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母源性leptin对肉鸡子代早期生长和肝脏脂肪代谢的影响
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摘要
本文以三黄肉鸡为实验对象,在研究母鸡日粮添喂半胱胺影响子代生长的过程中,首次在蛋中检测到leptin的存在,发现半胱胺导致母鸡leptin合成和分泌的改变,并使蛋中leptin沉积显著增加。因此,提出leptin可能作为一种信号物质介导半胱胺对肉鸡子代生长程序化影响的假说,认为蛋中沉积的leptin会改变子代肝脏leptin合成及其功能,并影响子代生长早期肝脏脂肪酸合成关键因子的表达,从而改变子代早期的生长和代谢模式。为验证这一假说,设计了蛋内注射leptin的试验,观察对胚胎、新生雏鸡和出雏后快速生长期肉鸡生长的影响,研究肝脏leptin含量及其受体表达的变化,并比较肝脏脂肪酸合成关键因子表达的差异,以探讨母源性leptin对肉鸡子代早期生长和脂肪代谢的影响,为揭示母源性leptin对禽类程序化影响的机制提供实验证据。1半胱胺对肉种鸡产蛋性能和对子代早期生长的影响及leptin勺介导作用
     本实验选用67周龄产蛋后期三黄肉种鸡480只,随机分为两组,对照组饲喂基础日粮,试验组在基础日粮中添加400 mg/kg半胱胺制剂CT2000,连续饲喂8周。结果显示:饲喂半胱胺对母鸡体重没有影响,试验组母鸡产蛋率升高2.24%(P<0.01),破蛋率降低40.55%(P<0.01),而畸蛋率升高20.15%(P<0.05);对平均蛋重、种蛋率、种蛋受精率和孵化率均无显著影响;显著增加蛋壳重量和降低蛋清重量(P<0.05)。随机选取种蛋在相同条件下孵化,子代在相同条件下饲养,结果显示:试验组子代出雏重显著降低,特别是试验组雌性子代出雏重显著低于对照组,并且生长迟缓,体重显著低于对照组,一直持续到42天。用Western blot芳法检测发现鸡蛋蛋黄和蛋清抽提物中含有leptin,试验组母鸡肝脏中leptin含量升高(P<0.05)。用放射免疫分析法检测发现试验组母鸡血清中leptin和T4水平下降(P<0.01),而肝脏(P<0.01)、种蛋蛋黄(P<0.01)和蛋清(P<0.05)leptin水平升高。另外,发现试验组子代12胚龄卵黄囊上LEPR mRNA水平显著下调(P<0.05)。以上结果提示:肉种鸡饲喂半胱胺后,母鸡肝脏分泌和鸡蛋中沉积的leptin含量增加,卵黄囊上LEPR mRNA表达下调,说明leptin可能作为信号物质介导了半胱胺对肉种鸡产蛋性能的影响和对肉鸡子代早期生长发育的程序化作用,并呈现性别特异性。
     2蛋内注射leptin对肉鸡胚胎发育和新生雏鸡肝脏脂肪代谢的影响
     为研究蛋内沉积的leptin对肉鸡胚胎发育的影响,本实验将三黄肉鸡种蛋随机分为两组,对照组在蛋清内注射100μL溶剂(PBS),实验组注射100μL含0.5μg重组小鼠leptin的PBS。分别于12、17胚龄和出雏当天(0日龄)称重并采取血样及肝脏组织样品。用放射免疫分析法测定血清和肝脏leptin浓度,实时荧光RT-PCR定量肝脏LEPR和脂肪代谢相关基因,用Western blot测定肝脏LEPR蛋白含量。结果表明,leptin处理组出雏重极显著降低(P=0.000),而肝体指数显著升高(P=0.018),特别是雌性差异极显著(出雏重和肝体指数均为P=0.000)。12胚龄和0日龄试验组肝脏leptin含量显著高于对照组(P=0.053,0.041),同时伴随0日龄试验组血清中leptin的含量极显著升高(P=0.009),然而0日龄时肝脏LEPR的mRNA表达和蛋白含量均未发现显著差异。试验组肝脏TG和Tch含量显著下降,而血清中TG和Tch浓度却显著升高,血清ApoB的水平升高,提示脂质转运出肝加强;试验组肝脏中ACC和FAS mRNA的表达没有差异,但是SREBP-1 mRNA的表达显著升高,提示肝脏脂肪酸合成潜力增强。并且这些肝脏脂质代谢指标的变化也表现性别特异性,leptin对雌性的影响更为显著。以上结果提示:蛋内注射leptin抑制肉鸡胚胎发育,降低出雏重,提高胚胎发育过程中肝脏leptin含量和血清leptin浓度,并影响新生雏鸡肝脏脂质代谢。
     3蛋内注射leptin对肉鸡出雏后早期生长和肝脏脂肪代谢的影响
     为了探讨母源性leptin对三黄肉鸡孵化后早期生长发育的影响及其剂量依赖关系,本实验挑选蛋重相近的三黄肉鸡种蛋,随机分为三组:对照组(control, Con)在蛋清内注射100μL溶剂(PBS), leptin处理组分别注射0.5μg/100μL PBS(低剂量组,LL)和5.0μg/100μL PBS(高剂量组,HL)的重组小鼠leptin,出雏后饲养至21日龄时采取肝脏和血清样品。用放射免疫分析法测定肝脏和血清中leptin和甲状腺激素的浓度,用Western blot测定雏鸡肝脏LEPR蛋白含量,实时荧光RT-PCR定量肝脏LEPR和脂肪代谢相关基因表达。结果表明,蛋内注射leptin影响肉鸡出雏后早期生长,并呈现性别特异性和剂量依赖性。无论低剂量还是高剂量,leptin处理组肉鸡的出雏重均低于对照组(P=0.042,0.154),低剂量组差异显著,并且主要针对雌性(P=0.005,0.094)。然而,两个处理组出雏后生长速度均高于对照组。21日龄时,蛋内注射leptin的肉鸡体重均显著高于对照组(P=0.042,0.003)。此外,HL组雄性肉鸡肌间脂肪带宽度显著增加(P=0.018),同时伴随肝脏leptin (P=0.036)、血清leptin (P=0.053)、FT3 (P=0.021)和T3(P=0.034)水平显著升高,肝脏与血清中Tch浓度升高,同时肝脏中SREBP-1、ACC、ApoB、GHR和IGF-I的表达均显著上调(P=0.052,0.017,0.010,0.028,0.020)。以上结果提示:蛋内注射leptin抑制肉鸡胚胎发育,但是出雏后生长速度加快,肝脏脂肪代谢模式发生改变,并且leptin对胚胎发育的影响以低剂量显著,主要针对雌性,而对出雏后生长的影响却主要表现在高剂量,主要针对雄性,说明不同剂量leptin的作用存在不同的时效性和性别特异性。此外,蛋内注射leptin影响出雏后肉鸡生长和代谢的机制可能有甲状腺激素和生长轴基因的参与。
