Sperm cryopreservation of silver barb (Barbodes gonionotus): cryoprotectants,cooling rate and storage time on sperm quality

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.     

Refrigerated storage and cryopreservation of sperm of red drum, Sciaenops ocellatus L.

To aid in artificial spawning of sciaenid fishes, the present authors developed techniques to collect, handle and cryopreserve sperm from red drum, Sciaenops ocellatus L. Sperm were collected by removing and slicing the testis, and adding Hanks' balanced salt solution (HBSS) or NaCl solution (each at 200-400 mOsm kg^?1) as an extender. Sperm were activated with 800 mOsm kg^?1 artificial sea water (ASW) to characterize motility. Sperm reached maximum motility (highest percentage motility observed for that sample) within 8 ± 1 s (mean ± SD) and remained at maximum motility for 33 ± 4 s. Sperm were exposed to graded osmotic pressures of ASW (8-800 mOsm kg^?1) to determine the range of osmolalities that elicited motility. Threshold activation (defined as ～10% motility) occurred at 351 ± 4 mOsm kg^?1 and complete activation occurred at 539 ± 2 mOsm kg^?1. Sperm stored at 200 mOsm kg^?1 retained motility for up to 13 days. Dimethyl sulphoxide (DMSO) was used as a cryoprotectant at concentrations ranging from 7.5% to 15% (v:v) in HBSS (200 mOsm kg^?1). There were no significant differences among post-thaw motilities of sperm cryopreserved at any concentration of DMSO. Sperm thawed on the benchtop at 21°C had lower post-thaw motility than did sperm thawed at 10, 20, 30, 40, 50 or 60°C in a water bath.     

Transport and cryopreservation of sperm of the common snook, Centropomus undecimalis (Bloch)

Sperm were collected in Florida from wild common snook, Centropomus undecimalis (Bloch), and were shipped to Louisiana State University for analysis and cryopreservation. Threshold activation of sperm (10% motility) occurred at 370 mOsmol kg^?1, and complete activation occurred at 680 mOsmol kg^?1. These values were significantly different. Sperm samples stored at 1°C in Hanks' balanced salt solution (HBSS) or in 0.6% NaCl solution at 200 mOsmol kg^?1 retained motility for as long as 22 days. Mean motility remained above 50% for 9 days for sperm stored in HBSS and for 7 days for sperm stored in NaCl solution. Sperm exposed to 5% dimethyl acetamide (62±10%; mean±SD), 10% dimethyl sulphoxide (DMSO) (39±16%), 5% glycerol (26±5%) or 10% glycerol (6±2%) for 30 min had significantly lower motility than did unexposed sperm (89±9%). When used as a cryoprotectant, samples frozen with 5% or 10% DMSO or 5% methanol had significantly higher post‐thaw motility than did samples frozen with other cryoprotectants. Sperm cryopreserved with 10% DMSO (38±12%) had significantly higher post‐thaw motility than did sperm cryopreserved with 15% DMSO (19±10%) or 20% DMSO (4±4%). There were no significant differences in hatch rates of eggs fertilized with fresh sperm (54±29%) or cryopreserved sperm (41±35%). Survival to first feeding was not different between fish produced with fresh sperm (37±30%; range, 0–86%) or with thawed sperm (24±29%; 0–77%). Transport of sperm to a cryopreservation laboratory and back to a hatchery for thawing and use enabled collaboration between groups with specific expertise and provides a model for the application of cryopreservation by transport of fresh and frozen samples.     

Cryopreservation of Mekong catfish,Pangasius bocourti Sauvage, 1880 spermatozoa

The effects of three extenders (Ginzburg fish ringer, Calcium‐free Hank's balanced salt solution, C‐F HBSS and sodium chloride, 0.9% NaCl) and four cryoprotectants (dimethyl sulphoxide, DMSO; dimethyl acetamide, DMA; methanol, MeOH and glycerol) in different concentrations (5%, 10% and 15%) on the motility, viability and fertilization rates of Mekong catfish (Pagasius bocourti) sperm were investigated. Sperm samples were transferred into 250‐μL French straws and sealed with a heated haemostat. The straws were then placed in a cryochamber. A computer‐controlled rate freezer (CL 3300) and programmable Cryogenesis, version 4 were used to regulate the freezing rate. The sperm samples were frozen at a rate of 10°C min^?1 from 4 to ?80°C and then evaluated after 72 h. Of the three extenders used with each cryoprotectant, C‐F HBSS had the highest fertilization rate of 75% (93% of control). This was not significantly different from the control treatment (fresh sperm) when tested with DMSO as the cryoprotectant. The lowest fertilization rate of 27% (38% of control) was resulting from the combination of 15% glycerol and C‐F HBSS. This study found that fertilization, motility and viability rates in all of the experiments had a positive significant correlation (P < 0.001).     

