Age-specific patterns in density-dependent growth of white crappie, Pomoxis annularis

The relationship between growth in white crappie, Pomoxis annularis Rafinesque, and climate, lake morphometry and population density was studied in 103 Texas reservoirs. Growth of age‐0 through age‐3 white crappie was significantly (P≤0.05) related to latitude, growing season and surface area. Growth of age‐1 through age‐5 white crappie also was related to annual precipitation. Multiple linear regression models with precipitation and mean catch per unit effort (CPUE) of white crappie as independent variables explained 14–22% of the variation in growth of age‐0, ‐1 and ‐2 white crappie. By contrast, growth of age‐3, ‐4 and ‐5 white crappie was unrelated to CPUE. Apparently, between age 2 and 3, the density of white crappie in Texas reservoirs falls below a density‐dependent growth threshold beyond which growth is no longer related to density. This density‐dependent threshold was most likely related to angler harvest of larger, older white crappie.     

A Strategy for Sperm Cryopreservation of Atlantic Salmon,Salmo salar,for Remote Commercial‐scale High‐throughput Processing

Sperm cryopreservation is an essential tool for long‐term storage of genetic resources for aquaculture fishes. The goal of this study was to develop an efficient and streamlined protocol for high‐throughput processing for sperm cryopreservation in Atlantic salmon, Salmo salar. The objectives were to evaluate: (1) osmolality of blood serum for determining extender osmolality, (2) effects of extenders for fresh sperm dilution and refrigerated storage, (3) effects of methanol and dimethyl sulfoxide (DMSO) on fresh sperm motility, and (4) motility and fertility after thawing. In this study, sperm samples were collected at a hatchery site in Canada and shipped to a freezing site located 2200 miles (3550 km) away in the USA. Evaluation of three extenders indicated that Mounib solution was suitable for diluting dry sperm for sample processing. Ten percent of methanol or DMSO was less toxic to sperm cells than was 15% within 30 min. Further testing with methanol at 5, 10, and 15%, and sperm solution : extender dilutions (v:v) of 1:1, 1:3, and 1:19 (at concentrations of 5 × 10⁷, 3 × 10⁸, and 1 × 10⁹ cells/mL) indicated that methanol at 5 and 10% showed less toxicity to fresh sperm within 1 h at sperm:extender dilutions of 1:1 and 1:3. Post‐thaw motility of sperm cryopreserved with 10% methanol was significantly higher than that with 10% DMSO, and fertility reflected those results (0–1% in DMSO vs. 38–55% in methanol). Further evaluation of sperm cryopreservation with 10 and 15% methanol at sperm dilution ratios of 1:1, 1:3, and 1:19 indicated that post‐thaw motility in 10% methanol was significantly higher than that in 15% methanol, and post‐thaw fertility in 10% methanol at 1:1 and 1:3 dilution ratios had fertilization rates similar to that of fresh sperm controls. Sperm samples from 12 males cryopreserved with 10% methanol showed male‐to‐male variation in post‐thaw motility (0–36%). Overall, a simplified standard protocol was established for cryopreservation of shipped sperm of Atlantic salmon using extender without egg yolk and yielded satisfactory post‐thaw motility and fertilization rates. This procedure can be readily adopted by aquaculture facilities to take advantage of high‐throughput cryopreservation capabilities at remote service centers. Most importantly, this approach lays the groundwork for an alternative commercial model for commercial‐scale production, quality control, and development of industrial standards. Control of male variability and sperm quality remain important considerations for future work.     

