Cryopreservation of sperm of common carp, Cyprinus carpio L.

Milt of common carp, Cyprinus carpio L. was cryopreserved in pellet form with the use of 12 extenders. Most efficient were: BE2 original extender (containing 85 mM NaCl, 50 mM KCl, 3 mm CaCl₂, 1 mm MgCl₂ with 10% dimethyl-acetamide (DMA) and 10% addition of hen's egg yolk) and Kurokura et al.'s extender with 15% DMA and 10% yolk (about 73% and 69% of eyed eggs, about 61% and 52% of swim-up larvae, respectively). Within the most effective treatments, survival from the eyed-egg stage to the swim-up stage was similar to that observed in the control group. Survival from the eyed-egg stage to the swim-up stage (percentage of eyed eggs was considered as 100%) was highly significantly and positively correlated with the actual rate of swim-up larvae.     

Factors Affecting Cryopreservation Efficiency and Enzyme Activity in Northern Pike,Esox lucius,Sperm

Abstract

Two freezing techniques (straws and pellets), three cryo-protectants (DMSO, glycerol, and DMA) in four concentrations, and several extenders were tested to determine their suitability for cryopreservation of northern pike, Esox iucius, sperm. Activity of aspar-tate aminotransferase (AspAT) and acid phosphatase (AcP) in cryo-preserved milt was determined. Fertilization ability of cryopreserved milt was affected by the freezing technique, by type and concentration of cryoprotectant, as well as by the kind of extender used. These factors also influenced AspAT and AcP activity assayed in cryopreserved sperm. Extender containing 0.6 M sucrose + 15% DMSO + 10% egg yolk was most suitable for cryopreservation of pike sperm in pellet form (90.5% of eyed eggs, as compared to control group, which was 89.1%).     

Computer-controlled freezing of rainbow trout Oncorhynchus mykiss (Walbaum) spermatozoa for routine programmes

The effects of extender composition and freezing rate on cryopreservation efficiency of refrigerated spermatozoa of rainbow trout Oncorhynchus mykiss (Walbaum) were evaluated in order to test the suitability of a computer-controlled ultrafreezer to cryopreserve milt samples obtained in field conditions and stored for several hours. A very highly significant first-order interaction between freezing rate and the type of extender was found. Six of the eight experimental variants did not differ significantly, resulting, after fertilization of eggs with cryopreserved sperm, in a range of 62.3–74.8% of eyed embryos. This procedure was effective for samples stored at 1 °C for 2 days.     

Characterization of rainbow trout milt collected with a catheter: semen parameters and cryopreservation success

Collection of fish milt by stripping risks the danger of milt contamination by urine. This may seriously influence milt characteristics and quality, including usefulness for cryopreservation. Urine contamination of milt may be avoided by using a catheter for sperm collection. The objectives of this study were to provide basic characteristics of milt collected with a catheter, to test the usefulness of this milt for cryopreservation, and to correlate characteristics of fresh and cryopreserved semen with sperm fertility rates. Milt from 25 rainbow trout Oncorhynchus mykiss (Walbaum) males were used. All samples were cryopreserved using the pellet method within 1 h of collection, using 0.6 m sucrose and 10% dimethyl sulphoxide (DMSO) as an extender. Catheterization resulted in semen of very good motility (> 90% motile spermatozoa) and high fertilization rates after cryopreservation (mean fertilization rate 81.8 ± 13.3% of control, at a sperm/egg ratio of 2.4 ± 0.3 × 10⁶). Osmolality of seminal plasma and concentrations of sodium, potassium and magnesium ions had low variability, which suggests that they are important for creating a stable environment for sperm storage in the sperm duct. Higher variability of certain seminal plasma characteristics, such as protein concentration and antiproteinase activity, suggests that these characteristics are related to individual semen features of particular males. A strong correlation of seminal plasma zinc concentration with protein concentration may reflect an importance of zinc in semen biology. Cryopreservation caused a significant release of protein and acid phosphatase from spermatozoa. Our results did not reveal any single characteristic of semen collected by catheter that could be used as a powerful predictor of cryopreservation success, presumably because all samples were of high quality.     