Cryopreservation of Persian sturgeon (Acipenser persicus) sperm: effects of cryoprotectants,antioxidant, membrane stabilizer,equilibration time and dilution ratio on sperm motility and fertility

Three experiments were performed to develop protocols for cryopreservation of Persian sturgeon Acipenser persicus, sperm. In the first experiment, sperm from six males was individually split in three subsamples and cryopreserved using Modified Tsvetkova's extender (mT) supplemented with dimethyl sulfoxide (DMSO), methanol (MeOH), glycerol (Gly) and ethylene glycol (EG) at concentration of 5%, 10%, 15% and 20%. In the second set of experiments, the effects of six equilibration times (0, 5, 10, 20, 40 and 60 min) and dilution ratios (volume sperm: volume extender 1:0.5, 1:1, 1:2, 1:3, 1:5 and 1:10) and the additive advantage of bovine serum albumin (BSA; 0, 2.5, 5 and 10 mg mL^?1) and ascorbic acid (0, 2.5, 5 and 10 U mL^?1), on the post‐thaw survival of sperm (triplicate set of six fish) were evaluated. Then, sperm was diluted in 1:1 mT extender with 10 mg mL^?1 BSA with selected cryoprotectants (15% MeOH and 10% DMSO) for 5 min. After a month of storage in liquid nitrogen, post‐thawed sperm motility; fertilization and hatching rate and viability of derived larvae were measured (Exp.3). Evaluation of cryoprotectants efficiency showed that MeOH 15% and DMSO 10% were suitable for cryopreservation of Persian sturgeon sperm. Gly and EG resulted in very low post‐thaw motility rates even at lowest concentration. No significant difference was observed among the four different equilibration times (0, 5, 10, 20 min) (P > 0.05) although higher equilibration times than 20 min resulted low post‐thaw motility (P < 0.05). The motility of frozen–thawed sperm did not significantly change when dilution ratio was increased from 1:0.5 to 1:3 (P > 0.05). However, higher dilution ratios (1:5 and 1:10) reduced the percentage of motile sperm. Supplementation of the cryoprotectant solution with 10 mg mL^?1 BSA significantly improved post‐thaw motility (P < 0.05), but ascorbic acid did not improve post‐thaw motility (P > 0.05). The results of experiment 3 showed that the highest fertilization (30.2 ± 5.75) and hatching rates (28.2 ± 5.25) were observed when samples were frozen with 15% MeOH (P > 0.05). Our study indicates that the use of mT extender consisting of 10 mg mL^?1 BSA in 15% MeOH diluted with sperm at 1:1 ratio for 5 min can be recommended cryopreservation method for Persian sturgeon sperm.     

Spermatological research of experimentally farmed Patagonian blenny (Eleginops maclovinus) (Perciformes: Eleginopsidae) in Chile

Spermatological research of the Patagonian blennie was carried, specifically biometric parameters, sperm density, sperm count and motility in different activation mediums (815, 716, 590 and 0 mOSm kg^?1), at different temperatures (5, 10 and 15°C) and pH levels (5, 7 and 9). The results indicate that Patagonian blennie spermatozoa have a primitive form, with a total length of 44.09 ± 3.36 μm, with a head length of 2.15 ± 0.28 μm and head width of 2.5 ± 0.31 μm. The mid‐piece had a length of 0.72 ± 0.12 μm, and its tail measures 41.21 ± 3.21 μm long. The motility pattern indicates that the spermatozoa are found immobile in the seminal plasma and only initiates its movement in a hypertonic medium from 590 to 815 mOsm kg^?1. The longest motility time that was registered at 10°C in 716 mOSm kg^?1 was of 245 ± 39 s and an optimum pH of 7 was observed.     

