【作者】 何绪刚；

【导师】 谢从新； 庄平；

【作者基本信息】 华中农业大学 ， 水产养殖， 2008， 博士

【摘要】 中华鲟（Acipenser sinensis Gray）是大型溯河洄游鱼类,国家一级保护动物,主要分布在我国东海、黄海和长江。成熟最大个体全长400cm,体重452kg。中华鲟淡水中出生后,本能地要洄游到大海中生活,待8～10龄性成熟后,再上溯洄游到出生地进行繁殖。在长江口,中华鲟幼鱼的集群时间为5～9月,这一水域所发现的中华鲟幼鱼以1龄个体为主。1981年长江葛洲坝水利工程的修建阻隔了中华鲟产卵洄游通道,加上过度捕捞,中华鲟物种数量锐减。调查证实葛洲坝下已形成中华鲟新的产卵场。到本世纪初,关于中华鲟产卵群体的个体形态结构、繁殖生物学习性,特别是葛洲坝下中华鲟产卵群体繁殖生态学和资源评估的研究,取得了明显的进展。但有关中华鲟渗透生理和盐度选择行为方面还未见报道。本课题围绕中华鲟1龄幼鱼在外界环境盐度刺激下渗透生理变化过程及其调控机制,以及在这一变化过程中中华鲟盐度选择行为的变化规律等内容展开工作,主要得到以下结果:1、海水适应过程中中华鲟血清渗透压、血清离子浓度及Na⁺/K⁺-ATPase活力变化将7月龄中华鲟幼鱼（128.8±15 g）从淡水环境(0%‰,46 mmol kg^-1)转入半咸水环境(10‰,273 mmol kg^-1)后:Na⁺、Cl^-对血清渗透压的贡献率增高;幼鱼血清渗透压最初12h升高,12～24h维持高渗状态(300 mmol kg^-1),24～216h逐渐下降到新的稳定水平(272 mmol kg^-1),此水平等渗于环境,而显著高于淡水对照组(264.14±0.72mmol kg^-1)（p＜0.05）;血清[Na⁺]、[Cl^-]和[Ca²⁺]变化趋势与血清渗透压相似,前24h浓度升高,24～216h浓度下降,216～480h重新稳定,且显著高于淡水对照组（p＜0.05）;血清[K⁺]变化明显滞后于血清[Na⁺]、[Cl^-]和[Ca²⁺],最初12h内浓度保持稳定,24～216h逐渐上升到最高值(3.62±0.10mmol L^-1)之后持续下降,480h时下降到淡水对照组水平;鳃Na⁺/K⁺-ATPase活力最初3h内受到抑制,3^-124h持续上升到最高值（3.20±0.20U）后至72h一直维持约为淡水对照组（1.45±0.03U）2倍的高活力水平,之后持续下降,216h以后保持稳定（2.39±0.082U）且显著高于淡水对照组水平（p＜0.05）;瓣肠Na⁺/K⁺-ATPase活力前3h酶活力有所升高,3～24h酶活力持续下降到最低值（0.12±0.02U）且显著低于淡水对照组水平（p＜0.05）,之后缓慢回升,480h时回升到淡水对照组水平;直肠Na⁺/K⁺-ATPase活力前3h酶活力保持稳定,之后酶活力下降,12～72h酶活力显著低于淡水对照组（p＜0.05）,216～480h酶活力低于淡水对照组,但差异不显著（p＞0.05）;肾Na⁺/K⁺-ATPase活力显著下降,各时间段酶活力均显著低于淡水对照组（p＜0.05）。2、海水适应过程中中华鲟血清激素水平变化从淡水环境(0‰,46 mmol kg^-1)转入半咸水环境(10‰,273 mmol kg^-1)后,机体迅速抑制了催乳素的分泌,加强了皮质醇和甲状腺素的分泌:最初12h内,血清催乳素水平直线下降近3倍,达0.24±0.03ng ml^-1,此后一直保持显著低于淡水对照组水平(0.85±0.11ng ml^-1)的低水平稳定状态（p＜0.05）;血清皮质醇最初3h内急剧上升到最高值(56.12±15.40 ng ml^-1),显著高于淡水对照组(8.66±1.56 ng ml^-1)（p＜0.05）,之后快速下降,24h以后回落到淡水对照组水平并保持稍高于淡水对照组的稳定状态;血清总甲状腺素（四碘甲腺原氨酸,TT₄）3h内上升到最大值(3.87±0.40ng ml^-1),显著高于淡水对照组(1.54±0.31 ng ml^-1)（p＜0.05）,之后逐渐下降,216h以后下降到淡水对照组水平;血清总三碘甲腺原氨酸（TT₃）3h内上升到最大值0.18±0.03 ng ml^-1,显著高于淡水对照组(0.04±0.01 ng ml^-1)（p＜0.05）,之后快速下降,但始终保持显著高于淡水对照组水平（p＜0.05）;血清游离甲状腺素（FT₄）和游离三碘甲腺原氨酸（FT₃）在最初3h内均迅速升高（p＜0.05）,之后快速下降,24h后回落到淡水对照组水平。3、不同盐度驯化下中华鲟幼鱼鳃泌氯细胞结构变化在淡水中,中华鲟幼鱼鳃中的泌氯细胞数量较少,且主要分布在近鳃小片基部,胞体与核均较大而明显,胞内含大量线粒体;泌氯细胞中有网管和囊管,但网管欠发达,囊管分布面积小,细胞表面有顶隐窝。表现为典型的淡水型泌氯细胞特征。与淡水组相比,半咸水组鳃泌氯细胞的分布和结构变化明显,泌氯细胞集中分布在鳃小片基部;超微结构显示,细胞内线粒体数量明显增加,胞质中网管颇为发达,囊管丰富,顶隐窝扩大,表面有微绒毛,表现为α-型（海水型）泌氯细胞特征。泌氯细胞具有分泌体内过多Na⁺、Cl^-以及调节体液渗透平衡的功能,其数量和结构变化与幼鱼所处的高渗环境相适应。4、海水适应过程中中华鲟主要消化酶活性的变化从淡水环境(0‰,46 mmol kg^-1)转入半咸水环境(10‰,273 mmol kg^-1)后,蛋白酶和淀粉酶在12h内降到最低,48h后酶活力持续回升,216h后酶活力回升到淡水对照组水平;脂肪酶活力在48h内降到最低,72h后持续上升,216h后酶活力上升到淡水对照组水平。脂肪酶活力受盐度影响较蛋白酶和淀粉酶更大。外界盐度环境的变化并未显著影响中华鲟肝脏对消化酶的合成与分泌。中华鲟消化器官蛋白酶和淀粉酶活性由高到低的顺序为:幽门盲囊＞十二指肠＞瓣肠＞胃＞肝脏;脂肪酶活性由高到低的顺序为:瓣肠＞十二指肠＞胃＞肝脏＞幽门盲囊。5、中华鲟盐度选择实验装置的设计当两种不同盐度的水相遇时产生垂直分层扩散,形成上层为低比重水、下层为高比重水的垂直分层,并出现一个稳定的分界面。据此盐度扩散规律,作者设计出六分室盐度选择实验装置用于中华鲟盐度喜好性实验。装置圆形,高1m,直径6m,中央区为过渡区域,含六个盐度分室,结构均匀。静态下各分室盐度可稳定10d以上;在不间断曝气和有实验动物扰动情况下,各分室盐度可稳定1d。实验时,受试鱼可单尾或多尾从中央区域放入,也可从各分室均匀放入。从受试鱼主动选择的盐度分室（或者在各盐度分室里活动频率、时间上的差异）即可直观了解鱼类的盐度喜好性。6、中华鲟幼鱼盐度喜好行为未接触过咸水的淡水8月龄幼鱼,显著趋向淡水;生活在长江口,还未完成渗透生理转变的野生中华鲟幼鱼,最喜5‰的盐度;而驯养于盐度10‰环境中一定时间的中华鲟幼鱼,最喜其驯化盐度10‰。环境盐度的持续刺激可改变中华鲟幼鱼盐度喜好性。正是这种可塑的盐度喜好性驱动着中华鲟幼鱼最终从河口半咸水水域进入到大海之中。更多还原

