Protocol For Ampoules Are Held In Liquid Nitrogen Vapor In The Neck Of A Dewar

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1. Gamete materials: sperm and a suspension of sperm cells prepared from testes.

Fresh eggs collected immediately before fertilization.

2. Solutions.

a. Extenders (solutions without cryoprotectant): the choice of appropriate extender depends on the species to be investigated (see Note 1).

b. Freshly prepared cryoprotectant solutions. The most commonly used cryoprotectants for fish sperm cryopreservation are dimethyl sulfoxide (DMSO), ethylene glycol, methanol, ethanol, glycerol, and dimethylacetamide. The concentration usually varies between 5 and 12% (w/v). Sucrose, polyethylenegly-col, albumin, and hen egg yolk have also been used (see Note 1).

c. Activating medium for fertilization: 25 mM Tris and 150 mM NaCl medium can be used for sturgeons and carp (12); 60 mM NaHCO3, 20 mM glycine, 5 mM theophylline, 50 mM Tris-HCl (pH 9.0) is used for Salmonid fishes (16).

3. Containers for milt collection: vials, flasks, tissue culture flasks. The type and size of container depends on species and volume of collected sperm (see Note 2).

4. Container with ice.

5. Cryocontainers: cryovials and straws.

6. Cryostorage equipment.

7. Water bath.

8. Refrigeration systems:

a. Programmable variable-rate cooler.

b. Wide-necked Dewar (10-15 L) with liquid nitrogen. The neck diameter of 10-15 cm.

c. Refrigerated alcohol cold baths (for -20 and -40°C).

d. Polystyrene box with dry-ice.

9. Thermocouple and device for monitoring temperature changes.

10. Storage system: crystorage containers with appropriate storage racks and inventory system suitable for holding cryovials.

11. Microscope with a computer-assisted semen analysis system.

12. Petri dishes for fertilization.

13. Low temperature-resistant markers for glass.

14. Hemocytometer.

3. Methods

1. Select the appropriate breeders and prepare them, if necessary, for spawning (see Note 3).

2. Stimulation of maturation. Unless naturally spawning fish are used, artificial stimulation is required. In order to obtain good quality sperm, keep fish under optimal conditions and avoid fluctuations and/or changes of temperature after stimulation and during spermiation. Inject chorionic gonadotropin or solution of the pituitary gland. The dose depends on species, fish weight, degree of maturation, and the quality and type of injected hormones. For examples of dosage refer to Table 1 (see Note 4). The injections are made intramuscularly in the cranial part of the body, or under the one of pectoral fins (see Note 5).

3. Collection of milt.

a. Collect sperm from mature males into clean and dry flasks applying gentle abdominal massage. For small males it can be useful to sedate the fish (with 25 mg/L tricaine methanesulfate [MS-222]) and use a capillary tube. The collection of sperm from large fish such as sturgeon is performed on a specially designed table. The table has two surfaces under a 90° angle and a 10° slope with soft cover. A tube with flowing water is inserted into the fish's mouth, while a transparent polyethylene tube is placed into its genital orifice. This method of sperm collection helps to prevent sperm contamination and trauma-tization of the fish (see Note 6).

b. Collection of sperm by sacrificing the fish. This approach is usually only necessary in the case of small aquarium fish or in some species where the collection of liquid sperm is not possible without extraction of testes (for example, Weather Loach Misgurnus fossilis). Anesthetize fish by placing them into a solution of 100 mg/L tricaine methanesulfate or 25-50 mg/L benzocaine. Extract testes from abdominal cavity and transfer them to a dry, clean dish.

4. Maintain the milt samples obtained at 4-5°C, or on ice in well-aerated flasks in a thin layer of sperm sample. It is preferably not to store sperm samples after collection, one should proceed immediately with cryopreservation (see Notes 7 and 8).