The present study was initiated upon the finding that dietary supplementation of cysteamine to Sanhuang broiler breeder hens affected offspring growth during early posthatch development, accompanied with significant alterations in leptin secretion in hens. The existence of leptin in the egg was proved and the yolk and albumin contents of leptin were found to be modified by cysteamine. We therefore put forward a hypothesis that leptin may be involved in mediating the maternal effect of cysteamine on offspring growth in broiler chickens, probably through modifying hepatic leptin synthesis and action, and altering mRNA expression of key factors involved in hepatic lipid metabolism. Leptin in ovo administration was employed then to verify the hypothesis. The effects of leptin in ovo administration on embryonic development, hatch weight, early posthatch growth rate were observed. The alterations in metabolic and endocrine parameters, hepatic leptin and LEPR expression, as well as the mRNA expression profiles for the key factors involved in lipid metabolism, including SREBP, FAS, ACC and ApoB, were characterized.
     1 Leptin is involved in the effect of cysteamine on egg laying of hens and posthatch growth of broiler offspring
     Cysteamine has been reported to modulate lipid metabolism and energy homeostasis and exert significant growth promoting effects on broiler chickens. However, little is known concerning its effects on egg production of hens and the growth rate of their offspring. In the present study,67-week-old broiler breeder hens were allotted at random to control and cysteamine-supplemented (400 mg/Kg) groups for 8 weeks. The hatchlings were fed under the same condition till 6 weeks of age. Cysteamine significantly increased the average laying rate by 2.24%(P<0.01), decreased dramatically the percentage of the broken eggs by 40.55%(P< 0.01), while increased that of the abnormal eggs by 20.15%(P<0.05). Cysteamine did not alter the egg weight, egg quality, fertility or hatchability, but significantly increased eggshell weight (P<0.05) and decreased albumin weight (P<0.05). Serum concentrations of thyroxine (T4) (P<0.01) and leptin (P<0.01) were significantly lower in cysteamine-treated hens, while triiodothyronine (T3), free T3 and glucagon were not affected. Western blot analysis with leptin-specific antibody detected a band of approximately 15-16 KDa in egg yolk and albumin extracts as well as liver homogenates of hens. Cysteamine did not affect the yolk content of T3, T4, E2 or glucagon, but significantly increased leptin content in liver of hens (P<0.05), as well as in yolk (P<0.05) and albumin (P<0.05) of eggs. These changes were accompanied by a significant down-regulation of leptin receptor mRNA expression(P<0.05) in yolk sac of day 12 embryos. Female offspring hatched from cysteamine-treated eggs demonstrated significantly lower body weight at hatching (P<0.01) and 42 days of age (P<0.01). The results indicate that cysteamine improves laying performance of hens and affects the early posthatch growth of broiler offspring, in a gender-specific fashion. The modified leptin secretion and egg deposition, together with altered yolk sac leptin receptor expression may be involved in such an effect.
