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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.)

The objective of this study was to determine the effects of various extenders, cryoprotectants and cryopreservation methods on post-thaw sperm motility and duration of sperm motility in common carp (Cyprinus carpio). We focused on freezing of common carp sperm utilizing a practical and inexpensive protocol for aquaculture. Sperm were diluted 1:1 in one of six extenders (common carp sperm extenders; CCSE 1-CCSE 6) containing three types of cryoprotectants (dimethyl sulfoxide; DMSO, methanol; MET and propylene glycol; PG) at a final concentration of 10%, and frozen at a rate of 10°C/min from an initial temperature 25 to -40°C before storage in liquid nitrogen. The results demonstrated that sperm diluted with CCSE 2 and DMSO had the best post-thaw motility (94.5 ± 3.3%), similar to that of the control (98.6 ± 0.7%; P>0.05). Duration of sperm motility from a treatment with CCSE 2 and DMSO (97 ± 20.8s) was not significantly different (P>0.05) from that of the control (73.3 ± 12.9s). A second experiment studied the effects of various cryopreservation methods on post-thaw sperm motility and duration of sperm motility, based on using CCSE 2 and DMSO in all treatments. Sperm were frozen using different cryopreservation methods: direct immersion into liquid nitrogen, controlled-rate programmable freezer, or exposure to liquid nitrogen vapor at different heights and time. Sperm frozen at a height of 2 cm above liquid nirogen surface for 10 min gave the highest post-thaw sperm motility (91.7 ± 7.8%) and longest duration of post-thaw sperm motility (105.7 ± 23.1s). Sperm frozen 2 cm above liquid nitrogen surface for 10 min produced the highest fertilization and hatching rate of about 73.6 ± 6.5% and 62.8 ± 5.9%, respectively, not significant different (P>0.05) from those of fresh sperm (75.6 ± 7.5% and 66.5 ± 4.8%, respectively). This study reports superior performance of the combination of CCSE 2 and DMSO for freezing common carp sperm that resulted in high fertilization capacity., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Effectiveness and efficiency of frozen sperm on fertilization and hatching success of eggs from silver barb was examined in relation to cryoprotectants, freezing rate and storage period. Sperm was diluted in calcium-free Hank's balanced salt solution, equilibrated with dimethylsulphoxide ( DMSO), propylene glycol, sucrose or methanol at 5%, 10%, 15% or 20% final concentrations, and frozen in 250-μL straws using a one-step freezing procedure (1, 5 and 8°C min−1 from 25 to −40°C). Highest post-thaw sperm motility was found from a treatment using 10% DMSO and 5°C min−1 (82.2 ± 2.1%), similar to that of 10% DMSO and 8°C min−1 (87.8 ± 3.2%). Post-thaw motility of sperm frozen at 5 or 8°C min−1 was significantly higher than 1°C min−1. Relative sperm motility declined significantly after 10 months of cryostorage while viability did not change during a 12-month cryostorage. Average fertilization rates of sperm after 1 and 4 months of storage were 64.5 ± 4.6% and 61.3 ± 3.4%, respectively, similar to those of fresh sperm (69.6-72.3%). Hatching rates of cryopreserved sperm (45.4-51.2%) were similar to those of fresh sperm (51.8-57.8%). This study developed suitable methods for cryopreservation of silver barb sperm that can be used to facilitate hatchery operation. [ABSTRACT FROM AUTHOR]

