【作者】 胡晓芬；

【导师】 袁宗辉；

【作者基本信息】 华中农业大学 ， 基础兽医学， 2008， 硕士

【摘要】 喹噁啉类为人工合成的、具有喹噁啉-1,4-二氧基本结构的动物专用药,具有提高饲料利用率、促进动物生长和广谱抗菌作用,曾作为抗菌促生长剂在世界范围内广泛应用。本类早期品种喹乙醇和卡巴氧由于对人和动物有不同程度的毒副作用已被限制使用。中国农业科学院兰州研究所八十年代研制的乙酰甲喹由于毒性大,主要作为抗菌剂使用。喹烯酮和喹赛多是新一代喹噁啉类抗菌促生长剂,毒性低,毒副作用小,对食品动物具有良好的促生长效果,在畜禽和水产养殖上具有广阔的应用前景。但目前的研究表明:超剂量使用喹乙醇导致鱼类出现中毒反应,引起鱼类抗应激力显著降低,受到应激因子刺激后,呈现“应激性出血症”。基于喹赛多、喹烯酮与喹乙醇的化学结构相似,抗菌促生长机制相同,且有研究表明喹噁啉类普遍具有肾上腺毒性,这两种添加剂会不会同样对鱼类的应激反应产生干扰作用,尚不清楚。同时,目前在喹乙醇对鱼类应激性毒性的研究基本停留在常规生理生化指标测定、器官病理切片观察上面,尚没有深入到下丘脑-脑垂体-肾间组织(HPI)轴的水平上,对应激激素皮质醇在血清中的含量变化及HPI轴组织结构的变化,尚未见报道。深入系统地了解喹乙醇、喹赛多和喹烯酮对鱼类的生理学、毒理学及其对鱼类应激轴的作用,对今后喹噁啉类在水产养殖中安全合理使用等方面提供了更可靠的科学依据。本试验以鲤(Cyprinus carpio L.)为试验对象,投喂添加不同剂量的喹乙醇、喹赛多和喹烯酮(50、150和500mg/kg)的饲料,于试验的4、8和12wk末,每组随机捞取鲤30尾分别于应激前、追逐及24h限制应激后采样。应激前测定鲤的增重、血液生理生化指标以及肝脏抗氧化指标,取鲤的头肾、中肾、肝脏、肠道进行了组织病理学研究,探讨了三种药对鲤生理学和毒理学的影响;追逐和24h限制应激后测定血清生化指标,探讨了这三种药对鲤应激的影响,研究结果如下:1.试验期间,用药各组平均增重与对照组相比均有不同程度提高。4wk后,喹赛多50、150、500mg/kg组比对照组分别提高25.7%(P＞0.05)、64.3%(P＜0.05)、92.9%(P＜0.05),喹乙醇50、150、500mg/kg组比对照组分别提高24.3%(P＞0.05)、48.6%(P＜0.05)、67.1%(P＜0.05),喹烯酮50、150、500mg/kg组比对照组分别提高34.3%(P＞0.05)、74.3%(P＜0.05)、91.4%(P＜0.05);8wk后,喹赛多50、150、500mg/kg组比对照组分别提高25.5%(P＞0.05)、56.2%(P＜0.05)、64.2%(P＜0.05),喹乙醇50、150、500mg/kg组比对照组分别提高22.6%(P＞0.05)、44.5%(P＜0.05)、40.9%(P＜0.05),喹烯酮50、150、500mg/kg组比对照组分别提高27.7%(P＜0.05)、40.9%(P＜0.05)、58.4%(P＜0.05);12wk后,喹赛多50、150、500mg/kg组比对照组分别提高14.6%(P＞0.05)、38.7%(P＜0.05)、45.7%(P＜0.05),喹乙醇50、150、500mg/kg组比对照组分别提高9.5%(P＞0.05)、31.2%(P＜0.05)、18.1%(P＞0.05),喹烯酮50、150、500mg/kg组比对照组分别提高14.1%(P＞0.05)、28.1%(P＜0.05)、37.2%(P＜0.05)。4和8wk各药物同剂量组相比均无显著差异(P＞0.05),仅12wk时喹乙醇500mg/kg组增重显著低于同剂量下的喹赛多和喹烯酮组(P＜0.05)。结果表明:添加剂量为50～500mg/kg的喹赛多和喹烯酮具有明显的促生长效果。50～500mg/kg喹乙醇也显著提高鲤的生产性能,但其500mg/kg剂量下,促生长效果下降。随着用药时间的延长,喹赛多、喹乙醇和喹烯酮的促生长效果均有所下降。2.随着剂量的增加,喹乙醇各组的红细胞总数(RBC)、血红蛋白(HGB)和红细胞压积(HCT)均呈下降的趋势,其500mg/kg组的RBC和HGB在各个时期均显著低于对照组及其他各药物组(P＜0.05)。喹赛多和喹烯酮50、150mg/kg组RBC、HGB和HCT呈上升的趋势,但500mg/kg组各指标都有下降,8和12wk时,喹赛多500mg/kg组RBC显著低于其50mg/kg组(P＜0.05),喹烯酮500mg/kg组RBC在各个时期均低于其50mg/kg组(P＜0.05)。结果表明:50～150mg/kg的喹赛多和喹烯酮对鲤的健康状态有促进作用,可促进红细胞生成从而增加氧的供应,而500mg/kg的长期作用会给鲤的血液生理带来负面作用。喹乙醇500mg/kg可能会干扰鱼体的造血机能导致贫血。3.4wk时,150和500mg/kg喹赛多和喹乙醇、500mg/kg喹烯酮能显著提高血清皮质醇含量(P＜0.05)。8和12wk后,鲤对药物有良好的适应。血糖含量在喹赛多和喹烯酮各组均有上升的趋势,但500mg/kg喹乙醇使其含量降低。喹乙醇500m/kg组的血清天门冬氨酸转氨酶(AST)、丙氨酸转氨酶(ALT)、碱性磷酸酶(ALP)、乳酸脱氢酶(LDH)活性在各个时期均上升,喹赛多500mg/kg和喹烯酮组和喹乙醇150mg/kg组偶尔也有上升的情况。4wk时总胆红素(TB)和直接胆红素(DB)均无显著变化(P＞0.05),至8和12wk时,其在喹乙醇500mg/kg组有显著增高(P＜0.05)。8和12wk时,血清白蛋白(ALB)含量在喹赛多150和500mg/kg组和喹烯酮150mg/kg组显著升高(P＜0.05),但在12wk喹乙醇500mg/kg组血清总蛋白(TP)和ALB含量显著降低(P＜0.05)。