Introduction
China is one of the largest producers and consumers of pork globally, with pig farming as the backbone of domestic animal husbandry (Li et al. Reference Li, Ozturk-Kerimoglu, He, Zhang, Pan, Liu, Zhang, Huang, Wu and Jin2022). Economic development has driven an increase in the consumption of high-quality pork products in China (Yu and Jensen Reference Yu and Jensen2022). The content and composition of fatty acids significantly influence the edible value and nutritional quality of meat products (Yi et al. Reference Yi, Huang, Wang and Shan2023; Zhang et al. Reference Zhang, Xu, Chu, Zong, Yang and Lou2019a). A previous study found that the fat from fat-type pigs exhibited a lower n-3/n-6 polyunsaturated fatty acid (PUFA) ratio, with the proportion of unsaturated fatty acids (UFA) ranging from 42% to 65%. These characteristics suggest promising applications for fat from fat-type pigs in the production of functional foods (Chernukha et al. Reference Chernukha, Kotenkova, Pchelkina, Ilyin, Utyanov, Kasimova, Surzhik and Fedulova2023). A recent study revealed that lard derived from subcutaneous fat (SF) contains higher levels of phosphatidylserine (PS) and offers superior health benefits as a dietary fat compared to lard from perirenal fat (PF) (Hou et al. Reference Hou, Ji, Chu, Wang, Sun, Wei, Zhang, Song and Wen2024). Chinese indigenous pig breeds are renowned for their higher intramuscular fat (IMF) content, better taste, and the nutritional value of pork. However, these breeds typically have a slower growth rate and a higher fat percentage (Zhang et al. Reference Zhang, Wang, Ma, Wang, Wang and Jiang2022). Therefore, regulating backfat thickness and lipid composition is crucial for enhancing the productivity and sustainability of the high-quality pork industry.
Dietary nutrient supplementation effectively regulates backfat thickness and fat percentage in pigs. Supplementation with glycine or betaine has been shown to significantly reduce the average backfat thickness of finishing Huan Jiang mini-pigs (Zhong et al. Reference Zhong, Yan, Song, Zheng, Duan, Kong, Deng and Li2021). Dietary supplementation with 500 mg/kg of hyodeoxycholic acid (HDCA) enhanced lipolysis and reduced backfat thickness in finishing pigs (Hu et al. Reference Hu, Sang, Wu, Pu, Yan, Luo, Zheng, Luo, Yu, He and Yu2024). Similarly, a low dietary n-6/n-3 PUFA ratio decreased total triglyceride and total cholesterol contents in the subcutaneous fat (SF) of Heigai pigs (Nong et al. Reference Nong, Wang, Zhou, Sun, Chen, Xie, Zhu and Shan2020). Conjugated linoleic acid (CLA) treatment markedly reduced fat deposition both in vivo and in vitro and altered lipid profiles in the longissimus dorsi (LD) muscle and serum of Heigai pigs (Wang et al. Reference Wang, Zhang, Huang, You, Zhou, Chen, Sun, Yi, Sun, Xie and Zhu2022, Reference Wang, Zhang, Huang, Zhou and Shan2023). Additionally, dietary supplementation with 1% glutamine (GLU) and 1% arginine (Arg) significantly reduced backfat thickness and saturated fatty acids (SFA) content in the backfat of growing-finishing pigs (Hu et al. Reference Hu, Jiang, Zhang, Yin, Li, Deng, Wu and Kong2017). Our previous study found that dietary supplementation with 1% leucine (Leu) reduced the ceramide (Cer) content in the fat tissue of Shaziling pigs (Zhang et al. Reference Zhang, Huang, Zheng, Wang, Zhou, Chen, Duan and Shan2024). Additionally, dietary GLU supplementation improved carcass traits, growth performance, and intramuscular fat (IMF) contents while decreasing fat percentage in Shaziling pigs (Zheng et al. Reference Zheng, Wan, Guo, Duan and Yin2024). However, it remains unclear whether GLU regulates the fatty acid profile and lipid composition of porcine fats.
In this study, we aimed to investigate the effects of GLU supplementation on fatty acids and lipid composition in SF and PF of Shaziling pigs. Our findings may serve as a reference for developing nutritional strategies to improve the nutritional quality of fat in Shaziling pigs.