This study was conducted to investigate the efficacy of dry shipper for the cryostorage of silver barb (Barbodes gonionotus) sperm, the subsequent risk of bacterial cross-contamination, and the effects of Aeromonas hydrophila on post-thaw sperm. Semen was diluted with calcium-free Hank's balanced salt solution containing 10% ME 2 SO, frozen at −8 °C/min and stored for 14 d in a dry shipper. A significant decline (P < 0.05) in the post-thaw sperm motility and viability of samples kept in the dry shipper for 14 d showed a reverse correlation (P < 0.05) with a slight increase in temperature within the dry shipper. The levels of contaminated bacteria in the compartments of the dry shipper were significantly (P < 0.05) lower than those detected in the liquid nitrogen tank. Bacteria from the atmosphere could recontaminate the chambers of the dry shipper and liquid nitrogen tank after 14 d. Bacillus was the most common bacteria isolated from the dry shipper, liquid nitrogen tank, circulating air, bench surface and outer surface of straws. There was no cross-contamination of A. hydrophila from contaminated straws to pathogen-free straws kept in either cryogenic tank. Post-thaw sperm motility and sperm viability significantly (P < 0.05) declined during cryostorage in the dry shipper and liquid nitrogen tank due to the introduction of A. hydrophila and the interaction effect of A. hydrophila and freezing. This study reports, for the first time, the efficacy of a dry shipper for the cryostorage of fish sperm for at least 14 d without a risk of bacterial cross-contamination. • Vapor phase of dry shipper can store silver barb sperm with high post-thawed quality. • Dry shipper had a low risk of bacterial contamination and cross-contamination. • Aeromonas hydrophila contamination is harmful to post-thawed sperm of silver barb. [ABSTRACT FROM AUTHOR]

Application of good sanitation practice in cryopreservation process is the key issue to improve the quality of cryopreserved fish sperm. This study implemented standard sanitation protocol with minimal contamination and evaluated the source of bacterial contamination associated with laboratory equipment and related materials across a series of cryopreservation process of silver barb ( Barbodes gonionotus) semen. The use of 16s rRNA sequencing and traditional biochemical methods were performed for bacterial identification. Animal origin (anal fin, culture water and semen contaminated with faeces and urine) and non-animal origin (liquid nitrogen from liquid nitrogen dewar, outer surface of straw, air circulation in cryopreservation laboratory and latex gloves used during cryopreservation procedure) were determined for bacterial contamination. Aeromonas punctata subsp. caviae was the most abundant species in anal fin, latex groves and semen contaminated with faeces and urine. Bacillus safensis and Bacillus sp. were found as frequently recovered species from liquid nitrogen dewar. Aeromonas hydrophila subsp . hydrophila and Pseudomonas fluorescens, fish pathogenic bacteria, were isolated from all animal origin samples. This was the first report indicating that standard sanitation and hygiene methodologies are recommended for sperm cryopreservation of B. gonionotus to prevent bacterial contamination. [ABSTRACT FROM AUTHOR]

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 °C min −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. [ABSTRACT FROM AUTHOR]

Disease prevention is a key issue in improving reproductive biotechnology for important and endangered aquatic species to minimize the risk of pathogen transmission and economic loss. This study investigated the effects of small-dose antibiotics on sperm viability and bacterial abundance in banana shrimp (Fenneropenaeus merguiensis) spermatophores during chilled storage and cryostorage. The experiment consisted of three treatments, no antibiotic (control), 0.1% penicillin-streptomycin (PS), and 0.1% penicillin-gentamycin (PG). Antibioics were added into a sperm extender containing banana shrimp spermatophores. In the chilled storage and cryopreservation trials, the most significant (P < 0.05) improvement in sperm survival was observed in 0.1% PS-treated spermatophores over a 30-day period at 2–4 °C and 6-month in a liquid nitrogen tank. Average sperm viability ranged from 46.3 ± 3.5 to 95.7 ± 0.6% and 78.8 ± 1.1 to 95.7 ± 1.0%, respectively. In the 0.1% PS-treated spermatophores, the numbers of culturable heterotrophic bacteria (CHB) fluctuated during 21-day chilled storage and decreased (P < 0.05) over 30-day storage compared to those in the control. The CHB count in cryopreserved spermatophores decreased (P < 0.05) with increasing storage time and was undetectable by day 7 of cryostorage. Pseudomonad bacteria were not observed in both chilled-stored spermatophores by 21 days post-storage and prefrozen spermatophores when 0.1% PS was incorporated into the sperm extender. The 0.1% PS treatment also inhibited the growth of culturable Vibrio in banana shrimp spermatophores during chilled storage and cryostorage. Several species of human and shrimp pathogens and spoilage bacteria, particularly Vibrio parahaemolyticus , V. metschnikovii , V. harveyi , Aeromonas media , A. sorbria , and Plesiomonas shigelloides were not found after 0.1% PS was added to the sperm extender. This study suggests that addition of 0.1% PS into the extender not only reduces the bacterial count of chilled and postthawed cryopreserved sperm of banana shrimp, but also had no adverse effect on sperm viability. • Penicillin-streptomycin (PS) was suitable for preservation of shrimp spermatophore. • 0.1% PS was effective on chilled storage and freezing of banana shrimp spermatophores. • 0.1% PS retained banana shrimp sperm viability during chilled storage and cryostorage. • PS supplement inhibited the growth of shrimp pathogens in preserved spermatophore. [ABSTRACT FROM AUTHOR]

