**2.1.4 Sperm fertilization and hatching experiments**

Fertilization capacity of post-thaw sperm frozen with DMSO (6–24% DMSO) was evaluated. The optimized sperm to egg ratio of 500:1 was selected for the following fertilization trials (Li et al., 2006). The artificial fertilization method was described in detail in Liu et al (2007b). Fertilization rates were evaluated within 6–8 h after insemination by counting the percentage of gastrula-stage embryos in relation to the total number of eggs used. Fortyeight hours after fertilization, the number of hatched larvae was counted in each experiment. The hatching rates were calculated as the percentage of hatched larvae in relation to the total number of eggs used in each experiment.