Materials and methods
Animals preparation and sample collection
The experiment was approved by the Zhejiang University Animal Care and Use Committee (ZJU20170466). A total of 48 150-day-old Shaziling pigs were randomly divided into two groups (control group and glutamate group), with each group having 6 replicate pens, with 4 pigs per pen. The control (CON) group was fed a basal diet (corn-soybean meal type diet), and the glutamate (GLU) group was fed a basal diet supplemented with 1% GLU (L-GLU). The supplementation dosage was based on our previous studies (Huang et al. Reference Huang, Wang, Zhang, Zheng, Duan, Wang, Zhou and Shan2025; Zhang et al. Reference Zhang, Huang, Zheng, Wang, Zhou, Chen, Duan and Shan2024; Zheng et al. Reference Zheng, Wan, Guo, Duan and Yin2024). The GLU levels in the CON and GLU-supplemented diets were 2.42% and 3.25%, respectively, as reported in our earlier work (Zheng et al. Reference Zheng, Wan, Guo, Duan and Yin2024). The basal diet met the nutritional requirements of pigs, and the detailed composition is shown in Supplementary Table 1. The trial period lasted a total of 51 days, with free food and water intake. At the end of the experiment, 6 pigs (1 pig per pen) were randomly selected from each group and slaughtered after 12 hours of fasting. The SF and PF were quickly collected and used for subsequent experiments.
Detection of carcass traits
After removing the head, legs, tail, and viscera, and retaining the suet and kidneys of the left half of the carcass, the left side of the carcass was weighed and then dissected into skeletal muscle and fat. The fat mass was weighed and recorded to calculate the fat percentage. Backfat thickness was determined by averaging the backfat thickness at the back edge of the left shoulder blade, the last rib, and the lumbar-sacral junction.
Hematoxylin-eosin staining
The paraffin section and H&E staining were performed according to our previous study (Liu et al. Reference Liu, Cai, You, Yang, Tu, Zhou, Valencak, Xiao, Wang and Shan2025). The sample was fixed with polyformaldehyde fixative for more than 24 hours, embedded, and made into paraffin sections. After dewaxing and rehydrating, the sections were added into a hematoxylin staining solution for 3–5 minutes and rinsed with tap water. After treating with a hematoxylin differentiation solution for a few seconds, the sections were treated with a blue returning solution to turn blue and then rinsed with tap water. After sequentially placing the sections in 85% and 95% alcohol and dehydrating them for 5 minutes, they were stained with an eosin staining solution for 5 minutes. The sections were dehydrated in anhydrous ethanol and xylene sequentially and sealed with neutral gum. The sections were examined under an upright microscope. Adipocyte area was measured using ImageJ (v 1.43), and three randomly selected fields of view on each slide were analyzed for cell size.
Fatty acid composition analysis
The fatty acid composition in adipose tissues was determined by Shanghai Applied Protein Technology Company, and the main steps were as follows. Using 51 fatty acid methyl ester mixed standard solutions and n-hexane, working standard solutions with 10 different mixed standard concentration gradients were prepared. Sample metabolites were extracted using a chloroform-methanol mixed solution with a volume ratio of 2:1. Mass spectrometry analysis used the Thermo Trace 1300/TSQ 9000 gas mass spectrometry. MSD Chemstation software was used to extract the chromatographic peak area and retention time. The standard curve was drawn, and the content of fatty acid in the sample was calculated.
Untargeted lipidomics analysis
The untargeted lipidomics in fat tissue was provided by Shanghai Applied Protein Technology Company, and the analysis was based on our previously published paper (Wang et al. Reference Wang, Zhang, Huang, You, Zhou, Chen, Sun, Yi, Sun, Xie and Zhu2022). To evaluate the system’s stability and data reliability, a pooled QC sample was created by combining the separate samples taken equally from each group. A 50 mg fat sample was weighed, and lipids were extracted using a 90% isopropanol (Thermo Fisher Scientific, USA)/acetonitrile (Thermo Fisher Scientific, USA) solution. LC-MS/MS analysis was performed using a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific, USA) coupled with a UHPLC Nexera LC-30A (SHIMADZU). Positive and negative ion modes were investigated using full-scan spectroscopy with mass-to-charge ratio (m/z) ranges of 200–1800 and 250–1800, respectively. Next, the mass-to-charge ratios of lipid molecules and their fragments were acquired using the following procedure: after each full scan, 10 fragment patterns (MS2 scan, HCD) were recorded. For lipid identification, peak extraction, peak alignment, and quantification, LipidSearch software version 4.1 (Thermo Scientific™) was employed. The extracted ion features were restricted to include only those that exhibited at least 50% nonzero measurement values across at least one experimental group.
Statistical analysis
The experimental data were initially processed using Microsoft Excel 2019. A two-tailed Student’s t-test was performed with IBM SPSS Statistics 20 to compare differences between groups. Data were represented as Mean ± SEM. Statistical significance was determined as follows: *P < 0.05, **P < 0.01, indicating significant differences. Differences were considered with trends at 0.05 ≤ P < 0.10. Data visualization was performed using GraphPad Prism (version 9.0.0) and R (version 4.4.0), with R packages ggplot2 (version 3.5.1) and ggprism (version 1.0.5).