血清肌酐(CRE)含量仅在12wk喹乙醇150和500mg/kg组显著增高(P＜0.05),尿素氮(BUN)在8和12wk喹乙醇500mg/kg组显著增高(P＜0.05)。血清离子在4和8wk时变化均不显著(P＜0.05),12wk时变化范围较大,喹乙醇500mg/kg组Na~+显著低于其他各组(P＜0.05),K~+在该组12wk时显著增高(P＜0.05),P在12wk时喹赛多500mg/kg组、喹乙醇和喹烯酮150和500mg/kg组显著升高(P＜0.05)。各个时期胆固醇(CHO)、甘油三酯(TG)和CL~-含量变化均不明显(P＞0.05)。结果表明,喹赛多和喹烯酮对鲤有较好的安全性,剂量为50～150mg/kg时,对鲤生理生化指标基本无明显影响:长期使用剂量高于500mg/kg的喹赛多和喹烯酮对鲤生理机能产生轻微的不良影响;整个试验周期内,500mg/kg喹乙醇对鲤的能量和物质代谢都有一定的破坏作用。4.应激后,皮质醇、血糖、天门冬氨酸转氨酶(AST)、丙氨酸转氨酶(ALT)、碱性磷酸酶(ALP)和乳酸脱氢酶(LDH)有显著变化(P＜0.05),可作为分析药物对应激影响的重点指标。4wk应激后,喹赛多、喹乙醇和喹烯酮500mg/kg组鲤血清皮质醇比对照组都有显著提高(P＜0.05),和基础水平的变化一致,表明鱼体HPI轴尚能进行正常的内分泌调控;8和12wk皮质醇基础水平无明显变化,但追逐应激后,150mg/kg喹乙醇组皮质醇显著升高(P＜0.05),500mg/kg组皮质醇显著降低(P＜0.05),可能由于500mg/kg喹乙醇组鲤肾间组织受到损伤,应激后不能正常地分泌皮质醇造成的。12wk时这种破坏表现得更为明显,追逐应激后,500mg/kg喹乙醇组皮质醇水平同样显著低于对照组(P＜0.05),但24h限制应激后其水平显著高于对照组(P＜0.05),这可能是皮质醇分泌和消除的能力均受到破坏造成的。血糖在应激后各组差异不显著(P＞0.05),但喹乙醇500mg/kg组有降低的趋势。各时期喹乙醇500mg/kg组应激后的血清AST、ALT和ALP活性与各对照组应激后相比普遍升高,表明应激会加强喹乙醇对肝细胞膜的通透性和细胞间的代谢作用的损伤。喹赛多500mg/kg组、喹烯酮500mg/kg组和喹乙醇150mg/kg组的血清AST、ALT和ALP活性在12wk应激后也有升高的情况。5.在本试验剂量范围内,这三种药都对超氧化物歧化酶(SOD)的活性表现为诱导作用,但随饲养时间延长,对SOD活性的诱导作用减弱;各药物150mg/kg能诱导谷胱甘肽-S转移酶(GST)活性,而500mg/kg对其活性有所抑制,喹乙醇500mg/kg作用最为显著(P＜0.05);而各组内二醛(MDA)含量无明显变化(P＞0.05)。上述变化表明,剂量高于500mg/kg的喹乙醇对鲤肝脏抗氧化机能有一定的破坏,而木试验剂量范围内的喹赛多和喹烯酮均能改善鲤肝脏抗氧化机能。6.剂量低于500mg/kg的喹赛多和喹烯酮,150mg/kg以下剂量的喹乙醇基本不会引起明显的病变。500mg/kg喹乙醇对这些组织结构会产生不同程度的病理改变,且随着饲养时间的延长,组织病变程度加深。长期使用500mg/kg的喹赛多和喹烯酮也会引起轻微的组织病变。综上所述,本文全面系统地研究了喹赛多、喹乙醇和喹烯酮对鲤生长性能、血液生理生化指标和组织器官病理学的影响,重点分析了应激前后血清皮质醇和应激相关指标的变化,结合头肾组织的病理变化,全面诠释了长期使用高剂量喹乙醇导致“应激性出血症”的原因,同时首次证实喹赛多和喹烯酮在水产养殖中长期应用比较安全,能够有效避免实际中由于捕捞、运输等引起的“应激性出血症”。为喹噁啉类在水产养殖中安全合理使用提供了可靠的科学依据。更多还原

【Abstract】 Quinoxalines,with the structure of quinoxaline-1,4-dioxides are synthetic antimicrobial agents,which have the characteristic for improving feed efficiency, promoting growth and increasing the rate of weight gain.For these reasons,they have been widely used as growth promoters in most countries of the world.However,carbadox and olaquindox,which the first were developed and used,have been banned or strictly limited to use due to their toxicities.Mequindox was synthesized by Lanzhou Institute of Animal and Veterinary Pharmaceutics sciences of CAAS in China,and used as antibacterial agent.Quinocetone and cyadox,which were new growth promoters of the quinoxalines family,had low toxicity,less side effect and obvious growth promotion in food-producing animals.But some researches showed that olaquindox can cause the stress hemorrhage syndrome with inappropriate use,and other researches considered that