Fertilizing capacity and motility of tench Tinca tinca (L., 1758) sperm following cryopreservation

Experiments were carried out to develop an optimal cryopreservation protocol for tench sperm by testing the fertilizing capacity and motility parameters including progressive motility, curvilinear velocity (VCL) and linearity (LIN) of cryopreserved sperm. Three experiments were designed to this aim: first experiment where we tested the effects of two extenders (sugar‐based Grayling and ion‐based Kurokura 180) and two cryoprotectants (DMSO and methanol) on fertilization and hatching success; second where we tested the effect of cryoprotectant type (methanol or DMSO) in different concentrations (5%, 10% and 15%) on fertilization and hatching success; and third where we tested the effect of two cryoprotectants (methanol and DMSO) on sperm motility parameters (progressive motility, VCL and LIN) after 4 h post‐thaw storage (4°C). Sperm prepared with the sugar‐based Grayling extender displayed better fertilization and hatching rates independently of the applied cryoprotectant most likely due to glucose present which acted as an external cryoprotectant. Concerning cryoprotectant concentrations, the use of 10% methanol yielded the highest fertilization (85 ± 15%) and hatching (80 ± 13%) rates, which were significantly higher than in all other groups. During the post‐thaw storage time, 5% methanol, 10% methanol and 5% DMSO groups had significantly higher motility parameters than other groups and we observed no significant decline in any of the parameters during the storage time. Overall, we found that a sugar‐based extender in combination with methanol as cryoprotectant is suitable for the cryopreservation of tench sperm and allows storage of cryopreserved sperm for up to 4 h post thaw.     

Hybridization between Haliotis rufescens and Haliotis discus hannai: evaluation of fertilization,larval development,growth and thermal tolerance

Two introduced abalone species are currently produced in Chile, red abalone Haliotis rufescens and Japanese abalone Haliotis discus hannai. However, red abalone accounts for 99% of total production, while the Japanese abalone has not adapted well to Chilean coastal waters. This study reports the hatching, growth and thermal tolerance performance in interspecific hybrids produced between red (R) and Japanese (J) abalone. Our results show that egg age and sperm concentration were critical factors to produce hybrids. The cross R♀ × J♂ showed a fertilization rate of 55.3 ± 3.5% using 20‐min‐old eggs and sperm concentrations of 14 × 10⁶ cells mL^?1, while the reciprocal cross (J♀ × R♂) was not successful. Further, larval development stages were similar in RR, JJ and RJ hybrid abalones. Among the experimental trials, settlement rate varied from 12.3% to 18.6% and final survival from 20.1% to 31.7%, being the RJ hybrid rates intermediate between parental species. The final shell lengths were similar between RR and RJ hybrids, but significantly higher in JJ abalones. In addition, thermal tolerance was ascertained due its pivotal role for the abalone physiology. Thus, RJ hybrids showed the highest HSP70 gene expression and offers new possibilities to expand Chilean abalone production in warm waters zones.     

Production and storage of sperm from the black sea bass Centropristis striata L

Black sea bass Centropristis striata L. are protogynous hermaphrodites that develop and spawn as females before changing sex to male. Since all fish eventually become males, determining the relationship between sperm production, sperm quality and seasonal changes in plasma levels of testosterone (T) and 11‐ketotestosterone (11‐KT) could be useful for identifying appropriate males to maintain as broodstock. Milt and blood samples were collected three times during an 8‐week spawning season. Milt volume (3.5±0.76 mL kg^?1), sperm density (3.2 × 10⁸± 0.31 cells mL^?1), sperm production [11 × 10⁸±3.4 cells kg^?1 body weight (BW)] and sperm motility (80±0.6%) were at their highest during the first sampling interval and coincided with the highest 11‐KT levels (1.0± 0.11 ng mL^?1). All of the sperm indices decreased to their lowest levels during the final 3 weeks of the study. Sperm viability was highly correlated (adjusted R²=0.84) with sperm motility. Sperm cryopreserved in modified Mounib's extender (MME) had the highest post‐thaw motility compared with two other extenders. Post‐thaw motility of sperm cryopreserved in MME was not different from fresh after 90 days of storage. There was no difference in fertilization rates between fresh (69±2.4%) and post‐thaw (67±4.1%) sperm samples taken from the same male or among males. These results demonstrate that the quality of black sea bass spermatozoa is higher earlier in the spawning season and that acceptable post‐thaw fertilization rates can be obtained from cryopreserved sperm.     

