To address the growing consumer demands for improved fish meat quality, desirable morphological traits, and sustainable production practices, researchers have intensified efforts in the selective breeding and genetic improvement of carp (Cyprinus carpio) varieties. However, traditional breeding methods are often time-consuming and inefficient, which poses challenges to the sustainable development of the carp aquaculture industry. The establishment of germ stem cell lines offers a crucial tool for the study of germ cells, genetic improvement, and species conservation. In this study, we successfully established a spermatogonial stem cell line (YRSSCs) from Yellow River carp (Cyprinus carpio haematopterus) that can be cultured in vitro for the long term. We optimized the culture conditions to maintain their self-renewal and differentiation capabilities. The results demonstrated that YRSSCs have a diploid karyotype and can stably proliferate for over a year in L-15 medium supplemented with 5 mmol/L HEPES, 50 μmol/L β-mercaptoethanol, 15% FBS, 2 ng/mL bFGF, 2 ng/mL LIF, 1% carp serum, 800 IU/mL penicillin, 0.8 mg/mL streptomycin, 2 μg/mL amphotericin B, 1% zebrafish embryo extract, and 1% glutamine at 30 °C in the absence of CO₂. The cells exhibited a typical germ stem cell gene expression profile, with strong expression of the vasa, plzf-a, and Oct4-a genes. Additionally, this study found that YRSSCs possess the ability to differentiate in vitro and functionally colonize in vivo within recipient bodies. This research explored the establishment of YRSSCs and their differentiation potential both in vitro and in vivo, providing a novel strategy for the genetic improvement of aquaculture fish species through germ stem cell-based gene editing and transplantation technologies.

The intermuscular-bone-free crucian carp (Carassius auratus, WUCI), developed through CRISPR/Cas9-mediated bmp6a and bmp6b knockout, offers advantages in consumer acceptance and processing efficiency. However, its effects on muscle texture and nutritional quality have not been fully elucidated. In this study, we compared the F3 generation of WUCI with its sibling wild-type crucian carp (Carassius auratus, WT), Songpu silver crucian carp (Carassius gibelio var. Songpu, SPYJ), and Fangzheng silver crucian carp (Carassius gibelio var. Fangzheng, FZYJ), focusing on muscle texture characteristics and nutritional attributes. WUCI exhibited significantly higher shear force but lower hardness than most comparison groups, with no substantial differences in muscle fiber morphology. Amino acid profiles were similar among all groups. WUCI showed lower crude fat content than WT but higher than FZYJ, and comparable levels of polyunsaturated fatty acids (PUFA), n-3 fatty acids, docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA) to WT and SPYJ. These findings indicate that the knockout of bmp6a and bmp6b does not alter muscle mass in crucian carp, supporting the potential of WUCI for commercial adoption as a gene-edited consumer-oriented fish product.

This study investigated the accumulation characteristics and associated health risks of 11 trace elements (Al, Rb, Cr, Ni, Mo, Sr, Pb, Ba, Ag, As, and Ga) in four crucian carp varieties: gene-edited intermuscular bone-free crucian carp (Carassius auratus, WUCI) and its sibling wild-type (Carassius auratus, WT), Fangzheng silver crucian carp (Carassius gibelio var Fangzheng, FZYJ), and Songpu silver crucian carp (Carassius gibelio var Songpu, SPYJ). Results showed that Al and Rb were the most abundant elements across all groups. WUCI exhibited distinct accumulation patterns, including significantly higher hepatic Mo concentrations (0.265 ± 0.032 mg/kg) and muscle/liver Rb levels (muscle: 8.74 ± 1.21 mg/kg; liver: 12.56 ± 2.05 mg/kg) compared to other varieties (p < 0.05), which supports the hypothesis of genotype-specific differences in heavy metal accumulation. Correlation analysis revealed that WUCI exhibited similar elemental interactions with WT and SPYJ (e.g., Al-Ni positive correlation, |rs| ≥ 0.8), while SPYJ displayed distinct patterns with fifteen negative correlations compared to three to five in others varieties, suggesting a potential alteration in elemental homeostasis. Pollution index (P_i) assessments indicated mild contamination for Pb in SPYJ liver (P_i = 0.265) and Cr/As in WUCI muscle (P_i = 0.247/0.218). Despite these values, all hazard indices remained below the established safety thresholds (THQ < 0.1, HI < 0.25, TCR < 10⁻⁶), reinforcing the overall safety of the tested fish. Notably, muscle As levels (0.86 ± 0.15 mg/kg) exceeded hepatic concentrations (0.52 ± 0.09 mg/kg), potentially due to differential detoxification mechanisms. These findings demonstrate the food safety of all tested varieties, while highlighting genotype-specific metabolic adaptations, providing critical data for evaluating gene edited aquatic products.

Elimination of intermuscular bones (IMBs) is vital to the aquaculture industry of cyprinids. In our previous study, we characterized bmp6 as essential in the development of IMBs in zebrafish. Knockout of bmp6 results in the absence of IMBs in zebrafish without affecting growth and reproduction. Therefore, we hypothesized that bmp6 could be used to generate new cyprinid strains without IMBs by gene editing. In this study, we established a gene editing strategy for knocking out the two orthologs of bmp6 in diploid crucian carp ( Carassius auratus ). We obtained an F ₃ population with both orthologs knocked out, in which no IMBs were detected by bone staining and X-ray, indicating that the new strain without IMBs (named WUCI strain) was successfully generated. Furthermore, we extensively evaluated the performance of the new strain in growth, reproduction, nutrient components, muscle texture and structure, and metabolites in muscle. The results showed that the WUCI strain grew faster than the wild-type crucian carp at 4-month-age. The reproductive performance and flesh quality did not show significant differences between the WUCI strain and wild-type crucian carp. Moreover, the metabolomics analysis suggested that the muscle tissues of the WUCI strain significantly enriched some metabolites belonging to the Thiamine metabolism, Nicotinate and Nicotinamide Metabolism pathway, which plays beneficial effects in anti-aging, anti-oxidant, and anti-radiation damage. In conclusion, we established a strategy to eliminate IMBs in crucian carp and obtain a WUCI strain whose performance was expected compared to the wild-type crucian carp; meanwhile, the WUCI strain enriched some beneficial metabolites to human health in muscle tissue. This study is the first report that a farmed cyprinid strain without IMBs, which could be stably inherited, was obtained worldwide; it provided excellent germplasm for the cyprinids aquaculture industry and a useful molecular tool for eliminating IMBs in other cyprinids.