Effect of different avian egg yolk types on fertilization ability of cryopreserved common carp (Cyprinus carpio) spermatozoa

In spite of the fact that egg yolk from different avian species has successfully been used as an additive for the cryopreservation of sperm in mammalian species, its efficacy for cryopreserving fish sperm has not previously been tested comparatively. Therefore, the present study was carried out to determine the effect of egg yolks from different avian species, namely domestic chicken (Gallus gallus domesticus), turkey (Meleagris gallopavo) and quail (Coturnix coturnix), on post-thaw motility and fertilization ability of cryopreserved common carp spermatozoa. Egg yolks from chicken, turkey and quail were analysed for moisture, total fat, protein, cholesterol and phospholipid profile. Total fat and cholesterol contents of the turkey egg yolk were higher than chicken and quail egg yolks (p < 0.05). Semen was frozen according to conventional slow freezing procedure. The extender contained 350 mM glucose, 30 mM Tris and 5 % glycerol supplemented with different ratios of avian egg yolk (10, 15 and 20 %). Semen was equilibrated at 4 °C for 15 min and placed into 0.25-ml straws and frozen in liquid nitrogen vapour (for 10 min at ?120 °C) and finally stored in liquid nitrogen (?196 °C) tank. The frozen spermatozoa were thawed in a water bath at 35 °C for 30 s. Fertilization was conducted using a ratio of 1 × 10⁵ spermatozoa/egg. Cryopreservation experiments resulted in higher post-thaw motility and fertilization rates. Mean post-thaw motility of cryopreserved spermatozoa was between 45 and 80 %, and fertilization rates, expressed as the percentage of eyed embryos, ranged from 70 to 95 %. In conclusion, the present study showed that turkey and quail egg yolks are suitable alternatives to the chicken egg yolk for the cryopreservation of common carp spermatozoa.     

Short-term storage and cryopreservation of milt from Atlantic salmon and sea trout

Experiments on short-term preservation of sperm were performed with Atlantic salmon (Salmo salar). Fertility was maintained for up to 10 days when 2 mm thick samples were stored at 0° C under an oxygen atmosphere in the presence of antibiotics (125 IU penicillin and 125 μg streptomycin per ml sperm). Fertility was completely lost after 24 days. Sperm stored without antibiotics fertilized 100% of eggs after 6 days.

Cryopreservation was carried out with milt from Atlantic salmon and sea trout (Salmo trutta). Semen mixed with extender was frozen on dry ice (pellets) with subsequent storage in liquid nitrogen. Sperm pellets were thawed in a 0.12-M NaHCO₃-solution (10° C) before insemination. The suitability of an extender as previously described by Stoss and Holtz and of a 0.3-M glucose solution with the addition of 10% DMSO, was tested on two different batches of sperm and eggs in Atlantic salmon and sea trout. In addition, the extender earlier reported by Mounib and an aqueous solution of 10% DMSO were only used in Atlantic salmon with one batch of gametes.

Insemination with cryopreserved Atlantic salmon sperm resulted in 36 to 91.3% eyed eggs (control = 100). The differences were caused by the type of extender and the batch of gametes employed. The very simple extender consisting of 0.3 M glucose and 10% DMSO only was the most successful. Results with cryopreserved sea trout sperm ranged between 38.6–54.8% eyed eggs, showing no difference between treatments.     

