四种不同食性的温水鱼类应激反应及其对糖代谢的影响
发布时间：2018-08-12 21:02
【摘要】：以四种不同食性的温水性鱼类草鱼Ctenopharyngodonidella、银鲫Carassius auratus gibelio、青鱼 Mylopharyngodonpiceus 和大 口 黑鲈 Micropterus salmoides为研究对象,分析了捕捞应激后鱼类下丘脑-垂体-肾间组织轴(Hypothalamic-pituitary-interrenal axis,HPI axis)、血浆和肝脏生理指标以及肝脏糖代谢酶活性的变化,并探讨了鱼类种类及食性、血浆葡萄糖水平和营养史(饥饿或正常摄食)对应激反应的影响。主要研究结果和结论如下:1、捕捞应激对草鱼HPI轴的影响:采用同源克隆方法获得草鱼HPI轴关键基因,包括促肾上腺皮质激素释放因子(Corticotropin releasing factor,CRF)、急性固醇调节蛋白(Steroidogenic acute regulatory protein,StAR)、11β-羟化酶(11β-hydroxylase,CYP11C1)和糖皮质激素受体(Glucocorticoidreceptor,GR)部分cDNAs序列:CRF部分片段长209 bp,编码69个氨基酸;StAR部分片段长314bp,编码104个氨基酸;CYP11C1部分片段长347bp,编码115个氨基酸;GR部分片段长259 bp,编码85个氨基酸。草鱼CRF、StAR、CYP11C1和GR与其它已知物种对应氨基酸序列的同源性分别为69%-99%、73%-86%、73%-89%和86%-98%。检测草鱼捕捞应激后1 h时下丘脑CRF、肾脏StAR和CYP11C1、脑垂体阿黑皮素原(Proopiomelanocortin,POMC)和肝脏 GRmRNA水平。结果显示应激后1 h时草鱼下丘脑CRF、肾脏StAR和肝脏GR mRNA表达水平较应激前显著升高(P0.05),而肾脏CYP11C1和脑垂体POMC mRNA表达量与应激前相比无显著差异(P0.05)。2、捕捞应激后草鱼血浆生理指标、肝糖原含量及肝脏糖代谢酶活性的变化:测定草鱼在应激后5min、30min、1h、4h和24h血浆皮质醇、葡萄糖和乳酸水平、肝脏糖原含量以及肝脏磷酸烯醇式丙酮酸羧激酶(Phosphoenolpyruvate carboxykinase,PEPCK)、葡萄糖-6-磷酸酶(Glucose-6-phosphatase,G6Pase)、已糖激酶(Hexoxinase,HK)和丙酮酸激酶(Pyruvate kinase,PK)活性。结果显示:与应激前Oh(对照组)相比,血浆皮质醇水平在应激后5 min显著升高(P0.05),在1 h达到最高值(P0.05)。血浆葡萄糖和乳酸水平在应激后30 min显著升高(P0.05),在1 h升至最高(P0.05)。应激后1 h肝脏糖原含量显著降低(P0.05),在24 h恢复至应激前的水平(P0.05)。应激后1 h肝脏PEPCK活性显著高于对照组(P0.05);应激后30 min肝脏G6Pase活性显著高于对照组(P0.05)。3.1、捕捞应激对草鱼(草食性鱼类)、银鲫(杂食性鱼类)和青鱼(肉食性鱼类)血浆皮质醇、葡萄糖和乳酸浓度、肝糖原含量以及肝脏HK和PK活性的影响:结果显示草鱼、银鲫和青鱼捕捞后血浆皮质醇、葡萄糖和乳酸浓度均显著升高(P0.05);草鱼和青鱼捕捞后2h时肝糖原含量呈下降趋势(P0.05),但银鲫捕捞前、后肝糖原含量未出现显著变化(P0.05);捕捞前、后青鱼血糖浓度显著高于草鱼和银鲫(P0.05)。银鲫肝糖原含量显著高于草鱼和青鱼(P0.05),其捕捞后血浆葡萄糖和乳酸浓度增加幅度较小。草鱼和银鲫捕捞后肝脏HK和PK酶活性未发生显著变化(P0.05),青鱼捕捞后2h时HK酶活性显著下降(P0.05)。3.2、血浆葡萄糖水平对草鱼血浆和脑生理指标及肝脏糖代谢应激反应的影响:对草鱼按浓度0、0.2、0.5和1.0 mg/g体重(Body mass,BM)分别腹腔注射葡萄糖溶液(命名为0G、0.2G、0.5G和1G组)。注射后1h进行捕捞应激(命名为0G-S、0.2G-S、0.5G-S和1G-S组)或不捕捞(命名为0G-NS、0.2G-NS、0.5G-NS和1G-NS组),检测草鱼应激前0 h,应激后1 h、2 h和4 h血浆生理指标[皮质醇、葡萄糖和乳酸浓度、乳酸脱氢酶(Lactic dehydrogenase,LDH)活性],脑生理指标(葡萄糖、糖原和乳酸浓度)以及肝脏糖代谢指标(糖原含量和PEPCK、G6Pase、HK和PK酶活性)的变化。结果显示,1)血浆生理:在应激前,1G-S组血浆皮质醇浓度显著高于OG-S、0.2G-S和0.5G-S组,且1G-NS组血浆皮质醇浓度显著高于OG-NS、0.2G-NS和0.5G-NS组(P0.05)。在应激后1h,0.5G-S组血浆皮质醇浓度显著低于其它三个应激组(P0.05)。在应激前和应激后1 h,应激组和不应激组血浆葡萄糖浓度都随着注射葡萄糖浓度的增加而升高(P0.05)。在应激前、应激后1h、2h和4h,1G-S组血浆葡萄糖浓度显著高于其它三个应激组(P0.05)。在应激后4h,血浆乳酸浓度和LDH酶活性都随着注射葡萄糖浓度的增加而升高(P0.05)。2)脑生理:在应激前,0.5G-NS和1G-NS组脑葡萄糖浓度显著高于OG-NS和0.2G-NS组,且0.5G-S和1G-S组脑葡萄糖浓度显著高于OG-S和0.2G-S组(P0.05)。在应激后1 h和2 h,1G-S组脑葡萄糖浓度显著高于其它三个应激组(P0.05)。0.5G-S组脑糖原含量在应激后1 h显著高于0G-S和0.2G-S组(P0.05)。在应激后2h,0.5G-S组脑乳酸浓度显著高于0.5G-NS组(P0.05)。3)肝脏糖代谢:在应激后2h和4h,1G-S组肝糖原含量显著低于其对应不应激组(P0.05)。在应激后1h,0G-S组肝脏G6Pase酶活性显著高于其它应激组(P0.05)。在应激后2h,0G-S组肝脏G6Pase酶活性显著高于1G-S组(P0.05)。0G-S组肝脏G6Pase酶活性在应激后1 h和2 h显著高于其对应不应激组(P0.05)。0.2G-S和1G-S组肝脏HK酶活性在应激后1 h显著高于其对应不应激组(P0.05)。3.3、不同营养史对银鲫和大口黑鲈应激反应及应激后肝脏糖代谢酶活性的影响:实验2×2设计,共设置银鲫饱食组、银鲫饥饿组、大口黑鲈饱食组和大口黑鲈饥饿组。在饱食或饥饿28天后,检测肝重指数(Hepatosomaticindex,HSI)及体成分。在捕捞应激后,检测0h(应激前)、1h、2h、4h和24h血浆生理指标(皮质醇、葡萄糖和乳酸浓度),脑生理指标(葡萄糖、糖原和乳酸浓度)以及肝脏糖代谢指标(糖原含量和PEPCK、G6Pase、HK和PK活性)的变化。结果显示,1)HSI和体成分:与初始组相比,银鲫在饥饿28天后粗脂肪下降61%(P0.05),灰分升高34%(P0.05);大口黑鲈在饥饿后HSI与初始组相比下降77%(P0.05)。2)血浆生理:在捕捞应激后,银鲫仅饱食组血浆皮质醇浓度在应激后2 h、4h和24 h显著升高(P0.05)。大口黑鲈饱食组血浆皮质醇浓度在应激后4 h和24 h显著升高,而饥饿组血浆皮质醇浓度在应激后lh显著升高(P0.05)。总体来说(0h-24h),银鲫饱食组血浆皮质醇浓度是饥饿组的3.5倍,而大口黑鲈饱食组血浆皮质醇浓度是饥饿组的1.2倍。银鲫饱食组和饥饿组血浆葡萄糖浓度分别在应激后2h和lh显著升高(P0.05)。而大口黑鲈仅饱食组血浆葡萄糖浓度在应激后1h、2h和4h显著升高(P0.05)。总体来说,大口黑鲈饱食组葡萄糖浓度是饥饿组的2.1倍,而银鲫饱食组葡萄糖浓度是饥饿组的1.4倍。银鲫和大口黑鲈血浆乳酸浓度在应激后都显著升高(P0.05),且饱食组和饥饿组之间没有显著差异(P0.05)。3)脑生理:银鲫饱食组和饥饿组脑葡萄糖浓度在应激后2 h都显著升高(P0.05)。大口黑鲈仅饱食组脑葡萄糖浓度在应激后lh、2h和4h显著升高(P0.05)。总体来说,大口黑鲈饱食组脑葡萄糖浓度是饥饿组的6.0倍,而银鲫饱食组脑葡萄糖浓度是饥饿组的1.2倍。银鲫和大口黑鲈脑糖原含量并没有受到捕捞应激的显著影响(P0.05)。总体来说,银鲫饱食组脑糖原含量是饥饿组的2.0倍,而大口黑鲈饱食组脑糖原含量是饥饿组的1.5倍。4)肝脏糖代谢:银鲫和大口黑鲈肝糖原含量在应激后并没有发生显著变化(P0.05)。