【作者】 谢全森；

【导师】 杨振才；

【作者基本信息】 河北师范大学 ， 动物学， 2011， 博士

【摘要】 本论文以水生爬行类模式动物中华鳖(Pelodiscus sinensis)为研究对象,利用生理生态、营养学以及生物化学等现代生物学技术,研究了禁食及低蛋白限制后恢复投喂对中华鳖补偿生长反应、中华鳖RNA/DNA比值的昼夜节律及其与投喂制度的关系以及低蛋白饲料胁迫下的中华鳖RNA/DNA比值变化及其与摄食生长的关系。研究内容包括:多重周期禁食-饱食、不同禁食时间及蛋白质限制后恢复对中华鳖补偿生长影响;中华鳖RNA/DNA比值的昼夜节律及其与投喂制度的关系;低蛋白饲料胁迫下的中华鳖RNA/DNA比值变化及其与摄食生长关系。通过研究中华鳖的补偿生长,为不同分类类群间补偿生长的比较和水产养殖的投饵制度研究提供资料,有助于了解补偿生长在野外种群生态和进化上的意义;通过研究中华鳖RNA/DNA比值是否存在昼夜节律,该节律是否受到投喂制度的影响,从而可以确定测定RNA/DNA比值适宜的取样时间和取样组织,为科学合理的投喂制度的确立提供参考;不同蛋白水平下SGR-R/D模型的建立,可以用R/D快速评价中华鳖在摄食不同蛋白饲料的瞬时生长率,为其它生物的生长模型建立提供基础资料。主要研究结果如下:1、在30±0.5℃下,对初体重17.61±0.46g的中华鳖进行了8周周期性禁食后饱食投喂养殖实验,对照组(C组)连续饱食投喂,处理组T1、T2、T3分别周期性禁食1d、2d和3d,再投喂6d、5d和4d。实验结束时,T1组最终体重和对照组差异不显著(P>0.05),而极显著高于T2和T3组(P<0.05);摄食率和特定生长率随每周禁食时间延长而降低,差异不显著(P>0.05);饵料系数随每周禁食时间延长而升高,各处理组之间差异不显著(P>0.05)。本实验结果表明,T1组中华鳖出现补偿生长效应是由于摄食率的提高,其禁食-饱食处理天数的适宜比例可能在1:6以上,禁食-饱食循环次数的适宜周期为5周。2、为探究中华鳖稚鳖补偿生长效应,对稚鳖(平均体重4.47g)进行6种不同时间短期饥饿处理:饥饿0(C组)、3(T1组)、6(T2组)、9(T3组)、12(T4组)和15d(T5组),然后进行饱食处理,实验共进行28d。研究结果表明:中华鳖稚鳖短期饥饿后具有补偿生长效应,T1组实验鳖通过提高摄食率和饲料转化率获得完全补偿, T2和T3组则通过提高摄食率获得部分补偿,而T4和T5组则由于饥饿时间过长不能获得补偿。3、在30±0.5℃下对初体重为6.59±0.22g的中华鳖稚鳖进行了继饲料蛋白质限制后恢复投喂对其补偿生长影响的研究。对照组(C组)连续11周饱食投喂含粗蛋白43.31%,能量为16.77KJ/g的饲料,蛋白限制阶段(第1-4周)处理组T1、T2、T3和T4的饲料蛋白含量分别为29.24%、31.42%、34.19%和37.16%,饲料含能与对照组相同;恢复阶段(第5-11周)各处理组均投喂对照组饲料。在蛋白限制阶段,随着饲料蛋白水平的降低,中华鳖饵料系数显著升高,T1和T2组限制期的末体重显著低于对照组。在恢复阶段,与对照组相比,T1组饵料系数显著降低,且连续6周其周增重高于对照组,恢复期结束时T1组中华鳖体重高于对照组,其它处理组体重则没有赶上对照组。实验结果表明蛋白限制程度影响中华鳖补偿生长,蛋白限制程度最高的T1组中华鳖获得了完全补偿生长;蛋白限制水平适宜时,限制4周,可获得6周左右的补偿生长。4、在光周期为14L:10D条件下连续48h测定了禁食和不同摄食次数的中华鳖肝脏和肌肉组织RNA/DNA比值。振幅F检验分析结果显示,中华鳖肝脏和肌肉RNA/DNA比值均表现出明显的昼夜变化规律,余弦节律分析表明,每次摄食后均会引起RNA/DNA比值昼夜节律的振幅和中值上升。与禁食组相比,日摄食一次组峰值相位略有提前,而日摄食两次组峰值相位延后,因此,摄食组的RNA/DNA比值所表现出的昼夜节律不完全符合余弦节律。此外,相关分析表明,肝脏和肌肉组织的RNA/DNA比值存在显著的相关性,通过比较两种组织RNA/DNA比值在两个连续周期的卡方值得知,肝脏组织的RNA/DNA比值的卡方值小于肌肉,说明肌肉的RNA/DNA比值更加敏感,但肝脏的稳定性或可靠性好,选用肝脏组织RNA/DNA比值更具代表性。能够较好地代表其摄食和生长特征的RNA/DNA比值测定取样时间建议采用节律的中值和峰值的时间点。5、研究了低蛋白饲料胁迫下的中华鳖生长性能和其RNA/DNA比值变化规律。采用三种蛋白水平饲料(蛋白含量分别为45%,40%,35%)投喂中华鳖,每隔15d称重,计算特定生长率(SGR)和相对增重率(Rw)并取样测定其肝脏(L)和肌肉(M) RNA/DNA比值。经过90d养殖试验,结果发现:1)随饲料蛋白水平的下降,其SGR、Rw、RNA/DNAM和RNA/DNAL比值均呈现下降趋势;2)肌肉和肝脏RNA/DNA比值与摄食率(FI)呈现直线上升趋势,说明两种组织的RNA/DNA比值与摄食率正相关。回归分析显示肝脏的相关关系更显著; 3)RNA/DNAL比值和饲料Pr含量分别与SGR、Rw存在明显的对数相关关系,RNA/DNAM比值和Pr含量分别与SGR、Rw有明显的线性相关关系,这说明用RNA/DNA比值替代SGR是可能的。但不同的组织的RNA/DNA比值与SGR的关系不同,相关关系的密切程度也不同,肝脏比肌肉的RNA/DNA比值与SGR和Rw的相关性更好。以上结果表明了低蛋白饲料胁迫下的中华鳖,其SGR、Rw、摄食率和RNA/DNA比值(肝脏和肌肉)均逐渐下降,RNA/DNA比值与SGR之间相关性较大,RNA/DNAL比值更加稳定、与SGR相关性更好,可以作为蛋白营养胁迫下敏感的瞬时生长速度指标。更多还原

【Abstract】 This study systematically investigated the effect of different feeding regime on compensatory growth (CG) of juvenile soft-shelled turtles, Pelodiscus sinensis; the diel rhythm in RNA/DNA ratio (R/D) of soft-shelled turtles and the relation between the feeding strategy and the R/D; the change regulation in R/D of soft-shelled turtles under low protein nutrition stress and the relation between the R/D and the feeding and growth performance. These studies were adopted modern biologicl techniques, such as physiological ecological, nutritional, biochemical methods. The study was consisted of five sections as followed: The effects of the cyclical feed deprivation and refeeding, different starvation period, protein restriction with subsequent realimentation on CG in juvenile soft-shelled turtles were investigated; The diel rhythm in RNA/DNA ratio (R/D) of soft-shelled turtles and the relation between the feeding strategy and the R/D; The change regulation in R/D