Effect of methanol concentration and thaw rate on the viability and fertility of cryopreserved Arctic char,Salvelinus alpinus (L.), spermatozoa

In two trials, Arctic char (Salvelinus alpinus) semen was frozen in 0.5 mL straws using extenders consisting of 0.3 M glucose and 10%, 12.5% or 15% methanol. Cryopreserved semen was thawed by immersing straws in 25 °C water for 17 s (11.6 °C s^?1) or in 5 °C water for 60 s (3.3 °C s^?1). The viability of the frozen–thawed semen was measured by determining post‐thaw motility and sperm membrane integrity. Two fertility trials were also conducted. There was no effect of trial or thaw rate on post‐thaw sperm viability or fertility. Use of 15% methanol in the extender resulted in the highest overall percentage of sperm motility and fertility. Use of 12.5% methanol as a cryoprotectant resulted in a higher per cent post‐thaw motility and a lower percentage of dead cells than did 10% methanol. Thus, levels of methanol higher than the commonly used 10% are beneficial for cryopreserving Arctic char sperm.     

Cryopreservation effects on the sperm quality of cachama blanca Piaractus brachypomus (Cuvier 1818)

The effects of straws volume, cryoprotectants and thawing temperatures were evaluated on the sperm quality of cachama blanca Piaractus brachypomus (Cuvier), an important Colombian fish species. Sexually mature fish were induced to ovulation or spermiation with a carp pituitary extract. A pool of suitable sperm samples was diluted in glucose, egg yolk, dimethyl sulphoxide (DMSO‐10%), methanol (MET‐10%) or ethylene glycol (ETG‐5%) and packed in 0.5, 2.5 or 5.0 mL straws and frozen in nitrogen vapour. The thawing process was performed in a 35 or an 80 °C water bath. The fertility was evaluated after 6 h post fertilization. The highest motility percentage (33 ± 3%) was observed with sperm cryopreserved with DMSO, packed in 5 mL straws and thawed at 35 °C. The treatments with DMSO and MET packed in 0.5 and 5.0 mL straws and thawed at 35 °C showed the highest fertility (higher than 71%) and the lowest fertility was obtained with MET‐2.5 mL (9 ± 5%). In all the treatments, a significant decrease in the sperm quality was observed at 80 °C. Sperm cryopreserved with DMSO‐10% or MET‐10%, packed in 2.5 or 5.0 mL straws are suitable to achieve acceptable fertilization and to fertilize high amounts of eggs.     

Cryopreservation of sea bass (Dicentrarchus labrax) spermatozoa in experimental and production simulating conditions

A sperm cryopreservation protocol adapted from turbot, was tested on sea bass using either 250-μL straws or 1.5-mL cryovials. A dilution to 1/3 in Mounib s extender and a cooling rate of −65 °C·min⁻¹ allowed frozen sperm to recover an initial motility similar to that of fresh sperm at thawing; however, significant differences in motility (P < 0.001, n = 10 fish semen) were observed at further post-activation times, the motility decrease being faster in thawed sperm. At the experimental scale, triplicate inseminations of 2-mL aliquots (approximately 2 000 eggs) showed a significant fertility decay of thawed sperm compared to that of fresh sperm (P < 0.01, n = 12 fish semen) when a discriminating 35·10³ spermatozoa to egg ratio was applied. When 70·10³ and 200·10³ spermatozoa per egg were provided in the same experimental conditions, no significant difference appeared between the fertilisation rates of fresh and thawed sperm. In order to validate the procedure for production or cryobank purpose, a scaled-up protocol was established. Two and 50 mL batches of eggs (approximately 2·10³ and 50·10³ eggs, respectively) were inseminated in triplicate using either fresh or thawed individual sperms of 5 males with 200·10³ spermatozoa per egg. The mean fertility decreased by 23.5 % due to cryopreservation. This decline was explained by the loss of fertility of only one sperm, and only in large-volume conditions, probably due to the delay of use after thawing.     

