Semen Properties and Spermatozoan Structure of Yellow Croaker, Larimichthys polyactis

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1 The Israeli Journal of Aquaculture - Bamidgeh, IIC: , 8 pages The IJA appears exclusively as a peerreviewed on-line Open Access journal at Sale of IJA papers is strictly forbidden. Semen Properties and Spermatozoan Structure of Yellow Croaker, Larimichthys polyactis Minh Hoang Le 1 *, Han Kyu Lim 2, Byung Hwa Min 2, Jung Uie Lee 2, Young Jin Chang 1 1 Department of Aquaculture, Pukyong National University, Busan , Korea 2 Aquaculture Management Division, National Fisheries Research and Development Institute, Gijang, Busan , Korea (Received , Accepted ) Key words: yellow croaker, Larimichthys polyactis, seminal plasma, spermatozoan structure, semen properties Abstract The aim of this study was to determine the properties of semen and the structure of spermatozoa of cultivated yellow croaker, Larimichthys polyactis. The volume of semen was 1.1±0.3 ml/fish, the sperm density 2.5±0.2 x 10 9 cells/ml, the spermatocrit 97.1±1.8%, and the sperm count 2.7±1.0 x 10 9 cells/fish. The seminal plasma contained 148.3±4.7 mmol/l sodium, 17.1±1.8 mmol/l potassium, 115.5±4.8 mmol/l chloride, 2.9±0.1 mmol/l calcium, 1.8±0.4 mmol/l magnesium, 0.1 mmol/l glucose, and 1.0 g/l total protein; osmolality was 342.5±3.6 mosm/kg and ph was 7.7±0.1. Regression analysis showed significant positive linear relationships between semen volume and fish weight, semen volume and fish length, sperm count and fish weight, and sperm density and spermatocrit. Thus, spermatocrit can be used as a rapid estimator of sperm density in yellow croaker. The spermatozoa consisted of three parts: a head without an acrosome, a mid-piece with three mitochondria, and a flagellum with a typical 9+2 arrangement. The longitudinal section of the head was kidney-shaped and µm. This study provides information on yellow croaker sperm physiology that can be used to improve sperm management efficiency in yellow croaker. The values and correlations between semen and seminal plasma can be used to formulate a species-specific extender for cryopreservation of yellow croaker semen. * Corresponding author. Tel: ; , mhle.vn@gmail.com

2 2 Le et al. Introduction Semen (or milt) contains spermatozoa and seminal plasma that mediates the chemical function of the ejaculate. Several types of spermatozoa have been distinguished in fish. Seminal plasma has a unique composition: some components support spermatozoa while others reflect functions of the reproductive system and the spermatozoa (Alavi and Cosson, 2006). Studies of semen properties help us understand the basic biochemical processes that occur in sperm motility and during fertilization (Ingerman et al., 2002), evaluate the reproductive ability of the semen of fish species (Alavi and Cosson, 2006; Rurangwa et al., 2004), and develop methods for short and long-term storage of fish semen (Alavi and Cosson, 2006). Information on the structure of fish spermatozoa helps us understand reproductive biology, taxonomic, and evolutionary relationships at family, (Jamieson, 1991), subfamily, and species levels (Lahnsteiner and Patzner, 2007), and optimize artificial reproduction, prevent polyspermy problems, and develop cryopreservation techniques (Billard et al., 1995). The yellow croaker, Larimichthys polyactis (also called Pseudosciaena polyactis), is a teleost that belongs to the Sciaenidae family and inhabits sea waters. It is an important traditional commercial fish species in Korea. It migrates out to the East China Sea in winter and returns to the Yellow Sea to spawn in spring (Kim et al., 1997). Basic reproductive and life-history information on this species is available (Trewavas, 1991). To have controlled and successful production in aquaculture systems, it is necessary to have adequate knowledge of semen properties and the spermatozoan structure of the cultivated fish. Therefore, we examined the semen properties and spermatozoan structure in cultivated yellow croaker during the peak spawning season. Such information will help to select good semen and improve protocols for cryopreservation and artificial propagation. Materials and Methods Fish and semen collection. The experiments were carried out at the Aquaculture Management Division of the National Fisheries Research and Development Institute, Korea, during June 2008, coinciding with the peak spawning activity for yellow croaker. Broodfish were raised in a 16-m 3 tank. Ten males were randomly caught, anesthetized in 100 ppm ethyl 3- aminobenzenoate methanesulfonate salt (Sigma, USA), and measured for weight and length. Before semen collection, they were held in a separate 2- m 3 spawning tank with flow-through sea water (32 psu salinity; C) at a flow rate of 0.2 l/s under a simulated natural photoperiod (12 dark/12 light). Fish were fed commercial feed (Suhyup Feed Co. Ltd., Uiryeong, Korea) once a day. Semen was collected by gentle abdominal massage from the anterior portion of the testes towards the genital papilla using a 1.5 ml Eppendorf tube to prevent contamination with urine, mucus, and blood cells. The semen of each fish was kept separately on ice for up to 30 min before semen properties were measured. Physical properties of semen. After the volume of the semen was determined, the density of the spermatozoa was counted. Semen was diluted

