【作者】 邱仁杰；

【导师】 成永旭；

【作者基本信息】 上海海洋大学 ， 水产养殖， 2011， 博士

【摘要】 中华绒螯蟹(Eriocheir sinensis)又称毛蟹,河蟹,属甲壳纲,十足目,方蟹科,绒螯蟹属,为我国特产。主要分布于中国东部海域沿岸及通海的河流、湖泊中,现为我国主要养殖的经济蟹类。近几年河蟹的养殖面积不断地增加,其养殖产量2005年高达57万吨。池塘养殖是我国河蟹养殖最主要的养殖形式,在池塘养殖过程中,特别是高温季节,大量的残剩饲料以及生物的排泄物等有机废物无法被及时分解而不断沉积,加之许多养殖池塘由于多年的养殖疏于清淤,造成池塘沉积过多的淤泥,这大大增加了池塘底部有机耗氧,使池塘底质恶化,致使主要底栖习性的河蟹长期处于低氧的环境胁迫下(hypoxia stress)。中华绒螯蟹在低氧胁迫下,一方面可能造成其免疫机能的下降,抗病力降低,继而感染疾病而招致死亡。另一方面免疫能力的下降又会降低其抗低氧能力,两者相互作用的叠加,是最终造成高温季节河蟹大量死亡的重要因素之一。改善中华绒螯蟹的营养,研究营养与其抗环境胁迫能力的关系,是提高其免疫性能和抗低氧能力的一个主要途径。必需脂肪酸(EFA)如:花生四烯酸(ARA)、二十碳五烯酸(EPA)和二十二碳六烯酸(DHA)等在水产动物体内的生理和生化合成中起着各种各样的功能。ARA,EPA和DHA等脂肪酸是细胞膜的重要组成部分,能够影响细胞吞噬能力及呼吸爆发强度,还可以通过控制与免疫相关的酶活性而增强水生动物机体的免疫能力。同时ARA和EPA是类二十烷酸的前体,类二十烷酸是一族在各种应激反应中具有高度活性的分子,对水产动物的免疫及抗胁迫能力有重要的作用。但是,关于脂肪酸营养对中华绒螯蟹幼蟹免疫和耐低氧胁迫的研究从未有过报道,在这个方面还是空白。本论文的目的,首先研究低氧对中华绒螯蟹幼蟹免疫及生理的影响;然后改变饲料中高不饱和脂肪酸(ARA,DHA,DHA/EPA)的含量,测定脂肪酸营养对幼蟹免疫和耐低氧能力的影响;最后,对中华绒螯蟹成蟹投喂含三种不同鱼油、豆油的饲料,测定不同油脂对成蟹免疫及耐低氧能力的影响。取得成果具体如下:1、低氧对中华绒螯蟹(Eriocheir sinensis)幼蟹免疫及生理的影响中华绒螯蟹幼蟹(1.4±0.43g)放入溶解氧为2.04±0.16(低氧,处理组)和8.01±0.22 mg/L(正常溶氧,对照组)的水体中24h,测量了其在0和24h后的总血细胞密度(THC),透明血细胞密度。在试验和对照组中,对0,2,8和24h时血淋巴中超氧化物歧化酶活性(SOD),乳酸,血蓝蛋白的浓度以及肝胰腺的代谢(葡萄糖,总胆固醇,尿酸,总蛋白,尿素和甘油三酯的浓度)进行了测定分析。总血细胞和透明血细胞密度受低氧影响显著,在低氧24h后比起始浓度分别降低了66%和49%。SOD酶活性在低氧8h后比起始时显著升高,乳酸浓度在低氧2,8和24h后都显著升高。血蓝蛋白浓度呈现一个先降低后上升的趋势,24h时与初始差异不大。试验组中,肝胰腺的葡萄糖,胆固醇和尿酸含量在24h内发生了显著变化。总蛋白,尿素和甘油三酯的浓度在对照和试验组中0,2,8,24h之间均没有显著差异。由此可见,低氧对中华绒螯蟹幼蟹的影响是全面的。2、投喂不同DHA或ARA含量的饲料对中华绒螯蟹幼蟹(Eriocheir sinensis)免疫和耐低氧能力的影响用含不同浓度的必需脂肪酸(EFA)——DHA(C22:6n-3)或ARA(C20:4n-6)饲料分别投喂中华绒螯蟹幼蟹3个半月和4个月,然后测定了其低氧24h(DHA和ARA试验)和正常溶氧下(ARA试验)的免疫和生理性能。五种不同DHA和ARA饲料(D1,D2,D3,D4,D5和A1,A2,A3,A4,A5)中DHA和ARA的添加比例为0%,0.25%,0.5%,1%,1.5%和0%,0.3%,0.6%,0.9%,1.2%(占饲料干重比例)。在DHA饲料试验中幼蟹低氧24h后血淋巴中THC(总血细胞密度)和透明血细胞密度在五个饲料组之间没有显著性差异。在五个饲料组间,Hc(血蓝蛋白)的变化呈现先降低后上升再下降的趋势,饲料中DHA的添加对Hc的浓度有显著影响。低氧24h后在添加DHA的饲料组SOD酶的活性显著高于0%DHA添加组。在肝胰腺中,随着饲料中添加DHA含量的增加总胆固醇和甘油三酯含量逐渐上升。在ARA试验中,低氧胁迫对幼蟹的生理和免疫指标的影响明显。在正常溶氧下,饲料中ARA的添加使幼蟹血淋巴中THC和透明血细胞密度显著上升,特别是适量添加时更明显(0.6,0.9%)。然而,在正常和低氧下血淋巴中SOD活性和Hc的含量不受饲料中添加ARA量的影响,各个饲料组之间没有显著差异。在两个溶氧下饲料中适当的添加ARA(0.6,0.9%)对血淋巴中乳酸和尿酸的浓度影响显著。低氧胁迫后肝胰腺中的免疫和生理性能在各ARA饲料组间没有显著差异。总之,低氧对幼蟹的免疫生理代谢有明显的负面影响。饲料中DHA的添加对中华绒螯蟹幼蟹的营养和生理性能及抗低氧的能力有改善作用。ARA试验结果表明饲料中添加适量水平的ARA(0.6,0.9%)能显著改善中华绒螯蟹幼蟹免疫和生理代谢功能及抗低氧的能力。3、投喂不同DHA/EPA比例饲料对雌雄中华绒螯蟹幼蟹(Eriocheir sinensis)免疫和耐低氧能力的影响中华绒螯蟹幼蟹(1.32-1.76g)投喂不同DHA/EPA(DHA,C22:6n-3;EPA,C20:5n-3)比例的饲料3个月,然后将其放入溶解氧为2.34±0.30mg/L和8.02±0.35mg/L的水体中24h,测定其生理和免疫指标的变化。含不同DHA/EPA比例的饲料分为5组(简称:DE1,DE2,DE3,DE4,DE5),其中DHA/EPA的比例分别为:0.3,0.6,1,2和3。测定结果发现,饲料中血液的免疫指标受饲料中DHA/EPA比例变化的影响不大,但是低氧后血蓝蛋白含量及正常溶氧下γ-GGT酶活性受饲料中DHA/EPA比例的影响显著。抗氧化酶SOD在正常溶氧下随DHA/EPA的增加呈现降低的趋势,但是低氧后各组之间的SOD活性没有显著差异。血淋巴中生理代谢指标受DHA/EPA比例变化影响显著,特别是乳酸脱氢酶的活性。另外,低氧对河蟹幼蟹的生理免疫影响显著。在肝胰腺中,受低氧胁迫后,γ-GGT酶活性和葡萄糖含量在DHA/EPA比例为2时显著高于其他组,而乳酸的含量在DHA/EPA比例为1的组最高。幼蟹性别对河蟹免疫和生理代谢指标基本没有影响。总而言之,饲料中DHA/EPA比例为1和2时对中华绒螯蟹幼蟹的免疫和生理更有益,对其抗低氧胁迫也更有帮助。4、投喂添加三种不同油脂(鱼油、豆油、鱼油豆油混合(1:1))饲料对中华绒螯蟹(Eriocheir sinensis)免疫及耐低氧能力的影响为了研究饲料中添加鱼油,豆油对中华绒螯蟹成蟹体重,免疫,代谢和耐低氧的影响,配制了添加不同鱼油和豆油比例的三种蟹用饲料,分别投喂中华绒螯蟹成蟹115d,然后测量了它们体重的变化,再将其放入溶解氧为2.57±0.44和9.06±0.06mg/L的水体中90h,测量其免疫和代谢指标的变化。结果发现:投喂含三种不同油脂饲料的中华绒螯蟹各组之间体重没有显著性差异;低氧胁迫对河蟹代谢指标影响较大;添加鱼豆混合油组饲料对中华绒螯蟹免疫和抗氧化能力有促进作用,相较于其他两组,此组河蟹耐低氧的能力更强。以上研究结果说明,低氧显著影响了中华绒螯蟹幼蟹的免疫和生理代谢性能;对幼蟹投喂含DHA或ARA不同的饲料,发现饲料中DHA的添加对幼蟹免疫系统有益,而饲料中ARA的适量添加(0.6,0.9%)能增强幼蟹的免疫和生理性能;当饲料中DHA/EPA比例为1,2时,对幼蟹的免疫和生理性能更有益;最后,鱼油与豆油在饲料中1:1比例混合添加会使成蟹的免疫和耐低氧能力更好。更多还原

