【作者】 孙得发；

【导师】 王建华；

【作者基本信息】 西北农林科技大学 ， 临床兽医学， 2011， 博士

【摘要】 水貂自咬症是水貂无意识的一种自我损伤行为,但是并不清楚这一种异常行为的生物学基础。本研究用当年繁殖和断奶后的育成水貂,通过自咬症水貂和临床健康水貂行为学的比较研究;机体抗氧化酶活性和甲基汞与总汞的测定;钙离子通道抑制剂对自咬行为的影响研究;抗氧化微营养维生素E以及亚硒酸钠对水貂自咬症发生的影响研究和水貂神经系统病理组织学研究,探讨导致水貂自咬症的原因。本项目通过以上研究取得以下成果:1水貂自咬行为规律及管理应激对水貂行为的影响随机选择当年断奶的60只自咬症水貂和60只临床健康水貂,公母各半,每个组3个重复,每个重复20只水貂,公母各半,每天观察水貂自咬行为发生的时间规律;通过转群以及连续3 d的模拟捕捉和注射,观察管理应激对自咬症水貂和临床健康水貂自咬频率和行为呆板症发生率的影响。结果表明,水貂自咬行为呈现间歇发作,发病时间主要集中在夜间和投喂食物前后,每年的7月和9月是自咬症的发病高峰期,自咬频率在该时期也最高。自咬症水貂和临床健康水貂均有行为呆板症发生,发生率分别为19.44%和11.67%;转群、连续3天的捕捉和注射会刺激自咬症水貂,水貂自咬频率显著升高,但随后的6 d和9 d呈现线性下降。管理应激同样也提高了健康水貂和自咬症水貂行为呆板症的发生率,但第6 d和第9 d也呈现下降的趋势。研究结果表明,水貂自咬行为是一种间歇性发作的神经行为疾病;行为呆板症在健康水貂和自咬症水貂群体中都有发生;转群、捕捉和注射等管理应激会刺激水貂自咬行为和行为呆板症的发生。2自咬症水貂机体抗氧化系统与汞残留的相关性研究随机选择健康和自咬症水貂雌雄各10只,心脏采血,用血液自动分析仪测定血液生理生化指标。随机选择自咬症和临床健康水貂各20只,空气心脏注射致死,快速去皮,采集肝脏、肾脏、大脑和肌肉样品,用于机体抗氧化酶活性、总汞和甲基汞等指标的测定。研究结果表明,自咬症水貂和临床健康水貂的红细胞数、血红蛋白含量、红细胞平均容积、血小板数等指标没有差异,但是白细胞数均极显著地高于临床健康水貂(p<0.01)。自咬症水貂肝脏和脑中的GSH水平明显低于健康水貂(p<0.01)。其中脑中的GSH水平只有正常水貂的76%。自咬症水貂体肝脏和大脑中VE水平分别比正常水貂低22.5%和21.1%。自咬症水貂两器官中总SOD和Cu/Zn-SOD,以及肝脏中的Mn-SOD显著或极显著地高于对照组(p <0.05或p <0.01)。自咬症水貂肝脏和大脑中的CAT酶活性均极显著地降低(p <0.01),其中肝脏降低34% ,大脑中降低21%。SBB水貂机体内GPx活性也有降低,但只有大脑中明显低于正常水貂。自咬症水貂大脑和肝脏中MDA分别比健康水貂高51%和22%。自咬症水貂大脑和肝脏中蛋白质羧基化程度明显高于健康组水貂。与健康组相比,自咬症水貂大脑和肝脏中PC分别高30 %和18.5 %。自咬症水貂大脑Ca2+-ATPase活性显著低于健康水貂,NOS活性也显著低于临床健康水貂组。自咬症水貂机体内甲基汞和总汞蓄积均显著高于健康组,其中自咬症水貂肝脏和大脑甲基汞分别比对照组高20%和48.8%,总汞分别比对照组高16%和44.8%。但自咬症水貂肾脏甲基汞和总汞含量与对照组没有显著差异。自咬症水貂甲基汞及总汞与GSH及MDA之间的相关性同健康组不同。在健康组,只有大脑中的MeHg及THg浓度与GSH水平成负相关。而肝脏、肾脏和大脑中氧化应激生物标记物MDA水平与MeHg及THg浓度之间均没有相关性。在自咬症水貂组,肝脏和大脑中的甲基汞浓度,以及大脑中总汞浓度均与同一组织中的GSH水平呈明显的负相关(p<0.01)。被测三器官中的MDA与MeHg及总汞均呈正相关。3 VE亚硒酸钠复合剂和尼莫地平对水貂自咬行为的影响研究试验选择72只标准色自咬症水貂,雌雄各半,随机分为3个组,每个组3个重复,每个重复8只自咬症水貂。试验分为3个处理,分别为对照组、尼莫地平组、维生素E+亚硒酸钠+尼莫地平组。对照组为基础日粮组;尼莫地平组:在基础日粮中添加25mg/kg的尼莫地平。每只水貂的饲料投喂量为200g左右/d。每只自咬症水貂每天尼莫地平的摄入量为5mg左右;维生素E+亚硒酸钠+尼莫地平组:在日粮中添加50ppm维生素E、5ppm亚硒酸钠和25ppm尼莫地平。研究钙离子通道抑制剂和抗氧化维生素对水貂自咬症的影响。试验结果表明,在日粮中添加钙离子通道抑制剂后,水貂自咬症的发生逐渐下降,但是到12d以后,这种逐渐消失,自咬症发生频率又有一定的升高,但在试验的21d中,自咬频率都低于对照组;尼莫地平和维生素E+亚硒酸钠复合剂共同作用显著地降低了自咬症水貂自咬发生频率,这种变化在投药的第9d基本保持较为稳定,而且自咬症的发生频率没有再次上升。4 VE和亚硒酸钠对水貂自咬症发病率及机体抗氧化酶水平和汞残留的影响选择当年断奶的1920只没有自咬行为的仔貂,随机分为2个组,每个组4个重复,每个重复240只水貂。试验组在养殖场常规日粮基础上额外添加50ppm维生素E和5ppm亚硒酸钠,研究其对自咬症的发生率的影响;随机选择实验组和对照组水貂各32只,空气注射法处死水貂,采肝脏、大脑和肾脏样品,测定GSH、VE和抗氧化酶活性和甲基汞残留。实验结果表明,维生素E和亚硒酸钠添加组均极显著地显著降低了水貂自咬症的发生率(p<0.01)。在水貂日粮中添加维生素E和亚硒酸钠复合剂极显著地提高了水貂肝脏维生素E的水平(p<0.01),实验组肝脏和大脑中的GSH水平都显著高于对照组(p<0.05)。维生素E+亚硒酸钠复合剂能够极显著地提高水貂大脑和肝脏组织中的GPx水平(p<0.01);同时也极显著提高了水貂肝脏CAT的活性(p<0.01),但对大脑的CAT活性影响不大。实验组肝脏和大脑中CuZn-SOD活性显著或者极显著地低于对照组(p<0.05 or 0.01),而T-SOD、Mn-SOD活性无显著差异。维生素E+亚硒酸钠复合剂能够显著降低水貂大脑和肝脏中甲基汞的含量(p<0.05),但是对肾脏中甲基汞的含量无显著影响。维生素E+亚硒酸钠复合剂极显著降低了水貂肝脏和大脑MDA的水平(p<0.01)。5自咬症水貂神经系统病理组织学研究试验采集20只临床健康水貂和20只自咬症水貂的大脑、小脑、延脑和脊髓等神经系统组织,用体积分数4%多聚甲醛固定后经选样、脱水、透明、石蜡包埋、切片和H.E染色,在200～400倍光学显微镜下进行观察并对比病理变化。显微观察结果表明,与临床健康水貂相比,自咬症水貂大脑中普遍存在神经细胞肿胀、溶解以及神经细胞卫星现象;部分神经锥体细胞固缩变形,细胞膜周边出现空泡化,神经纤维溶解消失,神经细胞核固缩浓染;部分自咬症水貂大脑的髓质出现空泡化,神经胶质细胞膜内侧浓染;小脑中的浦肯野氏细胞普遍存在肿胀、融解以及神经髓鞘溶解现象。延脑神经细胞肿胀,大胶质细胞膜溶解消失,神经椎体细胞周边出现严重的卫星现象;脊髓神经细胞明显肿胀,细胞膜消失,神经细胞核固化萎缩。病理组织学研究结果表明,水貂自咬症与神经细胞膜结构损伤、神经传导细胞以及神经髓鞘的病变有关。更多还原