Evaluation of selected metal nanoparticles on hatching and survival of larvae and fry of Indian major carp,rohu (Labeo rohita)

Different laboratory synthesized metal nanoparticles viz. Copper oxide (CuO), Zinc oxide (ZnO) and silver doped titanium dioxide (Ag‐TiO₂) were studied for their effect on hatching and survival of larvae and fry of Indian major carp, rohu, Labeo rohita both in direct application in tank water & coated onto tanks. Among these nanoparticles, CuO and ZnO nanoparticles exhibited highest percentage of hatching in both direct addition (78.0 ± 3.1% and 78.05 ± 4.2%, respectively) and coating onto tanks (58.6 ± 2.1% and 61.2 ± 2.7%, respectively) at 1 mg mL^?1 while least percentage of hatching was recorded in Ag‐TiO₂ nanoparticles irrespective of its concentration & mode of supplementation. Highest survival of L. rohita fry (50.13 ± 2.2%) was observed after 15 days post hatching in CuO coated tanks followed by ZnO coated tanks (38.6 ± 2.8%) while least was recorded in Ag‐TiO₂ coated tanks (22.53 ± 3.0%). However in control tanks coated with Poly‐Urethane base with hardener and uncoated control tanks, the survival was 42.4 ± 1.2% and 41.36 ± 1.8% respectively. Further, significantly lower microbial load of water was recorded in CuO nanoparticles coated tanks (1.5 × 10¹⁰ CFU L^?1) as compared to uncoated control tanks (1.1 × 10¹⁶ CFU L^?1) without affecting water quality parameters. On the other hand, in Ag‐TiO₂ coated tanks, significantly lower microbial load (1.0 × 10⁶ CFU L^?1) as compared to uncoated control tanks at 15 days post hatching was recorded. However, Ag‐TiO₂ was toxic to L. rohita larvae & fry both in direct application and coating onto tanks. Considering the beneficial effects of CuO nanoparticle application, it has the scope of being used in a more eco‐friendly way in hatchery operations.     

Efficacy of garlic (Allium sativum) extract applied as a therapeutic immersion treatment for Neobenedenia sp. management in aquaculture

Garlic, Allium sativum L., extract administered as a therapeutic bath was shown to have antiparasitic properties towards Neobenedenia sp. (MacCallum) (Platyhelminthes: Monogenea) infecting farmed barramundi, Lates calcarifer (Bloch). The effect of garlic extract (active component allicin) immersion on Neobenedenia sp. egg development, hatching success, oncomiracidia (larvae) longevity, infection success and juvenile Neobenedenia survival was examined and compared with freshwater and formalin immersion. Garlic extract was found to significantly impede hatching success (5% ± 5%) and oncomiracidia longevity (<2 h) at allicin concentrations of 15.2 μL L^?1, while eggs in the seawater control had >95% hatching success and mean oncomiracidia longevity of 37 ± 3 h. At much lower allicin concentrations (0.76 and 1.52 μL L^?1), garlic extract also significantly reduced Neobenedenia infection success of L. calcarifer to 25% ± 4% and 11% ± 4%, respectively, compared with 55% ± 7% in the seawater control. Juvenile Neobenedenia attached to host fish proved to be highly resistant to allicin with 96% surviving 1‐h immersion in 10 mL L^?1 (15.2 μL L^?1 allicin) of garlic extract. Allicin‐containing garlic extracts show potential for development as a therapy to manage monogenean infections in intensive aquaculture with the greatest impact at the egg and larval stages.     

