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Increased plasma 17-hydroxyprogesterone and milt production in response to gonadotropin-releasing hormone agonist in captive male starry flounder, Platichthys stellatus
Aquaculture 218 (2003) 703 – 716 www.elsevier.com/locate/aqua-online
Increased plasma 17-hydroxyprogesterone and milt production in response to gonadotropin-releasing hormone agonist in captive male starry flounder, Platichthys stellatus S.H. Moon a, H.K. Lim b, J.Y. Kwon c, J.K. Lee b, Y.J. Chang a,* a Department of Aquaculture, Pukyong National University, Pusan 608-737, South Korea Uljin Hatchery, National Fisheries Research and Development Institute, Uljin 767-860, South Korea c Department of Applied Biological Sciences, Sunmoon University, 100 Kalsan-ri, Tangjeong-Myeon, Asan-si, Chung Nam 336-708, South Korea b
Received 1 November 2001; received in revised form 15 November 2002; accepted 18 November 2002
Abstract In captivity, reproduction of male fish is often dysfunctional, resulting in abnormally viscous milt. Treatment with exogenous gonadotropin or gonadotropin-releasing hormone agonist (GnRHa) has been found useful in overcoming this reproductive failure in some fish species. In this study, captive male starry flounder (Platichthys stellatus) were treated with GnRHa at three different dosages (50, 100 or 200 Ag kg 1 fish body weight) by implantation with cholesterol pellets during their natural spawning season. The GnRHa treatment increased the levels of plasma testosterone (T) at the early spawning season and plasma 17-hydroxyprogesterone (17P4) throughout the spawning season. Both milt volume and sperm count were increased in a dose-dependent manner by GnRHa treatment, but the increase of milt volume was twice as rapid as the increase of sperm count. The treatment decreased sperm concentration. The increased milt volume was strongly correlated with the level of plasma 17P4 (r = 0.690), but not with T (r = 0.250). The level of 17P4 was also associated with sperm motility. The sperm obtained both from control and GnRHa-treated groups was used to fertilize starry flounder eggs, and the resultant embryos were grown until metamorphosis. No negative effects of the GnRHa treatment on fertilization, hatching and larval growth were found. The results suggest that GnRHa treatment can render captive male flounders to recover their ability to hydrate milt. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Fertilization; GnRHa; Larval growth; Milt volume; Sperm motility; 17-Hydroxyprogesterone; Testosterone
* Corresponding author. Tel.: +82-51-620-6135; fax: +82-51-628-7430. E-mail address: [email protected] (Y.J. Chang). 0044-8486/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0044-8486(02)00643-9
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1. Introduction In some fish species, captive males fail to spermiate or produce only a small volume of viscous milt. This reproductive failure hampers collection of the milt and subsequent fertilization of eggs (Donaldson and Hunter, 1983). This problem has been frequently pointed out in many marine flatfish species including plaice (Pleuronectes platessa) (Vermeirssen et al., 1998), yellowtail flounder (Pleuronectes ferrugineus) (Clearwater and Crim, 1998) and Atlantic halibut (Hippoglossus hippoglossus) (Vermeirssen et al., 2000). Reproduction of starry flounder (Platichthys stellatus), a marine fish species that occurs in countries surrounding the Pacific ocean, also appears to be dysfunctional in captivity. In preliminary studies in our laboratory (unpublished), both males and females of this species did not spawn spontaneously in captivity. When stripped manually during their natural spawning season, males produced a small amount of extremely viscous milt. Gonadotropin-releasing hormone agonists (GnRHa) have been tested on several marine and freshwater fish species to control reproductive processes (Garcia, 1993; Harmin and Crim, 1993; Pankhurst, 1994; Mylonas et al., 1996; Mylonas et al., 1997a,b; Mylonas et al., 1998; Clearwater and Crim, 1998; Vermeirssen et al., 1998; Pankhurst and Poortenaar, 2000; Vermeirssen et al., 