Effect of Bacillus velezensis substituting for feed antibiotics on the production, blood biochemistry and egg quality indices of laying hens

Background The excessive use of antibiotics in livestock feed industry caused inevitable side effect of microbial resistance. Beside this residual antibiotics in animal derived food stuff imposed serious health problems for humans. So this study aimed to investigate the potential use of Bacillus velezensis to substitute antibiotics for poultry production. A total of 468, 49-week-old Hy-Line Brown chickens, were randomly divided into four groups as control group (regular diet), experiment group I (0.1% B. veleznesis), experiment group II (0.2% B. veleznesis), and antibiotic group (50 mg/kg avomycin), with three replicates per group and trial period consisted on 42 days. Results The results showed that, compared with control group, the average egg production rate and daily feed intake of experimental groups I and II increased signicantly (P < 0.05), while the average egg weight was increased in experimental group II as compared to I. (P < 0.01). The feed conversion ratio was decreased (P > 0.05) in group II. Egg quality parameters such as yolk weight of the experiment group II was increased, but that of the antibiotic group and experiment group I was decreased, neither signicant (P > 0.05). Moreover, the eggshell strength, yolk color, albumen height and Haugh unit were signicantly increased (P < 0.05). Compared with the control group, probiotic groups can increase the progesterone and motilin (P > 0.05) but decrease the secretin and cholecystokinin in the blood plasma (P > 0.05). Conclusion This study suggested that B. velezensis can substitute in-feed-antibiotics and improved most of the study parameters signicantly. Which suggested that B. velezensis has potential future application value to replace the feed antibiotics.


Background
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 and development of poultry and reduce feed to egg ratio, but also increases economic pro ts, leading to further expanding of the modern intensi ed poultry industry [5,6].
However, with the continuous development of animal husbandry, the problem of antibiotic 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]. 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 the major health problem in 21st centuary [11]. In addition, a high proportion of antibiotics added to animal feed is excreted in animal's urine or manure, causing water pollution which could be a great harm to water sources and human health ultimately [12,13]. Effective intervention for all above problems in the countries where antibiotics are available without prescription, is to reform the regulatory environment, and to reclassify the antibiotics, by putting some antibiotics as a treatment choice and all others strictly restricted [14], as successful regulation of veterinary medicine in the Republic of Korea [15]. Also, antibiotics in animal feed will be completely banned by Chinese government from July 1, 2020. However, it is feared that banning the use of antibiotics may have adverse effect on animal health and farmers' pro ts. This has led efforts from all over the world to nd 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].
B. velezensis has been widely used as a biological control agent in agricultural elds due to their 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 Bacillus velezensis used as feed antibiotic replacement [24]. It was suggested that this bacteria can inhibit adherence, replication and virulence of intestinal pathogens [27]. In addition, they can play an important role to modulate immune system [28]. So, we prognosis that B. velezensis can 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 × 10 10 CFU/kg and 2.0 × 10 10 CFU/kg of B. velezensis as a feed additive instead of avomycin 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 rst time B. velezensis is assayed to replace antibiotics in hens feed. This study provides a practical basis for the application of Bacillus velezensis to laying hens feed instead of antibiotics.

Animals and grouping
A total of 468, 49-week-old healthy, Hy-Line Brown laying hens with similar weight were received from the Institute of Animal Husbandry and Veterinary Medicine of Anhui Province. They were randomly assigned into 1 of 4 groups (control group, experiment group I, experiment II, and antibiotic group), three replicates per group with 39 hens per replicate. A Completely randomized design was used to avoid any biasness of selection while allocating hens to the experimental groups.
Hens of 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). According to the results from our preliminary studies (unpublished data) [29], a new strain of B. velezensis was isolated from the manure of piglets. Hens of experiment group I and II were fed the regular diet plus 1 × 10 10 CFU/kg and 2 × 10 10 CFU/kg B. velezensis, respectively; and hens of antibiotic group were fed the regular diet plus 50 mg/kg avomycin.

