**2.1.1 Gametes collection**

Naturally matured fishes were obtained from Qingdao hatchery during the spawning season (From the middle of March to the end of May). Twenty males and 10 females (3 kg to 4 kg individually, 10 years old) were cultivated in a 20-m3 concrete rearing pond with flowthrough seawater and fed daily with cooked meat of bay mussel, *Mytilus edulis*. Prior to handling, males were firstly anesthetized in a 0.003% eugenol bath. Sperm was collected into petri dishes by gently hand-stripping the abdomen of the ripe males. Extreme care was taken to avoid the contamination of sperm with seawater, blood, urine and feces. The percentage of motile spermatozoa was checked with a Nikon-YS-100 light microscope (Nikon Corporation, Tokyo, Japan) at 250 × magnification. Sperm with motility > 85% was kept on crushed ice and transported to the laboratory for further use. Eggs were collected by abdominal pressure of the females at the time of ovulation. Good eggs were slightly yellowish, translucent and round-shaped. Eggs for fertilization trials were collected only from one female.

#### **2.1.2 General procedure for sperm freezing and thawing**

Sperm were diluted in Cortland extenders (Liu et al, 2006) containing DMSO with different concentrations (6–24% DMSO). After mixing thoroughly, 1.6 ml sperm was placed into 2-ml cryovials. The cryovials were transferred into a Kryo-360-1.7 programmable freezer (Planer Plc. Middlesex, UK), equilibrated for five minutes at 0oC, and frozen from 0 to −150oC at a cooling rate of 20oC min-1, then plunged into liquid nitrogen for storage. The frozen sperm were thawed in 40oC water bath after being preserved in liquid nitrogen for one month. After that, the thawed sperm was evaluated for motility and fertilizing capacity.