【Abstract】 Chinese mitten crab (Eriocheir sinensis) is called hair crab, river crab, is belong to crustacea class, decapoda order, square crab family, and mitten crab genus, is a specialty in China. It mainly lives the coast of Chinese eastern sea and the rivers, lakes connected with sea, now is a main aquaculture economic crabs species in China. Recent years, the annual yield of market-sized mitten crabs increased from 17 500mt in 1993 to 570 000mt in 2005. The pond aquaculture is the main form of farming crabs in China. In this kind aquaculture process, especially in hot weather, the organic waste such as abundant residual feed and excretion deposited at the bottom of the pond and could not be decomposed, meanwhile, the pond commonly did not clear up for several years. These all lead to the occurence of oxygen consumption at the bottom of the pond, the crabs lived in the benthic cave of pond were subjected to the hypoxia stress. At hypoxia condition, the immune abilities of crabs could reduce, and its resistance to disease would decrease, then they will die due to the infection. Another, the reduction of immune abilities would influence crabs’anti-hypoxia ability, the interaction of both factors led to massive death of mitten crabs in Chinese ponds.Improvement of crab nutrition, the research about the relationship of environmental stress tolerance and nutrition are an important way to enhancing the ability of immune and anti-hypoxia in crabs. Essential fatty acids (EFA) such as DHA, ARA and EPA et al. play a diverse role in the physiological and biochemical processes within aquatic animals. EFA is an important component in cell membrane, can influence the cellular phagocytosis and respiratory burst strength, and increase the aquatic animals’immune ability with regulating the enzymes relating to immune. In addition, the DHA and EPA is the precursors of eicosanoids, the eicosanoids is a kind of highly reactive molecules in a variety of stress response. But the reports about the relationship of nutrition and immune ability and resistance to hypoxia were not seen. The object of this study is to research the effect of hypoxia on the immunological and physiological ability in the juvenile Chinese mitten crab (Eriocheir sinensis); the effect of dietary DHA and ARA on immunological and physiological ability and response to hypoxia in juvenile Chinese mitten crab (Eriocheir sinensis); the effect of dietary different DHA/EPA ratio on immunological and physiological ability and response to hypoxia in juvenile Chinese mitten crab (Eriocheir sinensis); the effect of dietary different oil lipid on immunological and physiological ability and response to hypoxia in adult Chinese mitten crab (Eriocheir sinensis). Results obtains are as follow:1、The juvenile Chinese mitten crabs Eriocheir sinensis (1.40±0.43g) were cultured in water of 2.04±0.16 (hypoxia, treatment group) and 8.01±0.22mg/L (high dissolved oxygen (DO), control group) DO for 24h, respectively. The total haemocyte counts (THC), the hyaline haemocyte counts were measured at 0 and 24 h in the treatment group, and superoxide dismutase (SOD) activity, concentrations of lactic acid and hemocyanin (Hc) in haemolymph and the metabolism of hepatopancreas (concentrations