Feeding management
The laying hens were kept in 3-tier bird's cage (28 × 48 cm x 48 cm). To avoid contamination, 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 on daily basis, throughout the trail period and this strategy was used to reduce the effect of confounding variables. The laying hens were fed quantitatively twice a day (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 Ethics Committee of Anhui Agricultural University, Anhui, China.

Data acquisition and analysis
Eggs were collected once a day (2 pm). The data of egg production, egg weight, and feed consumption were recorded. 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 the phase 1 (day 2-21) and day 42 in the 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). 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 aseptic centrifuge tube and stored at -20 °C.
At the last day of the experiment, 2.0 mL blood sample from each randomly selected hen (1hen per cage, n = 39) was collected into a micro-anticoagulant tube. The blood sample was centrifuged at 3000 rpm for 20 min at 4 °C by high-speed freezing centrifuge to obtain the plasma then kept at -20 °C until 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, followed by 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 ANOVA procedures of SPSS 22.0.The data was presented as mean ± S.E.M, and the signi cance level was set up at p < 0.05 signi cant and, p < 0.01 extremely signi cant 3. Results

Production performance
As shown in Table 1, The average egg production rate in the experiment group I, and II were increased signi cantly 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 signi cantly in experimental group II as compared to the experimental group I. As for the average daily feed intake, antibiotic group, experiment I, and experiment II increased signi cantly (P < 0.01), (P < 0.01), (P = 0.045 < 0.05). There was no treatment effects on the feed conversion ratio of control group, antibiotic group and experimental group I, however it reduced signi cantly in group II. (P < 0.01). Table 1 The effect of Bacillus velezensis on production performance of laying hens. AEPR: average egg production rate; AEW, average egg weight; ADFI: average daily feed intake; FCR: feed conversion ratio.

Egg quality
The egg quality was determined twice in the whole period. As presented in Table 2, in the rst determination on day 21 of the phase one (2-21 days), compared with control group and antibiotic group EW, ESI and ESW in both experimental groups was slightly decreased, but not signi cant (P > 0.05). However a signi cant 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. In the second determination on day 42 of the phase two (22-42 days) compared with control group and antibiotic group,the EW, ESW and ESI had no signi cant difference in both experimental groups. And compared with the control group, the ESS and YC of experiment groups were not improved signi cantly (P > 0.05). ESC of the experiment I was increased, The YW of the experiment group II was increased, but that of the antibiotic group and experiment group I was decreased, neither signi cant (P > 0.05). Moreover, the ESS, AH and HU of the experiment groups were higher than that of the control group, but lower than that of the antibiotic group. As presented in Table 2, antibiotic and B. velezensis could increase triglyceride in the rst phase. However, triglyceride was reduced in the antibiotic group and experiment group I but increased in experiment group II in the second phase. Compared to antibiotic, feed with 0.2% B. velezensis could reduce the content of cholesterol in 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 experiment group I was increased, but reduced in experiment group II, however none was signi cant (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 signi cant (P > 0.05). 4. Discussion

