急性和慢性低氧胁迫对卵形鲳鲹鳃器官的影响

文章研究了窒息点以上的低氧胁迫对卵形鲳鲹(Trachinotus ovatus)鱼体鳃器官的影响,在常温条件下对(32.84±3.99)g的卵形鲳鲹进行了急性(0 h、3 h、6 h、12 h、24 h)和慢性(14 d)的低氧胁迫实验,采用常规石蜡切片和H-E染色以及电子显微技术,观察低氧胁迫对卵形鲳鲹鳃器官的影响。结果显示,卵形鲳鲹在急性低氧胁迫下鳃小片上皮肿胀和抬升;慢性低氧胁迫下随着时间延长,鳃小片上皮细胞与鳃小片分离,鳃呼吸表面积增加。结果表明低氧胁迫对卵形鲳鲹鳃器官具有较大影响,而且慢性胁迫的影响大于急性胁迫。     

低氧环境下卵形鲳鲹的氧化应激响应与生理代谢相关指标的研究

文章测定了急性和慢性低氧胁迫后卵形鲳鲹(Trachinotus ovatus)的氧化应激和生理代谢相关指标。结果显示,在(23±0.7)℃下,体质量(31.59±3.01)g的鱼在急性低氧胁迫条件下的个体成活率为100%,在慢性低氧胁迫条件下的成活率则不断下降,第11天时降为实验初始时的50%。急性低氧胁迫过程中随低氧胁迫时间延长鳃过氧化氢酶(CAT)活性下降,第3小时最低,随后升高,第24小时显著高于其他各组(P0.05)。还原型谷胱甘肽(GSH)、丙二醛(MDA)含量、超氧化物歧化酶(SOD)活性随低氧持续先上升后恢复至正常水平,慢性低氧胁迫下鳃CAT活性与对照组相比显著降低(P0.05)。MDA、GSH含量和SOD活性则均显著升高(P0.05)。肝糖原(LG)在急性低氧胁迫过程中随低氧呈先下降后恢复的趋势,第12小时降至最低,第24小时恢复至对照组水平,慢性胁迫后LG下降。乳酸脱氢酶(LDH)水平在急性低氧胁迫过程中随低氧先升后降,第3小时达最大值,第24小时下降为对照组水平,慢低氧胁迫后LDH水平显著高于对照组(P0.05)。     

环境胁迫对对虾免疫系统的影响研究

环境胁迫（environmental stress）是指环境对生物所处的生存状态产生的压力，分为急性环境胁迫和慢性环境胁迫。捕捞、干扰、水质的突变等可以引起急性环境胁迫，而水质逐渐恶化和高密度放养等会造成慢性胁迫。近年来随着人类活动范围的不断扩大和程度的加深，环境资源受到了很大的破坏，养殖对虾受到的环境胁迫日益严重。这些胁迫可使对虾产生应激反应，虽然这是一种保护性反应，但持续处于应激状态，机体的免疫防御体系的功能会受到抑制，就会影响到其抗病力。当前对虾的病害越来越严重，大规模的死亡已经成为制约养殖业发展的瓶颈。     

碱胁迫下罗非鱼鳃氨转运蛋白Rh基因的表达

为研究碱环境下广盐性鱼类氨转运途径,将尼罗罗非鱼同时进行急性碱度胁迫(2、4、6 g/L)和慢性碱度胁迫,检测胁迫后120 h内的血氨浓度与鳃组织中Rhag、Rhbg、Rhcg1和Rhcg2基因mRNA表达变化,并采用免疫组化技术观察鳃组织中Rh蛋白的阳性反应。结果表明,急性碱度胁迫下,血氨浓度在12 h内快速升高到达峰值,4种Rh基因表达量升高,并于24 h到达峰值;慢性碱胁迫组由于碱度不断升高,血氨浓度呈波动状态,4种Rh基因的表达量均维持在较高表达水平,表明几种Rh蛋白均可能参与血氨浓度调节。胁迫24 h,Rhcg1基因的表达量显著高于其他3种基因。免疫组化结果表明,急性与慢性胁迫组中Rhag、Rhbg和Rhcg在鳃组织中均发现阳性反应,且随碱度升高,阳性反应增强。本研究表明,在碱胁迫环境下,尼罗罗非鱼会增强Rh基因与蛋白表达量参与氨转运过程。     

