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

were completely damaged, 30% to 40% changed and only 10–20% showed an intact morphology. In this study, flow cytometric analysis, based on membrane integrity and mitochondrial function, was used to assess post-thaw sperm quality. After double staining with Rh123 and PI, we found 74.8% of post-thaw sperm showed membrane integrity and mitochondrial function. In rainbow trout (Ogier de Baulny et al., 1997), the plasma membrane and mitochondrial function were better protected with 10% DMSO.


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 .
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 www.intechopen.com 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.

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.

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 0 o C, and frozen from 0 to −150 o C at a cooling rate of 20 o C min-1 , then plunged into liquid nitrogen for storage. The frozen sperm were thawed in 40 o C water bath after being www.intechopen.com preserved in liquid nitrogen for one month. After that, the thawed sperm was evaluated for motility and fertilizing capacity.

Sperm motion characteristics analysis by using CASA
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 (18 o C to 20 o C). 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.

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.

Ultrastructure
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 sperm for each male).

Rhodamine 123, propidium iodide and flow cytometry
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 o C) with 5 mg/mL of Rhodamine 123 (Rh123, Sigma Chemical Co., St. Louis, MO, USA).

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Thereafter, sperm were incubated for 45 min in 1.5 mL of Cortland extender. With this staining method, only cells with functional mitochondria were stained, due to the negative potential of the inner membrane of the mitochondria. Samples were diluted and counterstained with 5 mg/mL of propidium iodide (PI, Sigma Chemical Co.). After 10 min, sperm samples were analyzed with flow cytometry (FACSvantage SE flow cytometer; Becton Dickinson, Mountain View, CA, USA) as previously described for trout sperm (Ogier de Baulny, et al, 1997). Sperm populations were identified according to their relative red and green fluorescence (staining with PI and Rh123, respectively). Sperm with red (stained with PI) DNA were interpreted as having a damaged plasma membrane, whereas those that were green (stained with Rh123) were interpreted as having intact mitochondrial function. Sperm that were only red (damaged plasma membrane and lacking mitochondrial function), were localized in Region 1, whereas those that were only green (intact membrane and functional mitochondria), were localized in Region 3. Sperm with both red and green fluorescence (damaged plasma membrane and functional mitochondria) were localized in Region 4, and those with no staining (intact plasma membrane, but no mitochondrial activity) were localized in Region 2.

Statistical analysis
To determine the effects of cryopreservation on sperm motility, fertilization capacity, structure and function, a paired-sample t-test was used to compare fresh versus frozenthawed sperm. All statistical analyses were performed with SPSS Version 11.0 software (SPSS Inc. Chicago, IL, USA) and P < 0.05 was considered significant. All data were expressed as mean±S.D.

Post-thaw sperm viability
The influence of cryopreservation on sperm motility and velocity was shown in Table 1. Percentages of motile post-thaw sperm frozen with 12-21% DMSO were higher than those with 6% DMSO, 9% DMSO and 24% DMSO. However, the procedure of cryopreservation has no significant (P>0.05) influence on the motile sperm velocity 10 s after activation compared with fresh sperm. In addition, the post-thaw sperm frozen with 12-21% DMSO showed similar types of straight trajectories. The motion characteristics of fresh and post-thaw sperm were evaluated by using computerassisted sperm analysis 10 s after activation. This table shows the percentages of locally motile (VAP range from 5 to 20μm s-1) and motile sperm (VAP > 20μm s-1) as well as their velocity (VAP) for fresh and post-thaw sperm. Values superscripted by the same letter are not significantly different (P>0.05, n=5).
The effect of time after activations on post-thaw sperm motility was shown in Fig. 1. The percentages of total motile sperm of both fresh (87. 2 ± 6.1%) and post-thaw sperm (81.9 ± 6.6%) frozen with 15% DMSO were not (P>0.05) different significantly 10 s after activation. However, 30 s after activation the percentage of total motile post-thaw sperm (72.3 ± 6.3%) was (P<0.05) lower than that of fresh sperm (82.7 ± 7.2%). Sixty seconds after activation, the percentage of post-thaw sperm motility drastically reduced to 38.7 ± 13.2%. Fig. 1. The influence of time after activation on the motility of fresh and post-thaw sperm in P. major. Ten seconds after activation, the total motilities of fresh and post-thaw sperm frozen with 15% DMSO were observed every 30 s using computer-assisted sperm analysis system. This figure describes the evolution of the total motilities of fresh ( ) and post-thaw sperm ( ) after activation respectively (n =5).