Results
Effect of GLU on fatty acid composition in SF of Shaziling pigs
Dietary supplementation with 1% GLU significantly reduced the fat percentage in Shaziling pigs compared to the CON group (Zheng et al. Reference Zheng, Wan, Guo, Duan and Yin2024) (Fig. 1A,B). Consistently, GLU tended to reduce the size of fat cells (Fig. 1C). Cell size analysis showed a significant increase in the ratio of adipocytes with an area of 0–2500 μm2 (P < 0.05) and a trend toward a decrease (P = 0.1) in the ratio of adipocytes with an area of 7500–10000 μm2 in the GLU group compared to the CON group (Fig. 1D). To explore the role of GLU in modulating the fatty acid composition of porcine fat, targeted fatty acid analysis was performed in SF and PF collected from the GLU and CON groups. The fatty acid analysis revealed a total of 12 SFAs, 20 MUFAs, and 14 PUFAs in porcine SF (Fig. 1E). Among them, C16:0, C18:0, C18:1n9c, C19:1n9t, C18:1n12, C18:2n6, and C18:3n3 were the most abundant. The results showed that dietary GLU did not affect the total content of fatty acids, SFAs, MUFAs, and PUFAs in SF (Fig. 1F,G). Similarly, GLU supplementation did not influence the content of total odd fatty acids (Odd-FAs) (Fig. 1H), trans fatty acids (Trans-FAs) (Fig. 1I), or the MUFA/PUFA ratio (Fig. 1J). However, GLU supplementation significantly increased the content of n3-PUFA (P < 0.05) compared with the CON group (Fig. 1K) and tended to reduce the n6/n3 ratio (P = 0.06) (Fig. 1L). Analysis of individual fatty acids showed that GLU significantly increased the accumulation of C18:3n3 (P < 0.05) while decreasing several other fatty acids, including C24:0 (P < 0.01), C15:1 t (P < 0.05), C14:1 (P < 0.05), C14:1 t (P < 0.05), C20:5n3 (P < 0.01), C22:6n3, C15:1 (P < 0.05), C22:2 (P < 0.01), C20:1 t (P < 0.01), and C18:1n7t (P < 0.01)(Fig. 1M).
Figure 1. The effect of GLU on the fatty acid composition of the subcutaneous fat in Shaziling pigs. (A) The backfat thickness of Shaziling pigs, n = 6. (B) The fat percentage in Shaziling pigs, n = 6. (C) Representative section of dorsal subcutaneous fat. (D) Frequency of adipocytes, n = 3. (E) Individual fatty acid proportion in SF, n = 5. the size of the circle represents the average percentage of fatty acid molecules in the total fatty acids. (F) The content of total fatty acid. (G) The content of fatty acids with different saturations: SFA, saturated fatty acid; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid. (H) The content of odd fatty acids. (I) the content of trans fatty acids. (J) The ratio of monounsaturated fatty acids to polyunsaturated fatty acids. (K) The content of n3, n6, n7, and n9 unsaturated fatty acids. (L) The ratio of n6 to n3 PUFA. (M) Heat map of differential fatty acids. The *P < 0.05 indicates a statistically significant difference.
Influence of GLU on the fatty acid composition of PF in Shaziling pigs
We next examined the effects of GLU on the fatty acid composition of PF in Shaziling pigs. A total of 8 SFAs, 20 MUFAs, and 12 PUFAs were detected in porcine PF (Fig. 2A). Among them, C16:0, C18:0, C18:1n7t, C18:1n12, C18:1n9c, C18:1n7, and C18:2n6 were the most abundant (Fig. 2A). Although the total fatty acid content was not affected by dietary GLU supplementation, MUFA levels were significantly decreased in the GLU group compared to the CON group (P < 0.05) (Fig. 2B,C). The contents of odd-FA, trans-FA, and the MUFA/PUFA ratio were not significantly influenced by GLU supplementation (Fig. 2D–F). Within the UFA pool, GLU supplementation significantly decreased the content of n-9 UFA (Fig. 2G) and reduced the n6/n3 ratio (Fig. 2H). Additionally, we identified 8 fatty acid species that were reduced in PF by GLU treatment, including the medium-chain fatty acid C10:0 and several long-chain fatty acids: C18:1n9t, C18:1n12t, C20:1, C20:1 t, C24:1, C22:6n3, and C22:5n6 (P < 0.05) (Fig. 2I).
