Pork possesses high nutritional value and is one of the most consumed meats worldwide (Matarneh et al., 2021). However, pork quality is declining due to the excessive pursuit of growth rate and lean percentage of pigs. Therefore, the current trend is to produce safe and high-quality pork as the consumer demands. Compared to the Western commercial pig breeds, the Chinese indigenous pig breeds are favored by consumers due to their premium meat quality and attractive flavor (Li et al., 2021). Among them, the Shaziling pig is such an indigenous breed known for superior meat quality and sensory attributes as manifested by high contents of intramuscular fat (IMF), amino acids and fatty acids (Duan et al., 2023; Song et al., 2022). However, what cannot be ignored is that these pigs grow slowly and have a lower lean percentage and a high fat content (Song et al., 2022), necessitating a simultaneous improvement in carcass traits and meat quality.

One promising strategy that may help achieve this objective is addition of certain amino acids to diets. Evidence for this is provided by observations that dietary inclusion of branched chain amino acids (Zhang et al., 2022), glycine (Zhong et al., 2021), arginine (Hu et al., 2017b; Madeira et al., 2015), or serine (Zhou et al., 2021) improves meat quality. Apart from these amino acids, glutamate (Glu) has gained our attention since Glu is the top ranked metabolite enriched in metabolic pathways for the Shaziling pig meat flavor. Moreover, the muscular Glu concentration is positively correlated with total essential amino acids, flavor amino acids, and sweet amino acids of Shaziling pigs (Duan et al., 2023). Based on these findings, it is hypothesized that Glu may play a key role in the muscle development and hence meat quality in Shaziling pigs.

Traditionally, Glu has been classified as a nutritionally “nonessential” amino acid (Wu, 2013). However, fresh insights into the effects of Glu on energy metabolism in the intestines of weaned piglets indicate that Glu is a nutritionally essential amino acid for maintaining intestinal and whole-body homeostasis in neonates (Hou et al., 2018; Qin et al., 2018; Sheng, 2015; Zhu et al., 2020). Although early research on Glu focused on the intestine (Duan et al., 2014; Rezaei et al., 2013), more recent studies have highlighted its impact on muscle and adipose tissues. Since the Glu requirement by muscle cannot be fully met through intramuscular synthesis of Glu (Ytrebø et al., 2006), it is necessary to add a large amount of Glu to the diet to support muscle growth. Indeed, recent research using a lipopolysaccharide-challenged piglet model (Duroc × Large White × Landrace) has provided direct evidence of the muscle growth-promoting effects of dietary Glu supplementation (1%) (Kang et al., 2017). Apart from its functions in muscle growth of pigs, Glu supplementation (1%) also reduces backfat thickness and increases IMF content in growing-finishing pigs (Duroc × Landrace × Yorkshire) (Hu et al., 2017a; Kong et al., 2015). These findings open up the possibility of adding appropriate Glu (1%) to diets of pigs to increase the lean percentage and improve meat quality. However, there is a paucity of data on the role of dietary Glu supplementation in Shaziling pigs.

Since 150 to 210 d of age seem to be the critical window for meat quality in Shaziling pigs (Song et al., 2022), we conducted a study using 150-d-old Shaziling pigs with a hypothesis that dietary Glu supplementation during the window period could improve growth performance, carcass traits, meat quality, amino acid and fatty acid composition of the longissimus dorsi muscle, and alter the colonic microbial community of Shaziling pigs.

Dietary Glu is extensively catabolized in various stages of pig growth (Hou et al., 2016). Increasing evidence suggests that dietary Glu supplementation at the levels of 1% to 4% is safe and can promote the growth of weaner and finisher pigs (Rezaei et al., 2013). The current study showed that Glu supplementation increased the ADG and final BW of Shaziling pigs without adverse effects on their health. Glu improves the intestinal morphology and maintains intestinal barrier integrity in pigs, thus promoting the absorption of nutrients (Qin et al., 2018). This suggests that the growth-promoting effects of Glu on Shaziling pigs may be due to its beneficial roles in intestinal functions.

Carcass traits, such as carcass yield, backfat thickness, loin-eye area, lean percentage and fat percentage, reflect the body composition and the production performance of animals. The loin-eye area is often considered as an indicator of meat yield, and the backfat thickness reflects the fat mass (Buck et al., 1962; Ferrucci et al., 2006). In this study, dietary Glu supplementation increased the loin-eye area and lean percentage and reduced the fat percentage of Shaziling pigs. Consistent with our results, a reduction in the average backfat thickness was also observed in Duroc × Landrace × Yorkshire pigs, although there were no significant impacts on the lean percentage (Hu et al., 2017a). These findings indicate that Glu supplementation has the potential to reduce the fat mass of both indigenous pigs and commercial lean pigs. However, the beneficial effects of Glu supplementation on the lean percentage were only observed in Shaziling pigs (indigenous pigs) but not in Duroc × Landrace × Yorkshire pigs (commercial lean pigs). Given the fact that lean percentages of indigenous pigs are lower than those of commercial lean pigs (Song et al., 2022), it is speculated that the different observations may be due to the inherent differences in the lean percentage between the two pig breeds. Another reason for the different observations may be attributable to the differences in the period of adding Glu to pigs' diets. In this study, Glu was added to the diets of Shaziling pigs during the critical period of pork quality development (150 d, 31.56 ± 0.95 kg), whereas the initial weight of pigs used in previous studies was 77.1 ± 1.3 kg. At this stage, pigs (Duroc × Landrace × Yorkshire) mainly deposit fat rather than lean meat. Therefore, Glu supplementation mainly plays a role in regulating lipid deposition.

