There is evidence to suggest a correlation between testis weight and semen quality capacity [1, 6]. Meanwhile, the testis is an important parameter for evaluating male reproductive capacity [33]. From our results, it can be perceived that the testis weight and semen quality of D Group roosters have been improved, which is consistent with previous research results [1]. In this study, 61 differentially expressed genes were identified by transcriptome sequencing of testis tissues from 350-day-old Houdan roosters, which underwent induced molting and recovery compared to those that did not undergo induced molting and recovery. GO functional and KEGG pathway enrichment analysis showed that these differentially expressed genes played important roles in the testis development and sperm generation process of roosters after induced molting. The differentially expressed genes were enriched in pathways related to cytokine-cytokine receptor interactions, neuroactive ligand-receptor interactions, ECM-receptor interactions, TGF-β signaling pathway, melanogenesis, mTOR signaling pathway, Wnt signaling pathway, focal adhesion, calcium signaling pathway, and MAPK signaling pathway. The differentially expressed genes detected in chicken testis tissue samples were mainly significantly enriched in steroid biosynthesis, lipid metabolism and glycerol lipid metabolism [34]. Li et al. found that cytokines and testosterone synergistically regulated the primary spermatocytes in sperm formation, sperm development, and the blood-testis barrier [35, 36]. Xiao et al. found that intercellular adhesion molecules had an antagonistic effect on the tight junction barrier of supporting cells, participated in the reconstruction of the supporting cell-blood-testis barrier, and regulated sperm adhesion in the epithelial cycle [37]. It was discovered by researchers Zhu et al. that the sperm motility and reproductive capacity of roosters were enhanced after induced molting. Through transcriptome sequencing analysis of rooster testicles, it was found that the regulation of cellular cytoskeleton is closely associated with spermatogenesis [1]. Cytokines play an important role in establishing and maintaining the immune characteristics of the testis [38, 39]. Testis cells produce various anti-inflammatory cytokines, which are important for the development of the testis and sperm generation.
The MAPK signaling pathway is one of the classical and important pathways in the eukaryotic signal transduction network. It is a key signaling pathway involved in cell proliferation, differentiation, apoptosis, and stress responses under normal and pathological conditions [40]. In this study, FGFR3 and TGFβ2 differentially expressed genes were significantly enriched in the MAPK signaling pathway in testis tissues. Several other studies [41–43] have shown that this pathway is involved in cell proliferation, differentiation, and apoptosis, making it an important signaling pathway. In mammals, MAPK can be translated as "mitogen-activated protein kinase" Regulating cell proliferation, differentiation, movement, and survival is one of the important pathways affecting testis development and spermatogenesis [44]. This suggests that the MAPK signaling pathway plays a critical role in regulating rooster testis development, spermatogenesis, and sperm quality. David M Ornitz et al. found that FGFR3 is a signal protein secreted by the FGF family, which mediates the intracellular signaling pathway with MAPK through the interaction between FGF ligands and their signal receptors [45]. FGF family members play a role in early embryonic development and organ development, and secreted FGF factors regulate basic cellular processes, including positive and negative regulation of proliferation, survival, migration, differentiation, and metabolism [45, 46]. FGFs also play an important role in adult tissues, often mediating metabolic functions, tissue repair, and regeneration by reactivating developmental signaling pathways [45, 47]. Elia Escasany et al. found that the transforming growth factor β (TGFβ) family of cytokines is a secreted multifunctional protein that is involved in cell proliferation and differentiation, wound healing, and fibrosis [47]. Study by Yang F et al. support the promoting role of TGF-β in neovascularization, which may be direct or indirect [48]. The Wnt signaling pathway plays an important role in development, regeneration, tumor development, and tissue repair [49]. The Wnt signaling pathway is usually divided into the canonical Wnt signaling pathway and the non-canonical Wnt signaling pathway, and Wnt ligands activate these two pathways by binding to different receptors on the cell surface [50]. The signals are transmitted to the cell nucleus through the Wnt and Ca2+ signaling pathways, activating downstream signaling molecules and regulating cell growth, morphology changes, and other processes [51]. WNT5A was typically involved in activating the non-canonical pathway and may inhibit the canonical pathway, thus participating in male germ cell development as a representative ligand involved in regulating male germ cell differentiation, and it was also a necessary ligand for the proliferation of testis cells [52]. In mammals, anti-Müllerian hormone (AMH) was produced by Sertoli cells that differentiate from the testis and by granulosa cells after birth [53]. Although AMH in birds is not identical to that in mammals [54, 55], it plays an important role in chicken gonadal development and maintaining testis morphology. This may also play an important role in the process of testis redevelopment and sperm production in roosters after fasting-induced molting in this study, but its specific functional role needs to be further explored.
Fibroblast growth factor receptor 3 (FGFR3), belonging to the fibroblast growth factor family, can specifically bind to the fibroblast growth factor receptor complex on the membrane of kidney and kidney tubular epithelial cells and regulate the reabsorption of calcium and phosphorus by activating the MAPK signaling pathway [37]. Ortiz-García CI et al. found that ROS produced by members of the NOX family play an important role in physiological processes such as sperm capacitation and acrosome reaction, and the ROS produced by NADPH oxidase (NOX) are associated with the activation of calcium-dependent proteinases and calmodulin-dependent proteinases [56]. Liu et al. found that ROS produced by NADPH oxidase can regulate the TGF-β signaling pathway through pathways including the MAPK pathway and the GTP-binding protein pathway [57]. Jankowska, A found that calcium-binding protein B (S100B) is present in most spermatogonia and sperm cells, and the Ca2+-regulated membrane-bound guanylate cyclase transduction mechanism plays a role in the biochemical action, molecular mechanism, and functional characteristics of spermatogenesis in the testis, mainly through S100B expression, transducing Ca2+-driven membrane-bound guanylate cyclase expression in germ cells [58]. This may be related to the significant improvement in sperm motility observed in this study. In summary, differential genes such as WNT5A, FGFR3, AMH, TGFβ2, S100B and NOX4 may play important roles in the process of testis redevelopment in aging laying hens after fasting-induced molting.