Figure 2. The effect of GLU on the fatty acid composition of the perirenal fat in Shaziling pigs. (A) Individual fatty acid s in PF, n = 5. the size of the circle represents the average percentage of fatty acid molecules in the total fatty acids. (B) The content of total fatty acid. (C) The content of fatty acids with different saturations: SFA, saturated fatty acid; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid. (D) The content of odd fatty acids. (E) The content of trans fatty acids. (F) The ratio of monounsaturated fatty acids to polyunsaturated fatty acids. (G) The content of n3, n6, n7, and n9 unsaturated fatty acids. (H) The ratio of n6 to n3 PUFA. (I) Heat map of differential fatty acids. The *P < 0.05, indicates a statistically significant difference.
GLU affected the lipid composition in the SF of Shaziling pigs
To further investigate the effects of GLU on lipo-nutritional quality, we analyzed the lipid composition of SF from GLU and control pigs using mass spectrometry-based lipidomic analysis. The lipidomics data showed that GLU had no substantial effect on total lipid content (Fig. 3A). However, the orthogonal projections to latent structure discriminant analysis (OPLS-DA) plot indicated a clear separation between the GLU and CON groups (Fig. S1A). Quantitative lipid analysis identified a total of 2,264 lipid molecules in 43 lipid subclasses, primarily including 777 triglycerides (TGs), 391 diglycerides (DGs), 168 phosphatidylcholines (PCs), 147 phosphatidylethanolamines (PEs), and 136 ceramides (Cers) (Fig. 3B). Lipid subclass composition analysis revealed that TGs were the predominant lipid subclass in both groups, accounting for 95.766% in the Con group and 96.039% in the GLU group (Fig. S1B,C). The dynamic distribution of lipid content between the two groups showed that the most abundant lipid molecule was TG (16:0/18:1/18:1) in both groups, while the least abundant lipid molecule was DG (8:0e/22:2) in the CON group and PC (20:3/14:1) in the GLU group (Fig. S1D). We further analyzed the composition of lipid subclasses in the SF of Shaziling pigs. A total of 43 lipid subclasses were categorized into 5 groups: glycerolipids (GLs), sterol lipids (SEs), fatty acyls (FAs), sphingolipids (SLs), and glycerophospholipids (GPs) (Fig. 3C–G). Although there was no significant difference in lipid groups between the CON and GLU groups, the lipid subclasses of lysophosphatidylinositol (LPIs) and lysophosphatidylcholines (LPCs) tended to decrease in the GLU group (Fig. 3H–J).
Figure 3. The effect of GLU on the lipid composition of the subcutaneous fat in Shaziling pigs. (A) The content of total lipid. (B) The types and quantities of different lipid molecules. (C–G) The content of different lipid classes in SF of shaziling pigs. (H) The content of glycerolipid subclasses. (I) The content of sphingolipid subclasses. (J) The content of glycerophospholipid subclasses. N = 5. Error bars represent SEM. *P < 0.05, two-tailed Student’s t-test.
Impact of GLU on the lipid composition of PF in Shaziling pigs
We further investigated the effect of GLU on the lipid composition of PF in Shaziling pigs. The OPLS-DA plot showed a clear separation between the GLU and CON groups (Fig. S2A). However, GLU had no significant effect on total lipid content (Fig. 4A). Quantitative lipid analysis identified a total of 2,382 lipid molecules across 40 lipid subclasses, including 787 TGs, 394 DGs, 169 Cers, 150 PEs, and 92 hexosyl-1-ceramides (Hex1Cers) (Fig. 4B). Analysis of lipid subclass composition revealed that TGs were the predominant lipid subclass in both groups, accounting for 97.399% in the CON group and 97.585% in the GLU group (Fig. S2B,C). Dynamic distribution analysis of lipid content showed that the most abundant lipid molecule in both groups was TG (16:0/18:1/18:1). Meanwhile, the least abundant lipid molecule was PC (36:2) in the CON group and LPC (17:0) in the GLU group (Fig. S2D). Next, we explored the effects of GLU on lipid subclass composition in PF. A total of 40 lipid subclasses were classified into 5 categories, including GLs, SEs, FAs, SLs, and GPs (Fig. 4C–G). Dietary GLU significantly decreased the content of total FAs compared to the CON group (P < 0.05) (Fig. 4G). Additionally, GLU had no significant effect on the contents of lipids in the GL pool (Fig. 4H) while inducing significant reductions in Cers (P < 0.05) in the SL pool (Fig. 4I), and PS and PIP in the GP pool (P < 0.05) (Fig. 4J).