【Abstract】 Self-biting behavior was unconscious self-injurious behavior in mink,but little was known about the biological basis of this disorder. All minks in this study were young mink weaned present year in Jinzhou mink farm in Dalian. The subject of this research was to compare the behavior of self-biting mink and health mink, to test anti-oxidative enzyme activity, methyl mercury (MeHg) and total mercury (THg) , to determine the effect of Ca2+ channel inhibitor on self-biting behavior, to research on the effect of VE+Na2SeO3 on self-biting behavior and oxidative enzyme activity. The nervous system pathological changes in mink with self-injurious behavior also were investigated in this study. The research result saw as below:1 Rules of self-biting behavior and the effect of management stress on behavior in farmed mink.60 self-biting minks and 60 clinical health minks (half male and half female) were choose from weaned mink in current year, each group 20 minks(half male and half female); investigated the self-biting behavior regular. Moved mink house, and caught and injected within 3 day to research the effect of managing stress on self-biting frequency and stereotypical behavior in mink. The result shown that self-biting behavior was intermittent occurrences; self-biting time was centered in night and the time before feeding. July and September is the peak of self-biting behavior, and self-biting frequency also reached the peak data. Both self-biting group and clinical health group have stereotypical behavior. The rate of incidence of stereotypical behavior was 19.44% and 11.67% respectively. Moved mink house, and catching and injecting within 3 day increased the self-biting frequency significantly, but it shown the down trend in next 6 and 9 day. This managing stress also increased stereotypical behavior rate in health and self-biting mink, it has same trend with self-biting frequency. Self-biting behavior is intermittent occurrence neurologic behavioral disease; stereotypical behavior happened both in self-biting mink and health mink. Moving house, catching and injecting stimulated the incidence of self-biting and stereotypical behavior.2 Anti-oxidative system and correlation with Hg residue of self-biting mink10 male and female minks were selected randomly from self-biting and health mink respectively, taken blood by cardiac puncture, to test hematological and biochemical Indexes of self-biting and health mink. 20 health and self-biting minks were selected randomly, heart-air-injection to death and take liver, kidney, brain and muscle samples to test