Tibetan sheep, a unique local breed in the Qinghai Tibet region of China, exhibits great adaptability to harsh environments, including cold weather, hypoxia and nutrient scarcity (Su et al., 2022). Studies have found that Tibetan sheep meat possesses desirable qualities in terms of its fresh and tender taste, its high protein but low fat content, its diverse types of amino acids as well as its rich aroma (Zhang et al., 2022a). These attributes actually fulfil the dietary requirements of modern consumers. However, in recent years, ecological factors, along with the limited availability of grassland areas, have gradually shifted the grazing practices in animal husbandry to housed or semi-housed feeding (Zhang et al., 2022b). As a result, this has led to an increased demand for protein feed to promote animal growth. In addition, the incomplete digestion and absorption of dietary proteins by animals increases the emission of nitrogen pollutants, thereby resulting in environmental pollution (Hristov et al., 2011) that subsequently restricts the development of animal husbandry (Qi et al., 2023). However, in an attempt to address the issue, researchers have found that supplementing animal diets with rumen-protected amino acids (RPAA) can lower the level of dietary proteins and hence, reduce nitrogen emission (Ma et al., 2010; Kamiya et al., 2021).

Sulfur containing amino acids (SAA) include methionine (MET), cysteine (CYS), cystine, taurine, etc. MET and CYS are considered to be the main SAAs because they are typical amino acids involved in protein synthesis (Brosnan and Brosnan, 2006). MET is the only sulfur-containing essential amino acid in animal growth, and it is involved in protein synthesis, methylation cycle, polyamine production, sulfur transfer pathway and other important metabolic reactions in animals (Finkelstein, 1990). CYS can be used as a precursor of important bioactive substances such as glutathione, hydrogen sulfide and taurine, thus playing antioxidant, digestive and immune roles (Blachier et al., 2020). Some studies have reported that dietary SAA supplementation has important nutritional significance for animal gastrointestinal development. Zhong et al. studied the effect of DL-Met addition on the intestinal development of young pigeons and found that 0.3% DL-Met could improve the intestinal morphology and structure of pigeons by activating Wnt/β-catenin signaling pathway (Zhong et al., 2022). Gu et al. (2021) added 20 gm/day of RPMET to the diet of dairy cows in the middle of lactation to promote rumen fermentation and increase the beneficial microorganisms such as Acetobacter, saccharofermentan, Thermoactinomyces and so on that promote milk synthesis. Similarly, Ding et al. (2019) found that adding 0.4% CYS to the piglet diet can improve the level of antioxidant stress metabolites in the plasma by increasing the abundance of Lactobacillus and pseudobutyrivibrio in the intestine. Ni et al. (2021) showed that providing 0.4 and 0.5% of CYS to the piglet mother would reduce the level of Firmicutes in the piglet colon and increase the level of Bacteroides. This change may affect the lipid metabolism of piglets. With further research, it was found that adding SAA also had an impact on meat quality. Lebret et al. (2018) found that adding 1.1% MET to the pig diet can improve the final pH value of pork and reduce drip loss, brightness and hue. Similarly, adding a high level of MET (21.36%) to the diet of arbor acres broilers also improved the pH value of broilers and significantly reduced the brightness and cooking loss (Wen et al., 2017). On the other hand, Conde Aguilera et al. found that providing sufficient SAA (0.46% MET and 0.70% MET+CYS) to piglets would increase the protein and water content of longissimus muscle of pig waist and reduce the content of fat, ash and total amino acids (Conde-Aguilera et al., 2010). The above results show that SAA supplementation in the diet will have an impact on gastrointestinal microorganisms and meat quality of livestock and poultry, However, there is a limited body of research examining the impact of dietary supplementation with MET and CYS on the gastrointestinal tract and meat quality of Tibetan sheep.

