Abstract Simple Summary Paper mulberry (PM) is an unconventional protein feed material, and silage is its main processing method. The present study aimed to investigate the health benefits and meat quality of supplementing Yangzhou geese with paper mulberry silage. Results indicated that paper mulberry silage supplementation had a promotional effect on the growth of Yangzhou geese, the sensory quality of the breast muscles improved, and the nutritional quality enhanced. Untargeted metabolomics analysis demonstrated that PM treatment enhanced guanidinoacetic acid levels in breast muscles and facilitated the metabolism of amino acids and the role of substances in lipid antioxidant pathways. Consequently, paper mulberry could be considered to be a novel protein feed option capable for geese. Abstract There have been few investigations into the health benefits and meat quality of supplementing Yangzhou geese with paper mulberry silage. One hundred and twenty 28-day-old Yangzhou geese were selected for the experiment and randomly divided into two groups: a control group (CON) and a paper mulberry silage group (PM), with six replicates in each group. The experiment lasted for a total of 6 weeks. The experiment found that compared with CON, PM had a promoting effect on the average daily weight gain of Yangzhou geese (p = 0.056). Sensory and nutritional analysis of breast muscles revealed a decrease in a* value (p < 0.05) and an increase in protein content (p < 0.05) following PM treatment. Through untargeted metabolomics analysis of breast muscle samples, it was found that 11 different metabolites, including guanidinoacetic acid and other substances, had a positive effect on amino acid metabolism and lipid antioxidant pathways of PM treatment. Overall, the strategy of feeding Yangzhou geese with paper mulberry silage is feasible, which can improve the sensory quality and nutritional value of goose meat. The experiment provides basic data for the application form of goose breeding, so exploring the impact of substances within paper mulberry on goose meat should be focused on in the future. Keywords: goose, paper mulberry, growth performance, carcass characteristic, meat quality 1. Introduction Goose (Anas cygnoides) meat has been recognized for its potential health benefits due to its high-quality protein and unsaturated fatty acid [[40]1,[41]2]. In 2020–2021, there was around 700 million producing animals/slaughtered (globally around 750 million) and the volume of goose meat (fresh or chilled) exceeded 4 million tons in China according to FAOSTAT statistical data. Geese, being herbivorous poultry, have a natural inclination towards consuming grass and possess the ability to digest various types of dietary fiber. However, the traditional small-scale feeding pattern of geese, which primarily relies on gazing, is insufficient to meet the demand for goose meat. As a result, the feeding system for geese has shifted from gazing to feedlot operations [[42]3]. Nevertheless, geese that are not fed with forage or roughage exhibit a decrease in the nutritional value of their meat compared to those supplemented with a moderate level of crude fiber [[43]4]. Therefore, it is crucial to incorporate green feed, such as forage, into the intensive feeding pattern to ensure the health of geese and improve the quality of their meat. Enhancing animal product quality is the primary focus of animal nutrition and feed science. Previous studies have shown that an optimal intake of crude fiber can contribute to improved geese growth performance and meat quality [[44]5]. This includes regulating amino acid and fatty acid profiles in the muscle tissue of geese [[45]6,[46]7,[47]8]. Therefore, many researchers have been studying the selection of suitable forage species and supplementation levels to enhance geese performance. However, the impact of adding dietary crude fiber still requires further investigation [[48]5]. With the rapid development of husbandry in China, the shortage of high-quality forage has become a significant challenge in improving animal production quality [[49]9]. Paper mulberry (Broussonetia papyrifera), a novel type of woody forage, possesses desirable characteristics such as strong stress resistance and high crude protein content (15–22% based on dry matter). Additionally, it contains abundant bioactive substances, including flavonoids [[50]10,[51]11]. Ensiling is commonly used for preserving fresh paper mulberry [[52]12]. While most research on feeding paper mulberry has focused on ruminants, particularly lambs, there is limited information available on its impact on poultry [[53]13,[54]14,[55]15]. Chen et al. reported that supplementing finishing pigs’ diet with 4% paper mulberry improved meat flavor by increasing the content of crude protein, amino acids, and total unsaturated fatty acids [[56]16]. However, the effect of feeding paper mulberry on the growth and meat quality of geese has not been investigated. Therefore, this experiment aimed to assess the effects of dietary paper mulberry supplementation on the growth performance, carcass characteristics, meat sensory quality, chemical components, and breast muscle metabolites in geese. 