Post-thaw Fertilization Rates Research Articles

Is catalase an effective additive to alleviate oxidative stress during cryopreservation of zebrafish sperm at the repository level?

The goal of this study was to investigate whether supplementation of cryoprotective medium with catalase (CAT), an antioxidation enzyme, is efficient for zebrafish sperm cryopreservation from the viewpoint of high-throughput genetic repository operations. Three cryoprotectants (10%, v/v), dimethylacetamide (DMA), dimethylformamide (DMF), and methanol were used. The objectives were to evaluate the effects of CAT on sperm motility, plasma membrane integrity, and concentration for: 1) fresh sperm at equilibration up to 60 min; 2) post-thaw sperm after cooling at 10, 20, and 40 °C/min), and 3) post-thaw fertilization and embryo survival rates. Catalase addition did not improve sperm motility, regardless of the cryoprotectants added. After 10-min exposure to DMA or methanol, membrane integrity was significantly decreased (70–75%) compared to controls. With catalase, sperm cells maintained membrane integrity and after 50 min equilibration, cell concentrations were maintained with CAT compared to cryoprotectant-only test groups. However, after cryopreservation and thawing, CAT did not affect the outcome of motility, membrane integrity, cell concentration, fertilization, or embryo survival assays. Analysis of cooling rates also indicated that CAT did not affect 3-hpf fertilization or 24-hpf survival rates. Overall, addition of CAT could provide some protection of sperm from oxidative stress before freezing, but not after thawing. We propose that decisions concerning routine use of CAT for repositories, especially those handling tens of thousands of frozen samples per year, would depend on whether efficient high-throughput operation, or specific research questions are programmatic goals.

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

In this study, a programmable freezing technique has been developed for strip spawned sperm in the blue mussel, Mytilus galloprovincialis. The optimized key parameters include cooling rate, endpoint temperature, thawing temperature, sugar addition and sperm to oocyte ratio. The sperm quality was assessed by the fertilization rate or the integrity of sperm component and organelle. The highest post-thaw sperm fertilization rate was 91%, which was produced with sperm cryopreserved in 8% dimethyl sulfoxide at the cooling rate of -4°C/min from 2°C to -30°C before being plunged into liquid nitrogen for at least 12 h, thawed in a 20°C seawater bath and fertilized at sperm to egg ratio of 50 000:1. The addition of glucose, sucrose or trehalose to 8% dimethyl sulfoxide could not further improve fertilization rates. The fluorescent assessments showed that the post-thaw sperm plasma membrane integrity and acrosome integrity were significantly damaged in comparison with fresh sperm.

Changes to Extender, Cryoprotective Medium, and In Vitro Fertilization Improve Zebrafish Sperm Cryopreservation.

Sperm cryopreservation is a highly efficient method for preserving genetic resources. It extends the reproductive period of males and significantly reduces costs normally associated with maintenance of live animal colonies. However, previous zebrafish (Danio rerio) cryopreservation methods have produced variable outcomes and low post-thaw fertilization rates. To improve post-thaw fertilization rates after cryopreservation, we developed a new extender and cryoprotective medium (CPM), introduced quality assessment (QA), determined the optimal cooling rate, and improved the post-thaw in vitro fertilization process. We found that the hypertonic extender E400 preserved motility of sperm held on ice for at least 6 h. We implemented QA by measuring sperm cell densities with a NanoDrop spectrophotometer and sperm motility with computer-assisted sperm analysis (CASA). We developed a CPM, RMMB, which contains raffinose, skim milk, methanol, and bicine buffer. Post-thaw motility indicated that the optimal cooling rate in two types of cryogenic vials was between 10 and 15°C/min. Test thaws from this method produced average motility of 20% ± 13% and an average post-thaw fertilization rate of 68% ± 16%.

