The milk flavor can be attributed to the presence of numerous flavor molecules and precursors. In this study, we employed widely targeted metabolomic analysis techniques to analyze the metabolic profiles of various milk samples obtained from goats, sheep, dairy cows, and buffaloes. A total of 631 metabolites were identified in the milk samples, which were further categorized into 16 distinct classes. Principal component analysis (PCA) suggested that the metabolite profiles of samples from the same species exhibit clustering, while separated patterns of metabolite profiles are observed across goat, sheep, cow, and buffalo species. The differential metabolites between the groups of each species were screened based on fold change and variable importance in projection (VIP) values. Five core differential metabolites were subsequently identified, including 3-(3-hydroxyphenyl)-3-hydroxypropanoic acid, inosine 5′-triphosphate, methylcysteine, N-cinnamylglycine, and small peptide (L-tyrosine–L-aspartate). Through multiple comparisons, we also screened biomarkers of each type of milk. Our metabolomic data showed significant inter-species differences in the composition and concentration of some compounds, such as organic acids, amino acids, sugars, nucleotides, and their derivatives, which may affect the overall flavor properties of the milk sample. These findings provided insights into the molecular basis underlying inter-species variations in milk flavor.

MicroRNA-26 (miR-26a and miR-26b) plays a critical role in lipid metabolism, but its endogenous regulatory mechanism in fatty acid metabolism is not clear in goat mammary epithelial cells (GMECs). GMECs with the simultaneous knockout of miR-26a and miR-26b were obtained using the CRISPR/Cas9 system with four sgRNAs. In knockout GMECs, the contents of triglyceride, cholesterol, lipid droplets, and unsaturated fatty acid (UFA) were significantly reduced, and the expression of genes related to fatty acid metabolism was decreased, but the expression level of miR-26 target insulin-induced gene 1 (INSIG1) was significantly increased. Interestingly, the content of UFA in miR-26a and miR-26b simultaneous knockout GMECs was significantly lower than that in wild-type GMECs and miR-26a- and miR-26b-alone knockout cells. After decreasing INSIG1 expression in knockout cells, the contents of triglycerides, cholesterol, lipid droplets, and UFAs were restored, respectively. Our studies demonstrate that the knockout of miR-26a/b suppressed fatty acid desaturation by upregulating the target INSIG1. This provides reference methods and data for studying the functions of miRNA families and using miRNAs to regulate mammary fatty acid synthesis.

Follicle development in dairy goats is lower after induced estrus during the non-breeding season, reducing conception rates and challenging year-round milk supply. This study investigated follicle development during the breeding and non-breeding seasons and explored molecular mechanisms for variations in the proportions of follicles of different sizes using ovarian RNA-seq and in vitro experiments. Induced estrus during the non-breeding season used a simulated breeding season short photoperiod and male effect methods, while the male effect method was used during the breeding season. This study identified an increase in follicle size during the breeding season and performed RNA-seq on ovaries to explore the underlying causes. The RNA-seq analysis elucidated pathways associated with cellular and hormonal metabolism and identified adenylyl cyclase 5 (ADCY5) as a key differentially expressed gene. In vitro experiments demonstrated that interfering with ADCY5 in ovarian granulosa cells (GCs) reduced steroid synthesis. Conversely, the overexpression of ADCY5 increased steroid synthesis. ADCY5 affects the biological function of GCs and consequently influences follicle development through the cAMP-response element binding protein (CREB) and p38 mitogen-activated protein kinase phosphorylation (MAPK) pathways. Overall, our findings demonstrate that follicle development in dairy goats differs between the breeding and non-breeding seasons and that the differential expression levels of the ADCY5 gene contribute to this discrepancy.