【作者】 王晶；

【导师】 许丽；

【作者基本信息】 东北农业大学 ， 动物营养与饲料科学， 2011， 博士

【摘要】 本文针对含黄素单氧化酶3(flavin-containing monooxygenase 3, FMO3)基因T239S位点,以海兰褐产蛋鸡为对象,日粮添加氯化胆碱作为前体物质,从遗传和营养两方面系统研究了蛋鸡鱼腥味综合征的影响,旨在探讨FM03基因型和胆碱及其互作对蛋黄三甲胺含量的影响及其作用机制。试验一：海兰褐蛋鸡鱼腥味综合征易感基因型的测定。采用PCR-RFLP法测定了FM03T329S突变位点在商品代海兰褐壳蛋鸡群体(731只)中的分布规律,分离筛选出鱼腥味综合征易感基因型的产蛋鸡,用于进一步试验。测序检验表明,试验所采用的方法能针对T329S位点,准确地分离筛选FM03基因型。结果表明：所选海兰褐蛋鸡(n=731)中,AA(野生型)、AT(杂合型)和TT(突变型)基因型频率分别为3.4%、76.6%和20.0%,T等位基因频率高于A等位基因。利用Hardy-Weinberg定律检验基因平衡状态,发现该位点在商品代海兰褐蛋鸡群体中极显著地偏离了平衡(P<0.001)。试验二：FM03基因型对蛋黄三甲胺(TMA)沉积的影响。采用单因素设计,选用已知FM03基因型海兰褐壳产蛋鸡132只,按基因型分为4个处理组,每组6个重复。其中AT、TT两种基因型各54只,每重复9只,饲喂试验日粮(添加2 960 mg/kg胆碱)；AA基因型24只,分为两个处理,每2只作为一个重复,分别饲喂对照日粮(添加370mg/kg胆碱)和试验日粮。预饲期7天,各组饲喂对照日粮。正试期42天。分别于预饲第3天和第6天,正式试验的第1、2、3、4、5、6、7、10、14、21、28、35和42天,每重复取3枚(AA基因型1-2枚)鸡蛋,分离蛋黄,混匀,比色法测定TMA。结果表明：(1)蛋黄TMA含量随时间呈间歇性变化。AT和TT基因型TMA含量从试验第3天起表现出间歇性上升趋势,分别于第35天和第21天时达到整个试验期最大值(5.02和10.53μg/g);AA基因型蛋黄TMA含量(1-2μg/g)无显著变化；从试验第3天起(35天除外),TT基因型TMA含量显著高于对照组和其他处理组(P<0.01)；(2)试验1-21天,TT基因型TMA含量快速增加,呈间歇性上升。将鸡蛋TMA沉积量(Y,μg/g)与时间(X,1-21天)进行曲线拟合,得到方程为：Y=0.0036X3-0.1349X2+1.7706X-0.6578(R2=0.9646)；(3)2 960 mg/kg胆碱处理下突变基因型蛋鸡鱼腥味鸡蛋通过感官检出时间为3-5天。试验三：基因型和胆碱对产蛋鸡生产性能、蛋品质的影响。试验按基因型和胆碱水平,采用3×2因子重复设计,每个基因型设2个胆碱添加水平(370和2 960 mg/kg),共6个处理组,每组6个重复,AT、TT两种基因型各108只,每个重复9只,AA基因型24只,每2只作为一个重复。预饲期1周,试验期6周。以重复为单位记录每天产蛋数、蛋重、死亡鸡数,每周记录鸡的耗料量一次。最后计算出1-3周,4-6周和1-6周的产蛋率、日采食量、平均蛋重、料蛋比和日产蛋重。试验第40-42天,每重复收集6枚鸡蛋(AA基因型收集4枚),4℃保存,其中3枚(AA基因型2枚)用于测定蛋品质(保证样品采集到测定间隔相同时间)：另外3枚用于制备冻干蛋黄粉,测定蛋黄营养成分。试验结果表明：(1)产蛋率受基因型显著影响(P<0.05),AA基因型高于TT和AT基因型；添加2 960 mg/kg胆碱显著降低了蛋鸡日采食量(P<0.01)、1-3周料蛋比(P=0.016),基因型与日粮胆碱添加量对产蛋性能无交互作用；(2)基因型对蛋品质无显著影响；日粮添加2 960 mg/kg胆碱极显著降低了蛋黄颜色(P<0.001),与370 mg/kg胆碱添加量相比,蛋壳厚度、蛋白高度和哈夫单位有所降低,但无显著性差异；基因型及其与胆碱的交互作用不影响鸡蛋品质。试验四：基因型和胆碱对产蛋鸡三甲胺代谢的影响。试验设计、试验条件同试验三。试验第7、21和42天,每个重复取3枚蛋(AA基因型2枚),分离蛋黄,混匀,-20℃保存,记录蛋重和蛋黄重,用于测定蛋黄中TMA。于试验第42天,各重复取一只接近平均体重的试鸡,颈静脉放血致死。采集血样,3 000 r/min离心10 min,分离血清,并加入0.01 M HCI固定TMA,-80℃保存。解剖试鸡,无菌棉线结扎盲肠后取出,4℃保存,当天测定TMA。蛋黄TMA和盲肠食糜TMA采用比色法测定,血清TMA采用顶空气相色谱法测定。结果表明：(1)FM03基因型显著影响蛋黄TMA含量,表现为2 960 mg/kg胆碱添加量下,TT基因型TMA含量极显著高于AA和AT基因型(P<0.001),但370 mg/kg胆碱处理下,各基因型产蛋鸡蛋黄TMA含量无显著差异；日粮胆碱添加水平对蛋黄TMA有显著作用(P<0.05),2 960 mg/kg胆碱处理下,各基因型组蛋黄TMA含量较370 mg/kg胆碱处理下同基因型组均有不同程度升高；FM03基因型和胆碱对蛋黄TMA有显著交互作用(P<0.05)；(2)基因型对盲肠食糜和血清TMA含量无显著影响,日粮胆碱水平极显著提高了产蛋鸡盲肠食糜和血清TMA含量(P=0.001),二者无交互作用；(3)盲肠食糜TMA含量与血清TMA含量正相关(P=0.061),与蛋黄TMA含量呈显著正相关(P=0.048)；蛋黄TMA含量与血清TMA含量无显著相关性(P=0.572),与蛋黄脂肪含量间呈显著正相关(P=0.013)。试验五：基因型与胆碱对FM03基因表达水平和FM03酶活的影响。试验设计、试验条件同试验三。于试验第42天清晨喂料前,各重复取一只接近平均体重的试鸡,颈静脉放血致死。解剖试鸡,立即取出肝脏、肾脏用冷冻的生理盐水洗去血汁,滤纸擦干称重,剪去结缔组织,液氮冷冻,置-80℃冰柜中保存,提取RNA和肝脏微粒体,分别以底物TMA和甲巯咪唑代谢产物的产率(nmol per mg microsomal protein per min)作为酶活性指标。结果表明：(1)基因型间肝脏FM03基因表达水平无显著性差异(P=0.079)；高剂量胆碱极显著降低了肝脏FM03 mRNA表达水平,AA、AT和TT基因型分别下降了19.48%(P>0.05)、38.10% (P<0.05)和54.29% (P<0.01),二者无交互作用。(2)基因型、胆碱水平及二者交互作用对肾脏FM03 mRNA表达量无显著影响。(3)FM03活性受基因型显著影响(P=0.003),AA基因型活性最高,其次AT基因型,TT基因型最低;与370mg/kg胆碱添加水平相比,2960 mg/kg添加量极显著降低了FM03对TMA和甲巯咪唑的代谢活性(P<0.01)。