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安徽野生猕猴实验动物化及其种质特异性研究
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摘要
安徽猕猴作为一种新的猕猴亚种种质资源,尚未被大规模开发利用。本研究通过对野生和笼养猕猴的生物学特征进行调查,明确其种质特异性,为新的实验动物模型开发奠定基础。通过建立一普通级实验猕猴封闭群,探讨野生猕猴实验动物化的标准路线和技术方法。本文对笼养状态下野生猕猴和子一代猕猴的行为特征、形态特征、生理特征和遗传特征分别做了调查,主要研究结论如下:
     采用扫描取样法,对猕猴的交配行为、攻击行为和友好行为观察结果表明,在交配期和非交配期,野生猕猴交配行为均大于子一代猕猴,差异性极显著(P<0.01);在交配期和非交配期,野生猕猴攻击行为均大于子一代猕猴,差异性显著(P<0.05);在交配期和非交配期,野生猕猴的友好行为与子一代猕猴无显著性差异(P>0.05)。野生猕猴的交配行为、攻击行为均大于子一代猕猴,主要由于子一代猕猴大多属于亚成年个体,尚未达到性成熟,加之性情较温驯所致。统计分析结果表明,笼舍内野生猕猴雌雄比为6:1、子一代猕猴雌雄比为1:1时,猕猴的攻击行为发生频次较低,友好行为发生频次较高,可见,成年猴1雄多雌、未成年猴1:1的性别配比较为合理。
     利用SPSS for Windows17.0对猕猴的形态特征进行抽样测量与统计结果显示,成年雄性形态学指标高于成年雌性,亚成年雄性形态学指标高于亚成年雌性。成年雄性、成年雌性和亚成年雌性的体重变量具有显著的性别差异(P<0.05),而躯干长变量具有显著的年龄差异(P<0.05),而野生猕猴和子一代猕猴形态指标之间差异性不显著(P>0.05),说明笼养状态下的环境条件和饲养管理未对猕猴的生长发育造成显著影响。
     非麻醉状态下测得的猕猴常规生理指标和血液生化指标表明,常规指标雌雄间差异不显著(P>0.05),与相同条件下所测得的其它亚种猕猴生理指标相比,均处于正常范围之内。血液生化指标碱性磷酸酶(ALP)、甘油三脂(TG)和谷氨酰基转移酶(GGT)野生猕猴雌雄间差异显著(P<0.05),其余指标无显著性差异。子一代猕猴碱性磷酸酶(ALP)、白蛋白(ALB)、血清Ca、甘油三脂青(TG)、谷氨酞基转移酶(GGT)雌雄间差异显著(P<0.05),且雌雄所得数值均比雄性低,其余指标雌雄间无显著性差异。将感染BV阳性的猕猴与感染BV为阴性的猕猴所测得的生化指标值相比较,除谷草转氨酶(AST)、尿素氮(BUN)、血清总胆固醇(CHOL)外,其余指标均有显著性差异,这可能与采样时猕猴所处的状态以及自身免疫和生理状况有关。
     利用微卫星分子标记技术,筛选出14对适合对安徽猕猴进行遗传学研究的位点。通过野生猕猴和子一代猕猴两个群体的遗传结构分析显示,安徽野生猕猴平均等位基因数为10.7,平均杂合度为0.7987,平均多态性信息含量为0.7713;子一代猕猴平均等位基因数为11.1,平均杂合度为0.7639,平均多态性信息含量为0.730。子一代种群与野生群相比,平均杂合度和多态性信息含量有所降低,经差异性检验(P>0.05),因此,遗传多样性并未显著降低,说明经人工驯养的遗传管理措施是合理的。为防止近亲交配,筛选出适合于安徽猕猴亲子鉴定和种群谱系构建的6对微卫星引物,利用这6对引物组成的鉴定体系对已知父母本的猕猴个体进行父权鉴定,鉴定正确率达到95%以上。
     在封闭群建立过程中,本研究分别对野生猕猴和子一代猕猴所感染的B病毒、寄生虫和病原菌进行检测发现,寄生虫和病原菌均达到普通级标准。因此,要建立普通级种群,B病毒是要排除的必要且唯一的项目。通过对环境参数和饲料营养成分检测发现,本中心猕猴的生活环境和营养状况基本达到普通级标准。为明确实验猕猴的遗传背景需构建准确的潜系档案,本文筛选出6对适合进行遗传监测和亲子鉴定的微卫星引物,通过对35只猕猴父权鉴定,摸索出一条适合本中心猕猴亲子鉴定的技术路线,采用此方法可以对更多个体进行亲权鉴定。
     总之,本研究摸索出了一条野生猕猴实验动物化的基本路线和技术方法。通过对安徽猕猴的生物学特征进行调查,对其行为特征、形态指标、生理生化和遗传特性有了一定的了解,为此亚种猕猴种质特异性的进一步明确和新的实验动物模型开发奠定了基础。
As a new genetic resource of macaque subspecies, rhesus macaques from Anhui province haven't been empoldered. Germ plasma characteristics were studied by investigating their biological characteristics, thus this can build the basis for experimental study models development. The technology pathway of laboratory rearing was studied when CV closed colony was established. Behavioral characteristics, morphological characteristics, physiological characteristics and hereditary characteristics were surveyed, the main results as follows:
     Copulation behavior, aggressive behavior and amicable behavior were observed by scan sampling in mating and no-mating season, there existed significant difference (P<0.01)in the copulation behavior of wild macaques outweigh that of the first filial generation. In mating and no-mating season, obvious difference existed in aggressive behavior (P<0.05) and no obvious difference existed in amicable behavior. There were significant difference between wild macaques and the first filial generation in copulation behavior and aggressive behavior, because the first filial generation macaques were mainly sub-adult, furthermore, they were good-natured. The aggressive behavior rate was lower and the amicable behavior rate was higher when the sex ratio of female to male was6:1and1:1in wild macaques and in the first filial generation cages, respectively. Therefore, one male and several females in the wild cage and the same number between males and females in the first filial generation cage were reasonable.