高pH急性和慢性胁迫对克氏原螯虾非特异性免疫和抗氧化能力的影响

为探讨高pH急性和慢性胁迫对克氏原螯虾(Procambarus clarkii)非特异性免疫和抗氧化能力的影响,分别设置对照(pH 7.6)和试验(pH 10.2)2个pH处理组,对克氏原螯虾进行急性(0 h、3 h、6 h、12 h、24 h、5 d)和慢性(10 d、15 d)pH胁迫试验。结果显示:高pH应激后,试验组血浆总蛋白(TP)含量和酸性磷酸酶(ACP)活性呈上升趋势。高pH急性胁迫下,24 h内试验组血浆碱性磷酸酶(AKP)呈先上升后下降趋势,5 d AKP活性显著上升;试验组血浆血糖(GLU)及皮质醇(CORTISOL)含量于pH胁迫5 d后达到峰值,且显著高于对照组;试验组肝胰腺超氧化物歧化酶(SOD)活性呈下降趋势,并于24 h开始显著低于对照组;试验组肝胰腺谷胱甘肽过氧化物酶(GSH-Px)活性在3～24 h显著低于对照组;试验组鳃乳酸脱氢酶(LDH)活性先下降后上升。高pH慢性胁迫下,试验组血浆AKP、ACP活性显著高于对照组;试验组血浆GLU及CORTISOL含量、鳃LDH活性呈下降趋势,肝胰腺SOD则呈上升趋势;肝胰腺GSH-Px、MDA水平于胁迫10 d时达到峰值。结果表明,高pH急性胁迫会对克氏原螯虾的免疫防御机制产生影响,导致氧化应激,随着胁迫时间的延长(慢性胁迫),机体部分非特异免疫和抗氧化指标逐步恢复。     

观赏鱼福利研究进展(下)

正(4)庇护场所。大多数鱼在远离自然条件下,很可能会导致急性或慢性胁迫,致使养殖鱼的福利水平降低。在家庭饲养观赏鱼的过程中,通过环境丰容技术适当地添加庇护物、水草等,增加栖息地的复杂性,可以尽量避免观赏鱼毫无遮掩地在环境中而产生焦虑等,提升观赏鱼的环境适应性,从而提高其存活率(秦传新等,2020)。人们为追求美观,对鱼缸环境进行设计,会为观赏鱼的健康与福利带来好处。     

盐度胁迫对许氏平鲉血液免疫酶活力的影响

研究了急性、慢性盐度胁迫对许氏平鲉体重及血液免疫相关酶活力的影响。慢性盐度胁迫实验表明,在盐度5的水体中,许氏平鲉体重增长率为负值,其余各盐度组(10、20、40)的生长指标与自然海水组(盐度33)差异不显著(P>0·05);随海水盐度的降低,许氏平鲉的溶菌酶活力逐渐上升,但当盐度降至5时,其活力与自然海水组无显著性差异;血液中超氧化物歧化酶(SOD)、过氧化氢酶(CAT)的活力随海水盐度降低呈逐渐上升趋势。急性盐度胁迫实验证实,在盐度5、10的急性胁迫初期,许氏平鲉血液的各项免疫酶活力波动较大。溶菌酶活力在胁迫24h时达到峰值,之后逐渐下降;血液SOD活力在96h检测过程中呈高低起伏变化趋势;血液CAT活力在胁迫初期持续降低,12h后逐步稳定在较低水平,显著低于胁迫前血液CAT活力(P<0·05)。     

