Morphology of the crayfish spermatozoon and of the spermatophore wall during three stages of final maturation including freshly ejaculated, post-mating, and after spermatozoa release was studied and compared. The crayfish spermatophore consists of a sperm mass enveloped by a three layered spermatophore wall. After mating, the thickness of the outer layer of the spermatophore is increased. The matrix in the middle layer of the spermatophore becomes reticulated, and granules inside this layer release their contents. Fibers in the inner layer degrade to small particles. The spermatozoon capsule swells and the space between the capsule and the spermatozoon appears. The area of the plasma membrane is increased by wrinkling of the surface and alteration from a single to a multilayered structure at the anterior part of the acrosome. The density of the subacrosome zone increases in the vicinity of the main body of the acrosome. With the onset of fertilization, the layers of the spermatophore are dissolved by female glair gland secretions. The spermatozoon extracellular capsule, plasma membrane, and membranous lamellae are eliminated, and bundles of filaments are released from anterior part of the acrosome. The subacrosome zone loses electron density and retracts. The electron-dense material of the innermost layer of the acrosome is discharged and, together with acrosome filaments, forms a filament/droplet structure at the anterior part of the spermatozoon. The most important change is observed in the subacrosome zone, which may play a key role in the fertilization. Also, morphological changes of the spermatozoon that occur after release from the capsule, especially formation of the filament/droplet structure, may contribute to the mechanism of egg-spermatozoon binding in the crayfish, representative of animals with non-motile spermatozoa.

Sperm density, mineral and organic composition of the seminal plasma and their physiological relationship were investigated in the Caspian brown trout (Salmo trutta caspius). To establish a rapid and accurate method for assessment of sperm density, three di¡erent techniques were used: sperm counting, spectrophotometry (at 480 nm) and determination of the spermatocrit. The seminal plasma contained 159.26 AE 8.84 mM sodium (Na), 33.72 AE 2.01mM potassium (K), 133.04 AE 5.96 mM chlorine (Cl), 1.68 AE 0.2 mM calcium (Ca) and 0.988 AE 0.13 mM magnesium (Mg). The following organic components were found: total protein 0.75 AE 0.14 mg100 mL À 1 , cholesterol 2.86 AE 0.58 mg L À 1 and glucose 3.81 AE 1.04 mM L À 1 . The mean sperm density was estimated to be 3.3 Â 10 9 spermatozoa mL À 1 . The spermatocrit (%) ranged from 25 to 52 in sperm samples. Highly signi¢cant linear relationships were found between sperm density and spermatocrit (R 2 5 0.703, Po0.001) and sperm density and optical density (R 2 50.909, Po0.001), indicating that optical density can be used as a quick and accurate method of estimating sperm density. Signi¢cant relationships were also found between sperm density and Ca, Mg and total protein of seminal plasma. A signi¢cant correlation was also observed between the Ca and Mg concentrations (R 2 50.774, Po0.01). The following correlations were observed between mineral and organic components: total protein and Ca (R 2 50.462, Po0.05), total protein and Mg (R 2 50.518, Po0.05) and glucose and Cl (R 2 50.374, Po0.05). These parameters should be considered when developing procedures for either arti¢cial fertilization or for cryopreservation of sperm. Sperm density and seminal plasma composition in Salmo trutta caspius A Hatef et al.

In this study, ovarian fluid composition and its effects on the motility and fertilizing ability of sperm were studied in endangered Caspian brown trout, Salmo trutta caspius, and were compared with a saline activation medium (125 mM NaCl, 30 mM Glycine, 20 mM Tris-HCl, pH = 9.0) and freshwater as the control. The ovarian fluid was composed of sodium 164.4 +/- 4.4 mM l(-1), potassium 1.8 +/- 0.1 mM l(-1), calcium 0.6 +/- 0.1 mM l(-1), magnesium 0.4 +/- 0.02 mM l(-1), chloride 127.4 +/- 5.9 mM l(-1), total protein 389.5 +/- 89.6 mg 100 ml(-1), cholesterol 9.3 +/- 1.2 mg dl(-1), and glucose 3.3 +/- 0.2 mM l(-1). The percentage of motile spermatozoa and the duration of sperm motility were significantly higher in ovarian fluid (62 +/- 3%, 74.6 +/- 0.8 s) than freshwater (35 +/- 4%, 44 +/- 1 s), but they did not differ significantly from saline medium (56 +/- 3%, 74.3 +/- 0.7 s) (P > 0.05). Higher eyeing rates were observed after the activation of sperm in ovarian fluid and saline solution than freshwater when 35,000 or 350,000 spermatozoa per egg were added into the activation media. However, no significant differences were observed at higher concentrations of spermatozoa per egg (730,000) (P > 0.05). Also, this study showed that the ovarian fluid composition can be considered as a species-specific character among salmonid fishes. As a conclusion, the results of this study recommend the use of ovarian fluid or the saline solution as an activation medium in the artificial reproduction of Caspian brown trout.

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Proteins of the signal crayfish Pacifastacus leniusculus egg and spermatophore were identified using in-gel digestion, mass spectrometry, and Mascot search. Forty-one and one-hundred-fifty proteins were identified in egg and spermatophore, respectively. The proteins were classified into nine categories including cell defence, cell signaling, cytoskeleton, DNA related activity, metabolism and energy production, protease and protease inhibitor, respiration, transportation, and others and unknown. Twenty-two proteins were found in both egg and spermatophore. The respiration and cytoskeleton groups are the most diverse categories in the protein profiles of the egg and spermatophore, respectively. No protein was assigned to DNA related activity and cell defence categories in the protein profile of the crayfish egg. Differences between protein profiles of the crayfish egg and spermatophore show different functional priorities for each of gametes. Several proteins having possible roles in gametogenesis, capacitation, acrosome reaction, and fertilization were identified. This proteomic profile of signal crayfish gametes provides a basis for further investigation of functional roles of the identified proteins in aspects of reproduction such as capacitation and fertilization.

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This study reports about the spermatozoal ultrastructure of three species of astacid crayfish, i.e., the stone crayfish Austropotamobius torrentium, signal crayfish Pacifastacus leniusculus, and noble crayfish Astacus astacus. The acrosome is a cup shaped and electron-dense structure at the anterior of the spermatozoon and comprises three layers of differing electron densities filled with parallel filaments that extend from the base to the apical zone. The acrosome was significantly longer in A. astacus than in P. leniusculus and the shortest acrosome belongs to A. torrentium. The width of the acrosome was significantly narrower in A. torrentium than in P. leniusculus and the widest acrosome belongs to A. astacus. The L:W ratio was significantly greater in A. torrentium than in P. leniusculus and the lowest ratio belongs to A. astacus. Radial arms are visible on each side of the acrosome or nucleus in sagittal view and wrap around the spermatozoon. Each radial arm comprises a parallel bundle of microtubules arranged along the long axis within a sheath. The nucleus, with decondensed material, is located in the posterior of the cell. All parts of the spermatozoon are tightly enclosed within an extracellular capsule. Despite a well-conserved general structure and similarity of pattern among these spermatozoa, differences in the dimensions of the acrosome within the studied species may be useful to help distinguish the different crayfish species.