【Abstract】 Chinese sturgeon, Acipenser sinensis Gray, a class I endangered species by the Chinese government, is an anadromous species that presently only remains in the Yangtze （=Changjiang） River, the East China Sea and Yellow Sea. Adult Chinese sturgeon, which are a maximum of 400 cm total length （TL） and 452 kg body weight （BW）, are one of the largest fish to enter fresh water. Juveniles hatched in the Yangtze River migrate to the sea and return to spawn at the age of 8-10 years. Chinese sturgeon juveniles concentrate at the river estuary during the period of May-September, and almost all of the young fish found in the estuary are younger than 1 year old. As a result of overfishing and construction of Gezhouba Dam in 1981 at Yichang, Hubei Province, 1766 km from the river estuary, blocked the spawning migration of Chinese sturgeon to the Yibin spawning reach, populations of Chinese sturgeon have greatly declined in abundance. Some successful spawning occurs in the short reach below the dam （Gezhouba spawning site） as verified by the capture of early-life stages in 1982 and during 1996-1999. By the beginning of this century, research on Chinese sturgeon has focused on the fields of morphology, reproductive biology and, especially, reproductive ecology and stock assessment of the spawning population below the Gezhouba Dam. To date, little information has been gathered concerning the osmoregulatory physiology and salinity preference of Chinese sturgeon. The author studied the development of osmoregulatory mechanisms and salinity preference behavior of 1-year-old juvenile Chinese sturgeon during seawater adaptation. Conclusions from the studies are as follows:1. Changes of serum osmolality, serum ion concentrations and Na⁺, K⁺-ATPase activities in juvenile Chinese sturgeon during seawater adaptationThe osmoregulation capabilities of 7-month-old juvenile Chinese sturgeon （128.8±15 g） transferred directly from freshwater (0%o, 46 mOsmol kg^-1) to brackish water (10‰, 273 mOsmol kg^-1) were studied over a 20-day period. Changes in serum osmolarity, chloride （Cl^-）, sodium （Na⁺）, potassium （K⁺） and calcium (Ca²⁺) ion concentrations, as well as gill, spiral valve, rectal and renal Na⁺, K⁺-ATPase activities were measured at 3, 12, 24, 72, 216 and 480 h after transfer to BW. The serum osmolarity and ion concentrations (Na⁺, Cl^- and Ca²⁺) increased immediately after the transference to BW, reaching maximum at 24 h and returned to a new steady state at 216 h, while the FW control group maintained basal levels which showed lower （p<0.05） than the BW group. Serum potassium ion concentration lagged behind the chloride, sodium, potassium and calcium ion concentrations after exposure to BW. K⁺ ion concentration of BW group maintained steady state in the first 12 h after transfer, but began to increase at 24 h, reaching maximum at 216 h after transference and returned to the levels of FW control at 480 h. Gill Na⁺, K⁺-ATPase activity of BW group exhibited an abrupt decrease in the first 3 h after transfer, but began to increase at 3 h, reaching