of soft-shelled turtles under low protein nutrition stress and the relation between the R/D and the feeding and growth performance. Results from the present study could make important contributions to promote the research work carried out the comparison for the CG among different species and understand the evolutionary significance of CG; We evaluated the diel variation of the R/D in this species and the effect of feeding strategy on this index, which could make sure the optimal sampling time and tissue for establish reasonable feeding strategy; R/D-Pr-SGR model can be used to evaluate nutrition value of formulate feed and the growth in the soft-shelled turtle species and to further perfect and develop growth evaluation system of aquatic reptiles, which would fill this subject crossing theory blank. The main results were as follows:1. An 8-week experiment was carried out to evaluate the CG of juvenile soft-shelled turtle, with the initial body weight of 17.61±0.46g, under four cyclical feeding regimes of feed deprivation and refeeding with the water temperature was 30.0±0.5℃. The deprivation and refeeding regimes included 1 day of deprivation followed with 6 days of refeeding (T1), 2 day of deprivation with 5 days of refeeding (T2), 3 day of deprivation with 4 days of refeeding (T3). The control group (C) fed apparent satiation daily throughout the whole experiment. At the end of the experiment, the final body weight, feeding rate and specific growth rate in group T1 and C were significantly higher than those of group T2 and T3. While there were no significant differences in feed conversion ratio among all groups. These results indicated that the turtles of group T1 show complete CG response mostly by enhanced the feeding rate. The optimal ratio of deprivation to refeeding period proportion may be below 1:6 and five cycles of deprivation and refeeding may be appropriate for the turtles.2. To ascertain CG capability of juvenile soft-shelled turtles, the turtles with an average body weight of 4.47g were treated with one of the following regimes: full food deprivation for 0 (group control), 3 (group T1), 6 (group T2), 9 (group T3), 12 (group T4) and 15 (group T5) days; and then the turtles in each group were fed to apparent satiation until the end of the 36-days experiment times. The result indicated that there was CG effect in juvenile Soft-shelled turtle following starvation, the T1 group of the completely CG which were obvious from significant increase of the feeding level and food conversion efficiency the early of re-alimentation phase. The T2 and T3 group showed partial CG, which was obvious from significant increase of the feeding level the early of re-alimentation phase. The T4 and T5 group of did not show CG, which were from long time full food deprivation.3. The effects of protein restriction with subsequent realimentation on compensatory growth (CG) of juvenile soft-shelled turtles were investigated. The turtles were fed five diets containing different crude protein content (T1: 29.24%, T2: 31.42%, T3: 34.19%, T4: 37.16%, C: 43.31%) while the energy content remained constant for 4 weeks (restriction period) and then re-fed a diet (43.31% crude protein) for 7 weeks (realimentation period). During the restriction period, feed conversion ratio (FCR) significantly increased in all protein-restricted groups (T1-T4) compared with the control group (C). At