【作者】 吕慎金；

【导师】 魏万红；

【作者基本信息】 扬州大学 ， 动物遗传育种与繁殖， 2008， 博士

【摘要】 本项研究以扬州市动物园及扬州平山堂养殖场梅花鹿为研究,采用扫描取样法、目标动物取样法研究了梅花鹿的昼间行为格局,分析了不同饲养条件下梅花鹿的行为差异以及对母性行为的影响,并探讨了游客密度对梅花鹿行为的影响;在分析不同饲养条件下梅花鹿行为格局的基础上,选择14个微卫星位点,研究了两个梅花鹿群体遗传多样性,并与梅花鹿行为性状进行关联分析;以与行为性状相关的BDNF和OPRK1两个基因为候选基因,利用PCR-SSCP技术研究了BDNF和OPRK1基因不同区段的SNP;并分析了SNP与梅花鹿群体行为性状之间的关系。研究结果如下:(1)梅花鹿昼间取食行为有2个高峰期,分别在7:30-9:30hr和16:30-17:30hr,其频次为7.35-10.10次/h之间;卧息行为高峰期在11:30-13:30hr,达到5.52-7.55次/h;观望行为主要发生在7:30-9:30及13:30-16:30hr之间;两地梅花鹿群体修饰行为和其它行为(排粪、排尿、饮水等)频次均较低。(2)不同饲养条件下行为比较表明,在7:30-8:30hr,鹿场公鹿取食行为极显著高于动物园(P=0.011),且在12:30-14:30和15:30-16:30hr公鹿取食行为频次也存在显著差异(P=0.002);公鹿的反刍行为除8:30-10:30hr不存在显著差异之外,其它时段均存在显著差异。鹿场公鹿在8:30-9:30hr卧息频次极显著高于动物园(P=0.002),在13:30-14:30及14:30-15:30hr存在显著差异(P=0.028, P=0.045),在10:30-11:30hr鹿场公鹿观望频次显著高于动物园(P=0.009),在12:30-16:30hr两群体观望频次一直表现为显著或极显著差异(P=0.002, P=0.044, P=0.046, P=0.000)。(3)两地母鹿取食行为频次在7:30-9:30hr显著差异,在12:30-15:30hr动物园母鹿取食频次上升,而鹿场母鹿取食频次下降,二者表现为显著差异(P=0.000, P=0.008, P=0.001)。鹿场母鹿反刍行为在9个时段均显著或极显著高于动物园。动物园梅花鹿在7:30-8:30hr观望频次较高,且和鹿场母鹿观望行为呈极显著差异(P=0.000)。(4)两地幼鹿取食行为频次在7:30-8:30hr呈现显著差异(P=0.038),在13:30-17:30hr鹿场幼鹿取食频次上升,而动物园幼鹿取食频次下降,二者表现为极显著差异(P=0.000, P=0.005, P=0.006)。幼鹿反刍频次集中在8:30-13:30hr,其中动物园幼鹿反刍行为主要集中在7:30-10:30hr,并有8个时段表现为显著或极显著差异。卧息行为有9个时段均呈显著或极显著差异,且鹿场幼鹿卧息行为频次一直低于动物园。(5)R型聚类分析表明,动物园梅花鹿行为性状简化为卧息和移动行为就基本能够判定个体的行为状态。而鹿场梅花鹿行为性状可以简化为卧息和修饰行为。(6)圈养条件下,仔鹿1-7日龄时母鹿的平均授乳时间约19min,且与其它两个时段的差异均达到显著水平;仔鹿1-7日龄和母鹿跟随相处时间平均达80min,且随日龄增加而下降,仔鹿跟随行为在三个日龄段的分析表明,两两之间都存在显著差异;母鹿对仔鹿的舔舐和修饰行为随日龄增加呈下降趋势,母鹿对仔鹿的寻找张望从最长每天4次左右下降到15-35日龄的每天不足1次。半散放条件下1-7日龄母鹿授乳时间平均为24min,且与其他两个时段的差异均达到显著水平;仔鹿1-7日龄和母鹿跟随相处时间为120min,跟随行为在三个日龄段两两之间都存在显著差异;分析表明不同饲养条件下仔鹿跟随行为以及母鹿对仔鹿的舔舐和修饰行为差异显著。(7)对公鹿、母鹿和幼鹿之间进行日行为差异分析表明,在游客高峰期,公鹿和母鹿的取食、卧息之间差异达到极显著水平(P<0.05),和母鹿的观望行为差异达到显著水平(P<0.05);母鹿和幼鹿的卧息、观望、移动行为之间差异都达到极显著水平(P<0.01),和幼鹿的取食行为差异达到显著水平(P<0.05)。统计5个游客密度梯度条件下公鹿、母鹿和幼鹿行为时间分配结果表明当游客密度对公鹿取食和观望行为产生显著或极显著影响,并随游客密度增加保持显著差异。游客密度也对母鹿观望行为产生显著影响(P<0.05),不同游客密度对幼鹿的行为的取食、观望、修饰行为产生显著或极显著影响(P<0.05)。(8)动物园梅花鹿群体14对微卫星位点的平均期望杂合度为0.441,观察杂合度为0.354,平均多态信息含量为0.332,各位点基因分化系数平均为0.231。鹿场梅花鹿14对微卫星位点分析表明,该群体平均期望杂合度为0.372,观察杂合度为0.300,平均多态信息含量为0.303,各位点基因分化系数平均为0.149。所检测位点为中度多态位点。(9)微卫星位点和梅花鹿行为性状进行最小二乘分析表明,TGLA53和BM4107位点与修饰行为显著相关;2AL2位点与观望行为有显著相关;2AL13和Mber14位点对卧息行为有极显著影响;BM6506位点对反刍和其它行为有显著影响;BL42位点对取食和卧息行为有显著影响。(10)在BDNF基因引物P11产物中检测到在其155bp发生C→T的突变;引物P12产物在其128bp处发生G→A的突变。OPRK1基因引物P21扩增片段在DNA序列72bp处发生G→A突变;P22扩增产物在132bp处发生T→A的突变。独立性χ2分析表明四对引物SSCP基因型均处于不平衡状态。(11)对BDNF基因SNP位点基因型与梅花鹿行为性状进行最小二乘分析表明,P11引物基因型对梅花鹿卧息行为有显著影响(P<0.05)。对移动行为有极显著影响(P<0.01);引物P12产物获得各基因型对卧息行为有显著影响(P<0.05),对观望行为性状有极显著的影响(P<0.01); P21引物基因型对梅花鹿修饰行为有极显著影响(P<0.01),引物P22各基因型对观望、卧息和修饰行为有极显著影响(P<0.01)。(12)综上所述,鹿场梅花鹿取食行为频次低于动物园,但其反刍和观望行为均显著高于动物园梅花鹿;综合分析认为动物园母鹿母性行为优于鹿场母鹿;游客密度对动物园梅花鹿的取食和观望行为产生显著影响;本研究所检测的14对微卫星位点均为中度多态位点,两地梅花鹿群体遗传多样性较丰富,分析表明有7个位点和行为性状存在显著相关;对BDNF和OPRK1基因的分析表明,4对引物的SSCP基因型均处于不平衡状态,初步确定BDNF和OPRK1基因可作为影响梅花鹿观望、卧息和修饰行为的候选基因,有待于进一步研究。更多还原

【Abstract】 The systematic investigation was conducted on the sika deer in the Yangzhou zoo and Pingshantang farm with scan sampling and focal animal sampling. The study observed the behaviour of sika deer, compared the behaviour rhythm and maternal behaviour among the male, female and young sika deer, compared the effect of visitor density on the behaviour in the two populations. The study also assayed the genetic diversity with 14 microsatellite loci in two populations, analyzed the correlation between the