2000; Mylonas and Zohar, 2001). In most studies so far, GnRHa treatment increased milt volume in male fish in captivity. Sometimes, GnRHa increases actual sperm production (Clearwater and Crim, 1998), but at times, it only increases milt fluidity (Garcia, 1993; Vermeirssen et al., 2000). In some fish, GnRHa treatment of male fish increased plasma levels of gonadal steroid hormones such as testosterone (T) and 11-ketotestosterone (11-KT) (Harmin and Crim, 1993; Pankhurst, 1994; Pankhurst and Poortenaar, 2000), but in others, it induced no clear changes (Clearwater and Crim, 1998) or even decreased the hormone levels (Vermeirssen et al., 2000). Progestogens such as 17,20a-dihydroxy-4-pregnene-3-one (17,20aP) and 17,20h-dihydroxy-4-pregnen-3-one (17-hydroxy-20h-dihydroprogesterone, 17,20hP) have been shown to respond to GnRHa treatment (Mylonas et al., 1997a,b; Vermeirssen et al., 1998; Vermeirssen et al., 2000). One of these progestogens, 17,20hP, has been suggested to play a key role in the process of spermiation in fish (Miura et al., 1991, 1992; Mylonas et al., 1997a,b; Vermeirssen et al., 1998). Recently, another progestogen, 17hydroxy-4-pregnen-3,20-dione (17-hydroxyprogesterone, 17P4) has also been suggested to be related to the process of spermiation in Atlantic salmon (Salmo salar), although the effect of this progestogen on milt production was attributed to its conversion to 17,20hP (King and Young, 2001). The changes of 17P4 level in response to GnRHa treatment, however, have not been studied in fish. The relationship between the level of 17P4 and milt production, therefore, has yet to be established. Few studies have, so far, measured sperm quality following GnRHa treatment (Mylonas et al., 1997a; Clearwater and Crim, 1998) despite the fact that the main interest of fish breeders is the viability of the fertilized eggs and embryos obtained using sperm produced by GnRHa treatment. In this study, we treated captive male starry flounder with GnRHa implants during their natural spawning season and investigated the individual response in milt volume, sperm count, sperm concentration, sperm motility and plasma T and 17P4. In addition, fertilization, hatching rate and larval growth were also studied in eggs inseminated with sperm produced by GnRHa-treated fish.
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2. Materials and methods 2.1. Fish Male starry flounder (caught from the wild) were kept in a circular tank (20 m3) for approximately 1 year at Uljin Hatchery, National Fisheries Research and Development Institute, Korea, until they were used for the experiment. At the beginning of their natural spawning season (January – March), 25 spermiating fish were randomly selected, placed into five plastic aquariums (200 l, five fish in each tank) with constant water flow and then subjected to GnRHa treatment. The size (means F S.D.) of fish used for the experiment was 551.7 F 109.4 g in body weight and 34.1 F 1.5 cm in total length. During the experiment, fish were kept under natural seawater temperature (means F S.D., 11.0 F 1.0 jC) and fed daily with frozen mackerel. All fish were tagged for identification with PIT tags (Pit-tag, Identification Devices). 2.2. GnRHa treatment The GnRH agonist, LHRHa (pGlu-His-Trp-Ser-Tyr-D-Ala-Leu-Arg-Pro-NHEt, Sigma) was dissolved in 50% ethanol (2 ml) and mixed with cholesterol powder (5.7 g). The mixture was dried, compounded with molten cocoa butter (0.3 g) and then compressed into pellets with a mold. Fish were weighed, and the pellets with different concentrations were prepared individually according to the weight of each fish, to achieve dosages of 50, 100 or 200 Ag GnRHa per kg of fish body weight. The pellets were implanted into the dorsal muscle. The fish (n = 5) received either no pellet (control), pellet that contains no GnRHa (sham), or a pellet containing GnRHa at three doses. 