As an important germplasm resource of fish, the cryopreservation of testis and ovary is of great significance to protect endangered species and increase genetic diversity. However, current methods of slow cooling and vitrification in gonad preservation require a specialized cooling equipment or a higher concentration of cryoprotectants to maintain cell viability. The short barbeled velvetchin (Hapalogenys nitens) is an important marine economic fish, and the germplasm resources have been degraded during long-term artificial breeding. Therefore, this study isolated the gonads of mature Hapalogenys nitens and investigated the cryopreservation effect of testis and ovary with three cryoprotectant combinations under four freezing procedures. The results showed that the gonad tissues were cut to blocks of 0.5 cm³, which could effectively cryopreserve the testes or ovaries with the cryoprotectant combinations of 15% ethylene glycol, 10% dimethyl sulfoxide, 0.2 M trehalose or 15% propylene glycol, 0.2 M trehalose, and 15% fetal bovine serum, respectively. The testes with cryoprotectants were only kept 5 cm above liquid nitrogen for 10 min and then immersed in liquid nitrogen, while the ovaries soaked in cryoprotectants were directly stored in the refrigerator at -80°C. After 7 days, the gonads were thawed in a water bath at 10°C for 8 min and analyzed by morphology, and the cell viability was measured by trypan blue or cell viability assay kits, resulting in a high survival rate (>90%). The present study successfully established cryopreservation protocols of gonad tissues in Hapalogenys niten. This was a convenient, rapid, and efficient method for the gonad cryopreservation of Sparidae fishes and provided reference for the preservation of other fish germplasm resources.

The cryopreservation of ovarian tissues from fish has recently been carried out for several endangered and commercially valuable species. However, previous studies in this context have focused on the cryopreservation of immature ovaries—mainly through slow freezing and vitrification—which requires specialized freezing equipment or higher cryoprotectant concentrations to keep cell viability. Therefore, the aim of this study was to explore a convenient, rapid, efficient and less toxic method for the cryopreservation of ovaries at the stage of vitellogenesis from yellow drum (Nibea albiflora), an economically important marine fish. The ovaries at the stage of vitellogenesis were isolated and cut into blocks of approximately 1 cm³, then cryopreserved with 15% propylene glycol (PG), fetal bovine serum (FBS) and 0.2 M trehalose as cryoprotectants. Finally, the samples were treated using three different freezing procedures, including a −80 °C refrigerator, liquid nitrogen, and their combination. After 7 days, the tissues were thawed and digested, and the cell survival rates and gene expression levels were detected using cell viability assay kits and qRT-PCR, respectively. The results of the viability assay showed that the procedure of ovarian tissue storage at −80 °C in a refrigerator for 1 h, followed by transfer to liquid nitrogen, resulted in the highest cell survival rate (>90%). Furthermore, the germ cells at various phases were of normal size; presented a full, smooth surface and regular shape; and did not show any signs of cell rupture, atrophy, depression, granulation or cavitation. Furthermore, the qRT-PCR results revealed that genes related to reproductive development, such as vasa, foxl2, zp3 and gsdf, were all down-regulated under the optimal protocol, while the expression of the nanos2 gene (which is specifically distributed in oogonia) maintained a higher level, similar to that in the control group. This indicated that the viability of germ stem cells (oogonia) was not weakened after freezing and that oogonia could be isolated from the cryopreserved ovaries for germ cell transplantation. The present study successfully establishes an optimal cryopreservation protocol for ovarian tissues from Nibea albiflora, providing reference for the preservation of ovaries at the stage of vitellogenesis from other species.

Visceral white spot disease caused by Pseudomonas plecoglossicida poses a severe threat to large yellow croaker (Larimichthys crocea) aquaculture. This study investigated the immunomodulatory effects and underlying mechanisms of Pseudostellaria heterophylla extract against P. plecoglossicida infection in L. crocea. Fish were fed a basal diet supplemented with 1% P. heterophylla extract for 30, 45, and 60 days, followed by intraperitoneal injection with 200 μL P. plecoglossicida (1 × 10⁴ CFU/mL). When the control group reached about 50% mortality, transcriptome sequencing of head kidney tissues was performed on the 45 and 60 days post-feeding of the treatment groups to analyze gene expression changes following bacterial infection. Survival rates of the treatment groups were 33.33%, 13.33%, and 20% higher than those of the control group at 30, 45, and 60 days post-feeding, respectively. Transcriptomic analysis revealed time-dependent transcriptional responses: in one group, 45 days post-feeding, 10 differentially expressed genes (DEGs) were identified (2 up-regulated and 8 down-regulated), whereas in another group, 60 days post-feeding, 893 DEGs were detected (417 up-regulated and 476 down-regulated). Functional enrichment analysis (GO, KEGG, and GSEA) demonstrated that DEGs were significantly enriched in immune-related pathways, including Toll-like receptor signaling, chemokine activity, Th1 and Th2 cell differentiation, hematopoietic cell lineage, and cytokine–cytokine receptor interaction. Key immune genes, including chemokines, Toll-like receptors, and T cell regulators, were significantly up-regulated. These findings indicate that P. heterophylla extract enhances both the specific and non-specific immune capabilities of L. crocea in a time-dependent manner, with prolonged supplementation eliciting more robust transcriptional activation of immune defense pathways. This study provides a scientific foundation for developing immunological prevention strategies against P. plecoglossicida infection in aquaculture.

Ubiquitination is an essential post-translational modification of proteins and involved in many physiological processes, including sperm differentiation, oocyte maturation, and embryonic development. Among them, the ubiquitin-conjugating enzyme E2 plays a crucial role in gametogenesis. However, the participation of E2 in the regulation of the gonad development of crustaceans is still very limited, especially for oogenesis. In this research, we isolated and identified full-length complementary DNA of ubiquitin-conjugating enzyme E2 from mud crab (Scylla paramamosain) (Sp-Ubc2), detected distribution in different tissues by real-time quantitative PCR, and described expression patterns during oogenesis by in situ hybridization. The results demonstrated that the deduced proteins with a ubiquitin-conjugating (UBC) catalytic domain was clustered with other arthropods. In addition, the Sp-Ubc2 was widely distributed in different tissues of female individuals and kept the highest expression level in the ovary. Moreover, the expression level of Sp-Ubc2 changed significantly during oogenesis and reached peak in secondary and tertiary vitellogenesis stages. At the same time, the Sp-Ubc2 signal in the ovary gradually concentrated in nuclei. These results indicated that Sp-Ubc2 might play important roles in the cell cycle of oogenesis. This study would provide insights into the regulatory mechanism of gonad development in S. paramamosain, and it also enriched the theoretical basis of ubiquitin family genes involved in crustacean gonad development.