Efficient method for cryopreservation of European huchen (Hucho hucho L.) and grayling (Thymallus thymallus L.) semen

Abstract The objective of this study was to test the effect of cryopreservation with the use of a methanol–glucose extender on sperm motility parameters and fertilization rates of European huchen (Hucho hucho) and grayling (Thymallus thymallus), belonging to the Salmonidae family of Salmoniformes. Distinct differences between those species in absolute fertilization rates, sperm concentrations and sperm motility parameters were noticed. Post-thaw sperm motility of huchen was low (about 45%) compared to sperm motility of cryopreserved grayling semen (about 68%). Absolute fertilization rates of cryopreserved huchen semen were high: 87–88% of eyed embryos and 75–80% of hatched larvae. On the other hand, absolute fertilization rates of cryopreserved grayling semen were low (about 50%), but the relative fertilization rates amounted to 100% of control with fresh semen. To the best of our knowledge, this study is the first report to demonstrate that the post-thaw fertilization rate of semen can be comparable to that of fresh semen at a ratio as low as 1.5 × 105 and 5 × 105 spermatozoa per egg for grayling and huchen, respectively. The described method seems to be universal for the cryopreservation of semen of Salmonidae. In our opinion, it can be a very useful tool for ongoing efforts for conservation of European huchen and grayling in Central Europe, for aquaculture of those species, as well as this procedure can be implemented in hatchery conditions after scaling-up.

Improvement in non-programmable sperm cryopreservation technique in farmed greenlip abalone Haliotis laevigata

This study assessed the effects of the addition of vitamins (l-ascorbic acid), amino acids (glycine and taurine), and monosaccharides (glucose, fructose and galactose) on sperm cryopreservation using a non-programmable freezing technique in farmed greenlip abalone. The results showed that the addition of taurine, glycine or l-ascorbic acid significantly improved the post-thaw sperm motility, whereas the post-thaw sperm fertilization rates were improved by the addition of glycine or l-ascorbic acid. Flow cytometry analysis demonstrated that the addition of glycine significantly enhanced the post-thaw sperm plasma membrane integrity and acrosome integrity. Results from the investigation on monosaccharides demonstrated that glucose, fructose and galactose had a similar cryoprotective effect, resulting in a similar level of the post-thaw sperm fertilization rate, plasma membrane integrity, mitochondrial membrane potential and acrosome integrity. In this study, the highest post-thaw fertilization rate of 96% was achieved by using the cryoprotective mediums containing 6% DMSO, 1% glucose and 0.6% glycine.

Cryopreservation of sperm from wild greenlip abalone (Haliotis laevigata) in Australia

Greenlip abalone (Haliotis laevigata) is one of the most economically valuable marine molluscan species in Australia and its aquaculture production comprises more than 50% of the total production of farmed abalone in this country. The development of a cryopreservation technique for this species will in particular enhance the efficiency of the established genetic improvement programs and thus the long-term development of this aquaculture industry. In this study, a series of experiments have been conducted to investigate factors important for the development of sperm cryopreservation techniques in this species, including the evaluation of (1) cryoprotectant toxicity; (2) pre-freezing temperature; (3) volume of sperm + cryoprotectant mixture in 2-mL cryovial; (4) sperm concentration; (5) cold storage duration; (6) thawing temperature; and (7) sperm to egg volume ratio on post-thaw sperm motility and/or fertility. The results show that 6% DMSO was the best among the cryoprotectants evaluated, resulting in a post-thaw fertilization rate similar to controls. Wild greenlip abalone sperm tolerated a reasonable range of conditions for most parameters crucial to the liquid nitrogen vapor cryopreservation method; pre-freezing temperature from − 60 to − 80 °C, duration of cold storage (for chemical equilibration) up to 1.5 h, and the thawed sperm/fresh egg ratio (by volume) up to 1:3. Although a 50 °C thawing temperature resulted in the highest fertilization rate of 95%, all the other temperatures evaluated (from 40 to 80 °C) produced a fertilization rate of 80% or higher. The use of the cryopreservation protocol established in this study will provide an alternative breeding technique to overcome key obstacles experienced in the existing genetic improvement programs.