In the current study, we first determined the amino acid profiles in the longissimus dorsi muscle to explore the mechanisms by which Glu enhances lean tissue deposition. Evidence from a pig model showed that Glu supplementation increased the amino acid content and downregulated the mRNA levels of genes involved in protein degradation such as muscle-specific RING finger-1 and muscle atrophy F-box in the longissimus dorsi muscle (Hu et al., 2019b). Furthermore, evidence for a relationship between Glu supplementation and protein synthesis comes from the finding that dietary Glu supplementation alleviates muscle protein loss by modulating mTOR signaling pathways in LPS-challenged piglets and increases the protein content of the longissimus dorsi muscle (Kang et al., 2017). These results are consistent with the current results since dietary Glu supplementation decreased the Asp content in the serum and longissimus dorsi muscle, and increased the Glu content in the longissimus dorsi muscle. Since Glu can be synthesized from the Ala, Asp, and branched-chain amino acids in animals (Li et al., 2009), it is assumed that in response to Glu supplementation, the Asp was converted to Glu to meet the need for muscle growth, thus increasing the lean percentage of Shaziling pigs. Considering that high concentrations of serum Glu are neurotoxic (Mark et al., 2001) and the skeletal muscle has a large requirement for Glu, the different results of Glu content between serum and muscle are reasonable. Another important reason for the increased lean percentage of Shaziling pigs fed a Glu-supplemented diet may be due to the upregulated mRNA expression of MyHC IIb and MyHC IIx. Generally speaking, the rapid growth of skeletal muscle is accompanied by a higher ratio of muscle fiber types II, especially MyHC IIb and MyHC IIx (Ruusunen et al., 2004). In this study, dietary Glu supplementation led to a higher mRNA expression of MyHC IIb and MyHC IIx. These results are in agreement with the literature where 30 g/kg monosodium L-Glu (MSG, an additive form of Glu) increased the relative mRNA expression of MyHC IIb and MyHC IIx in finishing pigs (Kong et al., 2015). In summary, Glu supplementation decreased the Asp content, increased the Glu content, and upregulated the mRNA expression of MyHC IIb and MyHC IIx in the longissimus dorsi muscle, leading to enhanced lean percentage of Shaziling pigs.

Appropriate increases in IMF concentrations can enhance the acceptability, eating quality, and nutritional value of pork. When the IMF content reaches more than 3%, the meat has a good flavor, taste, and tenderness (Daszkiewicz et al., 2005; Hou et al., 2023). In this study, Glu supplementation significantly increased the IMF content of Shaziling pigs to the level of 4.25%. Similar results were obtained in finishing pigs fed high-fat diets (Duroc × Large White × Landrace), in which dietary supplementation of 30 g/kg sodium Glu increased the IMF content to the level of 4.76% (Kong et al., 2015). However, the opposite was true when finishing pigs fed a control diet (Duroc × Large White × Landrace), where Glu supplementation had no significant influence on the IMF content (Hu et al., 2017b). The percentage of fat in the diet as well as the pig breeds used may offer an explanation for the inconsistency. Shaziling pigs, however, the increased fat deposition in response to Glu supplementation may be due to differences in Glu regulating lipid deposition in the adipose tissue and skeletal muscle.

In the present study, we also determined the fatty acid composition in the longissimus dorsi muscle of Shaziling pigs in response to Glu supplementation. Fatty acids not only affect the flavor and nutritional values of meat products, but also play a critical role in human health (Ma et al., 2015; Rhee et al., 2000). The n-3 PUFAs are important components of cell membranes and possess numerous physiological and health effects, such as esterizing cholesterol, decreasing the triglyceride (TG) content, and exerting anti-inflammatory roles; whereas the n-6 PUFAs (as the precursor of prostaglandin and leukotrienes) possess pro-inflammatory properties (Ferrucci et al., 2006). A diet with a lower n-6/n-3 PUFA ratio is thought to protect against cardiovascular diseases (Simopoulos, 2008). Moreover, PUFAs were negatively correlated with meat flavor, while MUFAs are positively correlated with meat flavor and exert a wide range of health-protective effects, such as lowering blood sugar content (Cameron et al., 2000). In this study, Glu supplementation increased the content of MUFAs and decreased the contents of n-6 PUFAs and PUFAs in the longissimus dorsi muscle. C18:1n9c and C20:1 belong to MUFAs, both of which in the longissimus dorsi muscle were significantly increased in response to Glu supplementation. We previously found that C18:1n9c was the most dominant MUFAs in the longissimus dorsi muscle of Shazilng pigs, and its content was positively correlated with IMF (Duan et al., 2023). Moreover, C18:1n9c can decrease the concentrations of low density lipoprotein, cholesterol, and lipid in blood (Su et al., 2022). C18:2n6c was the major PUFA in the longissimus dorsi muscle of Shaziling pigs and was inversely proportional to IMF (Duan et al., 2023). Therefore, the Glu supplementation-induced elevation in the IMF content may be associated with the increased C18:1n9c and decreased C18:2n6c levels in the longissimus dorsi muscle of Shaziling pigs. From the above studies, it can be inferred that Glu could improve the fatty acid composition in the longissimus dorsi muscle of Shaziling pigs, thus positively affecting the nutritional value, flavor, and eating quality of pork.

