蓝狐自咬症及遗传学基础研究
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摘要
蓝狐是世界上广泛饲养的珍贵毛皮动物,狐皮是制作高档裘皮服装和饰品的重要原料。自咬症的发生,严重影响了蓝狐的生长发育和毛皮质量,给蓝狐养殖业带来巨大的经济损失。本研究从行为学、环境因素、遗传因素等方面分析自咬症的发病机理,筛选自咬症蓝狐RAPD分子标记,探讨DRD1、DRD2、5-HT_1AR候选基因与自咬症的关联性。主要结果如下:
     1.选用体重相近的健康蓝狐90只(公母各半),其中试验组60只,对照组30只,模拟蓝狐在饲养管理过程中不良环境,采用摇床、转移栋舍、限制、拥挤、饥饿、休息等6种不同的应激诱导方式并结合10种血液生化指标探讨蓝狐自咬症的发病原因。应激诱导30d,试验组血清中GPT、ALP、GSH-PX和CAT活性极显著低于对照组(P<0.01),MDA含量显著低于对照组(P <0.05),SOD活性极显著高于对照组(P<0.01);应激诱导60d,ALP、CK、GSH-PX、CAT和MDA活性极显著高于对照组(P<0.01),SOD活性极显著低于对照组(P<0.01),GPT活性显著低于对照组(P<0.05);GOT、LDH、GLU活性在整个应激诱导过程中没有显著差异(P>0.05);应激诱导组与对照组蓝狐自咬症发病率没有显著差异(P>0.05)。
     2.选用4只健康蓝狐和4只自咬蓝狐相间放置,用监控摄像头进行行为学观察,在蓝狐的各类行为中,所占观测时间百分比由高到低依次为趴卧、走动、玩耍、采食、站立、梳理、抖毛;健康蓝狐的趴卧行为、玩耍行为明显高于自咬蓝狐;走动、抖毛和梳理行为明显低于自咬蓝狐。自咬蓝狐单次自咬持续时间在10秒钟以下的占70%以上,超过40秒的在5%以下;病情较重的自咬蓝狐自咬次数和持续时间都明显高于病情较轻的蓝狐。
     3.选用自咬蓝狐15只,健康蓝狐10只,分为三组进行繁殖,探讨自咬症与遗传的关系,并对试验基地蓝狐自咬发生情况进行统计分析。试验基地蓝狐自咬发病时间主要集中在8~10月份,基地蓝狐群的发病率为2.28%,发病较高的栋舍达4.17%,环境应激诱导试验群发病率为5.56%。30天分窝,自咬蓝狐存活的2窝后代全部自咬,发病率为100%,最早出现自咬的时间为44日龄,2窝健康蓝狐后代发病率为16.67%,蓝狐自咬症的发生受遗传影响比受环境影响要大。
     4.以DRD1、DRD2、5-HT_1AR基因为候选基因,采用直接测序方法探讨基因多态性与自咬症的关联。克隆测序得到蓝狐DRD1、DRD2、5-HT_1AR基因部分外显子序列长度分别为864bp、819bp和611bp。在DRD1、DRD2、5-HT_1AR基因部分外显子序列共检测到8个突变位点,其中DRD1基因T206C位点处碱基突变导致的基因多态与蓝狐自咬症有极显著关联(P<0.01);DRD2基因T356C位点、DRD2基因T457C位点、5-HT_1AR基因C351G位点处碱基突变导致的基因多态与蓝狐自咬症有显著关联(P<0.05);DRD1基因C314T位点、DRD1基因C668T位点、DRD2基因G115A位点、DRD2基因G400T位点处碱基突变导致的基因多态与蓝狐自咬症没有关联(P>0.05)。
     5.采用RAPD技术,研究与自咬症紧密连锁的遗传标记,从120条随机引物中,筛选出30条能产生清晰、稳定扩增产物且具多态性的随机引物,在自咬和健康蓝狐群中多态率为39.17%。其中6条随机引物在健康蓝狐和自咬蓝狐DNA池扩增出了差异条带,经个体验证,引物S472、S485在健康蓝狐和自咬蓝狐出现差异条带的个体数量差异达极显著水平(P<0.01),可以用作蓝狐自咬症的分子标记。
Blue fox is a precious fur-bearing animal widely distributed over the world,the pelt of blue fox is an important raw material for high-class fur garment. The self-biting disease of blue fox can cause serious influence on its growth and the pelt-quality, eventually cause considerable economic losses in blue fox farming. This study probed the mechanism of self-biting disease in terms of ethologic, environmental factor, genetic factor, screened RAPD molecular marker and explored the correlation between DRD1、DRD2、5-HT1AR candidate gene and self-biting disease. The results were as follows:
     1. 90 healthy blue foxes (half male and half female) with similar body weight were randomly divided into two groups, 60 in the stress-induced group, while 30 in the control group. The experimental group were given induced-stress imitating harmful environmental stress of rearing, bed swinging, shed transferring, confined in cage, overcrowded in cage, starvation and resting stress, combined with 10 serum biochemical indicator to explore the cause of self-biting. Induced for 30 days, enzyme activity in trial group were extra-significantly lower than the control group in GPT, ALP, GSH-PX, and CAT (P<0.01), MDA content was significantly lower than the control group (P<0.05), while SOD activity was significantly higher than the control group (P<0.01). The activity of ALP, CK, GSH-PX, CAT and MDA, induced for 60 days, were significantly higher than the control group (P<0.01), SOD activity was significantly lower than the control group (P<0.01) while GPT activity was significantly lower than the control group(P<0.05), the activity of GOT, LDH and GLU were not affected (P>0.05). Self-biting ratio showed no significantly difference between stress-induced and control blue foxes (P>0.05).
