Indian Journal of Animal Research

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Lipid Metabolomics Reveals Metabolic Responses of Subcutaneous Fat in Yaks under Different Feeding Conditions during the Cold Season

Ya-Qian Liu1,2, Yong-Gang Sun1,2,*, Yin-Cang Han1,2, Wei-Qin Ding1,2, Sheng-Wei Jin1,2, Jian-Yu Chen1,2, Fa-Jie Gou1,2
1Academy of Animal Husbandry and Veterinary Sciences, Qinghai University, Xining 810 016, China.
2Qinghai Key Laboratory of Plateau Livestock Genetic Resources Protection and Innovative Utilization, Xining 810 016, China.

ABSTRACT

Background: The aim of this experiment was to investigate the differences in subcutaneous fat metabolism expression in yaks under natural grazing and housed feeding conditions in the cold season. Eighteen 18-month-old yaks with similar body weights and non-significant differences in dorsal subcutaneous fat were randomly divided into three groups. 

Methods: One group (G18_SF) of yaks was selected for slaughter at the beginning of the experiment (October), one group continued to graze for 6 months and then slaughtered (G24_SF) and one group was housed for 6 months and then slaughtered (F24_SF) and subcutaneous dorsal fat of the three groups was collected for metabolomic analysis, screening of differential metabolites and enrichment of KEGG pathway.

Result: In this experiment, 110 differential metabolites were detected in G24_SF vs F24_SF and 83 metabolites were screened for down-regulation and 27 metabolites were up-regulated and the differential metabolites were mainly enriched in Alanine, aspartate and glutamate metabolism, Arachidonic acid metabolism and D-Glutamine and D-glutamate metabolism pathways. Ninety-seven differential metabolites were detected in G18_SF vs G24_SF and 41 metabolites were screened for down-regulation and 56 metabolites were screened for up-regulation, with the two groups of differential metabolites mainly enriched in Alanine, aspartate and glutamate metabolism, Caffeine metabolism and Starch and sucrose metabolism pathways. The results of this experiment revealed the information of differential metabolites and enrichment pathways of yak fat in different feeding methods and different months of age and laid the foundation for further in-depth study of subcutaneous fat deposition in yaks.

INTRODUCTION

Yaks live on the Qinghai-Tibetan Plateau and its surrounding areas and are the main domestic animals kept by plateau herders (Luo et al., 2018) and their milk, meat and their by-products not only greatly contribute to the development of the pastoral animal husbandry economy, but also increase the herders’ income. Yaks graze naturally and the growth of pasture and the climatic conditions of the plateau directly affect the growth performance of yaks. Their habitat has a cold season of 6-7 months in a year, during which the pasture grass is the most scarce and has the lowest nutrient value, which directly leads to insufficient nutrient intake of yaks and the energy stored in the body is used to maintain the basic activities of the life of yaks and the fat content of yaks is the lowest in this stage. As the pastureland becomes green and the quantity and nutritional value of the pasture gradually increase, the yaks consume enough nutrients and the body fat begins to be slowly deposited to make energy reserves for the long cold season and after the cold season the yaks begin to decompose the fat to provide energy and the cycle continues. In short, yak body fat undergoes the process of “decomposition~deposition~ maximum content~decom position”. Fat deposition in animals is a complex regulatory process that is influenced by a combination of neuromodulation, humoral regulation, a range of hormones and fat metabolizing enzymes (Newsholme et al., 2003; Du et al., 2018; Khan et al., 2020; Roy Kumar Ashwani et al., 2020; Singh Jeet Ravinder, 2018). The different nutrients absorbed by animals under housed and grazing conditions directly lead to changes in the corresponding physiological functions and metabolic mechanisms of animals. Generally, yaks can lose 25% of their body weight during the cold season (Long et al., 2005; Jayanthi et al., 2020). It was found that the capacity of fat deposition was greater in housed yaks than in grazing yaks and that triglycerides in yak subcutaneous fat were mainly regulated by AGPAT2 and DGAT2 genes (Xiong et al., 2023). The fatty acid composition of housed feeding yak meat is healthier for consumers in terms of nutritional value (Xiong et al., 2022). In summary, housed feeding yaks during the cold season can significantly improve their productive performance.
       