19. Cryopreservation and fertility of geoduck (Panopea zelandica) sperm and oocytes

Geoducks are large salt water clams. They are currently farmed in North America. In New Zealand, there is a small wild fishery for the native geoduck, Panopea zelandica . More recently, there is increasing interest in farming this high value species in New Zealand. Already a small batch has been reared and is ready to be experimentally on-grown on existing mussel farms. Cryopreservation can be a powerful tool for hatcheries and selective breeding programmes for aquaculture. In hatcheries, cryopreservation allows year-round spat supply without the need to condition broodstock for out of season production. This is particularly useful with new species where optimized conditioning regimes have not been established. Spawning techniques may also need development and cryopreservation can enable hatcheries to capitalize on single sex spawnings where gametes would otherwise be wasted. In selective breeding, cryopreservation enables breeders to make crosses on demand; providing them with complete flexibility and enabling faster genetic gains. Towards this goal, we investigated cryopreservation and fertility of gametes from the New Zealand geoduck, Panopea zelandica. Geoducks were induced to spawn during their natural spawning season using thermal shock and serotonin injection. Sperm was collected as concentrated as possible by aspiration and stored at 4 °C before being used in experiments. Oocytes were maintained at either room temperature or 4 °C. For both sperm and oocytes, three cryoprotectants, ethylene glycol, dimethyl sulphoxide and propylene glycol were made up in Milli-Q water and evaluated at final concentrations of 10% and 15% with or without 0.2 M trehalose. For sperm, a simple freezing method utilizing a rack attached to a polystyrene frame which floated the rack 3 cm above the liquid nitrogen surface was evaluated as a method of freezing. Sperm were diluted 1:1 with cryoprotectant solutions and loaded into 0.5 mL straws and frozen on the rack for 10 min before plunging into liquid nitrogen. For oocytes, two cooling regimes were evaluated. Oocytes were diluted 1:1, loaded into 0.25 mL straws and placed into controlled rate freezers that were programmed to hold at 0 °C for 5 min then cool at 1 °C min −1 to −10 °C, hold for 5 min then cool at either 0.3 or 0.5 °C min −1 to −35 °C. Upon reaching −35 °C, straws were plunged in liquid nitrogen. Straws containing sperm were thawed at room temperature. Straws containing oocytes were thawed at 28 °C until the ice had melted. Overall, unfrozen sperm obtained from serotonin injection had slightly higher fertility with fresh oocytes than sperm obtained from thermal shock (70% vs 50% at sperm concentration of 10 5 mL −1 ) The addition of bovine serum albumin to the fertilization assay improved sperm fertility by 30%. Sperm frozen in either 10% dimethyl sulphoxide or 10% or 15% propylene glycol gave the highest post-thaw fertilization rates (>70% at sperm concentration of 10 6 mL −1 ). Oocytes were lysed upon thawing prior to dilution for fertilization. Further work will investigate: optimization and development of cryopreservation methods for both gametes and larvae; the longevity of geoduck gametes and a larval assay to assess subsequent development.

Biophysics of zebrafish (Danio rerio) sperm

In the past two decades, laboratories around the world have produced thousands of mutant, transgenic, and wild-type zebrafish lines for biomedical research. Although slow-freezing cryopreservation of zebrafish sperm has been available for 30 years, current protocols lack standardization and yield inconsistent post-thaw fertilization rates. Cell cryopreservation cannot be improved without basic physiological knowledge, which was lacking for zebrafish sperm. The first goal was to define basic cryobiological values for wild-type zebrafish sperm and to evaluate how modern physiological methods could aid in developing improved cryopreservation protocols. Coulter counting methods measured an osmotically inactive water fraction (Vb) of 0.37±0.02 (SEM), an isosmotic cell volume (Vo) of 12.1±0.2μm3 (SEM), a water permeability (Lp) in 10% dimethyl sulfoxide of 0.021±0.001(SEM)μm/min/atm, and a cryoprotectant permeability (Ps) of 0.10±0.01 (SEM)×10−3cm/min. Fourier transform infrared spectroscopy indicated that sperm membranes frozen without cryoprotectant showed damage and lipid reorganization, while those exposed to 10% glycerol demonstrated decreased lipid phase transition temperatures, which would stabilize the cells during cooling. The second goal was to determine the practicality and viability of shipping cooled zebrafish sperm overnight through the mail. Flow cytometry demonstrated that chilled fresh sperm can be maintained at 92% viability for 24h at 0°C, suggesting that it can be shipped and exchanged between laboratories. Additional methods will be necessary to analyze and improve cryopreservation techniques and post-thaw fertility of zebrafish sperm. The present study is a first step to explore such techniques.

