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The objective of this study was to determine a simple and reliable ploidy identification protocol for the rainbow trout (RT), Oncorhynchus mykiss, in the field condition. To evaluate the ploidy level and compare different detection protocols, triploid RT and gynogenesis were induced by UV irradiation and/or heat shock. The hatching rate at day 30 was 85.2% and the survival rate at day 90 was 69.4% (fingerling). The sex ratio of female RT was 93.75% in the gynogenesis group, illustrating that the UV irradiation inactivated the sperm DNA. The hatching rate and survival rate were 82.0 and 74.7%, respectively, in the triploid-induced group. The triploid induction rate by heat shock procedure was 73.9%. Cytogenetic protocols for ploidy identification such as chromosome counting, erythrocyte nuclear size comparison, and analysis of nucleolar organizing regions (NORs) by silver staining were compared. Silver nitrate staining showed the greatest success rate (22/23 and 32/32 for the triploid-induced group and gynogenesis group, respectively), followed by erythrocyte nuclear size comparison (16/23 and 19/32 for the triploid-induced group and gynogenesis group, respectively) and, lastly, chromosome preparation (2/23 and 6/32 for the triploid-induced group and gynogenesis group, respectively) with the lowest success rate. Based on our findings, silver staining for RT ploidy identification is speculated to be highly applicable in a wide range of research conditions, due to its cost-effectiveness and simplicity compared to other numerous ploidy detection protocols.

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In an attempt to evaluate the function of MAP kinase in porcine oocytes and to develop a method of the assessment of its activity, myelin basic protein (MBP) was used as a substrate to detect the MAP kinase activity of porcine oocytes which had undergone maturation in vitro. The existence of MAP kinase and MAP kinase kinase (MAPKK) was verified in immature porcine germinal vesicle (GV) oocytes at 0 h culture via Western blotting. Porcine oocytes exhibited a low level of MAP kinase activity during the first 20 h of culture, which increased at 25 h, during which time a breakdown in the nuclear membrane occurred. Significantly higher increases (p<0.05) of MAP kinase activity were detected at 30 h of culture. Using the gel phosphorylation method, MBP was phosphorylated at two positions corresponding to mammalian MAP kinase-extracellular signal-regulated kinase (ERK 1) (44 kDa) and ERK 2 (42 kDa). The absolute levels of those proteins did not increase during 40 h of culture, suggesting that the detected increase in MAP kinase activity was the result of phosphorylation rather than changes in the total amount of protein. MAPKK and MAP kinase were dephosphorylated in first-stage (MI) meiotic oocytes by the addition of cycloheximide, a protein synthesis inhibitor. These results of this study indicate that the MAP kinase cascade does exists in porcine oocytes and that its activation leads to oocyte maturation.

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