2. Materials and Methods 2.1. Ethics Statement All the geese used in this experiment were treated in compliance with Chinese animal welfare regulations. The protocols for this study were approved by the Animal Ethics Committee of China Agricultural University (AW82203202-5-1). 2.2. Animals and Experiment Design One hundred and twenty 28-day-old healthy male Yangzhou geese with similar body weights were randomly allocated into either control (CON) or paper mulberry silage (PM) groups. The CON group was fed a basic diet, while the PM group was fed a diet with PM added. Each group contained 6 replicates with 10 geese per replicate. The pre-feeding period lasted for 7 days, followed by the feeding trial period within 42 days. The diets employed in this experiment were formulated to meet the nutritional requirements recommended by the National Research Council (NRC, 1994). The composition and nutrient levels of the basal diet are shown in [57]Table 1. The PM utilized in this study was purchased from Fengtang Ecological Agricultural Technology Co., Ltd. (Taian, China), and the nutritional composition of PM was shown in [58]Table S1. All geese had access to unlimited feed and water in the poultry feeding house. Table 1. Composition and nutrient level of experiment diets (air-dry basis) (%). Item Groups CON PM Ingredient Corn 60.00 57.30 Wheat bran 15.50 12.50 Soybean meal 20.50 18.25 Paper mulberry silage 0.00 8.00 CaHPO[4] 0.20 0.20 Shell 0.15 0.05 NaCl 0.30 0.30 Met 0.15 0.20 Premix 3.20 3.20 Total 100.00 100.00 Nutrient level ME/(MJ·kg^−1) 12.21 12.15 CP 16.50 16.50 CF 3.82 5.20 Ca 0.76 0.77 P 0.57 0.54 Lys 0.78 0.70 Met 0.77 0.76 [59]Open in a new tab The premix provided the following per kilogram of diets VA 6700 IU, VD3 1500 IU, VE 14 mg, VK3 1.75 mg, VB1 1.20 mg, VB2 4.35 mg, VB6 2.85 mg, VB12 0.002 mg, nicotinic 29.00 mg, D-calcium pantothenate 7.85 mg, folic acid 7.85 mg, D-biotin 0.08 mg, choline 3.00 mg, Ca 550 mg, P 370 mg, Fe 60 mg, Cu 5 mg, Mn 6.5 mg, Zn 60 mg, I 3.5 mg, and Se 0.25 mg; nutrient levels are calculated values. 2.3. Growth Performance and Sampling Geese underwent fasting before and prior to the morning feeding on the 1st and 42nd day of the feeding trial period. The initial weight measurement was recorded on the 1st experimental day, while the final weight measurement was taken on the 42nd experiment day. The average daily feed intake (ADFI), average daily gain (ADG), and feed-to-gain ratio (F/G) were calculated by recording the body weight, feed intake, and leftover feed of geese. On the final day of the feeding trial period, one goose in each replicate with the mean body weight of the replicate was selected and euthanized by electrical stunning and jugular vein bloodletting after fasting for 6 h. The geese carcass weight was measured after the feathers were removed. Then, the thorax was immediately opened to remove the viscera, and the eviscerated carcasses were weighed. The entire pectoral muscle was isolated and weighed post-excision of surplus adipose tissue. The mass index of carcass weight, eviscerated carcass, and breast muscle were then calculated according to the proper equation formulas [[60]17]. The entire right breast muscles were collected to determine meat quality at 4 °C and then kept at −20 °C for analyzing the chemical composition of the geese’s meat. The left breast muscles were sampled and quickly frozen in liquid nitrogen, then stored at −80 °C until further analysis. 