Metabolomics analysis can visualize the state of the organism and its overall effects, making the study of complex genes and proteins at the level of complex genes and proteins to become simple (Kokova et al., 2020) and roughly elaborating the mechanism of the organism’s response to stimuli (Middelkoop et al., 1993; Monaghan et al., 2009; Brand and Anderson, 2011). Previous studies on the metabolic mechanisms of subcutaneous fat deposition in yaks by housed feeding and grazing are scarce. The aim of this study was to compare the pattern of subcutaneous fat deposition in yaks under different feeding methods (grazing and housed feeding) and to detect and analyze the metabolites of yaks under different feeding conditions, with a view to revealing the mechanism of the effect of different feeding methods on subcutaneous fat deposition in yaks. After slaughtering the yaks at the end of the experiment, the subcutaneous fat on the back was collected, put into a liquid nitrogen tank and then transported back to the laboratory and stored at -80 m for reserve.

MATERIALS AND METHODS

Experimental design
 
The experimental yaks in this study were obtained from the Meilong Palm Livestock Management Specialized Cooperative (Qilian, Qinghai), which is located at an average altitude of 3.169 m above sea level and has a plateau continental climate. Eighteen newborn male calves (142.45±2.65 kg) at 18 months of age were selected to carry out the experiment and all the experiment cows were healthy and in good growth condition, with non-significant differences in body weight. At the beginning of the trial in October, 6 yaks were randomly slaughtered (G18_SF) and the remaining 12 yaks were randomly divided into 2 groups, one group was naturally grazed in the original group for 6 months to 24 months of age (G24_SF) and the other group was fully housed and fattened for 6 months to 24 months of age (F24_SF).
 
Feeding and management
 
Yaks in the grazing group grazed in the same pasture for 11 hours per day and the yaks had free access to food and water throughout the grazing period. The grazed pasture was dominated by dominant forages such as needlegrass, morning-glory, dwarf tarragon and small tarragon. Table 1 shows the nutrient levels of the forages in the free-grazing group. The TMR total mixed ration pattern was used to feed housed yaks once at 07:00 and 17:00, respectively. The pellet concentrates used in this experiment were obtained from a feed mill in Minle County, Gansu Province and the composition and nutrient levels of the basal rations are shown in Table 2.
 

Table 1: Nutrient levels of natural herbage (DM basis).


 

Table 2: Diet composition and nutrient levels (DM basis).


 
Test method
 
Sample handling
 
At the end of the experiment the yaks were slaughtered, samples were collected and stored at -80°C. Accurately weigh 50 mg of each sample into a 2 ml centrifuge tube, add a 6 mm diameter grinding bead; add 400 mL of extraction solution (methanol: water = 4:1 (v:v)) with 0.02 mg/mL of the internal standard (L-2-chlorophenylalanine); grind the samples for 6 min in a freezing tissue grinder (-10°C, 50 Hz); extract the samples by cryo-sonication for 30 min (5°C, 40 KHz); leave the samples at -20°C for 30 min; centrifuge for 15 min (13000 g, 4°C); transfer the supernatant into an injection vial with an insert tube for analysis; in addition, each sample is analyzed separately in an injection vial with an insert tube; and each sample is analyzed separately. The samples were allowed to stand at -20°C for 30 min, centrifuged for 15 min (13000 g, 4°C) and the supernatant was pipetted into an injection vial with an internal cannula for analysis. 20 mL of the supernatant was pipetted from each sample and mixed as the quality control sample.
 
LC-MS detection
 
The instrument platform for this LC-MS analysis was a Thermo Fisher Ultra High-Performance Liquid Chromatography Tandem Fourier Transform Mass Spectrometer (UHPLC-Q Exactive HF-X) system.
 
Chromatographic conditions: The column was ACQUITY UPLC HSS T3 (100 mm×2.1 mm i.d., 1.8 ìm; Waters, Milford, USA); mobile phase A was 95% water+5% acetonitrile (containing 0.1% formic acid) and mobile phase B was 47.5% acetonitrile+47.5% isopropanol+5% water (containing 0.1% formic acid), with an injection volume of 2 mL and the temperature of the column was 40°C (Table 3).
 

Table 3: Mobile phase elution gradients.


 
Mass spectrometry conditions: The samples were ionized by electrospray ionization and the mass spectrometry signals were collected in positive and negative ion scanning modes, respectively. The specific parameters are shown in Table 4.
 

Table 4: Mass spectral parameters.


