility coefficients of CF and NFE were insignificantly affected.

Table 5 Effect of dietary supplementation of Bacillus subtilis on digestibility coefficients basis on DM of Japanese quail birds at 6 weeks of age
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Discussion

Growth performance

Results of the present study showed that the use of B. subtilis spores supplementation at different levels in quail diets resulted in linear improvement in body weight gain and feed efficiency. Feeding quails with a B. subtilis-supplemented diet improved BW at all studied ages dependent on the spores’ concentration of the diet; the highest concentrations (BS7 and BS9) obtained better weight. The favorable effect of B. subtilis on DBWG and FCR was expressed throughout the brooding period and the overall one. B. subtilis can improve quail growth performance because of its ability to secret high-active protease, amylase, and lipase to decompose feed nutrients and provide more nutrients available to absorption (Li et al. 2014). Regarding feed efficiency, results of the present study proved that dietary probiotic decreased FCR significantly and had no effect on daily feed intake. Our previous results are consistent with the finding of Gao et al. (2017), Li et al. (2016), and Hossain et al. (2015). Therefore, it might be assumed that, in the present study, the dietary probiotic is involved in enhancing of protease, lipase, and amylase activities (Fig. 3) and increased serum levels of T3 and T4 (Table 4), which play very important role in growth rate, leading to enhancement the nutrients utilization to maintain optimum growth.

Blood biochemical indices

In the current study, dietary supplementation of B. subtilis did not alter serum levels of GLO, UA, and ALP and elevated TP and ALB values as well as decreased urea-N, CR, AST, and ALT values. In line with our results, Kasmani et al. (2012) and Yazhini et al. (2018) reported significant increase in serum proteins due to probiotic treatments. Furthermore, the findings of Kasmani et al. (2012), Hashemzadeh et al. (2013), and Ahmed et al. (2015) agreed with ours concerning the liver and renal function indicators. The increase in serum TP and ALB could be explained by the inhibition exclusion mechanism, where B. subtilis improves dietary protein utilization through its ability to inhibit pathogens growth, which reduces protein breakdown into nitrogen and diminishes dietary protein efficiency, and increases the surface area for nutrient absorption (Abd El-Moneim 2017, Abd El-Moneim et al. 2019, Saleh et al. 2017, Yazhini et al. 2018). In addition, liver protection role of probiotic was interpreted by Rishi et al. (2009) who postulated that probiotic supplementation reduces translocation of harmful bacteria in the liver, which decreases levels of serum transaminases.

Present results revealed that the hypolipidemic effect of feeding B. subtilis spores was greatly observed and enhanced by increasing its inclusion level in quail diet. These results are in line with earlier findings (Abd El-Moneim and Sabic 2019, Aluwong et al. 2013, Pourakbari et al. 2016, Yazhini et al. 2018). These negative impacts of probiotics on blood cholesterols and triglycerides may be due to their ability to incorporate cholesterol into their cellular membrane, produce hydrolyze bile salt enzymes (EC 3.5.1.24) for bile salt deconjugation in the enterohepatic circulation (Klaver and Van der Meer 1993), and the conversion of cholesterol by probiotics in the intestine into coprostanol, which is directly excreted with the feces (Ooi and Liong 2010) or inhibit the rate-limiting enzyme of cholesterogenesis, 3-hydroxyl-3-methylglutaryl-CoA reductase (Kalavathy et al. 2003, Pourakbari et al. 2016). Reduction in serum cholesterol level is mostly associated with reduced cholesterol content in poultry products. Probiotics administration in poultry diets could lead to the production of low-cholesterol eggs and meat. Feeding on probiotics strains enhanced internal egg quality criteria (Abdelqader et al. 2013, Fathi et al. 2018), reduced yolk cholesterol content (Abd El-Moneim and Sabic 2019), decreased TBARS content in broiler muscles, and elevated muscle contents of unsaturated fatty acids (Saleh et al. 2014b, 2011). Probiotics can therefore meet the growing global demand for low-saturated fat and low-cholesterol meat and eggs.