2.4. Meat Quality Determination The water holding capacity (WHC) was determined according to the method described by Faustman [[61]18]. The goose breast muscle sample of a 1 cm thick slice from goose breast muscle was taken for weighing. After pressurizing the steel ring of the soil permissible expansion and compression meter (B6101, Soil Instruments, Nanjing, China) to the maximum value three times and then decompressing it to zero on the micrometer, the meat sample was wrapped in gauze, placed on filter papers, and pressurized to 35 kg for 5 min on a soil allowable expansion and compression meter platform. After applying pressure, the meat was removed from the gauze and weighed promptly. Afterwards, the water loss rate under pressure was calculated. After the breast muscle sample was stored for 24 h at 4 °C, the pH and meat color values were measured via pH meter (PHBJ-260, Inesa Instruments, Shanghai, China) and 3NH Instruments colorimeter (NR20XE, Shenzhen, China), respectively. The lightness (L*), redness (a*), and yellowness (b*) were determined by the instrument under viewing geometry (45°/0°), measuring the aperture (Φ20 mm) and light source (D65) at room temperature and under normal light conditions. The pH and color values were measured in triplicate for each sample to obtain an average as the final values. An official methods of analysis (AOAC) was used for determining the chemical composition [[62]19]. The dry matter content was determined by using a vacuum method to freeze the samples at −50 °C for 5 days. Kjeldahl nitrogen was used to determine the content of crude protein, while Soxhlet extraction was used to determine the content of intramuscular fat. The crude ash was determined using the high-temperature burning method. 2.5. Analysis of Metabolomics 2.5.1. Extraction of Metabolites The metabolites were extracted using previously established methods and modified as needed [[63]20]. Accurately weighed 100 ± 2 mg meat samples were transferred into a 2 mL centrifuge tube containing 1000 μL tissue extract. After 3 steel balls were added, the samples were placed in a tissue grinder machine (50 Hz, 60 s) (MB-96, Meibi Instrument Co., Ltd., Jiaxing, China) and repeated twice. The sample was subjected to ultrasound at room temperature for 30 min followed by an ice bath for 30 min. Afterward, it was centrifuged for 10 min at 12,000 rpm and 4 °C, and the supernatant was carefully transferred into a new 2 mL centrifuge tube. The sample was concentrated and dried. To redissolve the sample, a 50% acetonitrile solution was accurately added (200 µL) and prepared with 2-chloro-l-phenylalanine (4 ppm), which was stored at 4 °C. The supernatant was then filtered with a 0.22 μm membrane and transferred into a detection bottle for LC-MS detection [[64]21]. 2.5.2. LC-MS Analysis Liquid chromatography conditions: the LC analysis was performed on an ultra-high-performance liquid phase system (Ultimate 3000, Thermo Fisher Scientific, Waltham, MA, USA). Chromatography was carried out with an ACQUITY UPLC^® HSS T3 (150 × 2.1 mm, 1.8 µm) (Waters, Milford, MA, USA). The column was maintained at 40 ℃. The flow rate and injection volume were set at 0.25 mL/min and 2 μL, respectively. For LC-ESI (+)-MS analysis, the mobile phases consisted of (A) 0.1% formic acid in acetonitrile (v/v) and (B) 0.1% formic acid in water (v/v). For LC-ESI (-)-MS analysis, the analytes were carried out with (C) acetonitrile and (D) ammonium formate (5 mM). Mass spectrum conditions: electrospray ion source (ESI), positive and negative ion ionization mode, positive ion spray voltage of 3.50 kV, negative ion spray voltage of 2.50 kV, sheath gas of 30 Arb, and auxiliary gas of 10 Arb. The capillary temperature was 325 °C, and the scanning range was 81~1000 with a resolution of 70,000. HCD was used for secondary cracking with a collision voltage of 30 eV, and the unnecessary MS/MS information was removed via dynamic elimination [[65]22,[66]23]. To effectively analyze the data, it was first converted to mzXML format using MSConvert, which is a component of the ProteoWizard software package (v3.0.8789). Next, we employed R software XCMS package (v4.1.3) to detect features, correct retention time, and align the data. To identify the metabolites, data was carefully scrutinized by considering the accuracy of the mass (ensuring it was less than 30 ppm) and cross-referenced the MS/MS data with a variety of online metabolite databases, including the Human Metabolome Database (HMDB), massbank, LipidMaps, mzcloud, and the Kyoto Encyclopedia of Genes and Genomes KEGG [[67]24]. The data were normalized using QC-RLSC signal correction to correct for bias. Only ion peaks with RSDs less than 30% in QC were kept for metabolite identification. 