【作者】 宋陈玲；

【导师】 王继文；

【作者基本信息】 四川农业大学 ， 畜牧生物工程， 2013， 博士

【摘要】 家禽属卵生动物,胚胎发育容易受到外界环境因素(营养、孵化温度、激素和运动等)的影响,其中孵化温度对禽类胚胎发育和机体代谢影响较大。本研究以鸭胚胎为研究对象,通过在鸭胚孵化中后期改变孵化温度,检测鸭胚及出壳后雏鸭组织器官发育、血液脂质代谢指标的变化,并继续重点以鸭腿肌为研究对象,通过转录组测序分析孵化温度影响胚胎组织发育的分子机制。取得的主要研究结果和结论如下：(1)孵化温度处理对鸭孵化效果有一定影响,但与对照组比差异不显著(P>0.05)。孵化温度对鸭胚胎腿肌发育具有抑制作用,在E25和出壳时达到显著差异(P<0.05)。孵化温度处理使雏鸭体重增加,在W2、W4和W6时处理组胸肌和腿肌重量都高于对照组,且在W6时两个组腿肌重差异达显著(P<0.05)。(2)孵化温度对鸭胚及雏鸭心脏发育影响不明显,整体表现抑制作用,但仅在E25时与对照组比差异达显著水平(P<0.05)。孵化温度升高对鸭胚胎期肝脏发育有抑制作用,但对出壳后肝脏发育有促进作用,在所研究的阶段差异显著(P<0.05)。(3)孵化温度提高后,降低了血糖水平,在鸭出壳后4W时差异达到显著水平(P<0.05)；孵化温度提高影响了血液脂质代谢指标,处理组血浆甘油三酯(TG)、总胆固醇(TC)和低密度脂蛋白(LDL)水平均高于对照组,其中LDL在胚胎期处理组和对照组间差异显著(P<0.05),TC在鸭出壳后对照组和处理组间差异显著(P<0.05)。(4)孵化温度升高对鸭胚胎期免疫器官发育也有一定影响,研究结果表明处理组鸭胚胎胸腺、脾脏和法氏囊器官指数降低,其中在E25法氏囊指数差异显著(P<0.05),在出壳时脾脏指数差异达显著(P<0.05)；孵化温度还影响了鸭胚胎及出壳后血液干扰素γ(IFN-γ)水平和免疫球蛋白/总蛋白比值,在2W时处理组和对照组两个指标差异皆达到显著水平(P<0.05)。(5)孵化温度对鸭胚胎发育的影响,也间接影响到了胚后鸭组织发育和血液脂质代谢,本研究结果表明：处理组血糖水平在E20和W4之间呈现显著负相关(P<0.01),且出壳和W4之间呈现显著负相关(P<0.01)。对照组血液中的TG水平在出壳和W2之间呈现显著负相关(P<0.05)。处理组TC水平在E20和W2之间呈现负相关,对照组TC水平在E20和W2之间呈现正相关。(6)孵化温度对鸭胚肌肉发育形态影响的分析结果表明：E20和W2时处理组胸肌和腿肌纤维均大于对照组,说明孵化温度对鸭E20鸭腿肌发育影响已经明显。因此,运用RNA-Seq转录本测序技术检测了处理组和对照组鸭E20时腿肌样品,分析结果显示：处理组和对照组clean reads占reads总数的98%以上,Reads在参考基因组上的分布均匀,建库策略的随机性好,基因数增长速度趋于平缓,检测到的基因数趋于饱和,基因覆盖度较高,说明测序质量满足后续分析。分析共注释了335个差异表达基因,其中182基因表达上调,153个基因表达下调。GO功能显著性富集性分析显示这些差异表达基因主要参与细胞组成、分子功能和生物学过程三个方面,涉及细胞形态结构、细胞免疫、能量代谢、信号传导等方面的变化。信号通路富集结果表明,孵化温度主要通过影响细胞蛋白质合成等过程,对鸭胚胎肌肉发育产生影响。其中,核糖体代谢过程和MAPK信号路径是孵化温度影响鸭胚骨骼肌发育的重要分子途径。更多还原

