Integrated analyses of transcriptomes, metabolomes, and proteomes unveil the role of FoXO signaling axis in buck semen cryopreservation

Sperm cryopreservation is a complex process involving gene expression, protein synthesis, membrane stability, and metabolic adaptation. However, molecular alterations in sperm cryopreservation and the mechanisms defending against freezing damage remain poorly understood. This study investigates these changes and defense mechanisms using transcriptomics, proteomics, and metabolomics data. During sperm cryopreservation, the expression level of G protein subunit alpha i3 (GNAI3) was significantly downregulated in post-thaw sperm (P < 0.001), while matrix metallopeptidase 9 (MMP9) was upregulated compared to FS groups (P < 0.01). Additionally, interleukin 6 (IL6) expression in the CS group showed an approximate increase (P < 0.05), whereas ribosomal protein S27a (RPS27A) expression decreased markedly (P < 0.05). Other important molecules such as macrophage stimulating 1 receptor (MST1R), hypoxia-inducible factor 1 subunit alpha (HIF1A), fibroblast growth factor 8 (FGF8), CD9 molecule (CD9), peptidase D (PEPD) and terminal nucleotidyltransferase 5B (TENT5B) also exhibited significant changes in expression (P < 0.05). Moreover, the study revealed the regulatory roles of metabolites such as glucose and glutamic acid during sperm cryopreservation. The involvement of catalase (CAT) protein in antioxidant defense was also noted. The interactions among mRNAs, miRNAs, proteins, and metabolites highlight the critical role of the FoxO signaling pathway in modulating responses to freezing. Our study reveals the molecular regulatory mechanisms of sperm during cryopreservation, emphasizing the importance of the FoxO pathway and specific metabolites in response to cryo-injury. These findings provide deeper insights into the complexity of sperm cryobiology and offer practical guidance for optimizing sperm cryopreservation.