The rapid development of metabolomics made it possible for scientists to delve deeper into research on metabolites and meat quality. For example, studies by Zhang et al. found that changes in the metabolic pathways of amino acids, lipids and carbohydrates in muscles altered metabolite levels that subsequently influenced the tenderness and flavor of meat (Zhang et al., 2022a,b). Similarly, MET, LYS and CYS are essential bioactive molecules that participate in key metabolic pathways in the body (Watanabe et al., 2020; Gondret et al., 2021), and as such, changes in their levels may also exert similar effects. In addition, rumen microorganisms are of great significance in animal nutrition digestion, absorption and metabolism (Zhang et al., 2020). Studies from Ding and Burrin showed that adding amino acids to animal diets can provide energy for the small intestine, increase the abundance of intestinal microbiota as well as maintain intestinal integrity (Burrin and Reeds, 1997; Ding et al., 2019). In this context, studies by Ma et al. (2023) revealed that feed additives could influence muscle metabolites and improve meat quality by altering the composition of rumen microorganisms in Tibetan sheep. In a similar vein, Zhiwei (Zhao et al., 2021) found that supplementing the diet of lactating yaks with RPMET and RPLYS could alter the composition of their rumen bacteria, influence the rumen fermentation environment and metabolism and ultimately alter the synthesis of metabolites in milk. Based on the above, the current study hypothesized that RPLYS, RPMET and RPCYS have the potential to modulate the gastrointestinal microbiota of Tibetan sheep, thereby affecting muscle metabolites and meat quality.

At present, the price of protein feed is becoming increasingly expensive, and high protein feed will increase animal nitrogen excretion and cause serious environmental pollution (Wei et al., 2023). In order to save the feeding cost, we need to reduce the protein level of feed, which will lead to the reduction of growth performance of livestock and poultry due to insufficient nutrition, and have a negative impact on meat quality (Zhang et al., 2024). The study found that maintaining a better amino acid balance at a low protein level can improve the utilization rate of protein raw materials, effectively relieve the current pressure of resource supply and demand, and achieve a win-win situation in productivity and environmental benefits (Liu et al., 2024). Therefore, this work applied 16S rDNA and metabolomics techniques to investigate the effects of rumen-protected sulfur-containing amino acids (RPSAA) on the gastrointestinal microbiota and muscle metabolites of Tibetan sheep. Correlation analysis was subsequently used to reveal the relationship between Tibetan sheep meat quality, gastrointestinal microbiota and muscle metabolism, thereby elucidating the mechanism through which RPSAA influences the meat quality. It is expected that the results of this study will be of significance to determine the optimal amount and specific types of RPAA to be added to Tibetan sheep feed, hence helping to reduce the level of feed protein, save feed protein resources, reduce feeding costs, while reducing environmental pollution and promoting the sustainable development of animal husbandry in the Qinghai Province.

MET and CYS are SAA, and there is a complex transformation relationship between them. MET is the precursor of CYS, which can transfer methyl through the intermediate metabolite S-adenosylmethionine to form S-adenosylhomocysteine, then it is hydrolyzed to homocysteine, and finally CYS is generated through sulfur transformation (Tang et al., 2010). In this context, research by Finkelstein (1990) suggested that CYS can replace more than half of the MET in the diet due to their turnover mechanism in the body. MET and CYS are precursors of glutathione, taurine and sub taurine, which have been proved to have strong antioxidant capacity (Wu et al., 2004; Kotzamanis et al., 2019). Good redox state can make animal meat show better quality characteristics in water holding capacity, color stability and anti lipid and protein oxidation (Estévez, 2015). Therefore, through the control of animal nutrition, MET and CYS can be reasonably used to improve meat quality. Adding amino acids to diets is beneficial for animal growth, production and reproduction. However, supplementing feeds with high levels of amino acids may also cause imbalance or antagonism, thereby inhibiting growth (Peng et al., 1973). Similarly, insufficient amounts of amino acids can reduce weight gain, feed efficiency and feed intake (Castro et al., 2020). This study set up three groups, namely the group without added RPSAA (AP-1 group), the group with added RPMET (AP-2 group), and the group with added RPMET and RPCYS (AP-3 group), in order to clarify the effects of RPSAA on rumen microbiota, meat metabolism, and meat quality in Tibetan sheep.