盐度胁迫对尼罗罗非鱼免疫相关指标的影响

为探究盐度胁迫对尼罗罗非鱼免疫的影响，对体质量(35.0±5.0) g的尼罗罗非鱼进行了急性和慢性的盐度胁迫实验，对免疫相关指标进行了检测和分析。在急性盐度胁迫中，设置0、5和15盐度组，分别在胁迫后6、12、24、48和96 h进行取样，检测血清SOD、CAT、GSH-Px和AKP的活性。在慢性实验中，设置0、10、20和30盐度组，胁迫8周后检测血清SOD、CAT、GSH-Px和AKP活性，并进行了无乳链球菌易感性实验。结果显示：①血清中SOD活性在急性盐度胁迫6、12和24 h时都有随盐度上升而上升的趋势，但在96 h时盐度15组酶活性显著低于盐度5组；在慢性盐度胁迫下，各组的酶活性呈现出随着盐度升高而显著性下降的趋势。②血清CAT活性在急性盐度胁迫下12和24 h时呈现出随着盐度升高而显著下降的趋势；在慢性胁迫下不存在显著性差异。③血清中GSH-Px活性在急性和慢性胁迫后，均呈现随着盐度升高而降低的趋势。④血清AKP活性在胁迫后6 h随盐度升高呈现出显著下降趋势；在慢性盐度胁迫下，盐度20组显著低于其他实验组。⑤尼罗罗非鱼对无乳链球菌易感性实验中，盐度10组的易感性和盐度0组之间无显著差异，盐度20和30组的易感性高于盐度0组。研究表明，两种盐度胁迫均会引起免疫相关指标的变化，急性盐度胁迫实验表明，盐度5和15可导致尼罗罗非鱼机体氧化损伤，但尼罗罗非鱼可以逐渐适应这一变化；慢性盐度胁迫实验表明，盐度高于20会抑制尼罗罗非鱼多种免疫指标活性，造成其对无乳链球菌的易感性升高。     

尼罗罗非鱼对碳酸盐碱度耐受性研究

该研究以2种规格[大:(21.56±0.27) g,小:(5.92±0.03) g]的尼罗罗非鱼(Oreochromis niloticus)幼鱼为研究对象,采用碳酸氢钠(NaHCO_3)分别配制不同浓度的碳酸盐碱水体进行3种胁迫实验:1)将鱼由淡水直接移入碳酸盐碱度为30或22 g·L~(-1)的水体中进行高浓度碱胁迫致死;2)将鱼由淡水直接移入碳酸盐碱度为6～10 g·L~(-1)的不同碱度组水体中进行96 h急性碱胁迫;3)分别以+2、+4和+6 g·(L·d)~(-1)的每日碱度增加进行慢性碱驯化,探索其对碳酸盐碱度的耐受能力。结果表明,尼罗罗非鱼急性胁迫96 h的半致死碱度为6.25～9.01 g·L~(-1),其耐碱能力虽弱于青海湖裸鲤(Gymnocypris przewalskii)等耐高碱鱼类,但仍强于大多淡水养殖鱼类;在幼鱼阶段,体质量增加3倍以上的尼罗罗非鱼对碳酸盐碱胁迫的耐受性显著增强;养殖中可通过2 g·(L·d)~(-1)的每日碱增加对罗非鱼进行碱驯化;互补重对数模型较好地拟合急性碱胁迫下"时间-碱度-死亡率"间关系。     

急性温度胁迫对妊娠期许氏平鲉血清皮质醇和血液生理指标的影响

为探究鱼类对温度突变水体的适应性和恢复性,将妊娠期许氏平鲉由正常水温(16℃)直接转入高温(25℃)和低温(7℃)水体,急性胁迫2h后再恢复至16℃,检测恢复期第0、6、12、24、48和72h时妊娠期许氏平鲉血清皮质醇和血液生理指标的变化情况。结果显示,25℃急性胁迫导致2尾鱼发生产仔行为;25℃和7℃急性胁迫后的恢复过程中,血清皮质醇(COR)、血糖(GLU)、球蛋白(GLB)、丙氨酸氨基转移酶(GPT)、天门冬氨酸氨基转移酶(GOT)和白细胞(WBC)水平均显著升高(P<0.05),总蛋白(TP)、白蛋白(ALB)、总胆固醇(TC)及甘油三酯(TG)水平显著降低(P<0.05),血清Ca₂₊、Mg₂ 和P水平无显著变化;红细胞(RBC)和血红蛋白(HGB)水平在25℃胁迫后明显上升(P<0.05),而在7℃胁迫后显著下降(P<0.05)。与急性低温胁迫相比,急性高温胁迫对妊娠期许氏平鲉机体生理情况的影响更显著。除COR和无机离子外,多个生理指标在72h后仍未恢复至正常水平。研究表明,急性温度胁迫导致妊娠期许氏平鲉生理状况和代谢水平受到显著影响,甚至发生提前产仔行为。在养殖生产中,对于进入生殖期的雌鱼,应密切监测水温变化,尤其是水温的上升,以减少温度应激导致的妊娠损伤。     