quinoxalines had the toxicity to impair kidney and adrenal tissues.But we don’t know if quinocetone and cyadox had the same deleterious effects on stress responses with the similar structure and mechanism of growth promotion as olaquindox.So the current study was carried out to evaluate the effects of quinoxalines(cyadox,olaquindox and quinocetone) on hematological and physiological response parameters,antioxidant defenses and histopathology in carp(Cyprinus carpio L.) with fed diets containing different doses(50,150 and 500 mg/kg) of cyadox,olaquindox and quinocetone respectively for 4,8 and 12wk.1.The growth performance of test groups were all increased.In 4 wk,the growth improvement in 50,150 and 500 mg/kg cyadox groups were 25.7%(P＞0.05),64.3% (P＜0.05),92.9%(P＜0.05),and 24.3%(P＞0.05),48.6%(P＜0.05),67.1%(P＜0.05) in 50, 150 and 500 mg/kg olaquindox groups,and 34.3%(P＞0.05),74.3%(P＜0.05),91.4% (P＜0.05) in quinocetone 50,150 and 500 mg/kg groups respectively.In 8 wk,there were 25.5%(P＞0.05),56.2%(P＜0.05),64.2%(P＜0.05) in 50,150 and 500 mg/kg cyadox groups,22.6%(P＞0.05),44.5%(P＜0.05),40.9%(P＜0.05) in 50,150 and 500 mg/kg olaquindox groups,and 27.7%(P＜0.05),40.9%(P＜0.05),58.4%(P＜0.05) in quinocetone 50,150 and 500 mg/kg groups respectively.In 12 wk,there were 14.6%(P＞0.05),38.7% (P＜0.05),45.7%(P＜0.05) in 50,150 and 500 mg/kg cyadox groups,9.5%(P＞0.05), 31.2%(P＜0.05),18.1%(P＞0.05) in 50,150 and 500 mg/kg olaquindox groups,and 14.1%(P＞0.05),28.1%(P＜0.05),37.2%(P＜0.05) in quinocetone 50,150 and 500 mg/kg groups respectively.Only in 12wk,the growth improvement in 500 mg/kg olaquindox group was lower than 500 mg/kg cyadox and quinocetone groups(P＜0.05).The results showed that 50～500 mg/kg cyadox and quinocetone had effective growth promotion. 50～500 mg/kg olaquindox can also increase growth performance,but decreases were found in 500 mg/kg group in 8 and 12 wk.The growth performance was decreased in all groups as feeding time prolonged.2.The hematological changes showed that the red blood cell counts(RBC), hematocrit,hemoglobin decreased with increased doses of olaquindox,and significantly dropped in olaquindox 500 mg/kg group in all periods(P＜0.05).The RBC,HGB and HCT in cyadox and quinocetone 50 and 150 mg/kg groups had an increased tendency, while it was decreased in 500 mg/kg groups.The RBC in cyadox 500 mg/kg group was significantly lower than cyadox 50 mg/kg group in 8 and 12 wk(P＜0.05).In quinocetone 500mg/kg group,it was lower than its 50 mg/kg group in