Development of sperm vitrification protocols for two endangered salmonid species: the Adriatic grayling, <Emphasis Type="Italic">Thymallus thymallus</Emphasis>, and the marble trout, <Emphasis Type="Italic">Salmo marmoratus</Emphasis>

Vitrification was applied to the sperm of two endangered fish species of So?a River basin in Slovenia, the Adriatic grayling (Thymallus thymallus) and marble trout (Salmo marmoratus) following testing different cooling devices and vitrifying media. Sperm was collected, diluted in species-specific non-activating media containing cryoprotectants, and vitrified by plunging directly into liquid nitrogen without pre-cooling. Progressive motility, curvilinear velocity, and straightness of fresh and vitrified-warmed sperm were evaluated with computer-assisted sperm analysis (CASA). Fertilization trials were carried out to test the effectiveness of vitrification in the case of grayling. A protocol utilizing a glucose-based extender, 30% cryoprotectants (15% methanol + 15% propylene glycol), 1:1 dilution ratio, and droplets of 2 μl on a Cryotop as cooling device yielded the highest post-thaw motility values for both Adriatic grayling (7.5?±?6.5%) and marble trout (26.6?±?15.8%). Viable embryos were produced by fertilizing eggs with vitrified grayling sperm (hatching 13.1?±?11.7%, control hatching 73.9?±?10.4%). The vitrification protocol developed in this study can be utilized in the conservation efforts for the two species as an alternative to slow-rate freezing when working in field conditions or when specific equipment necessary for slow-rate freezing is not available.     

Milt quality and spermatozoa morphology of captive Brycon siebenthalae (Eigenmann) broodstock

In order to develop artificial reproduction in freshwater fish for potential species to be developed in South American aquaculture, milt quality and sperm morphology were studied in yamú (Brycon siebenthalae) under captive conditions during the natural middle spermiation period. The volume of milt collected for each male was 1.8±1.2 mL and the sperm concentration was 13.9±4.0 × 10⁹ spermatozoa mL^?1. Spermatocrit (41.5±10.8%) was positively associated (r²=0.30) with sperm density calculated using a corpuscle counting chamber. Sperm motility was 88±9% and the average duration of forward motility was 41±7 s. Fertilization rate was 84±8% and there was no association between this trait and sperm motility (r²=0.009) or with sperm density (r²=0.073). These results suggest that captive B. siebenthalae broodstock can be reproduced successfully.     

Sperm fertility of the subtropical freshwater streaked prochilod Prochilodus lineatus (Characiformes) improved after dilution and cold storage

The aims of this study were to evaluate the efficiency of simple and complex extenders in prolonging the cold storage of sperm (Experiments 1 and 2) and to test the diluted‐cooled sperm in the best extender with regard to sperm quality parameters (Experiment 3) in the streaked prochilod, Prochilodus lineatus. In all the experiments, aliquots of 0.3 mL of sperm were diluted 1:10 in extenders and stored at 4–6 °C. Sperm diluted in simple extenders (NaCl and glucose solutions) yielded 0–26% sperm motility, whereas sperm diluted in complex extenders (BTS^?, M III^? and Androstar^?) yielded 62–81% sperm motility on day 4 after cold storage. When Androstar^? was further investigated, the following was observed on day 4: 53% motility with 94 s of duration; 47% live spermatozoa; 26–61% fertility rate; and 22–60% hatching rate. The use of Androstar^? improves the sperm fertility of the streaked prochilod during a 4‐day storage period and can therefore be used to facilitate artificial reproduction.     

Cryopreservation of summer flounder,Paralichthys dentatus L., sperm

The summer flounder, Paralichthys dentatus L., is a high‐value species and considerable research has been conducted to determine practices conducive for its culture. As milt can be limited in this species, experiments were conducted to develop a practical sperm cryopreservation protocol for hatchery use. Two dilution ratios (1:2 and 1:4; sperm:extender), 2 diluents (saline and sucrose‐based), 2 cryoprotectants (10% DMSO and 12% glycerol) and 3 freezing rates (?5, ?10 and ?15°C min^?1) were evaluated using differential staining to assess post‐thaw sperm survival. Seven combinations of the factors examined reduced post‐thaw viability by less than 30%. The average viability of sperm from fresh, pooled flounder milt (67.2 ± 2.9%) was not different from that of thawed milt diluted 1:4 with sucrose diluent (10% DMSO) frozen at ?5°C min^?1 (38.4 ± 7.7%) and fertilization and hatch success were not different in trials using fresh or thawed, cryopreserved sperm. From these experiments a practical sperm cryopreservation method was developed, but further refinement of the freezing protocol is necessary to optimize results.     

