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An investigation was conducted to clarify the role of oxidative stress and ultrastructural changes on the sperm of the silver barb, Barbonymus gonionotus caused by the cryopreservation. The sperm was cryopreserved using a dry shipper and 500‐μl straws loaded with 160 μl extended semen and 10% DMSO, frozen for 2 min within the upper chamber of the dry shipper, and immediately transferred into the lower chamber for cryostorage. Lipid peroxidation (LPO) levels of silver barb sperm were affected by the freezing and cryostorage period, progressively increasing (p < 0.01) throughout the 14‐day cryostorage. The ultrastructural defects, apoptotic, and necrotic cells were higher (p < 0.01) in the post‐thawed sperm. There were inverse correlations between ultrastructural damage and the sperm qualities (motility; r = −0.84, p < 0.0001 and viability; r = −0.72, p < 0.001). In conclusion, this cryopreservation procedure had the effect on the oxidative and ultrastructural damage in the sperm cells. There is a need for antioxidants supplement to avoid the oxidative damage and ultrastructural alteration during freezing–thawing process. [ABSTRACT FROM AUTHOR]

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 objective of this study was to investigate the effects of mixed Bacillus on growth, water quality and disease resistance against Vibrio harveyi in whiteleg shrimp (Litopenaeus vannamei). Postlarval shrimp (PL30) were fed with (a) a basal diet (the control), (b) a diet containing mixed freeze‐dried Bacillus probiotics (FB) and (c) addition of mixed microencapsulated Bacillus probiotics (MB) in culture water. Addition of FB and MB probiotics improved (p <.05) growth, feed efficiency, survival and culture water quality (ammonia and nitrite) compared to the control group although there was no difference (p >.05) between the two treated groups. Bacillus numbers in gastrointestinal tracts and culture water of FB‐ and MB‐administrated shrimp were higher (p <.05) than in the control. After a 30‐day culture, shrimp were infected with V. harveyi and monitored for 10 days. A significant reduction (p <.05) in cumulative mortality was observed in FB‐ and MB‐supplemented shrimp (43.24% and 45.05%, respectively), compared to the control (63.06%). This finding demonstrated that administration of microencapsulated probiotics was as effective as freeze‐dried probiotics for improving growth, feed efficiency, survival, Bacillus in gastrointestinal tracts, water quality (ammonia and nitrite) and conferring disease resistance to V. harveyi. [ABSTRACT FROM AUTHOR]

This study was designed to optimize the chilled storage method for banana shrimp (Fenneropenaeus merguiensis) spermatophores and evaluate the potential of moringa (Moringa oleifera Lam.) extract on the reduction in bacterial contaminants during spermatophore preservation due to the uncertainty of the broodstock. Spermatophores were suspended in five extenders: mineral oil, Ringer's solution, phosphate buffer, calcium‐free saline and 0.8% NaCl and stored at 2–4°C. During a 28‐day storage, spermatophores stored in mineral oil showed the highest sperm viability (89.1%) and intact morphology with a slight formation of hardened adhesive matrices. The effect of moringa extract was investigated on chilled spermatophores. Spermatophores were suspended in mineral oil (the control) and mineral oil containing either penicillin–streptomycin (0.1%) or moringa extract (0.1 mg/ml) during a 28‐day storage. Supplementation of moringa extract resulted in a significant increase (p <.05) in sperm survival, compared to the control, and a complete elimination of culturable Vibrio (Vibrio alginolyticus, Vibrio mimicus and Vibrio furnissii), Staphylococcus kloosii, Bacillus macerans, Listeria ivanovii, Corynebacterium paurometabolum and Corynebacterium bovis, in chilled spermatophores. Chilled storage of spermatophores in mineral oil containing moringa extract was a promising technique due to the inhibition of shrimp and human pathogens without the spermicidal effect on banana shrimp 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]