all periods(P＜0.05).The changes of hematological parameters revealed a possible anemic state and a toxic manifestation of 500 mg/kg olaquindox.While diets containing 50 and 150 mg/kg cyadox and 50 mg/kg quinocetone could enhance erythropoiesis to increase the oxygen supply to the major organs in response to the high metabolic demand.3.Effects of quinoxalines on cortisol and biochemical indices in serum of carp were investigated.150 and 500 mg/kg doses of cyadox and olaquindox,500 mg/kg quinocetone can increase cortisol level in serum in 4 wk(P＜0.05),while in 8 and 12 wk, all groups showed good accommodation.The glucose contents in groups of cyadox and quinocetone had increased tendencies,while in 500 mg/kg olaquindox group,it showed a descent(P＜0.05).The activities of AST,ALT,alkali phosphatase(ALP) and lactate dehydrogenase(LDH) in 500 mg/kg olaquindox group increased in all periods(P＜0.05). The increased phenomenon of total bulirubin(TB) and direct bulirubin(DB) was also found in olaquindox group in 8 and 12 wk.Total protein(TP) and albumin(ALB) decreased in 12 wk(P＜0.05).The contents of creatinine(CRE) increased in 150 and 500 mg/kg olaquindox groups only in 12wk(P＜0.05).Blood urea nitrogen(BUN) contents raised in 500 mg/kg olaquindox group in 8 and 12wk(P＜0.05).The concentrations of serum ion didn’t change greatly in 4 and 8 wk(P＜0.05).In 12 wk,a decrease of Na~+ content was found in 500 mg/kg olaquindox group.The content of K~+ was increased in 500 mg/kg olaquindox group,the concentrations of P raised in cyadox 500 mg/kg group, 150 and 500 mg/kg olaquindox and quinocetone groups(P＜0.05).Values of other biochemical indices in serum,such as cholesterol(CHO),triglyeride(TG) and CL~-didn’t change greatly(P＞0.05).4.Cortisol and glucose contents,AST,ALT,alkali phosphatase(ALP) and lactate dehydrogenase(LDH) activities in serum changed significantly after stress(P＜0.05),they can be used as indicators of stress response.In 4 wk,cortisol levels after stress in all groups were increased with the same tendency as basal levels.It revealed an undamaged HPI axis.In 8 and 12 wk,500 mg/kg olaquindox did not had effects on basal serum cortisol levels(P＞0.05).Hower,challenging fish with stress revealed an impairment of the capacity to raise cortisol.While eortisol level in olaquindox 500mg/kg group after 24h confined in 12 wk was not decreased as metioned after chasing stress,but an increase (P＜0.05),it might suggest that a decrease of plasma clearance rate of