Figure 4. The effect of GLU on the lipid composition of the perirenal fat in Shaziling pigs. (A) The content of total lipid. (B) The types and quantities of different lipid molecules. (C–G) The content of different lipid classes in PF of shaziling pigs. (H) The content of glycerolipid subclasses. (I) The content of sphingolipid subclasses. (J) The content of glycerophospholipid subclasses. N = 5. Error bars represent SEM. *P < 0.05, two-tailed Student’s t-test.
GLU-induced depot-specific changes in the molecular composition of lipids in fat tissues of Shaziling pigs
To further investigate the role of GLU in regulating the properties of PS, PIP, and Cer in porcine fats, we analyzed the individual acyl chain composition of these lipids. In the PS pool, GLU significantly decreased the content of C11:0 (P < 0.05) in SF (Fig. 5A) while reducing C18:0, C18:1, C19:0, C20:3, C40:5, C40:6, and C42:4 (P < 0.05) in PF (Fig. 5B), accompanied by decreased SFA and PUFA contents (Fig. 5C). For the PIP pool, GLU did not affect the fatty acyl chain composition in SF (Fig. 5D), but it significantly reduced the contents of C54:5 and C54:6 in PF (P < 0.05) (Fig. 5E). The saturation level of PIP was unaffected by GLU treatment in both SF and PF (Fig. 5F). In the Cer pool, GLU increased the contents of d36:2 in SF and d41:8 in PF (P < 0.05), while reducing d43:5 in SF and d15:1 in PF (P < 0.05) (Fig. 5G,H). Additionally, GLU significantly reduced SFA and PUFA contents in the Cer pool of PF (P < 0.05) (Fig. 5I). The heatmap of the top 30 differential lipid molecules in SF and PF revealed depot-specific effects induced by GLU supplementation (Fig. 5J,K). GLU broadly reduced GLs and increased GPs in SF, while reducing GPs and increasing GLs in PF.
Figure 5. The effect of GLU on the lipid molecules of the fat tissues in Shaziling pigs. (A,B) PS acyl chain contents at different saturation levels in SF (A) and PF (B). (C) Log2 fold change (GLU vs con) of SFA, MUFA, and PUFA contents in the PS pool. (D,E) PIP acyl chain contents at different saturation levels in SF (D) and PF (E). (F) log2 fold change (GLU vs con) of SFA, MUFA, and PUFA contents in the PIP pool. (G,H) Cer acyl chain contents at different saturation levels in SF (G) and PF (H). (I) Log2 fold change (GLU vs con) of SFA, MUFA, and PUFA contents in the cer pool. (J,K) Heat map of differential lipid species in SF (J) and PF (K) of Shaziling pigs. N = 5. Error bars represent SEM. *P < 0.05, two-tailed Student’s t-test.
Specific changes in the fatty acyl chain composition of porcine fat tissues induced by GLU supplementation
Next, we analyzed the individual fatty acyl chain composition associated with GLs, GPs, and SLs (Fig. 6 and Figs. S3,S4) in the SF and PF of Shaziling pigs. In SF, compared to the CON group, GLU supplementation broadly increased lipid molecules with 0 to 1 double bonds in the GL pool (Fig. S3A), lipid molecules with 3 to 5 double bonds in the GP pool (Fig. 6A) and lipid molecules with 0, 5, and 7 double bonds in the SL pool (Fig. S4A). Lipid molecules with 4 to 6 double bonds in the GL pool were decreased (Fig. S3A). Despite these changes, GLU had no significant effect on the total double bond composition in the GL, GP, and SL pools (Fig. 6B and Figs. S3B,S4B). In PF, GLU had minimal effects on the double bond composition of individual lipid species in the GL and GP pools (Fig. 6A and Fig. S3A). However, GLU increased SLs with 0 double bonds and decreased SLs with 4 double bonds (Fig. S4A). For total double bond composition, GLU significantly reduced lipids with 3 double bonds in the GP pool (P < 0.05) (Fig. 6C). Next, we explored the influence of GLU on the length of acyl chains (Fig. 6D–F and Figs. S3D–F, S4D–F). In SF, GLU induced a notable reduction in lipid molecules containing 25–50 carbon atoms in the GP pool. However, the total content did not reach statistical differences (P > 0.05) (Fig. 6D,E). In PF, GLU induced a notable reduction in lipid molecules containing 30–45 carbon atoms in the GP pool (Fig. 6D). GLU significantly reduced the total contents of lipid molecules containing 32 and 38 carbon atoms (P < 0.05) in the GP pool (Fig. 6F).
Figure 6. GLU affected the lipid acyl chain compositions in the GP pool of fat tissues. (A) GPS with double-bond contents. (B,C) Heatmap of acyl chain double bond contents in GP pools from SF (B) and PF (C) in the control and GLU groups. (D) GPS with different numbers of carbon atoms. (E,F) Heatmap of acyl chain carbon atoms in GP pools from SF (B) and PF (C) in the control and GLU groups. N = 5. Error bars represent SEM. *P < 0.05, two-tailed Student’s t-test.