Stress hormones and the cellular stress response in salmonids

The relationship between stress protein (SP) levels and the hormonal stress response in salmonids was examined through the measurement of gill SP70 and SP30 levels together with plasma cortisol, glucose and ion concentrations, in response to handling stress (45 s holding in a net), intraperitoneal cortisol implants, and heat shock (+10 °C). Handling and cortisol implants resulted in increased plasma cortisol and glucose levels. Heat shock following handling reduced plasma [Na⁺] below that observed in response to the handling stress alone, and heat shock following cortisol implant significantly lowered both plasma [Cl^–] and [Na⁺] below that of fish receiving the cortisol implant alone. Increased SP70 levels occurred 1 h following the 2 h +10 °C heat shock. Handling the fish prior to the application of heat shock suppressed the increase of SP70 levels in the gills. However, increased plasma cortisol concentrations alone did not attenuate gill tissue SP70 increase caused by heat shock. Physiological (10^–7 M) and pharmacological (10^–5 M) concentrations of adrenaline caused increased levels of SP70 in hepatocytes. Addition of the -blocker propanolol blocked this response to adrenaline. The results indicate that handling procedures do not result in an increase of hsp30 or hsp70 and may suppress hsp synthesis under certain circumstances.     

Confinement stress in sea bass (Dicentrarchus labrax) depresses peritoneal leukocyte cytotoxicity

Fish respond to stressful conditions via neuroendocrine responses (primary response) which result in increased levels of plasma cortisol which is considered immunosuppressive. Sea bass were confined at low (10 kg/m³) and high (60 kg/m³) density for 3–48 h. Plasma cortisol and glucose were evaluated and two principal cellular immune responses were assayed. A significant increase in plasma cortisol and glucose levels, as well as osmolarity, was found following stress. In addition, phagocytic activity, as shown by reactive oxygen species (ROS) production by challenged head kidney phagocytes and cytotoxic activity of eosinophilic granule cells from peritoneal cavity against K562 tumour cell lines appeared to be suppressed. Plasma levels of cortisol, glucose, osmolarity were correlated with cellular immunity, by the linear regression method. The suppression of cytotoxic activity was found to be significantly correlated with high plasma cortisol and glucose levels. These parameters could affect the eosinophilic granule cells of the peritoneal cavity.     

Depletion of serum alternative complement pathway activity in gilthead seabream caused by α-tocopherol and n-3 HUFA dietary deficiencies

Studies on the alternative complement pathway (ACP) activity in fish have suggested the particular relevance of this pathway as a non-specific immune mechanism being its activation directly related with the availability of certain nutrients. In the present study ACP activity in gilthead seabream fed different dietary levels of a-tocopherol and n-3 HUFA deficiencies was studied under different stress conditions (overcrowding and repetitive chasing).A reduction in ACP activity was found when diets with deficiencies in -tocopherol and n-3 HUFA were assayed at any of the experimental conditions assayed. Levels of plasma cortisol were similar to those of fish after crowding stress subjected to dietary deficiencies at low stocking density, suggesting that dietary deficiencies produced a chronic elevation of cortisol. When fish were subjected to repetitive chasing, plasma cortisol from fish fed control diet showed a progressive increase whereas plasma cortisol levels from both dietary deficient groups showed a transitory peak 4 days after the beginning of the repetitive chasing. However, ACP activity levels of fish fed deficient diets remained lower than the control group and constant during chasing period. These results suggest that both a-tocopherol and n-3 HUFA nutritional deficiencies rather than stress are an important determinant of fish complement activity.     

Plasmatic levels of cortisol in the response to acute stress in Nile tilapia, Oreochromis niloticus (L.), previously exposed to chronic stress

Many studies have been made about the physio-logical effects of isolated chronic or acute stress. However, few studies have been made to assess the combination of both responses. The fish submitted to chronic stress may be subjected to an additional acute stressor. The aim of the present work was to evaluate the acute stress response in Nile tilapia Oreochromis niloticus (L.) previously subjected to chronic stress. For this, two experiments were performed. In the first experiment, the fish were subjected to chronic stress followed by an additional acute stress. In the second experiment, the fish were submitted only to an acute stress. The data showed that Nile tilapia fingerlings can adapt to chronic stress situations, and this decreases, but does not eliminate, their capacity to respond to an additional acute stressor. In both experiments, plasma cortisol levels reached a peak 1 h after administration of the acute stressor. In fish previously submitted to chronic stress, the highest concentration of plasma cortisol measured was 196 ng mL^–1. This value was significantly different from the cortisol concentration obtained in the second experiment (267 ng mL^–1) with non-chronically stressed fish. The data also suggest that the chronic stress response can provoke a reduction in performance and growth rates compared with non-stressed fish.     