2.3. Sampling Fish were identified with the PIT tag before every sampling. Milt was collected individually from all fish in each group immediately before the implantation (0 day), and 1, 4, 9 and 14 days post implantation (dpi), and then every week up to 85 dpi when the majority of fish ceased spermiating. Milt was stripped into Eppendorf tubes by pressing the abdomen of the fish gently and was stored at 4jC until analysis. Blood samples were collected from three fish in each group every 2 weeks from 0 to 85 dpi and also at 4 dpi to see the response of T and 17P4 to the GnRHa treatment. Blood was taken from the vasculature at the caudal peduncle using a 3-ml heparinized syringe. The blood was centrifuged briefly and the plasma was stored at 73jC until analyzed for T and 17P4 using radioimmunoassay. 2.4. Milt variables Milt volume of individual fish was recorded after collection, and part of the milt was used to determine other variables. Sperm count and sperm concentration were measured using a haemocytometer after staining the sperm with 2% eosin solution. Sperm motility index was determined under a light microscope by the method of Stru¨ssmann et al. (1994)
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with slight modifications. Briefly, sperm with rapid movement, slow movement, moderate vibration and no movement were scored as 3, 2, 1 and 0, respectively. Milt sampled before (0 dpi) and soon after (1 dpi) the implantation was extremely viscous and small in quantity, causing difficulties in measuring sperm count, sperm concentration and sperm motility. Hence, the earliest data available for these parameters were from 4 dpi. The remaining milt was centrifuged at 5600 g for 30 min, and the pH was measured from the seminal fluid using an electronic pH meter (pH/Ion Meter EP-880).
Fig. 1. Responses of plasma testosterone (A) and 17-hydroxyprogesterone (B) to GnRHa treatment during the natural spawning season in captive male starry flounder. Sham: treated with sham pellet. 50GnRHa, 100GnRHa and 200GnRHa: treated with the dosages of 50, 100 and 200 Ag GnRHa per kg fish, respectively. The data points underlined indicate that significant differences exist between treatments at each time point (Kruskal – Wallis test, P < 0.05). Different alphabetical superscripts indicate significant difference by multiple comparison between treatments (Tukey’s test, P < 0.05). *Significant difference from sham control (Dunnett’s t-test, P < 0.05).
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2.5. Steroid radioimmunoassay Testosterone and 17P4 were extracted from 250 Al of plasma using 2 ml of diethyl ether, and the hormone levels were determined after Aida et al. (1984), using tritiated T and 17P4 (Amersham Pharmacia, UK). Antibodies for both T and 17P4 were purchased from SigmaAldrich. The cross-reactivity of the T antibody with 17P4 was 0.5%, and of the 17P4 antibody with T was 0.2%. Extraction efficiencies, calculated as recovery of 3H-labeled steroids extracted with plasma, were 95% and 97% for T and 17P4, respectively. The intra-
Fig. 2. Effects of GnRHa treatment on milt volume (A, B). The data points underlined indicate that significant differences exist between treatments at each time point (one-way ANOVA, P < 0.05). Different alphabetical superscripts indicate significant difference by multiple comparison between treatments (Tukey’s test, P < 0.05). Sham: treated with sham pellet. 50GnRHa, 100GnRHa and 200GnRHa: treated with the dosages of 50, 100 and 200 Ag GnRHa per kg fish, respectively.
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and interassay coefficients of variation were 9.4% and 7.4% for T, and 9.7% and 8.8% for 17P4, respectively. 2.6. Sperm quality—fertilization, hatching and larval growth Sperm obtained from each group at 35 dpi was used to fertilize eggs from a female starry flounder that was also treated with GnRHa at the dosage of 200 Ag/kg. Sperm from each
Fig. 3. Effects of GnRHa treatment on sperm count (A) and sperm concentration (B). The data points underlined indicate that significant differences exist between treatments at each time point (one-way ANOVA, P < 0.05). Different alphabetical superscripts indicate significant difference by multiple comparison between treatments (Tukey’s test, P < 0.05). Sham: treated with sham pellet. 50GnRHa, 100GnRHa and 200GnRHa: treated with the dosages of 50, 100 and 200 Ag GnRHa per kg fish, respectively. nd: not determined.