     Physiological parameters were measured by SPSS for Windows17.0, the weight of adult males, adult females and sub-adult females have significant sexual difference (P<0.05). the trunk length had significant difference (P<0.05) among different age groups. There was no obvious difference between wild macaques and the first filial generation macaques (P>0.05). which can explained that the macaques were not influenced bv environmental conditions.
     Routine physiological Indexes and biochemical parameters indicated that, there was no obvious difference between males and females (P>0.05). They were on normal level in the same conditions. There existed significant difference in ALP, TG and GGT between wild males and females (P<0.05), and no obvious difference in other parameters. There existed significant difference in ALB, Ca, TG and GGT between the first filial generation males and females (P<0.05). There existed obvious difference between BV positive individuals and BV negative individuals except AST, BUN and CHOL, which mainly because physiological indexes and biochemical parameters were influenced by their immunity and physiological state.
     14microsatellite loci primers were selected by PCR-based typing. Genetic structure of wild macaques and the first filial generation were analyzed. The average number of allete was10.7and11.1, respectively. The average heterozygosity was0.7987and0.7639, respectively. The average polymorphic information content was0.7713and0.730, respectively. Compared by wild macaques, the genetic diversity of the first filial generation was decreased. In order to prevent close mating, we selected6primers for paternity verification, the accurate rate exceeded95%.
     In the process of closed colony establishment, BV, parasite and pathogenic bacteria of the wild macaques and the first filial generation macaques were detected. Results showed that all the indexes had come to CV level. Therefore, BV was the only and necessary exclusion. The environmental quality parameters and indexes of main nutrition were up to CV standard. Pedigree was needed to be constructed so as to define the genetic background.35macaques were tested by6loci, and the technology pathway was found, thus large-scale individuals could be identified.
     On the whole, the basic technology pathway and methods were found. Behavioral characteristics, morphological characteristics, physiological characteristics and hereditary characteristics were learned about. This could provide a basic theory for further definition of genetie resources and development of new experimental animal models.
引文
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