a peak value at 24 h, and returned a new steady state at 216 h. The differences between gill Na⁺, K⁺-ATPase activity of BW and FW fish were significant （p< 0.05） after 12 h. In contrast, Na⁺, K⁺-ATPase activity of the spiral valve showed transient increase after transference from FW to BW, and then decreased rapidly at 3 h, reaching the lowest at 24 h after transference. At 216h after exposure to BW, Na⁺, K⁺-ATPase activities of the spiral valve increased slowly to the levels of FW control. Rectal Na⁺, K⁺-ATPase activity of BW group maintained steady state in the first 3 h after transfer, but began to decrease at 3 h, and returned slowly to the levels of FW control at 216 h. Renal Na⁺, K⁺-ATPase activity decreased rapidly after exposure to BW, and the differences between renal Na⁺, K⁺-ATPase activity of BW and FW fish were significant （p< 0.05）.2. Changes in serum hormone levels in juvenile Chinese sturgeon during seawater adaptationAfter exposure to BW, the fish bated rapidly its secretion of prolactin （PRL）, and accelerated to secrete cortisol and thyroxine. The serum PRL level decreased immediately after the transference to BW, and reached a new steady low state at 12 h which was significantly lower than in the FW control fish （p<0.05）. The serum cortisol level of BW group exhibited an abrupt increase after transfer, reaching a peak value at 3 h and returned rapidly to the steady level at 24 h. The new steady level of cortisol in BW group was higher than the level of the FW control group, and did not show significant differences. After transference from FW to BW, Serum total thyroxine （TT₄） level showed transient increase, reaching maximum at 3 h and returned slowly to the level of the FW control group at 216 h. Serum total triiodothyronine （TT₃） increased immediately after the transference to BW, reaching a peak value at 3 h and returned to a new steady state which was significantly higher than in the FW control fish （p<0.05）. Serum free thyroxine （FT₄） and free triiodothyrinine （FT₃） increased immediately in the first 3 h after the transference to BW, and returned rapidly to the level of the FW control group at 24 h.3. Structural changes of gills chloride cell of juvenile Chinese sturgeon acclimated to various salinitiesModifications in the chloride cells of gill epithelia of juvenile Chinese sturgeon in FW and BW were examined by light and transmission electron microscopy. In freshwater, a few chloride cells were present on the base of lamellae and in the interlamellar region of the filament with undeveloped tubular network and vesicle-tubular as freshwater-type chloride cells. While in brackish water, fish showed a marked increase in the number and size of chloride cells. On the lamellae of these fish, chloride cells were generally centralizing to the base of lamellae. Ultrastructural modifications included: presence of a more compact tubular network, a greater development vesicle-tubular and an enlarged apical crypt bearing some short microvilli asα-subtypes （seawater-type） chloride cells. The gill chloride cell, with increasing in the number and modifying structure, participate in extruding excess Na⁺, Cl^- and adjusting osmolality of body fluid in the hyperosmotic medium.4. The activities of digestive enzymes of juvenile Chinese sturgeon during seawater adaptationThe protease, amylase and lipase activities of the alimentary canal in 7-month-old juvenile Chinese sturgeon were increased in the few hours after exposure to BW, and then decreased rapidly. The minimum activities of protease and amylase occurred at 12h after exposure to BW. The protease and amylase activities were increased continually after 48h after exposure to BW, and were the same as activities of freshwater control group at 216h. The minimum activity of lipase occurred at 48h after exposure to BW. The lipase activity was increased continually after 72h, and was the same as lipase activity of freshwater control group at 216h after exposure to BW. The activity of lipase was affected strongly by salt. Environment salinity didn’t affect secrete of digestive enzymes in liver. The activities of protease and amylase were from high to low: pylorus vesica > duodenum > ileum > stomach > liver. The lipase activity was from high to low: ileum > duodenum > stomach > liver > pylorus vesica.5. Design a device for the study of salinity preference in Chinese sturgeonWhen two kinds of different salinity water met, there showed vertical stratification phenomenon and appeared a stable interface, forming the light water in the upper layer and the heavy water in the lower layer. According to the law of salinity diffusion, a six-chambered device for the study of salinity preference in Chinese sturgeon was designed. The device, including six salinity chambers, was circular, 1-m-high, 6-m-diameter, and the center area was the transition region. The structure was even. Salinities in the six chambers could steady 10 days without disturbing, while those salinities only steadied one day in the state of continuing bubbling or fish turbulence. To test salinity preference, fish were placed in the testing device from six chambers or the center area. We could perceive salinity preference, or behavioral selection of a particular concentration of dissolved salts, according to the difference of the frequency or duration in six salinity chambers.6. Salinity preference behavior of juvenile Chinese sturgeonJuvenile Chinese sturgeon which was strictly confined to freshwater showed preference for fresh water. While, wild juvenile Chinese sturgeon which concentrated at the river estuary without completing conversion from hyper-osmoregulatory to hypo-osmoregulatory chose salinity 5%o. In contrast, juvenile Chinese sturgeon which had been acclimated in 10‰BW "preferred" salinity 5‰. In the present study, condition salinity appeared to have affected the salinity preference of juvenile Chinesesturgeon. It is the plasticity of salinity preference to drive juvenile Chinese sturgeoninto the sea.更多还原