2.5.3. Multivariate Statistical Analysis To analyze the impact of PM treatment on goose meat metabolites, a multivariate statistical analysis was conducted. Orthogonal projection to latent structures–discriminatory analysis (OPLS-DA) was used to evaluate the key metabolites via SMICA 14.1 (UMETRICS, Umeå, Sweden). The metabolites with a VIP greater than 1 from OPLS-DA, fold change (FC) > 2.0 or <0.5 and p < 0.05 were regarded as significantly differential metabolites. The differential metabolites and their respective metabolic pathways were analyzed using MetPA, which is part of Metaboanalyst 5.0 ([68]www.metaboanalyst.ca assessed on 15 January 2021) and is mainly based on the KEGG metabolic pathway. 2.6. Statistic Analysis All data were statistically processed with one-way ANOVA using SPSS 26.0 (SPSS Inc., Chicago, IL, USA). Results were displayed as mean and standard error of the mean (SEM). Values of p < 0.05 were considered to be statistically significant. 3. Results 3.1. Growth Performance and Carcass Characteristics The effect of PM supplement on the growth performance and carcass characteristics of geese was shown in [69]Table 2. Following a 7-day adaptation period, the initial body weight did not show significant difference between CON and PM groups (p > 0.05). The dietary PM supplementation had no significant impact on the final body weight compared with CON treatment, as well as ADFI and F/G (p > 0.05). And no differences were observed in the dressing percentage, eviscerated percentage, and breast muscle percentage between CON and PM groups (p > 0.05). However, a trend (p = 0.056) was observed for increased ADG (4.81%) for the supplementation with PM. Table 2. Effects of CON and PM diet supplementation on initial body weight, final body weight, average daily gain, dressing percentage, eviscerated percentage, and breast muscle percentage. Item Groups SEM p-Value CON PM Live weight, g D[1] 632.00 612.89 8.65 0.421 D[42] 3168.5 3277.56 34.17 0.095 ADFI, g 323.64 338.65 8.35 0.690 ADG, g 60.39 63.44 0.79 0.056 F/G 5.58 5.60 0.08 0.841 Dressing percentage, % 84.15 83.12 0.51 0.343 Eviscerated percentage, % 68.06 68.50 0.65 1.000 Breast muscle percentage, % 7.65 8.08 0.17 0.486 [70]Open in a new tab D[1], the initial live weight of the geese (28-day-old). D[42], the final live weight of the geese (70-day-old). 3.2. Meat Quality Parameters The satisfaction of consumers with meat is greatly influenced by its sensory and nutritional qualities. As shown in [71]Table 3, there is no significant difference in WHC and pH[24h] of the breast muscles between the PM group and the CON group (p > 0.05). When considering the color of the meat, there were no significant differences in the L* and b* values between the CON and PM groups (p > 0.05). However, the a* value of the PM group was notably lower than that of the CON group (p < 0.05). Table 3. Effects of CON and PM diet supplementation on breast meat quality. Item Groups SEM p-Value CON PM WHC, % 28.25 27.81 0.61 0.765 pH[24h] 5.85 5.87 0.02 0.629 Meat color L* 49.59 47.77 0.62 0.157 a* 22.36 19.69 ** 0.63 0.005 b* 10.44 10.63 0.10 0.407 Dry matter, % 22.07 23.55 0.44 0.089 Crude protein, % 79.35 80.42 * 0.28 0.041 Intramuscular fat, % 9.05 8.91 0.11 0.588 Ash, % 1.53 1.55 0.03 0.728 [72]Open in a new tab Calculation of crude protein, ether extract, and ash based on dry matter, * p < 0.05 and ** p < 0.01, compared with the CON group. The PM group had a higher content of crude protein compared to the CON group (p < 0.05). No significant differences were observed in dry matter, ether extract, and ash between the two groups (p > 0.05). 3.3. Untargeted Metabolomic 3.3.1. General Information for Metabolomics Data The data for metabolomics were obtained using an internal mass spectrometry-based method. After analyzing the metabolites of MS/MS, a total of 572 metabolites were found across all the samples, with no missing observations. Out of these, 381 compounds were identified for metabolomics in positive ionization mode, while 191 were identified in negative ionization mode ([73]Tables S2 and S3 provide more information). The application of OPLS-DA reduced the complexity of the model and improved its explanatory capacity, while maintaining its predictive ability. As shown in [74]Figure 1, this analysis was also conducted on metabolites, and the validity of the permutation analysis was confirmed. Figure 1. [75]Figure 1 [76]Open in a new tab Multivariate analysis of metabolites in breast muscle of CON and PM groups. (A) Orthogonal projection to latent structures–discriminatory analysis (OPLS-DA) score plot. (B) statistical validation of OPLS-DA via 7× cross-validation and 200× permutation testing of metabolites. 