试验六：基因型和胆碱对产蛋鸡脂质代谢和血清激素水平的影响。于试验第42天,每重复随机选取1只鸡,空腹翅静脉采血,离心分离血清,-20℃保存,测定血清和肝脏脂质,血清激素水平,血清一氧化氮水平和丙二醛含量。通过相关分析对血液生理生化指标筛选,探讨基因型、胆碱可能的作用方式。结果表明：(1)血清甘油三酯(P=0.038)、游离脂肪酸水平受基因型显著影响(P=0.001)；日粮中添加2 960 mg/kg胆碱改善了机体脂质代谢,表现为降低了血清TG(P=0.038)、NEFA(P=0.001)水平,增加了HDL-C水平(P=0.06),降低了产蛋鸡肝脏中总脂肪(P=0.015)、甘油三酯含量(P=0.006),二者无交互作用。(2)产蛋鸡血糖、血清一氧化氮含量受胆碱水平影响,随胆碱水平增加分别下降27.49%(P<0.001)和20.60%(p=0.15),T4水平增加26.05%(P=0.008),T4/T3提高。(3)FM03 mRNA表达水平与血清胰岛素(P=0.004)、T4(P=0.041)、血糖(P=0.017)含量显著相关。综上所述,FM03基因型对生产性能和蛋品质无明显作用,蛋黄TMA含量受基因型、胆碱添加水平及其交互作用影响。其中,T329S突变导致酶活性的下降是造成基因型间TMA代谢差异的主要原因；添加高剂量胆碱增加了TMA的产生和吸收,同时抑制了肝脏FM03mRNA表达水平和酶活性,加剧了TMA代谢的负担。更多还原

【Abstract】 To evaluate the interaction between FM03 genotype and diet supplementation of choline (TMA precursor), genotypic and allelic frequencies of FMO3 for commercial Hyline (brown-shelled strain) were tested firstly. Then the effects of FM03 genotype and diet supplementation of choline on production performance, egg quality, deposition of TMA in egg yolk, and TMA metabolism were studied. Further, FMO3 mRNA levels and FM03 activity were investigated to explain the effect of genotype on TMA and lipid metabolism. Hormone levels were also examined to discuss the mechanism of choline on FM03 mRNA levels.In Trial 1, genotyping of fishy-egg tainting hens in HyLine strains were studied. A total of 731 HyLine Brown hens were genotyped for a A/T polymorphism at position nt 1034 of the chicken FMO3 cDNA sequence (T329S for amino acid sequence). A PCR-RFLP method was developed to analysis the distribution of the mutation in Hyline Brown hens. Genotype of AA (wild type), AT (heterozygous type) and TT (mutant type) were found with percentage of 3.4%,76.6% and 20.0%, respectively. The allelic frequencies of T was higher than that of A, and the allelic frequencies were not in Hardy-Weinberg equilibrium (P<0.001) in the Hyline Brown laying hens strain.The purpose of Trial 2 was to examine the effect of FMO3 genotype on TMA deposition in egg yolks from hens fed with high levels of choline. A total of 132 hens were allotted four treatments by genotype. A total of 54 hens per genotype (AT and TT) were fed diet supplemented with 2 960 mg/kg choline, and each treatment consisted of 6 replicates with 9 hens. A total of 24 hens with AA genotype (only 24 hens of this genotype were present in the population) were fed either a control diet supplemented with 370 mg choline /kg or a diet supplemented with 2 960 mg choline /kg, and each treatment consisted of 6 replicates with 2 hens. Hens were fed control diet for a one-week of adapting period followed by a six-week of trial period. Two or three eggs per replicate (1-2 eggs for AA) were collected at d 3,6 in adapting period, and d 1,2,3,4,5,6,7,10, 14,21,28,35 and 42 in trial period. The yolk was separated for TMA analysis. (1) The changes of TMA concentrations in egg yolk were intermittent. TMA concentration in