总体来说,大口黑鲈饱食组肝糖原含量是饥饿组的5.0倍,而银鲫饱食组肝糖原含量是饥饿组的1.3倍。在捕捞应激后,仅银鲫饱食组肝脏PEPCK酶活性在应激后2h和24h显著升高(P0.05)。总体来说,银鲫饱食组肝脏PEPCK酶活性是饥饿组的1.4倍,而加州饥饿组肝脏PEPCK酶活性是饱食组的2.1倍。在应激后4h,银鲫饱食组肝脏G6Pase酶活性显著高于饥饿组(P0.05)。在应激前、应激后2h、4h和24h,大口黑鲈饥饿组肝脏HK酶活性显著高于饱食组。3.4、不同营养史对银鲫应激反应及应激后肝脏糖代谢酶活性的影响:银鲫设饱食组、饥饿14天组和饥饿28天组,对其进行捕捞应激,检测应激前0h、应激后1h、2h、4h和24h血浆生理指标(皮质醇、葡萄糖和乳酸浓度),脑生理指标(葡萄糖、糖原和乳酸浓度)以及肝脏糖代谢指标(糖原含量和PEPCK、G6Pase、HK和PK活性)的变化。结果显示,1)血浆生理:银鲫仅饱食组血浆皮质醇浓度在应激后1h、2h、4h和24h显著高于应激前水平(P0.05)。在应激后2 h、4h和24h,饱食组血浆皮质醇浓度显著高于饥饿14天和28天组(P0.05)。银鲫所有处理组血浆葡萄糖和乳酸浓度在应激后1 h和2h都显著升高(P0.05)。2)脑生理:银鲫饱食组和饥饿28天组脑葡萄糖浓度在应激后2h显著升高(P0.05)。在应激后2h,饱食组和饥饿28天组脑葡萄糖浓度显著高于饥饿14天组(P0.05)。应激前和应激后1h、2 h、4h和24 h,银鲫饥饿28天组脑糖原含量显著低于饱食组(P0.05)。3)肝脏糖代谢：饥饿14天组肝糖原含量在应激后2 h显著高于饥饿28天组(P0.05)。饱食组肝脏PEPCK酶活性在应激后2h、4h和24h显著升高(P0.05)。根据上述结果得出以下结论:1、捕捞应激后草鱼HPI轴中CRF、StAR和GR途径被激活,肝脏糖原分解和糖异生活动加强。2、与草鱼和青鱼相比,捕捞后银鲫应激反应强度相对较低。捕捞后血糖升高未导致草鱼、银鲫和青鱼的肝脏糖酵解酶活性增强。3、腹腔注射1.0 mg/gBM的葡萄糖可以在短时间内刺激草鱼血浆皮质醇浓度升高,而注射0.5 mg/g BM的葡萄糖会抑制应激后血浆皮质醇浓度的升高。应激和血浆葡萄糖浓度都会促使草鱼脑葡萄糖浓度的升高。外源葡萄糖处理抑制了应激后的肝脏生糖作用,表现为应激后早期血糖浓度升高趋势慢,乳酸堆积加剧以及肝脏G6Pase酶活性升高受到抑制。4、营养史影响银鲫和大口黑鲈血浆生理、脑生理和肝脏糖代谢的应激反应。饥饿抑制银鲫应激后血浆皮质醇和血浆葡萄糖浓度的升高,抑制大口黑鲈应激后血浆葡萄糖和脑葡萄糖浓度的升高,表明饥饿在一定程度上降低应激反应。饥饿对银鲫应激后肝脏糖异生途径产生抑制作用,而在大口黑鲈中则作用相反。5、无论短期还是长期饥饿均会降低银鲫应激后血浆皮质醇和葡萄糖浓度。短期和长期饥饿后银鲫脑糖原含量显著降低,表明饥饿导致其脑糖原分解。短期和长期饥饿都会抑制银鲫应激后肝脏糖异生能力的增强。
[Abstract]:The hypothalamic-pituitary-interrenal axis (HPI axis) of four warm-water fishes (Ctenopharyngodonidella, Carassius auratus gibelio, Mylopharyngodon piceus and Micropterus salmoides) with different feeding habits were analyzed. The main results and conclusions are as follows: 1. The effects of fishing stress on the HPI axis of grass carp were obtained by homologous cloning method. Bond genes, including Corticotropin releasing factor (CRF), Steroidogenic acute regulatory protein (StAR), 11beta-hydroxylase (CYP111C1) and glucocorticoid receptor (GR), encode 209 BP of the CRF fragment. 69 amino acids; part of StAR was 314 BP long, encoding 104 amino acids; part of CYP111C1 was 347 BP long, encoding 115 amino acids; part of GR was 259 BP long, encoding 85 amino acids. The levels of CRF, StAR, CYP111C1, POMC and GR mRNA in hypothalamus, kidney, pituitary and liver at 1 h after stress were significantly higher than those before stress (P 0.05). There was no significant difference (P 0.05). 2. Changes of plasma physiological indexes, hepatic glycogen content and hepatic glycometabolism enzyme activity in grass carp after fishing stress: plasma cortisol, glucose and lactic acid levels, liver glycogen content and liver phosphoenolpyruvate carboxykinase (Phosphoelpyruvate carboxyk) were measured at 5 min, 30 min, 1 h, 4 h and 24 h after fishing stress. Inase, PEPCK, Glucose-6-phosphatase (G6Pase), Hexoxinase (HK) and Pyruvate kinase (PK) activities. The results showed that plasma cortisol levels increased significantly at 5 minutes after stress (P 0.05) and reached the highest level at 1 hour (P 0.05) compared with pre-stress oh (control group). Liver glycogen content decreased significantly at 1 hour after stress (P 0.05), and returned to pre-stress level at 24 hours (P 0.05). Liver PEPCK activity at 1 hour after stress was significantly higher than that of control group (P 0.05); liver G6Pase activity at 30 minutes after stress was significantly higher than that of control group (P 0.05). The plasma cortisol, glucose and lactate concentrations, hepatic glycogen contents, and liver HK and PK activities of grass carp, silver crucian carp and black carp were significantly increased after harvesting (P 0.05). The glycogen content of liver showed a downward trend (P 0.05), but there was no significant change in glycogen content of liver before and after harvesting (P 0.05); before and after harvesting, the glycogen concentration of black carp was significantly higher than that of grass carp and silver crucian carp (P 0.05). The glycogen content of liver of silver crucian carp was significantly higher than that of grass carp and blue carp (P 0.05), and the plasma glucose and lactic acid concentration increased slightly after harvesting. The activity of HK and PK in liver did not change significantly after harvesting (P 0.05). The activity of HK decreased significantly at 2 hours after harvesting (P 0.05). The effects of plasma glucose levels on plasma and brain physiological indices and liver glucose metabolism stress of grass carp were studied. Grass carp were intraperitoneally injected with glucose at concentrations of 0,0.2,0.5 and 1.0 mg/g body mass (BM), respectively. The plasma physiological indices (cortisol, glucose and lactate concentration, lactate dehydrogenase (Lactic hydrogenase, LD) and lactate dehydrogenase (Lactic dehydrogenase, LD-NS) of grass carp were measured at 1 hour after injection under fishing stress (named 0G-S, 0.2G-S, 0.5G-S and 1G-S groups) or non-fishing (named 0G-NS, 0.2G-NS, 0.5G-NS and 1G-NS groups). The results showed that: (1) Plasma physiology: before stress, the plasma cortisol concentration in 1G-S group was significantly higher than that in OG-S, 0.2G-S and 0.5G-S groups, and the plasma cortisol concentration in 1G-NS group was significantly higher than that in OG-NS, 0.2 G-S group. G-NS and 0.5G-NS groups (P 0.05). The plasma cortisol concentration in 0.5G-S group was significantly lower than that in the other three stress groups at 1 hour after stress (P 0.05). The plasma glucose concentration in stress group and stress group increased with the increase of glucose concentration at 1 hour, 2 hours and 4 hours after stress (P 0.05). After 4 hours of stress, plasma lactate concentration and LDH activity increased with the increase of glucose concentration (P 0.05). 2) Cerebral physiology: Before stress, the concentration of glucose in 0.5G-NS and 1G-NS groups was significantly higher than that in OG-NS and 0.2G-NS groups, and the concentration of glucose in 0.5G-S and 1G-S groups was significantly higher than that in OG-S and 0.2 G-NS groups. Cerebral glucose concentration in 1G-S group was significantly higher than that in other three stress groups at 1 h and 2 h after stress (P 0.05). Cerebral glycogen content in 0.5G-S group was significantly higher than that in 0G-S and 0.2G-S groups at 1 h after stress (P 0.05). Cerebral lactate concentration in 0.5G-S group was significantly higher than that in 0.5G-NS group (P 0.05). 3 Hepatic glucose metabolism in 1G-S group was significantly higher than that in 0G-S and 0.5G-S group at 2 h and 4 h after stress (P 0.05). The activity of G6Pase in liver of 0G-S group was significantly higher than that of other stress groups (P 0.05). The activity of G6Pase in liver of 0G-S group was significantly higher than that of 1G-S group at 2 hours after stress (P 0.05). The activity of G6Pase in liver of 0G-S group was significantly higher than that of 1G-S group at 1 hour and 2 hours after stress (P 0.05). The activity of HK enzyme in liver of group-S was significantly higher than that of stress stress group (P 0.05). 