Effect of Different Doses of TF on The Lymphoid Organs
The body weight and immune organ index were measured. After treatment with different doses of TF for a week, the body weight, as well as spleen and thymus indices increased in laying hens, compared to the control group (P > 0.05) (Fig. 1a, b). Moreover, after treatment termination with TF for a week, the weight, as well as spleen, bursal, and thymus indices were still higher, compared to those in the control group (P > 0.05) (Fig. 1a, c).
Effect of Different Doses of TF on Intestinal Mucosal Morphology in Laying Hens
The data obtained from the small intestinal villus height (VH) and crypt depth (CD), as well as the ratio (V/C) that represent the absorption capacity (15) were analyzed statistically. Moreover, after treatment with different doses of TF for a week, the VH and V/C from the duodenum to ileum sections increased significantly, especially in the TF-0.25mL and TF-1.00mL group, the VH in TF-1mL group was the highest and was higher by 10.64% in duodenum (P = 0.019), 15.44% in jejunum (P = 0.001) and 13.17% in ileum (P = 0.002) than control group (Fig. 2a). The V/C increased most significantly, by 41.11% of TF-1.00mL group in duodenum (P = 0.000), 33.93% of TF-1.00mL group in jejunum (P = 0.000) and 24.60% of TF-0.25mL group in ileum (P = 0.004) (Fig. 2f). However, a significant decrease was observed in the CD, the CD was the lowest in TF-0.25mL and TF-1mL group and was lower by 15.87% of TF-1.00mL group in duodenum (P = 0.001), 20.82% of TF-0.25mL group in jejunum (P = 0.000) and 16.78% of TF-1.00mL group in ileum (p = 0.001) than control group (Fig. 2d).
After the treatment termination with TF for one week, the VH, CD, and V/C maintained the same trend in TF-0.25mL and TF-1.00mL groups (Fig. 2c, e, g). Therefore, oral TF could increase the VH and decrease the CD with a significantly rising ratio of V/C.
Effect of Different Doses of TF on The Intraepithelial Lymphocytes, IL-10, and TNF-α in Different Intestinal Tissues
The number of Intraepithelial lymphocyte (IELs) was analyzed, and the contents of IL-10, as well as TNF-α, were detected by ELISA. After one week of continuous treatment with TF, the number of IELs and the concentration of IL-10 were significantly higher in TF-0.25mL and TF-1.00mL groups, compared to the control. The IELs proliferation was the highest in the TF-1.00mL group, which increased by 19.50% in duodenum (P = 0.000), 31.82% in jejunum (P = 0.000), 27.47% in ileum (P = 0.000) than the control group (Fig. 3b). The IL-10 content was the greatest in the TF-1.00mL group, which was greater by 14.91% in duodenum (P = 0.001), 10.43% in jejunum (P = 0.000), 18.71% in ileum (P = 0.000) and 37.01% in rectum (P = 0.000) than the control group (Fig. 3d). However, the TNF-α content showed a significant decrease in this regard, the TNF-α content of the duodenum in the TF-0.25mL group was the lowest, which was 10.80% (P = 0.002) lower than the control group. In jejunum and ileum of the TF-0.05mL group were the lowest, which was 15.06% (P = 0.000) and 14.98% (P = 0.000) respectively. In rectum of the TF-1.00mL group was the lowest, which was 11.72% (P = 0.006) lower than the control group (Fig. 3f).
After the treatment termination with TF for one week, the number of IELs were still on the rise from the duodenum to the ileum, especially in the TF-1.00mL group (P < 0.05) (Fig. 3c). The IL-10 content from the duodenum to rectum increased only in the medium and high dose group (Fig. 3e). Furthermore, the TNF-α content in the four TF dose groups from the duodenum to rectum was still significantly lower, compared to the control group (P < 0.05) (Fig. 3g). After the treatment termination with TF for a week, the effect of TF on the IL-10 was weakened from the duodenum to the rectum.
Effect of Different Doses of TF on The Number of Goblet Cells and Their Expression of Muc2 in Different Intestinal Tissues
The number of goblet cells was analyzed by PAS staining, and the Muc2 expression was detected by the RT-PCR. The detection of the intestinal segments of 12-day-old laying hens showed an increase in the number of goblet cells from the duodenum to the ileum in a dose-dependent manner, especially in the TF-1.00mL group, which increased by 41.08% in duodenum (P = 0.000), 30.52% in jejunum (P = 0.000), 37.02% in ileum (P = 0.000) than the control group (Fig. 4b).