Yolk of AA and TT hens increased intermittently as deposition time increased from d 3, and reached the peak at d 35(5.03μg/g) and d 21(10.53μg/g),respectively;while TMA concentration of AA hens were relatively stable in trial period. From d 3(except d 35), yolk TMA concentration of TT hens were significantly higher than that of AA hens. (2) The fitting curve showed that TMA concentrations of egg yolk from TT genotype hens, fed with 2960 mg choline /kg diet, intermittently increased as deposition time increased. The relationship between concentration of TMA in the egg yolks (μg/g, Y) and desposition time (1～21 day, X)is Y=0.0036X3-0.1349X2+1.7706X-0.6578 (R2=0.9646). (3) The fishy eggs were detected at 3～5 day of supplementing 2 960mg/kg choline in the diet.Trial 3 was used to study the effect of FMO3 genotype and choline supplementation on performance of laying hens and egg quality. A 3×2 two-factorial design was applied in this study. The 3 genotypes of AA, AT and TT and 2 levels of 370 and 2 960mg choline addition/kg were included. At 43 wk of age, a total of 240 hens were fed experimental diets for 6 weeks.108 hens of per genotype (AT and TT) were randomly fed either a control diet supplemented with 370 mg choline/kg or a diet supplemented with 2 960 mg choline/kg, and each dietary treatment consisted of 6 replicates with 9 hens. A total of 24 hens with AA genotype were allotted to one of two treatments consisted of 6 replicates with 2 hens. Hens were fed control diet for a one-week adapting period followed by a six-week trial period. The results showed that:(1) Hens fed the diet supplemented with 2 960mg/kg choline had a decreased feed intake (P< 0.01) and feed efficiency (1-3w, P=0.016). Laying performance was not affected by the genotype excepting that egg production was higher in AA hens (P<0.05). No interaction between genotype and dietary choline concentration was found. (2) Interaction between genotype and dietary choline concentration had no effect on egg quality. Decreased tendency in yolk colour (P<0.001) was observed at the dietary supplemental choline with 2 960 mg/kg. Eggshell thickness, albumen height and haugh unit were also decreased by higher dietary choline addition, while no significant effects were found.Trial 4 was aimed to investigate the effect of genotype, choline and their interaction on TMA metabolism. A same experimental design was used as in trial 3. (1) The significant effects of genotype (P<0.05), choline (P<0.005), and their interaction (P<0.05) on yolk TMA concentration were observed at d21 and d 42. Based on the main effect of the genotype, the TMA contents in egg yolks of TT hens were higher than those of other hens (P<0.05). When dietary choline level was increased up to 2 960 mg/kg, significant differences among the TMA content of the three FMO3 genotypes were found. No significant differences (P>0.05) were