Introduction

Antibiotics are chemical substance produced by microorganisms which can resist pathogens to improve human and animal health as well as improve the quality of food products. Since the discovery of antibiotics in 1929, they have contributed to treat infectious diseases previously known to kill many humans and animals. Therefore, antibiotics are called guardians of human beings [1,2,3]. A class of antibiotics (Spiramycin, Tetracycline, Virginiamycin, Erythromycin) has been added to poultry feed for enhancing growth and production in large-scale intensive farming environments [4]. Adding a certain amount of feed antibiotics can not only promote the feed conversion rate, growth, development of poultry and reduce feed to egg ratio but also increases economic benefits, leading to expand the modern intensified poultry industry [5, 6].

However, with the continuous development of animal husbandry, the problem of antibiotics abuse has also emerged. Antibiotics remaining in livestock and poultry products (such as meat, eggs, and milk) may induce abnormal reactions such as disturbances in physiological and biochemical processes [7]. Some studies, for example, have shown that the abuse of antibiotics has a certain degree of correlation with the increased risk of cancer [8, 9]. The Use of antibiotics in animal production imposes a serious selection pressure on microbes which are exposed to sub-inhibitory doses of antibiotics. This has raised the problem of antibiotic resistance through mutation and gene transfer [10]. Controlling the propagation of antibiotic-resistant bacteria has become a major health problem in the 21st century [11]. Besides, a high proportion of antibiotics added to animal feed is excreted in animal’s urine or manure, causing water pollution which could be great harm to water sources and human health ultimately [12, 13]. Effective intervention are required, to reform the regulatory environment and to reclassify the antibiotics, by putting some antibiotics as a treatment choice and all others strictly restricted is the suggested solution for all above problems in the countries where antibiotics are available without a prescription[14]. Similar to the successful regulation of veterinary medicine in the Republic of Korea [15]. Also, antibiotics in animal feed have been completely banned by the Chinese government from July 1, 2020. However, it is feared that banning the use of antibiotics may have adverse effects on animal health and farmers’ profits. This has led efforts from all over the world to find an animal growth promoter that can effectively replace antibiotics to prevent antibiotic residues in poultry products, such as eggs [16, 17]. Currently, probiotic preparations, Oligosaccharide preparations, enzyme preparations, and Chinese herbal medicine are effective antibiotic substitutes [18,19,20,21]. Probiotic preparations have become an emerging growth-promoting additive due to the enormous amount of research and good effect on animal growth [22, 23].

Bacillus velezensis has been widely used as a biological control agent in agricultural fields due to its excellent ability to suppress plant diseases [24, 25]. It is considered as a potential rhizobacterial organism with extraordinary biosynthetic machinery, which can trigger innate immunity in plants [26].

However, there are few experiments on the B. velezensis used as feed antibiotics replacement [24]. It was suggested that this bacteria can inhibit adherence, replication, and virulence of intestinal pathogens [27]. In addition, it can play an important role to modulate the immune system [28]. So, we prognosis that B. velezensis have the potential to be developed as a probiotic agent. In the present experiment, to prove that the B. velezensis can be a good antibiotics substitute, we investigated the effect of adding 1.0 × 1010 CFU/kg and 2.0 × 1010 CFU/kg of B. velezensis as a feed additive instead of flavomycin on the production performance, egg quality, blood physiological and biochemical indices of Hy-Line Brown laying hens. To the best of our knowledge, this is the first time B. velezensis is assayed to replace antibiotics in hens feed. This study provided a practical basis for the application of B. velezensis to the laying hens feed instead of antibiotics.

Results

Production performance

As shown in Table 1, The average egg production rate in the experiment group I, and II were increased significantly as compared to the control group (P < 0.05), however, it decreased as compared to the antibiotic group. The average egg weight (AEW) was increased significantly in experimental group II as compared to the experimental group (I) The average daily feed intake of the antibiotic group, experimental group I, and experimental group II increased significantly (P < 0.01), (P < 0.01), (P = 0.045 < 0.05). There were no treatment effects on the feed conversion ratio of the control group, antibiotic group and the experimental group I, however, it reduced significantly in group (II) (P < 0.01).

Table 1 The effect of Bacillus velezensis on the production performance of laying hens
Full size table

Egg quality

The egg quality was determined twice in the whole period. As presented in Table 2, in the first determination on day 21 of the phase one (2–21 days), compared with the control group and antibiotic group EW, ESI and ESW in both experimental groups was slightly decreased, but not significant (P > 0.05). However, a significant decrease in ESS was observed in both experimental groups as compared to the control and antibiotics group. And the eggshell color ESC, YW, YC, AH, and HU were higher than that of the control group but lower than that of the antibiotic group.

Table 2 The effect of Bacillus velezensis on the egg quality of laying hens
Full size table

In the second determination on day 42 of phase two (22–42 days) compared with the control group and antibiotic group the EW, ESW, and ESI had no significant difference in both experimental groups. And compared with the control group, the ESS and YC of experimental groups were not improved significantly (P > 0.05). ESC of the experimental group I was increased, The YW of the experimental group II was increased, but that of the antibiotic group and experimental group I was decreased, neither significant (P > 0.05). Moreover, the ESS, AH, and HU of the experimental groups were higher than that of the control group but lower than that of the antibiotic group. As presented in Table 2, antibiotics and B. velezensis could increase triglyceride in the first phase. However, triglyceride was reduced in the antibiotic group and experimental group I but increased in experimental group II in the second phase. Compared to the antibiotic, feed with 0.2% B. velezensis could reduce the content of cholesterol in the second phase.

