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MMEETTHHOODDSS IINN MMOOLLEECCUULLAARR BBIIOOLLOOGGYYTTMM Volume 253 GGeerrmm CCeellll PPrroottooccoollss VVoolluummee 11:: SSppeerrmm aanndd OOooccyyttee AAnnaallyyssiiss EEddiitteedd bbyy HHeeiiddee SScchhaatttteenn Cryopreservation of Semen of Salmonidae 1 1 Cryopreservation of Semen of the Salmonidae with Special Reference to Large-Scale Fertilization Franz Lahnsteiner 1. Introduction 1.1. Importance of Semen Cryopreservation in Fish Fish semen cryopreservation has important applications in the following fields: (1) in aquaculture, for synchronization of artificial reproduction, for efficient utilization of semen, and for maintaining the genetic variability of broodstocks; (2) in biodiversity, for gene banks of endangered species and of autochthon fish populations; and (3) in temporary unlimited supply with mate- rial for research as, for example, for toxicological tests or interspecific breeding. Salmonidae are the traditionally cultured fish in many parts of the world. As a favorite game in sport fishing, restocking and conservation is necessary for many populations. Therefore, the semen cryopreservation is of particular importance in these species. 1.2. The Spermatozoa of Salmonid Fish Salmonidaehave simple, constructed spermatozoa (1). They are acrosome- less and have a slightly ovoid head (length: 1.37 ± 0.15 µm; diameter 1.21 ± 0.13 µm), a cylindrical midpiece (length: 0.55 ± 0.08 µm; diameter: 0.74 ± 0.20µm), and a flagellum that is about 40 µm long (1). The midpiece contains three to five mitochondria that are fused with each other to a so-called chon- driosome (1). The sperm motility is inhibited by 20–40 mM potassium (2). After motility activation, semen has high motility rates up to 100% and swimming velocities of 120–140 µm/min (3,4). However, the sperm motility dura- tion is very short. In 15–20 s, motility decreases for more than 50%, and it stops completely within 1 min (3,4). Fertilization is external and also a very From:Methods in Molecular Biology, vol. 253: Germ Cell Protocols: Vol. 1 Sperm and Oocyte Analysis Edited by: H. Schatten © Humana Press Inc., Totowa, NJ 1 2 Lahnsteiner quick process. It occurs within 15–20 s after the gametes have been released into water (5). For successful fertilization, spermatozoa have to swim into the micropyle of the egg (5). Therefore, sperm motility and fertility are correlated and motility is often used for viability determination (3,4), especially as fertilization assays and the subsequent hatching of eggs is very time-consuming in theSalmonidae(6). 1.3. Specific Problems in the Cryopreservation of Salmonid Spermatozoa The cryopreservation of spermatozoa of the Salmonidae and of fish in general faces the following problems: 1. Fish semen cryopreservation is not a laboratory method but has to be applied under field conditions with a minimum of technical supply. Therefore, the basic cryopreservation protocol must be adapted for easy and reliable outdoor use (6,7). 2. As external fertilizing species with a limited annual reproduction period, Salmonidae have a high egg production. During natural spawning and in artificial insemination, several thousand eggs are fertilized simultaneously. There- fore, the cryopreservation methods also have to be adapted for large-scale fertilization by developing special techniques for freezing of large semen volumes and for insemination of numerous eggs (6). 3. Fish semen reveal wide quality differences depending on fish age, spawning state, and general conditions (8). To obtain consistent and good cryopreservation results, it is necessary to test semen for suitability for cryopreservation. Useful parameters have been calculated from regression models (3) and are (for >50% postthaw fertility) as follows: fresh semen motility rate >80%, average path swimming velocities between 80 and 100 µm/s, seminal plasma pH < 8.2, and seminal plasma osmolality >330 mosmol/kg (3). 