microsatellite locus and behaviour traits. The BDNF(Brain drive neuro-factor) and OPRK1(Kappa Opoioid Receptor 1) was detected as candidate gene which had correlation with behaviour traits. With the PCR-SSCP method to find the SNP of BDNF and OPRK1 in the different length, we analyzed the correlations between SNP and behaviour traits in the sika deer populations. The results were as follows:(1) The eating behaviour of sika deer had two peak periods, it was 7:30-9:30 and 16:30-17:30, and its frequency was 7.35-10.10 per hour in the peak time. The peak of relaxing behaviour was 11:30-13:30, the frequency was 5.52-7.55 per hour. The observing peak was 7:30-9:30 and 13:30-16:30, but the frequency of grooming and others was very low in all of the time. (2)The eating behaviour of farm male sika deer was different significantly from that of the zoo sika deer during 7:30-8:30, 12:30-14:30 and 15:30-16:30(P=0.002). The relaxing behaviour of farm male sika deer had higher significant difference than zoo. And during 13:30-15:30, there was significant difference(P=0.028, 0.045). The observing frequency behaviour of farm sika deer had higher than that of zoo sika deer. And the 12:30-16:30 periods, the observing behaviour had significant difference all the time(P=0.002, 0.044, 0.000).(3)The eating behaviour of female sika deer in the two populations had significant difference at the 7:30-9:30. The farm sike deer eating frequency in the zoo raised at the time of 12:30-15:30, but descended in the farm at same time(P=0.000, 0.008, 0.001). The ruminating behaviour of farm female sika deer had significant difference among 9 periods with the zoo population. The observing behaviour of sika deer in the zoo frequency was high and had high significant difference with farm female sika deer(P=0.000).(4)The eating behaviour of young sika deer had significant difference at 7:30-8:30 in two populations(P=0.038). At 13:30-17:30, the eating frequency of young sika deer in the farm ascended and the zoo descended(P=0.000, 0.005, 0.006). The ruminating frequency main happened at 8:30-13:30, and the young sika deer of zoo mainly happened at 7:30-10:30, there had significant difference among 8 periods. There were 9 periods that the ruminating frequency of young sika deer in the farm had higher than the zoo. There were 9 periods that the relaxing behaviour had significant difference, and the relaxing behaviour of young in the farm always was less than the zoo.(5) The R cluster results showed that the behaviour character of zoo sika deer can be summed with relaxing and moving, and the sika deer in the farm can be summed with relaxing and grooming.(6) The lactating time was about 19 minutes when the young sika was 1-7 days old in the farm, and the lactating time had significant difference with the other two periods. The young sika deer following behaviour was about 80 minutes at the age of 1-7 days, there had significant difference among the three periods. The time of female licking and grooming the young descended with the age of young sika deer ascended. The female searching and looking frequency was about 4 times at the young was 1-7 days old, but it was only below 1 times when the young was 15-35 days old. The lactating behaviour was about 24 minutes when the young was 1-7 days old in the zoo. And there was significant difference among three periods. The young following behaviour was about 120 minutes at the age of 1-7 day, and there was had significant difference among three periods. There was had significant difference with the following behaviour, grooming and licking behaviour with the different feeding conditions.