Table 1

Examples of Hormone Doses Used for Stimulation of Fish Spermiation

Examples of Hormone Doses Used for Stimulation of Fish Spermiation

Table 1

Species

Injected material

Dose

Sturgeon (Acipenser baeri)

Powder of sturgeon pituitary

2 mg/kg

gland dissolved in water

Carp (Cyprinus Carpio)

Powder of carp pituitary gland

4 mg/kg

dissolved in water

Loach (Misgurnus fossilis)

Chorionic hormone

100 U/fish

5. Inspection of cell quality under the microscope (see Note 9). Activate cell movement by addition of the appropriate activating medium. Fresh sperm can be activated by water from the aquarium tank after dilution not less than in 40 times followed by good, rapid mixing. Sperm with low motility rate and with low cell density should not be used for cryopreservation. For activation after cryopreserva-tion, see step 15 (see Note 10). If necessary, cell density can be established with the help of a hematocytometer.

6. Preparation of milt suspension if step 2 was used. Prepare sperm suspension from extracted testes. Care must be taken to avoid milt contact with water, urine, blood, and feces to prevent activation and contamination of sperm. Testes are cut into small pieces and homogenized with the extender.

7. Dilute sperm with appropriate cryoprotectant (see Notes 11 and 12). Establish the appropriate ratio of dilution for each species (see Note 13). Cryoprotectant solutions that causes more than a 50% decline in sperm motility rate must not be used. Cryoprotectants should be added very slowly and gradually (see Note 14) with a constant mixing of sperm with the added solution. The concentration of cryoprotectant usually varies in the range between 5 and 12% (v/v).

8. Equilibration. Immediately after sperm collection, it is cooled down to 5°C and diluted with isothermal cryoprotectant medium. The duration of exposure to the cryoprotectant solution at 5°C is generally in the range of 20 to 60 min. However, for some species equilibration might not be necessary (see Note 15).

9. Load samples in 1-2 mL vials. The volume of the cryopreserved sample should not usually exceed 2 mL (see Note 2). The smaller the volume that is used, the easier it is to achieve the desired freezing regime within the sample. Leave at least one-fourth of ampoule volume free of sample (see Note 16).

10. Freezing.

a. Freezing in vapor-phase liquid nitrogen (see Note 17). Place vials or straws above the liquid nitrogen horizontally on the rack at a predetermined height. The height of sample placement and the time of exposure at this height depend on sample volume, type of container, type of the Dewar, and temperature at that height. For example, samples of sturgeon sperm in 12% (v/v) DMSO placed in vials in 0.7- or 1.5-mL volumes are kept at 20-cm above the surface of liquid nitrogen for 20 min (17). During this time sperm is cooled down to -20°C. After that samples are moved at 2-cm above the liquid nitrogen level for 5 min followed by plunging into liquid nitrogen.

Sperm of salmonid fish is cryopreserved in straws with volumes of 0.5 or 1.2 mL (16). Straws containing 0.5 and 1.2 mL are frozen at 1.5- and 2.5-cm above the level of liquid nitrogen (LN) respectively, with a holding time of 10 min (16).

b. Freezing in alcohol baths. Similar results can be obtained by freezing sperm in cold baths that are capable of maintaining a set temperature. Prepare two cold baths precooled to -20 and -40°C. Vials (0.7 or 1.5 mL) are placed in the bath at -20°C for 2.5 min and then are transferred to a bath at -40°C for 2.5 min. The samples are then plunged into liquid nitrogen.

c. Freezing in dry-ice. Place small aliquots (100-250 pL) of sperm diluted with cryoprotectant directly onto dry ice and allow them to cool for 4-5 min. Then transfer to cryovials and store in liquid nitrogen.

d. Controlled-rate cooling. The following protocol can be applied for sperm of different species. The first step from 0 to -15°C involves cooling at 1-5°C/min (1-2°C/min for Carp); second step from -15 to -70°C cooling at 15-20°C/min, and the third step plunging into liquid nitrogen (see Note 18).