3.3. The effects of different nutritional history on stress response and activity of glucose metabolic enzyme in liver of silver crucian carp and perch were studied in Experiment 2 *2 design. A total of three groups were set up, i.e. the satiety group of silver crucian carp, the starvation group of silver crucian carp, the satiety group of big mouth black bass and the starvation group of big mouth black bass. After 28 days of starvation, liver weight index (HSI) and body composition were measured. Changes of plasma physiological indexes (cortisol, glucose and lactic acid concentration), brain physiological indexes (glucose, glycogen and lactic acid concentration) and liver glucose metabolic indexes (glycogen content and PEPCK, G6e, HK and PK activity) were detected at 0 h (before stress), 1 h, 2 h, 4 h and 24 h after fishing stress. The results showed that: 1) HSI and body composition: compared with the initial group, crude fat decreased by 61% (P 0.05) and ash increased by 34% (P 0.05) after 28 days of starvation; HSI decreased by 77% (P 0.05). 2) plasma physiology: after fishing stress, the plasma cortisol concentration of the only full-fed group increased significantly at 2, 4 and 24 hours after stress (P 0.05). The plasma cortisol concentration in the satiety group increased significantly at 4 h and 24 h after stress, while that in the starvation group increased significantly at 1 h after stress (P 0.05). Overall (0 h-24 h), the plasma cortisol concentration in the satiety group was 3.5 times higher than that in the starvation group, while that in the satiety group was 1.2 times higher than that in the starvation group. The plasma glucose concentration of satiety group and starvation group increased significantly at 2 h and 1 h after stress (P 0.05), while that of satiety group increased significantly at 1 h, 2 h and 4 h after stress (P 0.05). The concentration of lactic acid in the plasma of silver crucian carp and sea bass increased significantly after stress (P 0.05), and there was no significant difference between the satiety group and the starvation group (P 0.05). In general, the brain glucose concentration in the satiety group was 6.0 times higher than that in the starvation group, while the brain glucose concentration in the satiety group was 1.2 times higher than that in the starvation group. Glycogen content in brain of satiety group was 1.5 times as much as that of starvation group. 4) Liver glycogen metabolism: liver glycogen content of silver crucian carp and big black bass did not change significantly after stress (P 0.05). Overall, liver glycogen content of satiety group was 5.0 times as much as that of starvation group, while liver glycogen content of satiety group of silver crucian carp was 1.3 times as that of starvation group. After fishing stress, the activity of PEPCK in the liver of the satiated crucian carp group increased significantly at 2 h and 24 h after stress (P 0.05). Overall, the activity of PEPCK in the satiated crucian carp group was 1.4 times higher than that of the starved crucian carp group, while the activity of PEPCK in the starved California group was 2.1 times higher than that of the satiated crucian carp group (P 0.05). (3.4) The activity of HK enzyme in liver of starvation group was significantly higher than that of satiety group 2 h, 4 h and 24 h before and after stress. The effects of different nutritional history on stress response and activity of glycometabolism enzyme in liver of Gilted crucian carp were studied: the satiety group, the starvation group for 14 days and the starvation group for 28 days. Changes of plasma physiological parameters (cortisol, glucose and lactic acid concentration), brain physiological indexes (glucose, glycogen and lactic acid concentration) and liver glycometabolism indexes (glycogen content and PECK, G6Pase, HK and PK activity) were observed. The results showed that 1) plasma physiology: the plasma cortisol concentration in the full-fed group was