In this study, the proportion of RPSAA added in this study has no adverse effect on the carcass quality of Tibetan sheep. The color of meat is closely related to its freshness as it directly reflects the quality of meat and influences consumers’ acceptance (Ripoll and Panea, 2019). Meat that is popular among consumers generally exhibits a bright cherry red color (Sawyer et al., 2009). However, when the values of a^* and b^* are not significantly different, a significant decrease in L^* can result in a dark red state. In this context, research has found that the L^* of meat was related to its muscle fat content and WHC. For instance, Silva et al. found that a decrease in beef meat’s brightness was accompanied by a lower intramuscular fat (Silva et al., 2019). In this study, the L^* value of AP-2 decreased significantly, while its fat content was the lowest. As already pointed out, the amount and distribution of water in muscle tissues can also affect the color of meat. Muscles contract when they become stiff after death, and water, from the muscle fiber compartment, enters the intercellular space followed by the extracellular space (Honikel et al., 1986). An increase in muscle contraction therefore leads to an increase in the water leakage which subsequently affects the refractive index of the muscle surface and leads to an increase in L^*. In this study, AP-2 also had the lowest cooking loss and the highest moisture content, and hence the lowest L^*.

Linda et al. found that in color-stable lamb loin, a decrease in inosinemonphosphate (IMP) was accompanied by a significant increase in inosine concentration (Samuelsson et al., 2022). This is because after sheep are slaughtered, ATP in the meat forms IMP through ADP and AMP, and then further forms hypoxanthine through inosine (Yano et al., 1995). During the process of muscle conversion into meat, the initial concentrations of AMP and IMP in the meat are high, while the inosine and hypoxanthine content is low, resulting in poor tenderness of the meat. At later stages, ATP reserves are depleted and secondary energy consumption begins. This involves the decomposition of AMP and IMP into inosine and hypoxanthine, resulting in an increase in meat tenderness (Graham et al., 2012; Beldarrain et al., 2023). Interestingly, in the correlation analysis, there was a significant correlation between L^* and SF with purine metabolism and metabolites in its surrounding pathways, as well as with the microbiota and SCFAs in the rumen and jejunum (Figure 7C). So far, research has revealed that the gastrointestinal microbiota could regulate meat quality by producing SCFAs (Zhang et al., 2022a; Dou et al., 2023). Therefore, it was speculated that feeding Tibetan sheep with RPSAA could influence purine metabolism and its nearby pathways by altering the gastrointestinal microbiota and increasing the production of SCFAs, thereby reducing L^* and increasing SF.

In recent years, people have been paying attention to both the nutritional needs of animals as well as the tenderness of their meat. Tenderness is an important quality attribute that also affects consumers’ purchase of meat, and it is generally determined through a sensory evaluation or measurement of SF (Warner et al., 2021). In their study of meat tenderness, Haiqing et al. found that adding different concentrations of RPMET to the diet of Tan sheep did not significantly affect the SF (Li et al., 2020). In fact, the study by Zhiyuan et al. showed that low to medium concentrations of RPLYS and RPMET significantly reduced the SF of yak meat (Ma et al., 2021). On the other hand, in this study, the addition of RPSAA was found to significantly increase the SF of Tibetan sheep meat. The above findings suggest that the role of RPAA may vary according to the animal species, types of amino acids and feeding background. The tenderness of meat is also related to its final pH value (pHu), with values ranging from 5.8 to 6.19 corresponding to a higher meat SF compared with those which are ≤5.79 or ≥ 6.2. This could be attributed to the fact that, within this pH range, small heat shock proteins are not easily degraded. At the same time, cathepsin B and μ-Calpain have low activities which further increase the toughness of meat and delay its tenderness (Lomiwes et al., 2013). In this study, the pHu of the RPSAA group was between 5.8 and 6.19. This could explain why the SF of this group was greater compared with that of the ordinary amino acid group. However, it should be noted that the maximum SF of the LL samples did not exceed 11 kgF, with Bickerstaffe suggesting that this value was an acceptable upper limit for cooked meat tenderness (Bickerstaffe et al., 2001).