the anti-oxidative enzyme activity, THg and MeHg. The results shown that, RBC, Hgb, MCV, MCH and BPC had no difference between self-biting mink and health mink. But WBC of self-biting mink was higher significantly than health mink(p<0.01). GSH level in self-biting mink was lower than health mink( p<0.01). The content of GSH was only 76% compare with normal mink. VE level in liver and brain of self-biting mink also was 22.5% and 21.1% lower than health mink. SBB minks had higher total superoxide dismutase (SOD), Cu/Zn-SOD and Mn-SOD activities in both tissues (p <0.05 or p <0.01). Catalase (CAT) activities in liver and brain tissues of SBB minks were markedly lower than those of normal group (p <0.01). GPx activities also lower than normal mink, but only have significant different in brain. SBB minks produced more MDA with 51% increase in brain (p <0.01) and 22% increase in liver (p <0.01) compared to normal minks. Similarly, the hepatic and cerebral PC in SBB minks were 18.5% (p <0.01) and 30% higher (p<0.001), respectively. Ca2+-ATPase and NOS activities in the brain of SBB minks were lower than health mink (p <0.01). SIB minks accumulated more cerebral MeHg and hepatic THg compared to the normal minks (p <0.05 or p <0.01) with 48.8% increase in brain and 20% increase in liver of MeHg, 16% increase in liver and 44.8% increase in brain of THg. But there were no significant differences in kidney between the two groups. The correlation between MeHg or THg accumulation and GSH or MDA levels in SIB group was not consistent with that in normal group. In normal group, only cerebral GSH had negatively correlation with MeHg or THg concentrations. While in SIB group, both cerebral and hepatic GSH level had negative effect on MeHg accumulation, and a significantly positive correlation between MDA and MeHg or THg was found in all of the three tissues.3 The effect of VE+Na2SeO3 and Nimodipine on self-biting behavior in mink72 standard color minks with self-biting behavior were selected, half male and half female, divide into 3 group, 3 repeats in each group and 8 self-biting minks in each repeat. 3 treatments were control, Nimodipine and Nimodipine+VE+Na2SeO3 respectively. In Nimodipine group, 25ppm nimodipine was add in mink feed. The feed supply was 200g each mink each day. Nimodipine intake was 5mg each mink each day. 50ppm VE, 5ppm Na2SeO3 and 25ppm nimodipine was added in mink feed in Nimodipine+VE+Na2SeO3 group. The study of this research was to determine the effect of Ca2+ channel inhibitor and Vitamin E and Na2SeO3 on the self-biting behavior. The result shown that, self-biting frequency decreased remarkable in the beginning, but this effect reduced day by day after 12 d in Nimodipine group, the self-biting frequency increased again. But the self-biting frequency