ABSTRACT: Background: Intramuscular fat (IMF), the white adipose tissue deposited between skeletal muscle fibers, is a key determinant of beef quality due to its contribution to meat, flavor, juiciness and tenderness. However, IMF develops later and grows more slowly, compared to other fat depots such as subcutaneous fat (SF) in cattle. The cellular and molecular mechanisms underlying the delayed development and slower growth of IMF compared to other fat depots remain poorly understood. Results: We performed single-cell RNA sequencing (scRNA-seq) on the stromal vascular fractions (SVFs) from IMF and SF of adult Angus crossbred steers as well as the mononuclear cell fractions (MCFs) from skeletal muscles of newborn Angus crossbred bull calves, with each tissue type collected from two animals. A total of 14,802 cells from 6 animals were sequenced. A clustering analysis revealed that these cells comprised ten cell types, including adipose progenitor cells (APCs), muscle satellite cells (MuSCs), myoblasts, smooth muscle cells, and various immune cell populations. The SF SVF from adult cattle harbored a significantly higher proportion of APCs than the IMF SVF. The MCFs from newborn calves did not contain detectable APCs. A subclustering analysis revealed that the APCs comprised six subpopulations (C0–C5), among which C3 and C5 were absent in the IMF SVF while C1 was markedly less abundant in the IMF SVF than the SF SVF. A gene set variation analysis and a pseudotime trajectory analysis showed that C1 and C3 represented more differentiated APCs, with higher expression of genes involved in adipogenesis, such as PPARG, ADAM12, and PPARGC1A, whereas subclusters C0 and C4 represented undifferentiated, uncommitted APCs, with higher expression of genes involved in DNA replication and cell adhesion, compared to the other subclusters. Conclusions:Overall, this single-cell transcriptomics study suggests two potential differences in APCs between IMF and SF in adult cattle: (1) IMF contains fewer APCs than SF; (2) APCs in IMF are adipogenically less committed and differentiated compared to APCs in SF. These differences may explain why IMF develops later and grows more slowly than SF in cattle. This study also suggests that, in cattle, intramuscular fat begins to develop postnatally, challenging the widely held belief that it forms during late gestation.