significantly higher than that before stress at 1, 2, 4 and 24 hours after stress. The plasma cortisol concentration in the satiety group was significantly higher than that in the starvation group at 2 h, 4 h and 24 h after stress (P 0.05). (P 0.05). Glucogen concentration in brain was significantly higher in starvation group than in starvation group (P 0.05). Glucogen content in brain was significantly lower in starvation group (P 0.05). Glucogen content in liver was significantly higher in starvation group (P 0.05) than in starvation group (P 0.05). Glucogen content in starvation group (P 0.05). The activities of PEPCK in the liver of the predator group were significantly increased at 2, 4 and 24 hours after stress (P 0.05). According to the above results, the following conclusions were drawn: 1. CRF, StAR and GR pathways were activated in the HPI axis of grass carp after fishing stress, and liver glycogen decomposition and gluconeogenesis activity were enhanced. 2. Compared with grass carp and black carp, the stress response intensity of silver crucian carp after fishing was relatively low. Glucolytic enzymes activities in liver of grass carp, crucian carp and herring were not increased. 3. Intraperitoneal injection of 1.0 mg/gBM of glucose could stimulate the plasma cortisol concentration of grass carp in a short time, but 0.5 mg/gBM of glucose could inhibit the increase of plasma cortisol concentration after stress. Exogenous glucose treatment inhibited the glycogenesis of the liver after stress, which was manifested by the slow increase of blood glucose concentration, the aggravation of lactic acid accumulation and the inhibition of G6Pase activity in the liver. 4. Nutritional history affected the plasma physiology, brain physiology and liver glucose metabolism of silver carp and black bass. Starvation inhibited the elevation of plasma cortisol and glucose levels after stress, and inhibited the elevation of plasma glucose and brain glucose levels after stress, suggesting that starvation reduced stress response to a certain extent. Starvation inhibited the hepatic gluconeogenesis pathway after stress in Carassius auratus, whereas in Perch macrocephalus, the effect was opposite. 5. Short-term and long-term starvation decreased plasma cortisol and glucose levels after stress. Short-term and long-term starvation
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S917.4
本文编号：2180359
[Abstract]:The hypothalamic-pituitary-interrenal axis (HPI axis) of four warm-water fishes (Ctenopharyngodonidella, Carassius auratus gibelio, Mylopharyngodon piceus and Micropterus salmoides) with different feeding habits were analyzed. The main results and conclusions are as follows: 1. The effects of fishing stress on the HPI axis of grass carp were obtained by homologous cloning method. Bond genes, including Corticotropin releasing factor (CRF), Steroidogenic acute regulatory protein (StAR), 11beta-hydroxylase (CYP111C1) and glucocorticoid receptor (GR), encode 209 BP of the CRF fragment. 69 amino acids; part of StAR was 314 BP long, encoding 104 amino acids; part of CYP111C1 was 347 BP long, encoding 115 amino acids; part of GR was 259 BP long, encoding 85 amino acids. The levels of CRF, StAR, CYP111C1, POMC and GR mRNA in hypothalamus, kidney, pituitary and liver at 1 h after stress were significantly higher than those before stress (P 0.05). There was no significant difference (P 0.05). 