This work aimed to identify the key members of the bacterial community growing on common carp (Cyprinus carpio) fillets during chilled storage with next-generation sequencing (NGS) and cultivation-dependent methods. Carp fillets were stored for 96 h at 2 °C and 6 °C with and without a vacuum package, and an additional frozen-thawed storage experiment was set for 120 days. Community profiles of the initial and stored fish samples were determined by amplicon sequencing. Conventional microbial methods were used parallelly for the enumeration and cultivation of the dominant members of the microbial community. Cultivated bacteria were identified with 16S rRNA sequencing and the MALDI-TOF MS method. Based on our results, the vacuum package greatly affected the diversity and composition of the forming microbial community, while temperature influenced the cell counts and consequently the microbiological criteria for shelf-life of the examined raw fish product. Next-generation sequencing revealed novel members of the chilled flesh microbiota such as Vagococcus vulneris or Rouxiella chamberiensis in the vacuum-packed samples. With traditional cultivation, 161 bacterial strains were isolated and identified at the species level, but the identified bacteria overlapped with only 45% of the dominant operational taxonomic units (OTUs) revealed by NGS. Next-generation sequencing is a promising and highly reliable tool recommended to reach a higher resolution of the forming microbial community of stored fish products. Knowledge of the initial microbial community of the flesh enables further optimization and development of processing and storage technology.
Common carp (Cyprinus carpio) is one of the most cultured fish species over the world with many different breeds and plenty of published protocols for sperm cryopreservation. However, data regarding preservation of gonadal tissue and surrogate production is still missing. A protocol for freezing common carp spermatogonia was developed through varying different factors along a set of serial subsequent experiments. Among the six cryoprotectants tested, the best survival was achieved with dimethyl sulfoxide (Me2SO). In the next experiment, a wide range of cooling rates (0.5-10°C/min) and different concentrations of Me2SO were tested resulting in the highest survival achieved using 2 M Me2SO and cooling rate of -1°C/min. When testing different tissue sizes and incubation times in the cryomedia, the highest viability was observed when incubating 100 mg tissue fragments for 30 min. Finally, sugar supplementation did not yield significant differences. When testing different equilibration (ES) and vitrification solutions (VS) used for needle-immersed vitrification, no significant differences were observed between the tested groups. Additionally, varied exposure time to VS did not improve the vitrification outcome where the viability was 4-fold lower than that of freezing. The functionality of cryopreserved cells was tested by interspecific transplantation into sterilized goldfish recipients. The exogenous origin of the germ cells in gonads of goldfish recipient was confirmed by molecular markers and incorporation rate was over 40% at 3 months post-transplantation. Results of this study can serve for long-term preservation of germplasm in carp which can be recovered in a surrogate recipient.
