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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., (Copyright © 2017. Published by Elsevier Inc.)

This objective of the study was to evaluate the effects of A. hydrophila subsp. hydrophila and P. fluorescens on sperm motility, sperm viability and sperm morphometry of cryopreserved silver barb (Barbodes gonionotus) semen and survival of tested bacteria after cryostorage. Semen was diluted in a calcium-free Hank's balanced salt solution (Ca-F HBSS) supplemented with or without 0.25% penicillin-streptomycin (PS) after which A. hydrophila subsp. hydrophila or P. fluorescens was immediately added into extended semen prior to freezing. Extended semen and cryostored semen kept for 20 min, 24 h, 7 d, 14 d and 28 d were assessed for sperm motility, sperm viability, sperm morphometry, survival of challenged bacteria and the relationship between bacteria and sperm. Bacterial-exposed semen with or without 0.25% PS supplementation showed a significant reduction (P < 0.05) in sperm motility and viability during a cryostorage of 28 d, compared to semen without bacterial supplementation (control groups). Addition of A. hydrophila subsp. hydrophila and P. fluorescens resulted in a significant (P < 0.05) alteration of sperm morphometry of cryopreserved semen, especially flagellum width. The two pathogens were detected at a level of 10(5) CFU ml(-1) in cryostored semen with or without antibiotic supplementation. There were significant correlations among bacterial number, percentage of sperm motility and viability and flagellum width. In conclusion, the presence of A. hydrophila subsp. hydrophila and P. fluorescens had a deleterious effect on cryopreserved silver barb sperm based on a reduction in sperm motility and viability and alteration of sperm morphometry, especially flagellum width., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Disease prevention is a key aspect in developing cryopreservation procedures for fish sperm and in improving the reproductive biotechnology for commercially important aquatic species. The purpose of this study was to evaluate the effects of an antibiotic supplementation (0.25% penicillin-streptomycin, PS and 0.25% penicillin-gentamicin, PG) on sperm motility and viability, bacterial profile and fertilization capacity of cryopreserved silver barb (Barbodes gonionotus) semen. The experimental protocol involved three treatments: addition of PS alone; addition of PG alone; and no addition of antibiotics (Control). Semen samples were frozen and cryostored for 12-mo. Administration of 0.25% PS significantly (P<0.05) improved sperm motility and viability and reduced (P<0.05) total heterotrophic bacteria, Gram negative bacteria and pseudomonads bacteria in cryopreserved semen. Post-thawed semen treated with 0.25% PS did not contain contaminating bacteria including Bacillus subtilis subsp. inaquosorum, Bacillus safensis, Aeromonas punctata subsp. caviae, Serratia plymuthica, Pseudomonas azotoformans and Pseudomonas sp. Post-thawed semen supplemented with 0.25% PG showed degenerative changes in motility and viability of sperm. Eggs fertilized with 0.25% PS or antibiotic-free cryopreserved semen had similar fertilization rates, lower (P<0.05) than those of fresh semen. Incorporation of 0.25% PS was suitable for cryopreservation of silver barb semen based on the presence of good quality of post-thawed sperm and elimination of bacterial contaminants: B. subtilis subsp. inaquosorum, B. safensis, A. punctata subsp. caviae, Ser. plymuthica, P. azotoformans and Pseudomonas sp. This is the first study to investigate the antibiotic effect on the number of bacteria and their profile in cryopreserved semen of fish., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Biosafety issue associated with the risk of pathogenic contamination of cryopreserved semen is a common concern because of associated declines in sperm quality, storage period and disease transmission. This study was conducted to evaluate the effects of methods of semen collection on sperm quality and bacterial composition of post-thawed semen of silver barb (Barbodes gonionotus). Semen collection methods consisted of four treatments: (1) hand-stripping of abdomen without rinsing of urogenital area with water, (2) hand-stripping of abdomen after rinsing of urogenital area with water, (3) catheterization without rinsing of urogenital area with water and (4) catheterization after rinsing of urogenital area with water. Semen diluted with calcium-free Hank's balanced salt solution containing 10% dimethylsulfoxide (DMSO) was frozen at a freezing rate of -8°Cmin(-1) before plunging in liquid nitrogen. Post-thawed semen collected by catheterization after rinsing urogenital area had the lowest bacterial number, about 2-log reduction of total heterotrophic, Gram negative and pseudomonad bacteria, compared with the other three collection treatments. However, percentages of motile and viable sperm were not significantly (P>0.05) different among treatments. This method eliminated Flavobacterium aquatile, Bacillus megaterium, Kocuria varians, Staphylococcus haemolyticus and Aeromonas media in cryopreserved semen. This is the first report demonstrating the effects of semen collection methods on bacteriological quality of frozen-thawed fish semen., (Copyright © 2016 Elsevier B.V. All rights reserved.)