11. Store in liquid nitrogen (see Note 19).

12. Thawing. Transfer the vials or straws from liquid nitrogen directly into a water bath at 40°C (see Note 20). Rotate the vials while they are immersed in a water bath until the appearance of the liquid phase. Then shake the vials in the air until no ice remains.

For samples frozen as pellets, add them directly to eggs along with the activator, the temperature of which would be the same as river (or tank) water from which the fish originated. In the case of carp, the thawed sperm samples could be stored on ice and the fertility rate could be improved after some additional treatment (see Note 21).

13. "Rehabilitation" of sperm. Aerate sperm from carp for 5-15 min after thawing before fertilization (see Note 21).

14. Fertilization. Ensure that the eggs are ready for fertilization. Place sperm samples directly into a container with eggs together with activating medium. Use 42 mM NaHCO3 or NaCl as an activating media (see Note 10). If available use a goose feather for mixing eggs if a large quantity is to be used.

15. Assessment of sperm quality after cryopreservation. Use microscope assessment as outlined in step 5, plus fertilization and hatching rate for assessment of sperm quality.

16. Keep detailed comprehensive records of the cryopreserved materials and protocol. 4. Notes

1. The choice of extender depends on the species from which the sperm is obtained (see Table 2).

2. Good results are usually obtained when fish sperm is frozen in granules, straws, and/or plastic vials of 0.05-2 mL volume. Larger volumes create difficulties in achieving uniformed freezing of the sample. Containers larger than 5 mL are not normally recommended for the cryopreservation of fish sperm.

Table 2

Example of Cryoprotectant Media for Some Species

Fish species Medium ingredients Quantity

Carp (Cuprinus Carpio) (18) NaCl 42 mg

KCl 6 mg

CaCl26H2O 18 mg

MgSO47H2O 62 mg

NaHCO3 280 mg

Sucrose 137 mg d-Mannitol 1.5 g

Tris-oxymethyl- 1.697 g aminomethane basis

Glutathione red 56 mg

Polyvinyl alcohol 5 mg

Hen egg yolk 12.0 mL

HCl Adjust pH 8.1

H2O up to 100 mL

Ethylene glycol 19.6 mL

Sturgeon fish (17) Tris-HCl buffer 0.05 M

Egg yolk 20%

DMSO 25% (after dilution 1:1

final concentration 12.5%)

Salmonid fish (16) NaCl 600 mg

KCl 315 mg

CaCl22H2O 15 mg

MgSO47H2O 20 mg

HEPES 470 mg

H2O Up to 100 mL

Methanol 10 mL

Bovine serum albumin 1.5 g

Sucrose 0.5 g

Hen egg yolk 7 mL

3. The success of cryopreservation strongly depends on the initial quality of the sperm. Higher quality material can be obtained from matured breeders in the middle of a breeding season (19,20). The quality of sperm drops substantially by the end of the spawning period in trout, herring, and others species (21). The recommended motility rate of sperm that can be used for cryopreservation should ideally not be lower than 80%. For example, the sperm of sturgeon with a motility rate lower than 40% should not be used for cryopreservation. Sturgeon sperm with low motility and cell concentration is not recommended to be used for fertilization, even without cryopreservation (2). Better quality sperm can usually be obtained in the morning, when natural spawning takes place for the majority of species.

4. A variety of hormones and doses may be employed (see Table 1).

5. The injecting is performed slowly and gentle rubbing to the site of injection is applied afterward to avoid injected hormone leaking out because of muscle constriction. It is optimal to inject the fish in the morning. After injection place the fish back into water at their optimal light and temperature regime. Spermiation normally occurs 12-43 h after injection, depending on species and spawning temperature.