 
Data processing
 
After the raw data were imported into the metabolomics processing software ProgenesisQI (WatersCorporation, Milford,USA), a series of pre-processing was carried out and a combination of multidimensional and unidimensional analyses was used to screen for differential metabolites in yak subcutaneous fat under different feeding conditions for volcano diagram analysis, using a combination of multidimensional and unidimensional analyses, with the VIP value of the first principal component of the OPLS-DA model>1 and the P value returned by the t-test (Wilcox-test)<0.05 were used as criteria to screen the differential metabolites of yak subcutaneous fat under different feeding conditions for volcano plot analysis. Online cloud platform (https://report.majorbio.com/wholerna/geneset_overview/task_id/3u9i_gd0m9blvcljkgt4jsbc2a9.html) was used to compare the differential metabolites with KEGG and HMDB databases and KEGG pathway enrichment analysis was performed after obtaining the annotation information of metabolites in the databases.

RESULTS AND DICUSSION

Principal component analysis
 
From the principal component analysis in Fig 1, it can be seen that there is a certain grouping trend of subcutaneous fat under different feeding conditions and the difference between the G24_SF and F24_SF groups is large and the difference with the G18_SF group is small, in which the QC samples are clustered together, which indicates that the samples have good reproducibility, the quality of the QC is high and the data are reliable and can be subjected to further analysis of variance.
 

Fig 1: PCA analysis of the population sample.


 
Screening for differential metabolites
 
Venn diagrams show the metabolites that are shared or unique between metabolic sets. To obtain the differential metabolites between metabolic sets, differential metabolite analysis was performed. As shown in Fig 3, there were 97 differential metabolites between the G24_SF and G18_SF sets and 110 differential metabolites between the F24_SF and G24_SF sets (Fig 2).
 

Fig 2: Differential metabolite venn plot.


 

Fig 3: Compound classification of the differential metabolite KEGG.


 
Bioinformatics analysis
 
Classification of KEGG compounds
 
The analyzed metabolites were compared to the KEGG Compound database to obtain an overview of metabolite classification, as shown in Fig 3a and it was found that the metabolites differing between the F24_SF and G24_SF groups were classified into a total of seven major types, including vitamins and cofactors, peptides, nucleic acids; the different metabolites produced were mainly peptides and lipid compounds. It was found that the differential metabolites of the G18_SF and G24_SF groups were categorized into five major types, including peptides, nucleic acids, lipids, hormones and transmitters and carbohydrates; the differential metabolites produced were mainly peptides and carbohydrates (Fig 3b).
 
Functional analysis of the KEGG database
 
The experiment searched the KEGG database for the differential metabolites of each sample group and obtained 31 and 18 signaling pathways belonging to 6 and 5 classes, respectively and the results showed (Fig 4) that the differential metabolites of each sample group were significantly annotated to the metabolism of amino acid metabolism, carbohydrate metabolism and lipid metabolism, among others; Digestive system and Nervous system in the system of organismal systems, etc.; Cancer: overview in the human disease, etc.; Membrane transport in the environmental information processing, etc.
 

Fig 4: Functional annotation of the differential metabolite KEGG.


 
Hierarchical cluster analysis of differential metabolites
 
Cluster analysis can provide a better understanding of the relationship between the differential metabolites screened above. Fig 5 Horizontal coordinate is the sample name, vertical coordinate is the differential metabolite, red color indicates high expression of the substance content and blue color indicates low expression of the substance content. As shown in Fig 5a, the G24_SF group showed high expression of phosphatidyl ethanolamine (PE) and phosphatidylcholine (PC) related metabolites and N-acetylneuraminic acid and low expression of LysoPE, LysoPC, Beta-D-Glucosamine and Methycholine. Low expression of docosapentaenoic acid, ophthalmic acid, N-acetylneuraminic acid and PE and PC related metabolites and high expression of LysoPE, LysoPC, Beta-D-Glucosamine and Methycholine in the F24_SF group.As can be seen in Fig 5b, in the G24_SF group, the expression of PE and PC related metabolites and LysoPE-related metabolites was low and the expression of Palmitoyl-L-carnitine and 2-Pentyl-3-phenyl-2-propenal was high. The expression of PE and PC related metabolites, LysoPE-related metabolism and Beta-D-Glucosamine was high in the G18_SF group;low expression of Palmitoyl-L-carnitine and 2-pentyl-3-phenyl-2-propene.
 

Fig 5: Metabolite expression profiles and VIP values.


 
Volcanic composition
 
Each dot in Fig 6 represents a specific metabolite. The dots on the left are metabolites that are differentially down-regulated in expression and the dots on the right are metabolites that are differentially up-regulated in expression. In Fig 6a, 83 metabolites were down-regulated and 27 metabolites were up-regulated in the G24_SF relative to the F24_SF group and in Fig 6b, 41 metabolites were down-regulated and 56 metabolites were up-regulated in the G24_SF relative to the G18_SF group. Table 5 and Table 6 show that products with annotations were detected in the G24_SFvsF24_SF and G24_SFvsG18_SF groups, respectively.
 