Moreover, the relative levels of the Muc2 gene from the duodenum to rectum were significantly increased in the medium and high-dose groups, especially in the TF-1.00mL group which increased by 117.94% in duodenum (P = 0.000), 105.85% in jejunum (P = 0.000), 45.09% in ileum (P = 0.000) and 50.59% in rectum (P = 0.000) than the control group (Fig. 4d). After treatment termination with TF for one week, on the 19th day of age, the number of goblet cells in each intestine segment continued to increase, especially in the TF-1.00mL group (P < 0.05) (Fig. 4c). However, the relative levels of the Muc2 gene were only higher in the TF-1.00mL group, compared to the control group, especially in the ileum and rectum (P < 0.05) (Fig. 4e). Therefore, after a week of treatment termination with TF, the effect of TF on the Muc2 gene was relatively weakened.
Effects of Different Doses of TF on The Expression of TLR2 and TLR4 in Different Intestinal Issues
To explore the regulation role of TF in the intestinal mucosal immunity, the expression of toll like receptor (TLR-2 and TLR-4) was detected by the RT-PCR. The detection of the intestinal segments of 12-day-old laying hens showed that TF-1.00mL groups could significantly increase the relative levels of TLR-2 and TLR-4 genes from the duodenum to rectum. For TLR-2 genes, which increased by 42.31% in duodenum (P = 0.000), 46.85% in jejunum (P = 0.000), 44.48% in ileum (P = 0.000) and 57.71% in rectum (P = 0.000) than the control group (Fig. 5a); For TLR-4 genes, which increased by 31.98% in duodenum (P = 0.000), 24.10% in jejunum (P = 0.000), 6.34% in ileum (P > 0.05) and 15.69% in rectum (P = 0.003) than the control group (Fig. 5c). After treatment termination with TF for one week, on the 19th day of age, the relative levels of TLR-2 were higher in the jejunum, ileum, and rectum, not the duodenum, compared to the control group (Fig. 5b). The relative levels of the TLR-4 gene obtained an increased trend from the duodenum to rectum (Fig. 5d). These data show that the extent of the effect of TF on the TLR-2 and the TLR-4 was reduced on the 19th day of age.
Effect of Different Dose of TF on Antioxidant Function of Intestine in Laying Hens
Antioxidant enzymes (i.e., SOD, CAT, and GSH-PX), total antioxidant capacity of T-AOC, and lipid peroxidation products of MDA were detected to explore the effect of TF on the antioxidant function of the intestine. As shown in Fig. 6, after a week of continuous treatment with different doses of TF, it was found that the antioxidant enzymes of SOD, CAT, and GSH-PX, as well as the total antioxidant capacity of T-AOC from the duodenum to rectum were significantly increased in the TF-1.00mL groups, compared to the control group. For the antioxidant enzymes of SOD which increased by 25.73% in duodenum (P = 0.000), 17.07% in jejunum (P = 0.002), 31.54% in ileum (P = 0.000) and 22.06% in rectum (P = 0.000) than the control group (Fig. 6a); For the antioxidant enzymes of CAT which increased by 25.77% in duodenum (P = 0.000), 18.49% in jejunum (P = 0.000), 16.98% in ileum (P = 0.001) and 17.79% in rectum (P = 0.000) than the control group (Fig. 6c); For the antioxidant enzymes of GSH-PX which increased by 13.99% in duodenum (P = 0.014), 15.15% in jejunum (P = 0.000), 20.25% in ileum (P = 0.000) and 21.62% in rectum (P = 0.000) than the control group (Fig. 6e); For the total antioxidant capacity of T-AOC which increased by 25.64% in duodenum (P = 0.000), 23.1% in jejunum (P = 0.000), 16.44% in ileum (P = 0.000) and 43.16% in rectum (P = 0.000) than the control group (Fig. 7a); However, the content of MDA which decreased by 62.47% in duodenum (P = 0.000), 28.30% in jejunum (P = 0.000), 20.95% in ileum (P = 0.001) and 15.81% in rectum (P = 0.000) than the control group (Fig. 7c). On the other hand, on the 19th day of age, there was no significant difference between the different dose groups and the control group regarding the antioxidant enzymes of SOD, CAT, and GSH-PX, total antioxidant capacity of T-AOC, and lipid peroxidation products of MDA (Fig. 6b, d, f; Fig. 7b, d). Therefore, continuous treatment with TF for a week could significantly improve the function of intestinal antioxidants. After the treatment termination with TF, the effect of TF on intestinal antioxidant function was weakened in this study.