Development of cryopreservation methods for silver barb (Barbodes gonionotus,Bleeker) spermatozoa by manipulation of cooling rates in dry shipper

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.     

A uniform method for cryopreservation of semen of the salmonid fishes Oncorhynchus mykiss (Walbaum), Salmo trutta f. fario L., Salmo trutta f. lacustris L., Coregonus sp.

The present study describes a uniform method for cryopreservation of semen of Salmonidae (Oncorhynchus mykiss (Walbaum), Salmo trutta f. fario L., Salmo trutta f. lacustris L., Coregonus sp.). It presents a new type of extender and experiments demonstrating that warming of frozen/thawed semen to 20°C prior to fertilization significantly increases the fertilization rate. Freezing is performed in straws in the vapour of liquid nitrogen and for insemination a diluent technique is used. The consistency of the method was tested by repeating the experiments with different batches of semen and eggs. The following fertilization rates (% of control) were obtained: Oncorhynchus mykiss: 89.6 ± 16.0% (mean ± standard deviation, n= 25, n of control = 20, sperm/egg ratio of 1.6 ± 0.2 × 10⁶ spermatozoa/ egg). Salmo trutta f. fario: 93.8 ± 6.4% (n= 12,9.9 ± 1.2 × 10⁶spermatozoa/egg), Coregonus sp.: 92.8 ± 2.4% (n= 6, 0.5 × 10⁶ spermatozoa/egg), Salmo trutta f. lacustris: 85.0 ± 8.4% (n= 12, 4.8 ± 1.4 × 10⁶ spermatozoa/egg).     

Improved Cryopreservation of Sperm of Paddlefish (Polyodon spathula)

Experiments were performed to improve protocols for sperm cryopreservation of paddlefish (Polyodon spathula), a species for which there has been limited study. The first experiment was conducted to investigate the effects of two extenders (modified Tsvetkova’s extender: mT and modified Hanks’ balanced salt solution: mHBSS) in combination with methanol (MeOH) and dimethyl sulfoxide in two concentrations (5 and 10%) on the postthaw motility and fertilization rates of cryopreserved sperm. The highest postthaw motility (85 ± 5%) was observed when sperm were frozen using mT extender with 10% MeOH as cryoprotectant. Extenders (P = 0.0018) and cryoprotectants (P = 0.0040) each had a significant effect on the postthaw motility of paddlefish sperm. The highest fertilization (80 ± 3%) was found when eggs were fertilized with sperm frozen with mT extender in combination with 10% MeOH. However, there was no significant difference among fertilization rates when MeOH was used as a cryoprotectant in either concentration or in combination with either mT or mHBSS extenders. In the second experiment, 4000 eggs were fertilized with the pooled contents of five straws of thawed sperm (total volume of 1.25 mL) using mT extender in combination with 5% MeOH, and hatch rates as high as 79 ± 5% were observed. A third experiment was also conducted to clarify the role of MeOH concentration; however, no significant difference was found among fertilization and hatch rates when either 5 or 10% MeOH was used as a cryoprotectant. These results suggest that MeOH is a safe and reliable cryoprotectant for freezing of paddlefish sperm and obtaining viable postthaw sperm for consistent fertilization and hatch rates. Further, this experimental protocol is relatively simple and applicable for commercial hatchery production of paddlefish.     