Discussion
Shaziling pig is characterized by high IMF content, superior meat quality, but a high fat percentage (Song et al. Reference Song, Zheng, Zheng, Zhang, Zhong, Guo, Li, Long, Xu, Duan and Yin2022). These characteristics underscore the need to evaluate the lean carcass percentage, fattening effect, reproductive performance, and other important traits (Liu et al. Reference Liu, Brewster, Gilmour, Henman, Smits, Luxford, Dunshea, Pluske and Campbell2021). Nutritional strategies are effective for regulating fat percentage in pigs and improving fatty acid composition (Liu et al. Reference Liu, Du, Tu, You, Chen, Liu, Li, Wang, Lu, Wang and Shan2023; Nong et al. Reference Nong, Wang, Zhou, Sun, Chen, Xie, Zhu and Shan2020; Wang et al. Reference Wang, Zhang, Huang, You, Zhou, Chen, Sun, Yi, Sun, Xie and Zhu2022, Reference Wang, Zhang, Huang, Zhou and Shan2023; Zhang et al. Reference Zhang, Huang, Zheng, Wang, Zhou, Chen, Duan and Shan2024), which directly influence the edible value and nutritional quality of meat (Zhou et al. Reference Zhou, Ling, Wang, Xu, You, Chen, Nong, Valencak and Shan2024; Zhang et al. Reference Zhang, Xu, Chu, Zong, Yang and Lou2019a). As a significant reservoir of lipids, the lipo-nutritional quality of porcine fat has become a growing concern. GLU, abundant in muscle, has been shown to promote muscle growth when supplemented exogenously or produced endogenously (Hou and Wu, Reference Hou and Wu2018). Our previous study demonstrated that dietary GLU supplementation significantly improved growth performance and IMF contents, while reducing the half-carcass fat percentage of Shaziling pigs (Zheng et al. Reference Zheng, Wan, Guo, Duan and Yin2024). However, the effect of GLU on the lipid profile of porcine fat remains unclear. In this study, we revealed the regulatory roles of GLU in enhancing n3 PUFA accumulation and reconfiguring the lipid profile in the fat tissues of Shaziling pigs. Furthermore, we clarified the depot-specific characteristics of porcine fat tissues in response to dietary GLU supplementation.
Nutritional strategies effectively remodel the fatty acid profile in the skeletal muscle and fat tissues of pigs. For example, dietary supplementation with 1% CLA induced SFA accumulation and MUFA reduction in the SF of Heigai pigs (Wang et al. Reference Wang, Zhang, Huang, Zhou and Shan2023). Similarly, supplementation with 1% GLU and 1% Arg significantly reduced total SFA content in the SF of Duroc × Landrace × Yorkshire (DLY) growing-finishing pigs (Hu et al. Reference Hu, Jiang, Zhang, Yin, Li, Deng, Wu and Kong2017). In contrast, 1% Leu promoted the accumulation of SFA, MUFA, and PUFA in the LD muscle without affecting their contents in the SF and PF of Shaziling pigs (Zhang et al. Reference Zhang, Huang, Zheng, Wang, Zhou, Chen, Duan and Shan2024). In the current study, we observed that, in parallel with a reduced fat percentage, 1% GLU significantly decreased MUFA content in PF without altering SFA and UFA contents in the SF of Shaziling pigs. However, GLU demonstrated a superior ability to regulate the position of the first unsaturated bond in PUFA molecules. Specifically, GLU increased n3 PUFA in SF and decreased n9 PUFA content in PF. Additionally, GLU reduced the n3/n6 ratio in both SF and PF. Both n3 and n6 PUFAs are crucial in promoting human health and reducing disease risk. Numerous studies have highlighted the health benefits of n3 PUFAs in mitigating cardiovascular disease, diabetes, cancer, and various mental diseases (Shahidi and Ambigaipalan, Reference Shahidi and Ambigaipalan2018). Likewise, n6 PUFA is essential for preventing heart disease (Wang Reference Wang2018). The n6/n3 PUFA ratio is particularly important in regulating lipid metabolism and deposition, ultimately influencing the composition of fatty acids (Duan et al. Reference Duan, Li, Li, Fan, Sun and Yin2014). Research has demonstrated that a higher n3 PUFA content and a lower n6/n3 PUFA ratio are more beneficial for human health (Mariamenatu et al. Reference Mariamenatu, Abdu and Kostner2021). For instance, Nong et al. reported that dietary supplementation with a lower n6/n3 PUFA ratio significantly increased n3 PUFA content and reduced the n6/n3 PUFA ratio in the backfat of Heigai pigs (Nong et al. Reference Nong, Wang, Zhou, Sun, Chen, Xie, Zhu and Shan2020). Our results showed that GLU tended to reduce the n6/n3 PUFA ratio in fat tissues, suggesting that GLU might enhance the nutritional quality of fat in Shaziling pigs.