     2 Effects of in ovo leptin administration on embryo development and hepatic lipid metabolism in newly hatched broiler chickens
     Leptin in ovo administration was used in the present study to investigate the effect of leptin deposited in eggs on embryonic development and hepatic lipid metabolism in the chicken. Eggs purchased from Sanhuang broiler breeding farm were injected with either 0.5μg of recombinant mice leptin in 100μL of phosphate buffered saline (PBS) or 100μL PBS before incubation. Serum and liver samples were taken and fetal body weights were recorded on embryonic days 12 (E12) and E17 as well as at hatching (DO). Serum concentration of leptin was measured with radioimmunoassay. Hepatic expressions of leptin receptor and lipid metabolic genes were determined with Real-time RT-PCR, and LEPR protein content was detected with Western blot analysis. Chicks hatched from leptin-treated eggs showed lower hatch weight but higher liver index than the control group (P=0.000,0.018). Hepatic leptin content was increased in E12 and DO and so was the serum leptin concentration in DO. However, no differences were found in hepatic LEPR expression at either mRNA or protein levels. Liver contents of TG and Tch were decreased, whereas the opposite was true for serum levels of TG and Tch. This, combined with higher serum level of ApoB, suggests higher lipid transport from liver in leptin-treated group. Hepatic expression of ACC and FAS mRNA was not altered, but SREBP-1 mRNA expression was up-regulated significantly, suggesting a potential increase of fatty acid synthesis in the liver. It was noteworthy that in ovo leptin injection affected female chicks more significantly. These results suggest that in ovo leptin injection inhibits embryo development and decreases hatch weight, which may be associated with alterations in hepatic leptin synthesis and secretion, as well as hepatic lipid metabolism in newly hatched broiler chickens.
     3 Effect of in ovo leptin administration on early posthatch growth and hepatic lipid metabolism in broiler chickens
     Leptin in ovo administration was employed to investigate the dose-dependent effect of leptin deposited in eggs on early posthatch growth and hepatic lipid metabolism in the chicken. Breeder eggs were allocated to three groups and injected with 0.5μg (low leptin, LL),5.0μg (high leptin, HL) of recombinant mice leptin in 100μL of PBS or 100μL PBS (Control, Con) before incubation. The hatchings were raised under the same condition till 21 days (D21) of age when serum and liver samples were taken for analysis. Serum concentrations of leptin and thyroid hormones were measured with radioimmunoassay, hepatic expression of leptin receptor and lipid metabolic genes were determined with Real-time RT-PCR, and LEPR protein content was detected with Western blot analysis. Chicks treated with high or low doses of leptin in ovo had lower hatch weight than the control group, the differences being more significant in LL group and female chicks being more susceptible (P=0.042). However, the posthatch growth rate was higher in both leptin-treated groups compared to control group. At D21, chicks treated with leptin in ovo demonstrated significantly higher body weight than control group (P=0.042,0.003). Furthermore, male chickens in HL group showed higher width of intermuscular fat cingulum, which was accompanied with significantly higher hepatic and serum leptin (P= 0.036,0.053), increased serum FT3 (P=0.021) and T3 (P=0.034), as well as elevated hepatic and serum Tch. Hepatic expressions of SREBP-1, ACC, ApoB, GHR and IGF-I mRNA were found significantly up-regulated in male chickens of HL group (P=0.052, 0.017,0.010,0.028,0.020). These results indicate that in ovo administration of leptin inhibits embryo development but increase posthatch growth rate in broiler chickens with modifications in hepatic lipid metabolism. These effects were dependent on dose, gender and developmental stage. The females were more susceptible to low dose of leptin and affected more significantly before hatching, while the males were more susceptible to high dose of leptin and the effect were more pronounced after hatching. In addition, thyroid hormones and growth axis genes may be involved in the mechanism of leptin's effects on posthatch growth and hepatic lipid metabolism in the chicken.
引文
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