the end of the restriction period, body mass in groups T1 and T2 was significantly lower than the control. During the realimentation phase, the weekly body mass gain (WG) in group T1 was greater than that of the control from week 5 to week 10. FCR significantly decreased in group T1 compared with the control. No significant differences in final body mass between C and T1 groups were found at the end of the realimentation period. These results indicated that soft-shelled turtles receiving a diet in which protein was reduced from 43.31% to 29.24% (approximate 33% reduction) for 4 weeks achieved a 6-week complete CG response.4. The effect of starvation and feeding rhythm (feeding once daily at 8:00, feeding twice daily at 8:00 and 18:00) on the diel variation in R/D was studied in Soft-shelled turtles, Pelodiscus sinensis, under the photoperiod was 14L:10D. Sampling to investigate the diel variation in muscle and hepatic tissue of R/D of turtles was carried out for 48h. The amplitude F text analyzed suggested that there was significant diel rhythm in R/D of these tissues. The cosine rhythm software analyzed the rhythm parameter suggested the amplitude and mesor increased by feeding action, the acrophase in feeding once and twice group was ahead and postpone, respectively. The diel rhythm of R/D in feeding group did not complete accordance with the cosine rhythm. The correlation analysis indicated that the coefficient of correlation was 0.85 between the R/D ratio of hepatic tissue and that of muscle tissue. The chi-square value of two continual periods of hepatic tissue were lower than those of the muscle tissue, suggested that the better stability and reliability in hepatic tissue. The optimal sampling time was rhythm mesor and acrophase time.5. A 90-day experiment was carried out to determine the regulation of growth performance and R/D in Soft-shelled turtles, under low protein nutrition stress. Three protein contents diets (45%, 40%, 35%) were prepared and every 15d, a subsample of four individuals was taken, which were used to produce a range of specific growth rates (SGR) and relative weight gain ratio (Rw). An ultraviolet absorption assay was used to measure nucleic acid concentrations in muscle and liver tissue. The results were as followed: 1). The value of SGR and R/D were decreased with the dietary protein content decreased. The value of R/D in muscle were continuance higher than that of the liver tissue, suggested that the better stability and reliability in liver tissue; 2). The R/D and feeding rate (FI) present linear up trend, indicated the R/D and FI are positive correlation. Regression analysis revealed that the more significantly correlation in liver tissue; 3) The liver and muscle pf R/D, Pr and the SGR and Rw was obvious linear correlation, respectively, suggested that the possibility of the SGR and Rw was substituted by R/D. The relation and the degree of correlation between the different organizations of R/D differ with the SGR and Rw values, the degree of correlation in liver was higher than that of the muscle. The results showed that the SGR, Rw FI and R/D (the liver and muscle) of the soft-shelled turtles under low protein feed stress are gradually decline. The degree of correlation and stability between R/D and SGR (and Rw) in liver was higher than that of the muscle, which could be as a sensitive index under protein nutrition stress.更多还原