2. Changes of plasma physiological indexes, hepatic glycogen content and hepatic glycometabolism enzyme activity in grass carp after fishing stress: plasma cortisol, glucose and lactic acid levels, liver glycogen content and liver phosphoenolpyruvate carboxykinase (Phosphoelpyruvate carboxyk) were measured at 5 min, 30 min, 1 h, 4 h and 24 h after fishing stress. Inase, PEPCK, Glucose-6-phosphatase (G6Pase), Hexoxinase (HK) and Pyruvate kinase (PK) activities. The results showed that plasma cortisol levels increased significantly at 5 minutes after stress (P 0.05) and reached the highest level at 1 hour (P 0.05) compared with pre-stress oh (control group). Liver glycogen content decreased significantly at 1 hour after stress (P 0.05), and returned to pre-stress level at 24 hours (P 0.05). Liver PEPCK activity at 1 hour after stress was significantly higher than that of control group (P 0.05); liver G6Pase activity at 30 minutes after stress was significantly higher than that of control group (P 0.05). The plasma cortisol, glucose and lactate concentrations, hepatic glycogen contents, and liver HK and PK activities of grass carp, silver crucian carp and black carp were significantly increased after harvesting (P 0.05). The glycogen content of liver showed a downward trend (P 0.05), but there was no significant change in glycogen content of liver before and after harvesting (P 0.05); before and after harvesting, the glycogen concentration of black carp was significantly higher than that of grass carp and silver crucian carp (P 0.05). The glycogen content of liver of silver crucian carp was significantly higher than that of grass carp and blue carp (P 0.05), and the plasma glucose and lactic acid concentration increased slightly after harvesting. The activity of HK and PK in liver did not change significantly after harvesting (P 0.05). The activity of HK decreased significantly at 2 hours after harvesting (P 0.05). The effects of plasma glucose levels on plasma and brain physiological indices and liver glucose metabolism stress of grass carp were studied. Grass carp were intraperitoneally injected with glucose at concentrations of 0,0.2,0.5 and 1.0 mg/g body mass (BM), respectively. The plasma physiological indices (cortisol, glucose and lactate concentration, lactate dehydrogenase (Lactic hydrogenase, LD) and lactate dehydrogenase (Lactic dehydrogenase, LD-NS) of grass carp were measured at 1 hour after injection under fishing stress (named 0G-S, 0.2G-S, 0.5G-S and 1G-S groups) or non-fishing (named 0G-NS, 0.2G-NS, 0.5G-NS and 1G-NS groups). The results showed that: (1) Plasma physiology: before stress, the plasma cortisol concentration in 1G-S group was significantly higher than that in OG-S, 0.2G-S and 0.5G-S groups, and the plasma cortisol concentration in 1G-NS group was significantly higher than that in OG-NS, 0.2 G-S group. G-NS and 0.5G-NS groups (P 0.05). The plasma cortisol concentration in 0.5G-S group was significantly lower than that in the other three stress groups at 1 hour after stress (P 0.05). The plasma glucose concentration in stress group and stress group increased with the increase of glucose concentration at 1 hour, 2 hours and 4 hours after stress (P 0.05). After 4 hours of stress, plasma lactate concentration and LDH activity increased with the increase of glucose concentration (P 0.05). 2) Cerebral physiology: Before stress, the concentration of glucose in 0.5G-NS and 1G-NS groups was significantly higher than that in OG-NS and 0.2G-NS groups, and the concentration of glucose in 0.5G-S and 1G-S groups was significantly higher than that in OG-S and 0.2 G-NS groups. Cerebral glucose concentration in 1G-S group was significantly higher than that in other three stress groups at 1 h and 2 h after stress (P 0.05). Cerebral glycogen content in 0.5G-S group was significantly higher than that in 0G-S and 0.2G-S groups at 1 h after stress (P 0.05). Cerebral lactate concentration in 0.5G-S group was significantly higher than that in 0.5G-NS group (P 0.05). 