(7)We compared the behaviours among male, female and young in two phase times, Male and female had a high significant difference of eating, relaxing behaviour in high visitor density(P<0.05), the result between male and young of observing behaviour were significant difference(P<0.05), female and male showed high significant difference of relaxing, observing and locomotion behaviour(P<0.01) and results between female and young of eating behaviour are similar(P<0.05). The results showed significant effect on male’s observing behaviour when visitor density reached 6 per minute, and with the visitor density ascended, the highly significant effect always had on the male. The visitor density had significant effect on the observing behaviour of female (P<0.05). The different visitor density had significant effect on the behaviour of eating, observing, grooming with the young(P<0.05).(8) The average of expected H (Heterozygosity) and PIC (Polymorphism Information Content) in the zoo of sika deer were 0.441 and 0.332 respectively, and the values of observed H was 0.354. The gene differentiation value was 0.231. For the sika deer in the farm, the average of expected H and PIC were 0.372 and 0.303 respectively, and observed H was 0.300. The gene differentiation was 0.149.(9) The loci of TGLA53, BM4107 had significant or highly significant effect on the grooming behaviour, as well as 2AL2 locus on the observing behaviour, 2AL13 on the relaxing behaviour, BM6506 locus on the behaviour of ruminating and others, BL42 on the eating and relaxing and Mber14 on the behaviour of relaxing.(10)We detected the mutation of C→T at the 155bp site with primer P11 of BDNF, and mutation of G→A was founded at the site of 128bp with P12 primer. With the primer P21 of OPRK1 gene, the mutation of G→A was discovered at the site of 72bp and T→A at the 132bp site was taken out with the primer of P22.(11)The results showed that the SNP loci of BDNF gene had significant or high significant effect on the relaxing and moving(P<0.05, 0.01) with the primer P11. In the primer P12, the gene type of SNP had significant effect on the relaxing and observing(P<0.05). The results showed that the gene type of primer P21 had high significant effect on the grooming behaviour(P<0.01), and there had significant effect on the observing, relaxing and grooming behaviour with the primer of P22(P<0.01).(12)The eating frequency of farm was less than the zoo sika deer, but the ruminating and observing was more than the zoo. The maternal behaviour of zoo sika deer overmatched the farm. The visitor density had significant effect on the eating and observing behaviour of sika deer in the zoo. The 14 microsatellite loci were all in middle polymorphism, and the genetic diversity was just in high, 7 loci was significant correlation with the behaviour traits. The four primers of the BDNF and OPRK1 gene were all in disequilibrium state in the sika deer. We suggest that the BDNF and OPRK1 gene are worthy of being further studies as candidate genes having effects on the behaviour traits.更多还原