3.3.2. Different Metabolites of Breast Muscle between PM and CON Groups To identify potential biomarkers among the metabolites, the VIP obtained from the OPLS-DA was used, focusing on distinguishing the important metabolites between the PM and CON groups. A total of five metabolites were found to be upregulated, while six metabolites were downregulated, based on the established criteria of FC > 2.0 or <0.5, p < 0.05, and OPLS-DA VIP > 1 ([77]Figure 2A). Further analysis revealed that these metabolites belong to various categories, including purine nucleotides, organic oxides, fatty acyls, lactams, carboxylic acids and derivatives, cinnamic acids and derivatives, pyrimidine nucleotides, and others ([78]Figure 2B, [79]Table 4). These metabolites significantly contribute to the observed differences between the PM and CON groups. Figure 2. [80]Figure 2 [81]Open in a new tab Identification of differential metabolites between CON and PM groups. (A) Volcano plot for differential metabolites. (B) Biochemical categories of identified differential metabolites are shown in a pie chart. Table 4. The detailed results of differential metabolites in the geese breast muscle (OPLS-DA VIP > 1, FC > 2 or <0.5, and p-value < 0.05). Name Adduct m/z rt(s) VIP FC Variation p-Value Xanthylic acid [M − H]^− 363.0339 74.2 2.5624 0.43003 ↓ 0.0027389 3′-Ketolactose [M − H]^− 339.0944 345.9 2.4382 2.47560 ↑ 0.0036448 Ethyl icosapentate [M + H]^+ 331.2631 846.6 1.9362 3.81320 ↑ 0.0115820 Epsilon-caprolactam [M + H]^+ 114.0918 423.6 1.9165 0.49254 ↓ 0.0121200 9,12,13-TriHOME [M − H]^− 329.2334 642.8 1.8918 0.31053 ↓ 0.0128290 Guanidinoacetate [M − H]^− 116.0494 366.1 1.8054 3.10280 ↑ 0.0156530 gamma-Glutamylalanine [M + H]^+ 219.0952 774.9 1.7771 0.23896 ↓ 0.0167070 N-Alpha-acetyllysine [M]^+ 118.0710 399.1 1.7316 0.28660 ↓ 0.0185530 4-Hydroxycinnamoylagmatine [M]^+ 276.1447 237.4 1.6351 3.05340 ↑ 0.0231710 dTMP [M − H]^− 321.0482 123.1 1.6030 0.42068 ↓ 0.0249450 trans-1,2-Cyclohexanediol [M]^− 115.9194 931.1 1.3355 5.64660 ↑ 0.0461810 [82]Open in a new tab ↓ means down-regulation, and ↑ means up-regulation, compared with the CON group. 3.3.3. Elucidating the Metabolites Difference and Pathway In order to compensate for the limited significant metabolites between the PM and CON groups, MS/MS metabolite data was utilized for MSEA and KEGG pathway analysis with the help of Metaboanalyst. The findings from the metabolite set enrichment analysis (MSEA) revealed significant enrichment in tryptophan metabolism, sphingolipid metabolism, phenylalanine metabolism, and phenylalanine, tyrosine, and tryptophan biosynthesis (p < 0.05) ([83]Figure 3A). The study revealed that the metabolites L-tryptophan and 2-oxoadipate were found to be enriched in tryptophan metabolism. Additionally, L-serine and ethanolamine were enriched in sphingolipid metabolism, while phenylacetic acid was enriched in phenylalanine metabolism. Interestingly, L-phenylalanine was found to be enriched in both phenylalanine metabolism and phenylalanine, tyrosine, and tryptophan biosynthesis (p < 0.05). These findings emphasize the close association between amino acid metabolism and biosynthesis, as depicted in [84]Figure 3B,C and [85]Table S5. Figure 3. [86]Figure 3 [87]Open in a new tab Function analysis for the metabolites. (A) Metabolite set enrichment analysis. (B) KEGG pathway analysis. (C) The significantly enriched KEGG pathway is involved via different metabolites (in the red background). 4. Discussion 4.1. Growth Performance, Carcass Characteristics, and Meat Quality of Yangzhou Geese Geese are herbivorous poultry capable of efficiently digesting and utilizing green forage. The addition of PM to their diet can meet their grazing needs while increasing dietary fiber intake, which enhances their health and well-being, ultimately reducing the risk of diseases and undesirable behaviors [[88]25]. There was no significant effect on the body weight between the PM group and the CON group at 28 days of age; however, the PM group’s weight was slightly lower than that of the CON group. This may be attributed to the dietary adjustment in the PM group during the pre-test period. The impact of the diet on the growth performance of geese can be assessed through parameters such as the ADFI and ADG. Liu et al. showed that with an increase in dietary crude fiber levels, geese exhibited an upward trend in ADFI, and a pattern of initial increase followed by a decrease in ADG [[89]26]. However, it significantly impacted the ADFI, with the geese’s feed intake gradually decreasing as the proportion of paper mulberry leaf powder increased. In this experiment, there was no significant effect on the ADFI between the PM group and the CON group. Nevertheless, the addition of PM had a promoting effect on the ADG of geese, which demonstrates