6. It is of paramount importance to avoid sperm contamination with water, urine, and feces during fish stripping. As it was demonstrated in salmon (Salmo salar) (22), the collection of sperm by abdominal pressure causes urine contamination that can dilute milt by as much as 80% (v/v). This causes substantial variability of sperm osmolality, a decrease of K+ concentration, and motility. The contamination of sperm with up to 25% (v/v) urine causes the one-third decrease in motility after cryopreservation in comparison with uncontaminated samples. Other authors (23) also reported the decrease of survival in cryopreserved sperm of Atlantic salmon (Salmo salar) after mixing it with different amounts of urine. Therefore, it is recommended (24) that one wipes the abdominal part of a male thoroughly before collecting a sperm sample to clear any water and to expel urine by gentle abdominal pressure. It is more reliable to use catheters for sperm collection, this minimizes contamination. Feces in sperm also affects the motility rate. Clean, uncontaminated, sperm samples from the Walleye (Stizostedion vitreum) could be stored almost six times longer (11.7 d) than sperm contaminated with feces (2 d) (25). Contamination of sperm with blood also decreases the duration of possible storage, as well as the motility rate. Bacterial contamination has also been demonstrated, in our laboratories, to have a negative impact on the functional stability of cells. Antibiotic treatment helps significantly to extend the duration of hypother-mal storage in Stellate sturgeon (Acipenser stellatus) (26). The risk of sperm contamination can be one of the reasons why cryopreservation is recommended straight after sample collection without any preliminary storage.

7. The container for holding sperm samples can also affect the quality of sperm. Tall and narrow containers must be avoided to minimize the possibility of hypoxic conditions developing in the holding container. The conditions for short-term storage/holding of sperm samples need to provide the best possible conditions for appropriate access of oxygen, and the use of wide-neck containers is optimal. Depending on sperm concentration the depth of sperm sample should be in a range of 5 to 15 mm. For example, sturgeon sperm placed into a fridge in closed vials, without access of oxygen, loses motility in 3 h. Samples kept in refrigerators in open humidifying containers, with a depth of less than 15 mm, preserve the sperm motility for as long as several days (26). Experiments with carp sperm also indicates that decrease of storage temperature and thickness of samples increase sperm survival (Kopeika, unpublished data). The layer of air above the sperm sample should be 7-10 times thicker than the layer of sperm. These conditions are especially important when sperm is not going to be used immediately for cryopreservation.

8. In order to decrease the energy lost of spermatozoa during maintenance, the temperature of temporary storage needs to be approx 0-5°C. If ice is used for temporary storage, direct contact between ice and sample needs to be avoided. Sperm of sturgeon (Acipenser guldenstadti) stored at 1-4°C maintained fertilizing ability for 5-6 d (27), whereas when stored in ice fertilizing ability was maintained for up to 8 d (2). Prolonged storage at 1-4°C was also shown to be effective for many other species of fish (28). However, storage at temperatures of 1-4°C is not adequate for the long-term preservation of sperm.

9. Motility assessment. The percentage of motile sperm can be assessed by light microscopy. However, the operator requires experience to assess the motility rate accurately. Therefore, where available, a computer-assisted semen analysis system should be used wherever possible to standardize measurement of motility. This system not only allows the accurate estimation of sperm motility, but also a range of other valuable measurements for the assessment of sperm quality. Adequate assessment of sperm quality also depends on their appropriate dilution. It has been demonstrated for sperm of trout, carp (29), and Siberian sturgeon (30) that after low levels of dilution not all sperm were activated simultaneously and the process of activation can last from 15 s to 3 min after dilution. It is advised that in some cases two-step dilution should be used; first dilute sperm with nonactivating medium and then dilute sperm with activating medium. The degree of dilution needs be optimized for each species. As a general approach we recommend that 5 pL of sperm should be activated by 2 mL H2O for "native" sperm, or activating medium for freeze-thawed sperm. If larger volumes of sperm need to be assessed, then two-step dilution is preferable (30). First, dilution with a 1:20 ratio in nonac-tivation solution of 400 mM sucrose, 20 mM Tris (pH 8.0) (sperm remains immotile); second, activation of sperm using 30 mM Tris (pH 8.0).

