In vitro inoculation of Aeromonas hydrophila and Pseudomonas fluorescens in cryopreserved silver barb (Barbodes gonionotus) milt: Effect on fertilization capacity and transmission potential to embryos.

Purposive use of cryopreserved sperm contaminated with pathogenic agents has increased the risk of spreading of fish diseases. The objective of this study was to investigate the effects of A. hydrophila subsp. hydrophila and P. fluorescens inoculations into cryostored milt on fertilization capacity and transmission potential to embryos of silver barb (Barbodes gonionotus) with or without 0.25% penicillin-streptomycin (PS) administration. The experiment comprised six treatments: addition of milt into T1) extender only, T2) extender with 0.25% PS, T3) extender with A. hydrophila subsp. hydrophila (BG19), T4) extender with A. hydrophila subsp. hydrophila (BG19) and 0.25% PS, T5) extender with P. fluorescens (BG20) and T6) extender with P. fluorescens (BG20) and 0.25% PS. Milt were loaded into 0.25-mL straws and cryostored in the controlled-rate programmable freezer. After a cryostorage for 28 d, post-thawed sperm were evaluated for the fertilization capacity and risk of pathogen transmission to embryos. Inoculation of A. hydrophila subsp. hydrophila and P. fluorescens into extended milt (T3 and T5) caused a reduction (P < 0.05) in fertilization capacity of cryopreserved sperm. Cryopreserved sperm inoculated with the two pathogenic bacteria and 0.25% PS (T4 and T6) did not fertilize the eggs. The two pathogenic bacteria could be transmitted into embryos after artificial insemination of eggs with bacterial-inoculated cryopreserved sperm, suggesting that the risks of disease transmission via cryopreserved fish sperm would exist. This is the first study reporting pathogenic bacterial transmission on in vitro fish embryos through artificial insemination of cryopreserved sperm.