In recent years, the relationship between cell apoptosis and tenderness attracted much attention. In this context, the study by Cheng Chen et al. revealed that cell apoptosis, mediated by apoptosis inducing factor (AIF), could increase the tenderness of beef muscle (Chen et al., 2020). Similarly, Zhang et al. found that inducing higher cell apoptosis through diet could increase the tenderness of meat, while delaying the apoptotic process resulted in a tougher meat texture (Ma et al., 2020; Zhang et al., 2022). After animal slaughter, muscle cells enter a state of hypoxia during which they produce a hypoxic stress response that activates various signaling pathways. This, in turn, gradually leads to a change from oxidative respiration to anaerobic glycolysis to provide energy for cells (Wang et al., 2022). However, hypoxic stress also inevitably produces ROS and induces HIF-1α accumulation through the PI3K/AKT–mTOR signaling pathway (Tang and Zhao, 2020; Chen et al., 2022). HIF-1 is an important regulatory pathway for cell apoptosis, and it can regulate metabolic adaptation, metastasis and anti-apoptosis of hypoxic cells (Paik et al., 2017). It was found that an increase in HIF-1α expression was accompanied by an increase in sarcoplasmic calcium (Ca²⁺) during postmortem maturation. Ca²⁺, through calmodulin CaMKK β activating AMPK, promotes the transfer of GLUT1 to the cell membrane and the phosphorylation of transcription factors to induce glucose uptake and transport. As a result, this process maintains energy production in cells under hypoxic conditions while inhibiting cell apoptosis (Gao et al., 2019; Xin et al., 2023). Therefore, it can be inferred that the AMPK pathway can regulate the post mortem glycolysis process. In this study, the PI3K-Akt, mTOR, HIF-1 and AMPK signaling pathway as well as the metabolites in the RPSAA groups were upregulated compared with the AP-1 group. Hence, it was speculated that, after slaughter, the activation and upregulation of these pathways in the RPSAA group of Tibetan sheep could delay cell apoptosis and meat tenderization due to hypoxic stress. However, further research would be required to indeed ascertain the above speculation.

On the other hand, it was found that, in the RPCYS group, glycolysis/gluconeogenesis and the citrate cycle (TCA cycle) were upregulated, hence indicating that aerobic respiration and glycolysis were the two mechanisms through which energy was provided to muscle cells at this time. These findings also suggested that the muscle was in an early postmortem stage (Matarneh et al., 2018). Upregulation of the glycolysis pathway generally increases lactate concentration, thereby reducing pH and increasing meat hardness (Chen et al., 2019). However, in the RPCYS group, the meat texture was harder although the pH value was higher than for the other two groups. This could be due to the slower glycolysis rate that prolonged the zombie stage and slowed down the softening rate of the meat (Honikel, 2014). Therefore, this study suggests that the acceleration of glycolysis rate, followed by a rapid decrease in pH, can be beneficial for the hydrolysis of muscle fiber proteins as well as for improving the tenderness of meat (Barón et al., 2021).

The composition and fatty acids content of meat not only affect its flavor but also affect human health. For example, research has shown that PUFA possessed anti-obesity and anti-inflammatory properties. Similarly, the intake of n-3 and n-6 PUFA is essential for normal human growth and development due to their regulatory role in cell functions, signal transduction and immune response (Liu et al., 2023). Of these, C20:5 N3 (EPA), C22:6 N3 (DHA) and C22:5 N3 (DPA) are particularly important n-3 PUFAs as they are functional fatty acids which are involved in regulating cholesterol, while reducing risks of human neurodegeneration, coronary heart disease and thrombosis (Montenegro et al., 2022). Similarly, n-6 PUFA such as arachidonic acid (AA) and linoleic acid (LA) are of significance as an increase in LA intake can improve plasma lipids, blood glucose control and insulin resistance (Marangoni et al., 2020). This work applied GC–MS to determine the composition and fatty acids content of three groups of Tibetan sheep meat and found that the n-3 PUFA, EPA, DHA, DPA and C20:3 N6 content in AP-2 significantly decreased. In fact, the overall fatty acids content also showed a downward trend. Non-target metabolomics detection further showed that fatty acids such as linoleic acid, arachidonic acid, palmitic acid, γ-linolenic acid and eicosenoic acid were downregulated in AP-2 (AP-2 vs. AP-1), with their involvement in the biosynthesis of unsaturated fatty acids, linoleic acid metabolism and butanoate metabolism also downregulated (Table 4). In contrast, the biosynthesis of unsaturated fatty acids and linoleic acid metabolism (AP-3 vs. AP-2) in AP-3 were upregulated, with the amount of γ-linolenic acid and linoleic acid also increased (Table 4). Research has found that butanoate metabolism, through the β-Hydroxyl group-β-Methylglutaryl CoA pathway, increased lipid synthesis (Liu et al., 2018). However, due to the significant correlation between these fatty acids, metabolites and the microbiota in the rumen and jejunum (Figure 7B), it was speculated that in AP-2, the downregulation of pathways related to lipid synthesis, and hence a decrease in the fatty acids content, was caused by changes in the gastrointestinal microbiota of Tibetan sheep (Figure 8). These results were also reflected in the works of Wen et al. and Conde-Aguilera et al. who found that the addition of sufficient MET to diets resulted in a lower fat content in chicken breast compared to the MET-deficient group (Conde-Aguilera et al., 2010; Wen et al., 2017). This observation could be attributed to a MET-induced increase in carnitine content that not only promoted the oxidation of fatty acids but also reduced the amount of constant fatty acids that could be stored in adipose tissues (Zhan et al., 2006). Finally, being precursors of flavor substances, fatty acids can influence the flavor of meat through reactions such as lipid oxidation. A decrease in the fatty acids content, as it was the case for AP-2, would therefore affect its flavor.