Fig 6: Volcanic map of differential metabolites.


 

Table 5: Details of differences between F24_SF and G24_SF groups.


 

Table 6: Details of differences between G18_SF and G24_SF groups.


 
KEGG pathway enrichment
 
By comparing the F24_SF with the G24_SF group (Fig 7a and Table 7), KEGG pathway enrichment was found to involve metabolic pathways such as Alanine, aspartate and glutamate metabolism, Arachidonic acid metabolism, D-Glutamine and D-glutamate metabolism and others;By comparing the G18_SF with the G24_SF group (Fig 7b and Table 8), KEGG pathway enrichment was found to involve metabolic pathways such as Alanine, aspartate and glutamate metabolism, Caffeine metabolism, Starch and sucrose metabolism and others.
 

Fig 7: KEGG pathway enrichment bubble map.


 

Table 7: G24_SFvsF24_SF (top 10) KEGG pathway enrichment table.


 

Table 8: G18_SFvsG24_SF (top 10) KEGG pathway enrichment table.


       
In this study, yaks were grazed and housed during the dry grass period and yak subcutaneous fat was used as the object of study to investigate the effects of different feeding methods and different ages on yak fat metabolism by analyzing the metabolomic changes of yak subcutaneous fat at the ages of 18 months and 24 months.
 
Differential metabolite analysis
 
2-Pentyl-3-phenyl-2-propenal, N-Acetylneuraminic acid and Xanthine were the most abundant in F24_SF, followed by G24_SF and the least abundant in G18_SF.N-acetylneuraminic acid (Neu5Ac), also known as sialic acid, is an acetylated derivative of the acidic sugar neuraminic acid, Neu5Ac enhances intestinal absorption of minerals and vitamins and promotes bone development; and Neu5Ac has also been associated with neurotransmission, promotion of brain development, cosmetic whitening, promotion of the growth of probiotics, anti-tumor, anti-viral or bacterial infections and anti-Alzheimer’s disease, among other functions (Bavaresco et al., 2008).Studies have shown that Neu5Ac enhances immunity in mice. Neu5Ac prevents HFD-induced hyperlipidemia in rats by regulating lipid-associated and coagulation-associated genes, which in turn induce changes in metabolites and proteins (Yida et al., 2015). Hypoxanthine, also known as 6-hydroxypurine, which can be obtained biologically by the removal of one amino group from adenine, is an important alkaloidal purine that is widely distributed in the body and is involved in the regulation of the body’s physiological functions. Hypoxanthine may be used to assess health status (Mahanty and Xi, 2020). Arginine is an essential amino acid for young animals, which can regulate protein synthesis and metabolism, promote growth and development, improve intestinal development and the number of intestinal villi and increase the immunity of animals (Nieves and Langkamp-Henken, 2002; Peranzoni et al., 2007). It was shown that feeding 1.15% L-arginine to ZDF rats significantly reduced abdominal and perirenal fat in rats (Clapés and Rosa Infante, 2009; Fu et al., 2005). Therefore, it is hypothesized that the nutritional value of yaks housed at 24 months of age may be somewhat higher; and the above differential metabolites may affect fat deposition in yaks, which can be promoted later by regulating them.
       
