番鸭产蛋各期肝脂肪酸组成及AMPK信号通路基因表达研究

旨在探究产蛋各期番鸭肝腺苷酸活化蛋白激酶（AMPK）信号通路基因表达和肝脂肪酸组成，为番鸭肝脂质代谢应答产蛋提供机理研究。本研究选取开产前22周龄、产蛋初期30周龄、产蛋中期40周龄和产蛋末期60周龄母番鸭各15羽，全自动生化仪测定血脂水平，苏木精-伊红（HE）染色和油红O染色观察肝组织学结构，实时荧光定量PCR （qRT-PCR）检测肝AMPK通路基因表达，气相色谱-质谱联用法（GC-MS）检测产蛋各期肝脂肪酸组成。结果表明，总胆固醇（TC）、低密度脂蛋白胆固醇（LDL-C）、甘油三酯（TG）和极低密度脂蛋白胆固醇（VLDL-C）水平在40周龄显著高于22、30和60周龄（P<0.05）；高密度脂蛋白胆固醇（HDL-C）水平在30和40周龄显著低于22和60周龄（P<0.05）。肝HE和油红O染色切片显示，肝在22周龄呈实质状，至产蛋40和60周龄，肝脂滴沉积明显（P<0.05）。AMPKα1在22、30、40和60周龄呈本底低水平表达，并显著低于产蛋各期肉碱脂酰转移酶1（CPT1）和脂肪酸合成酶（FAS）的表达量（P<0.05），FAS在22、40和60周龄均呈高水平表达（P<0.05）；羟甲基戊二酰辅酶A还原酶（HMGR）、肝细胞核因子4α（HNF4α）、乙酰辅酶A羧化酶1（ACC1）和固醇调控元件结合蛋白-1（SREBP1c）在40周龄表达量显著高于22和30周龄（P<0.05）。肝中饱和脂肪酸（SFA）、单不饱和脂肪酸（MUFA）和多不饱和脂肪酸（PUFA）主要由C16∶0、C18∶0、C18∶1和C20∶2 n-6构成，分别占总脂肪酸含量的32%、16%、30%和9%。C14∶0和C16∶0含量在40周龄显著高于22周龄（P<0.05）；C24∶0、C20∶2 n-6和PUFA含量在60周龄显著高于22和40周龄（P<0.05）。综上，番鸭产蛋期肝通过上调FAS等脂质合成基因表达，合成大量长链脂肪酸，沉积于肝，并增加血脂水平。

Fatty Acid Composition and Gene Expression of AMPK Signaling Pathway in Liver of Muscovy Duck at Different Egg-laying Stages

The aim of this study was to investigate the gene expression in AMP-activated protein kinase (AMPK) signaling pathway and fatty acid composition in the liver of Muscovy duck at different egg-laying stages, so as to illustrate the mechanism of lipid metabolism in response to egg-laying. Fifteen female Muscovy ducks were selected at pre-laying (22 weeks), early laying (30 weeks), laying (40 weeks) and post-laying (60 weeks) stages, respectively. Serum lipid levels were determined by automatic biochemical analyzer. Liver histological structure was observed by hematoxylin-eosin (HE) staining and Oil Red O staining, gene expression in AMPK pathway was determined by quantitative real-time PCR (qRT-PCR), and fatty acid composition was measured by gas chromatography-mass spectrometry (GC-MS). The results showed that the levels of total cholesterol (TC), low density lipoprotein cholesterol (LDL-C), triglyceride (TG) and very low density lipoprotein cholesterol (VLDL-C) were significantly higher at 40 weeks than those at 22, 30 and 60 weeks (P<0.05). The levels of high density lipoprotein cholesterol (HDL-C) at 30 and 40 weeks were significantly lower than those at 22 and 60 weeks (P<0.05). Liver sections stained by HE and Oil Red O showed that the reticular formation was intact at 22 weeks, and lipid deposition significantly increased at 40 and 60 weeks (P<0.05). AMPKα1 was in a low expression level at 22, 30, 40 and 60 weeks, and was significantly lower than the expression of carnitine palmitoyltransferase-1 (CPT1) and fatty acid synthase (FAS) at different egg-laying stages(P<0.05). FAS expressed in a significantly high level at 22, 40 and 60 weeks (P<0.05). The expression levels of hydroxy-methylglutaryl coenzyme A reductase (HMGR), hepatocyte nuclear factor 4α (HNF4α), acetyl-CoA carboxylase 1 (ACC1) and sterol regulatory element binding protein 1 (SREBP1c) at 40 weeks were significantly higher than those at 22 and 30 weeks (P<0.05). Liver saturated fatty acids (SFA), monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) were mainly composed of C16:0, C18:0, C18:1 and C20:2 n-6, accounted for 32%, 16%, 30% and 9% of the total fatty acids, respectively. The contents of C14:0 and C16:0 at 40 weeks were significantly higher than those at 22 weeks(P<0.05). The contents of C24:0, C20:2 n-6 and ΣPUFA at 60 weeks were significantly higher than those at 22 and 40 weeks (P<0.05). In conclusion, the liver synthesized large amount of long-chain fatty acids by up-regulating the expression of lipid synthesis genes, including FAS,during egg-laying. The synthesized lipids were deposited in liver and transported into serum thus increased the serum lipid level during laying.