Production performance
The use of bacilli-based probiotic feed-formulations was observed to be a promising health-promoting approach. Bacillus spp. are widely used in the poultry industry [30].
At the late stage of feeding, due to the change of metabolism in the body, the absorption of nutrients in feed is weakened, which leads 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 bene ts. Bacillus, such as Bacillus 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 [31]. Numerous studies have shown that adding Bacillus sp. to laying hen feed can signi cantly increase egg production rate, average egg weight, reduce feed conversion ratio, and improve egg quality and immunity [32][33][34][35].
Eggs are the main products in the laying hen farm, and the egg production rate determines the breeding e ciency. Therefore, improvement of the egg production has an important economic value [36]. In this study, compared with the control group, the average egg production rate in the antibiotic group, experiment group I and experiment group II were increased signi cantly (P < 0.01). It was reported that laying hens fed with B. subtilis OFMCC 1.921 had an improved egg production between week 5 to 8 and week 9 to 12 [35]. A similar result was reported by [37]. In addition, the results from that study also proved that B. amyloliquefaciens B-1895 improved the average egg production rate. However, Li et al. (2006) reported that hens fed with 3 × 10 9 CFU/g, 6 × 10 9 CFU/g, and 9 × 10 9 CFU/g of B. subtilis had no signi cant effect on egg production rate, but could signi cantly reduce average daily feed intake and feed conversion ratio with an improved production performance. It might be related to the Bacillus sp., treatment level, duration, variety and age of laying hens. [33] As for the ADFI of antibiotic group, experiment I, and experiment II increased signi cantly (P < 0.05). And the feed conversion ratio of experimental group II was reduced signi cantly as compared to the control group (P < 0.01). Ribeiro et al. showed that feeding with B. subitils can increase the egg production rate by 2.63%, but there was no signi cant change in the feed conversion ratio [38]. In this study, supplementation of B. velezensis could increase the average daily feed intake of experimental group I and reduce the feed conversion ratio of experimental group II. This may be due to the fact that probiotic can consume excess oxygen in the intestine, to produce bacteriocins and volatile bacteriostatic substances [39]. It is conducive to the degradation of nutrients in feed and the improvement of feed conversion rate, thereby improving animal feed intake and reducing feed conversion ratio.

Egg quality
The egg quality was determined twice in the whole period. The strength and thickness of eggshell are two important indicators to measure the quality of eggs. In addition, the strength and thickness of eggshell also affect the freshness of eggs. Improving eggshell quality is important to the laying hen production, and it is also a research hotspot for domestic and foreign researchers at present. Adding Bacillus sp. to the diet of hens can increase the eggshell thickness and eggshell strength, improve the quality of eggshell, reduce the rate of broken eggs, and improve economic bene ts of farms [36,40]. Similarly, it was also reported that addition of Bacillus subtilus to the diet improved the Haugh unit and protein index signi cantly, and the thickness of egg shell was also improved [37]. It was proved that the usage of 0.05% B. subtilis culture in the diet had the greatest effect on the egg physical characteristics and the yolk weight, shell weight, Haugh unit and color all increased (P < 0.05) [41]. Similar results were obtained by [42]. However, in our study, except for the eggshell strength, yolk color, albumen height and Haugh unit in the second determination, there was no signi cant change in other indicators. It may be related to the late laying stage of hens. The mechanism of improving egg quality by B. velezensis still needs further study.
From Table 2, it can be noted that the supplementation of B. velezensis can increase triglyceride in yolk of the rst phase. Triglycerides were increased in all three test groups but the maximum increase was observed in experimental group II. While the content of cholesterol in yolk was decreased in all three test groups as compared to the control group. Park et al. pointed that the fermented buckwheat as a feed additive could reduce the yolk triglyceride [43]. It was also reported that there was a decrease in yolk cholesterol in hens fed with the B. subtilis supplemented diet [33]. In the second determination of egg quality, the results of some indices were not identical with that of the rst determination, it might be related to the age of laying hens.

