Abstract 

This experiment aimed to explore the effects of dietary supplementation of LIQI99 on the production performance, intestinal health of laying hens, and the reduction of Salmonella contamination in eggs, and to determine its appropriate supplementation level and mechanism of action. A total of 288 45-week-old Hy-Line Brown laying hens were selected and randomly divided into 4 groups. The groups were fed with a basal diet (control group) and experimental diets supplemented with 1g/kg, 2g/kg, and 4g/kg of LIQI99 respectively. A 12-week feeding trial and intestinal health detection were conducted for verification. The results showed that LIQI99 could significantly improve the average daily feed intake of laying hens and enhance serum antioxidant capacity (indicated by increased GSH-Px content); it could improve intestinal morphology (increased villus-to-crypt ratio and decreased crypt depth). After Salmonella challenge, LIQI99 could target and kill pathogenic bacteria in the intestine, regulate beneficial bacteria, up-regulate the immune organ index (mainly thymus and spleen), and reduce the adhesion and invasion of Salmonella to intestinal epithelial cells. In conclusion, the appropriate supplementation level of LIQI99 is 4g/kg, which can reduce egg contamination by enhancing the intestinal barrier function and inhibiting Salmonella colonization.

01 Materials and Methods of the Experiment

1.1 Experimental Animals and Grouping A total of 288 healthy 45-week-old Hy-Line Brown laying hens with similar body weights were selected and randomly divided into 4 groups, with 6 replicates in each group and 12 hens in each replicate. The grouping was as follows:  Control group (Con): Fed with a basal diet;  Experimental Group 1 (T1): Basal diet + 1g/kg LIQI99;  Experimental Group 2 (T2): Basal diet + 2g/kg LIQI99;  Experimental Group 3 (T3): Basal diet + 4g/kg LIQI99. The pre-test period lasted for 2 weeks, and the formal test period lasted for 12 weeks. 

1.2 Experimental Diets and Feeding Management The basal diet was formulated with reference to NRC (1994) and NY/T33-2004. Its main components included corn (62.5%), soybean meal (24.0%), soybean oil (0.5%), stone powder (8.0%), and premix (5.0%), with a metabolic energy of 11.15 MJ/kg and crude protein of 16.05%. The laying hens were raised in a three-layer stepped cage house, with free access to feed and water. The temperature was controlled at 15-26°C, and the relative humidity was 40%-65%. The number of dead and culled hens, egg production, and egg weight were recorded daily, and regular immunization and disinfection were carried out. 

1.3 Determination Indicators and Methods 1.3.1 Production Performance and Egg Quality Production performance: The average daily feed intake, egg production rate, average egg weight, and feed-egg ratio were counted monthly;

Egg quality: At the 4th, 8th, and 12th weeks, 3 eggs per replicate were taken from each group. The eggshell thickness, eggshell strength, and Haugh unit were measured using an EGGTESTER ULTIMATE™ eggshell thickness gauge. 

1.3.2 Serum Antioxidant Indicators At the 12th week of the formal test, 6 laying hens were selected from each group for blood collection. The serum was separated by centrifugation at 3000 r/min for 10 minutes, and antioxidant indicators (GSH-Px, MDA, T-SOD) were determined. 

1.3.3 Intestinal Health Detection At the 12th week, the laying hens were slaughtered for sampling, and the following determination was conducted: Intestinal morphology: HE staining was performed on the duodenum, jejunum, and ileum tissues. The villus height (VH) and crypt depth (CD) were measured, and the villus-to-crypt ratio (V/C) was calculated. 

1.3.4 Salmonella Challenge Test At the 11th week, 18 laying hens were selected from the Con group, T1 group, T2 group, and T3 group respectively, and divided into the Sal group (control group challenged with Salmonella), Sal+T1 group (challenged with Salmonella + 1g/kg LIQI99 group), Sal+T2 group (challenged with Salmonella + 2g/kg LIQI99 group), and Sal+T3 group (challenged with Salmonella + 4g/kg LIQI99 group), with 6 replicates in each group and 3 hens in each replicate. On the day of challenge, 1 mL of Salmonella Typhimurium suspension containing 1×10⁹ CFU was intragastrically administered to each hen. On the 3rd and 7th days after the challenge, the thymus, spleen, bursa of Fabricius, liver, and heart were collected. The fat around the organs was peeled off, the organs were rinsed with normal saline, dried with absorbent paper, weighed, and recorded. The organ index was calculated as follows: Organ index (g/kg) = Fresh weight of the organ (g) / Live weight before slaughter (kg). Flora detection: 0.1g of fresh fecal samples were obtained using a sterile rectal probe. Lactobacillus, Escherichia coli, and Salmonella were detected on selective media. 

1.4 Statistical Analysis SPSS 26.0 software was used for one-way analysis of variance (ANOVA) and Duncan's multiple comparison test. A P-value < 0.05 was considered statistically significant, and the data were expressed as "mean ± standard error"

02 Experimental Results 2.1 Effects of LIQI99 on Production Performance and Egg Quality Indicators of Laying Hens 

Table 1 Experimental Results of the Effects of LIQI99 on Production Performance and Egg Quality Indicators of Laying Hens

The results showed that with the progress of the experimental cycle, the egg production rate of the experimental groups (supplemented with LIQI99) was gradually higher than that of the control group, and the difference was extremely significant at the 12th week (P ≤ 0.001), indicating that long-term supplementation had a significant improvement effect; at the 4th week, the feed-egg ratio of the T groups was significantly lower than that of the control group (P = 0.002), and it continued to decrease from the 8th to the 12th week, reflecting that LIQI99 could optimize the feed conversion efficiency; Eggshell thickness and strength: from the 4th to the 12th week, the eggshell thickness and strength of the experimental groups were higher than those of the control group, and the difference was significant in the later stage of the experiment (P ≤ 0.001); at the 12th week, the Haugh unit of the experimental groups was significantly higher than that of the control group (P = 0.016), indicating that LIQI99 could improve the protein quality and the freshness of eggs.