Thyroid hormones activities

Reducing blood glucose level in the present study may be related to the suppressive effect of probiotics on glucagon, which decrease blood glucose value (Abd El-Moneim 2017, Aluwong et al. 2013). Moreover, dietary inclusion of B. subtilis increased serum levels of T3 and T4. This elevation in thyroid hormones may be explained by the probiotic-enhanced activity of hypothalamus hormone (thyroid-stimulating hormone-releasing hormone; TSH-RH), consequently activating the release of thyroid-stimulating hormone (TSH) from the anterior pituitary (Aluwong et al. 2013). Also, probiotics might stimulate thyrotropin secretion and, hence, T4 secretion by enhancing the activity of corticotrophin-releasing factor (CRF) (Geris et al. 1999, Klieverik et al. 2009). Therefore, it seems more likely that dietary inclusion of B. subtilis may enhance the growth performance in Japanese quail, by taking into account our results in serum T3 and T4 concentration.

Antioxidative status and lipid peroxidation

Results of our study showed linear increase in GSH content and CAT activity and decrease in lipid peroxidation. These findings revealed that probiotic possesses the physiological role of enhancing antioxidant defense system of birds. This promotion impact might be attributed to the ability of probiotics to produce certain factors chelate free radicals, capture reactive oxygen species, and inhibit their cytotoxic activity (Lin and Yen 1999). Moreover, probiotics can promote antioxidant system of the bids via the augmentation of antioxidant enzymatic activities (e.g., superoxide dismutase (SOD) and glutathione peroxidase) as well as total antioxidant status of the host (Wang et al. 2017). The antioxidant enzymatic system of probiotics itself plays major role in promoting host’s antioxidative status. LeBlanc et al. (2011) found that Lactobacillus casei produced SOD which accelerates weight loss recovery, increases gut enzymatic activity, and reduces intestinal inflammation in mice. Zheng et al. (2016) cleared that Enterococcus faecium stimulates the antioxidant capacity of broiler chickens through its reaction with hydroxyl radicals which in turn promotes the oxidation resistance of biological macromolecules. Our results are in close agreement with findings by many investigators (Abd El-Moneim and Sabic 2019, Abd El-Moneim 2017, Abudabos et al. 2016).

Digestive enzyme activities

Gastrointestinal enzymes activities play a fundamental role in nutrient digestion and, eventually, reflected on poultry performance, health and nutrients retention. In the present study, duodenal amylolytic, proteolytic and lipolytic activities were higher in B. subtilis treated groups. These results are in correspondence with the finding of Jin et al. (2000) who reported that the inclusion of Lactobacillus elevated amylase and lipase activities. Moreover, Wang and Gu (2010) observed significant increase in protease and amylase activities in Arbor Acres broilers fed diets supplemented with Bacillus coagulans NJ0517. Contrarily, Zhang et al. (2016) and Zhi-gang et al. (2014) demonstrated that probiotics had no effect on protease activity. Rodjan et al. (2018) and Palamidi et al. (2016) noticed non-significant alternations in amylolytic, lipolytic, or proteolytic activities as influenced by probiotic administration. The present augmentation in digestive enzymes activities may be due to the contribution of exoenzymes secreted by probiotic bacteria along with the endogenous enzymes produced by the host (Bedford and Schulze 1998, Saleh et al. 2014a, Wang and Gu 2010). Higher activity of protease, amylase, and lipase improved the digestion of protein, starch, and lipids, which might consider an explanation for the growth enhancement observed in the present study.

Nutrients digestibility coefficients

Our results revealed significant increase in the digestion of DM, OM, CP, and EE as well as numerical increase in the digestion of CF and NFE. These results synchronize and harmonize with the increase in duodenal amylolytic, proteolytic, and lipolytic activities observed in the present study. Results of the present study are in agreement with previous reported findings noticed that B. subtilis can significantly increase digestibility coefficients of CP, EE, OM, and DM (Gao et al. 2017, Hossain et al. 2015, Li et al. 2014, Mountzouris et al. 2010).

Conclusion

It can be concluded that different levels of dietary B. subtilis spores supplementation could enhance antioxidative status and digestive enzyme activities. However, the high concentrations (BS7 and BS9) of B. subtilis spores could improve the nutrient digestibility and growth performance of growing Japanese quail birds.