**1. Introduction**

Approximately 80 million tons of chicken eggs are produced every year, and they are a crucial source of animal protein in many developing countries [1,2]. Globally, 821 million people are malnourished [3], so international e fforts to increase egg production are of high priority. In more developed countries, consumers are increasingly concerned with the quality of eggs and the bioavailability of favorable functional ingredients [4,5]. Increasing the quantity and quality of eggs around the world is key not only to alleviating hunger, but also to providing important dietary nutrients and keeping up with a rapidly changing international livestock industry. Currently, huge scientific e ffort is devoted to modifying agricultural produce traits to create "designer foods" [6]; eggs are no exception, and considerable e ffort is currently underway to modify egg traits and produce "designer eggs" to meet consumer demand [7,8].

Both genetic and environmental factors influence the quantity and quality of eggs [9–11]. Heritability estimates of egg traits such as overall weight, albumen weight, and yolk weight, are around 0.30–0.70 [12–14], which indicates that genetic factors are crucial to the regulation of egg traits. These general egg traits, which are mass and quality of egg components (yolk, albumen, and eggshell), are important for quality of egg itself as the product of egg market. Environmental factors including age, nutrition, stress, disease, medication, and production system also have important roles in modifying egg traits [9,10]. Thus, studies seeking to enhance egg traits should be cognizant of both genetic and environmental factors.

Egg enrichment with omega-3 polyunsaturated fatty acids (n-3 PUFA), which provide various human health benefits, is of current interest [15]. Evidence suggests that fatty acids quantity can be altered through changes to hens' diet [16]. Other research suggests that responses to dietary enrichment and conversion into egg metabolites may be breed-specific [17]. Both breed and feed appear to regulate the abundance of metabolites in egg yolk and albumen. Recently, we also have reported that egg yolk amino acid was modified by both breed and feed [18]. This is very likely to be the tip of the iceberg regarding the e ffects of genetic and environmental factors on egg composition.

Metabolomics, the study of the set of metabolites present in various tissues, is used to identify novel metabolites or changes in metabolite ratios in tissues [19]. Metabolomic analyses are used in a variety of fields, including biomedicine [20,21], plant science [19], food science [22,23], and ecological/environmental science [24,25]. Typical metabolite analyses use gas chromatography– mass spectrometry (GC–MS), liquid chromatography–mass spectrometry (LC–MS), capillary electrophoresis– mass spectrometry (CE–MS), and nuclear magnetic resonance (NMR). Metabolome analyses have previously been used to identify changes caused by stress and/or feed in mice using CE–MS [26] and GC–MS [27], so we expect a metabolomics approach to provide useful insight into the role of breed and feed in determining egg composition.

As studies into the metabolomes of livestock animals accumulate, a central database—the Livestock Metabolome Database—has been developed to compile this information. Research into cattle metabolic constitutes a large proportion of this work (76 of 149 articles), as does research into animal health, nutrition, and production (97 of 149 papers; [28]). For instance, e ffects of long-distance transportation in serum metabolites have been studied in cattle [29] and heat stress-induced metabolomic changes have been investigated in chicks [30]. Relatively few reports target non-major livestock breeds, and there is a need to populate the Livestock Metabolome Database with basic metabolome data for all livestock breeds.

In this study, we analyzed the yolk and albumen metabolome of eggs and general egg traits from two di fferent breeds of hens under two di fferent feed conditions. We used the GC–MS/MS technique to measure metabolome content, and tested how egg metabolites are influenced by specific genetic and environmental factors, i.e., breed and feed, respectively.