Sperm cryoconservation in Anarhichas sp., endangered cold-water aquaculture species with internal fertilization

This study contributes to the identification of an adequate cryopreservation medium for wolffishes sperm (Anarhichas minor and A. lupus). Our results also confirm the presence of antifreeze proteins in the seminal fluid of the Atlantic wolffish (Anarhichas lupus) that could facilitate the process of cryoconservation in association with increased post-thaw motility and fertilization rates. Cryo-Fish, a commercial diluent was the most efficient in our trials, with 80% motility post-thaw.

Survival of Pacific oyster, Crassostrea gigas, oocytes in relation to intracellular ice formation

The effect of IIF in Pacific oyster oocytes was studied using cryo and transmission electron microscopy (TEM). The viability of oocytes at each step of a published cryopreservation protocol was assessed in an initial experiment. Two major viability losses were identified; one when oocytes were cooled to −35 °C and the other when oocytes were plunged in liquid nitrogen. Although the cryomicroscope showed no evidence of IIF in oocytes cooled with this protocol, TEM revealed that these oocytes contained ice crystals and were at two developmental stages when frozen, prophase and metaphase I. To reduce IIF, the effect of seven cooling programmes involving cooling to −35 or −60 °C at 0.1 or 0.3 °C min −1 and holding for 0 or 30 min at −35 or −60 °C was evaluated on post-thaw fertilization rate of oocytes. Regardless of the cooling rate or holding time, the fertilization rate of oocytes cooled to −60 °C was significantly lower than that of oocytes cooled to −35 °C. The overall results indicated that observations of IIF obtained from cryomicroscopy are limited to detection of larger amounts of ice within the cells. Although the amount of cellular ice may have been reduced by one of the programmes, fertilization was reduced significantly; suggesting that there is no correlation between the presence of intracellular ice and post-thaw fertilization rate. Therefore, oyster oocytes may be more susceptible to the effect of high solute concentrations and cell shrinkage than intracellular ice under the studied conditions.

Cryopreservation of Semen from Lake Sturgeon

AbstractLake sturgeon Acipenser fulvescens, once abundant and the only species of sturgeon endemic to the North American Great Lakes, is now rare and threatened with extinction as a result of overfishing and habitat destruction. Sperm cryopreservation can be useful for preserving the genetic variability of local populations of lake sturgeon. In this study, we show the conditions of extended storage of unfertilized gametes and successful cryopreservation of lake sturgeon semen using methanol as a cryoprotectant. We demonstrate that unfertilized eggs can be transported for 8–12 h, provided that their initial quality is good. Our results indicate that cryopreservation of lake sturgeon milt caused increased damage to sperm DNA (measured by comet assay); however, some spermatozoa maintained their fertilizing ability. Fertilization with cryopreserved semen caused an 87% reduction in postthaw fertilization rates of prehatched larvae compared with control fertilizations with fresh semen.