of glucose, total cholesterol, uric acid, total protein, urea, triglyceride in hepatopancreas) were assayed at 0, 2, 8 and 24 h in both treatment and control groups. The hyaline haemocyte counts and THC decreased significantly by 66% and 49% after exposure to hypoxic water for 24 h, respectively. SOD activity, Hc and lactic acid content were significantly affected in treatment group, and there were significant differences between treatment and control groups. In the treatment group the concentration of glucose, cholesterol and uric acid in hepatopancreas had a significant variation for 24 h. The concentration of total protein, urea and triglyceride between treatment and control groups was not significantly different after 0, 2, 8, 24 h. However, there were significant differences between treatment and control groups in terms of total protein, glucose and uric acid concentration. Taken together, the effect of hypoxia is comprehensive in juvenile Chinese mitten crab Eriocheir sinensis.2、The juveniles of Chinese mitten crab (Eriocheir sinensis) were raised with diets including different contents of the essential fatty acids (EFA)――docosahexaenoic acid (DHA, C22:6n-3) or arachidonic acid (ARA, C20:4n-6) for about 3.5 and 4 months, and their immunological and physiological ability after acclimated to hypoxia 24 h (DHA and ARA diet) and normal DO (dissolved oxygen) level (ARA diet) were evaluated. Five different DHA and ARA dietary preparations (group D1, D2, D3, D4, D5 and A1, A2, A3, A4, A5) were compared: 0%, 0.25%, 0.5%, 1%, 1.5% and 0%, 0.3%, 0.6%, 0.9%, 1.2% (d.w.). In DHA diet experiment the THC (total haemocyte counts), the hyaline haemocyte counts had no significant difference in five dietary groups in hypoxia water for 24 h, the hemocyanin (Hc) appeared an interesting fluctuation trend, higher supplement of DHA in diets significantly influenced the Hc content. SOD activities in supplement of DHA groups were significantly higher than 0% supplement group exposed to hypoxia for 24h. In hepatopancreas total cholesterol and triglyceride content increased with the enrichment of DHA in diets. In ARA diets experiment the immunological and physiological parameters were considerable affected by hypoxia stress. The THC and the hyaline haemocyte counts were significantly increased by the supplementation of ARA, especially in moderate supplementation (0.6%, 0.9% d.w.) in normal DO water. However, there were no differences in both SOD activity and Hc content of haemolymph among dietary treatments in normal and low DO level respectively. The LD and UA content of haemolymph in moderate supplementation ARA (0.6%, 0.9% d.w.) were significantly different with the no supplementation ARA in both DO level. In hepatopancreas the immunological and physiological ability had no difference among the dietary groups in low DO water. Thus, the hypoxia had a considerable negative impact on the immunological and physiological parameters of crabs. Enriching DHA in diets has significant improved the nutrition, physiological ability and adaptation to hypoxia stress in juveniles of E. sinensis. And the results from ARA experiment suggested that dietary ARA, especially moderate ARA level (0.6%, 0.9% d.w.), significantly improved immune and physiological