鲑鳟鱼类精子超低温保存研究进展

鱼类精子超低温冷冻保存技术发展至今已有60年的历史,取得了巨大的成就,尤其在鲑科鱼类的精液冷冻保存及应用方面。经过统计发现,国外学者在精子超低温冷冻方面取得的主要突破都集中在鲑鳟鱼类领域。本文综述了鲑鳟鱼类精子超低温保存技术的发展史、相应的关键技术和冷冻保存研究的意义、基本生物学原理以及有关精液冷冻保存的研究现状与进展。     

Design and Cost Analysis of a Self‐contained Mobile Laboratory for Commercial‐scale Aquatic Species Cryopreservation

Although aquatic species cryopreservation protocols have been studied around the world over the past 60 yr., germplasm repository development efforts and commercialization have begun only recently. The goal of this project was to develop a self‐contained mobile laboratory for on‐site high‐throughput cryopreservation of aquatic species. The objectives of this study were to: (1) identify how a mobile laboratory would function in different operational scenarios, (2) customize an enclosed cargo trailer to function as a mobile laboratory, (3) evaluate the laboratory layout and ability of cryopreservation equipment to operate from generator power, and (4) document the investment costs for private and public groups to integrate a mobile laboratory into an existing cryopreservation facility at three levels of automation and estimate the total cost per trip based on hypothetical assumptions for two scenarios (aquaculture production and repository development). There were three operational designs identified for the mobile laboratory: (1) self‐contained work inside the unit using generator power, (2) work inside the unit using external facility power, and (3) using the equipment inside of a host facility. The investment costs for a base‐level mobile laboratory ranged between US$5670 and US$5787 for private groups and between US$5208 and US$5315 for public groups. With the addition of a range of automated processing equipment, total investment costs ranged from US$13,616 to US$103,529 for private groups and US$12,494 to US$94,891 for public groups. The total cost per trip to cryopreserve sperm of 59 blue catfish, Ictalurus furcatus, males to produce 6300 0.5‐mL French straws was estimated to range from US$6089 to US$14,633 for private and between US$5703 and US$16,938 for public groups depending on the level of automation. Total cost per trip to cryopreserve sperm of 500 males of five different species in the genus Xiphophorus to produce 641 0.25‐mL French straws was estimated to range from US$6653 to US$7640 for private and US$7582 to US$8088 for public groups depending on level of automation. Overall, a commercial‐scale mobile laboratory was developed that can assist current germplasm activities and support future repository and industry development, and the layout information provided can help others to design and build comparable units.     

乌克兰鳞鲤精子超低温冷冻保存方法研究

为研究适用于乌克兰鳞鲤精子的超低温冷冻保存方法,分析比较3种稀释液[Hank′s、Cortland、Freezefish冻精稀释液,精子与每种稀释液均设置3种比例(1∶1、1∶3、1∶5)]及3种体积分数为10%的抗冻剂(二甲基亚砜、1,2-丙二醇和丙三醇)对乌克兰鳞鲤精子低温(4 ℃)保存活力的影响;运用筛选出的冷冻保护液及稀释比例,分析比较3种“3步冷冻法”以及3种解冻温度(20、30、40 ℃)对乌克兰鳞鲤精子活力的影响。试验结果表明,采用Hank′s作为稀释液,10%二甲基亚砜为抗冻剂,精子与稀释液比例为1∶3,平均降温速率为12 ℃/min,解冻温度为30 ℃时,精子活力最高(>68%)。通过对稀释液、抗冻剂、稀释比例、降温速率和解冻温度的层层筛选,建立了适宜乌克兰鳞鲤精子超低温冷冻保存的方法,在其种质保护方面具有重要意义,为开展其他鱼类精子超低温冷冻保存提供参考。     

史氏鲟精子超低温冷冻保存研究

比较了史氏鲟精子在3种不同配比浓度稀释液的保存效果。试验结果表明,配方Ⅲ作为稀释液,8%甲醇作为抗冻剂,二步法超低温(-196℃)冷冻保存,5h后取出,38℃水浴解冻取得最好的冻后激活率,解冻后激活率为(52.3±3.5)%。解冻精子分别采用井水和激活液D(10mmol/L Tris+10mmol/L NaCl+25mmol/L Glu,pH 8.0)激活,进行人工授精。结果显示配方Ⅲ冻精采用激活液D激活授精获得最高受精率为68.56%,最高孵化率为52.91%。本次试验表明,1～2mmol/L范围内,低浓度K+比高浓度K+对史氏鲟精子保存有利;52～82mosmol/kg范围内,高渗稀释液有利于史氏鲟精子的保存;且激活授精方法是影响冻精受精率和孵化率的关键因素之一。     