Abstract: Microbial contamination in sperm banks is of concern to sperm bank managers to prevent the perceived risk of disease transmission during cryostorage. The objective of this study was to examine the bacterial profile of cryopreserved spermatophores during long-term storage in liquid nitrogen in order to assess the risk of microbial flora during cryostorage. Spermatophores (N =216) collected from males (N =108) were equilibrated in calcium-free saline containing 5% dimethyl sulfoxide for 30 min and frozen at a cooling rate of −2 °C min−1 prior to storage in liquid nitrogen tanks. Cryopreserved spermatophores were sampled for bacterial profiles after 1 h and 30, 60, 90, 120, 150, 180 and 210 days using Plate count Agar, Thiosulphate Citrate Bile Salts Agar and Pseudomonas Isolation Agar. The incubation temperature was 37 °C for 24–48 h. Long-term storage of spermatophores in liquid nitrogen resulted in the reduction of the bacterial numbers. The number of total heterotrophic bacteria and total Vibrio decreased significantly (P <0.05) to 2.1±0.4×104 CFU/g (95.5% reduction) and 18.7±4.1×103 CFU/g (93.8% reduction), respectively, after 30 days in storage, while those of total Pseudomonas declined significantly (P <0.05) to 1.11±5.9×105 CFU/g (62.8% reduction) within 1 h of storage. Average sperm viability of cells from fresh spermatophores (96.4±1.8%) was not significantly different (P >0.05) from those of cryopreserved spermatophores held in liquid nitrogen for 1 h, 30 or 60 days (94.2±2.1, 89.8±3.5 and 86.7±3.9%, respectively, however it decreased significantly (P <0.05) after 90 days of storage. There were 23 bacterial species identified from fresh and cryopreserved spermatophores. The bacterial species least able to survive cold storage were Proteus mirabilis, Enterobacter spp., Vibrio spp., Kocuria varians, Bacillus cereus, B. amyloliquefaciens, B. circulans, Pseudomonas aeruginosa and Burkholderia cepacia because they were not detected from cryopreserved spermatophores after 90 or 120 days. The normal flora remaining at the end of the experiment (day 210) were Staphylococcus non aureus, Micrococcus spp., Proteus spp., Ochrobactrum anthropi, Corynebacterium spp., B. licheniformis and Pseudomonas spp. Cryopreservation of P. monodon spermatophores nearly eliminated the occurrence of pathogenic bacteria (Vibrio spp. and Ps. aeruginosa) culturable at 37 °C during long-term storage in liquid nitrogen. [Copyright &y& Elsevier]

