**1. Introduction**

Long-term storage of sperm in liquid nitrogen is a valuable technique for genetic resources preservation (Kopeika et al. 2007). The research on fish sperm cryopreservation has achieved great advances since the first successful sperm cryopreservation in herring (Blaxter 1953). It provides many benefits such as ease of global germplasm shipping and supply (Tiersch et al. 2004), selective breeding and hybridization with desirable characteristics (Henderson-Arzapalo et al. 1994), and conservation of genetic diversity (Van der Walt et al. 1993; Tiersch et al. 2000; Ohta et al. 2001). Furthermore, a frozen sperm bank could maintain the continuous and stable supply of gametes for hatchery seed production or laboratory experimentation. Because of the advantages of this technique, fish sperm of over 200 freshwater and 40 marine species have been cryopreserved successfully (Gwo 2000).

Most of fish sperm cryopreservation researches have focused on freshwater species such as cyprinids (Babiak et al. 1997; Lahnsteiner et al. 2000), salmonoids (Conget et al. 1996; Cabrita et al. 2001), catfishes (Christensen and Tiersch 1997; Viveiros et al. 2000) and loach (Kopeika 2003a, b; Dzuba & Kopeika 2002). In recent years, with the rapid development of marine fish aquaculture, some experiments on germplasm cryopreservation have also been conducted in marine fish species, especially the great commercial value ones such as red seabream (Liu, et al. 2006;Liu, et al. 2007a,b;Liu, et al. 2010 a,b) turbot (Dréanno et al. 1997; Chen et al. 2004), flounder (Richardson et al. 1999; Zhang et al. 2003), and halibut (Billard et al. 1993).

Damage to sperm morphology and function usually occurs during the process of freezing and thawing. Cellular damage may greatly decrease motility, impair velocity, and reduce fertilizing capacity, even lead to DNA strand breakage or mutation (Dréanno et al, 1997; Lahnsteiner et al, 1996a; Warnecke & Pluta 2003; Kopeika et al, 2004). Although motility and fertilizing capacity are usually assessed in frozen-thawed sperm, these methods have limitations. Many factors affect the validity of these assessments, including subjectivity, microscope performance, the quality of eggs, and fertilization protocols. Some new

Marine Fish Sperm Cryopreservation and Quality Evaluation in Sperm Structure and Function 241

preserved in liquid nitrogen for one month. After that, the thawed sperm was evaluated

Sperm motion characteristics were assessed by using a computer-assisted sperm motion analysis system (CASAS-QH-Ш, Tsinghua Tongfang Inc., Beijing, China) at room temperature (18oC to 20oC). The method for computer-assisted sperm motion analysis was describied in Liu et al (2007b). The designation of the motility status was based on the level of the average path velocity (VAP). Sperm with average path velocity <5 μm s-1 were considered immotile, with average path velocity >20 μm s-1 were defined as motile, and 5– 20 μm s-1 as locally motile. Therefore, in the present study sperm motility includes the percentage of local motile sperm and motile sperm. Motility and velocity of fresh and postthaw sperm were quantitatively recorded by CASA immediately 10 s after activation, and changes of motility of post-thaw sperm frozen with 15% DMSO were observed every 30 s.

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

Prior to scanning electron microscopy, sperm were fixed in 2.5% glutaraldehyde diluted in PBS (pH 7.6), dehydrated in a series of increasing concentrations of ethanol, critical-point dried, evaporated with gold, and examined with a scanning electron microscope (KYKY-2800B; KYKY Technology Development Ltd., Beijing, China) For transmission electron microscopy, spermwas prefixed in 2.5% glutaraldehyde, post-fixed in 1% osmium tetroxide, and embedded in Epon 812. Ultrathin sections were prepared, counterstained with 2%uranyl acetate followed by lead citrate, and examined with a transmission electron microscope (HITACHI H- 7000; Hitachi Ltd., Tokyo, Japan), and the number of sperm with various categories (normal, slightly damaged, and seriously damaged sperm) of cryodamage was determined. One-hundred sperm were randomly selected for observation each time; this was repeated three times on different sections (total of 300 frozen-thawed

The staining method used was described in (Liu et al. 2007a). An aliquot of mixed fresh or frozen-thawed sperm with 15%DMSO was incubated for 20 min (in the dark, temperature 4 oC) with 5 mg/mL of Rhodamine 123 (Rh123, Sigma Chemical Co., St. Louis, MO, USA).

for motility and fertilizing capacity.

**2.1.3 Sperm motion characteristics analysis by using CASA** 

**2.1.4 Sperm fertilization and hatching experiments** 

relation to the total number of eggs used in each experiment.

**2.1.6 Rhodamine 123, propidium iodide and flow cytometry** 

**2.1.5 Ultrastructure** 

sperm for each male).

technologies have been used in fish sperm quality analysis, such as computer-assisted sperm analysis (CASA), being used to objectively evaluate sperm motility (Lahnsteiner et al., 1996b; Lahnsteiner et al., 1998; Kime et al., 1996) ; Electron microscopy, being used to detect cryodamage (ultrastructural changes) in frozen-thawed sperm (Zhang et al, 2003, He &Woods 2004); In addition, flow cytometry of fluorescent-stained sperm have been used in mammals (Graham et al, 1990; Gravance et al, 2001) and turkeys (Donoghue et al ,1995), providing rapid, precise information regarding the viability of thousands of individual sperm. In recent years, flow cytometry has also been successfully used to assess both fresh and cryopreserved fish sperm (Ogier de Baulny et al, 1999; Segovia et al, 2000).

Red seabream is one of the most commercially important marine fish species for aquaculture in China. However, the decline of wild red seabream population has occurred due to over fishing and marine pollution in recent years. The use of cryopreserved sperm can provide an efficient method to increase its genetic population size and to help maintain genetic diversity. The aims of this study were to establish efficient methods for cryopreservation of red seabream sperm with 2-mL cryovials and to objectively measure the post-thaw sperm motility characteristics by means of CASA, to evaluate the post-thaw sperm fertilization capacity, and the cryodamage by electron microscopy and flow cytometry.