Post-thaw sperm fertilizing capacity and hatchability
Fertilization rates and hatching rates of fresh and post-thaw sperm were shown in Fig. 2 with 12-21% DMSO. However, lower (P<0.05) fertilizing capacity of post-thaw sperm frozen with 6% DMSO, 9% DMSO and 24% DMSO were observed. In addition, the percentages of motile of post-thaw sperm and fertilization rates showed a high positive linear regression (r = 0.876). Similarly, the percentages of motile spermatozoa and hatching rates of post-thaw sperm showed a high positive linear regression (r = 0.878). Fig. 2. Fertilization rates and hatching rates of fresh and post-thaw sperm in P. major. Cryopreserved sperm was thawed and activated for the artificial fertilization with sperm to egg ratio 500:1. This figure describes the fertilization rates and hatching rates of post-thaw sperm frozen with 6-24% DMSO. For fertilization rates of fresh and post-thaw sperm; For hatching rates of fresh and post-thaw sperm. Columns marked with the same letter are not significantly different (P>0.05, n=5).

Sperm ultrastructure
Ultrastructure of fresh and intact frozen-thawed red seabream sperm are shown in Fig. 1. These sperm had a head, midpiece, and tail. The head was ovoid and contained the nucleus and centriolar complex; the latter consisted of two centrioles. The midpiece was approximately cylindrical and contained mitochondria. The flagellum consisted of nine peripheral doublets and two central microtubules; the axoneme was a typical 9 + 2 structure ( Fig. 3 A, B). The proportion of fresh sperm with normal morphology was 77.8 ± 5.6%, whereas after cryopreservation, 63.0 ± 7.2% of the sperm had normal morphology (Fig. 3 C), 20.7 ± 3.1% were partly damaged (e.g. swelling or rupture of head, midpiece and tail region, as shown in Fig. 3  severely damaged; the plasma membranes was completely ruptured and only nuclei, mitochondria, or some fragments of cellular organelles were found (Fig.3 E). Completely damaged plasmalemma and nuclear envelop. (h, head; m , mid-piece; t, tail region. nu, nucleus; ne, nuclear envelope; bb, basal body; mi, mitochondrion; pm, plasmalemma; f, flagellum; v, vacuole). Scale bar = 0.5 μm.

Fluorescent staining and flow cytometry
Sperm populations were localized into four distinct regions according to their relative green and red fluorescence after staining with PI and Rh123 (Fig. 4). For fresh sperm, 83.9% had an intact membrane and functional mitochondria, 5.1% had nonfunctional mitochondria, 9.8% had nonfunctional mitochondria, and 1.2% had both a damaged membrane and nonfunctional mitochondria; whereas for frozen-thawed sperm, the percentages of sperm localized in four regions were 74.8%(Region 3), 12.7%(Region 4), 9.9% (Region 2), and 2.6%(Region 1), respectively.  Region 1, sperm with a damaged plasma membrane but normal mitochondrial function. Region 2, sperm with an intact plasma membrane but lacking mitochondrial function. Region 3, sperm with an intact plasma membrane and functional mitochondria. Region 4, sperm with a damaged plasma membrane and functional mitochondria.