lower than control group in experiment period. Nimodipine+VE+Na2SeO3 decreased self-biting frequency significantly after 9 d of treatment, this effect continued in all experiment period.4 The effect of VE+Na2SeO3 on accidence rate of self-biting and anti-oxidative enzyme activity and Hg residue in mink.1920 weaned minks were selected randomly and divided into 2 groups, each group has 4 repeats, 240 minks in each repeats. 50ppm VE and Na2SeO3 add in feed of treatment group to research the effect on accident rate of self-biting. Selected 32 minks in every group before take fur in winter, heart-air-injection to death and taken liver, kidney and brain samples to test GSH, VE level, anti-oxidative enzyme activity and MeHg residue in tissues. Experiment result shown that, VE+Na2SeO3 group decreased the self-biting accident rate of mink significantly(p<0.01). VE level increased in liver in treatment group(p<0.01), GSH level in liver and brain also higher than control group(p<0.05). VE +Na2SeO3 improved GPx activities in brain and liver(p<0.01), and CAT in liver also increased in treatment group(p<0.01), but have no effect on CAT activity in brain tissue. CuZn-SOD activities in liver and brain were lower than control group( p<0.05 or 0.01),but no effect on T-SOD、Mn-SOD. VE+Na2SeO3 decreased MeHg level in brain and liver significantly (p<0.05), but have no effect on HeHg level in kidney. VE+Na2SeO3 treatment decreased MDA level in liver and brain significantly (p<0.01).5 Histopathological studies on nervous system in self-biting behavior minkThe cerebrum, cerebellum, medulla oblongata and medulla marrow from each of 20 healthy minks and 20 self-injurious minks were collected in current study. All samples were fixed in 4% paraformaldehyde, followed by dewatering, hyalining, paraffin embedding, slicing and hematoxylin eosin staining. All sections were observed at 200- 400 times light microscope. Compared with clinical health mink, histopathological changes were found in nervous system of minks with self-injurious behavior. Swelling, lysing and microsatellite phenomenon of nerve cells were observed in the cerebrum. Many nuclei in nerve cells became pyknotic (shrunken and dark). Vacuolizations were observed in cerebrum of some minks with self-injurious behavior. Intracellular membrane of neuroglia cell was deeply stained. Swollen and lysed Purkinje cells were investigated in cerebellum, and some of the myelin sheaths were lysed in this region. Neurocyte swelling, membrane ablation of macroglia and serious microsatellite phenomenon were observed in medulla oblongata of SIB mink. Nerve cells pyknotic and membrane lysed also found in spinal marrow. Self-injurious behavior of minks has correlation with pathological changes of membrane of nerve cell and nerve myelin sheath.更多还原