10. By the time sperm is thawed and ready to be used for fertilization they have gone through a range of stresses. Further treatments, such as activation of motility in pure water, can also affect functional activity of weak sperm cells post-thaw. Therefore, care has to be taken during handling of sperm after thawing and pure water should not be used as an activator for cryopreserved-thawed sperm during fertilization. Better activation will be attained in activation media that have higher osmolarity than pure water. However, the increase in osmolarity in the activating medium has to be within the range that is safe for the eggs. There are different media used for sperm activation (40 mM NaHCO3, or 60 mM NaCl + 50 mM Tris-HCl (12), Tris/NaCl/CaCl210/20/2 mmol/L, pH 8.5) (31). There is also some evidence that the addition of certain chemicals during, or immediately after, fertilization (32) with cryopreserved fish sperm can enhance the hatching rate.

11. Choice of extender. The osmolarity of the extender solution appears to be one of the most important factors in preparation of an appropriate extender. Because large interindividual differences have been demonstrated in fish semen osmotic responses, in order to maximize the outcome of cryopreservation the osmolarity of extender should be adjusted on the individual bases if possible. Increased concentration of buffer or K+ may be used to inhibit sperm motility in extenders for some species such as sturgeon.

12. Both permeable (glycerol, DMSO, ethylene glycol, dimethylacetamide, methanol) and nonpermeable (sucrose) cryoprotectants may be used in protocols to cryopre-serve fish sperm. The absence of an ideal cryoprotectant, as well as the lack of a full understanding of their mechanisms in cell protection, make selection of a single cryoprotectant difficult for different species. However, the optimal cryoprotec-tant can be determined empirically. The concentration usually varies in the range between 5 and 12% (v/v). Better cell protection can be achieved on employing higher concentrations, but this has to be balanced with toxicity effects of the cry-oprotectant. Addition of nonpenetrating agents, such as sucrose, is generally considered to be beneficial; however, direct mixing of fish sperm with cryoprotectants inevitably leads to the death of all cells (33). Clearly, extenders play an important role in sperm protection. It has also been demonstrated for salmon sperm that mul-ticomponent salt media are much more effective for cryopreservation (16). For marine species, the first successful protocol described by Blaxter (3), utilized a mixture of seawater and distilled water with 12.5% (v/v) glycerol as a cryoprotectant. The authors used the carp medium detailed in Table 1 with 8% (v/v) DMSO (final concentration) for freezing marine species.

13. The level of dilution of the cryoprotectant medium is equally important and it is species sensitive (16). High-density semen needs a greater level of dilution. It was found for sturgeon sperm that the optimal ratio was 1:1 sperm:medium (0.1 M Tris-HCl buffer, 10% [v/v] egg yolk, 25% [v/v] DMSO] (17); however, the ratio can vary between species and even individuals. It has also been demonstrated that the concentration of sperm cells in beluga (Huso huso) semen may vary between males in the range of 0.58 to 6.4 billion of cells/mL and in stellate (Acipenser stellatus) from 0.9 to 10.4 billion of cells/mL (2). Studies on the effect of cell concentration showed that increasing carp sperm concentration from 0.5-0.8 billion/mL to 5-8 billion/mL, or 11-16 billion/mL in freezing medium resulted in a significant reduction of the temperature of ice nucleation from -4.73 ± 0.23°C to -5.75 ± 0.56°C, and -7.16 ± 2.04°C, respectively (34). We believe that a standard dilution level cannot be applied for such a broad range of sperm concentrations. For salmonids, sturgeon, carp and tilapia, milt is generally diluted between 1- and 20-fold.