Simulation modelling of high‐throughput cryopreservation of aquatic germplasm: a case study of blue catfish sperm processing

Emerging commercial‐level technology for aquatic sperm cryopreservation has not been modelled by computer simulation. Commercially available software (ARENA, Rockwell Automation, Inc. Milwaukee, WI) was applied to simulate high‐throughput sperm cryopreservation of blue catfish (Ictalurus furcatus) based on existing processing capabilities. The goal was to develop a simulation model suitable for production planning and decision making. The objectives were to: (1) predict the maximum output for 8‐h workday; (2) analyse the bottlenecks within the process, and (3) estimate operational costs when run for daily maximum output. High‐throughput cryopreservation was divided into six major steps modelled with time, resources, and logic structures. The modelled production line processed 18 fish and produced 1164 ± 33 (mean ± SD) 0.5‐mL straws containing one billion cryopreserved sperm. Two such production lines could support all hybrid catfish production in the United States and 15 such lines could support the entire channel catfish industry if it were to adopt artificial spawning techniques. Evaluations were made to improve efficiency, such as increasing scale, optimizing resources, and eliminating underutilized equipment. This model can serve as a template for other aquatic species and assist decision making in industrial application of aquatic germplasm in aquaculture, stock enhancement, conservation and biomedical model fish.     

Cryopreservation of Atlantic cod (Gadus morhua) sperm in large‐volume straws: applications for commercial production and gene banking

In our study, we used a full factorial analysis of variance design to examine the effects of diluent [Mounib's sucrose‐based diluent+hen's egg yolk (EY) and Hanks' balanced salt solution (HBSS)+EY], freezing rate (?2.5, ?5.0 and ?7.5 °C min^?1) and thawing rate (2.5, 5.0 and 7.5 °C min^?1) on motility and velocity of Atlantic cod sperm cryopreserved in 2.5 mL cryogenic straws. We found that post‐thaw sperm performance was strongly influenced by the presence of higher‐order interactions of the factors we tested. For all models broken down by diluent, the 2.5 °C min^?1 thawing rate had the lowest sperm motility recovery index. Mounib's sucrose‐based diluent+EY had the highest motility recovery index at all thawing rates. Mean per cent motility for fresh sperm (87.7±2.9%) was not significantly different than of sperm cryopreserved using Mounib's sucrose‐based diluent+EY, frozen at ?2.5 °C min^?1 and thawed at 5.0 °C min^?1 (77.1±2.9%). For Mounib's sucrose‐based diluent+EY, velocity was significantly higher with sperm thawed at 7.5 °C min^?1, than sperm thawed at 2.5 °C min^?1, while thawing rate had no effect for HBSS+EY. Our findings have implications for cod mariculture and aiding in conservation efforts for a dominant marine fish species.     

The effect of cryoprotectants on the motility and fertilizing capacity of cryopreserved African catfish Clarias gariepinus (Burchell 1822) sperm

The effect of six cryoprotectants was investigated on the cryopreservation of African catfish Clarias gariepinus (Burchell) sperm. Fructose (6%) solution buffered with NaHCO₃‐CO₂ was used as the diluent in the experiments. Glycerol (5–11%), ethylene glycol, methanol and propylene glycol (5–15%) and, finally, dimethyl sulphoxide (DMSO) and dimethylacetamide (DMA) (10%) were tested using various equilibration times (2–30 min). Sperm was frozen in 250‐μL straws in a programmable freezer (Cryocell‐15, BLS, Hungary) from 3 °C to ?4 °C at 4 °C/min and from ?4 °C to ?80°C at 11 °C/min. Thawing was carried out in a 40 °C water bath for 5 s. Fertilization and hatching trials were performed only with DMSO and DMA using 200 and 100 μL of diluted sperm (100 and 50 μL of pure sperm) and the dry and the wet fertilization methods. Ethylene glycol, glycerol, methanol and propylene glycol yielded poor results. An average post‐thaw motility rate of 44.0 ± 9.7% and 22.6 ± 18.1% was achieved after 10 min equilibration using DMSO and DMA respectively. Highest average fertilization (86.8 ± 3.1%) and hatching (67.1 ± 11.9%) rates were achieved with DMA and DMSO, respectively, 200 μL of diluted sperm and the wet fertilization technique. The use of cryoprotectants increased the percentage of malformed larvae compared with the control groups. We found that DMA at a 10% concentration was equally as suitable for the cryopreservation of African catfish sperm as DMSO.     

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.     

