【作者】 张自强；

【导师】 陈耀星；

【作者基本信息】 中国农业大学 ， 基础兽医学， 2014， 博士

【摘要】 禽类具有特殊的视觉系统,能比人类感知更宽的频谱范围,所以对光的波长(颜色)变化非常敏感。本研究是我们实验室前期研究发现在肉鸡生长前期绿光能促进其细胞免疫和体液免疫和在生长后期蓝光的促进作用更明显的基础上,进一步探讨在肉鸡饲养过程中采用不同光色的光照组合是否能够更好地改善其细胞免疫和体液免疫功能,及其作用的相关机制。为此选用刚出壳的AA肉鸡,分别饲养在LED灯为光源的白光(W,400-700nm)、红光(R,660nm)、绿光(G,560nm)和蓝光(B,480nm)下,26日龄时转换光色组成12个光色组合(WR、WG、WB、RW、 RG、RB、GW、GR、GB、BW、BR、BG),同时以4个单色光(WW、RR、GG、BB)为试验对照,继续饲养到49日龄。主要研究结果如下：1、不同光色组合对肉鸡免疫功能的影响49日龄时,GB光色组合组的抗新城疫病毒和牛血清白蛋白的抗体水平最高,比其他光色组合提高了11.66%～51.43%(P=0.000-0.044)和11.65%-43.33%(P=0.000～0.009),但与BG组无显著差异(P=0.489和0.452)。外周血的T和B淋巴细胞增殖率也是GB组最高,其中T淋巴细胞的增殖率比其他光照组高12.38%～36.19%(P=0.000-0.013),但与GW组(P=0.112)、BG组(P=0.714)和BB组(P=0.161)无显著差异；B淋巴细胞增殖率高于其他光照组12.96%～50.01%(P=0.000～0.004),但与BG组差异不显著(P=-0.677)。GB组的血浆IL-2含量高于单独单色光组9.68%～60.27%(P=0.000～0.025),但TNF-α含量低于单独单色光组16.88%～40.49%(P=0.000-0.002),且它们都与BG组间无显著差异(P=0.382和0.402)。2、不同光色组合对肉鸡脾发育和淋巴细胞增殖的影响49日龄时,在GB光色条件下饲养的肉鸡,脾器官指数高于单独单色光组11.61%～36.26%(P=0.000～0.019),但与BG组无显著差异(P=0.398)；脾小结的直径分别高于WW组37.76%(P<0.001)、RR组64.84%(P<0.001)和GG组10.72%(P=0.033),而与BG组(P=0.628)和BB组(P=0.212)间差异不显著；脾的动脉周围淋巴鞘面积分别高于WW组41.01%(P<0.001)、RR组75.36%(P<0.001)和GG组19.84%(P=0.003),但与BG组无显著差异(P=0.671)；脾细胞增殖细胞核抗原(PCNA)的累计光密度(IOD)值高于WW组23.09%(P<0.001)、RR组27.84%(P<0.001)和GG组14.64%(P=0.007),但与BG组(P=0.459)和BB组(P=0.058)之间差异不显著；脾T淋巴细胞的增殖率高于WW组、RR组和GG组18.33%～42.85%(P=0.000～0.003),但与BB和BG组无显著差异(P>0.05)；脾B淋巴细胞增殖率高于其他所有光色组11.90%～34.28%(P=0.000～0.018)。3、不同光色组合对肉鸡褪黑激素分泌的影响及其在调节脾淋巴细胞增殖中的作用35日龄,GB组肉鸡的血浆和脾中褪黑激素含量均高于其他单独的单色光组21.97%～55.03%(P<0.001)和23.22%-71.93%(P=0.000～0.017),但与BG组之间无显著的差异(P=0.334和0.234)。脾淋巴细胞增殖相关性分析显示,血液和脾中褪黑激素的水平与脾淋巴细胞增殖的相关系数分别为r=0.947和r=0.909,具有高度的相关性。体外细胞试验进一步证实,外源性生理浓度的褪黑激素刺激使不同光色组合条件下的肉鸡脾T淋巴细胞增殖率升高12.44%～19.26%(P<0.05),且升高趋势与不同光色处理组之间褪黑激素的分泌相关。4、不同光色组合对肉鸡脾淋巴细胞抗氧化功能的影响49日龄时,GB组肉鸡脾的GSH-Px和SOD酶活性最高,分别比其他光色组高11.27%～58.66%(P=0.000～0.003)和12.18%～32.14%(P=0.001～0.026),但与BG组间差异不显著(P>0.05)；GB组的T-AOC的酶活性也是最高,高于WW组36.84%和RR组71.69%(P<0.001),但与GG组、BB组和BG组之间无显著的差异(P>0.05)。相反,GB组的MDA含量最低,低于WW组、RR组和GG组21.18%～40.89%(P=0.003-0.044),但与BB和BG组之间无显著的差异(P>0.05)。5、不同光色组合对肉鸡脾淋巴细胞褪黑激素受体表达的影响RT-PCR和Western blot检测显示,GB组肉鸡脾褪黑激素受体Mel1a、Mel1b和Mel1c的mRNA和蛋白表达显著高于其他的单色光照组,但与BG组间无显著差异,且这三种受体的mRNA或蛋白水平都是Mel1b>Mel1c>Mel1a。免疫组化染色定位显示,Mel1a受体主要分布在动脉周围淋巴鞘和椭球周围淋巴鞘；Mel1b受体广泛分布于整个脾组织,包括白髓、红髓,边缘区；Mel1c受体也广泛分布于椭球、边缘区、红髓。6、褪黑激素介导的光色组合诱导肉鸡脾的淋巴细胞增殖作用中的信号通路体外试验结果显示,外源性褪黑激素促进35日龄GB组肉鸡脾T淋巴细胞增殖的作用被褪黑激素受体阻断剂抑制。与对照组相比,Mel1b受体特异性阻断剂(4P-PDOT)降低T淋巴细胞增殖率13.29%(P=0.028),Mel1c受体特异性阻断剂(prazosin)降低了11.05%(P=0.035),但Mel1a和Mel1b非特异性受体阻断剂(luzindole)的作用不显著,仅降低6.34%(P=0.136)。进一步的胞内信号通路试验发现,添加MEL+PD98059(ERK通路阻断剂)组和MEL+PDTC (NF-κB通路特异性通路阻断剂)组的T淋巴细胞增殖率分别低于MEL组13.05%(P=-0.033)和13.97%(P=0.029),表明两种信号通路阻断剂都能够显著抑制肉鸡脾T淋巴细胞的增殖。结论：在肉鸡饲养过程中,与采用单独的单色光照相比,生长前期采用绿色光照(0-26日龄)和生长后期(27-49日龄)改为蓝色光照,能够更好地促进肉鸡脾的发育,提高细胞免疫和体液免疫应答水平,改善免疫功能。其作用机理显示：光色通过影响肉鸡体内褪黑激素的分泌而影响肉鸡免疫功能；褪黑激素一方面提高肉鸡脾组织抗氧化酶GSH-Px和SOD的活性和抗氧化功能；另一方面主要通过与Mel1b和Mel1c受体结合,经细胞内ERK及其下游NF-κB信号途径促进脾淋巴细胞增殖而发挥免疫调控的作用。更多还原

【Abstract】 Chickens have a special visual system and can detect a broader spectrum of colors than humans. Therefore, the chicken is extremely sensitive to subtle differences in light color. Our previous study showed that green light promotes cellular and humoral immune responses during the early stage, and blue light enhances immune responses during the later growth stages in the broilers. However, whether a combination of different monochromatic lights will result in better immune responses and its correlative mechanism needs to be confirmed.In this study, Arbor Acre male broilers were exposed to white light (W,400-700nm), red light (R,660nm), green light (G,560nm) and blue light (B,480nm) from0to26days by a LED system, then, the broilers were respectively switched to others color monochromatic lights until day49. The12kinds of