14. The osmotic potential of media is inevitably substantially increased by the addition of cryoprotectant. One option that may be employed to avoid dramatic osmotic shock is the addition of egg yolk, which we have found to improve sperm survival (17). An additional factor that may be manipulated to minimize osmotic shock is the way dilutions are performed. We have observed that rapid mixing of sperm with cryoprotectant medium can damage substantive amount of cells or affect their motility. For that reason slow addition of cryoprotectant is essential for obtaining optimal results. The best sperm survival is obtained when cooled cryoprotectant medium is slowly added to sperm of equal temperature by pouring it to the wall of a slowly swirled container.

15. Equilibration time period is another important factor. Spermatozoa of salmon are relatively small and readily permeable to cryoprotectants (16). Therefore, the exposure time of sperm to cryoprotectants can be short to avoid any potential toxic effect. However, this is not the case for all species. When the motility level of freeze-thawed sturgeon sperm was compared following different equilibrating durations, it was observed that the samples that had a 40-min exposure to the cryoprotectant medium at 5°C had a significantly higher motility rate than the ones that were frozen almost immediately after placing them in cryoprotectants (12).

16. During freezing the volume of the sperm suspension expands and may consequently increase the pressure in the cryovials, potentially causing cryodamage. The increased pressure can also disrupt the hermetically sealed ampoules and thus lead to contamination of the samples. Therefore, it is important not to completely fill the vials leaving enough space for sample expansion.

17. In order to minimize the fluctuations in cooling rate when freezing in liquid nitrogen vapor, it is important to make sure that conditions of freezing are controlled appropriately. To estimate the optimal height above liquid nitrogen, where the samples are going to be held, several sperm samples should be placed at different levels above liquid nitrogen surface. The higher the sample is above the surface, the longer the freezing time should be. All samples should then be moved to approx 1 cm above liquid nitrogen for a further 10-15 min before plunging into liquid nitrogen. Assessment of motility rates in the thawed samples will indicate which holding height is optimal, and this should be used for routine cryopreserva-tion. The cooling rate in vapor can be estimated using a thermocouple, one end of which is placed in the sample and the other in ice.

18. The cooling rate can be finely controlled by employing a controlled-rate freezer. However, it is not always practically possible to have it on the site of sperm collection. Fish sperm freezing is often performed under field conditions, where other methods of freezing, such as freezing in liquid nitrogen vapor or on dry ice, are more practical. The majority of modern controlled-rate freezers provide linear cooling rates and this may not always be the optimal cooling regime as some workers have found exponential cooling to be more effective (34). The closest easily achieved approximation to an exponential cooling regime can be achieved by using liquid nitrogen vapor-phase freezing or dry ice.

19. The authors have noted that storage of cryopreserved sperm in liquid nitrogen is possible for a long period of time without any detectable decline in sperm quality.

20. Studies on optimization of the thawing regime have demonstrated that the best thawing regime for 1-2 mL vials is using a 40°C water bath. As soon as sperm samples of sturgeon or salmon are thawed they should be immediately used for fertilization. Granules (see Fig. 1) can be thawed in a different way; granules are placed directly into eggs along with activating medium, the temperature of which should be the same as river water. In the case of carp, the thawed sperm samples could be stored on ice and the fertility rate could be improved after some additional treatment as described in Note 21.

Fig.1. Sperm granules after cryopreservation.

21. Ice nucleation/crystallisation results in the removal of gases from the suspension of frozen cells. Saturation of sperm with oxygen has been reported to improve the quality of spermatozoa for cryopreserved-thawed sperm of carp (35). Significant increases in fertility were observed in all sperm samples that were aerated over 5 and 15 min at 0°C. To explain this effect the dynamics of energetic components of sperm cells was studied in fresh, sperm cryoprotectants treated and cryopreserved samples (36,37). It was found that exposure to cryoprotectants, as well as cryopreservation, affects almost all elements of the energetic system of sperm including a decrease of ATP and an increase of creatine phosphate levels (36,37).

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