3 Semen properties and spermatozoon structure of yellow croaker times by pipetting 10 µl semen into 990 µl 0.2% eosin. A hemocytometer counting chamber was used to determine sperm density. One droplet of diluted semen was placed on a hemocytometer slide (depth 0.1 mm) with a coverslip, the sperm was allowed to settle for 3-5 min, sperm cells were counted in 16 chamber cells using light microscopy ( 400), and the number was calculated according to Le et al. (2007). Spermatocrit was defined as the ratio of white packed material volume to the total volume of semen multiplied by 100 (Rurangwa et al., 2004). Glass microhematocrit capillary tubes ( mm) were filled with semen and one end was sealed with clay. The capillary tubes were centrifuged at 15,000 rpm for 10 min. Spermatocrit counts were read in a Hawksley micro-hematocrit reader (Sons Ltd., England). Sperm count was calculated as the product of semen volume and sperm density. Biochemical properties of seminal plasma. To analyze biochemical properties in seminal plasma, semen was separated from the seminal plasma by centrifugation (15,000 rpm for 10 min). Seminal plasma was centrifuged twice to avoid possible contamination with spermatozoa. The supernatants were frozen and stored in a refrigerator for 3 days until analysis. Biochemical components of seminal plasma were determined by Fuji Dri-Chem 3500 (Fujifilm Co. Ltd., Japan). The ph and osmolality of seminal plasma was measured by ph meter (Istek, Korea) and osmometer (Wescor Inc., USA). Structure of spermatozoa. The structure of spermatozoa was studied using transmission electron microscopy (TEM). Fresh semen samples were fixed for 3 h in 2.5% glutaraldehyde solution in 0.1 M phosphate buffer solution (PBS, ph 7.2) at 4 C, washed in the same buffer, immersed for 2 h in 1.0% osmium tetroxide (OsO 4 ) in PBS at 20 C, washed again with PBS, serially dehydrated with ethanol from 50% to 100%, and embedded in Epon 812. Hardened blocks were sectioned at a thickness of µm and the sections were mounted on copper grids. These were post-stained with 2% uranylacetate in 50% ethanol and lead citrate solution. Finally, the grids were examined and photographed using TEM (JEM-1230, Japan and JEOL 1010, JEOL Ltd., Japan). Statistical analysis. Data were expressed as means±standard deviation. Data were analyzed using SPSS 16.0 for Windows software package. Differences with a probability of 0.05 were considered significant. Results Physical and biochemical properties of semen. Semen volume, density, spermatocrit, and total number of sperm per fish are shown in Table 1. Sodium and chloride were the dominant ions. Correlations between fish weight, fish length, spermatological properties, and biochemical properties are presented in Table 2. There were significant polynomial correlations between semen volume and fish weight (r = 0.937), semen volume and fish length (r = 0.924), sperm count and fish weight (r = 0.928), and sperm density and spermatocrit (r = 0.782). Structure of spermatozoon. The spermatozoa of yellow croaker consists of three distinct parts: a head without an acrosome, the mid-piece, and the