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group was pooled (from all five fish) and 0.5 ml of the sperm was mixed with 20 ml of eggs (about 6000 eggs). Fertilized eggs from each group (n = 3) were placed in 50-l plastic tanks with gentle aeration. Water temperature and illumination were maintained at 12.5 F 1 jC and 130 – 150 lx, respectively. The seawater was partially exchanged everyday to maintain good water quality. Approximately 24 h after fertilization, eggs were sampled from each tank (in triplicate) and the number of fertilized eggs was counted under a microscope. By this time, the survived eggs reached the gastrula stage. Eggs showing clear cleavage were counted as ‘‘fertilized,’’ whereas eggs with no sign of cleavage were regarded as ‘‘unfertilized’’ (these eggs were mostly opaque). Five days after fertilization, hatching rate (%) was calculated by counting floating larvae and dead eggs. The hatched larvae were grown until metamorphosis (32 days post hatching, dph). They were fed with Chlorella spp. and rotifers (8 –10 individuals ml 1) from 4 to 24 dph and, thereafter, with rotifers and Artemia nauplii.
Fig. 4. Relationships between milt volume ( Y) and the levels of plasma testosterone (X) (A) and 17hydroxyprogesterone (X) (B) that were elevated by GnRHa treatment. r = Pearson’s Correlation Coefficient.
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2.7. Statistics All data were expressed as means F S.E. Data for steroid hormones were analyzed using Kruskal – Wallis test, followed by nonparametric Tukey-type multiple comparisons ( P < 0.05) (Zar, 1984) and Dunnett’s t-test to show difference between sham groups and treatments ( P < 0.05). Data for milt variables, fertilization, hatching and larval growth were analyzed using one-way ANOVA, followed by Tukey’s test ( P < 0.05). Additionally, Pearson’s Correlation Coefficient (r) was calculated to find out the relationships between variables measured. The data for milt volume and sperm count were subjected to regression analysis to determine whether the main response to GnRHa is the increase of milt hydration or actual sperm production.
3. Results 3.1. Response of plasma T and 17P4 to GnRHa treatment Implantation with GnRHa induced increases of both plasma T and 17P4 levels (Fig. 1A and B). The increase of plasma T level was faster and stronger at higher doses, showing dose-dependency in responding to GnRHa. Plasma 17P4 levels increased with a similar tendency, but with greater individual variation among fish. High levels of both hormones after the implantation also appeared to last longer at higher GnRHa doses. In control and sham groups, T level increased slightly in the middle of the experiment, but 17P4 level remained lower than 0.5 ng/ml throughout the experiment without any surge. 3.2. Effects of GnRHa treatment on milt volume, sperm count, sperm concentration All milt variables studied here showed either dose-dependent increase or decrease after the implantation with GnRHa (Figs. 2 and 3). The treatment also advanced the timing of the major milt production within the period studied (Fig. 2A and B). The peak of milt
Fig. 5. Regression analysis of milt volume (X) and sperm count ( Y). r = Pearson’s Correlation Coefficient.
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production was observed at around 21 dpi in all treated groups and at 56 dpi in control groups. In treated groups, the milt production started to increase sharply from 9 dpi and became significantly different from control groups mainly between 14 and 28 dpi at higher doses (100GnRH and 200GnRH) ( P < 0.05, Fig. 2A and B). In control and sham groups, the milt production was low throughout the experiment. The number of spermatozoa produced during the experiment responded to the GnRH treatment, but the differences were statistically significant at 9, 14 and 21 dpi only at the highest dose ( P < 0.05, Fig. 3A). In contrast, sperm concentration decreased significantly in all treated groups at 14 and 21 dpi ( P < 0.05, Fig. 3B). The concentration in treated groups tended to be constantly lower than that in sham groups throughout the experiment. Sperm motility index was high from 4 to 49 dpi in all experimental groups including controls, and it declined after 49 dpi in all groups, except the 200GnRH group where it
Fig. 6. Relationship between sperm motility and the level of 17-hydroxyprogesterone (A) and seminal plasma pH (B).