Influence of Egg Yolk on the Quality of Cryopreserved Spermatozoa of Common Carp,Cyprinus carpio

Cryopreservation of fish gametes can help in producing quality fish seeds. Success of cryopreservation is evaluated by the post-thaw motility of the spermatozoa. The changes in the seminal plasma during cryopreservation would alter the energy supply for the motility of the spermatozoa, and thus energy supplementation is found to be useful during cryopreservation. Cyprinus carpio spermatozoa were cryopreserved along with egg yolk as a co-cryoprotectant after 1:100 dilution with 0.85% physiological saline as extender and DMSO as cryoprotectant (85:15). The diluents contained egg yolk at three different concentrations, viz., T₁ (5%), T₂ (10%), and T₃ (15%). The diluted milt was equilibrated for 10 min at 5°C and loaded into 0.25 ml straws. The loaded straws were then frozen with LN₂ vapor for 5 min and immersed in liquid nitrogen. Observations were made once in 7 days for 42 days on motility parameters based on which the duration, score, pattern, and percentage were determined. There were significant differences in the motility duration between treatments, and egg yolk at 5% (T₁) concentration was found to support the cryopreserved spermatozoa better than the other concentrations; the difference in motility duration was statistically significant (P > 0.005).     

Cryogenic and Refrigerated Storage of Rainbow Smelt Osmerus mordax Spermatozoa

The effects of extender composition, cryoprotectant, and freezing rate on post-thaw rainbow smelt Osmerus mordax sperm motility were examined, and the fertilization capacity of fresh and post-thaw sperm were compared. The highest post-thaw motility (75%) was obtained when milt was diluted 1:3 with an extender containing 600 mM sucrose supplemented with 10% dimethyl sulfoxide and 1.5% bovine serum albumin and frozen at a rate of –20 C/min. Post-thaw motility for sperm stored in this extender was similar to fresh sperm and did not change after 90 d of storage. Furthermore, there were no differences in fertilization rate or embryo survival to the eyed stage between fresh and post-thaw sperm frozen in this extender. The lowest post-thaw motility was observed when sperm were frozen with methanol at a rate of -30 C/min. Refrigerated sperm diluted 1:3 with the 600 mM sucrose extender remained motile for 30 d. These data demonstrate that rainbow smelt spermatozoa can be effectively used following short and long-term storage using a simple, sucrose-based extender.     

Cryopreservation of scaly carp (Cyprinus carpio) sperm: effect of different cryoprotectant concentrations on post-thaw motility, fertilization and hatching success of embryos

The aim of the present study was to determine the effect of various cryoprotectants on post-thaw sperm quality and fertilizing capacity of cryopreserved scaly carp (Cyprinus carpio) semen. The present study focused on freezing of scaly carp sperm utilizing a practical and inexpensive protocol for aquaculture. Semen was diluted with Kurokura’s extender composing 3.6 g/l NaCl, 10 g/l KCl, 0.22 g/l CaCl₂, 0.08 g/l MgCl₂ and 0.2 g/l NaHCO₃. The extender contained three different cryoprotectants (DMSO, DMA and egg yolk) at ratios of 5, 10 and 15 %. Semen was placed into 0.25-ml straws and exposed to liquid nitrogen vapor (?120 °C) using an insulated box with an adjustable tray for 10 min and then plunged into liquid nitrogen (?196 °C) tank. The thawing process was performed in a water bath at 40 °C for 10 s. The results indicated that type of cryoprotectants and their concentrations are rather effective in scaly carp sperm cryopreservation on post-thaw sperm quality, while they are very important in order to obtain high fertilization rates. The highest fertilization rate was determined as 96.4 ± 0.15 % with 15 % egg yolk, while the highest hatching rate was determined as 99.3 ± 0.80 with 15 % DMA. In conclusion, the applied cryopreservation method for scaly carp sperm is suitable to fertilize high amounts of eggs.     