Nutritional strategies play an important role in regulating lipid dynamics in porcine meats. In this study, we found that GLU supplementation tended to decrease the contents of LPG and LPI in SF, while significantly reducing Cer, PS, and PIP levels in PF compared to the CON group. As an important factor influencing meat nutritional quality, more research focuses on how dietary strategies regulate lipid composition in meat. For example, supplementation with 1% CLA significantly altered the content of TGs, LPGs, phosphatidic acid (PAs), and phytosphingosine (phSMs) in the LD muscle, while decreasing the serum Cer level of Heigai pigs (Wang et al. Reference Wang, Zhang, Huang, You, Zhou, Chen, Sun, Yi, Sun, Xie and Zhu2022). Similarly, 5% Bacillus subtilis and Enterococcus faecium co-fermented feed increased the levels of PC and phosphatidylethanolamine (PE) in the LD muscle of finishing pigs (Liu et al. Reference Liu, Du, Sun, Tu, Gu, Cai, Lu, Wang and Shan2024), and an 8% mulberry leaves diet in Yuxi black pigs caused significant accumulation of TGs and Cers, while reducing PC and PS levels in the LD muscle (Hou et al. Reference Hou, Ji, Chu, Wang, Sun, Wei, Zhang, Song and Wen2024). In our previous study, we found that 1% Leu supplementation led to a reduction in PEs, cardiolipins (CLs), and phosphatidylglycerols (PGs) in the LD muscle, and in lysophosphatidylethanolamines (LPEs), Cers, and phosphatidylinositols (PIs) in the adipose tissue of Shaziling pigs (Zhang et al. Reference Zhang, Huang, Zheng, Wang, Zhou, Chen, Duan and Shan2024). These findings suggested that lipid classes in GPs, SLs, and GLs are more sensitive to nutrient-induced remodeling in both muscle and fat tissues.
In the backfat of GLU-supplemented Shaziling pigs, we observed a reduction in the levels of LPC and LPI. LPCs are major components of oxidized low-density lipoprotein (oxLDL), which is involved in the development of atherosclerosis and inflammatory factors (Liu et al. Reference Liu, Zhu, Chen, Yan, Zhu, Chen and Peng2020). LPC has also been identified as a potential biomarker for obesity, given its association with chronic low-grade inflammation (Bellot et al. Reference Bellot, Moia, Reis, Pedrosa, Tasic, Barbosa and Sena‐Evangelista2023; Rasouli et al. Reference Rasouli, Yao-Borengasser, Varma, Spencer, McGehee, Peterson, Mehta and Kern2009; Zhu et al. Reference Zhu, Zong, Zhu, Jiang, Ma, Zhang, Zhang, Bai, Yang, Ben and Li2014). LPIs act as ligands for GPR55 and are implicated in conditions such as obesity and cancer (Alhouayek et al. Reference Alhouayek, Masquelier and Muccioli2018). The reduction of LPC and LPI levels through GLU supplementation could, therefore, have beneficial effects on lipid metabolism and inflammation. Furthermore, Cers in porcine fat tissues exhibited a strong response to dietary amino acid supplementation. Sphingolipids are ubiquitous building blocks of cell membranes that have key functions in membrane structure, cellular signaling, and mitochondrial function (Boyd et al. Reference Boyd, Majumder, Stiban, Mavodza, Straus, Kempelingaiah, Reddy, Hannun, Obeid and Senkal2023; Maceyka and Spiegel, Reference Maceyka and Spiegel2014). As the central molecule of sphingolipid metabolism, Cer regulates a diverse range of cellular processes that are important in immunity, chronic inflammation, and inflammatory disorders (Maceyka and Spiegel, Reference Maceyka and Spiegel2014). Elevated Cer levels are characteristic of obesity (Huang et al. Reference Huang, Sulek, Stinson, Holm, Kim, Trost, Hooshmand, Lund, Fonvig, Juel and Nielsen2024). Cers in adipose tissues influence energy metabolism and nutrient regulation, and recent studies have shown that promoting Cer catabolism can reduce atherogenesis and inflammation (Chaurasia et al. Reference Chaurasia, Kaddai, Lancaster, Henstridge, Sriram, Galam, Gopalan, Prakash, Velan, Bulchand and Tsong2016; Zhang et al. Reference Zhang, Zhang, Wang, Zhang, Dong, Zeng, Yan, Sun, Wu, Liu and Liu2019b). The decrease in Cer content in the PF of GLU pigs suggested potential health benefits for the pigs and consumers.
