**1. Introduction**

Polyunsaturated fatty acids (PUFAs) are essential nutrients that have a range of biological e ffects such as on brain function, cardiovascular disease, obesity, cancer, and bone health in humans [1,2], and also a ffect the reproduction quality of livestock animals [3]. Among PUFAs, arachidonic acid (ARA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) are known as long chain PUFAs, and their physicochemical properties and metabolisms provide various functions in mammalian cells. ARA and EPA/DHA are also known as ω6 and ω3 polyunsaturated fatty acids, respectively, and their biological functions and the importance of ω3/ω6 fatty acid balance in maintaining homeostasis have attracted increasing attentions [1,2]. Mammals cannot synthesize ARA, EPA, and DHA from their

precursors in sufficient quantities, i.e., linoleic acid (LA) and alpha-linoleic acid (ALA), and they must rely on dietary intake. Therefore, the dietary intake of ARA, EPA, and DHA is essential to maintain human health, and it is well known that livestock animals and marine foods are the primary nutrient sources for ARA and EPA/DHA, respectively.

The lipid profiles in tissues are affected by different balances of dietary PUFAs [4–9]. In fact, the intake of ω3 PUFAs increases tissue levels of ω3 PUFAs and their oxidized forms, namely oxylipins, while the amount of ω6 PUFAs and their oxidized forms is decreased [4,5]. Moreover, a previous study revealed that dietary intake of ω3 PUFA inhibits ARA biosynthesis from LA by the suppression of fatty acid desaturase 2 (FADS2) expression [6], and competes with ARA for membrane phospholipid remodeling. In addition, several ω3 PUFA intervention studies have shown that the supplementation of ω3 PUFA increases ω3-PUFA-derived oxylipins while decreasing ARA-derived oxylipins in human peripheral blood [7]. Although oxylipin profiles under different dietary conditions have been reported [4,5,8,9], there have been few studies that comprehensively investigated the lipid profile after the dietary intake of ARA, EPA, or DHA side-by-side in various tissues, which would be necessary to grasp the effects of different PUFA-containing diets.

After dietary intake, PUFAs are actively incorporated into cells as acyl chains of membrane phospholipids and other lipid classes, such as triacylglycerol and cholesteryl esters. Intake of ARA, EPA, and DHA affects several physiological functions, such as membrane scaffold formation to harmonize biomolecule interactions [10], energy storage and related functions to maintain metabolism [11], and signal transduction via bioactive lipid mediators produced by cyclooxygenases (COX), lipoxygenases (LOX), and cytochrome P450 (CYP) [12]. Since these functions are accomplished by a molecular diversity of lipid species, the comprehensive profiling of lipids in cells and tissues is important to understanding the mechanisms underlying the effect of different PUFA supplementations.

Liquid chromatography coupled with high resolution tandem mass spectrometry (LC-HR-MS/MS) and triple quadruple mass spectrometry (LC-QqQ/MS) are popular techniques for untargeted and targeted lipidomic analysis, respectively [13,14]. LC-QqQ/MS-based targeted lipidomics has high sensitivity, and has often been used for the profiling of oxylipins, where the concentrations in plasma range from 10 pM to 100 nM [14]. In contrast, LC-HR-MS/MS, which has high resolution and scanning speed, has often been used for the profiling of glycerol (phospho) lipids, sphingolipids, and sterols, where concentrations in plasma range from 10 nM to 100 μM [14]. The current informatics technique for untargeted lipidomics enabled us to characterize more than 90 lipid classes by untangling the MS/MS spectrum [15]. Mass-spectrometry-based lipidomics, therefore, has the potential to comprehensively examine tissue lipid profiles in detail, and this lipidomic profiling could give us new insights into dietary PUFA distribution and metabolism in the body.

In this study, we investigated the lipid profiles in plasma and tissues including the liver, kidney, white adipose, skeletal muscle, heart, small intestine, lung, brain, and spleen after dietary intake of the ethyl ester form of ARA (ARA-E), EPA (EPA-E) or DHA (DHA-E) in mice. In total, 1026 molecular species of lipid were identified, of which 915 and 111 were annotated in untargeted and targeted analyses, respectively.

#### **2. Materials and Methods**