     2. 4 healthy foxes and 4 self-biting foxes were alternately put in the cages for ethologic study, video-recorded by cameras. In all behaviors of blue foxes, the percentage of recording time from high to low was as follows: lying behavior, slow pace, playing behavior, grazing behavior, standing behavior, grooming behavior and shaking behavior. Healthy blue foxes were more likely to do lying behavior, playing behavior than in self-biting ones and slow pace, grooming behavior, shaking behavior were obviously lower than self-biting ones. More than 70% self-biting foxes took less than 10s to bite themselves each time, The ones that spent more than 40s biting themselves only took less than 5%.The more serious the illness was, the more time the foxes spent on biting themselves.
     3. 15 self-biting and 10 healthy foxes were divided into three groups to breed in order to investigate the relationship between self-biting and heredity. The result showed that self-biting happened mainly from August to October, the self-biting rate in experimental fur farming was 2.28%, the highest shed was 4.17%, the stress-induced foxes was 5.56%.Two den generations of self-biting were all self-biting weaned at 30 days, the rate was 100%, the earliest time was 44 days, while the self-biting rate of healthy generations was 16.67%. the occurrence of self-biting influenced by heredity was more likely than environment.
     4. DRD1, DRD2 and 5-HT1AR gene were chosen as the candidate genes to study the correlation between gene polymorphism and self-biting disease by direct sequencing. Part of DRD1, DRD2 and 5-HT1AR gene exon of blue fox were cloned, the length of whole sequences is 864bp, 819bp and 611bp respectively. gene polymorphism of T206C in the DRD1 gene had a distinguished tendency to self-biting disease (P<0.01), gene polymorphism of T356C in the DRD2 gene, T457C in the DRD2 gene, C351G in the 5-HT1AR receptor gene have a relationship with self-biting disease (P<0.05), gene polymorphism of C314T in the DRD1 gene, C668T in the DRD1 gene, G115A in the DRD2 gene, G400T in the DRD2 gene had no relationship with self-biting disease (P>0.05).
     5. RAPD technique was used to screen close linkage molecular marker of self-biting disease. Among 120 random primers, 30 primers showed polymorphism and can amplified stable and clear PCR products, the percentage of polymorphism was 39.17%. 6 random primers amplified specific fragments in healthy cisterna DNA and self-biting cisterna DNA. Detected by individuals, the numbers with specific fragments amplified by primer S472 and primer S485 in the healthy foxes and self-biting foxes showed significantly difference (P<0.01), theses two primers could be used as molecular marker of self-biting disease.
引文
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