【Abstract】 As the oviparous, Poultry embryonic development is susceptible to external environmental factors, including nutrition, incubation temperature, hormones and exercise, in particular the incubation temperature has great impact on poultry embryonic development and metabolism. In this study the duck embryo was selected as the research, by changing the incubation temperature in duck middle and late embryonic period we detected changes of tissue and organ development, blood lipid metabolism of duck embryo and hatched ducklings. Meanwhile, we further investigated the molecular mechanism of the effects of incubation temperature on embryonic tissue development by Transcriptome sequencing, in which duck leg muscle was selected as the research object.The main results and conclusions are listed as follows:(1)Incubation temperature treatment had a certain influence on incubation effect, However, compared with control group no significant difference was found(P>0.05). Leg muscle development was inhibited by elevated incubation temperature, there were significant differences on day E25and the day of hatching between the two groups (P<0.05). Ducklings weight increased by incubation temperature in treatment group. The weight of breast and leg muscle in treatment group were heavier than control group on W2, W4and W6, and there was significant difference between the two groups’leg muscle weight on W6(P<0.05).(2) The impact of incubation temperature on duck embryos and ducklings heart development was not obvious, the overall performance was inhibition, only a significant difference on the E25was found between the two groups (P<0.05). We found temperature could inhibit liver development in the embryonic period, but facilitate its developmen in the postnatal period, and significant differences were found in the research stages (P<0.05).(3) The duck blood glucose levels were reduced by raising the incubation temperature, and significant difference between the two groups was found at4weeks of age (P<0.05). Higher Incubation temperature had an impact on blood lipid metabolism indicators. The plasma TG, TC and LDL levels of treatment group were higher than the control group. The difference between two groups’LDL levels was significant in embryonic period (P<0.05), and TC was significant in postnatal period (P<0.05).(4)Incubation temperature also had some impacts on the duck immune organs development in embryonic stage. The results showed that thymus, spleen and bursa organ index of the treatment group ducks decreased in embryonic period, in which the bursa index differences between the two groups was significant on E25(P<0.05), the spleen index differences of two groups on the day of hatching was significant (P<0.05).Higher incubation temperature also had an impact on the content of IFN-γ and immunoglobulin/total protein ratio, and significant differences of the both indexes between the two groups were found at W2(P<0.05).(5) The impact of incubation temperature on the duck embryonic development, also indirectly affect the duck embryo tissue development and blood lipid metabolism during the postnatal period. The results showed that:in the treated group, blood glucose levels revealed a significant negative correlation between the E20and W4(P<0.01), as well as the day of hatching and W4(P<0.01). TG levels in the blood of control group between the day of hatching and W2showed a significant negative correlation (P<0.05), TC levels showed a negative correlation between E20and W2in treatment group, with a positive correlation between E20and W2in the control group.(6)The results of analysis of the impact of incubation temperature on duck embryo muscle development showed that the breast and leg muscle of treatment group were larger than that in the control group on the day of E20and W2, which indicated that effect of incubation temperature on the E20duck leg muscle development was obvious. Therefore, the Illumina/Hiseq-2000RNA-seq deep sequencing analysis was used to analysis transcriptome data of duck E20leg muscle, and the analysis showed as follows:clean reads of two groups occupied over98%of the total number of reads. Distribution of Reads in the reference genome database was uniform and random. Growth rate of gene number was gentle, the number of genes detected tended to be saturated. All these results suggested the quality of this sequencing could meet the need of further analysis. In total, we detected335differently expressed genes, in which182genes were up-regulated and153genes were down-regulated. GO significant enrichment analysis showed these differently expressed genes mainly involved in cell composition, molecular function and biological process, which related with changes in cell morphology, cell-mediated immunity, energy metabolism and signal transduction. Signal pathway enrichment results showed that incubation temperature affected the duck embryonic muscle development by affecting the process of protein synthesis. Ribosomal metabolic processes and MAPK signal pathway were important molecular pathways for the effects of incubation temperature on duck embryo skeletal muscle development更多还原