胚蛋注射丙酮酸肌酸调控肉鸡能量代谢和肌肉发育的作用机理研究

The deficiency of energy reserves during the late hatching stage always increases embryo mortality, and even blocks the subsequent growth of chickens. Meanwhile, the newly hatching chicks cannot get access to the feed within 24 to 72 hours posthatch owing to a large span of pipping time and other factors including management and transportation etc, which further aggravates their energy depletion. In poultry, in ovo feeding (IOF) of exogenous active agents has been proposed as an effective means of regulating early nutrition supply of chick embryos and subsequent growth of chickens. Some studies reported that feeding creatine pyruvate (CrPyr) played an important role in regulation of energy metabolism and muscle development of broilers. However, the effect of injecting CrPyr during late embryogenesis on the energy metabolism and subsequent muscle growth of broilers and its mechanism are still not fully elucidated. Thus, the objective of this project is to study the effect of injecting CrPyr into embryonic chicks on the CrPyr-induced energy buffering ability of creatine pool, the activities of key enzymes involved in energy metabolism, the muscle satellite cell mitotic activity and the gene expression of muscle regulatory factors. We further investigate the expression of LKB1/AMPK and Wnt signal pathways to unearth the regulatory mechanism of CrPyr injection. The study will reveal the effect of injecting CrPyr during late embryogenesis on the energy metabolism and subsequent muscle growth of broilers and its regulatory mechanism. These results will provide the important theoretical basis for solving the deficiency of energy reserves during the late embryogenesis, and also provide a new approach for early nutritional regulation of broilers.

孵化后期能量储备不足是导致鸡胚死亡和雏鸡生长发育迟缓的主要原因之一。同时，出壳时间、管理和运输等因素致使雏鸡难以在出壳后24-72h及时开食，加剧了雏鸡能量的消耗。胚蛋注射外源活性物质是调控家禽早期营养继而影响后期生长的有效技术手段。已有研究表明，饲喂丙酮酸肌酸（CrPyr）可调控肉鸡能量代谢和肌肉发育，但胚蛋注射CrPyr对孵化后期能量代谢和随后肌肉生长发育的影响与机理不明。本项目拟选取爱拔益加肉鸡种蛋，采用孵化后期胚蛋注射CrPyr技术，研究其对肉鸡肌酸代谢的能量缓冲效应、能量代谢关键酶活性、卫星细胞有丝分裂活性、肌肉发育关键调控基因表达的影响；探讨CrPyr对LKB1/AMPK和Wnt信号通路关键因子表达的影响与机制。本研究可阐明CrPyr对肉鸡孵化后期和出雏早期能量代谢和随后肌肉生长发育的影响及其机制，可为解决雏鸡能量储备不足问题提供重要科学依据，为肉鸡早期营养调控提供研究新思路。

胚蛋注射（In ovo feeding，IOF）营养素或活性物质是一种调控家禽早期营养的有效技术手段。丙酮酸肌酸（Creatine pyruvate，CrPyr）是一种新型营养素，它兼具肌酸的特性和丙酮酸的功能。研究表明，CrPyr对肉鸡能量代谢有积极作用，能增强机体的能量供应、促进肌肉发育。但目前关于胚蛋注射CrPyr调控肉鸡能量代谢和肌肉发育的研究还未见报道。本研究发现：胚蛋注射12 mg的CrPyr可提高肉鸡42日龄的体重，改善肉鸡的生长性能。胚蛋注射CrPyr提高了孵化后期鸡胚和雏鸡肝脏葡萄糖和糖原的浓度，这可能与提高了肝脏糖异生关键酶的活性、糖异生途径和糖原合成关键酶基因的表达，促进了肝脏的糖异生和糖原合成过程有关；注射CrPyr提高了胸肌和腿肌肌酸和磷酸肌酸的浓度，增强了鸡胚和雏鸡肌肉的能量储备；注射CrPyr提高了出壳当天肉鸡胸肌葡萄糖转运载体的表达，促进了胸肌对血液葡萄糖的摄取，同时提高了糖酵解过程中关键酶的活性，促进了胸肌中的葡萄糖分解供能，这可能与高浓度的肌酸激活了肉鸡胸肌LKB1/AMPK信号通路有关。胚蛋注射12 mg的CrPyr可提高胸肌重量和相对胸肌重量，有提高新生仔鸡腿肌重及腿肌率的趋势，促进肌肉发育。胚蛋注射CrPyr可上调雏鸡胸肌MRFs基因家族及Pax7基因的表达水平，抑制Myostatin基因的表达，提高3日龄肉鸡胸肌卫星细胞有丝分裂活性指数，促进卫星细胞的增殖和分化，这可能与肌酸激活了Wnt/β-catenin信号通路有关。同时通过激了mTOR通路和抑制泛素蛋白酶体途径降解肌肉蛋白，有利于腿肌肌肉的发育。蛋白质组学分析发现，胚蛋注射CrPyr主要通过调控ATP相关酶活性、调节碳水化合物代谢、脂代谢等过程，通过甲状腺激素信号通路和AMPK信号通路来影响机体的能量代谢过程。此外，胚蛋注射CrPyr可以通过影响肌纤维的发育、细胞增殖分化和蛋白质合成等过程来影响胸肌的发育过程。胚蛋注射CrPyr改善了肉鸡空肠的结构和功能，增强了空肠的消化和吸收能力，有利于肉鸡的生长发育。本项目初步揭示了胚蛋注射CrPyr对肉鸡能量代谢和肌肉发育的调节机制，为解决雏鸡机体能量储备不足问题提供相关参考，也为家禽早期营养调控提供新的研究思路。