2.2 Effects of LIQI99 on Serum Biochemical Indicators of Laying Hens 

Table 2 Experimental Results of the Effects of LIQI99 on Serum Biochemical Indicators of Laying Hens

The results showed that the SOD activity increased significantly with the increase of the supplementation level (P = 0.003), the GSH-Px activity showed an upward trend (P = 0.456), and the MDA content showed a downward trend (P = 0.782), indicating that LIQI99 has potential in enhancing the serum antioxidant capacity of laying hens

2.3 Effects of LIQI99 on Intestinal Tissue Morphology of Laying Hens 

Table 3 Experimental Results of the Effects of LIQI99 on Intestinal Tissue Morphology of Laying Hens

The results showed that LIQI99 had a tendency to reduce the intestinal crypt depth of laying hens, and the villus-to-crypt ratio was extremely significantly increased (P < 0.001), which was beneficial to enhance absorption. It significantly reduced the jejunal crypt depth (P < 0.001) and extremely significantly increased the villus-to-crypt ratio (P < 0.001), optimizing the absorption function. It also significantly reduced the ileal crypt depth (P = 0.001) and significantly increased the villus-to-crypt ratio (P = 0.027), improving intestinal health.

2.4 Effects of LIQI99 on Organ Indices of Laying Hens Under Salmonella Challenge 

Table 4 Experimental Results of the Effects of LIQI99 on Organ Indices of Laying Hens Under Salmonella Challenge

The results showed that for immune organs: the thymus and spleen indices in the Sal+T3 group were significantly increased (P ≤ 0.004), and the bursa of Fabricius index was significantly increased only in the Sal+T3 group (P = 0.456, but the difference in the T3 group was significant), suggesting that high-dose LIQI99 could enhance the function of immune organ indices and resist Salmonella infection. For metabolic organs: the liver index in the Sal+T3 group was significantly higher than that in the Sal group (P = 0.004), which might be due to LIQI99 assisting the liver in metabolizing toxins; there was no significant difference in the heart index among the groups (P = 0.451), indicating that LIQI99 had little effect on the heart. In conclusion, LIQI99 can enhance the ability of laying hens to resist Salmonella infection by up-regulating the immune organ indices (mainly thymus and spleen), and the high-dose group (T3 group) has a more prominent effect.

2.5 Effects of LIQI99 on Intestinal Flora of Laying Hens Under Salmonella Challenge 

Table 5 Experimental Results of the Effects of LIQI99 on Intestinal Flora of Laying Hens Under Salmonella Challenge

This experiment explored the effect of LIQI99 on the intestinal flora of laying hens when challenged with Salmonella.

The results showed that there was no significant difference in Lactobacillus among the groups (P = 0.417), indicating that LIQI99 had little effect on the number of Lactobacillus, but had a certain tendency to promote the growth of Lactobacillus; the number of Escherichia coli in the three groups treated with LIQI99 was extremely significantly lower than that in the control group (P < 0.001), indicating that LIQI99 could effectively inhibit the proliferation of harmful bacteria; for Salmonella: the number in the Sal+T3 group was significantly lower than that in the Sal group (P = 0.003), indicating that high-dose LIQI99 could reduce the colonization of Salmonella in the intestine after challenge

03 Conclusion and Analysis 

In the experiment, LIQI99 significantly reduced the colonization amount of Salmonella in the intestine of laying hens, which was directly related to the product's characteristic of targeted adsorption of pathogenic bacteria. Through electrostatic interaction, it selectively adsorbs pathogenic bacteria such as Salmonella with CS31A protein, and H⁺ glycans penetrate the bacterial cell wall to coagulate its proteins. In vitro experiments showed that the antibacterial rate against Salmonella reached 99.9%, which confirmed the result that the number of Salmonella in the high-dose group decreased sharply in the challenge experiment. At the same time, the product had no antagonistic effect on beneficial bacteria such as Lactobacillus, which was consistent with the stable number of Lactobacillus in the experiment and ensured the balance of intestinal flora. The improvement of intestinal morphology was one of the core findings of the experiment, and the intestinal repair function of LIQI99 was the key reason. Acid-hydrolyzed glycan montmorillonite forms a nanostructure in the gastrointestinal chyme, repairs the intercellular junctions of intestinal cells, and forms a mucosal protective layer, thereby reducing the crypt depth and increasing the villus-to-crypt ratio. This is consistent with the result that the villus-to-crypt ratio of the duodenum and jejunum was significantly increased in the experiment, which further promotes nutrient absorption and explains the optimization of the feed-egg ratio of laying hens. In addition, the enhancement of serum antioxidant capacity and the increase of immune organ indices in the experiment were related to the product's immune-enhancing characteristics. Oligosaccharides stimulate the activation of T lymphocytes, improve the body's antioxidant and immune functions, and help laying hens resist Salmonella infection. As an alternative product to antibiotics in the post-antibiotic era, the effects of LIQI99 in improving the production performance of laying hens and reducing egg contamination shown in the experiment fully verify its product positioning of "efficient sterilization and safe replacement of antibiotics"