detected on TMA contents in egg yolks from AA, AT and TT hens fed with 370mg choline /kg feed. The concentrations of TMA in yolk from TT hens were affected by choline levels (P<0.001). There was a slight increase in yolk TMA concentrations from AA and AT hens fed with higher levels of choline. (2) Supplementing 2 960mg/kg choline to diets resulted in an increased TMA concentration of caecal chime (P<0.001) and serum (P=0.017), whereas both genotype and their interaction was without an effect. (3)TMA contents of caecal chime (P=0.048) and serum (P=0.051) were positivly related to yolk TMA concentration, but no significant relation existed between TMA contents of caecal chime and serum.Trial 5 was conducted to investigate the effects of genotype, choline and their interaction on FMO3 mRNA levels and FMO3 activity. A same experimental design was used as in trial 3. (1) The genotype did not significantly influence the relative expression of FMO3 mRNA in the liver (P=0.079). The FMO3 mRNAs in liver was significantly down-regulated by the addition of 2 960mg/kg choline to diet (P< 0.001). Hepatic FMO3 mRNA level of TT hens was dramatically suppressed by 54.29%(P<0.001), while decreased modestly by to 19.48% and 38.10%(P<0.05) for AA and AT hens, respectively. No interactions on hepatic FMO3 mRNA between genotype and choline were observed. (2) Genotype, choline and their interaction did not affect FMO3 mRNA in kidney. (3) Activity of liver microsomal FMO3 towards TMA and MMI were significantly affected by genotype (P<0.01) and dietary choline (P<0.01). No interactions on hepatic FMO3 mRNA between genotype and choline were observed. The FMO3 activity was listed from high to low as follows:AA> AT>TT; 370mg/kg>2 960mg/kg.Trial 6 was carried out to assess the effects of genotype, choline and their interaction on lipid metabolism and endocrine. Supplementing 2 960mg/kg choline to the diets resulted in improvement on lipid metabolism. (1) Decreased tendency in serum TG (P=0.038) and NEFA (P =0.001) levels, total fat (P=0.015) and TG (P=0.006) in liver were observed with increasing the HDL-C levels (P=0.06). (2) The contens of blood glucose and serum NO were significantly decreased by 27.49%(P<0.001) and 20.60%(P= 0.15),while the levels of serum T4 increased by 26.05%(P=0.008). The data of T4/T3 was also increased by choline of 2 960mg/kg. (3) The levels of serum IRS (P=0.004), T4 (P=0.041) and glucose (P=0.017) were related to FM03 mRNA levels.In summary, no significantly effects of FMO3 genotype on egg performance and egg quality were found, while the significant effects of genotype, choline and their interaction on yolk TMA concentration were observed. On the one hand, T329S mutation in the FMO3 gene causes a reduction of enzyme activity;on the other hand, high amounts of choline increased TMA production, but decreased FMO3 mRNA levels and activity.更多还原