Abstract

   Identifying potential gut microbes and metabolites that can influence
   muscle fiber type is gaining interest in meat quality research. In this
   study, muscle fiber characteristics, muscle metabolite profiles, and
   gut microbiota and metabolome were compared among three pig breeds
   (Taoyuan black, TB; Xiangcun black, XB; and Duroc pigs). The results
   showed that the slow-twitch fiber percentage was higher (P < 0.05) in
   native pigs (TB and XB pigs) compared to Duroc pigs. The differences
   were mainly regulated by Lactobacillus abundance and tryptophan
   metabolism. Further, fecal microbiota transplantation from XB pigs
   transferred a higher slow-twitch fiber percentage, Lactobacillus
   abundance, kynurenic acid level, and AMPK/PGC-1α expression to mice.
   These findings suggest that Lactobacillus in the colon of TB and XB
   pigs, through kynurenic acid production, may promote slow-twitch fiber
   formation via the AMPK/PGC-1α signaling pathway.

   Subject terms: Microbiome, Applied microbiology

Introduction

   Skeletal muscle is characterized by high heterogeneity and plasticity,
   attributed to its composition of different types of muscle fibers. The
   muscle fibers can be classified into four types based on the isoforms
   of myosin heavy-chain (MyHC), including type I (slow-oxidative), type
   IIa (fast-oxidative), type IIx (intermediate), and type IIb
   (fast-glycolytic)^[36]1. These muscle fibers can be transformed under
   specific physiological conditions in the order of type I→type IIa→type
   IIx→type IIb, or vice versa^[37]2. In livestock meat quality research,
   it is widely accepted that animals with a higher proportion of
   oxidative muscle fibers (type I and IIa) have better meat
   quality^[38]3. Previous studies have found that native pig breeds have
   a higher proportion or expression of oxidative muscle fibers compared
   to commercial pigs. For instance, compared with Landrace pigs, the mRNA
   expressions of MyHC I and MyHC IIa were higher in Jinhua pigs^[39]4;
   the expression of MyHC I was also higher in Bama Xiang pigs^[40]5.
   Furthermore, the Korean native black pigs have shown a higher
   expression of MyHC I than the Landrace pigs^[41]6. Therefore, exploring
   the mechanism of slow-twitch fiber formation through the
   characteristics of native pigs could provide valuable insights for
   strategies aimed at improving meat quality.

   The gastrointestinal tract of large animals harbors a vast and complex
   microbial community that plays a crucial role in the digestion and
   absorption of nutrients, and regulates gut health^[42]7. Additionally,
   the gut microbiota influences peripheral tissue metabolism and
   metabolic diseases through the production of different bioactive
   compounds, such as branched-chain amino acids, short-chain fatty acids,
   and bile acids^[43]8. The gut microbiota and gut microbiota-derived
   metabolites also significantly influence muscle physiology and
   function. Studies have shown that germ-free mice exhibit muscle atrophy
   and downregulation of genes associated with muscle growth and
   mitochondrial function, compared to specific pathogen-free mice^[44]9.
   Probiotic Lactobacillus casei Shirota administration to mice has been
   shown to mitigate age-induced muscle mass loss, strength reduction, and
   mitochondrial dysfunction^[45]10. In pigs, germ-free pigs have been
   found to exhibit reduced muscle mass, while fecal microbiota
   transplantation (FMT) from normal pigs can partially restore muscle
   growth and development^[46]11. These findings suggest that gut microbes
   and their metabolites are important for the maintenance of skeletal
   muscle growth and function.

   Native pigs exhibit significant differences from commercial pigs not
   only in muscle fiber composition but also in their gut microbiota. For
   instance, Jinwu pigs have a higher abundance of fiber-digesting genera
   in the colon, such as Treponema and Terrisporobacter, compared to Duroc
   × Landrace × Yorkshire (DLY) pigs under roughage feeding^[47]12.
   Similarly, Shaziling pigs showed a higher abundance of beneficial
   bacteria, such as Lactobacillus johnsonii and Lactobacillus amylovorus,
   in the ileum than Yorkshire pigs^[48]13. Moreover, the abundances of
   Firmicutes, Bacteroidetes, Tenericutes, Spirochaetes, Fusobacteria, and
   Kiritimatiellaeota in the ileum of Ningxiang pigs are lower than those
   in DLY pigs^[49]14. Xiangcun black (XB) pig is a crossbreed derived
   from Taoyuan black pigs (TB, the maternal line) and Duroc pigs (the
   paternal line). Our previous study indicated that TB and XB pigs have
   higher slow-twitch fiber percentages and better meat quality than the
   Duroc pigs during the finishing stage^[50]15. Additionally, the
   microbiota composition in the ileum of TB and XB pigs differs from that
   in Duroc pigs during suckling and weaning periods^[51]16. However, the
   mechanistic links between gut microbiota and muscle fiber type
   development, which are critical for meat quality traits, remain
   unexplored. Therefore, studying these three breeds (TB, XB, and Duroc
   pigs) during the growing stage allows us to uncover the potential
   mechanisms by which gut microbiota in Chinese native pig breeds shape
   superior meat quality during development.

   Given the presence of the gut-muscle axis, we hypothesized that the gut
   microbiota of TB and XB pigs may regulate the formation of slow-twitch
   fibers. Thus, the present study selected growing TB, XB, and Duroc pigs
   to identify core gut microbes and metabolites that might regulate the
   formation of muscle slow-twitch fibers and further validate their
   effects through FMT. The findings will provide references for the