1.4. Cryopreservation The described semen cryopreservation method was tested onHucho hucho, Oncorhychus mykiss, Salvelinus alpinus, Salvelinus fontinalis, Salmo trutta f. lacustris, Salmo trutta f. fario, Thymallus thymallus, and Coregonus lavaretus. The method requires the following working steps: the dilution of semen in the extender, filling of semen in freezing vessels, freezing and thawing, and semen handling for fertilization (6). An equilibration of semen in the extender is not required, as the sperm cells are small and permeable and the cryoprotectants penetrate the cells in less than 1 min (6). The extender composition was determined in a series of motility and fertility tests (6). The extender inhibits the sperm motility as a result of high potassium concentrations, maintains the sperm viability because of its balanced ionic composition, and protects the spermatzoa during freezing and thawing by a combination of cryoprotectants and additives (methanol, hen egg yolk, bovine serum albumin). Cryopreservation of Semen of Salmonidae 3 Semen must be frozen as concentrated as possible, as high amounts are necessary for large-scale insemination. Therefore, low dilution ratios of semen in the extender should be used. However, too low dilution rates lead to cell com- pressions (critical concentration [2.0–3.0] × 109 cells/mL extender) during freezing and thawing and, subsequently, to a loss of semen viability (6). Addi- tionally, semen density and therefore also the dilution rates are species-specific in the Salmonidae(6). As salmonid semen cryopreservation is a field technique for freezing, a simple procedure is required. It is done in the vapor of liquid nitrogen in an isolated box whereby the distance of straws from the level of liquid nitrogen determines the freezing rates (6). Freezing rates are species-specific (7). In practice, semen volumes of at least 20–30 mL must be handled at once. However, only in straws with volumes of 0.5 mL and 1.2 mL were the freezing and thawing rates optimal. In larger straws and plastic bags, the results were very inconsistent. Therefore, a method was developed to build straw packages consisting of 1.2 mL straws by connecting them in flexible racks (7). Thawing is performed in water of adequate temperature. To recover fertility, frozen salmonid semen must be warmed to 20°C, a temperature higher than the physiological optimum (4–6°C). The membranes or the metabolism are possibly better stabilized by this thawing procedure (6). The thawed semen has several types of alteration (9). Therefore, it is nonstable after thawing (3) and must be handled very accurately, and in com- parison to fresh semen, modified insemination procedures have to be applied (6,7): To compensate cell lesions originating during freezing and thawing and to reactivate sperm motility and fertility, special saline solutions are necessary (6,9). Also, the insemination itself is of importance: The wet fertilization (eggs and semen are placed in fertilization solution and mixed) is only suited for fertilization of small egg quantities up to 50 g (7). To inseminate larger egg quantities, dry fertilization (eggs and semen are mixed before fertilization solution is added) has to be applied (7), as it results in more homogeneous mixing of gametes (7). 1.5. Quality and Fertilizing Capacity of Frozen–Thawed Semen There exist no species-specific differences in semen quality after cryopreservation. Representative changes in motility parameters and fertilizing capacity of rainbow trout semen after cryopreservation have been investigated (9)and are shown in Table 1. When compared to untreated semen, the percentage of immotile spermatozoa is significantly increased and the rate of motile spermatozoa is decreased. At low sperm-to-egg ratios also, the fertilization rate (evaluated in the embryo stage before hatching) is significantly decreased (see Table 1). However, the decrease in fertilizing capacity can be completely com- 4 Lahnsteiner Table 1 Motility and Fertility