4 4 Le et al. Table 1. Physical and biochemical properties of semen of yellow croaker Larimichthys polyactis (n = 10). Size of fish Length (cm) 23.3±0.5 Wt (g) 128.8±1.8 Physical properties of semen Spermatocrit (%) 97.1±1.8 Semen volume (m/fish) 1.1±0.3 Sperm density (cell x 10 9 /ml) 2.5±0.2 Sperm count (cell x 10 9 /fish) 2.7±1.0 Biochemical properties of seminal plasma Sodium (mmol/l) 148.3±4.7 Potassium (mmol/l) 17.1±1.8 Chloride (mmol/l) 115.5±4.8 Calcium (mmol/l) 2.9±0.1 Magnesium (mmol/l) 1.8±0.4 Glucose (mmol/l) 0.1±0.0 Total protein (g/l) 1.0±0.0 Osmolality (mosm/kg) 342.5±3.6 ph 7.7±0.1 flagellum (Fig. 1). In the head, there is a round nucleus covered by a thin layer of cytoplasm. The nucleus is µm long and µm wide. There are three round mitochondria surrounding the mid-piece sleeve. The nucleus has an invagination, where the proximal centriole and distal centriole are located. The proximal centriole is perpendicular to the distal centriole which connects with the flagellum. The proximal centriole is unusual not only in being in the same axis as the basal body but also in having its longitudinal axis similarly oriented, near but not at, the anterior limit of nuclear fossa. At least one, apparently the distal centriole, consists of nine triplets. The 9+2 flagellum has long lateral fins and its plane slightly tilts relative to that of the two central singlets. Discussion The volume of semen of the yellow croaker was lower than in turbot (Suquet et al., 1992), scaly carp (Bozkurt, 2006), or European perch (Alavi et al., 2007), but higher than in filefish (Le et al., 2007). The semen of the yellow croaker had a very low density compared to other teleosts such as rainbow Table 2. Correlation between spermatological properties and seminal plasma composition of yellow croaker Larimichthys polyactis semen. FL FW SV ST SD SC Na + K + Cl - Ca Mg OSM Fish length (FL) Fish wt (FW) * Semen volume (SV) * * Spermatocrit (ST) * * * Sperm density (SD) * * * * Sperm count (SC) * * * * * Sodium (Na + ) Potassium (K + ) Chloride (Cl - ) * Calcium (Ca) * * Magnesium (Mg) Osmolality (OSM) * * ph * Correlation is significant at p<0.05.

5 Semen properties and spermatozoon structure of yellow croaker 5 Fig. 1. The structure of a spermatozoon in yellow croaker: (A) longitudinal section of fresh spermatozoa, (B) cross-section of mid-piece with three mitochondria, (C) cross- section of flagellum with 9+2 arrangement; d = distal centriole, f = flagellum, m = mitochondrion, n = nucleus, p = proximal centriole (scale bars = 0.2 µm). trout, whitefish, yellow perch (Ciereszko and Dabrowski, 1993), marbled sole, brown sole, olive flounder (Chang and Chang, 2002), and sea and brook trout (Dziewulska et al., 2008). On the other hand, the spermatocrit of yellow croaker was higher than in Masou salmon (Lim et al., 2008) and sea and brook trout (Dziewulska et al., 2008). Differences in semen production can be related to factors such as age and weight of males (Suquet et al., 1994, 1998), rearing conditions, nutrition, breeding seasonality, methods of spawning induction, spawning behavior of broodfish (Rurangwa et al., 2004), sampling period, and sampling method (Suquet et al., 1994). The relationship between semen volume and fish weight in yellow croaker was similar to those of scaly carp (r = , p<0.05; Bozkurt, 2006) and brown trout (r = , p<0.05; Bozkurt et al., 2006). The relationship between semen volume and fish length in yellow croaker compares with that of brown carp (r = , p<0.05; Bozkurt et al., 2006). There was a strong positive relationship between sperm density and spermatocrit. Similar correlations have been reported for rainbow trout, whitefish, yellow perch (Ciesreszko and Dabrowski, 1993), Atlantic halibut (Tvedt et al., 2001), haddock (Rideout et al., 2004), and Atlantic cod (Rakitin et al., 1999). There was a significant relationship between spermatocrit and optical density in turbot, but no significant correlation between spermatocrit and sperm density (Suquet et al., 1992). Spermatocrit can be used as a quick and easy