Overall, compared with the ordinary amino acid group, the WHC and texture of the groups fed with RPSAA increased, resulting in better food quality. In addition, when comparing the two groups of RPSAA, the fatty acid metabolism pathway was significantly upregulated in AP-3. This could be because, in AP-2, the endogenous CYS synthesized by MET through the sulfur transfer pathway was insufficient to meet the needs of Tibetan sheep. On the other hand, the exogenous CYS added to feed in AP-3 helped to meet the dietary requirements, hence increasing the production of antioxidant substances such as glutathione and taurine, and preventing the oxidation of fatty acids (Medina et al., 2022). As a result, AP-2 has the best edible quality, but its fatty acid nutrition is reduced.

Amino acids are immediately decomposed by microorganisms in the rumen to produce ammonia. In this case, RPAA can reduce the degradation within the rumen, thereby allowing the amino acids to enter the small intestine. This not only improves the utilization rate of amino acids but also increases animal production (Ma et al., 2021; Liu et al., 2023). However, RPAA decomposed in the rumen and entering the small intestine may also alter the gastrointestinal microbiota. As such, this decomposition process can influence fermentation parameters and even regulate some metabolism in animals (Zhang et al., 2022a; Liu et al., 2023; Wei et al., 2023). Previous studies found that the addition of RPLYS and RPSAA did not significantly affect the rumen pH, ammoniacal nitrogen and total concentration of SCFAs in yaks (Liu et al., 2023) and brown lambs (Liu et al., 2021), with these findings being consistent with those of the current study. However, the ammonia-nitrogen concentration in the jejunum of the RPSAA group significantly decreased, while the concentrations of butyric acid and valeric acid significantly increased, thereby indicating that RPSAA improved the utilization rate of AA in the jejunum and promoted jejunal fermentation (Wang et al., 2022) This could also explain the improved carcass quality in both RPSAA groups.