Successful cryopreservation of Pacific oyster ( Crassostrea gigas) oocytes

Protocols for cryopreservation of sperm and oocytes would provide the ultimate control over parental crosses in selective breeding programmes. Sperm freezing is routine for many species, but oocyte freezing remains problematic, with virtually zero success in aquatic species to date. This paper describes the development of a successful protocol for cryopreserving high concentrations of Pacific oyster ( Crassostrea gigas) oocytes. Ethylene glycol (10%) and dimethyl sulfoxide (15%) were found to be the most effective cryoprotectants resulting in post-thaw fertilization rates of 51.0 ± 8.0 and 45.1 ± 8.3%, respectively. Propylene glycol was less effective and methanol resulted in zero fertilization post-thaw. The use of Milli-Q water rather than seawater as a base medium significantly improved fertilization (20.4 ± 3.0 and 8.7 ± 2.2%, respectively) as did the inclusion of a 5 min isothermal hold at −10 or −12 °C (35.9 ± 5.0 and 31.9 ± 4.6%, respectively). The optimal cooling rate post-hold was 0.3 °C min −1, with virtually zero post-thaw fertilization with cooling rates of 3 and 6 °C min −1. Using an optimized protocol, post-thaw fertilization rates for oocytes from eight individual females ranged from 0.8 to 74.5% and D-larval yields from 0.1 to 30.1%. For three individuals, larvae were reared through to spat. Development of D-larvae to eyed larvae and spat was similar for larvae produced from unfrozen (24.8 ± 4.1% developed to eyed larvae and 16.5 ± 3.2% to spat) and cryopreserved (28.4 ± 0.6 and 18.7 ± 0.5%, respectively) oocytes. The ability to cryopreserve large quantities of oyster oocytes represents a major advance in cryobiology and selective breeding.

Birth of mice after in vitro fertilization using C57BL/6 sperm transported within epididymides at refrigerated temperatures

The transportation of cryopreserved spermatozoa is an economical, efficient, and safe method for the distribution of mouse strains from one facility to another. However, spermatozoa from some strains, including C57BL/6 (B6), are very sensitive to freezing and thawing and frequently fail to fertilize eggs by conventional in vitro fertilization methods at the recipient mouse facility. Since many genetically engineered mice have the B6 genetic background, this sensitivity poses a major obstacle to studies of mouse genetics. We investigated the feasibility of transporting spermatozoa within epididymides under non-freezing conditions. First, we examined the interval that B6 and B6D2F1 (BDF1) spermatozoa retained their ability to fertilize when stored within epididymides at low temperatures (5 °C or 7 °C). Fertilization rates were >50%, irrespective of the spermatozoa used, when epididymides were stored for 3 d at 7 °C. B6 spermatozoa, but not BDF1 sperm, had better retention of fertilizing ability at 7 °C versus 5 °C. We then transported freshly collected B6 and BDF1 epididymides from a sender colony to a recipient colony using a common package delivery service, during which the temperature was maintained at 5 °C or 7 °C for 2 d. Sufficiently high fertilization rates (68.0–77.5%) were obtained for all experimental groups, except for B6 spermatozoa transported at 5 °C. These spermatozoa were successfully cryopreserved at the recipient facility and, yielded post-thaw fertilization rates of 27.6–66.4%. When embryos derived from the B6 spermatozoa that were transported at 7 °C were transferred into recipient females, 52.7% (38/72) developed to term. In conclusion, transportation of epididymides at refrigerated temperatures is a practical method for the exchange of mouse genetic resources between facilities, especially when these facilities do not specialize in sperm cryopreservation. For the B6 mouse strain, the transportation of epididymides at 7 °C rather than 5 °C, is recommended.

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.

Cryogenic and refrigerated storage of Atlantic cod (Gadus morhua) and haddock (Melanogrammus aeglefinus) spermatozoa

The aim of this study was to improve methods for short- and long-term preservation of Alantic cod (Gadus morhua) and haddock (Melanogrammus aeglefinus) spermatozoa. The effects of extender composition, cryoprotectant concentration, freezing rate, and dilution ratio on post-thaw sperm motility were examined. The fertilization capacity of fresh and post-thaw sperm were compared and sperm viability, as determined by differential fluorescent staining (propidium iodide/SYBR 14®), was correlated with motility. The highest post-thaw motility was obtained when sperm was diluted 1:3 with an extender containing glutathione, sucrose, and potassium bicarbonate (modified Mounib's extender, MME), supplemented with 10% DMSO and frozen at a rate of −5 °C min−1. The post-thaw motility of cod (66±2.1%) and haddock (53±2.1%) sperm did not differ between 5 and 90 days of storage but was lower than fresh sperm. No difference in fertilization rate was found between fresh and post-thaw cod sperm, but fertilization was lower using post-thaw haddock sperm when compared to fresh ones. Spermatozoa motility was highly correlated (Adjusted R2=0.94) with the proportion of cells that incorporated SYBR 14®. Refrigerated cod and haddock sperm diluted 1:3 with MME remained motile for 40 and 38 days, respectively. These results demonstrate that spermatozoa of cod and haddock can be refrigerated or cryopreserved and yield acceptable sperm motility and post-thaw fertilization rates.