Physio-Chemical Characteristics of Seminal Plasma and Development of Media and Methods for the Cryopreservation of European eel Sperm

The high sperm density, together with the short spermatozoa swimming time, makes European eel sperm manipulation and assessment for quality difficult. Two diluting media (K15 and K30) previously designed for Japanese eel sperm were tested. After 24 h, European eel sperm showed significant reduction in the percentage of motile spermatozoa after activation and different motility parameters (VAP, angular velocity; VCL, curvilinear velocity; VSL, straight line velocity; BCF, beating cross frequency), concluding that these media are not suitable to preserve the sperm of this species. After a hormonal treatment to induce spermiation, sperm volume, density and motility were recorded at weekly samplings. The variation of the osmolality (325–330 mOsm kg⁻¹), pH (8.4–8.6) and the ionic composition (concentration of Na⁺, K⁺, Mg²⁺ and Ca²⁺) of the seminal plasma were registered. Physio-chemical results were related with sperm quality throughout the treatment, to determine which must be the suitable characteristics of one extender for the sperm of this species, and to find the best conditions to obtain suitable cryopreservation media for European eel sperm. K⁺ concentration increased, while Ca²⁺ and Mg²⁺ concentrations showed a progressive reduction in correlation with the sperm quality improvement. Na⁺ showed a decreasing, but not significant tendency. P1 and P2 freezing media were designed considering the physio-chemical parameters as well as the ionic composition shown by the best quality sperm samples, and then compared with the previously described solutions, TNK and K30. Sperm quality was determined, checking the percentage of motile spermatozoa and motility parameters using computer-assisted sperm analysis (CASA) software. Samples were frozen after dilution (1:5, 1:20, 1:100) in different freezing media supplemented with 10% dimethyl sulfoxide (DMSO). After thawing, samples frozen with low dilution ratio (1:5) in TNK and P1 media showed higher, although not significant, spermatozoa survival (35.5 ± 14.5 and 36.6 ± 6.7%). The addition of l-α-phosphatidylcholine to the media seems to have a positive effect, as reported in the Japanese eel.     

Cryopreservation of filefish (Thamnaconus septentrionalis Gunther, 1877) sperm

The present study examined the possibility of long‐term storage, by cryopreservation in liquid nitrogen, of the sperm of filefish (Thamnaconus septentrionalis). Changes in motility, survival rate, ultrastructure and fertilization rate of the sperm after freezing and thawing were tested. For selection of the immobilizing solution, artificial seawater (ASW) of 250, 350 and 450 mOsmol kg^?1 were tested. Sperm motility was significantly inhibited in 350 mOsmol kg^?1 ASW, and restored entirely after 100% ASW (1200 mOsmol kg^?1) was added. Two cryoprotectants, dimethyl sulphoxide and glycerol, were employed. The sperm was diluted at the ratio of 1:6 with the extenders, and frozen at a freezing rate of ?40°C min^?1 to ?100°C after equilibration for 10 min at room temperature, followed by plunging into liquid nitrogen. The highest post‐thawed sperm motility and survival rate were obtained with 5% glycerol. Afterwards, the effect of different freezing rates was examined using 5% glycerol as a cryoprotectant, and the rate of ?30°C min^?1 to ?100°C showed the best result.     

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.     

