| 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. |
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