Dried seafood products are one of the most popular seafood items in Thailand. This study was divided into two experiments. In first experiment, a survey in intrinsic and bacteriological characteristics of twenty dried seafood products distributed in Chon Buri province, Thailand was performed. Values of water activity, pH and NaCl content were 0.35±0.01-0.72±0.00, 5.47±0.03-9.24±0.01, and 0.66±0.06-2.26±0.03%, respectively. Based on the Thai regulatory standard, half of dried seafood products contained total viable count over acceptable level. Predominant isolated bacteria in dried seafood products were Staphylococcus spp. (39.7%), Bacillus spp. (30.1%), and Pantoea sp. (19.2%). Foodborne pathogens, i.e., B. cereus and Salmonella enterica serovar Weltevreden were found in dried seafood products. In second experiment, two forms of antimicrobials (concentrated (CBV) and partially purified (PBV) substances) prepared from B. velezensis BUU004 and two commercial preservatives (nisin and lactic acid) were introduced into crushed dried seasoned squids to investigate inhibitory activity against pathogens, B. cereus TISTR 687, S. aureus ATCC 25923, Salmonella Typhimurium TISTR 292, and Escherichia coli ATCC 25922. PBV had broader antagonistic activity than CBV, nisin and lactic acid against tested pathogens. Scanning electron microscopy analysis represented bacteriostatic action of PBV on tested pathogens through cell lysis evidenced by an irregular cell shape along with pore formation on cell membranes. Our study demonstrates that PBV has a preservative potential to control pathogen growth and improve biosafety of dried seafood products. [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]

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]

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: Development of probiotics for replacement of antibiotics is one of the key factors for management in sustainable aquaculture. This study examined the effects of probiotic forms (microencapsulated and freeze-dried cells), compositions of probiotics (bacteria, yeast and microalgae) and mode of probiotic administration (probiotic supplementation in either water additives or through probiotic-enriched artemia) on the growth, survival rate and microbial number of Litopenaeus vannamei at various stages in two separate experiments: (1) larval stages from zoea 3 to mysis 3 and (2) postlarval (PL) stages from PL 1 to PL 21. The treatments of both experiments included addition of: (T1) artemia enriched with mixed microencapsulated Bacillus (MB), (T2) artemia enriched with mixed MB and microencapsulated yeast (MY), (T3) artemia enriched with mixed MB, MY and microencapsulated Chaetoceros (MC), (T4) mixed MB, (T5) mixed MB and MY, (T6) mixed MB, MY and MC, (T7) mixed freeze-dried Bacillus (FB), (T8) mixed FB and freeze-dried yeast (FY), (T9) mixed FB, FY and freeze-dried Chaetoceros (FC) and (T10) control (no addition of probiotics). Results demonstrated that two probiotics forms and modes of probiotic administration did not affect (P>0.05) growth and survival of zoea 3 to PL 21. The compositions of probiotics did not affect (P>0.05) growth and survival of zoea 3 to mysis 3. However, PL 1 to PL 21 treated with either bacteria and yeasts or bacteria, yeasts and Chaetoceros exhibited increases (P<0.05) in growth and survival as compared to controls. Numbers of total heterotrophic bacteria (THB), Bacillus and yeasts in shrimp from zoea 3 to PL 21 or culture water of all treatments were higher (P<0.05) than in controls. This investigation suggested that incorporation of bacteria and yeasts or bacteria, yeasts and Chaetoceros as microencapsulated or freeze-dried forms in water additives or probiotic-enriched artemia possessed as effective probiotics for rearing of L. vannamei based on the enhancement of survival and growth rate and concomitant increase in the beneficial microbial probiotics in shrimp larvae and postlarvae as well as in culture water. [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]

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]

Abstract: Epidemics of epizootics and occurrence of multiresistant antibiotics of pathogenic bacteria in aquaculture have put forward a development of effective probiotics for the sustainable culture. This study examined the effectiveness of forms of mixed Bacillus probiotics (probiotic A and probiotic B) and mode of probiotic administration on growth, bacterial numbers and water quality during rearing of white shrimp (Litopenaeus vannamei) in two separated experiments: (1) larval stages and (2) postlarval (PL) stages. Forms of Bacillus probiotics and modes of probiotic administration did not affect growth and survival of larval to PL shrimp. The compositions of Bacillus species in probiotic A and probiotic B did not affect growth and survival of larvae. However, postlarvae treated with probiotic B exhibited higher (P <0.05) growth than probiotic A and controls, indicating Bacillus probiotic composition affects the growth of PL shrimp. Total heterotrophic bacteria and Bacillus numbers in larval and PL shrimp or culture water of the treated groups were higher (P <0.05) than in controls. Levels of pH, ammonia and nitrite of the treated shrimp were significantly decreased, compared to the controls. Microencapsulated Bacillus probiotic was effective for rearing of PL L. vannamei. This investigation showed that administration of mixed Bacillus probiotics significantly improved growth and survival of PL shrimp, increased beneficial bacteria in shrimp and culture water and enhanced water quality for the levels of pH, ammonia and nitrite of culture water. [Copyright &y& Elsevier]