Gastrointestinal microbiota plays a crucial role in the nutrition and health of ruminants. As far as RPSAA is concerned, Bacteroidetes and Proteobacteria are particularly affected within the rumen. In this study, the three groups did not differ significantly in terms of their relative proportion of the top five main phylum in the jejunum. Bacteroidetes, the largest portal in the rumen, is responsible for protein hydrolysis, carbohydrate degradation and the fermentation of amino acids into acetate (Hinsu et al., 2017). On the other hand, Firmicutes, the largest portal in the jejunum. Play an important role in the degradation of fiber and cellulose (Crisol-Martínez et al., 2017). The structure of the two is different at the phylum level, with differences also noted in their fermentation modes. Correlation analysis was performed on the microbial communities and fermentation parameters of the rumen and jejunum, with the results showing a close connection between the rumen and jejunum. Prevotelaceae_ UCG-003 uses various sugars to produce acetic acid and succinic acid while enhancing fiber digestion (Li et al., 2019). Lachnospiraceae_ NK3A20 can alleviate intestinal inflammation by producing butyric acid to inhibit the production of pro-inflammatory cytokines by intestinal neutrophils (Li et al., 2021). Hence, it is also referred to as a probiotic. Finally, Turicibacter is a pathogen that is positively correlated with colitis (Rettedal et al., 2009). The abundance of Prevotellaceae_ UCG-003 and Lachnospiraceae_NK3A20 increased in the AP-2 and AP-3 groups, especially with the addition of RPCYS (AP-3 group), while the proportion of Turicibacter decreased significantly in the two groups. The first two bacterial genera are positively correlated with butyric acid, isobutyric acid, valeric acid, isovaleric acid and acetic acid, but negatively correlated with the Turicibacter in the jejunum. Therefore, feeding RPAA can be useful to increase the abundance of beneficial bacteria and SCFAs in the rumen of Tibetan sheep. As it flows from the rumen to the jejunum, it further inhibits the production of harmful bacteria and maintains the healthy development of the jejunum. The influence of diet on gastrointestinal microbiota can also regulate muscle metabolites and affect meat quality (Peng et al., 1973). In this study, the abundance of microbiota in the rumen and jejunum was closely related to the amount of fatty acids and purine metabolism as well as to the L^* and SF of the LL meat. Therefore, it was speculated that feeding RPSAA promotes fermentation in the rumen and jejunum of Tibetan sheep, thereby affecting fatty acids and purine metabolism in the LL, and ultimately altering L^* and SF (Figure 8). However, these changes in meat quality may also be caused by the complex transformation relationship between MET, cysteine, and CYS, leading to metabolic changes in Tibetan sheep and synergistic effects on their living environment. As noted before, further research would be required to ascertain the above speculation, and especially to verify the specific mechanism through which rumen and jejunal microbiota influence the quality of Tibetan sheep meat.

A number of studies have shown that reducing the level of crude proteins in feed and adding RPAA can reduce the emission of nitrogenous pollutants from animals without affecting their growth. In this study, feeding the RPMET group (AP-2) improved the carcass quality of Tibetan sheep by increasing their carcass weight, abdominal fat, and back fat; Compared with the AP-3 group, the AP-2 group achieved the best edible quality by reducing brightness, cooking loss, and shear force, but the nutritional value of fatty acids in the AP-2 group decreased. Further analysis indicates that the addition of RPSAA affects important metabolic pathways and metabolites in Tibetan sheep muscles. The downregulation of unsaturated fatty acid biosynthesis, linoleic acid metabolism, and butanoate metabolism in AP-2 could have been responsible for the decrease in the fatty acids content. Finally, correlation analysis showed that the increase of beneficial microbiota (Prevotella 1, Lachnospiraceae NK3A20 group, Prevotella UCG-003, Lachnospiraceae ND3007 group) in the rumen of AP-2 and AP-3 could increase the abundance of beneficial microbiota (Eubacterium nodatum group, Mogibacterium group) in the jejunum and inhibit the growth of harmful ones (Turicibacter). In addition, an increase in the abundance of gastrointestinal microbiota and SCFAs also influenced the key metabolites of LL (inosine, hypoxantine, AMP, ADP, L-glutamate, etc.), thereby regulating purine metabolism and ultimately affecting the brightness and tenderness of LL. In a word, the results suggested that RPAA can improve the overall quality of Tibetan sheep’s meat.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary material.

The animal studies were approved by Institutional Animal Care and Use Committee Guidelines of Qinghai University (protocol number 0515). The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent was obtained from the owners for the participation of their animals in this study.

JL: Conceptualization, Data curation, Formal analysis, Methodology, Software, Validation, Visualization, Writing – original draft. LH: Conceptualization, Formal analysis, Funding acquisition, Methodology, Project administration, Supervision, Visualization, Writing – original draft, Writing – review & editing. SH: Conceptualization, Investigation, Resources, Validation, Writing – review & editing. LG: Formal analysis, Software, Writing – review & editing. ZY: Data curation, Writing – review & editing. SS: Software, Writing – review & editing. ZW: Investigation, Writing – review & editing. BY: Investigation, Writing – review & editing.