Abstract The differences of fatty acids in yak’s meat under graze feeding (GF) and stall feeding (SF) regimes and the regulation mechanism of the feeding system on the fatty acids content in yak ’s meat was explored in this study. First, the fatty acids in yak’s longissimus dorsi (LD) muscle were detected by gas liquid chromatography (GLC). Compared with GF yaks, the absolute content of ΣSFAs, ΣMUFAs, ΣUFAs, ΣPUFAs and Σn-6PUFAs in SF yak’s LD were higher, whereas Σn-3PUFAs was lower; the relative content of ΣMUFAs, ΣPUFAs, Σn-3PUFAs and ΣUFAs in SF yak’s LD were lower, whereas ΣSFAs was higher. The GF yak’s meat is healthier for consumers. Further, the transcriptomic and lipidomics profiles in yak’s LD were detected by mRNA-Sequencing (mRNA-Seq) and ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS), respectively. The integrated transcriptomic and lipidomics analysis showed the differences in fatty acids were caused by the metabolism of fatty acids, amino acids, carbohydrates and phospholipids, and were mainly regulated by the FASN, FABP3, PLIN1, SLC16A13, FASD6 and SCD genes in the PPAR signaling pathway. Moreover, the SCD gene was the candidate gene for the high content of ΣMUFA, and FADS6 was the candidate gene for the high content of Σn-3PUFAs and the healthier ratio of Σn-6/Σn-3PUFAs in yak meat. This study provides a guidance to consumers in the choice of yak’s meat, and also established a theoretical basis for improving yak’s meat quality. Keywords: yak’s meat, candidate gene, feeding system, fatty acids, n-3PUFAs 1. Introduction With living standards improving, consumers are more and more focused on meat quality [[34]1], and request to get more superior meat. Meanwhile, the meat industry focuses on developing valuable nutritional properties in meat. Moreover, meat safety incidents frequently happen in the world, and meat safety becomes a focus too [[35]2]. The content and type of fatty acids in livestock meat are among the key factors which affect the sensory qualities of meat such as tenderness, color, and cooking loss [[36]3,[37]4]; on the other hand, the composition of fatty acids in livestock meat is a critical index in evaluating the meat nutrition. Saturated fatty acids (SFAs) in livestock meat can increase the risk of cardiovascular disease for consumers [[38]5], while more polyunsaturated fatty acids (PUFAs), especially n-3 PUFAs like eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), can protect blood vessels and prevent cardiovascular and cerebrovascular diseases [[39]6,[40]7]. The livestock meat with a lower ratio of Σn-6/Σn-3 PUFAs is a benefit to human health [[41]8], so is very popular with consumers. Researchers in livestock husbandry are attempting to alter the composition of fatty acids in livestock meat by increasing the content of unsaturated fatty acids (UFA), especially n-3 PUFAs in recent years. There are significant differences in digestion and absorption characteristics between monogastric and ruminant animals, and the rumen bacteria plays an important part in digestion and absorption for ruminant animals. In production practice, the proportion of concentrate feed for monogastric animals is usually higher, while the proportion of roughage for ruminants is higher. The rumen microbiome through the dietary ingredients under different feeding systems greatly affects the composition of fatty acids. Due to the hydrogenation of ruminal microorganisms in ruminants, the content of SFAs in ruminants’ muscle is usually higher than that of monogastric animals and the ratio of PUFA to SFA is lower [[42]9,[43]10]. The variation in nutrient composition in livestock meat is controlled by the genetics of the animal and the production environment of the livestock [[44]11]. The effect of genetics, diet, gender and feeding system on the content and composition of fatty acids in livestock meat were proved [[45]12,[46]13]. The feeding system is one of the most important factors affecting the fatty acids in livestock meat [[47]14]. Compared with livestock meat from stall feeding (SF), livestock meat from graze feeding (GF) tends to possess a higher percentage of n-3 PUFAs and conjugated linoleic acid [[48]15]. The yak (Bos grunniens) is a unique animal species and one of the dominant animal breeds on the Tibetan Plateau [[49]16]. Nowadays, the yield of yak’s meat in the world exceeds 300 thousand tons per year. Because of high protein content, plenty of functional fatty acids, a unique flavor and an absence of pollutants, yak’s meat has widely attracted the attention of the market [[50]17,[51]18]. The content of SFAs in yak muscle was lower than the values in cattle, while the contents of PUFAs and monounsaturated fatty acids (MUFAs) in yak muscle were higher than the values in cattle [[52]19,[53]20]. The ratio of n-6 PUFAs/n-3 PUFAs in muscle of yak calf was 1.15 [[54]21]. The traditional grazing system is still predominant in yak industry [[55]22], so the yak’s growth is greatly affected by the natural conditions including temperature, precipitation, and grass status. The emergence rate and production performance of yaks are lower by contrast with other cattle breeds. The optimum slaughter ages for yaks is 3.5–5 years longer than 1.5–2 years for beef cattle [[56]23]. Moreover, grass in the Tibetan Plateau has a very short growth period due to the cold weather, and overgrazing has led to the serious grassland degradation at present [[57]24], which seriously threatens the fragile ecology of Tibetan Plateau. In recent years, the SF is being applied in yak production to improve production efficiency and protect the environment [[58]25]. The change of feeding system for yaks maybe