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 .
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 . 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 [36-39]. Because eggs are the main products in the laying hen farm, and the egg production rate determines the breeding efficiency. Therefore, improvement of the egg production has an important economic value . In this study, compared with the control group, the average egg production rate in the antibiotic group, experimental group I, and experimental group II were increased significantly (P < 0.01). Similar results reported that laying hens fed with B. subtilis OFMCC 1.921 had an improved egg production between week 5 to 8 and weeks 9 to 12 . Another study suggested the similar result . 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 3×109 CFU/g, 6×109 CFU/g, and 9×109 CFU/g of B. subtilis had no significant effect on egg production rate, but could significantly reduce average daily feed intake and feed conversion ratio with an improved production performance. However, it might be related to the Bacillus sp., treatment level, trial duration, variety, and age of laying hens. 
As for the ADFI of the antibiotic group, the experimental group I, and experimental group II increased significantly (P< 0.05). And the feed conversion ratio of experimental group II was reduced significantly as compared to the control group (P < 0.01). However, Ribeiro et al. (2014) showed that feeding with B. subitils can increase the egg production rate by 2.63%, but there was no significant change in the feed conversion ratio . In our study, the 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 probiotics can consume excess oxygen in the intestine, to produce bacteriocins and volatile bacteriostatic substances . Which 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.
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. Because, 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. Many studies suggested that 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 the economic benefits of poultry farms [40, 44]. Similarly, it was also reported that the addition of B. subtilus to the diet improved the haugh unit and protein index significantly, and the thickness of eggshell was also improved . Forte et al. (2016) proved that the using of 0.05% B. subtilis culture in the diet had the greatest effect on the egg's physical characteristics and the yolk weight, shell weight, haugh unit, and color all increased significantly (P< 0.05) . Similar results were obtained by . However, in our study, at the second determination level, except for the eggshell strength, yolk color, albumen height, and haugh unit, there was no significant change in other indicators. It may be related to the late laying stage of hens. As the bird’s age is very important to the overall quality of eggs. However 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 the yolk of the first 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 out, that the fermented buckwheat as a feed additive could reduce the yolk triglyceride . It was also reported that there was a decrease in yolk cholesterol in hens fed with the B. subtilis supplemented diet . In the second determination of egg quality, the results of some indices were not identical with that of the first determination, it might be related to the age of laying hens, as reported by Travel et al. (2011) that the low quality eggs can arise in young birds, due to early ovulation .
The effect of Bacillus velezensis on biochemical indices in plasma
Results showed that triglyceride in the plasma of the 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. (2018 supplementation of fermented brown seaweed in the feed could significantly increase triglyceride and cholesterol in the blood of laying hens (P < 0.05) . But Zhao et al. (2013) reported that fed with fermented Ginkgo-leaves has an increased effect on triglyceride and cholesterol in blood plasma . 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 . Cholesterol is a precursor of many important hormones and vitamin synthesis. It is also a component of the animal brain, liver, and other important cells . Studies have confirmed 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 beneficial to human health . 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 [54, 55], which could be helpful for improving the quality of eggs.
The study found that the dietary supplementation of 0.1% B. velezensis and 0.2% B. velezensis increased the progesterone and motilin in the blood plasma of laying hens compared to those of the control group, but not significant (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, breasts, and ovary . 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 [57, 58] Kim et al. proved that adding B. subtilis to the heifers feed could obtain similar results as presented in our study. In addition, the results also confirmed that progesterone, induced the level of cholesterol, and progesterone is relative to the feed efficiency , which is consistent with our experimental results. But the mechanism of action is not fully investigated. Motilin is a gut peptide, produced in the upper intestinal mucosa that induces strong contraction in the small intestine which can prolong the time of gastric emptying . Tack et al. (2016) have confirmed 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 . According to the results of this experiment, we can conclude that supplementation with B. velezensis could increase the average daily feed intake, which was 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 . Since it was further reported that secretin is a kind of enterogastrone, it has attracted more and more attention [63, 64]. However studies confirmed that secretion has a strong inhibitory effect on gastric acid secretion in humans, dogs, and rats [65, 66], and hence should be reduced. In this experiment, adding B. velezensis to the laying hens feed could decrease secretin content and increase average daily intake, we inferred that secretin could inhibit gastric motility, if secreted abnormally in the plasma of the laying hens. Cholecystokinin was discovered in 1928 and it can induce gallbladder contraction and promote pancreatic enzyme secretion. Cholecystokinin acts as a satiety neurotransmitter to regulate the termination of feeding . 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 first 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 significant comparing to the control group. But, these hormones could play a great amplified role on the body, and also have a positive effects on the productivity of hens and the quality of eggs.