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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.)

Summary: The development of a chilled storage protocol of fish sperm requires an understanding of sperm biology and function as the activation/inhibition of fish sperm is greatly affected by several environmental factors. This study aimed to determine the effects of ionic and non‐ionic solutions, extender types (Ringer's solution, Ca‐F HBSS solution, HBSS solution, He and Wood solution, Saline solution, and Modified Cortland solution), and chilled‐storage period on sperm quality and fertilization ability of Asian seabass, Lates calcarifer semen. Regulation of Asian seabass sperm motility was dependent on the osmolality of both ionic and non‐ionic activation media. The threshold levels on the initiation of sperm motility were detected in KCl (>100 mM), NaCl (>50 mM), CaCl2 (>50 mM), glucose (>300 mM), and mannitol (>100 mM) solutions. Relatively high percentages of sperm motility (>80%) were observed when activated with KCl, NaCl, CaCl2, glucose, and mannitol solutions at above 700, 600, 350, 1,000, and 1,000 mM, respectively. Ringer's solution was the most optimal extender for chilled storage of Asian seabass semen at 2–4°C supported by the retention of sperm motility and viability for 6 days. Semen diluted in Ringer's solution and chilled‐stored for 2 days exhibited acceptable fertilization (66.1% ± 6.2%) and hatching (56.4% ± 2.9%) rates. This report, for the first time, describes the ionicity and non‐ionicity effects on the motility of Asian seabass sperm. [ABSTRACT FROM AUTHOR]

The aim of this study was to develop a simple cryopreservation protocol for silver barb, Barbodes gonionotus, semen using a dry shipper. Freezing rates within the upper and lower chambers of dry shipper were recorded for 14 days post liquid nitrogen loading (dpl). To regulate freezing rates, straws (250 and 500 µl) wrapped with various insulators (polystyrene foam box, oxygen tube, silicone tube and electric wire) were frozen within the upper chamber. Straws containing semen diluted with Calcium‐free Hank's Balanced Salt Solution (Ca‐F HBSS) and 10% dimethyl sulphoxide were cryopreserved with or without insulators. Appropriate protocols were selected based on sperm quality during a 45‐day cryostorage. The upper chamber had potential as a freezing chamber within 9 dpl due to no significant (p > 0.05) change in freezing rates. High percentages of sperm motility and viability (p < 0.05) were observed when 250 µl straws with silicone tube (T4) frozen for 5 min, non‐insulated 500 µl straws (T9) and 500 µl straws with polystyrene foam box (T12) frozen for 1–5 min, having freezing rates of 43.1 ± 1.3, 71.3 ± 1.4 and 14.7 ± 0.4°C/min respectively. Dry shipper can be used as a freezing tool to cryopreserve silver barb semen. [ABSTRACT FROM AUTHOR]

Abstract: 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 2cm above liquid nirogen surface for 10min 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 2cm above liquid nitrogen surface for 10min 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 &y& Elsevier]

Abstract: The aim of the present study was to determine changes in sperm quality of walking catfish (Clarias macrocephalus) during its spawning season, and develop a simple and efficient protocol for the chilled storage of semen at 4°C. Semen samples were evaluated monthly during April–November in 2006. Percentage of spermiating males and sperm motility peaked during the middle of the spawning season, June–September. Sperm density and seminal plasma osmolality increased significantly (P <0.05) throughout the spawning season, while semen pH did not change during this period. The effects of extender, dilution ratio and timing of chilled storage on successful storage period of extended semen were examined during the years 2006–2007. Fresh semen was diluted 1:1 with several sperm extenders, calcium-free Hanks'' balanced salt solution (Ca–F HBSS), Hanks'' balanced salt solution (HBSS), extender 7 (NaCl 5.780g, KCl 2.558g, CaCl2·2H2O 0.103g, NaHCO3 0.235g, MgCl2·6H2O 0.220g, pyruvate 6.0g, citric acid 0.100g, HEPES buffer 2.380g in 1000mL distilled water), extender 13 (NaCl 8.760g in 1000mL distilled water), and modified Cortland, in tissue culture flasks. Longest successful storage period of sperm was 10days with Ca–F HBSS. Semen diluted with Ca–F HBSS at 1:1, 1:2 or 1:4 ratios significantly lengthened (P <0.05) the storage period over those at dilutions of 1:9 or 1:19. To determine the effect of season on the success of chilled storage, semen diluted with Ca–F HBSS at a ratio of 1:4 was prepared at three sampling intervals in the beginning (May), middle (August) and end (November) of spawning season in 2007. Extended semen prepared in August remained motile (20±3.1%) for as long as 10days, whereas those prepared in May or November were immotile on days 9 and 7 of storage, respectively. Extender-preserved semen was capable of fertilizing eggs as efficiently as fresh semen within two days of storage. A significant decrease in fertilization and hatching rates of extender-preserved semen compared to fresh semen on days 4 and 6 was due to low sperm motility. These results demonstrate that semen of walking catfish can be stored for short term at 4°C without appreciably affecting motility and fertilization. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]