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Notes: The present study describes a uniform method for cryopreservation of semen of Salmonidae (Oncorhynchus mykiss (Walbaum), Salmo trutta f. fario L., Salmo trutta f. lacustris L., Coregonus sp.). It presents a new type of extender and experiments demonstrating that warming of frozen/thawed semen to 20°C prior to fertilization significantly increases the fertilization rate. Freezing is performed in straws in the vapour of liquid nitrogen and for insemination a diluent technique is used. The consistency of the method was tested by repeating the experiments with different batches of semen and eggs. The following fertilization rates (% of control) were obtained: Oncorhynchus mykiss: 89.6 ± 16.0% (mean ± standard deviation, n= 25, n of control = 20, sperm/egg ratio of 1.6 ± 0.2 × 106 spermatozoa/ egg). Salmo trutta f. fario: 93.8 ± 6.4% (n= 12,9.9 ± 1.2 × 106spermatozoa/egg), Coregonus sp.: 92.8 ± 2.4% (n= 6, 0.5 × 106 spermatozoa/egg), Salmo trutta f. lacustris: 85.0 ± 8.4% (n= 12, 4.8 ± 1.4 × 106 spermatozoa/egg).

Notes: Using the cryopreservation method of Lahnsteiner, Berger, Weismann & Patzner (1995, Aquaculture Research26, 801-807) the influence of allowable variations of methodical parameters (storage of semen before cryopreservation, dilution ratios in the extender, equilibration in the extender, cooling rates, storage of deep-frozen semen in liquid nitrogen, storage of frozen/thawed semen, minimal sperm/egg ratio) was investigated under the aspect of routine utilization.Under optimized experimental conditions, fertilization rates were 90-100% of controls in Oncorhynchus mykiss (Walbaum), Salmo trutta L. f. lacustrisSalmo truttaL.f. fario and Salvelinus fontinalis (Mitchill). The following results were obtained: 1. Storage of untreated semen for more than 1 h before cryopreservation decreased the postthaw fertility. 2. Equilibration of semen up to 20 min in the extender did not affect the postthaw fertility. 3. Optimal dilution ratio of semen in the extender was threefold in Oncorhynchus mykiss and Salvelinus fontinalis. Lower dilution ratios decreased the postthaw fertility, higher dilution ratios did not affect the postthaw fertility. In Salmo trutta f. lacustris and Salmo trutta f. fario, which have a higher sperm density, optimal dilution ratio of semen in the extender was fivefold to sevenfold. 4. In Oncorhynchus mykiss, as in Salmo trutta f lacustris and Salmo trutta f. fario, the optimal freezing height was at 1.5 cm above the level of liquid nitrogen (-110 ± 2oC); in Salvelinus fontinalis it was 2.5 cm above the level of liquid nitrogen (-92 ± 2oC). Changes in the freezing height of 0.5 cm (about 10oC) resulted in a significant decrease of postthaw fertility. 5. Storage of deep-frozen semen for up to 370 days in liquid nitrogen had no influence on its postthaw fertilization rate. 6. Storage of frozen/thawed semen for 30 s before insemination significantly decreased its postthaw fertility. 7. Reliable minimal sperm:egg ratio to obtain fertilization rates of 90-100% of control was 3-5 X 105 spermatozoa egg-1.

Notes: The present study investigated motility, acrosome reaction, fertility and cryobiological parameters of the semen of the sterlet, Acipenser ruthenus L. Sperm motility persisted for about 4 min in water, and the main swimming type was the linear motion. Motility was prolonged at osmolalities of 12.5 mosmol kg−1 and in the presence of magnesium ions, while calcium had no effect. Also a pH in the range of 7.0–9.0 had no effect on ` motility. At osmolalities of 25–50 mosmol kg−1 the sperm motility was partly inhibited, at osmolalities of 100 mosmol kg−1, completely and irreversibly. In 50 mosmol kg−1 solutions with 2.5–5 mM L−1 KCl the motility inhibition was total, but reversible. The acrosome reaction was not induced by one of the described solutions, but the percentage of spermatozoa with reacted acrosomes was low (〈20%) and highly variable in all experiments. The optimal extender base for cryopreservation was a solution consisting of 50 mM L−1 NaCl, 5 mM L−1 KCl, 10 mM L−1 Tris (pH 8.5). From the tested cryoprotectants only dimethylsulphoxide (DMSO) and methanol provided sufficient cryoprotection. After freezing and thawing, the motility rates and swimming velocities were higher with DMSO than with methanol. However, the fertility was very significantly reduced with DMSO (10.3±0.5%) while with methanol fertilization rates in a similar range (32.7±4.4%) as with fresh semen (33.90±0.8%) could be obtained. Optimal freezing conditions for sterlet semen were in the vapour of liquid nitrogen 3–5 cm (−95°C to −85°C) above its surface, the optimal thawing conditions at 25°C for 30 s. The acrosome reaction was not induced by these cryopreservation protocols.

Notes: 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.

Notes: 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.