lead to differences in the fatty acids in yak’s meat. But as far as we know, there are no reports on the effect of the feeding system on the fatty acids in yak’s meat and the reasons for the differences. The content of fatty acids in meat is the result of the metabolism of fatty acids in livestock muscle. Compared with monogastric animals, the fat metabolism in ruminants is more complex. The rumen microorganisms in ruminants can translate fatty acids from forage or feed into new fatty acids [[59]26], and finally these new fatty acids deposit in subcutaneous, muscle and the abdomen. It is difficult to explore the effect of the feeding system on fatty acids in yak’s meat using a single molecular biology technique. Existing research suggests that the feeding system adjusts the content of fatty acids in livestock meat by rescheduling the metabolite concentration and the expression level of genes involving in fatty acids metabolism (like LIPC, ERFE, FABP3, PLA2R1, LDLR, and SLC10A6 and so on) [[60]27]. Transcriptomics can study the transcription and regulation of all genes in cells at the global level and has been applied to the studies of fatty acids in livestock like cattle [[61]28,[62]29], pig [[63]30], goat [[64]31] and yak [[65]32]. The newest RNA-sequencing (RNA-seq) analysis can examine the mRNA level in yak muscle under different feeding system and estimate gene expression profile. Lipidomics is a kind of high-throughput analysis technology and can systematically analyze the changes of lipid composition and expression in organisms [[66]33] and explore the functions of lipid families and lipid molecules in various biological processes [[67]34]. There are some reports on the studies of the characterization of fatty acids in dairy [[68]35,[69]36], cattle [[70]37,[71]38], goat [[72]39] and pork [[73]40,[74]41] by lipidomics. The lipidomics of yak muscle can help to identify the metabolic networks of fatty acids, which can affect the content and composition of fatty acids. The fatty acids phenotype-related gene functions and lipid metabolism pathways in yak muscle under different feeding system can be revealed by the approach combined lipidomics and transcriptomics. In this study, we hypothesized that the feeding system could affect the fatty acids metabolism in yak muscle by the differential expression of genes associated with fatty acids, and ultimately cause a change to the content and composition of fatty acids in it. The absolute and relative content of fatty acids in yak’s longissimus dorsi (LD) muscle were determined, and the differences in the characteristics of fatty acids in yak muscle between GF and SF were observed. Further, the differentially expressed genes (DEGs) and significantly different lipids (SDLs) in GF and SF yak’s LD were identified by RNA-Seq transcriptomics and non-targeted lipidomics, respectively. The DEGs and SDLs were annotated by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genome (KEGG), then the molecular mechanism of the feeding system affecting fatty acids in yak muscle was explored. This study investigated the differences of fatty acids in yak’s LD under SF and GF and established a theoretical basis for the evaluation of yak’s meat under different feeding system and provided a reference for improving intramuscular fat (IMF) content in yaks. 2. Materials and Methods 2.1. Animals and Sample Collection The animal experiment was approved by the Ethics Committee of the Lanzhou Institute of Husbandry and Pharmaceutical Sciences, Chinese Academy of Agricultural Sciences (Permit No. SYXK-2020-0166). A total of twelve healthy male yaks (the same genetic background, two-year old, 210.33 ± 10.23 kg) were selected in May and were divided into two groups in the study. The GF group: six yaks (n = 6) were only grazed in natural pasture with no supplements; the SF group: six yaks (n = 6) were fed with total mixed ration (TMR) food in a stall. The GF and SF yaks were fed in Qinghai province in China, and the yaks in each group can freely eat grass or MTR and drink water by themselves, respectively. All experimental animals were dewormed before the test. By September, all yaks were humanely slaughtered by professional technicians at a commercial abattoir. The slaughter procedure was conducted in accordance with European Commission Regulation. The LD samples from between the 12th and 13th ribs of the left side of the carcass were immediately collected after the yaks were slaughtered. Part of the LD samples were put in liquid nitrogen immediately, and the rest were frozen at −20 °C. The grass sample was collected in September too. The composition of TMR and the content of common nutrition and major fatty acids in grass and TMR are shown in [75]Table 1. Table 1. The composition of the total mixed ration (TMR) and the content of common nutrition and major fatty acid in natural grass and TMR (air-dry basis). Item TMR Natural Grass Ingredient (%) Corn 19.20 - Wheat bran 9.20 - Whole corn silage 32.00 - Oat Hay 28.00 - Rapeseed meal 8.10 - NaHCO[3] 1.00 - NaCl 1.50 - Premix 1.00 - Total 100.00 - Common nutrition (%) Crude fat 4.52 2.63 Crude protein 16.96 11.93 Neutral detergent fiber 23.24 76.14 Acid detergent fiber 13.84 10.09 Calcium 0.79 5.22 Phosphorus 0.37 0.07 Fatty acids (%) C16:0 0.68 0.30 C18:0 0.21 0.09 C18:1 0.18 0.07 C18:2n6 0.58 0.29 C18:3n3 1.42 0.68 [76]Open in a new tab The ingredient of premix was 3000 IU VA, 500 IU VD3, 5 IU VE, 0.1 mg Se, 30 mg Fe, 18 mg Mn, 18 mg Zn and 3 mg Cu in per kg of the diets. 2.2. Determination of Fatty Acids The absolute content of fatty acids in the yak’s LD was detected according to the method described in references [[77]42,[78]43,[79]44]