of Untreated and Cryopreserved Rainbow Trout Semen Parameter Fresh Frozen/Thawed Immotile (%) 4.8 ± 3.3a 71.3± 9.4b Local motile (%) 9.5 ± 4.4a 5.3 ± 3.6a Motile (%) 85.7 ± 12.2a 23.5± 5.5b Circular motile (%) 52.8 ± 10.6a 20.1± 10.3b Nonlinear motile (%) 29.1 ± 8.0a 17.1± 8.1b Linear motile (%) 18.1 ± 11.1a 62.8± 14.0b Average path sperm velocity (µm/sec) 90.3 ± 17.2la 94.5± 16.5a Fertility at sperm-to-egg ratio (3–4) × 106 79.6± 12.2a 76.3± 7.4a Fertility at sperm-to-egg ratio (1.5–2) × 106 79.8± 10.5a 62.4± 6.6b Note: Data are mean ±SD,n= 20. Data in one row superscripted by the same letter are not significantly different. pensated by higher sperm-to-egg ratios (see Table 1) (6) and then fertilization rates in the range of fresh semen control are obtained (6,7). The percentage of embryonic malformations is similar with cryopreserved and untreated semen (6). 2. Materials All chemicals are of analytical grade. Distilled water is used. 1. Hen egg yolk is prepared freshly and carefully separated from the white, which causes agglutination of spermatozoa. 2. Extender: Dissolve 600 mg NaCl, 315 mg KCl, 15 mg CaCl ·2H O, 20 mg 2 2 MgSO ·7H O, and 470 mg HEPES (sodium salt) in approx 80 mL water. Adjust to 4 2 pH 7.8 with NaOH or HCl; fill with water to 100 mL. Add the following cryoprotectants and additives: 10% (v/v) methanol, 0.5% (w/v) sucrose, 1.5% (w/v) bovine serum albumin, 7% (v/v) hen egg yolk. Measure required egg yolk volumes in plastic syringes without needles. At < –20°C, the extender without hen egg yolk is stable for an unlimited time; the extender with egg yolk is prepared freshly. 3. Fertilization solution: Dissolve 500 mg NaHCO and 600 mg Tris in approx 80 mL 3 of water, adjust pH to 9.0 with NaOH or HCl, and fill with water to 100 mL. 4. Freezing vessels: Straws with volumes of 0.5 mL and 1.2 mL are commercially available. Preparation of straw packages (seeFigs. 1 and 2): Use 1.2-mL straws and 0.45 to 0.55-mm-thick plastic foil from commercially available plastic bags that remains flexible at liquid-nitrogen temperature (test!). Cut two plastic ribbons to a width of 1.5 cm and a length depending on the desired number of straws that should be connected (required length per straw = 1.3 cm) (see Fig. 1A). Place the ribbons on top of each other (seeFig. 1A). Seal them together at their wide side with a commercial plastic bag sealing apparatus (sealing width of 6 mm) in a way that a 6-mm sealed portion is followed by a 7-mm unsealed Cryopreservation of Semen of Salmonidae 5 Fig. 1. Steps in the production of flexible plastic racks. ous = opened unsealed portion, pr = plastic ribbons, s = sealed portion, us = unsealed portion. (A) Plastic ribbons are placed one above each other; (B)plastic ribbons are sealed together; (C)the unsealed portions are opened and straws are fitted in. portion (seeFig. 1B). Place the 1.2-mL straws in the unsealed portions with their plugged side (see Fig. 1C). Fit them tightly. In case the fit is not proper, the openings must be adjusted by additional sealing or enlargement. The distance between the single straws is 0.5 cm (seeFig. 2A,B). 5. Freezing apparatus: Freezing is done in a self-constructed insulated box (inner dimensions: base-27 ×18 cm, height = 33 cm) on a tray (seeFig. 3). This tray can 6 Lahnsteiner Fig. 2. Straw packages consisting of 1.2-mL straws: (a)plastic rack during fitting in the straws; (b)plastic rack loaded with straws; (c)straw package, placed horizon- tally on a plane as done for freezing; (d) straw package rolled together as used for storage and for cutting open; (e)straw package in the can of a liquid-nitrogen storage container;(f) straw package during cutting open. 6 Cryopreservation of Semen of Salmonidae 7 Fig. 3. Freezing box: A = insulated wall; B = freezing chamber; C = tray for straws; D = cover; E = overflow trap for liquid nitrogen. (a) Freezing box ready for use; (b)scheme of the freezing box. Left: cross-section, right: longitudinal section; E = set screw; F = tray holder. 