In animals, fat deposition is related to TG storage, fatty acid synthesis, lipid mobilization and the β-oxidation of fatty acids. SREBP-1C is a key nuclear transcription factor of lipid metabolism and can upregulate the mRNA expression of acetyl-CoA carboxylase (ACC) and FAS to promote fatty acid synthesis (Sakakibara et al., 2006). HSL is the key rate-limiting enzyme that initiates the hydrolysis of TG stored in adipose tissue (Enevoldsen et al., 2001). CPT1 is a key enzyme responsible for transporting long-and medium-chain fatty acids to enhance the β-oxidation of fatty acids (Zhao et al., 2017). AMPK, as a cellular energy sensor, is an important regulator of energy homeostasis in the body and cells (Kim et al., 2016). Numerous studies have shown that the AMPK/SIRT1 signaling pathway can activate the PGC-1α, which subsequently promotes mitochondrial biogenesis and enhances fatty acid β-oxidation (Cantó et al., 2009). In the current study, dietary Glu supplementation increased the mRNA expression of FAS and SREBP-1C in the longissimus dorsi muscle. These results indicated that Glu could increase the lipogenesis in the longissimus dorsi muscle. These findings are consistent with the result of increased IMF content in the longissimus dorsi muscle.

The literature suggests a possible role of the gut microbiota in the regulation of fat deposition in indigenous pigs (Zheng et al., 2021b). The normal gut microbiota exerts a wide range of health-promoting effects, ie., resisting enterobacter infection, producing vitamins, improving intestinal function, promoting digestion and absorption, regulating intestinal immune function, and maintaining energy metabolism homeostasis (Cho et al., 2012; Kau et al., 2011). We found that Glu supplementation increased the relative Spirochaetota abundance and the acetic acid content in the colon. Interestingly, the acetic acid content was positively associated with the relative Spirochaetota abundance and IMF content and tended to be negatively correlated with the fat percentage. However, different results were reported when looking at Duroc × Landrace × Yorkshire pigs, with Glu supplementation increasing the relative Actinobacteria abundance and the contents of butyrate and propionate (Hu et al., 2019a). An important reason for the different observations may be because of the different diets used in each experiment. The diets used in the above mentioned study had a higher crude fiber level (3.09% vs. 2.42%) and a lower protein content (13.14% vs. 14.88%) and fat (1.87% vs. 5.94%) compared with ours. In addition, acetic acid is the main SCFA, accounting for approximately 60% of the total SCFAs, and can be absorbed and utilized by many tissues (Frampton et al., 2020). Studies have shown that acetic acid is partially (approximately 40%) metabolized by the liver, and a large proportion is transported to the skeletal muscle and adipose tissue (Giron et al., 2022). Indeed, acetic acids are a possible regulator of lipid metabolism. For instance, studies in rats have shown that acetate could upregulate the mRNA expression of lipogenesis-related genes, such as ACC and FAS (Sun et al., 2023). Similarly, studies in ruminants and mice show that acetate supplementation promotes milk fat accumulation and upregulates the mRNA expression of lipogenesis-related genes of mammary epithelial cells (Qi et al., 2023; Wang, 2023). Apart from these in vivo studies, in vitro studies with 3T3-L1 preadipocytes have also shown that the peroxisome proliferator activated receptor γ (PPARγ)expression and lipid drops were significantly increased in response to acetate treatments (Hong et al., 2005). Studies in porcine adipocytes have shown that acetate treatment increased the expression of PPARγ and CCAAT/enhancer-binding protein alpha (C/EBPα), thus promoting adipogenic differentiation (Xu et al., 2014). Therefore, considering the described effects of Glu on the gut microbiota composition and the acetic acid content, it may be speculated that Glu supplementation reduces fat percentage and increases the IMF content of Shaziling pigs via the Spirochaetota-acetic acid axis.

Our results showed that dietary Glu supplementation increased the lean percentage and the IMF content, decreased fat percentage, and upregulated the mRNA expression of MyHC IIb and MyHC IIx in the longissimus dorsi muscle of Shaziling pigs; moreover, it increased the Spirochaetota relative abundance and acetic acid content in the colon, which was associated with increased IMF content and decreased fat percentage. These findings will facilitate better understanding of the effects of Glu on the growth and development of Shaziling pigs, and contribute to the application of Glu as a safe and efficient functional additive in Shaziling pigs.