Fat tissues, primarily divided into visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT), are highly flexible and heterogeneous organs with distinct metabolic properties (Nahmgoong et al. Reference Nahmgoong, Jeon, Park, Choi, Han, Park, Ji, Sohn, Han, Kim and Hwang2022; Sakers et al. Reference Sakers, De Siqueira, Seale and Villanueva2022; Zhou et al. Reference Zhou, Xu, Wang, Ling, Nong, Xie, Zhu and Shan2022). As the major component of fat tissue, the lipid profile plays a critical role in determining the nutritional quality of fat (Hou et al. Reference Hou, Ji, Chu, Wang, Sun, Wei, Zhang, Song and Wen2024). Recent studies have confirmed that fat tissues exhibit remarkable adaptability to dietary changes (Nilaweera and Cotter, Reference Nilaweera and Cotter2023; Sárvári et al. Reference Sárvári, Van Hauwaert, Markussen, Gammelmark, Marcher, Ebbesen, Nielsen, Brewer, Madsen and Mandrup2021). However, the effects of nutritional strategies on depot-specific alterations in lipid composition in porcine fat have not been thoroughly explored. Our previous study found that 1% Leu supplementation led to a significant reduction in lipid classes in PF but not in SF. Similarly, the current study demonstrated that lipid dynamics were more active in PF following GLU supplementation than in SF. Our previous research also showed that short-term cold exposure induced broad changes in fatty acid species in visceral fat compared to subcutaneous fat (Zhou et al. Reference Zhou, Xu, Wang, Ling, Nong, Xie, Zhu and Shan2022). This evidence suggests that porcine visceral fat is not merely an inert energy storage tissue. Instead, it is highly active in lipid metabolism and capable of lipid remodeling in response to dietary and environmental changes. Additionally, we observed that after GLU treatment, the lipids that were differentially elevated in SF were mostly GP, while those that were differentially reduced were predominantly GL. In contrast, the opposite trend was observed in PF. We hypothesize that this could be due to the stronger glycerophospholipid metabolic activity in SF (Hou et al. Reference Hou, Ji, Chu, Wang, Sun, Wei, Zhang, Song and Wen2024). Recent studies showed that acyl chains in lipids exhibit diverse structures, which influence the elongation, desaturation, and transport of fatty acids (Ho et al. Reference Ho, Zheng and Ali2022; Vanni et al. Reference Vanni, Riccardi, Palermo and De Vivo2019). In this study, we found that GLU specifically decreased the GP species containing three double bonds, 32 and 38 carbon atoms in PF. However, further research is needed to determine how this alteration affects the nutritional quality of fat.
Conclusion
In conclusion, this study reveals the effects of dietary GLU supplementation on fatty acids and lipid dynamics of fat tissue in Shaziling pigs. In SF, GLU significantly increased the deposition of n3 PUFA (P < 0.05), especially C18:3n3 (P < 0.05). In PF, GLU significantly decreased the content of MUFAs and n9 UFAB (P < 0.05), with a tendency to decrease the n6/n3 PUFA ratio (P = 0.06), and significantly decreased C18:1n9t, C24:1, and C22:6n3 content (P < 0.05). The lipidomic analysis showed that GLU significantly reduced Cer, PS, and PIP content (P < 0.05), and altered the acyl chain saturation, length, and composition within GP pools in PF. These results suggest that GLU supplementation regulates lipid metabolism and alters the fatty acids and lipid composition in fat tissues of Shaziling pigs.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/anr.2025.10007.
Acknowledgements
The project was partially supported by the National Natural Science Foundation of China (U19A2037) and the “Hundred Talents Program” funding from Zhejiang University to T.Z.S., and we thank members of the Shan Laboratory for their comments and support.
Author contributions
Y.Z.: data curation, investigation, methodology, visualization, writing – review & editing; Y.H.: data curation, investigation, writing – original draft; X.X.: investigation; L.W.: writing-review & editing; S.Z.: investigation; C.Z.: methodology; Y.D.: methodology; T.W.: methodology, supervision; T.Z.S.: conceptualization, funding acquisition, project administration, resources, supervision, writing – review. All authors reviewed the results and approved the final version of the manuscript.
Conflict of interest
The authors declare no conflicts of interest, financial or otherwise.
Ethical standards
All procedures were reviewed and preapproved by the Zhejiang University Animal Care and Use Committee (ZJU20240229) and the Institute of Subtropical Agriculture, Chinese Academy of Sciences (ISA-2020-023).
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