6 6 Le et al. technique to estimate spermatozoa density in haddock (Rideout et al., 2004). Likewise, it can be used to determine spermatozoa density in yellow croaker. Important biochemical criteria include the presence or absence of inorganic and organic components in the semen and the osmolality and ph of seminal plasma (Alavi and Cosson, 2006). Sodium, potassium, and chloride (the dominant ions in yellow croaker seminal plasma) were 144.0, 18.0 and mm/l in Masou salmon (Lim et al., 2008), 136.6, 30.5, and mm/l in brook trout (Dziewulska et al., 2008), and 159.2, 33.7, mm/l in Caspian brown trout (Hatef et al., 2007). Calcium and magnesium levels in yellow croaker seminal plasma are higher than in sea or brook trout (Dziewulska et al., 2008) and lower than in filefish (Le et al., 2007) and Masou salmon (Lim et al., 2008). Although the origin and functions of protein in fish seminal plasma are not completely known, part of the proteins in seminal plasma may originate from disrupted spermatozoa (Lahnsteiner et al., 2004). Sperm motility is influenced by hypo and hyper-osmotic pressure in freshwater and marine fishes, respectively (Alavi and Cosson, 2006; Alavi et al., 2007) and osmolality in freshwater fishes is generally lower than in marine (Alavi and Cosson, 2006). Osmolality of seminal plasma in yellow croaker were higher than in European perch (298.1 mosm/kg; Alavi et al., 2007) and jundiá (274.8 mosm/kg; Borges et al., 2005). The ph value in seminal plasma of fish may be representative of high spermatozoa viability during the spawning period (Dziewulska et al., 2008). The ph of the seminal plasma in the present study was similar to values obtained by Le et al. (2007) and Lim et al. (2008), higher than those of Bozkurt et al. (2006), and lower than those of Chang and Chang (2002) and Dziewulska et al. (2008). The head of yellow croaker spermatozoa is smaller (2-4 µm) than in other teleosts. The spermatozoon of the yellow croaker has a kidney-shaped head, like the damselfish, that differs from the shape in teleosts with external fertilization: northern pike has a ball-shaped head, while cardinal fish has an ovoid-shaped head (Jamieson, 1991; Le et al., 2007). The cross-section of the mid-piece of teleosts has different numbers of mitochondria: there are eight in marbled sole, seven in brown sole and starry flounder, and six in olive flounder (Chang and Chang, 2002), five in filefish (Le et al., 2007), and three in yellow croaker. In other teleosts, the mitochondria surround the mid-piece (Jamieson, 1991) but in the yellow croaker they are found on only one side. Differences between yellow croaker and other fish could be related to spawning periodicity, contamination of semen by urine during stripping (Suquet et al., 1994), period of spermiation during the breeding season, or endocrine parameters such as steroids during spermatogenesis (Alavi et al., 2007). In conclusion, our data could be useful for estimating the number of males needed in relation to the number of available mature females in yellow croaker. The values and correlations between semen properties and seminal plasma composition could be used to formulate a species-specific extender solution (diluent) that mimics seminal plasma for cryopreservation of yellow

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