Studies on cryopreservation of eggs from rainbow trout (Salmo gairdneri) and coho salmon (Oncorhynchus kisutch)

The effect of pre-freezing treatments as well as freezing of inseminated, not water-activated eggs from rainbow trout Salmo gairdneri, and coho salmon, Oncorhynchus kisutch, was investigated in relation to survival and further development.Effects above freezing temperatures included: the temperature at insemination, viability of inseminated and unactivated eggs after storage, suitability of an incubation medium and the tolerance of eggs to various levels of the cryoprotectant dimethylsulfoxide (DMSO). Freezing experiments included: investigating the action of DMSO (0, 1, 2 mole) and the tolerance of coho eggs to temperatures between ?4.6 to ?30°C. Insemination temperatures between 0.5°C and 9.8°C (coho eggs) as well as incubation in an artifical medium (1-0°C) for 80 min (rainbow trout eggs) and 170 min (coho eggs) did not influence subsequent fertility. Storage of inseminated and unactivated rainbow trout eggs for 135 min and beyond reduced egg fertility. DMSO at 2 and 4 mole was detrimental to coho eggs (1-0°C). One mole DMSO had no (coho) or reduced (rainbow trout) influence on egg fertility when it was added gradually.In the presence of 1 mole DMSO most eggs remained unfrozen (67–89%) when kept for 10 min in frozen artificial medium (?4.6%) and 27–32% subsequently reached the eyed stage (control = 100). Further cooling (0.3°C/min) to ?10°C was still tolerated (62% unfrozen, 22% eyed eggs) but not to ?20°C (6% unfrozen, no development) and ?30°C (no survival). Use of 2 mole DMSO did not improve the results.     

The effect of individual male potency on fertilization ability of fresh and cryopreserved milt of rainbow trout, Oncorhynchus mykiss (Walbaum)

The goal of the present study was to examine individual male potency in rainbow trout, Oncorhynchus mykiss (Walbaum), expressed as the fertilization ability of fresh and cryopreserved sperm. One female and four males bearing genetic markers enabling determination of the progeny paternity were chosen as gamete donors. Samples of eggs were inseminated with sperm from separate individuals or with pooled sperm. Genetic examination of the progeny obtained after fertilization of eggs with pooled milt showed differences in male potency. The proportions of offspring sired by four individual males after fertilization of eggs with the fresh milt were similar to those obtained after fertilization with cryopreserved milt (correlation r = 0.95; n = 4; P < 0.05). These proportions did not correlate with the proportions of progeny resulting from fertilization of eggs when sperm was not pooled.     

Fertility of rainbow trout (Salmo gairdneri) gametes: Gamete viability in artificial media

A series of experiments was performed to test the effect of potassium ions (K⁺) on the storage of rainbow trout (Salmo gairdneri) sperm cells and eggs. Storage of eggs for up to 10 min in saline solutions, extender with K⁺ concentrations of ≤3.4 mM and coelomic fluid did not affect fertility. Fish Extender #6 had a detrimental effect on eggs within 15 s of application and reduced fertility to near zero within 10 min. Variation in the K⁺ concentration indicated that high (≥6.8 mM) concentrations decreased egg fertility while low (≤3.4 mM) concentrations had no effect on fertility. Insemination of eggs before the addition of Fish Extender #6 allowed storage for 14 h. Semen diluted (1:10) in coelomic fluid maintained fertility for 45 s, whereas semen diluted (1:1) in coelomic fluid maintained fertility for 60 min. Fish Extender #6 at K⁺ concentrations between 0 mM and 68 mM had no effect on fertility of semen diluted 1:2.5 after 1 h of storage.     

添加氯化钾和蛋黄对虹鳟精液冷冻保存的效果

为筛选出更有效的虹鳟(Oncorhynchus mykiss)精液冷冻保存液,在以10％ 甲醇和0.3 M葡萄糖作为主要成分的基础上,分别或同时添加0.9 g/L氯化钾和10％ 蛋黄形成四种冷冻保存液,用于虹鳟精液冷冻保存试验,分别形成4个处理组:Ⅰ#(不添加氯化钾和蛋黄)、Ⅱ#(添加氯化钾)、Ⅲ#(添加蛋黄)和Ⅳ#(...     

