iTRAQ-based quantitative proteomic analysis provides insights into strong broodiness in Muscovy duck (Cairina moschata) combined with metabolomics analysis☆
Graphical abstract
Introduction
The Muscovy duck is a special kind of waterfowl that belongs to a different genus than domestic duck, and it has the characteristics of high heat resistance and high meat yield but strong broodiness and low egg-laying performance. It is quite challenging to reduce or eliminate the broodiness of the Muscovy duck. The broodiness of poultry has attracted great attention, and it is commonly regulated by the environment, nutrition, heredity and endocrinology of the ducks [[1], [2], [3], [4]]. There is a low heritability of broodiness, and it is difficult to eliminate broodiness by conventional breeding methods [5]. Many studies and our own previous studies have shown that the hypothalamic-pituitary-gonadal axis is a key regulatory axis for the regulation of reproductive performance in birds, in which ovaries play an important role. Ovaries secrete a variety of hormones, such as progesterone, estradiol, follicle-stimulating hormone, luteinizing hormone, androgen, and prolactin, which are considered to be the key hormones associated with broodiness, and most of these are steroid hormones [6,7]. These hormones affect the development and maturation of oocytes and follicles and ultimately promote or inhibit egg laying. Therefore, those genes, proteins, metabolites, growth factors and signaling pathways related to the synthesis, metabolism, and transport of steroid hormones have become the focus of current research on broodiness. Steroid biosynthesis, Steroid hormone biosynthesis, the GnRH signaling pathway, Calcium signaling pathway, PI3K-Akt signaling pathway, Progesterone-mediated oocyte maturation and Oxytocin signaling pathway are all key signaling pathways related to reproductive performance [[8], [9], [10]]. Studies have shown that the inhibition of steroid synthase in steroid biosynthesis causes ovarian function degeneration and triggers broody behavior [11]; the cholesterol side-chain cleavage enzyme gene in the steroid hormone biosynthesis pathway is associated with hormone release and follicular development [7,12]. In addition, the large number of follicular atresia and the degeneration of follicles during the broody period of birds is also related to the autophagy and apoptosis of granular cells [13,14]. However, how these factors affect the ovarian structure and function of Muscovy ducks to subsequently cause the broody behavior remains unclear.
In the recent years, Gel-free proteomic approaches based on mass spectrometry (MS) have been widely used in livestock and poultry breeding studies [15]. Some studies using proteomics techniques have achieved good results by iTRAQ methods, such as studies of the egg-laying performance of Huoyan geese [9], the molecular mechanisms for hyperprolificacy of Small Tail Han sheep [16], the diversity of wool fibers in sheep and goats [17], as well as studies in the plant and medical fields [[18], [19], [20]]. At the same time, an untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based global metabolomics method was used to identify metabolites [21,22], combing univariate and multivariate statistical analyses to screen DEMs and to conduct pathway analysis [23]. Important metabolites selected as biomarkers have important research significance for the study of some physiological or pathological characteristics. Given our interest in obtaining a deeper understanding of the broodiness of Muscovy ducks, we undertook a combined proteomic and metabolomic investigation in ovary tissue of Muscovy ducks during the broody and laying periods. This study aims to reveal molecular mechanisms of Muscovy duck broodiness and to provide a new method for researching the broodiness of poultry.
Section snippets
Ethics statement
All animal experiments in this study were reviewed and approved by the Institutional Animal Care and Use Committee at the College of Animal Science, Fujian Agriculture and Forestry University. All of the following procedures were strictly performed according to the regulations and guidelines established by this committee.
Animal and tissue collection
In this study, small white feather female Muscovy ducks (MB lines, The MB lines were obtained by crossing two lines with divergent egg production rates by the Putian Guangdong
Identification of ovary tissue protein in Muscovy duck during the broody and laying periods
In the present study, a total of 297,472 spectra were generated from the iTRAQ experiment using the laying period as the control group and the broody period as the experimental group. Mascot search against the NCBI nr mallard sequence database and blast compared with several animal protein databases identified a total of 24,599 spectra, 22,044 unique spectra, 11,708 peptides, 11,217 unique peptides and 2759 proteins (Supplementary Table S2). Approximately 89% of these proteins had a coefficient
Interesting differentially expressed proteins between broody and laying periods
In this study, a total of 335 DEPs were identified by iTRAQ quantification between the broody and laying periods, and several (APOV1, GAL, SAA, GNB5, VLDLR and CDK1) important and significantly up-regulated or down-regulated proteins were selected that may be associated with the broodiness of Muscovy duck. APOV1 was the most up-regulated protein in the broody period. This small, hormonally regulated protein was found to play an important role in productive performance in quail [30]. It is a
Conclusion
The iTRAQ-based quantitative proteomics analysis and untargeted LC-MS/MS-based global metabolomics method utilized here identified many DEPs, DEMs and pathways, which have provided some insight into the molecular mechanisms of strong broodiness in Muscovy ducks. Among them, APOV1, GAL, SAA, GNB5, VLDLR and CDK1 are obviously up-regulated or down-regulated proteins, which are mainly involved in the pathways related to reproductive performance and may regulate the development and maturation of
Declaration of interest
All authors declare that they have no conflict of interest.
Acknowledgements
This project was supported by the National Natural Science Foundation of China (31001005), the Fujian Provincial Natural Science Foundation (2016J01092), the Agricultural Industrialization Project of Fujian Province (fjzycxny2017011), the Earmarked Fund for Modern Agro-industry Technology Research System of China (CaRS-43).
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Biological significance: The Muscovy duck is a commercially and nutritionally important meat duck that is widely raised in the southeast coastal areas of China. However, it has the characteristics of strong broodiness, and poor egg-laying performance has restricted the rapid development of Muscovy duck breeding. Although previous studies have obtained some molecular mechanisms of broodiness by transcriptome and Long Non-Coding RNAs analysis, the global profiles of proteins and metabolites in tissue of Muscovy duck between broody and laying periods are still not available. Proteins and metabolites as functional implementers have potential research significance physiologically. The work herein also represents the first time that the combination analysis of proteomics and metabolomics was used to reveal the molecular mechanisms of broodiness in Muscovy ducks. The results of this study will provide more insights into the broodiness of Muscovy ducks at the level of proteins and metabolites.
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These authors contributed equally to this work.