Dimethylglycine, niacinamide and glutamine were lower than in the housed group. Dimethylglycine (DMG) is a chemical complex that appears in the pathway of choline to glycine metabolism and has the emulsifying effect of a surfactant, which can effectively emulsify oil-water mixtures, break down fat particles in the mixtures, promote the chemical action of digestive enzymes in the intestinal tract and increase the contact area between the villi of the small intestine and the nutrients to promote the digestive absorption of the nutrients. Nicotinamide (NAM) is the amide form of vitamin B3.NAM can regulate fat metabolism, enhance body immunity and promote fat deposition in lambs; it was found that peri rumen nicotinamide can promote the growth and development of fattening lake sheep and increase the average daily weight gain; and Li suggested that NAM, by inhibiting the activity of SIRT1 dehydrogenase to promote lipid synthesis in animals (Li et al., 2015). Glutamine (GIn) is a non-essential amino acid, on the one hand, it can promote the proliferation of intestinal cells and intestinal development, enhance the activity of small intestinal enzymes and promote the absorption of nutrients; on the other hand, it can promote the proliferation of immune cells and improve the immunity of the body.It was found that the addition of 0.8% GIn significantly increased the mean daily feed intake and mean daily weight gain of 1- and 4-week-old chicks; the addition of GIn to the diet increased the mean daily weight gain and apparent digestibility of weaned piglets (Hwang et al., 2015). The above DMG, hypoxanthine, NAM, GIn and GIcN all promoted fat deposition, which was lower in the G24_SF group, presumably because yaks are in a cold environment and the body must provide sufficient energy to resist the cold, resulting in excessively low fat deposition compared to housed yaks.DMG, NAM, arginine, GIn, GIcN (Shmagel et al., 2019; Bowman and Wolfgang, 2019), PRO, CAR (Salem et al., 2015; Brown et al., 2014; Bao et al., 2015), AA (Demetz et al., 2014; Zheng and Hasegawa, 2016) and Neu5Ac all improved the immunity of yaks and were lower in the G24_SF group. Lower temperatures in the cold season on the Tibetan Plateau, a decrease in drinkable water resources and an uneven distribution of pasture grasses resulted in a lower nutrient intake by yaks during the cold season, which reduced the immunity of yaks. Therefore, grazing may affect the expression levels of the above differential metabolites, which in turn affects fat deposition and immunity in yaks; secondly, the nutritional value of housed yak meat is a little bit better compared to grazed yak meat.
 
KEGG enrichment pathway analysis
 
The Alanine, aspartate and glutamate metabolism pathways are thanked as significantly different metabolic pathways common to G24_SF, F24_SF and G24_SF. The metabolites of alanine (Ala) can provide the body with carbon shelf, nitrogen source and energy. Aspartate (Asp) not only regulates the immune system, but also plays an important role in the proliferation of immune cells. Glutamate (Glu) regulates normal physiological activities such as cell proliferation, differentiation, secretion and contraction through glutamate receptors (Skerry and Genever 2001; Newsholme et al., 2003). Glutamine is synthesized from glutamate, NH3 and ATP in skeletal muscle by the action of glutamine synthetase. It has been found that glutamine promotes the cytokine TNF to enhance immunity (Calder and Yaqoob 1999). Differential metabolites enriched to this pathway may be related to fat deposition, immunocompetence and growth and development in yaks and are important for resolving the mechanisms of yak adipocyte proliferation and differentiation as well as immunoregulation.
       
In summary, these metabolites regulate fat metabolism and may promote fat deposition by inhibiting adipocyte proliferation, promoting intestinal development and increasing nutrient absorption. Therefore, different feeding practices as well as different months of age will affect fat deposition in yaks and these significantly enriched pathways and differential metabolites will be the main metabolites and metabolic pathways that are highly affected during yak growth and development.

CONCLUSION

Using yak dorsal subcutaneous fat as test material, non-targeted metabolomics was used to investigate the effects of different feeding methods on fat deposition in yaks during the cold season and to further determine the changes of differential metabolites during production in different feeding methods and at different months of age, with the main conclusions as follows:
       
Feeding practices as well as growth and development may directly affect the type and amount of subcutaneous fat metabolites in yaks. Metabolic pathways of subcutaneous fat deposition in yaks are influenced by different feeding practices and different months of age. Differential metabolites such as 2-pentyl-3-phenyl-2-propene and N-acetylneuraminic acid may affect fat deposition in yaks, which could be promoted later by modulating them. Yak cold-season fattening can effectively improve the problem of yak cold-season fat loss and can promote the efficient production and fat deposition of yaks and improve the quality of yak meat.

​ACKNOWLEDGEMENT

Thank you to all the members of the group.
 
The animal welfare statement
 
The authors confirm that the ethical policies of the journal, as noted on the journal’s author guidelines page, have been adhered to and the appropriate ethical review committee approval has been received. 
 
Funding
 
This study was supported by the National Natural Science Foundation of China under Grant No. 32060750, the Qinghai Provincial Science and Technology Program under Grant No. 2024-NK-P41 and 2024-NK-109 and the “Kunlun Talents - High-end Innovative and Entrepreneurial Talents” Project of Qinghai Province.
 
Institutional review board statement
 
The animal handling procedures in this study were approved by the Ethics Committee for the Use of Laboratory Animals, College of Animal Husbandry and Veterinary Science, Qinghai University (Permit No.2023-QHMKY-001).
 
Informed consent statement
 
Not applicable.
 
Data availability statement
 
The data that support the findings of this study are available from the corresponding author upon reasonable request.

CONFLICT OF INTEREST

The authors declared that they had no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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