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remained high until 85 dpi. No clear changes in sperm motility or seminal plasma pH were observed between experimental groups (data not shown). 3.3. Correlation between milt variables and plasma T and 17P4 To identify any correlation between variables measured in this study, the data from individual fish were further analyzed by calculating Pearson’s Correlation Coefficient (r). Plasma T level was not correlated with milt volume (r = 0.250, Fig. 4A) or any other milt variables (data not shown). Interestingly, however, a strong positive correlation was found between plasma 17P4 level and milt volume (r = 0.690, P < 0.01, Fig. 4B). Plasma 17P4 levels were also positively correlated with spermatozoa number (r = 0.470, P < 0.01) (data not shown). Milt volume showed a very strong positive correlation with sperm count (r = 0.860, P < 0.01, Fig. 5), indicating that the increase of milt production after GnRHa treatment was due to not only increasing milt volume but also increasing spermatozoa production. Regression analysis of the correlation between sperm count ( Y) and milt volume (X) projected a model ( Y = 0.5598X + 0.1507), where the increase of sperm number is predicted to be nearly 50% slower than the increase of milt volume in response to GnRHa treatment, indicating the decrease of the density of spermatozoa. Sperm motility index measured from individual fish did not show a linear correlation with plasma 17P4 level, but did show a very interesting relationship. Notably, when the plasma 17P4 level was high, the sperm motility index was almost always the highest (Fig. 6A). Additionally, when the individual data for sperm motility index were plotted against seminal plasma pH, the pH tended to be high when the motility index is high (r = 520, P < 0.01, Fig. 6B). 3.4. Sperm quality—fertilization, hatching and larval growth No differences were found in fertilization rate and larval growth between different groups (Table 1). Hatching rate in the 100GnRHa group was higher than that in control
Table 1 Effect of GnRHa treatment to male broodstock on fertilization and hatching rates and growth of the resultant larva Treatments
Control Sham pellet 50GnRHa 100GnRHa 200GnRHa
Fertilization rate (%)
Hatching rate (%)
Total length (mm) 18 dph
32 dph
59.1 F 1.4 60.5 F 2.6 52.9 F 2.7 60.2 F 2.8 52.8 F 2.5
32.0 F 1.3 41.1 F 2.8 38.9 F 2.6 48.7 F 3.8* 37.7 F 2.8
6.11 F 0.11 6.60 F 0.09 6.14 F 0.19 6.14 F 0.15 6.39 F 0.18
10.12 F 0.16 10.18 F 0.08 10.01 F 0.12 10.08 F 0.23 10.32 F 0.19
No significant differences were observed between treatments (Tukey’s test, P>0.05) except the hatching rate of 100GnRHa group that was significantly different only from control (marked by *) ( P < 0.05). dph: days post hatching. Each data is mean F S.E. (n = 3).
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group ( P < 0.05). Overall, no negative effects on sperm quality were found after GnRHa treatment up to the stage of metamorphosis.