Discussion
Motility is an important characteristic for estimating the quality of fresh as well as cryopresrved sperm (Lahnsteiner et al., 1996a). In this study, the freezing-thawing process did not significantly change the main motility pattern and swimming velocity of motile sperm 10 s after activation, and the progressive linear motion was still the dominant pattern. Moreover, for the sperm cryopreserved with 12-21% DMSO, the freezing-thawing process also didn't significantly influence their motility and motility pattern, although it significantly reduced their motility period. However, different results were obtained from the sperm cryopreservation of turbot (Dréanno et al., 1997), which the percentage of motile post-thaw sperm was significantly lower than that obtained from fresh sperm while the velocity and the duration of motion were not significantly modified.
No significant difference in the fertilization rates and hatching rates were observed between sperm cryopreserved with 12-21% DMSO and fresh sperm. However, Lahnsteiner et al. (2003) reported that in cyprinids sperm, the post-thaw fertilization ratios obtained with sperm to egg ratios of 1.3-2.6×10 6 :1 did not reach that of the fresh sperm. Similar results have also been reported in turbot Suquet et al., 1998) and flounder (Zhang et al., 2003). These may be due to the species specific or un-ideal protocols used in sperm cryopreservation. In this study, for the post-thaw sperm a high positive correlation was observed between the percentage of motile sperm and fertilizing capacity. This was consistent to the results that obtained from turbot (Dréanno et al., 1999), common carp (Linhart et al., 2000) and African Catfish (Rurangwa et al., 2001).
In the present study, the data from ultrastructural investigation and flow cytometric analysis demonstrates that more that 60% of post-sperm were normal in morphology and mitochondrial function. These results further confirmed the high performance of the protocols established for red seabream. In addition, the high fertilization capacity of postthaw sperm implies that some of the slightly damaged spermatozoa can still fertilize eggs and develop into larvae. However, it remains to be determined whether the larvae from cryopreserved sperm develop into healthy adults.
During the process of cooling, freezing and thawing, spermatozoa are subjected to a series of damages (Oehninger et al., 2000). In ultrastructural investigation, we found 20.7 ± 3.1% were slightly damaged in some way and 16.4 ± 4.2% were severely damaged. One of the causes may be the ice crystal formation during the freezing process and some researchers agree that intracellular ice formation is the major injury mechanism at rapid cooling rates (Toner et al.,1993;Chao & Liao 2001). Other causes of cryodamages include pH fluctuation, cold shock, osmometric effect, and cryoprotectant toxicity (Chao & Liao 2001). The swelling and rupture of the plasmalemma after thawing may be due to the damage to the unit membrane which is very sensitive to freezing and thawing (Lahnsteiner et al., 1992). Similar morphological changes were reported in post-thaw sperm of ocean pout (Yao et al., 2000), rainbow trout (Lahnsteiner et al., 1996c), and atlantic croaker (Gwo et al., 1991). For example, in grayling sperm (Lahnsteiner et al., 1992), a marked decrease in sperm quality was observed, about 40% to 50% of the spermatozoa were completely damaged, 30% to 40% changed and only 10-20% showed an intact morphology. In this study, flow cytometric analysis, based on membrane integrity and mitochondrial function, was used to assess postthaw sperm quality. After double staining with Rh123 and PI, we found 74.8% of post-thaw sperm showed membrane integrity and mitochondrial function. In rainbow trout (Ogier de Baulny et al., 1997), the plasma membrane and mitochondrial function were better protected with 10% DMSO.
Plasma membrane integrity and mitochondrial function are the two most important attributes for fertilizing an egg. The damage to membrane integrity and mitochondrial function could destabilize the sperm membrane and affect mitochondrial energy metabolism, thereby affecting spermatozoa viability. However, what interested us most is that although about 30% of spermatozoa were damaged in some way or even totally ruptured, the fertilization capacity of post-thaw sperm were not affected significantly in the standardized artificial fertilization experiment (Oehninger et al., 2000). Three hypotheses can be considered. The first hypotheses is that the sperm that survived freezing-thawing with normal morphology and mitochondrial function as shown in Fig. 3 and Fig. 4 region 3 should be similar to fresh sperm in fertilization capacity. The second is that the sperm cryopreserved with the established method could provide adequate numbers of motile spermatozoa with normal sperm parameters to fertilize the eggs in artificial fertilization experiment. The third is that the process of freezing-thawing may result in a population of partially damaged yet motile spermatozoa, which can fertilize eggs and develop into larva normally. Such a population usually exhibits a certain degree of plasma and mitochondrial membrane leakiness as shown in Fig. 3 and Fig. 4 region 2, 4.

Conclusion
In conclusion, the fertilizing capacity and egg hatchability were not significantly reduced by the post-thaw sperm treated with 12-21%DMSO, although the post-thaw sperm quality was influenced during the freezing and thaw process in motility, ultrastructure and mitochondrial function. The cryopreservation protocol used for red seabream sperm should be of great value for the establishment of sperm banks and assessment of ultrastructure and flow cytometry facilitated identification of damaged sperm; However, the exact nature of cryodamage to fish sperm are not yet fully understood. Sperm motility, structure integrity and mitochondrion function were damaged with different extent, although the fertilization capacity of cryopreserved sperm was not changed. There are many questions need to answer, how does the cryodamage reduce the sperm motility duration? If the cryodamages influence the gene expression and the embryo and larvae development? how to improve the post-thaw sperm quality by optimize the cryopreservation method?