3 Hepatic glucose metabolism in 1G-S group was significantly higher than that in 0G-S and 0.5G-S group at 2 h and 4 h after stress (P 0.05). The activity of G6Pase in liver of 0G-S group was significantly higher than that of other stress groups (P 0.05). The activity of G6Pase in liver of 0G-S group was significantly higher than that of 1G-S group at 2 hours after stress (P 0.05). The activity of G6Pase in liver of 0G-S group was significantly higher than that of 1G-S group at 1 hour and 2 hours after stress (P 0.05). The activity of HK enzyme in liver of group-S was significantly higher than that of stress stress group (P 0.05). 3.3. The effects of different nutritional history on stress response and activity of glucose metabolic enzyme in liver of silver crucian carp and perch were studied in Experiment 2 *2 design. A total of three groups were set up, i.e. the satiety group of silver crucian carp, the starvation group of silver crucian carp, the satiety group of big mouth black bass and the starvation group of big mouth black bass. After 28 days of starvation, liver weight index (HSI) and body composition were measured. Changes of plasma physiological indexes (cortisol, glucose and lactic acid concentration), brain physiological indexes (glucose, glycogen and lactic acid concentration) and liver glucose metabolic indexes (glycogen content and PEPCK, G6e, HK and PK activity) were detected at 0 h (before stress), 1 h, 2 h, 4 h and 24 h after fishing stress. The results showed that: 1) HSI and body composition: compared with the initial group, crude fat decreased by 61% (P 0.05) and ash increased by 34% (P 0.05) after 28 days of starvation; HSI decreased by 77% (P 0.05). 2) plasma physiology: after fishing stress, the plasma cortisol concentration of the only full-fed group increased significantly at 2, 4 and 24 hours after stress (P 0.05). The plasma cortisol concentration in the satiety group increased significantly at 4 h and 24 h after stress, while that in the starvation group increased significantly at 1 h after stress (P 0.05). Overall (0 h-24 h), the plasma cortisol concentration in the satiety group was 3.5 times higher than that in the starvation group, while that in the satiety group was 1.2 times higher than that in the starvation group. The plasma glucose concentration of satiety group and starvation group increased significantly at 2 h and 1 h after stress (P 0.05), while that of satiety group increased significantly at 1 h, 2 h and 4 h after stress (P 0.05). The concentration of lactic acid in the plasma of silver crucian carp and sea bass increased significantly after stress (P 0.05), and there was no significant difference between the satiety group and the starvation group (P 0.05). In general, the brain glucose concentration in the satiety group was 6.0 times higher than that in the starvation group, while the brain glucose concentration in the satiety group was 1.2 times higher than that in the starvation group. Glycogen content in brain of satiety group was 1.5 times as much as that of starvation group. 