cortisol was happened as time prolonged.The glucose content had a decent tendency in 500 mg/kg olaquindox group,and in this group,AST,ALT and ALP activities were induced after stress in all periods(P＜0.05),it indicated that stress enhanced the damage to cell membrane and intercellular metabolism caused by olaquindox.While inductions of AST, ALT and ALP activities were also found in 500 mg/kg cyadox,500 mg/kg quinocetone and 150 mg/kg olaquindox groups in 12wk(P＜0.05).5.Activities of superoxide dismutase(SOD) and glutathione-S transferase(GST), malondialdehyde(MDA) level in liver of carp pre-stress were also determined.There was a clear increase of SOD activity at the doses of 150 and 500mg/kg of the three quinoxalines,500 mg/kg olaquindox especially(P＜0.05).GST activity was decreased only in olaquindox 500 mg/kg group in 4 and 12 wk,while it was increased in 50 mg/kg cyadox and 150 mg/kg quinocetone groups in 12 wk(P＜0.05).All the three additives had little influence on MDA level(P＞0.05),an increase was only found in 500 mg/kg cyadox group in 12 wk(P＜0.05).From the results,we can conclude that olaquindox at the dose up to 500 mg/kg had oxidative damage on carp,while at the dosage range of out test, cyadox and olaquindox can improve the function of antioxidant defense systems. 6.The histopathological effects of cyadox,olaquindox and quinocetone on head kidney,kidney,liver,intestine of carp were investigated.Results showed time-dependent histopathological changes of all the tissues in fish in 500 mg/kg olaquindox group.Slight damage was found in 500 mg/kg cyadox and quinocetone groups with the time prolonged, while histological structure was less influenced by cyadox and quinocetone below the dose of 500 mg/kg,and olaquindox below 150 mg/kg.In conclusion,this study had evaluated the effects of cyadox,olaquindox and quinocetone on hematological and physiological response parameters,antioxidant defenses and histopathology in carp.It demonstrated that high dose of olaquindox had adverse effects on physiological status accompanied with alterations of physiological stress response of fish,and firstly gave the reason for stress hemorrhage syndrome caused by olaquindox.While cyadox and quinocetone were less toxic,deleterious effects were only found at the dose of 500 mg/kg for long-term use,stress hemorrhage syndrome would not happened after appropriate use.These results give scientific guidances for reasonable and safe use of quinoxalines in aquiculture.更多还原