different monochromatic lights combination (WR, WG, WB, RW, RG, RB, GW, GR, GB, BW, BR, BG) and4control groups (WW, RR, GG, BB) were formed. The results as followed:1. Effect of different monochromatic light combination on immune response in the broilersAt49days, the levels of anti-Newcastle disease virus (NDV) and anti-bovine serum albumin (BSA) IgG were the highest in GB group, and were elevated by11.66%～51.43%(P=0.000～0.044) and11.65%～43.33%(P=0.000～0.009)ascompared to single monochromatic lights,but there was no significant difference between the GB and BG groups.The peripheral blood T lymphocyte proliferation in the GB group showed an increased in vitro (12.38%～36.19%, P=0.000～0.013) compared with the other groups.However, there was little difference between GB and GW (P=0.112), BG (P=0.714) or BB groups (P=0.161). Theperipheral blood B lymphocyte proliferation in the GB group was significantly increased by12.96%～50.01%(P=0.000～0.004) than in other monochromatic light groups. No differences were found between the GB and BG group broilers.The IL-2concentration in the peripheral blood was the highest in the GB group, and was increased by9.68%～60.27%(P=0.000～0.025) over the single monochromatic light groups. But the TNF-α concentration in the peripheral blood in the GB group consistently remained lower than in the other groups, and it was lower16.88%～40.49%(P=0.000～0.002) than those in the single monochromatic light groups. No remarkable differences in the IL-2and TNF-α concentration of the GB and BG groups were observed.2. Effect of different monochromatic light combination on spleen development and lymphocyte proliferation in the broilersThe organ index of spleen in the GB group was higher by11.61%～36.26%(P=0.000～0.019) at49days compared to the broiler reared under the other single monochromatic light conditions. However, no significant difference was observed between the GB and BG groups. The diameter of splenic nodule in the GB group was higher by37.76%(P<0.001),64.84%(P<0.001) and10.72%(P=0.033) in comparison with broilers grown with WW, RR and GG, but the GB group was not significantly different from the BG (P=0.628) and BB (P=0.212) groups. The areas of periarterial lymphatic sheath in the GB group were raised by41.01%(P<0.001),75.36%(P<0.001)and19.84%(P=0.003) in comparison with WW, RR and GG groups. However, no significant difference was found between the GB and BG groups. The IOD of PCNA in the GB group was higher by23.09%(P<0.001),27.84%(P<0.001) and14.64%(P=0.007) than that of WW, RR and GG light groups. No significant differences were observed between the GB group and BG (P=0.459) or BB groups (P=0.058).The spleen T lymphocytes proliferation in GB group was higher18.33%～42.85%(P=0.000～0.003) than in WW, RR and GG light groups. However, spleen T lymphocyte proliferation in the GB group was not significantly different from the BG and BB group (P>0.05). The GB group showed enhanced B lymphocyte proliferation by11.90%～34.28%(P=0.000～0.018) compared to other light treatments.3. Effect of different monochromatic light combination on melatonin secretion and regulating lymphocyte proliferation in the broilersThe melatonin concentration of blood plasma or spleen in the GB group was the highest, and increased by21.97%～55.03%(P<0.001) and23.22%～71.93%(P=0.000～0.017) compared to the birds reared under all of the other light conditions at35d of age. The correlationanalysis indicate that a significant correlation (r=0.947and r=0.909) was observed between spleen lymphocyte proliferation with plasma or spleen melatonin level in different monochromatic light combination groups.The experiment in vitro showed that physiological level melatonin stimulated spleen T