4. Discussion GnRHa treatment increased milt production in a dose dependent manner and elevated the levels of plasma T and 17P4 in starry flounder male during the natural reproductive season. The increase of milt volume by GnRHa has been consistent in all previous studies on flatfish species (Harmin and Crim, 1993; Clearwater and Crim, 1998; Pankhurst and Poortenaar, 2000; Vermeirssen et al., 2000) and also non-flatfish species (Garcia, 1993; Garcia-Abiado et al., 1996; Mylonas et al., 1997a,b; King and Young, 2001). Increased milt volume induced by GnRHa, sometimes resulted from increased milt fluidity. In many studies, however, the increase of milt volume induced by GnRHa was not only caused by an increase in milt fluidity, but also in sperm production (Harmin and Crim, 1993; Mylonas et al., 1997a; Clearwater and Crim, 1998; Pankhurst and Poortenaar, 2000). Clearwater and Crim (1998) observed a decrease in spermatocrit from yellowtail flounder after GnRHa treatment, but explained it as the result of a seasonal decrease in sperm concentration rather than a GnRHa effect. On the contrary, a significant decrease of spermatocrit in response to GnRHa treatment has been reported in rabbitfish (Siganus guttatus) (Garcia, 1993), plaice and Atlantic halibut (Vermeirssen et al., 1998, 2000). Supporting the latter findings, the results from the present study also demonstrate that GnRHa treatment decreases sperm concentration. The regression analysis of sperm count and milt volume indicates that sperm count increases as milt volume increases, but the increase of milt volume is nearly twice as rapid as the increase of sperm count. This suggests that GnRHa treatment can be an effective way to ease the problem of extremely viscous milt in the captive male starry flounder. The effect of GnRHa on sperm motility is not clear in this study, since the motility measured was high in all groups regardless of GnRHa treatment from the early stages of the experiment. Differences were only noticeable at the highest dose of GnRHa near the end of the spawning season when the motility declined in all other groups. Seminal plasma pH was also little affected by the treatment. However, sperm motility appears to be positively related with seminal plasma pH when individual data were examined. Morisawa and Morisawa (1988) suggested that fish spermatozoa acquire the mobility during the transition from the testis to sperm duct where the pH is high. In support of this, a positive linear relationship between seminal plasma pH and percentage of motile spermatozoa has been shown in a cyprinid fish Alburnus alburnus (Lahnsteiner et al., 1996). Clearwater and Crim (1998) has also shown that GnRHa treatment increase both sperm motility and seminal plasma pH. The increased level of plasma T in response to GnRHa in this study is in agreement with results from other fish species, including winter flounder (Pseudopleuronectes americanus) (Harmin and Crim, 1993), plaice (Vermeirssen et al., 1998), and greenback flounder (Rhombosolea tapirina) (Pankhurst and Poortenaar, 2000). These results, however, contradict with the results from other studies where the GnRHa induced no clear changes in plasma T level as in striped bass (Morone saxatilis) (Mylonas et al., 1997b), white bass (Morone chrysops) (Mylonas et al., 1997a) and yellowtail flounder (Clearwater and Crim,
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1998); or decreased the hormone levels as in Atlantic halibut (Vermeirssen et al., 2000) and striped bass (Mylonas et al., 1998). It has been suggested that plasma T is more likely associated with spermatogenesis than spermiation in fish (Borg, 1994; Kokokiris et al., 2000). In the present study, T was elevated in response to GnRHa, but was not correlated with any milt variables, implying that the spermiation process in this species may not be dependent on plasma T. Plasma 17P4 has not been measured previously in response to GnRHa treatment in male fish. However, other progestogens such as 17,20aP and/or 17,20hP have been shown to increase after GnRHa treatment in other fish species (Mylonas et al., 1997a,b; Vermeirssen et al., 1998; Vermeirssen et al., 2000). It has also been shown that, in an in vitro study, testicular somatic cells produce 17P4 in response to gonadotropin, and