453 CRYOPRESERVATION OF SEABREAM SEMEN J-C. Gwo 1. INTRODUCTION The Sparidae family contains a number of economically important species throughout the world. Four seabreams (Acanthopagms latus, A. scheligeli, Pagrus major and Rhabdosargus sarba) are the most popular for aquaculture in Taiwan. They all are protandrous, being a functional male at an early age (usually one year old) but reversing sex to become a functional female in later years. Self-fertilization is possible for these four species. Such studies could facilitate genetic understanding and quickly lead to the development of inbred lines. The cryopreservation of sperm would also aid broodstock management, gene conservation and genetic selection of the beneficial traits for intensive commercial farming. Kurokura et al. [1] succeeded in using frozen crimson seabream (Evonnis japonica) semen to fertilize female red seabream and produced hybrids with increased growth rate and desired body color. 2. PROTOCOL FOR FREEZING AND THAWING 2.1 Fish handling and semen collection  Four seabreams ( Acanthopagms latus, A. scheligeli, Pagrus major and Rhabdosargus sarba) were obtained from local fish farms in Peng-Hu, Taiwan. Semen was collected during the breeding season (October to Feburary) by abdominal massage from single males weighing between 250 and 500 g. The semen was placed on crushed ice immediately after collection; freezing was accomplished and sperm motility was determined within 30 min.  The activation of fresh sperm motility, the measurement of semen osmolality, the protocol of freezing and thawing and the fertilization tests were examined as the same as those of grouper semen in this book. 2.2 Freezing and thawing procedures  Sperm motility was evaluated for 5 cryoprotectants (DMSO, glycerol, propylene glycerol, ethylene glycerol and methanol). The effects of cryoprotectant concentrations of 10, 20 and 30% and an equilibration time of 5, 30 and 60 min were surveyed.  Five extenders were compared: 150 mM unbuffered NaCI, 300 mM glucose, marine teleost Ringer, 150 mM sodium citrate, and distilled water. Ten percent dimethyl sulfoxide (DMSO; Sigma) was chosen as the sole cryoprotectant. © 2009 by Taylor & Francis Group, LLC