The effect of Bacillus velezensis on biochemical indices in plasma
Results showed that triglyceride in the plasma of antibiotic group and experiment group I was increased, but decreased in the experimental group II (P > 0.05). In the study of Choi et al. supplementation of fermented brown seaweed in the feed could signi cantly increase triglyceride and cholesterol in blood of laying hens (P < 0.05) [44]. But Zhao et al. (2013) reported that fed with fermented Ginkgo-leaves has an increased effect on triglyceride and cholesterol in serum [45]. The triglyceride and cholesterol in animal serum are important to animal cells. Most tissues in the body can use triglyceride decomposition products to provide energy for metabolism [46]. Cholesterol is a precursor of many important hormones and vitamins synthesis. It is also a component of animal brain, liver and other important cells [47]. Studies have con rmed that high levels of blood plasma cholesterol in animals could increase the risk of atherosclerosis. Therefore, the intake of animal meat products with low triglyceride and cholesterol content is bene cial to human health [48]. In our study, fed with 0.2% B. velezensis could decrease the content of triglyceride and cholesterol in blood plasma. This is consistent with the results of [49,50], which could be helpful for improving the quality of eggs.
The study found that he dietary supplementation of 0.1% B. velezensis and 0.2% B. velezensis increased the progesterone and motilin in the blood plasma by increase of laying hens compared to those of the control group, but not signi cant (P > 0.05). However, progesterone and motilin level in the antibiotic group were decreased (P > 0.05) ( Table 3), which indicated the usage of B. velezensis in the feed was prevalent. It is known that progesterone is an important steroid hormone and its main target organ is the uterus. In addition, it also acts on other tissues, including the brain, pituitary, breast and ovary [51]. Its function is to promote the growth, development and differentiation of these organs, and participate in their functional regulation. Moreover, progesterone is also involved in regulating and transforming the proliferation and differentiation of some abnormal cells, such as breast cancer cells and ovarian cancer cells [52,53] Kim et al. proved that adding B. subtilis to the heifers feed could obtain similar results as in presented in this study. In addition, the results also con rmed that progesterone induced the level of cholesterol, and progesterone is relative to the feed e ciency [54], which is consistent with our experimental results. But the mechanism of action is not fully investigated. Motilin is gut peptide produced in the upper intestinal mucosa that induces strong contraction in the small intestine which can prolong the time of gastric emptying [55]. Tack et al. have con rmed that motilin-induced gastric phase III contraction may be a starvation signal during the digestive interval, which explains the cause of hunger in humans to some extent. Lack of motilin and gastric phase III contraction may be associated with unexplained loss of appetite [56]. According to the results of this experiment, we can conclude that supplementation with B. velezensis could increase the average daily feed intake, these results are correlated with the increase of motilin.
In addition, B. velezensis in the feed can decrease the secretin and the cholecystokinin (P > 0.05). Secretin is the earliest discovered animal hormone, mainly distributed in the duodenal mucosa, a small amount in the jejunum, ileum and antrum [57]. Since it was further reported that secretin is a kind of enterogastrone, it has attracted more and more attention [58,59]. It has been con rmed that secretion has a strong inhibitory effect on gastric acid secretion in human, dog and rat [60,61] Consent to Publish inferred that secretin could inhibited gastric motility in plasma of the laying hens. Cholecystokinin was discovered in 1928 and it has the ability to induce gallbladder contraction and promote pancreatic enzyme secretion. Cholecystokinin acts as a satiety neurotransmitter to regulate the termination of feeding [62]. Under modern production conditions, inadequate feeding of animals is a common phenomenon. In order to develop the production potential of animals and to further improve the performance of animals, the rst consideration is how to increase feed intake. Due to the important role of secretin and cholecystokinin in feeding regulation, reducing the content of secretin and cholecystokinin in animals has become a worth considering way to improve the feed intake.
It should be emphasized that although the alteration of the hormone in the plasma was not signi cant comparing to the control group, because the hormone could play a great ampli ed role on the body, it also have positive effect on the productivity of hens and the quality of eggs.

Conclusion
According to the above results, it can be concluded that dietary supplementation with B. velezensis could improve the production performance, egg quality and the plasma biochemical index in poultry. On the whole, the effect of supplementation with 2.0 × 10 10 CFU/kg B. velezensis was better than that of 1.0 × 10 10 CFU/kg B. velezensis.
As this was the rst feed trial to use B. velezensis as a replacement of antibiotics and hence the results of experimental groups were not better than those of the antibiotic group, but to a certain extent, B. velezensis can replace the feed antibiotics, which has potential future application values to be used as antibiotic replacement in animal feed industry.