Cryopreservation of spermatozoa of the barramundi,Lates calcarifer (Teleostei: Centropomidae)

Three cryoprotectants, dimethylsulphoxide (DMSO), glycerol and methanol, were tested for their ability to cryopreserve viable sperm from Lates calcarifer. The dilution ratio of milt to diluent was 1:4 (v:v) and the initial freezing rate was 31°C/min. DMSO at a concentration of 5% with either 15% milk powder or 20% egg yolk gave the best results (post-thaw motility of spermatozoa: 70–100% for 7 min). Glycerol gave acceptable results when used at high concentration (20%) with 15% milk poweder. Methanol provided little protection against freezing damage.     

Distribution of lipid droplets is an indicator for egg quality in brown trout, Salmo trutta fario

In the eggs of the brown trout, Salmo trutta fario, lipid droplets are clearly visible beneath the oolemma on the surface of the protein yolk. When examining the distribution of lipid vesicles in egg batches of different quality, four categories of lipid vesicle distribution could be distinguished: In the first category, the lipid droplets were evenly distributed throughout the egg. These egg batches had a high quality (percentage of eggs developing to eyed stage embryos: 96.2 ± 5.2). Some lipid droplets had the tendency to coalesce in one pole of the egg in the second category. This coalescing of lipid droplets was accompanied with a reduction in egg quality to about 50% of the first category (% of eyed stage embryos: 40.6 ± 15.2). In the last two categories, the lipid droplets were mostly coalesced in one or two poles of the egg and the egg quality was very low (% of eyed stage embryos: 3.3 ± 2.9 and zero, respectively). In conclusion, examination of lipid droplets distribution, in brown trout, can distinguish between high and low quality eggs in an easy and reliable way.     

Individual variation in progeny size and quality in rainbow trout, Oncorhynchus mykiss (Walbaum)

Early life history traits of fish are very variable as a result of both genetic and environmental factors. In this study, we examine individual variation in early life history traits in progeny of seven females crossed with one male rainbow trout, Oncorhynchus mykiss (Walbaum). Individual eggs were followed from fertilization through hatching until death of the larvae by starvation. Larvae and yolksac size (size was shown to be a good indicator of energy content) increased with increasing egg size, but there were still differences between families after variation in egg size was accounted for. Incubation time was not correlated with egg size, and did not differ between families. The progeny from the different families utilized the available energy differently, as both longevity and growth-rate without food was independent of yolk-sac size, but strongly dependent on family. The observed between-family differences in early life history traits in rainbow trout were mainly caused by genetically based effects; egg size and thus probably egg quality, which differed strongly bet ween females, could not adequately explain these differences.     

Cryopreservation of YamúBrycon siebenthalae Milt

The yamú Brycon siebenthalae is an endemic fish of the Orinoco river basin, but wild stocks are decreasing because of the disruption of their habitat. We evaluated a protocol for the cryopreservation of yamú sperm to contribute to the preservation of this endangered genetic resource. Milt was mixed with a cryoprotectant medium (5.5% glucose, 12% egg yolk, and 5%, 10%, or 15% dimethyl sulfoxide - DMSO) in a ratio 1:4 (milt:medium), stored in 0.5-mL French straws, frozen in nitrogen liquid vapor (-76 C), then immersed and stored in liquid nitrogen for 10 d or 12 mo. Motility of thawed spermatozoa was higher ( P < 0.001) in 10% DMSO medium than 5% DMSO or 15% DMSO mediums; but lower than the control ( P < 0.001). With sperm cryopreserved, the highest level of fertilization was achieved with 10% DMSO ( P < 0.001) after 10 d or 12 mo of cryopreservation. Fertilization of eggs inseminated with 6.4 × 10⁹ spermatozoa per g of eggs was higher ( P <0.05) than with 1.6 × 10⁹ spermatozoa per g of eggs. There was no difference (P > 0.05) in fertilization between insemination doses of 3.2 × 10⁹ and 6.4 × 10⁹ spermatozoa per g of eggs. Cryopreservation of yamu milt can be performed successfully with a simple medium combined with 10% of DMSO as cryoprotectant. The highest level of fertility was achieved using between 3 × 10⁹ and 6 × 10⁹ spermatozoa per g of fresh eggs.     