Cryopreservation of Arctic charr, Salvelinus alpinus (L.), semen in various extenders and in three sizes of straw

Cryopreservation of Arctic charr Salvelinus alpinus (L.) semen was investigated using three diluents, three cryoprotectants [10% dimethyl sulphoxide (DMSO), 10% dimethylacetamide (DMA) or 20% glycerol] and three sizes of straw. The three diluents and three cryoprotectants were combined, resulting in nine extenders. One part semen was added to three parts extender, and motility was evaluated to assess the toxicity of six of the extenders. Semen in nine extenders was frozen in 0.5‐mL straws using liquid nitrogen vapour. Semen extended in 0.3 m glucose and each of the cryoprotectants was also frozen in 0.5‐mL, 1.7‐mL (flat) or 2.5‐mL straws. The freezing rate in each size of straw was measured. Fertility trials were conducted to determine the post‐thaw viability of the frozen semen. The motility of activated spermatozoa was higher in the DMA and DMSO extenders than in the glycerol extender. For the trial using 0.5‐mL straws, post‐thaw fertility results were higher for all extenders containing DMSO, or 0.3 m glucose and DMA, than for all other combinations of diluent and cryoprotectant. For the straw size comparison, the highest fertility was obtained for the 1.7‐mL straw using either DMSO or DMA and for the 2.5‐mL straw using DMSO. For all cryopreservation trials, fertility was low for extenders containing glycerol.     

Effect of cryoprotectants, extenders and freezing rates on the fertilization rate of frozen striped catfish, Pangasius hypophthalmus (Sauvage), sperm

The effects of four cryoprotectants (methanol, MeOH; dimethyl sulphoxide, DMSO; dimethyl acetamide, DMA; and ethylene glycol, EG), three extenders (calcium‐free Hanks' balanced salt solution, C‐F HBSS, Hanks' balanced salt solution, HBSS and sodium chloride, NaCl) and two different freezing procedures (one‐ and two‐step) on the cryopreservation of striped catfish (Pangasius hypophthalmus (Sauvage)) sperm were investigated. Sperm were frozen using a controlled‐rate freezer in 250 μL straws and stored for 2 weeks in a liquid nitrogen (LN₂) container. They were then airthawed at room temperature, and fertilization, motility and viability were assessed. The highest fertilization rate of 41% (81% of control) was achieved with the combination of 12% DMSO and 0.9% NaCl using a one‐step freezing procedure (10°C min^?1). Also, DMA resulted in a higher fertilization rate (30% or 51% of the control) than MeOH (18% or 38% of the control) or EG (8% or 12% of the control). In addition, the three extenders used did not affect fertilization rates after cryopreservation with each cryoprotectant. There were no significant differences among the three cryoprotectant concentrations and between the one‐ and two‐step freezing procedures. However, fertilization rates of cryopreserved sperm were significantly lower than the controls (P<0.05). The results of this study indicate that high fertilization rates of striped catfish eggs can be achieved using cryopreserved sperm when frozen at 10°C min^?1 in DMSO or DMA with either 0.9% NaCl or C‐F HBSS.     

Effect of extender composition and freezing rate on post-thaw motility and fertility of Arctic char, Salvelinus alpinus (L.), spermatozoa

