Effects Of Different Light Wavelengths On Development Of Small Intestine And Bursa In Chick Embryos During The Late Period And Its Modulation Mechanisms

Small intestine is an important place to digest and absorb and the first defensive line of mucosal barrier for birds. Previous studies found different wavelength lights could influence the mucosal mechanical and immunological barriers in the small intestine of broilers, but little is known about the effects of different wavelength lights on the development of the small intestine in chicken embryos during the late incubation. However, the late incubation of chick embryo is the best period for plasticity of small intestine, meanwhile the perfect development of small intestine is good to get the maximum development and growth after posthatching. In order to explore the effects of different wavelength lights on the development of small intestine and bursa of chick embryos during the late period, chicken embryos were exposed to white light (400-700nm), blue light (480nm), green light (560nm), red light (660nm), and dark as a control, and were studied by the methods of histology, histochemistry, immunohistochemistry, qRT-PCR, ELISA and cell culture. The results would be provided a theoretical basis for excavation of improving the growth potential and immune function of just post-hatch chicks with lights stimulating during chicken embryos incubation. The results as followed:1. Development of morphological structure and physiological function of small intestine in chick embryos during the late incubationDuring the late-stage of chicken incubation, the length of villus, thickness of muscle layer, the expression of PCNA, and density of goblet cells changed significantly in the small intestine with the embryonic age increasing. Up to E21, the villus height and thickness of muscle layer of duodenum, jejunum and ileum were increased150-180%and1.2-1.7times compared with E15, respectively. The average optical density value of small intestinal mucosa PCNA in duodenum, jejunum and ileum was higher38-53%than that of E15. The sulfated goblet cells were the most at E15and neutral goblet cells are prodominent at E18, and at E21, a large number of sialylated subtype goblet cells emerged, the goblet cell density of duodenum, jejunum and ileum were higher3.3,4.5and7.1fold than those of E18, and the expressions of Muc2mRNA in duodenum, jejunum and ileum were higher26.63,11.67and7.75fold than those of E15, and MUC2positive cells emerged largely. At this time, mosaic connection between intestinal epithelial cells was decreased, and tight junctions, gap junctions and desmosomes were developed.SI and PepT1mRNA began to express on E15. With the increase of embryo age, the expression of SI mRNA in duodenum and jejunum, PepTl mRNA in jejunum and ileum increased on E18and then decreased on E21. While the expression of SI mRNA in ileum decreased continually and the expression of PepTl mRNA in duodenum increased continually. The antioxidant function of jejunum and the ability of T lymphocyte proliferation of ileocecal junction increased continually and there was no obvious difference in B lymphocyte proliferation of ileocecal junction among different embryo ages.2. Development of morphology and function of bursa of Fabricus during the late incubation of chick embryosThe lymphoid follicles of bursa began to appear on E15, and the PCNA positive rate of intrafollicilar lymphocytes was high, reaching97.7%. The bursa had some antioxidant function, but the ability of lymphocyte proliferation was low after stimulation by ConA and LPS. With the increase of embryo age, the area of lymphoid follicle of bursa increased rapidly and the PCNA positive rate of intrafollicilar lymphocytes decreased. Until E21, the area of lymphoid follicle of bursa was increased6times than that of E15, and the PCNA positive rate of intrafollicilar lymphocytes was similar as that of E18. However, the antioxidant function and the ability of T lymphocyte proliferation further improved, but ability of B lymphocyte proliferation had no obvious change after antigen simulation.3. Effect of white light on mucosa structure and physiological function of small intestine of chick embryos during the late incubationOn E15and E18, there were no significant difference between white group and dark group in the morphological structure and physiological function of small intestine. Until E21, villus height, Muc2, PepTl and SI mRNA expression in white group were higher than those of in dark group. However, there was no obvious difference in antioxidant ability between the two groups. The ability of T lymphocyte transformation was higher in white group than that of in dark group (p<0.05) after stimulation of ConA, while there was no significant difference between the two groups in the ability of B lymphocyte transformation after stimulation of LPS.4. Effect of white light on growth and physiological function of bursa of Fabricus in chick embryos during the late incubationOn E15, there was no significant difference between white and dark groups in follicular area of bursa, the expression of PCNA in follicle, the antioxidant ability and the ability of lymphocyte transformation. On E21, CAT, SOD activity and T-AOC was significantly higher in white group than that of in dark group (p<0.05), and content of MDA in jejunum was higher24.9%in white group than that of in dark group. After antigen stimulating, the ability of T lymphocyte proliferation was significantly higher than that of in dark group, while there was no significant difference between the two groups in the ability of B lymphocyte proliferation.5. Effect of monochromatic light on mucosa structure and physiological function of small intestine in chick embryos during the late incubationOn E15, there was no significant difference among different treatment groups