Application of nanosilver-coated zeolite as water filter media for fungal disinfection of rainbow trout (Oncorhynchus mykiss) eggs

This study investigated the indirect use of silver nanoparticles (AgNPs) for reduction of fungal infections during incubation period of fertilized rainbow trout eggs. Different concentrations of nanosilver-coated zeolite (0.5, 1, and 1.5 % AgNPs) were compared with unmodified zeolite as water filter media in semi-recirculation systems. For testing the effect of AgNPs on reduction of fungal infection, fertilized eggs were transferred in incubators receiving water from filters coated with nanosilver. The eggs in each incubator were inoculated with Saprolegnia-infected trout eggs. Any dead or infected eggs and embryos were periodically removed, while the performance of the filters was assessed by calculating the survival rates from fertilization up to completion of the yolk–sac absorption stage. The results showed that the filters containing 0.5 % AgNPs increased the survival rate by 4.56 % from fertilization to the swim up stage compared to the control (p < 0.05). Also, the additional application of activated carbon (as absorbent media) along with AgNP-coated media in filters caused an increase of about 11.24 % in the survival rate for the larval stage (p < 0.05). In contrast to the control group with about 6 % fungal infection, no infections were observed during the incubation period in the incubators containing nanosilver-coated filters. Therefore, the final results confirmed that the indirect use of AgNPs in the aforementioned filters were significantly effective for preventing fungal infections in semi-recirculation systems for rainbow trout, making them a candidate for replacing the chemical fungicides currently used during egg incubation in hatchery systems.     

Are environmental conditions in Finnish streams limiting to early life-history survival in the nonnative rainbow trout?

The nonnative rainbow trout Oncorhynchus mykiss has been an unsuccessful invader in North European streams, although it has been widely introduced. Here we studied whether early life history stages (egg incubation and hatching, first overwintering) act as filters for the establishment of hatchery rainbow trout. Survival of hatched alevins was approximately 80%, whereas only 47% of the embryos survived. However, the latter value was impacted by the high number of unfertilized eggs. Correlation coefficients with embryo survival rate and environmental variables (pH and temperature) were statistically insignificant. In the overwintering experiments, the survival of rainbow trout was 93%. The growth was generally slowed during the winter, but in the spring the growth of rainbow trout exceeded that of the native brown trout. Our data demonstrated that the survival and growth of rainbow trout during early life-history stages were relatively high and comparable to those of the native brown trout. Based on the variables considered in our study, our results suggest that environmental conditions during early life-history stages are not detrimental for rainbow trout in the study streams.     

Application of sperm cryopreservation in selective breeding of the Pacific oyster,Crassostrea gigas (Thunberg)

The robustness of Pacific oyster, Crassostrea gigas (Thunberg), sperm cryopreservation in the context of selective breeding based on family lines was investigated. Irrespective of egg density, high fertilization success was achieved with cryopreserved sperm when sperm:egg ratios of 1000:1 to 10 000:1 were used. Variation among replicate runs on the same oyster batches was minimal, indicating that cryopreservation and larval rearing procedures were repeatable. Twenty independent single male–female crosses were made to assess the utility of cryopreserved sperm in selective breeding. The fertility of unfrozen sperm was generally a poor predictor of cryopreserved sperm fertility. Based on D‐larval yields, 17 of the 20 crosses were likely to yield adequate spat for selective breeding (>10⁵ D‐larvae from 1 million eggs), two were marginal (5 × 10⁴ D‐larvae) and one was inadequate (4 × 10³ D‐larvae). An alternative fertilization strategy to improve D‐yield from a given number of sperm was then tested. Fertilizing 10 million eggs at a sperm:egg ratio of 200:1 increased the total D‐yield when compared with fertilizing 1 million eggs at a sperm:egg ratio of 2000:1 for the same male–female pair. We conclude that, despite wide variation in fertility, cryopreserved sperm is useful for family production.