this 17P4 is converted to 17,20hP by sperm 20h-hydroxysteroid dehydrogenase (Sakai et al., 1989). Ueda et al. (1985) suggested that the effect of 17P4 on milt volume is not direct, but via its conversion to 17,20hP. King and Young (2001) have shown that injection of GnRHa in combination with 17P4 to Atlantic salmon males resulted in a consistent increase of milt volume. The present finding also demonstrates a strong positive correlation between plasma 17P4 level and milt production (both milt volume and sperm count). Taken together, these data suggest that 17P4 is in the pathway of GnRH stimulation of milt production in fish. Earlier studies demonstrated that spermiation in teleosts involves a hormone-dependent hydration of seminal plasma (Clemens and Grant, 1965; Billard et al., 1982). This indicates that one of the most likely functions of the increased progestogens in response to GnRHa or gonadotropin is stimulating this hydration process. In other studies, it has been suggested that 17,20hP stimulates the ‘‘sperm duct’’ to increase seminal plasma pH by which sperm acquires motility (Miura et al., 1991, 1992; Yaron, 1995). In accordance with this, plasma 17P4 level in the present study was shown to be almost always high when the motility of sperm was highest. Together, these data suggest that progestogens increased after GnRHa treatments, may stimulate sperm duct to induce the hydration of semen and to increase the pH, resulting in increased milt volume and sperm motility. No negative effects of GnRHa treatment on fertilization and hatching rate, and subsequent growth of the larva were found in this study. In other studies, the sperm produced from pike (Esox lucius) treated with either gonadotropin or pituitary extracts has also been proven to have good fertilizing ability (Billard and Marcel, 1980). In other flatfish species, GnRHa treatment did not cause a negative effect on the fertilizing ability of sperm (Clearwater and Crim, 1998). These data, including the present findings, indicate that GnRHa treatment during the natural spawning season has no negative effect on sperm quality and subsequent larval growth in fish. In conclusion, GnRHa treatment during the natural spawning season, especially by implantation, can stimulate milt hydration and sperm production in captive male flounder, and 17P4 may be in the pathway of the GnRH action on this process.
Acknowledgements This study was funded in part by the Fisheries and Marine Bioresources Development Center, Pukyong National University, Korea.
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gonadal steroids on milt volume in the New Zealand snapper, Pagrus auratus (Sparidae). Aquaculture 125, 185 – 197. Pankhurst, N.W., Poortenaar, C.W., 2000. Milt characteristics and plasma levels of gonadal steroids in greenback flounder Rhombosolea tapirina following treatment with exogenous hormones. Mar. Freshw. Behav. Physiol. 33, 141 – 159. Sakai, U., Ueda, H., Suzuki, N., Nagahama, Y., 1989. Involvement of sperm in the production of 17a20h dihydroxy-4-pregnen-3-one in the testis of spermiating rainbow trout, Salmo gairdneri. Biomed. Res. ( Tokyo ) 10, 131 – 138. Stru¨ssmann, C.A., Renard, P., Ling, H., Takashima, F., 1994. Motility of pejerrey, Odontesthes bonariensis spermatozoa. Fish. Sci. 60, 9 – 13. Ueda, H., Kambegawa, A., Nagahama, Y., 1985. Involvement of gonadotropin and steroid hormone in spermiation in the salmon, Oncorhynchus rhodurus and goldfish, Carassius auratus. Gen. Comp. Endocrinol. 59, 24 – 30. Vermeirssen, E.L.M., Scott, A.P., Mylonas, C.C., Zohar, Y., 1998. Gonadotropin releasing hormone agonist stimulates milt fluidity and plasma concentrations of 17,20h-dihydroxylated and 5h-reduced, 3a-hydroxylated C21 steroids in male plaice (Pleuronectes platessa). Gen. Comp. Endocrinol. 112, 163 – 177. Vermeirssen, E.L.M., Shields, R.J., Mazorra de Quero, C., Scott, A.P., 2000. Gonadotrophin-releasing hormone agonist raises plasma concentrations of progestogens and enhances milt fluidity in male Atlantic halibut (Hippoglossus hippoglossus). Fish Physiol. Biochem. 22, 77 – 87. Yaron, Z., 1995. Endocrine control of gametogenesis and spawning induction in the carp. Aquaculture 129, 49 – 73. Zar, J.H., 1984. Biostatistical Analysis. Prentice-Hall, Englewood Cliffs, NJ.