7 8 Lahnsteiner Table 2 Optimal Dilution Ratios of Semen in the Extender and Sperm Density in the Salmonidae Dilution ratio Sperm density Species (semen:extender) (cells/mL) Salvelinus alpinus 1:2 8.0 × 108–2.5× 109 Hucho hucho, 1:3 4.0 × 109–8× 109 Oncorhynchus mykiss, Salvelinus fontinalis, Thymallus thymallus Salmo trutta f. fario 1:5 9.0 × 109–1.5× 1010 Salmo trutta f. lacustris 1:7 1.0 × 1010–2× 1010 be adjusted to different distances (0–10 cm) above the surface of liquid nitrogen (see Fig. 3A,B), allowing the application of various freezing conditions (6). Adjustable trays are of advantage when freezing levels have to be changed fre- quently. Otherwise, floating trays are also useful (8). 3. Methods 3.1. Semen Collection Dry the genital papilla of the fish from adhering water. Place a collection tube of adequate size under the genital pore and collect the semen by pressure on the abdomen (seeNote 1). Store the semen on ice. 3.2. Semen Dilution and Filling of Semen into Straws Dilute the semen in 4°C cold extender at the required ratio. Reliable dilution ratios and maximal and minimal sperm densities are listed in Table 2 (seeNote 2). A 1-min equilibration is sufficient. Do not extend equilibration to >10 min. Cool straws to 4°C. Fill straws with micropipets. As the straws have a stopper that avoids liquid penetration, the sperm suspension can be sucked in by mouth. As this is the quickest way to fill the straws, it is of advantage in routine fieldwork. 3.3. Freezing of Straws Cooling of the freezing box and equilibration to stable conditions requires between 15 and 30 min at room temperature. This must be considered for semen processing. 1. Cool the interior of the box with liquid nitrogen. 2. Fill up with liquid nitrogen until the overflow trap is reached. When ready for freezing, the box contains a volume of 2.67 L liquid nitrogen. Cryopreservation of Semen of Salmonidae 9 Table 3 Freezing and Thawing Conditions for Semen of the Salmonidae Freezing level Species Straw type and temperature Thawing Hucho hucho, 0.5 mL 1.5 cm (–110 ± 2°C)x 25°C, 30 sxxx Oncorhynchus mykiss, 1.2 mL 1.0 cm (–130 ± 2°C)x 30°C, 30 sxxx Salmo trutta f. fario, Salmo trutta f. lacustris, Thymallus thymallus, Esox lucius Salvelinus fontinalis, 0.5 mL 2.5 cm (–92 ± 2°C)xx 25°C, 30 sxxx Salvelinus alpinus 1.2 mL 2.0 cm (–100 ± 2)xx 30°C, 30 sxxx Note: Similar superscripts indicate that freezing rates or thawing rates were similar under these conditions. Freezing temperature was measured with a thermoelectrode inserted in the straws. 3. Adjust the tray to the desired freezing level and equilibrate for 5 min to reach the appropriate temperature. 4. Place the straws or straw packages on the tray (see Fig. 2C) and freeze for 10 min. Freezing levels and freezing temperatures are species-specific and are shown in Table 3; freezing rates are shown in Fig. 4. 5. Cover to avoid extensive nitrogen evaporation (seeNote 3). 6. Plunge straws into liquid nitrogen. When using an adjustable tray, the whole tray can be immersed into liquid nitrogen. 3.4. Storage of Straws Transfer the single straws into the cans of commercial liquid-nitrogen containers. Because the straw packages remain flexible in liquid nitrogen, they can be rolled together (see Fig. 2D) and are placed in the cans also (see Fig. 2E). 3.5. Thawing of Straws 1. Thaw the 0.5-mL straws in 25°C water for 30 s and the 1.2-mL straws in 30°C water for 30 s (for thawing rates, seeFig. 4) (seeNote 4). Thawing is not species- specific. Take the single straws out of the liquid-nitrogen container and transfer immediately into water. Gently agitate during thawing. After thawing, cut away the straw stopper and release the sperm suspension onto the eggs. 2. Process straw packages in a similar way. Take them out of the container, roll them out quickly, and place in water. Thereafter, roll together again and cut away the plugs of the straws with scissors (see Fig. 2F). Release sperm suspension onto the eggs.