**2. Results**

## *2.1. Egg Traits*

To test the e ffects of breed and feed on egg traits, 10 traits were investigated (Table 1). Two-way mixed design analysis of variance (ANOVA) revealed a significant e ffect of breed on yolk weight (F1,12 = 8.098, *P* = 0.0147), eggshell weight (F1,12 = 7.287, *P* = 0.0193), lightness of eggshell color (F1,12 = 6.022, *P* = 0.0304), redness of eggshell color (F1,12 = 10.818, *P* = 0.0065), and yellowness of eggshell color (F1,12 = 14.394, *P* = 0.0026). Rhode Island Red (RIR) eggs showed higher yolk weight and lower eggshell weight than Australorp (AUS) eggs. Eggshell color in RIR eggs had less lightness, and more redness and yellowness than that of AUS. There were no significant e ffects of feed or interaction terms between breed and feed on any of the egg traits.


**Table 1.** Egg traits at two stages in Rhode Island Red and Australorp.

#### *2.2. Egg Metabolite Traits*

Egg metabolite traits from yolk (138 metabolites) and albumen (132 metabolites) were semi-quantified using GC−MS/MS. Full results were shown for yolk and albumen in Supplementary Tables S1 and S2, respectively. Controlling for multiple comparisons in each sample, some metabolites were found to be metabolome-wide significantly altered by breed and feed ( *Q* < 0.1).

Albumen ribitol was significantly a ffected by breed (Table 2), with RIR eggs containing significantly higher ribitol levels than AUS eggs. Three metabolites in yolk and 12 metabolites in albumen had significant e ffects of feed (Table 3). Erythritol and threitol were significantly altered by feed in both the yolk and the albumen. Urea was altered in the yolk samples only, whereas isoleucine, dihydrouracil, linoleic acid, 4-hydroxyphenyllactic acid, alanine, glycine, N-butyrylglycine, pyruvic acid, ribitol, and valine were altered in albumen samples only. For threitol, erythritol, dihydrouracil, linoleic acid, pyruvic acid, and ribitol, the fermented feed group in both chicken breeds showed significantly higher metabolite content than mixed feed. On the other hand, the fermented feed group had significantly lower contents of urea, isoleucine, 4-hydroxyphenyllactic acid, alanine, glycine, N-butyrylglycine, and valine than the mixed feed group. There was a significant interaction between breed and feed on N-butyrylglycine in the albumen; N-butyrylglycine content in RIR chickens was higher with mixed feed than with fermented feed, but this e ffect was reversed in the AUS samples (Table 4).


**Table 2.** Metabolite with breed-induced changes (*Q* < 0.1).

**Table 3.** Metabolites with feed-induced changes (*Q* < 0.1).

1 The Human Metabolome Database [31]. \* *Q* < 0.1.


1 The Human Metabolome Database [31]. \* *Q* < 0.1.

*Metabolites* **2019**, *9*, 224


**Table 4.** Metabolite with breed × feed-induced changes (*Q* < 0.1).