The effect of B. velezensis on biochemical indices in blood plasma

As shown in Table 3, triglyceride in plasma of antibiotic group and experimental group I was increased, but reduced in experimental group II, however none was significant (P > 0.05). The level of progesterone and motilin in the blood plasma of laying hens was increased in experimental group I and II as compared to those of the control group and antibiotic group, which recommended the successful usage of B. velezensis. The results also showed that the, the level of secretin and cholecystokinin was decreased in all three test groups as compare to the control group. All the above increase and decrease in the results were not significant (P > 0.05).

Table 3 The effect of Bacillus velezensis on the hormonal level in blood plasma of laying hens
Full size table

Discussion

Production performance

The use of bacillus-based probiotic feed-formulation was observed to be a promising health-promoting approach. Bacillus spp. were widely used in the poultry industry [29].

At the late stage of feeding, due to the change of metabolism in the body, the absorption of nutrients in the feed was weakened, which lead to the decrease in production performance and the deterioration of egg quality, even the decrease of immunity and the deterioration of anti-stress ability, thus affecting economic benefits. Bacillus sp. such as B. coagulans has the functions of regulating or maintaining intestinal micro-ecological balance, enhancing immunity, promoting the absorption of calcium, phosphorus, vitamin D, and so on [30]. Numerous studies have shown that adding Bacillus sp. to the laying hen’s feed can significantly increase egg production rate, average egg weight, reduce feed conversion ratio, and improve egg quality and immunity [63] to avoid any biasness of selection while allocating hens to the experimental groups.

Hens of the control group were fed a regular diet (59% yellow corn, 27.5% bean pulp, 8.5% limestone powder, and 5% premix, with 16% Crude protein, and 2556.0 Kcal/Kg Metabolic energy) without any active probiotic [32]. According to the results from our preliminary studies (unpublished data) [64], a new strain of B. velezensis was isolated from the manure of piglets. Hens of experimental group I and II were fed the regular diet plus 1 × 1010 CFU/kg and 2 × 1010 CFU/kg B. velezensis, respectively; and hens of the antibiotic group were fed the regular diet plus 50 mg/kg flavomycin.

Feeding management

The laying hens were kept in a 3-tier bird’s cage (28 × 48 cm x 48 cm). To avoid contamination by pathogens aviary system was used [65], in which the cages were separately housed based on the treatment, under the same conditions, including room temperature, humidity, light, and ventilation, there was no enrichment provided for any environmental condition. Similar weight hens were used, and their health condition, feeding, and drinking were recorded daily, throughout the trial period and this strategy was used to reduce the effect of confounding variables. The laying hens were fed quantitatively twice a day [32], (7 am and 2 pm) for 42 days. This study was conducted at the Dongshan Chicken Farm of Anhui Academy of Agricultural Sciences; and the experimental protocol was approved by the Animal Care and Use Committee, the Anhui Academy of Agricultural Sciences, and the Ethics Committee of Anhui Agricultural University, Anhui, China.

Data acquisition and analysis

Eggs were collected once a day (2 pm). The data for egg production, egg weight, and feed consumption were recorded. The trial period was divided into two phases (Phase 1: day 2–21 & Phase 2: day 22–42) to examine the production parameters based on the duration of feeding. Then, the average egg production rate (AEPR), average egg weight (AEW), average daily feed intake (ADFI), and feed/egg ratio (FCR) were calculated. On day 21 of phase 1 (day 2–21) and day 42 in phase 2 (day 22–42), 39 eggs from each treatment (balanced with egg weight) were collected and the egg quality indices including egg shape index (ESI), eggshell color (ESC), eggshell strength (ESS), yolk weight (YW), yolk color (YC), eggshell weight (EW), albumen height (AH), and Haugh Unit (HU) were determined. ESI was measured with a Vernier caliper (Measuring & Cutting Tool Work Co. China). ESC was measured with the eggshell color tester (Robotmation Com., Japan). ESS was tested with the Egg Shell Force Gauge MODEL-III (EGG-0530, Robotmation Com., Japan) [66]. Yolk weight, yolk color, eggshell weight, albumen height, and Haugh Unit were determined by using the Egg Multi Tester (EMT-5200 Robotmation Com., Japan). The egg yolk was collected in an aseptic centrifuge tube and stored at -20 °C.

On the last day of the experiment, a 2.0 mL blood sample from each randomly selected hen (1hen per cage, n = 39) was collected into a micro-anticoagulant tube [65]. The blood sample was centrifuged at 3000 rpm for 20 min at 4 °C by high-speed freezing centrifuge to obtain the plasma then stored at -20 °C until further analysis.

Blood concentrations of cholesterol and triglyceride were determined using cholesterol and triglyceride assay kits according to the manufacturer’s instructions (Changchun Huili Biotech, China). The progesterone, motilin, secretin, and cholecystokinin of egg yolk were determined using progesterone, motilin, secretin, and cholecystokinin kits, respectively, following the manufacturer’s instructions (Shanghai Enzyme-Union Biotech, China).

Since the study conducted, was based on a feeding trial and birds were not harmed during the whole trial period. After carefully collecting the blood sample, all the birds were healthy and returned to the cage again. No animals were killed at humane endpoints.

All the data were analyzed by using one-way analysis of variance (ANOVA) to determine whether any significant difference occurred within study groups or not. The data were presented as mean ± S.E.M, and the significance level was set up at p < 0.05 significant. The SPSS software, version 22.0 was used to analyze the data, and determine the effect of dietary treatments.