This study investigated the effects of pathogenic bacteria, Aeromonas hydrophila , contamination on the changes in sperm oxidative damage, ultrastructure, and quality in the silver barb (Barbonymus gonionotus) following cryopreservation. Suspensions of silver barb sperm were in vitro inoculated with A. hydrophila (0, 103, and 105 CFU/mL; named as control, T1, and T2, respectively) and incubated at 25 ± 1 °C for 5 min, before equilibration with 10% DMSO. Sperm samples were frozen and cryostored within a dry shipper at the upper and lower chamber, respectively. The number of A. hydrophila incubated with sperm caused sperm quality decrease and was inversely correlated with unfrozen sperm motility (r = −0.5987) and viability (r = −0.5277) and post-thaw sperm motility (r = −0.5710) and viability (r = −0.7573). The lipid peroxidation (LPO) levels were significantly (p < 0.05) affected by the in vitro inoculation of A. hydrophila and freezing condition (unfrozen or frozen). Significant (p < 0.05) inverse correlations were observed between LPO levels and sperm quality (motility and viability) of silver barb sperm. Ultrastructural damage of silver barb sperm (plasma membrane, mitochondria, flagella, and nucleus) was significantly (p < 0.05) affected by the freezing-thawing process and A. hydrophila contamination. In addition, the inoculation of A. hydrophila and cryopreservation process caused the alteration of sperm morphology at the ultrastructure level including swelling and rupture of the plasma membrane, vesicular to swollen-mitochondria, tail coiling, disarranged flagellum, chromatin condensation, and nuclear fragmentation. Our study revealed that a high level of A. hydrophila contamination and freezing-thawing process during cryopreservation process can activate the necrosis and apoptosis-like phenomenon and autophagy pathway in silver barb sperm. • Post-thaw sperm quality and Aeromonas hydrophila numbers exhibited an inverse correlation. • Lipid peroxidation levels were increased in the post-thaw sperm and A. hydrophila -inoculated sperm. • Inverse correlations were found between lipid peroxidation levels and post-thaw sperm quality. • Freezing-thawing process and A. hydrophila can trigger sperm ultrastructural damage. • Necrosis/apoptosis/autophagy of post-thaw sperm could be induced by A. hydrophila. [ABSTRACT FROM AUTHOR]

The objectives of this study were to generate the optimal cryopreservation protocols (programmable controlled-rate and practical methods) for banana shrimp (Fenneropenaeus merguiensis) spermatophores and investigate the efficacy of plant extracts (moringa, Moringa oleifera Lam and ginger, Zingiber officinale Roscoe) on sperm survival and bacterial abundance in cryostored spermatophores. Spermatophores were exposed to various cryoprotectants at different concentrations and equilibrium times prior to examining sperm viability. A minimal toxicity to banana shrimp sperm was observed in spermatophores suspended in 5% DMSO. The optimal freezing protocol using a programmable controlled-rate freezer included exposure of spermatophores in calcium-free saline (Ca–F saline) containing 5% DMSO with 20 min equilibration time, and then utilization of a one-step freezing at the rate of −2 °C min−1 from 25 °C to −80 °C and holding for 30 s before storage in a liquid nitrogen tank. Highest sperm viability of post-thawed spermatophores was obtained after thawing at 30 °C for 2 min in a water bath. Cryopreservation of F. merguiensis spermatophores using a practical method was achieved by suspending spermatophores in Ca–F saline containing 5% DMSO with equilibration time of 20 min at 25 °C and placing at 4 cm over liquid nitrogen surface (a freezing rate of about −2.5 °C min−1) prior to plunging into liquid nitrogen. In order to evaluate the efficacy of plant extracts on sperm survival and bacterial abundance of cryostored spermatophores, two freezing protocols (programmable and practical methods) with ethanolic moringa and ginger extracts (0.1 mg mL−1 each) were developed for long-term cryostorage. Moringa and ginger extracts had no detrimental effect on F. merguiensis sperm due to retaining high viable sperm without significant differences (P > 0.05) compared to the control after a 12-month cryostorage. Supplementation of moringa or ginger extract into sperm extender resulted in a reduction of bacterial abundance in cryostored spermatophores. This report, for the first time, indicates the promising potential of plant extracts as an alternative method for controlling bacterial contaminants in cryopreserved spermatophores of penaeid shrimp. • This study reports the cryopreservation protocol using a programmable controlled-rate freezer for banana shrimp spermatophore. • This study also develops a practical method for cryopreservation of banana shrimp spermatophore using a Styrofoam box. • The extracts from moringa and ginger can be used as antimicrobial agents in cryopreservation of banana shrimp spermatophore. [ABSTRACT FROM AUTHOR]