Notes: The present paper summarizes the data on a semen cryopreservation method for the Salmonidae (Oncorhynchus mykiss, Salmo trutta f. lacustris, Salvelinus fontinalis, Salvelinus alpinus, Salmo trutta f. fario, Hucho hucho, Coregonus lavaretus, Thymallus thymallus) and for the Northern pike (Esox lucius) published during recent years. It describes (1) methods used for the determination of sperm viability; (2) the protective efficiency of substances specifically for protection of internal and external parts of cells and the process of extender development; (3) the freezing, thawing and fertilization conditions; and (4) the tolerable deviations from the freezing protocol for more easy application. Finally, biomarkers are reported that predict the suitability of semen for cryopreservation and the quality of frozen–thawed semen.

Notes: Semen of the African catfish, Clarias gariepinus (Burchell, 1822), was investigated with respect to its cellular composition, sperm cell density, maturation grade, motility and fertility. Storage conditions were tested, whereby sperm viability was assessed by measurement of the motility after activation and by fertility tests. Testicular semen differed in its composition, i.e. the sperm density and numbers of spermatids, according to the maturity grade of the testis. Two semen types could be distinguished: semen type I was characterized by high sperm densities and low numbers of spermatids and semen type II had lower sperm densities and higher numbers of spermatids. Two semen types did not differ in motility and fertility (when adjusted for differences in sperm density). During storage, the sperm viability was influenced by the sodium concentration of the storage medium, temperature, membrane stabilizers as bovine serum albumen (BSA) or hen egg yolk, antibiotics and oxygen. Semen viability was maintained best when it was diluted at a ratio of 1:5 in storage solution (150 mmol L−1 NaCl, 2.5 mmol L−1 KCl, 1 mmol L−1 CaCl2, 1 mmol L−1 MgSO4, 20 mmol L−1 Tris (pH 8.5) and 0.5% BSA or 0.5% hen egg yolk) and stored at 4 °C. Oxygen gassing and addition of antibiotics (1 mg mL−1 gentamycine sulphate) to the storage solution affected the two semen types in different ways. Antibiotics had no effect on type I semen, but had a positive effect on type II semen. Oxygen gassing had a positive effect on type I semen but a negative effect on type II semen.

Notes: The fine structure and motility of spermatozoa and the composition of the seminal plasma of the perch Perca fluviatilis are investigated by electron microscopy, computer assisted cell motility analysis (CMA) and biochemical methods. The spermatozoon is asymmetrical as the flagellum inserts mediolateral on the nucleus. It lacks an acrosome, has an ovoid head and a small midpiece with one mitochondrion. Sperm motility–initiated in distilled water (10° C)–is characterized as follows: 85·0 ± 2·7% of the spermatozoa are motile, the main swimming type (10 ± 1 s after motility initiation) is the linear motion (61·4 ± 24·4%) and the average swimming velocity is 122·4 ± 21·9 μm s–1. When motility is initiated with NaCl, glucose or sucrose solutions of 100 mosmol kg–1 the percentage of motile spermatozoa and the swimming types are similar as in water, but the swimming velocity (174·0 ± 22·3 μm s–1) is significantly higher. Motility is inhibited by high osmolality of the diluent: when increasing the osmolality of the saline solutions to 350 mosmol kg–1 sperm motility is totally suppressed while potassium (10–40 mmol 1–1) does not affect motility parameters. pH optimum for sperm motility is between pH 7·0 and 8·5. The seminal fluid contains 124·01 ± 21·68 mmol 1–1 sodium, 10·22 ± 1·11 mmol 1–1 potassium and 0·72 ± 0·26 mmol 1–1 calcium. pH is 8·25 ± 0·09, and osmolality 283·90 ± 37·19 mosmol kg–1. The following organic components were determined: monosaccharides (glucose 63 ± 19 μmol 1–1, fructose 54 ± 28 μmol 1–1, galactose 59 ± 25 μmol 1–1), lipids (cholesterol 5·51 ± 6·42 μmol 1–1, triglycerides 72 ± l00 μmol l–1, cholesteryloleate 15–150 μmol 1–1, phosphatidylcholine 26 · 31 μmol 1–1, glycolipids 1–10 mg 100 m1–1), lactate 108 ± 99 μmol 1–1, hydroxybutyrate 102 ± 99 nmol 1–1, choline 59 ± 159 μmol 1–1, protein 344·75 ± 59·06 mg 100m1–1, enzymes (β-d-glucuronidase l.4 ± 0.7 μmol h–1 100 ml–1, protease (caseolytic activity) 1·0 ± 0·6 μmol h–1 100 ml–1, alkaline phosphatase 2520·0 ± 861·0 μmol h–1 100 ml–1, acid phosphatase 44.0 ± 16.0 μmol h–1 100 ml–1, glucose-6-phosphate dehydrogenase 38·9 ± 86·9 μmol h–1 100 ml–1, lactate dehydrogenase 134·4 ± 69·6 μmol h–1 100 ml–1, butyrylcholine esterase 0·014 ± 0·010 μmol h–1 100 ml–1, adenosine triphosphatase 562·8 ± 665·4 μmol h –1 100 ml–1).

Notes: The composition of the organic compounds of the seminal fluid, pH values and osmolalities were investigated in three cyprinid species, the bleak (Alburnus alburnus), the chub (Leuciscus cephalus) and the zaehrte (Vimba vimba). The seminal fluid contains monosaccharides (glucose, fructose, galactose, xylose), lipids (cholesterol, fatty acids, phosphatidylcholine, glycolipids) and proteins, and exhibits activities of acid phosphatase, β-glucuronidase, proteases and to some extent of alkaline phosphatase. The composition of free amino acids reveals species specific differences.