Storage of unfertilized eggs of rainbow trout ( Oncorhynchus mykiss) in artificial media

Abstract

Unfertilized eggs of rainbow trout ( Oncorhynchus mykiss) were stored in coelomic fluid and in Cortland medium (devoid of bicarbonate and phosphate), which was buffered with 20 mM Hepes (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) or Tris–HCl (Tris hydroxymethyl aminomethane hydrochloride) and adjusted to a pH of 8.5. Eggs stored in these solutions for 24 and 48 h, were fertilized using sperm derived from 4–6 males. The sperm was applied in a dilutant (127 mM NaCl, 20 mM Tris–HCl, pH 9.5). The percentage of embryos reaching the eyed and hatching stages was recorded as an index of fertility. Unfertilized trout eggs stored for 48 h at 12–13°C in Cortland medium buffered with Hepes or Tris exhibited the same fertility as eggs fertilized prior to storage. In fact, the percentage of embryos reaching the eyed stage was significantly greater in eggs stored in the modified Cortland solutions than in eggs stored for the same amount of time in coelomic fluid. It appears that fertility may be better in eggs stored in Cortland medium containing Hepes than in medium containing Tris. Since serine protease inhibitors have been shown to enhance fertility of goldfish eggs, the effect of a soybean trypsin inhibitor on egg fertility was investigated. Trout eggs stored for 24 h in Cortland medium containing 2.0 mg soybean trypsin inhibitor/ml were almost completely infertile. Eggs stored in modified Cortland medium and fertilized in the direct presence of soybean trypsin inhibitor (0.01 mg/ml sperm dilutant) exhibited the same percentage of eyed and hatched embryos as eggs stored in Cortland medium alone. This suggests that the inhibitor did not directly block fertilization but had an effect during storage. The results indicate that modified Cortland medium can be used for the storage of trout eggs for at least 2 days. Furthermore, washing and storage for a short time in this medium may actually enhance the fertilization of the eggs of some females.