The effects of extender composition and freezing rate on motility and fertility of frozen‐thawed Arctic char, Salvelinus alpinus, spermatozoa were investigated. Three freezing rates, two semen diluents and three cryoprotectants were tested. Semen frozen in 0.3 mol L^?1 glucose diluent with 10% methanol as a cryoprotectant or in a diluent described by Lahnsteiner with 10%N,N‐dimethylacetamide (DMA) resulted in the highest sperm motility. Fertility was the highest for semen frozen in a glucose–methanol extender but was not significantly different than that for semen frozen in Lahnsteiner's diluent with 10% DMA. Dimethyl sulphoxide (DMSO) at 10% was a relatively ineffective cryoprotectant with either semen diluent. Semen frozen at 6 cm above the surface of liquid nitrogen resulted in a higher post‐thaw sperm motility and fertility than semen frozen at 5 cm. The addition of 7% fresh egg yolk to glucose diluent containing methanol or DMSO did not improve the fertility of frozen‐thawed spermatozoa. However, the addition of 7% fresh egg yolk to glucose–DMA extender significantly improved the fertilization percentages of frozen‐thawed spermatozoa. In conclusion, dilution of semen 1:3 in 0.3 mol L^?1 glucose with 10% methanol and freezing 6 cm above the surface of liquid nitrogen (freezing rate of 40±8°C min^?1, mean±SD from ?5 to ?55°C) is a promising protocol for cryopreservation of Arctic char semen.     

Evaluation of cryodamage in small abalone,Haliotis diversicolor,spermatozoa by using a flow cytometer

We used flow cytometry to determine the quality of small abalone, Haliotis diversicolor, sperm before freezing and after thawing. We investigated the effects of cryopreservation on the characteristics of small abalone semen and determined the motility and fertilization capacity of the pre-freezing and post-thawed sperm. The percentages of motility and fertility were 61 ± 2% and 67 ± 1%, respectively, for the post-thawed sperm and 90 ± 4% and 92 ± 0%, respectively, for the pre-freezing sperm. Sperm cells were stained with specific fluorescent dyes to measure plasma and acrosomal membrane integrity, mitochondrial status, oxidation level, DNA compaction, and viability through flow cytometry. The frozen–thawed sperm exhibited significantly higher mitopotential activity (p < 0.05, damaged mitochondria; 25.01 ± 1.18%) and oxidation value (p < 0.01, free radicals; 63.79 ± 3.93%) compared with the pre-freezing sperm. The oxidation level was the most sensitive signal of the cryopreservation-induced small abalone sperm damage. Flow cytometry is valuable for the objective, accurate, and rapid assessment of pre-freezing and post-thaw small abalone sperm quality.     

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 sea cucumber Apostichopus japonicus (Selenka) sperm

A simple and convenient method for the cryopreservation of sea cucumber Apostichopus japonicus (Selenka) sperm was tested in the present study. The highest motility (76.7±2.9%) of post‐thawing sperm was obtained in 15% dimethyl sulphoxide (DMSO) with a 1:9 dilution (semen volume to DMSO volume) when 0.5 mL semen–DMSO mixture was frozen at 6 cm above liquid N₂ in a closed styrofoam box. After thawing, sperm cryopreserved in glycerol almost lost motility entirely. Although there was no significant difference in percentage of motile sperm between 15% and 20% DMSO, the duration of sperm motility of 15% DMSO group was longer than that of 20% DMSO group. The motility of post‐thawing sperm enhanced when the dilution ratio of semen increased from 1:1 to 1:9. Morphological changes such as the loss of mitochondria, swollen plasma membrane and broken or rolled‐up tails were observed in post‐thawing sperm using an eosin–nigrosin staining. The fertility of cryopreserved sperm was significantly lower than that of unfrozen sperm. The 10‐fold increase in sperm to egg ratio resulted in double fertility for cryopreserved sperm, and about 70% fertility relative to the control.     

Improvement in wild endangered Persian sturgeon,Acipenser persicus (Borodin, 1897) semen cryopreservation by 2‐hydroxypropyl‐beta‐cyclodextrin (HβCD)