response and the anti-hypoxia ability of Chinese mitten crab juveniles.3、The juveniles of Chinese mitten crab (Eriocheir sinensis) (1.32-1.76g) were raised with diets with different DHA/EPA ratios (docosahexaenoic acid (DHA, 22:6n-6); eicosapentaenoic acid (EPA, 20:5n-3)) for 3 months, and their immunological and physiological variables after acclimated to hypoxia (2.34±0.30mg/L) 24 h and at normal DO (DO: dissolved oxygen; 8.02±0.35mg/L) water were measured. Five different DHA/EPA ratio dietary preparations (group 1, 2, 3, 4, 5) were compared: 0.3, 0.6, 1, 2, 3. We found the immunological ability in haemolymph was not significantly affected by the different DHA/EPA ratio diet except the Hc content after exposure to 24 h hypoxia and the activity ofγ-GGT (γ-glutamyltransferase) at normal DO level. The antioxidase activity SOD showed a decreasing trend from diet 1 to 5 at normal DO level but there were no significant difference in the five diets after exposure to 24 h hypoxia. In haemolymph, the physiological metabolism indices were evidently affected by the different dietary DHA/EPA ratio, especially the activity of LDH (lactate dehydrogenase) in diet 4 was significant higher than other diet groups’after exposure to 24 h hypoxia. Moreover, hypoxia significantly influenced the most immunological and physiological indices. In hepatopancreas, the activity ofγ-GGT and Glu (glucose) content in diet 4 were distinctly higher than other diet groups, and the content of LD (lactic acid) in diet 3 was the highest in the five diets after exposure 24h hypoxia. Thus, feeding diet 3 and 4 was better for the immune ability, transport oxygen and metabolism physiology of intermolt juveniles of E. sinensis after exposure to 24 h hypoxia.4、The objective of this study is to measure the effect of supplement of fish and soybean oil in diets on the weight, immune, metabolism and resistance to hypoxia in Chinese mitten crab (E.sinensis). The three diets for adult crabs with different ratio fish oil and soybean oil were prepared. These diets were fed crab for 115 days. Their weight changes were measured. And then the crabs were placed into hypoxia water (2.57±0.44mg/L) and normal DO level (9.06±0.06mg/L) for 90 hours,after that their immune and metabolic indices were measured. We found that the hypoxia stress influence the metabolism of crab in some ways. The weights of crab were not significantly affected by the variety of oil lipid in diets; the supplement of fish and soybean oil (1:1) in diets evidently improved the crabs’immune and antioxidant ability, comparing with other two groups, the crabs in mix oil group had a better ability of resistance to hypoxia.The above results demonstrated that hypoxia significantly influenced the immunity and physiological ability in the juvenile of Chinese mitten crab; the supplement of DHA in diets improved the immunity system of juvenile crabs, the proper supplement of ARA (0.6, 0.9%) in diets would enhance the immunity and physiological ability of juvenile crabs; when the DHA/EPA in diets is 1 and 2, the juvenile crabs’immunity and resistance to hypoxia is better; and last, the supplement of fish oil and soy oil (1:1) in diets is better for the immunity and resistance to hypoxia in adult crabs.更多还原