Stress responses and disease resistance in salmonid fish: Effects of chronic elevation of plasma cortisol

Basal levels of plasma cortisol in unstressed salmonid fish are normally in the range 0–5 ng ml⁻¹. An acute stress such as handling or 1 h confinement caused a temporary elevation of the plasma cortisol levels of both brown trout,Salmo trutta L., and rainbow trout,Salmo gairdneri Richardson, in the range 40–200 ng ml⁻¹ with a return to basal levels within 24–48 h. The extent of the cortisol elevation in response to an acute stress was dependent upon both the species and strain of trout. Chronic stresses, such as prolonged confinement or crowding, resulted in an elevation of plasma cortisol levels to approximately 10 ng ml⁻¹. Under these circumstances, blood cortisol levels remained elevated for periods of up to 4 weeks before acclimation finally occurred. It is shown, by means of intraperitoneal implantation of cortisol, that chronic elevation of plasma cortisol levels in the brown trout results in a dose-dependent increase in mortality due to common bacterial and fungal diseases. This effect is apparent at plasma cortisol levels as low as 10 ng ml⁻¹, levels below those often reported as being representative of ‘unstressed’ fish. These findings are discussed in relation to the known immunosuppressive effects of corticosteroids in teleost fish.     

Development of a modified cortisol extraction procedure for intermediately sized fish not amenable to whole-body or plasma extraction methods

The corticosteroid hormone cortisol is the central mediator of the teleost stress response. Therefore, the accurate quantification of cortisol in teleost fishes is a vital tool for addressing fundamental questions about an animal’s physiological response to environmental stressors. Conventional steroid extraction methods using plasma or whole-body homogenates, however, are inefficient within an intermediate size range of fish that are too small for phlebotomy and too large for whole-body steroid extractions. To assess the potential effects of hatchery-induced stress on survival of fingerling hatchery-reared Spotted Seatrout (Cynoscion nebulosus), we developed a novel extraction procedure for measuring cortisol in intermediately sized fish (50–100 mm in length) that are not amenable to standard cortisol extraction methods. By excising a standardized portion of the caudal peduncle, this tissue extraction procedure allows for a small portion of a larger fish to be sampled for cortisol, while minimizing the potential interference from lipids that may be extracted using whole-body homogenization procedures. Assay precision was comparable to published plasma and whole-body extraction procedures, and cortisol quantification over a wide range of sample dilutions displayed parallelism versus assay standards. Intra-assay  %CV was 8.54 %, and average recovery of spiked samples was 102 %. Also, tissue cortisol levels quantified using this method increase 30 min after handling stress and are significantly correlated with blood values. We conclude that this modified cortisol extraction procedure provides an excellent alternative to plasma and whole-body extraction procedures for intermediately sized fish, and will facilitate the efficient assessment of cortisol in a variety of situations ranging from basic laboratory research to industrial and field-based environmental health applications.     

The effect of cortisol administration on blood plasma immunoglobulin M (IgM) concentrations in masu salmon (Oncorhynchus masou)

Immunoglobulin M (IgM) is known as a main factor in the humoral immune system of teleosts. In the present study, the effect of cortisol on plasma IgM concentrations was investigated using a specific antibody to IgM in masu salmon (Oncorhynchus masou). Cortisol was orally administered each day for 2 weeks at a dose of 1 mg g⁻¹ in the diet, and for the following week the fish were fed a non-treated diet. Blood plasma samples were collected at 0, 1, 2 and 3 weeks after the initiation of treatment. Oral administration of cortisol elevated plasma cortisol concentrations to about 40 ng/ml for 2 weeks after administration and slightly reduced plasma IgM concentration; the suppression was statistically significant one week after the period of hormone administration. However, treatment with cortisol did not affect plasma concentrations of total protein or α1-protein, one of the major serum proteins, during the experimental period. These results indicate that cortisol specifically suppresses plasma IgM concentrations.     