In this study, cryopreservation feasibility of Persian sturgeon (Acipenser persicus) and the effect of different doses of 2‐hydroxypropyl‐beta‐cyclodextrin on thawed spermatozoa quality (motility duration and motility percentage) were investigated. For freezing, semen of seven male individuals was pooled in equal volumes and diluted with 4°C [Tris‐HCl (100 mM), pH = 8, DMSO 10%] extenders containing 0, 5, 10, 15 mM of HβCD in a ratio of 1:1(semen/extenders). Then semen was filled into 0.5‐mL straws, and was frozen with vapour of liquid nitrogen at 4‐cm above surface of liquid nitrogen. After 3 min, straws were plunged in to liquid nitrogen. Thawing was performed at 40°C water baths for 15 s. Motility duration of the 10 mM HβCD treated spermatozoa at days 14 (228.98 ± 16.39) and 56 (199.66 ±21.78) were longer than other treatments. In day 56, the motility percentage in treatment with 10 mM was significantly higher (16.14 ± 2.54) (P < 0.05) compared with 5 mM treatment (8.75 ± 2.47) (P < 0.05). Therefore, it is recommended that 10 mM of HβCD can be used as an additive cryoprotectant for increasing cryopreserved spermatozoa quality in this species.     

Early out-of-season induced spawning of channel catfish Ictalurus punctatus (Rafinesque) conditioned in heated earthen ponds

This study documents early out-of-season induced spawning of channel catfish Ictalurus punctatus. During the early spring (February–April) of 1999, 2000 and 2001, ponds containing (1) male and female channel catfish (mixed-sex ponds) or (2) male channel and blue catfish I. furcatus only, or female channel catfish only (single-sex ponds) were heated to 24–30°C to encourage gonadal maturation and spawning. Unheated ponds were stocked with males and females and were monitored during the duration of heating. When natural spawning occurred in the heated ponds, the fish were captured by seining and unspawned females were injected with 100 μg kg⁻¹ of synthetic leutenizing hormone-releasing hormone. Injected females were either paired with males or held in communal all-female groups, and monitored for ovulation. Eggs were collected and fertilized with sperm of channel catfish or blue catfish. Females paired with males were induced to spawn 44 days (mixed-sex ponds) and 50 days (single-sex ponds) before natural spawning occurred in unheated ponds. Spawning latency (the time between injection and ovulation) and the percentage of neurulated embryos from eggs fertilized using channel catfish sperm was not different between spawning before the natural season (P=0.68) and during the natural season in fish from mixed-sex ponds (P=0.57). Females held in all-female groups produced eggs 34 days before the onset of spawning in unheated ponds. Spawning latency was not different between spawns before and during the natural season (P=0.16), and the percentages of neurulated embryos from eggs fertilized with channel catfish sperm (P=0.76) or blue catfish sperm (P=0.77) before or during the natural season were not different. This study demonstrates the feasibility of conditioning of channel catfish females for early out-of-season induced spawning in the laboratory.     

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).     

Early Innate Immune Response of Mannose‐binding Lectin and Lysozyme in Juvenile Channel Catfish,Ictalurus punctatus

Innate immune proteins mannose‐binding lectin (MBL) and lysozyme were studied in channel catfish, Ictalurus punctatus, during two consecutive years at 2, 4, 6, 9, and 12 mos of age. Both groups maintained in indoor tanks in light/dark photoperiod at mean temperature 27 C. Dot‐blot enzyme linked immunosorbent assay (ELISA) for MBL and turbidometry lysozyme assay done to quantify the two innate proteins and means determined for two consecutive years. An increase in mean MBL was seen in 4‐mo catfish (26.9 ± 0.8 µg/mL) when compared with all other age groups. A decrease in mean MBL was seen in 6‐ and 9‐mo catfish when compared with all other age groups. Both 2‐mo (21 ± 1.4 µg/mL) and 12‐mo (19.9 ± 0.4 µg/mL) catfish were very similar in mean MBL concentrations. The greatest increase in mean lysozyme was seen at 4 mo (15 ± 5.0 µg/mL). Juvenile channel catfish could produce lysozyme at 2 mo equivalent to lysozyme concentrations found in 9‐ and 12‐mo catfish. The greatest increases for MBL and lysozyme were seen in 4‐mo catfish. Mean total protein of 26.7 mg/mL and mean albumin/globulin ratio of 0.7 were found for the two groups of channel catfish sera.