Commercial‐scale Validation of Temperature‐step Rearing on Growth Physiology in Turbot,Scophthalmus maximus

The aim of this study was to investigate the possible benefit of “temperature‐steps” (T‐steps) rearing for juvenile turbot (initial weight 15.1 g) under realistic production scale and to determine whether initial growth advantage is maintained throughout the rearing period to market size. One group (called T‐step 22‐19‐16) of juvenile turbot was reared at three different temperatures, that is, 22 C (from 17 to 60 g) followed by 19 C (from 60 to 100 g) and 16 C (>100 g); another group (called T‐step 19‐16) at two temperatures, that is, 19 C (from 17 to 100 g) and lowered to 16 C (>100 g); and the third group (called C16) at one constant temperature, that is, 16 C. Relative growth was significantly higher in the two T‐step groups, with the T‐step 19‐16 showing the highest overall growth. Feed conversion efficiency was highest in the 19‐16 group. Only minor effects of the experimental rearing on blood physiology were found, with one notable exception of inverse relationship between plasma glucose and growth. Overall, these findings indicate that a short interval of rearing fish at high temperature during the early juvenile phase may have a long‐term effect on biomass increment in turbot. This is an important finding for the turbot industry.     

条纹锯精液超低温冷冻保存研究

为建立条纹锯精液超低温冷冻保存方法,实验采用计算机辅助精子分析系统（CASA）分析了采用6种抗冻保护剂（GLY[甘油]、DMSO[二甲基亚砜]、PG[丙二醇]、EG[乙二醇]、METH[甲醇]、DMA[二甲基乙酰胺]）在4种浓度（5%、10%、15%、20%,v/v）下对条纹锯精液的冷冻保存效果。结果发现,以HBSS为稀释液,采用程序降温仪分步降温冷冻保存条纹锯精液,37℃水浴解冻后的精子中,15% PG 作为抗冻保护剂的精子运动率最高,达到（93.1±0.9）%,与鲜精差异不显著（P>0.05）,15% PG 作为抗冻保护剂的精子水浴解冻后精子的运动速度最高,平均直线速度、平均曲线速度、平均路径速度分别达到了（88.3±0.3）μm/s、（76.2±0.5） μm/s、（86.7±0.7） μm/s,与鲜精差异不显著（P>0.05）。在不同种类及不同浓度抗冻保护剂保护下,15% PG 作为抗冻保护剂的精子解冻后 1 min内运动率变化与鲜精差异不显著（P>0.05）。研究表明,15% PG为条纹锯最佳抗冻保护剂,可用于条纹锯精液的超低温冷冻保存。     

High‐throughput Cryopreservation of Sperm from Sex‐reversed Southern Flounder,Paralichthys lethostigma

The Southern flounder, Paralichthys lethostigma, is a valuable aquaculture fish with established markets in the USA. All‐female production in this species is an important technology for aquaculture because the females usually have body sizes twice those of males at the same age, and sex‐reversed males (genotypic XX neomales) are used for all‐female production by crossing with genetically normal females. However, sperm volume from the neomales is usually small (<0.5 mL) and limits their application for all‐female fish production. Cryopreservation of sperm from these sex‐reversed neomales will provide access on demand with increased efficiency to extend the application of neomales. The goal of this study was to develop a protocol for cryopreservation of sperm from the Southern flounder by using an automated high‐throughput processing system. The objectives were to: (1) determine the effect of osmolality on activation of sperm motility; (2) evaluate the effect of extender solutions on sperm motility capacity; (3) evaluate the acute toxicity of cryoprotectants (dimethyl sulfoxide [DMSO], propylene glycol, and polyethylene glycol) on sperm motility, and (4) estimate the effect of cooling rate on sperm cryopreservation and post‐thaw fertilization. Sperm motility was activated when osmolality was 400 mOsmol/kg or higher. Of the three extender buffers tested, HEPES4‐(2‐hydroxyethyl)‐1‐piperazineethanesulfonic acid (HEPES) at 300 mOsmol/kg resulted in better protection for sperm motility than did Hanks' balanced salt solution and Mounib solution at 300 mOsmol/kg during 7 d of refrigerated storage. After 30 min equilibration with the cryoprotectant of 15% DMSO, sperm motility was 24 ± 21% (fresh sperm motility without any cryoprotectants was 42%). After cooling at a rate of 20 C/min, post‐thaw sperm motility was 8 ± 5% and fertilization was 63 ± 40% evaluated at the 32–64 cell stage (5 × 10⁵ sperm per egg). Overall, a protocol was developed for sperm cryopreservation in the Southern flounder with high‐throughput processing, which provides a tool to preserve the valuable genetic resources from neomale flounders, and enables germplasm repository development for the Southern flounder.     