Effect of some repetitive factors on turbot stress response

The stress response of the turbot, Scopthalmus maximus (L.), to repetitive factors including netting, air exposure, blood sampling and hand-stripping, were tested using two different tank sizes as well as two blood sampling techniques. Exposure of juvenile fish to air for 1-4 min had no immediate effect on plasma cortisol concentrations or haematocrit values. Similarly, the serial netting of immature fish from tanks did not significantly modify plasma cortisol concentrations, haematocrit or osmolarity values. Hand-stripping of mature males was more disturbing than air exposure. The cumulative effect on plasma cortisol levels and osmolarity of stress factors such as netting, air exposure, blood sampling and stripping applied simultaneously to mature males in a 16 m3 tank and repeated twice daily for several days was recorded. The level of cortisol increased from 5 ng ml-1 to 300 ng ml-1 after 10 days of treatment, while an osmoregulatory imbalance and fish death were observed. Moreover, adaptation of fish to smaller tanks seemed to improve the increased plasma cortisol levels and death rate. Reduction in the number of stress factors applied greatly decreased both the range of physiological responses and the death rate. In order to avoid a cumulative stress response, handling of fish should therefore be reduced to a minimum.     

Interactive effects of a high-quality protein diet and high stocking density on the stress response and some innate immune parameters of Senegalese sole Solea senegalensis

Amino acids (AA) regulate key metabolic pathways, including some immune responses. Therefore, this study aimed to assess whether an increased availability of dietary AA can mitigate the expected increase in plasma cortisol and metabolites levels due to high stocking density and its subsequent immunosuppression. Senegalese sole (Solea senegalensis) were maintained at low stocking density (LSD; 3.5 kg m^?2) or high stocking density (HSD; 12 kg m^?2) for 18 days. Additionally, both treatments were fed a control or a high protein (HP) diet (LSD, LSD HP, HSD and HSD HP). The HP diet slightly increased the levels of digestible indispensable AA, together with tyrosine and cysteine. HSD was effective in inducing a chronic stress response after 18 days of treatment since fish held at HSD presented higher plasma cortisol, glucose and lactate levels. Moreover, this increase in stress indicators translated in a decrease in plasma lysozyme, alternative complement pathway (ACP) and peroxidase activities, suggesting some degree of immunosuppression. Interestingly, while plasma glucose and lactate levels in HSD HP specimens decreased to similar values than LSD fish, plasma lysozyme, ACP and peroxidase activities increased, with even higher values than LSD groups for ACP activity. It is suggested that the HP diet may be used as functional feed since it may represent a metabolic advantage during stressful events and may counteract immunosuppression in sole.     

Effects of dietary inclusions of red beet and betaine on the acute stress response and muscle lipid peroxidation in rainbow trout

This study evaluates the effects of red beet (RB) and betaine on rainbow trout submitted to an acute stress challenge. A control diet was compared with four experimental diets in which red beet (14 and 28%) and betaine (0.9 and 1.63%) were incorporated in different concentrations according to a factorial design. Cortisol in plasma and fin, glucose and lactate plasma levels, and malondialdehide (MDA) in muscle were all measured before the stress challenge and 30 min and 6 and 12 h after the stress challenge as parameters to determine the diet effects. RB and betaine had no effect on cortisol, glucose, and MDA basal levels. However, lactate basal levels were significantly lower on fish fed with RB and betaine. Thirty minutes after the stress challenge, there was a significant increase in plasma and fin cortisol, glucose and lactate concentrations, although fish fed with diets containing RB and betaine showed significantly higher plasma cortisol values. MDA values of fish fed with 14% RB and 0.9% betaine were significantly higher than MDA values from fish fed with 28% RB and 1.63% betaine. After 6 and 12 h, plasma and fin cortisol and lactate levels recovered in a similar trend. Glucose plasma levels recovered in almost all groups 12 h after the stress. Also, MDA values recovered basal levels after 6 and 12 h. RB and betaine did not enhance the tolerance to the stress challenge compared to the control group, although the presence of these ingredients had no negative effect on any of the stress indicators.