脯氨酸对中华鳖生长性能、抗氧化能力和抗嗜水气单胞菌感染能力的影响

Effects of Proline on Growth Performance,Antioxidant Capacity and Resistance to Aeromonas hydrophila Infection of Pelodiscus sinensis

本研究旨在探讨脯氨酸对中华鳖生长性能、抗氧化能力和抗嗜水气单胞菌感染能力的影响。试验选取初始体重为200 g左右的健康中华鳖幼鳖48只,随机分为对照组(P0组)和脯氨酸组(P4组),分别饲喂添加0(基础饲料)和40 g/kg脯氨酸的饲料,每组3个重复,每个重复8只。试验期为42 d。养殖试验结束后,从各组随机选择3只中华鳖经腹腔注射1 mL的嗜水气单胞菌(Ah)菌液,分别标记为P0+Ah组和P4+Ah组;再各组随机选择另3只中华鳖经腹腔注射等量无菌生理盐水,分别标记为P0+NS组和P4+NS组。攻毒24 h后,取样测定相关指标。结果表明:1)与对照组相比,脯氨酸组中华鳖存活率、增重率和特定生长率显著提高(P<0.05),但肝体比和脏体比无显著差异(P>0.05)。2)与P0+NS组相比,P4+NS组中华鳖裙边组织中谷胱甘肽过氧化物酶(GSH-Px)和超氧化物歧化酶(SOD)活性显著提高(P<0.05),且线粒体活性氧(ROS)产生速率显著降低(P<0.05)。在Ah应激作用下,与P0+Ah组相比,P4+Ah组裙边组织中GSH-Px和SOD活性显著提高(P<0.05),而线粒体ROS产生速率显著降低(P<0.05)。3)与P0+NS组相比,P4+NS组中华鳖裙边组织中脯氨酸和羟脯氨酸含量显著提高(P<0.05)。在Ah应激作用下,与P0+Ah组相比,P4+Ah组裙边组织中脯氨酸和羟脯氨酸含量显著提高(P<0.05)。4)与P0+NS组相比,P4+NS组中华鳖裙边组织中Ⅰ型胶原蛋白A1(COL1A1)、Ⅰ型胶原蛋白A2(COL1A2)、脯氨酰4-羟化酶A1(P4HA1)、脯氨酰4-羟化酶A3(P4HA3)、转化生长因子-β2(TGF-β2)和转化生长因子-β3(TGF-β3)mRNA相对表达量显著提高(P<0.05),组织金属蛋白酶抑制因子2(TIMP2)和组织金属蛋白酶抑制因子4(TIMP4)mRNA相对表达量显著降低(P<0.05)。在Ah应激作用下,与P0+Ah组相比,P4+Ah组裙边组织中COL1A1、COL1A2、P4HA1、P4HA3、TGF-β2、TGF-β3、基质金属蛋白酶1(MMP1)、基质金属蛋白酶2(MMP2)、TIMP2和TIMP4 mRNA相对表达量均显著提高(P<0.05)。5)组织学研究发现,P4+NS组中华鳖裙边组织中胶原层界限清晰,胶原纤维排列整齐,而P4+Ah组裙边组织中胶原层分界趋于模糊,胶原纤维紊乱无序,且胶原纤维出现向内部降解的趋势。综上可知,饲料中添加40 g/kg的脯氨酸可以改善中华鳖幼鳖的生长性能,且可能通过增强抗氧化酶活性,降低ROS产生,并平衡胶原蛋白物质代谢,发挥抵抗Ah应激的作用。

The aim of this study was to investigate the effects of proline on growth performance,antioxidant capacity and resistance to Aeromonas hydrophila infection of Pelodiscus sinensis.Forty-eight healthy young Pelodiscus sinensis with an initial body weight of about 200 g were randomly divided into control group(P0 group)and proline group(P4 group),which fed diets supplemented with 0(basal diet)and 40 g/kg proline,respectively,and each group had 3 replicates and each replicate had 8 turtles.The experiment lasted for 42 days.After the feeding experiment,3 turtles were randomly selected from each group to be intraperitoneally injected with 1 mL Aeromonas hydrophila(Ah)bacteria solution,and labeled as P0+Ah group and P4+Ah group,respectively;another 3 turtles were randomly selected in each group to be intraperitoneally injected with the same amount of sterile normal saline and labeled as P0+NS group and P4+NS group,respectively.After 24 h of challenge,the relevant indices were determined by sampling.The results showed as follows:1)compared with the control group,the survival rate,weight gain rate and specific growth rate of Pelodiscus sinensis in proline group were significantly increased(P<0.05),but there were no significant differences in hepatosomatic ratio and viscerosomatic ratio(P>0.05).2)Compared with P0+NS group,the activities of glutathione peroxidase(GSH-Px)and superoxide dismutase(SOD)in calipash tissues of Pelodiscus sinensis in P4+NS group were significantly improved(P<0.05),and the production rate of mitochondrial reactive oxygen species(ROS)was significantly decreased(P<0.05).Under Ah stress,compared with P0+Ah group,the activities of GSH-Px and SOD in calipash tissues in P4+Ah group were significantly increased(P<0.05),while the production rate of mitochondrial ROS was significantly decreased(P<0.05).3)Compared with P0+NS group,the content of proline and hydroxyproline in calipash tissues of Pelodiscus sinensis in P4+NS group was significantly improved(P<0.05).Under Ah stress,compared with P0+Ah group,the contents of proline and hydroxyproline in calipash tissues in P4+Ah group were significantly increased(P<0.05).4)Compared with P0+NS group,the mRNA relative expression levels of collagen typeⅠA1(COL1A1),collagen typeⅠA2(COL1A2),prolyl 4-hydroxylase A1(P4HA1),prolyl 4-hydroxylase A3(P4HA3),transforming growth factor-β2(TGF-β2)and transforming growth factor-β3(TGF-β3)in calipash tissues of Pelodiscus sinensis in P4+NS group were significantly increased(P<0.05),and the mRNA relative expression levels of tissue metalloproteinase inhibitor 2(TIMP2)and tissue metalloproteinase inhibitor 4(TIMP4)were significantly decreased(P<0.05).Under Ah stress,compared with P0+Ah group,the mRNA relative expressions of COL1A1,COL1A2,P4HA1,P4HA3,TGF-β2,TGF-β3,matrix metalloproteinase 1(MMP1),matrix metalloproteinase 2(MMP2),TIMP2 and TIMP4 in calipash tissues in P4+Ah group were significantly increased(P<0.05).5)Histological studies had shown that the collagen layer in calipash tissues of Pelodiscus sinensis in P4+NS group was clearly delimited and the collagen fibers were orderly arranged,while that in P4+Ah group tended to be blurred and disordered,and the collagen fibers tended to be degraded internally.In conclusion,dietary supplementation of 40 g/kg proline can improve the growth performance of young Pelodiscus sinensis,and also play a role in resisting Ah stress by enhancing the activity of antioxidant enzymes,reducing ROS production and balancing collagen metabolism.