Use of ACP‐104 at Different Dilutions for Sperm Cryopreservation of Dourado,Salminus brasiliensis

The dourado, Salminus brasiliensis, is an important fish in South American river basins. The fish‐farming potential and concerns for its conservation in watersheds has stimulated studies with this species. This study compared the effect of different dilutions of two cryoprotectant solutions on the quality of cryopreserved dourado sperm (duration of motility/motility rate) post‐thawing using three different activating solutions. The cryoprotectant solution ACP‐104 was compared with the cryoprotectant solution most commonly used for dourado, which consists of a mixture of dimethyl sulfoxide, glucose, egg yolk, and distilled water. The semen was mixed following dilutions of semen : cryoprotectant solution: 1:3, 1:9, 1:15, 1:21, and 1:27. The cryopreserved samples were activated using distilled water, 0.45% NaCl or 1% NaHCO₃. The results showed differences in thawed semen using the different activating solutions and cryoprotectant solutions. The glucose‐based cryoprotectant not only resulted in sperm motility equal to or better than that produced by ACP‐104, but the quality was not affected by the dilution. However, the sperm quality improved with increasing dilutions for ACP‐104. Under the conditions tested, the standard cryoprotectant solution is recommended for the cryopreservation of dourado semen because it requires a lower solution volume and, therefore, reduced storage space for the same volume of semen.     

Cryopreservation of strip spawned sperm using non‐programmable freezing technique in the blue mussel Mytilus galloprovincialis

In this study, a non‐programmable freezing technique has been developed with the strip spawned blue mussel (Mytilus galloprovincialis) sperm. The key parameters optimized including (1) cryoprotectant agents (CPAs); (2) cooling temperature; (3) thawing temperature; (4) sugar and amino acid supplementation and (5) sperm to egg ratio. The fertilization rate and/or integrity of plasma membrane and acrosome were used as sperm quality assessment indicators. The highest post‐thaw sperm fertilization rate of 95% was achieved, when sperm were cryopreservated in 8% dimethyl sulfoxide at 7.8 cm above the liquid nitrogen surface and thawed in a 60°C seawater bath. The addition of glucose, sucrose or trehalose in dimethyl sulfoxide did not, whereas 0.8% glycine did significantly improve the post‐thaw sperm fertilization rates. The fluorescent evaluation has demonstrated that the addition of glycine significantly improved the post‐thaw sperm acrosome integrity, revealing a positive role of glycine in the improvement of post‐thaw sperm quality in blue mussels.     

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.     

Cryopreservation of sperm in farmed blacklip abalone (Haliotis rubra Leach, 1814)

This study developed a technique of sperm cryopreservation using liquid nitrogen (LN) vapour in farmed blacklip abalone Haliotis rubra through evaluating the following five key factors: (1) cryoprotectant agent (CPA) toxicity; (2) cooling temperature; (3) thawing temperature; (4) sperm to egg ratio and (5) sugar addition, using sperm motility or fertilization rate as quality assessment indicators. The results demonstrated that 6% dimethyl sulfoxide (DMSO) was the best single CPA for sperm cryopreservation in this species. The highest post‐thaw sperm motility was achieved when sperm were exposed to LN vapour for 10 min at 5.2 cm above the LN surface and thawed at 60°C and recovered at 16°C in seawater baths. Post‐thaw sperm motility was found to be significantly higher when 6% DMSO was used in combination with 1% or 2% glucose than 6% DMSO alone. Further evaluation of fertilization rate between these CPAs showed that 6% DMSO+2% glucose achieved the highest fertilization rate of 70% at a sperm to egg ratio of 10 000:1.