lymphocyte proliferation in different monochromatic light combination groups. Comparing to without melatonin treatment, the T lymphocyte proliferation was enhanced by12.44%～19.26%(P<0.05) in melatonin treatment groups, and thisproliferation trend was accordant with different light groups melatonin secretion.4. Effect of different monochromatic light combination on spleen lymphocyte antioxidative function in broilersAt49days, the activity of antioxidative enzymeGSH-Px and SOD in the GB groups were the highest, and increased by11.27%～58.66%(P=0.000～0.003) and12.18%～32.14%(P=0.001～0.026) compared to other light groups, but there was no difference between GB and BG group. T-AOC level in GB groups was also the highest, and it was higher than that of WW group (36.84%, P<0.001) and RR group (71.69%, P<0.001), but no significant difference among GB, BG, GG and BB groups. However, MDA content in GB groups was the lowest, and decrease21.18%～40.89%(P=0.003～0.044) compared to that of WW, RR and GG light conditions, but no significant difference among GB, BB and BGgroups (P<0.05).5. Effect of different monochromatic light combination on expression of melatonin receptor in thespleen ofbroiler.RT-PCR and Western blot results suggested that mRNA and protein expressionof Mel1a, Mel1b or Mel1c in the GB group were significant higher than that in othermonochromatic light,but no significant difference between GB and BG groups. ThemRNA or protein expressionin this three kinds ofmelatonin receptor showed Mel1b>Mel1c>Mel1a. Immunohistochemical localization in melatonin receptor showed that Mel1a-positive cells were primarily present in periellipsoid lymphatic sheaths and periarterial lymphatic sheath; Mellb-positive cells were found in the entire spleen tissue, including white pulp, red pulp and marginal zone; Mellc-positive cells were widely observed in ellipsoid, red pulp and marginal zone.6. Effect of signal pathway of melatonin on light color induced spleen T lymphocyte proliferation in the broilersThe results in vitro showed that extrinsic melatonin stimulating spleen T lymphocyte proliferation in the GB group was inhibited by melatonin receptor antagonist at35days. Compared to the control group, spleen T lymphocyte proliferation was decreased13.29%(P=0.028) by Mel1b selective antagonist (4P-PDOT), and was reduced11.05%(P=0.035) by Mel1c selective antagonist (prazosin), but it only descend6.34%(P=0.136) by Mel1a/Mel1b non-selective antagonist (luzindole) and no significant roles was observed in the spleen T lymphocyte proliferation. Further intracellular signal pathway study found that T lymphocyte proliferation in MEL+PD98059(ERK signal pathway blocking agent)and MEL+PDTC (selective antagonist of NF-κB) groups wererespectively decreased by13.05%(P=0.033) and13.97%(P=0.029) compare with MEL group. The result showed spleen T lymphocyte proliferation was obviously inhibited by two kinds of signal pathway blocking agent in broilers.Conclusion:Compared to single monochromatic light regimen, the light regimen, which green light was use in the early stage (0～26days) and blue light was adopted at a later age (27～49days), can better promote spleen development and enhance cellular and humoral immunity response, improving the immune function in broilers. The relative mechanism showed light color can affect broiler immune function through melatonin secretion in vivo. Howeveron the one hand, melatonincan enhance broiler spleen antioxidative ability by improving the enzyme activity of GSH-Px and SOD,on the other hand, melatonin would promote T lymphocyte proliferation and regulate immune through Mellb and Mellc receptor, which may be involved in ERK and downstream NF-κB signaling pathway更多还原