4) Liver glycogen metabolism: liver glycogen content of silver crucian carp and big black bass did not change significantly after stress (P 0.05). Overall, liver glycogen content of satiety group was 5.0 times as much as that of starvation group, while liver glycogen content of satiety group of silver crucian carp was 1.3 times as that of starvation group. After fishing stress, the activity of PEPCK in the liver of the satiated crucian carp group increased significantly at 2 h and 24 h after stress (P 0.05). Overall, the activity of PEPCK in the satiated crucian carp group was 1.4 times higher than that of the starved crucian carp group, while the activity of PEPCK in the starved California group was 2.1 times higher than that of the satiated crucian carp group (P 0.05). (3.4) The activity of HK enzyme in liver of starvation group was significantly higher than that of satiety group 2 h, 4 h and 24 h before and after stress. The effects of different nutritional history on stress response and activity of glycometabolism enzyme in liver of Gilted crucian carp were studied: the satiety group, the starvation group for 14 days and the starvation group for 28 days. Changes of plasma physiological parameters (cortisol, glucose and lactic acid concentration), brain physiological indexes (glucose, glycogen and lactic acid concentration) and liver glycometabolism indexes (glycogen content and PECK, G6Pase, HK and PK activity) were observed. The results showed that 1) plasma physiology: the plasma cortisol concentration in the full-fed group was significantly higher than that before stress at 1, 2, 4 and 24 hours after stress. The plasma cortisol concentration in the satiety group was significantly higher than that in the starvation group at 2 h, 4 h and 24 h after stress (P 0.05). (P 0.05). Glucogen concentration in brain was significantly higher in starvation group than in starvation group (P 0.05). Glucogen content in brain was significantly lower in starvation group (P 0.05). Glucogen content in liver was significantly higher in starvation group (P 0.05) than in starvation group (P 0.05). Glucogen content in starvation group (P 0.05). The activities of PEPCK in the liver of the predator group were significantly increased at 2, 4 and 24 hours after stress (P 0.05). According to the above results, the following conclusions were drawn: 1. CRF, StAR and GR pathways were activated in the HPI axis of grass carp after fishing stress, and liver glycogen decomposition and gluconeogenesis activity were enhanced. 2. Compared with grass carp and black carp, the stress response intensity of silver crucian carp after fishing was relatively low. Glucolytic enzymes activities in liver of grass carp, crucian carp and herring were not increased. 3. Intraperitoneal injection of 1.0 mg/gBM of glucose could stimulate the plasma cortisol concentration of grass carp in a short time, but 0.5 mg/gBM of glucose could inhibit the increase of plasma cortisol concentration after stress. Exogenous glucose treatment inhibited the glycogenesis of the liver after stress, which was manifested by the slow increase of blood glucose concentration, the aggravation of lactic acid accumulation and the inhibition of G6Pase activity in the liver. 4. Nutritional history affected the plasma physiology, brain physiology and liver glucose metabolism of silver carp and black bass. Starvation inhibited the elevation of plasma cortisol and glucose levels after stress, and inhibited the elevation of plasma glucose and brain glucose levels after stress, suggesting that starvation reduced stress response to a certain extent. Starvation inhibited the hepatic gluconeogenesis pathway after stress in Carassius auratus, whereas in Perch macrocephalus, the effect was opposite. 5. Short-term and long-term starvation decreased plasma cortisol and glucose levels after stress. Short-term and long-term starvation
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S917.4
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