A protocol for the on-site use of frozen milt from rainbow trout ( Oncorhynchus mykiss Walbaum) applied to the production of progeny groups: comparing males from different populations

Progeny testing could have a potential in aquaculture particularly in species where the males spawn over several years, and also in the monitoring of genetic changes between generations and sub-populations of salmonids, where the males are used for only one season. But these potentials can only be realised by the use of frozen sperm. Cryopreserved sperm could also be a means of exchange of genetic material between locations and populations when transportation of live fish is restricted. With the present protocol for the on-site use of frozen milt, post-thaw fertilization rates were >80% of controls with Norwegian rainbow trout males at a mean number of motile spermatozoa to egg ratio of 3.8×10 6. Milt samples were diluted either 1:3 or 1:5 in an ice cold extender containing methanol as cryoprotectant and aliquots were transferred to 0.5 ml straws, which were equilibrated at 4 °C for 10 min and then frozen on racks in a programmable freezer unit; the freezing curve had a freezing rate of 30 °C min −1 from +4 to −110 °C. Fertilization rates differed between sires within groups (Italian sires in Italy, Norwegian sires in Norway). Norwegian sires also gave significantly higher fertilization rates than did the Italian sires. Milt was sampled in the Italian stock rather late in the season, which is known to influence sperm quality in a negative way. But differences may also have been as much a function of quantity of the spermatozoa. The Italian males produced on average only half the number of spermatozoa of the Norwegian sires per unit volume, 704 compared with 1633, and had half the fraction of post-thaw motile spermatozoa (17.9% compared with 35.1%). Thus, there was four times the number of motile spermatozoa per cryopreserved dose from Norwegian sires. The between groups and sires within group differences in fertilization rates were substantiated with the production of the progeny groups. The fertilization rates obtained with Italian males and females decreased substantially when the numbers of eggs increased even when mean spermatozoa to egg ratio was kept constant: with 150 eggs the mean fertilization rate was (64.8±3)% or about 70% of controls, whereas with 1000 eggs it was (40.9±5.6)% or about 45% of controls. A minor reduction in mean fertilization rate to (69.9±1.2)% was observed with Norwegian males and females when the number of eggs was raised from 150 to 1000 at constant spermatozoa to egg ratio. The findings indicate that the protocol can be applied on a semi-commercial scale. But the mixing of eggs with spermatozoa could indeed be a crucial variable, and particularly so when milt is not used in excess. Results further indicate that the same sires perform either good or bad in both environments.

Effect of extender composition and equilibration time on fertilization ability and enzymatic activity of rainbow trout cryopreserved spermatozoa

The effects of extender composition and equilibration time on fertilizing ability of cryopreserved spermatozoa from rainbow trout, Oncorhynchus mykiss , were investigated. In addition, enzyme activity in supernatants from thawed sperm was assessed. The use of the two extenders: Erdahl & Graham's + 10% DMA (dimethyl acetamide) + 10% egge yolk and 0.3 M glucose + 10% DMA yielded the highest post-thaw fertilization rates. We observed interactions between extender constituents and the equilibration of diluted semen. This indicates a multifactorial effect of the extender constituents on spermatozoal resistance against injuries. The 10-min equilibration of spermatozoa in extender before freezing generally lowered the fertilization ability of spermatozoa, except for DMA-based extenders. The addition of egg yolk to the extender was generally beneficial, especially in DMA- and DMSO-based extenders. The use of low-density lipoprotein fraction showed no advantage to full-yolk or free-of-yolk extenders. Aspartate aminotransferase and lactate dehydrogenase leakage from damaged spermatozoa correlated negatively with the ability of cryopreserved spermatozoa to fertilize eggs. Each factor tested, when analyzed separately, did not give general information about its effect on the fertilization ability of cryopreserved sperm. The multifactorial analysis of the important factors in cryopreservation of trout spermatozoa showed their cumulative effect. This is the most likely reason for divergent information reported elsewhere on the effect of various factors in the cryopreservation of rainbow trout spermatozoa.