in morphological structure and physiological function of small intestine in chicken embryos. With the increase of embryo age, the change pattern of small intestinal mucosal morphology and function were similar among different treatment groups, but the magnitude of the change was not the same in different treatment groups. Until E21, villus height and muscle thickness in the three segments of small intestine were higher in green light group than that of in dark group (p<0.05) The expression of intestinal mucosal PCNA in jejunum and ileum was higher significantly in green group than that of other treatment groups. The expression of PepTl and SI mRNA in duodenum, jejunum and ileum was higher in green group than that of in red and dark groups. Goblet cell density in jejunum and ileum of green and blue groups were higher than that of in dark and red groups. And the ratio of sulfated and sialylated goblet cells in ileum were lower in green group than that of other treatment groups. The expression of Muc2gene and MUC2of the three segments of small intestine was higher in green group than that of other treatment groups. The antioxidant function was higher in green group than other treatment groups. After simulation of ConA, lymphocyte proliferation index of ileocecal junction in green group was the highest, showing significant difference compared with that of red and dark groups (p<0.05). While simulated with LPS, there was no significant difference among different treatment groups.6. Effect of monochromatic light on development and physiological function of bursa in chick embryos during the late incubationOn E15, the area of follicle of bursa in red group was the smallest, which were significant difference compared with other treatment groups. With increasing of embryo age, the area of follicles of bursa in each treatment group increased, the expression of PCNA in follicle decreased and antioxidant function of bursa enhanced. Until E21, the area of follicular in green and blue groups were35499.0±1775.4and26586.5±5419.0, respectively, and there were significant difference compared with that of darkness group (23875.1±2388.4)(p<0.05). And the area of follicular in red group was the smallest,15288.1±307.2, which was significant difference among the other treatment groups (p<0.05). The positive rate of PCNA in follicle in green group was the maximum (77.9%), which was significant difference as compared to red group (71.7%) and dark group (73.2%)(p<0.05). The activity of CAT, SOD and T-AOC in green group was higher significantlythan that of dark group (p<0.05). While content of MDA in dark group was higher than that of green group. When stimulated with ConA, lymphocyte proliferation index in green group was higher by12.1%and5.1%than red and dark group. When stimulated with LPS, the ability of lymphocyte proliferation was higher in green group than red and black group. However, there was no difference was observed between green and blue group.7. Effect of light on IL-6and TNF-α in jejunum tissue of chick embryos during the late incubationOn E15, there was no significant difference among different treatment groups in the level of IL-6 and TNF-a in jejunum. With the increase of embryo age, on E21, the level of IL-6and TNF-a in jejunum in green group was329.21±19.69ng/L and374.62±23.35ng/L, respectively. There was significant difference between green and dark group (248.21±71.20ng/L;267.86±26.03ng/L)(p<0.05). The level of IL-6in jejunum in red group was the lowest (238.01±11.16ng/L), but there was no significant difference between red and dark groups (p>0.05).8. Effect of light on expression of Mella, Mellb and Mellc in jejunum in chick embryos during the late incubationVarious monochromatic lights and white light stimulus had a certain effect on the expression of Mel1a、Mel1b and Mellc mRNA in chicken embryo jejunum during the stage of incubation. With the age increasing, the expression of Mella, Mellb and Mellc mRNA in different treatment groups increased. On E21, the expressions of Mel1a、Mel1b and Mel1c mRNA of jejunum in chicken embryos were the highest in green group, it was1.5-1.7,1.1and1.2-2.5folds as high as that of dark and red groups. There was significant difference in Mella mRNA expression in green group compared with that of dark and red group (p<0.0400), and the expression of Mellc mRNA was higher significantly in green group than that of red light (p=0.0228).9. Effect of light on the expression of Mella, Mellb and Mellc in bursa of chick embryos during the late incubationWith the increase of embryo age, the expression of Mel1a, Mel1b and Mel1c in green and blue groups continued to increase. The expression of Mel1a, Mel1b and Mel1c on E21were higher8.1,16.3and4.4times that of E15in green group, and the expression of Mel1a, Mellb and Mel1c were higher7.7,2.7and4.4times than that of on E15in blue group. The expression of Mella, Mellb and Mel1c in white and dark groups increased on E18and then decreased on E21, which was decreased in red group from E15to E21.Conclusion:(1) The late stage of chicken embryos incubation (from E35to E21) was the stage of rapid development for morphological structure and function of small intestinal mucosa. The ontogeny and differentiation of epithelial cells in broiler embryos display temporal and spatial differences.(2) Lights had a certain effects on development and function of small intestine and bursa. Green light enhanced significantly the development of small intestinal mucosa and bursa, and promoted the maturity of small intestinal mucosa and bursa earlier during the late incubation.(3) The effects of light on the development and function of small intestine and bursa might be associated with the secretion of melatonin in chick embryos and further influencing the cytokine, antioxidant function and expression of melatonin receptor in jejunum and bursa.