黄羽肉鸡胚期卵黄微生物动态变化及对早期肠道微生物定殖的影响

Dynamic Changes of Yolk Microbiota in Yellow-Feathered Broiler and Its Role on Early Colonization of Intestinal Microbiota During the Embryonic Stage

【目的】研究黄羽肉鸡种蛋孵化过程中卵黄微生物区系的动态变化和功能,及卵黄微生物对肉鸡胚胎肠道早期微生物定殖的影响。【方法】选取35周龄黄羽肉鸡种蛋100枚,放置在全自动孵化箱中孵化。种蛋入孵前对孵化箱进行清洁消毒,检查内部通风系统和湿度控制系统是否正常工作。孵化温度控制在(37.5±1)℃,湿度控制在(60±10)%。在种蛋孵化的第7、11、15和19天随机选取6枚种蛋在无菌操作台中剥去蛋壳及尿囊膜,使胚胎露出卵黄囊后通过无菌注射器刺入,收集卵黄样品。在孵化的第19和21天在超净工作台中使用经灭菌的手术刀和镊子解剖鸡胚,切开鸡胚腹部后收集胚胎中全肠道样品。样品采集后迅速放入液氮中速冻,随后转入-80℃冰箱中保存待测。对卵黄微生物进行DNA提取,并进行16s rDNA测序和序列分析。【结果】1)黄羽肉鸡种蛋卵黄中存在丰富的微生物区系,共注释到4305个OTU(operational taxonomical unit),在不同孵化时间点卵黄和胚胎肠道中微生物共具有18个门(Phylum),34个纲(Class),53个目(Order),89个科(Family),126个属(Genus)。门水平上主要包括变形菌门(Proteobactreia)、厚壁菌门(Firmicutes)、拟杆菌门(Bacteroidetes)、疣微菌门(Verrucomicrobia)等,属水平上以嗜糖假单胞菌属(Pelomonas)、罗尔斯通菌属(Ralstonia)、水杆菌属(Aquabacterium)、粪杆菌属(Faecalibacterium)等为主;2)黄羽肉鸡种蛋卵黄微生物区系在不同孵化阶段的Alpha多样性中Chao1指数(P=0.027)和Shannan指数(P=0.043)出现显著差异。随着孵化时间的推移,卵黄微生物区系趋于稳定,同时卵黄中阿克曼氏菌属(Akkermansia)相对丰度不断提高,在E07、E11、E15和E19分别为0.53%、0.84%、1.43%和3.10%。卵黄微生物区系在属水平上主要包括嗜糖假单胞菌属(Pelomonas),罗尔斯通菌属(Ralstonia)、水杆菌属(Aquabacterium)等;3)黄羽肉鸡种蛋卵黄微生物功能主要包括碳水化合物代谢、氨基酸代谢、脂类代谢、辅助因子和维生素代谢等;4)黄羽肉鸡种蛋卵黄微生物区系和胚胎肠道微生物区系在门水平上均以变形菌门(Proteobacteria)、厚壁菌门(Firmicutes)、拟杆菌门(Bacteroidetes)、疣微菌门(Verrucomicrobia)为主,在属水平上均以嗜糖假单胞菌属(Pelomonas)、罗尔斯通菌属(Ralstonia)、水杆菌属(Aquabacterium)、阿克曼氏菌属(Akkermansia)为主。【结论】黄羽肉鸡种蛋卵黄中含有丰富的微生物种群,其对卵黄营养物质吸收代谢及胚胎肠道早期微生物区系的建立有重要作用。

【Objective】 The purpose of this experiment was to study the dynamic changes and functions of the yolk microbiota during the hatching of yellow-feathered broiler fertilized eggs, and the influence of yolk microbiota on the early intestinal colonization of embryos. 【Method】 One hundred fertilized eggs of 35-week-old yellow-feathered broilers were selected and placed in automatic incubators for incubation. Before hatching, the incubator was cleaned and disinfected, and the internal ventilation system and humidity control system were checked for normal operation. The incubation temperature was controlled at(37.5±1) ℃and the humidity at(60±10)%. Six breeder eggs were randomly selected on days 7, 11, 15 and 19 of incubation to collect the yolk samples by stripped the eggshell and allantoic membrane in a sterile operation table, exposed the embryo to the yolk sac and then pierced it through a sterile syringe. Samples were rapidly snap frozen in liquid nitrogen after collection and subsequently transferred to a-80 ℃ refrigerator for storage pended test. The yolk microorganisms were subjected to DNA extraction, and 16s rDNA sequencing and sequence analysis were performed. 【Result】 1) A total of 4 305 OTUs were annotated in the yolk of fertilized eggs, and a total of 18 Phyla, 34 Classes, 53 Orders, 89 Families and 126 Genera were found in the yolk and intestine at different incubation time points. The phylum level mainly included Proteobactreia, Firmicutes, Bacteroidetes, Verrucomicrobia, etc. The genus level is dominated by Pelomonas, Ralstonia, Aquabacterium, Faecalibacterium, etc;2) Significant differences in the Chao1index(P = 0.027) and Shannan index(P = 0.043) were observed in the Alpha diversity of yolk microbiota at different incubating stages. The yolk microbiota tended to stabilize with incubation time, while the relative abundance of Akkermansia in the yolk increased continuously to 0.53%, 0.84%, 1.43% and 3.10% at E07, E11, E15 and E19, respectively. At the genus level, the yolk microbiota mainly included Pelomonas, Ralstonia, Aquabacterium and so on;3) The functions of yolk microbiota in yellow-feathered broiler breeding eggs mainly include carbohydrates metabolism, amino acids metabolism, lipids metabolism,cofactors and vitamins metabolism;4) Both the yolk microbiota and intestinal microbiota of fertilized eggs were dominated by Proteobacteria, Firmicutes, Bacteroidetes, and Verrucomicrobia at the phylum level, and Pelomonas, Ralstonia, Aquabacterium and Akkermansia at the genus level. 【Conclusion】There are abundant microbial populations in the yolk of yellow-feathered broiler fertilized eggs, which play an important role in the absorption and metabolism of yolk nutrients and the early microbial colonization of embryo intestine.