Effects of supplementary fennel (Foeniculum vulgare) essential oil in diet on growth and reproductive performance of the ornamental fish,Convict cichlid (Cichlasoma nigrofasciatum)

This study evaluated the effects of diet containing 0 (C: control), 75 (D₁), 100 (D₂), 125 (D₃) and 150 (D₄) mg kg^?1 F. vulgare essential oil on growth and reproductive performance of C. nigrofasciatum. A total of 225 Convict cichlids female with mean weight (1.65 ± 0.02 g) were distributed into 15 glass aquaria (15 fish tank^?1) in triplicate treatments. During 40 days of experimental period, fish fed at 3% of their body weight daily. At the end of experiment, growth indices, gonadosomatic index and reproductive indices were determined. The best feed conversion ratio was obtained in D₄ (1.19 ± 0.03, P < 0.05). The addition of F. vulgare essential oil did not have any statistical effects on other growth indices (specific growth rate, weight gain and condition factor). Total survival rates in all treatments and control were uniformly high, ranging from 93% to 97% (P > 0.05). F. vulgare essential oil has no negative effect on survival rate of C. nigrofasciatum. There were no significant differences among protein content of fish body (P > 0.05). The lowest value of fat content (26.5 ± 0.5%) was observed in D_2, and it had significant difference with control and other groups (P < 0.05). The ash percentage in treatments fed with F. vulgare essential oil were higher than control fish (P < 0.05). The highest moisture content was detected in D₄ (72.68 ± 0.19%) and D₂ (71.23 ± 2%) groups. Among the used dosage, D₄ was the most effective dosage that could significantly increase GSI (11.06 ± 1.55%), fecundity (340 ± 21) and hatching ratio (92.33 ± 1.63%). There were no significant differences in diameter of eggs among various treatments (P > 0.05).     

Sperm motility and seminal plasma characteristics in Barbus sharpeyi (Günther, 1874)

Spermatozoa concentration, ionic composition, osmolality, glucose and total protein contents of seminal plasma and sperm motility were determined in Barbus sharpeyi (Cyprinidae, Teleosotei). Spermatozoa concentration ranged from 9.77 to 20.20 × 10⁹ spermatozoa mL^?1. Osmolality (mOsmol kg^?1) and ionic contents (mM L^?1) of the seminal plasma were 274.5±9.0, 70.0±3.4 Na⁺, 28.8±0.9 K⁺, 101.7±3.1 Cl^?, 0.9±0.1 Mg²⁺ and 2.1±0.1 Ca²⁺ respectively. Total protein and glucose were 5.3±0.2 g L^?1 and 76.7±4.3 mM L^?1 respectively. Sperm motility was initiated in a hypo‐osmotic condition, composed of either an ionic (KCl or NaCl) or a non‐ionic (sucrose) activation medium. Duration of sperm motility was very short: <2 min after activation in distilled water. Percentage of motile spermatozoa was significantly higher in an activation medium containing NaCl compared with that of distilled water. An activating medium containing NaCl or KCl higher than 150 mM or sucrose higher than 275 mM totally inhibited the activation of sperm motility. Immediately after sperm activation, wave(s) propagated along the flagellum, but waves were restricted to the proximal part of the flagellum (close to the head) at 1 min post activation. Studied characteristics in the present study were compared with those of other cyprinids for understanding inter‐species differences.     

Effect of dietary supplementation of periphyton on growth,immune response and metabolic enzyme activities in Penaeus monodon

