Artificial Seminal Plasma Research Articles

Diluent for cool storage of the Japanese eel ( Anguilla japonica) spermatozoa

The appropriate ionic composition for the Japanese eel ( Anguilla japonica) sperm diluent for cool storage was examined. Eel spermatozoa were immotile in isotonic NaCl, KCl, and mannitol solutions, and the motility was initiated when osmolality was increased. Spermatozoa were diluted 50 times with various isotonic solutions and stored for 3 h at 5 °C. Subsequently, this mixture was diluted 21 times with 450 mM NaCl to examine the percentage motility. Spermatozoa diluted with artificial seminal plasma, consisting of 149.3 mM NaCl + 15.2 mM KCl + 1.3 mM CaCl 2 + 1.6 mM MgCl 2 buffered with 5 mM NaHCO 3-NaOH at pH 8.2, or with isotonic NaCl + KCl solution, showed high percentage motility after storage. However, spermatozoa diluted with isotonic NaCl, mannitol, NaCl + CaCl 2, or NaCl + MgCl 2 solutions showed significantly lower motility compared to the spermatozoa stored in the former two solutions. In subsequent experiments, spermatozoa were diluted with isotonic solutions of various potassium concentrations (0, 5, 15, 25, 35, 45, 75, and 164.5 mM) at 50 times and stored at 3 °C for up to 28 days to examine the optimal composition of diluent. Spermatozoa that were diluted with the 5–35 mM KCl solutions showed a decrease of percentage motility at 24 h, but they showed a marked recovery of motility by the seventh day. Increasing the potassium concentration in the isotonic diluent reduced the rate of decline in motility observed at 24 h of storage. Spermatozoa diluted with the 45, 75 or 164.5 mM KCl isotonic solutions did not show the temporal decrease of motility at 24 h. However, the percentage motility indices (percentage motility/initial percentage motility × 100) of spermatozoa in 75 or 164.5 mM KCl solutions at day 21 (26.2% and 0.6%, respectively) were significantly lower than those of 15–45 mM KCl solutions (47.9–64.8%). These results indicate that the potassium ion is an essential constituent of the isotonic diluent for cool storage of the Japanese eel spermatozoa. The optimal KCl concentration for short term storage (up to 7 days) is 45–75 mM, and that for longer storage is 15–45 mM.

Androgenetic diploids of rainbow trout (Oncorhynchus mykiss) produced by fused sperm

Androgenesis is a technique that produces individuals with all of their chromosomes from male parents. It can be useful in generating homozygous lines of fish, and in the recovery of species from cryopreserved sperm. We produced androgenetic diploids by using fused sperm to fertilize eggs. Eggs of albino "Yellow" rainbow trout (Oncorhynchus mykiss) were exposed to 60Co γ-ray irradiation to inactivate the maternally-derived nuclear DNA, and were then fertilized with wild-type rainbow trout sperm fused using an artificial seminal plasma containing 85 mM CaCl2. Embryos that reached the hatching stage (average 0.11%) were pigmented. The nuclear DNA content of cells from newly hatched fry was the same as the DNA content of erythrocyte nuclei of normal diploids.

Roles of pH and cyclic adenosine monophosphate in the acquisition of potential for sperm motility during migration from the sea to the river in chum salmon.

In the natural process of the migration of chum salmon from the sea to the river, spermatozoa moved from the testis to the sperm duct, and the pH value of seminal plasma, concentration of cyclic adenosine monophosphate (AMP) in the sperm cells, and potential for sperm motility increased. Cyclic AMP levels and the potential for motility gradually increased when testis spermatozoa with no capacity for movement were incubated in the artificial seminal plasma of which the pH was much the same as, or higher than, the pH of natural seminal plasma from the sperm duct. Such correlation in motility, pH, and cyclic AMP suggests that the increases in seminal pH and intracellular cyclic AMP level during passage of spermatozoa from the testis to the sperm duct cause the acquisition of potential for motility. Motility of testicular spermatozoa demembranated with Triton X-100 was very low in fish caught in the sea, while motility of spermatozoa from the posterior portion of the sperm duct was much higher in fish caught in the river. Furthermore, nondemembranated, intact spermatozoa showed a lag in the timing of the acquisition of potential for motility vs. demembranated spermatozoa: The demembranated sperm exhibited the potential earlier than the nondemembranated sperm. These data suggest that increase in activity of the motile apparatus, the axoneme, is a prerequisite, in part, for the acquisition of sperm motility, whereas the development of some function of the plasma membrane also contributes to this phenomenon.

Induction of potential for sperm motility by bicarbonate and pH in rainbow trout and chum salmon.

Spermatozoa of rainbow trout and chum salmon, which have no potential for motility in the testis, acquire that potential in the sperm duct. This paper demonstrates that there is little difference between the levels of sodium, potassium, calcium, magnesium, chloride and osmolality of the seminal plasma in the testis and in the sperm duct. However, the bicarbonate concentration of the seminal plasma and the pH value of semen were higher in the sperm duct than in the testis. When immotile spermatozoa obtained from the testis were incubated in artificial seminal plasma with a high pH and containing HCO3-, spermatozoa became motile within 1 h. These results suggest that spermatozoa of salmonid fish acquire the potential for motility as a result of the increase in seminal bicarbonate concentration and pH that occurs as spermatozoa pass from the testis to the sperm duct.