| The process of African ostrich(Struthio camelus) farming is the most important and difficult activity and the brooding stage that is up to 90 days is the critical period for the economic success. During this phase, the intestinal development and mucosal immune function is imperfect, and vulnerable to a variety of gastrointestinal diseases, leaving ostrich with reduced growth performance and even can cause death, consequently ostrich farming enterprises may face losses. Boron(B) is a kind of trace elements with a variety of biological activities. Due to that, boron has effects on animal growth, organ development, immune function and metabolism of other trace elements, therefore it attracted more and more attention. So far, the effect of Boron on gut physiology in African ostrich has been poorly reported. In order to investigate the role of Boron on intestinal physiology, forty eight 10-days-old African ostrich chicks were randomly divided into 6 groups(n= 8) and each group was fed with the same premix at a fixed 4 times a day while supplemented with different concentration of boric acid water(0, 40, 80, 160, 320 and 640 mg/L) for different groups until 90 days. By using HE and cell special staining, immunohistochemistry, Tunel, ELISA and real time quantitative PCR techniques, in this study, we investigated the effects of boron on African ostrich chicks intestinal development, intestinal mucosal immune function and Notch1/Jagged1/Hes-1 signal pathway which not only provide reliable data and theoretical guidance for the brooding of ostrich feeding and management, but also help in understanding of gut physiology for further research and disease prevention. The detailed results are as follows: 1. Effect of Boron on intestinal development and morphology in ostrich chicksThe relative weight of duodenum, jejunum and ileum in 40, 80 and 160 mg/L groups has increased but did not show statistically significance. In the high dose group, especially 640 mg/L group, the relative weight of the intestine(duodenum, the jejunum and ileum) decreased significantly(P <0.05). With the increase in boron concentration, the intestinal villus height and villus height / crypt ratio increased and reach a maximum in 80 mg/L group; on the contrary, in high doses 320 and 640 mg/L groups, the villus height and villus height/crypt ratio slightly decreased, while significantly decreased in the 640 mg/L group. In terms of morphology, structure of intestinal tissue in the low dose groups was well developed, but in the high dose groups, villi morphology of duodenum and jejunum was irregular and the striated borders became fuzzy, top villus epithelium was damaged and showed swelling, epithelial tissue was lost significantly, and some tissues fall off and assembled into pieces. The connective tissue in lamina propria exhibited obvious edema, subepithelial basement membrane was ruptured, and its epithelium also showed swelling. However, the effect of high dose boron on ileum was relatively lower but the villus showed irregular shape and striated border was fuzzy, with less loss of epithelial tissue. 2. The effect of Boron on intestinal mucosal immunity in ostrich chicksHE staining, toluidine blue staining and PAS staining were adopted to count the number of immune related intestinal cells( intraepithelial lymphocytes, goblet cells and mast cells) and the contents of SIg A in intestinal mucosa were measured by ELISA, the result showed that the number of i IEL, goblet cells and mast cells and contents of SIg A increased with increased concentration of boron, and goblet cells, mast cells and contents of SIg A were significantly(P <0.05 or P < 0.01) increased in 80 mg/L group. However, the number of i IEL, goblet cells and mast cells and contents of SIg A increased with increased boron concentration, and goblet cell, mast cell and contents of SIg A decreased in high dose of boron concentration, and significantly decreased in 640 mg/L group.The above experiments show that the optimal boron could increase the number of associated immune cells in the intestine(significantly increased goblet cells and mast cells) and the SIg A contents in the intestinal mucosa, thereby strengthening the ostrich chicks intestinal mucosal immune function and 80 mg/L is most appropriate. The high doses of boron suppresses mucosal immunity. 3. Effect of Boron on ostrich jejunum cell proliferation and apoptosisThe result showed that PCNA expression was mainly scattered in the cell nucleus of the jejunal crypt and glandular cells and a little expression in the nucleus of lamina propria in the control group and 40, 80 and 160 mg/L group by PCNA immunohistochemistry. However, PCNA positive cells not only expressed abundantly in the intestinal crypt and glandular cells nuclei, but also expressed in the nucleus of lamina propria and epithelial tissue in 320 and 640 mg/L group. The proliferation index at 80, 160, 320 and 640 mg/L groups was significantly increased(P <0.05 or P < 0.01), compared to the control group.The apoptotic cells mainly distributed in lamina propria in 0?40?80 and 160 mg/L group, while, in 320 and 640 mg/L group, in addition to the apoptotic cells expressed in lamina propria and a large number of apoptotic cells expressed in the basal epithelial cells. By IOD test, the result show that the expression of apoptotic cells decreased in low doses of boron, especially in 80 mg/L. The expression of apoptotic cells significantly increased in 320 and 640 mg/L(P <0.05 or P < 0.01). Caspase-3 m RNA expression levels which is a key apoptosis gene was performed by the relative quantitative PCR and the result showed that compared with the control group, the caspase-3 m RNA expression level were significantly decreased(P <0.05) in 40?80 and 160 mg/L group, and the caspase-3 m RNA expression level was significantly(P <0.05) increased in the 320 and 640 mg/L group. 4 Effect of Boron on Notch1 / Jagged1 / Hes-1 signaling pathwayIn order to clarify whether the Notch1/Jagged1/Hes-1 signaling pathway was involved during the supplementation of boron on intestinal cells differentiation and proliferation, the current study examined gene expression levels in Notch1/Jagged1/Hes-1 signaling pathway by Real-time quantitative PCR. Cmpared with the control group, Notch1, Jagged1 and Hes-1 gene decreased in different level in 40?80 and 160 mg/L group, while Notch1, Jagged1 and Hes-1 gene significantly(P <0.05 or P <0.01) increased in 320 and 640 mg/L groups. This suggests that low doses of boron could suppress the overexpression of Notch1/Jagged1/Hes-1 signaling pathway that drive epithelial cells differentiation into secretory lineage. High doses of boron could promote the expression of Notch1/ Jagged1/ Hes-1 signaling pathway that drive epithelial cell differentiation into absorptive lineage, which may compensate for the absorptive function, due to the loss and apoptosis of the epithelial cells that can further trigger the loss of absorptive cells, and this may be an adaptive compensatory mechanism. |
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