A 60‐day indoor trial was conducted to study the effect of periphyton supplementation on metabolic and immune responses in tiger shrimp, Penaeus monodon. Periphyton developed over bamboo substrate in outdoor tanks (15 m²) was used as dietary supplement for P. monodon (2.02 ± 0.04 g) reared in 1000 L FRP tanks. Graded levels of periphyton were included in shrimp basal diets: 0% (P0), 3% (P3), 6% (P6), 9% (P9) and P0 diet with natural periphyton (NP) over bamboo substrate. At the end of the trial, P6 and NP showed significantly higher (P < 0.01) growth rate, 23.9% and 20%, respectively, compared with control, P0. Comparatively, lower level of metabolic enzymes, such as lactate dehydrogenase, malate dehydrogenase, aspartate aminotransferase and alanine aminotransferase, was recorded in treatments P3, P6 and NP compared with control, P0. The periphyton‐supplemented group, P3 had significantly higher (P < 0.05) superoxide dismutase (15.83 ± 0.96) and catalase activity (15.73 ± 0.69) compared to 6.88 ± 2.84 and 9.15 ± 0.67 unit mg^?1 protein min^?1_, respectively, in P0. Similarly, higher total haemocyte counts, 32.58 ± 1.30, 28.51 ± 3.12 and 27.26 ± 4.43 × 10⁶ cells mL^?1_, were recorded in P6, NP and P3, respectively, compared to P0, 23.57 ± 1.80 × 10⁶ cells mL^?1. After challenge with Vibrio harveyi, P3 recorded the highest relative percentage survival 67% followed by NP (58%) and P6 (42%) compared with control. However, treatment with highest periphyton inclusion (P9) did not differ significantly with P0 on growth and immunological parameters. This study indicates that periphyton supplementation at 3–6% level improves growth, immune response and metabolic activities in P. monodon.     

A comparison between water exchange and settling tank as a method for suspended solids management in intensive biofloc technology systems: effects on shrimp (Litopenaeus vannamei) performance,water quality and water use

Biofloc systems rely on microbial processes in the water column to recycle animal waste products, reducing the need for water exchange. These increases biofloc concentration in the water and some form of removal is needed. An experiment was carried out to evaluate two management practices to control biofloc in Litopenaeus vannamei culture. Six tanks (48 m³) were divided into two treatments: water exchange and solid settler. Shrimp were stocked at 164 shrimp m^?2 and with 0.67 g of weight. After 61 days, shrimp under solid settler treatment demonstrated mean weight of 12.7 ± 0.5 g with survival of 73.8 ± 1.4%, and those under water exchange had a final weight of 10.1 ± 0.2 g and survival rate of 57.8 ± 11.1%. Total suspended solids did not differ between the treatments: 326.8 ± 24.9 mg L^?1 for water exchange and 310.9 ± 25.3 mg L^?1 for solid settlers. Settleable solids and productivity/respiration ratio was higher (P < 0.05) in water exchange treatment, indicating differences in physical and biological characteristics of bioflocs. Solids removal method influenced the water use, in which 1150 ± 249 L of water was necessary to produce one kilogram of shrimp using water exchange strategy, and 631 ± 25 L kg^?1 with the use of settlers. Our results indicate that continuous operation of settlers can reduce variability in solids characteristics and water quality variables such as ammonia. Both strategies are efficient in controlling biofloc concentrations of the water; however, settlers can reduce water use and improve shrimp production.     

Sperm cryopreservation of tiete tetra Brycon insignis (Characiformes): effects of cryoprotectants,extenders, thawing temperatures and activating agents on motility features

The aim of this study was to test the effects of cryoprotectants [dimethyl sulphoxide (DMSO) and methylglycol], extenders (0.9% NaCl, 5% glucose, Beltsville Thawing Solution^? and Merck III^?), thawing temperatures (30 and 60 °C) and activating agents (0.29% NaCl and 1% NaHCO₃) on the cryopreservation process of tiete tetra Brycon insignis sperm. Sperm was loaded in 0.5 mL straws, frozen in nitrogen vapour at ?170 °C and stored in liquid nitrogen. Post‐thaw sperm quality was evaluated in terms of subjective motility rate, quality motility score (0=no movement; 5=rapidly swimming spermatozoa), duration of motility and vitality (eosin–nigrosin staining). Post‐thaw sperm motility rate was greater in methylglycol (76–88%), compared with DMSO (23–59%). In general, the highest quality motility scores were observed when sperm was thawed at 30 °C and triggered in 1% NaHCO₃ (3.5–4.3). Duration of motility was longer when triggered in 1% NaHCO₃ (95–120 s) compared with 0.29% NaCl (69–107 s). Sperm vitality was not affected by any of the parameters tested and varied from 51% to 69% intact sperm. Brycon insignis sperm frozen in methylglycol combined with any of the extenders tested and using the methods described above yields motility above 57% and that should last long enough to fertilize oocytes.