Xenogenesis has been identified as a potential technique for hybrid catfish (channel catfish, Ictalurus punctatus ♀ × blue catfish, I. furcatus ♂) embryo production. This process can be accomplished by transplanting germline stem cells from a diploid donor fish into a sterile recipient. Currently, freshly extracted stem cells are used, which presents challenges as germline stem cell production depends on the donor’s size, age, and seasonal cycles. Thus, culturing stem cells would solve this issue by providing a year-round supply which can significantly enhance the efficiency of the xenogenesis process. The present study aimed to compare the effectiveness of using fresh versus short-term cultured stem cells for germ cell transplantation. During the present study, triploid channel catfish fry were injected at 5 days post-hatch (DPH) with either freshly isolated or cultured blue catfish stem cells labeled with PKH26 dye. Stem cells cultured as both attached and unattached to the culture container were evaluated separately. Growth performance and survival of recipient fish were assessed at 45 and 90 DPH. Additionally, colonization of donor cells in the recipients was quantified using PKH26 dye fluorescence to calculate the percentage of cell and cluster areas. PCR and fluorescence image data were used to determine the percentage of xenogens.
At 45 and 90 DPH, no significant differences in body weight or total length of fry were observed between treatments (P > 0.05). However, fluorescence image analysis revealed that recipient fish injected with cultured-attached stem cells exhibited the highest percentage of cluster area (P < 0.05) compared to those injected with freshly extracted or cultured-unattached stem cells at both sampling periods at 45 and 90 DPH. Additionally, a significant increase in percentage of cluster area was observed from 45 to 90 DPH in both cultured-attached and freshly transplanted treatments. The percent cell area at 45 DPH was significantly higher in cultured-attached treatment (P < 0.05) than other treatments, while no significant difference was noted at 90 DPH between freshly transplanted and cultured-attached treatments (P > 0.05). PCR analyses showed that a higher proportion of xenogens (>80%) were found in recipient fish injected with cultured-attached or freshly extracted stem cells, compared to those injected with cultured-unattached cells (< 60%). Overall, the findings from the current study demonstrate that cultured-attached stem cells exhibit comparable performance to freshly extracted stem cells across all measured variables. Therefore, cultured-attached stem cells present a promising option for future germ cell transplantation to support xenogenesis, as they can be readily available under controlled culture conditions, enhancing the efficiency of germ cell transplantation for commercial-scale hybrid catfish production.