the good quality of PM. The carcass characteristics, such as dressing percentage and eviscerated percentage, serve as reliable indicators of an animal’s nutrient intake from the diet. Among these characteristics, the quality of breast muscle plays a crucial role in determining poultry meat production performance [[90]27]. Aslan et al. demonstrated that the inclusion of corn silage in geese diets did not significantly impact their dressing percentage [[91]28]. In line with previous studies showing higher breast muscle percentages in geese that were fed a combination of grass and grain compared to those on a single diet, our study found that supplementing with PM also increased breast meat percentage [[92]29]. Desbuards et al. demonstrated that augmenting the dietary fiber content in the maternal diet during pregnancy and lactation resulted in amplified colon length and body weight in the offspring, particularly an increase in leg muscle weight [[93]30]. It is postulated that this alteration is associated with the absorption of dietary fiber by the gut microbiota, encompassing the secretion of hormones such as insulin and growth hormone, which are acknowledged to stimulate protein synthesis and foster muscle development. Consequently, it can be inferred that the elevation in breast muscle percentage observed within our experiment’s PM group may be attributed to incorporating paper mulberry into their diet, thereby leading to a heightened intake of fiber by geese. The assessment of meat quality encompasses both sensory and nutritional aspects. Sensory quality primarily comprises pH value, water holding capacity, and meat color. The pH level directly reflects muscle acidity and significantly influences the heat resistance, cooking loss, and processing performance of muscles. An elevation in pH levels leads to a reduction in the rate of anaerobic glycolysis of muscle glycogen, thereby minimizing water exudation loss, promoting greater protein structure stability, and enhancing overall meat freshness [[94]31]. In this experiment, both the PM group and the CON group exhibited similar pH values within the normal range (5.8–6.2). Water holding capacity can affect the edible quality of meat, including its color, aroma, and taste, etc. Xiong et al. showed that adding a certain amount of PM to the diet has no effect on the pH of goat meat and reduces dripping loss in goat meat, while our results indicated that there is no significant difference in water holding capacity of the breast muscles between the PM group and the CON group [[95]9]. This may be related to the different muscle compositions among various species. Meat color is influenced by factors such as myoglobin content, residual hemoglobin content, myohemoglobin amount, oxidation state, and surface light reflection ability [[96]32]. Goose meat is classified as white meat, and a slightly pinkish hue can promote consumer purchases. In this experiment, the a* value, which represents the redness of meat, was lower in the PM group; this indicated that the addition of PM to the diet has a positive impact on the color of goose breast meat. The nutritional value of goose meat is influenced by the levels of chemical components like protein and fat, and the higher the protein content, the better the nutritional value of meat. In this experiment, the addition of PM to the diet improved the nutritional quality of goose breast meat, with significantly higher protein content in the PM group compared to the CON group. Liu et al. showed that the effect of alfalfa-mixed silage can increase muscle protein content and decrease the crude fat content of geese meat [[97]33]. This may be related to the presence of a significant amount of digestible protein and an appropriate amino acid composition in the forage, thereby promoting the deposition of protein in goose meat. Overall, the use of PM feed can improve the sensory and nutritional quality of goose meat to some extent. 4.2. Untargeted Metabolomic (LC-MS/MS) Analysis of Breast Muscle in the CON and PM Groups Metabolomics is employed for the rapid screening of small molecule metabolites in tissues, fluids, and cells under specific conditions. The dietary factor plays a pivotal role in determining the quality of raw meat. Numerous animal studies utilize metabolomics to evaluate the dietary value of raw meat. Although direct comparisons between individual experiments are not feasible due to inherent variations in methodologies and experimental setups, these findings can serve as valuable references for future investigations [[98]34]. To characterize