An efficient method for cryopreservation of testicular sperm from the Northern pike, Esox lucius L.

The cryopreservation of semen from the Northern pike, Esox lucius L., was investigated with a method that was originally developed for the Salmonidae. Because the amounts of semen obtained by stripping were insufficient, the suitability of testicular sperm was tested for cryopreservation. Frozen-thawed testicular sperm had fertilization rates similar to frozen-thawed semen obtained by stripping (74.2-84.7%), and at sperm to egg ratios of S= 4.5 × 105 spermatozoa per egg, the post-thaw fertilization rates were also similar to fresh, untreated semen controls. Out of all the fertilization solutions investigated, a 100-mm NaCl, 10-mm Tris (pH 9) solution resulted in the highest post-thaw fertilization rates. To facilitate the fertilization of large egg batches, 1.2-mL straws were used for cryopreservation with a similar efficiency to 0.5-mL straws.

Methanol as cryoprotectant and the suitability of 1.2 ml and 5 ml straws for cryopreservation of semen from salmonid fishes

For salmonid semen, the cryoprotective action of 10% methanol was compared with a 5% dimethyl sulphoxide (DMSO), 1% glycerol mixture, until now one of the most effective cryoprotectants. In Oncorhynchus mykiss (Walbaum), Salmo trutta L. f. fario, Salmo trutta L. f. lacustris and Salvelinus alpinus (L.), semen cryopreserved with both cryoprotectants yielded post-thaw fertilization rates of 90-100% of control with untreated semen at sperm-to-egg ratios of 1.8 × 106-2.4 × 106 spermatozoa per egg. However, at sperm-to-egg ratios of 0.9 × 106-1.2 × 106 spermatozoa per egg, semen cryopreserved with methanol had significantly higher fertilization rates than semen frozen with the DMSO/glycerol mixture. In other studies we obtained similar data for Coregonus sp., Salvelinus fontinalis (Mitchill), Thymallus thymallus (L.) and Hucho hucho (L.), proving that methanol is the most effective and generally applicable cryoprotectant for semen of the studied salmonid species. To facilitate the insemination of large egg batches we investigated the suitability of 1.2 ml and 5 ml straws for deep freezing of semen of Oncorhynchus mykiss, Salmo trutta f. fario, Salmo trutta f. lacustris and Salvelinus alpinus. With 1.2 ml straws the fertilization rates were similar to 0.5 ml straws when using lower freezing and higher thawing temperatures. The 5 ml straws resulted in a fertilization success of only about 40% of fresh semen control.

Physiological and Biochemical Determination of Rainbow Trout,Oncorhynchus mykiss, Semen Quality for Cryopreservation

In rainbow trout, Oncorhynchus mykiss, parameters to determine semen fitness for cryopreservation and quality control of cryopreserved semen were investigated. The following parameters can be used to evaluate semen fitness for cryopreservation as they are statistically significant (P < 0.01) correlated to the post-thaw fertilization rate: motility rate of fresh semen (y = 4.996x - 0.0958x2 + 0.0006x3 - 5 1.7363); sperm velocity of fresh semen (y = 6.741x - 0.036x2 - 268.37); seminal plasma osmolality (y = 0.539x - 125.59); seminal plasma pH (y = -82.768x + 728.133); seminal plasma triglyceride levels (y = 0.069x + 29.863); seminal plasma ß-D-glucuronidase activity (y = -1.112x + 0.0058x2 + 82.229); seminal [lasma lactate dehydrogenase activity (y = -0.096x + 0.00006x2 + 583.80); spermatozoan acid phosphatase activity (y = -132.51x + 126.38x2 + 66.48); spermatozoan adenylate kinase activity (y = 3.474x + 4.925). Quality of deep-frozen semen can be evaluated by motility parameters (P < 0.01): frozen/thawed semen motility rate and post-thaw fertilization rate: y = 1.943x + 28.002; sperm velocity and post-thaw fertilization rate: y = 0.8812x - 0.0059x2 + 24.9686.