Increased plasma 17-hydroxyprogesterone and milt production in response to gonadotropin-releasing hormone agonist in captive male starry flounder, Platichthys stellatus S.H. Moon a, H.K. Lim b, J.Y. Kwon c, J.K. Lee b, Y.J. Chang a,* a Department of Aquaculture, Pukyong National University, Pusan 608-737, South Korea Uljin Hatchery, National Fisheries Research and Development Institute, Uljin 767-860, South Korea c Department of Applied Biological Sciences, Sunmoon University, 100 Kalsan-ri, Tangjeong-Myeon, Asan-si, Chung Nam 336-708, South Korea b
Received 1 November 2001; received in revised form 15 November 2002; accepted 18 November 2002
Abstract In captivity, reproduction of male fish is often dysfunctional, resulting in abnormally viscous milt. Treatment with exogenous gonadotropin or gonadotropin-releasing hormone agonist (GnRHa) has been found useful in overcoming this reproductive failure in some fish species. In this study, captive male starry flounder (Platichthys stellatus) were treated with GnRHa at three different dosages (50, 100 or 200 Ag kg 1 fish body weight) by implantation with cholesterol pellets during their natural spawning season. The GnRHa treatment increased the levels of plasma testosterone (T) at the early spawning season and plasma 17-hydroxyprogesterone (17P4) throughout the spawning season. Both milt volume and sperm count were increased in a dose-dependent manner by GnRHa treatment, but the increase of milt volume was twice as rapid as the increase of sperm count. The treatment decreased sperm concentration. The increased milt volume was strongly correlated with the level of plasma 17P4 (r = 0.690), but not with T (r = 0.250). The level of 17P4 was also associated with sperm motility. The sperm obtained both from control and GnRHa-treated groups was used to fertilize starry flounder eggs, and the resultant embryos were grown until metamorphosis. No negative effects of the GnRHa treatment on fertilization, hatching and larval growth were found. The results suggest that GnRHa treatment can render captive male flounders to recover their ability to hydrate milt. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Fertilization; GnRHa; Larval growth; Milt volume; Sperm motility; 17-Hydroxyprogesterone; Testosterone
* Corresponding author. Tel.: +82-51-620-6135; fax: +82-51-628-7430. E-mail address: [email protected] (Y.J. Chang). 0044-8486/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0044-8486(02)00643-9
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1. Introduction In some fish species, captive males fail to spermiate or produce only a small volume of viscous milt. This reproductive failure hampers collection of the milt and subsequent fertilization of eggs (Donaldson and Hunter, 1983). This problem has been frequently pointed out in many marine flatfish species including plaice (Pleuronectes platessa) (Vermeirssen et al., 1998), yellowtail flounder (Pleuronectes ferrugineus) (Clearwater and Crim, 1998) and Atlantic halibut (Hippoglossus hippoglossus) (Vermeirssen et al., 2000). Reproduction of starry flounder (Platichthys stellatus), a marine fish species that occurs in countries surrounding the Pacific ocean, also appears to be dysfunctional in captivity. In preliminary studies in our laboratory (unpublished), both males and females of this species did not spawn spontaneously in captivity. When stripped manually during their natural spawning season, males produced a small amount of extremely viscous milt. Gonadotropin-releasing hormone agonists (GnRHa) have been tested on several marine and freshwater fish species to control reproductive processes (Garcia, 1993; Harmin and Crim, 1993; Pankhurst, 1994; Mylonas et al., 1996; Mylonas et al., 1997a,b; Mylonas et al., 1998; Clearwater and Crim, 1998; Vermeirssen et al., 1998; Pankhurst and Poortenaar, 2000; Vermeirssen et al., 2000; Mylonas and Zohar, 2001). In most studies so far, GnRHa treatment increased milt volume in male fish in captivity. Sometimes, GnRHa increases actual sperm production (Clearwater and Crim, 1998), but at times, it only increases milt fluidity (Garcia, 1993; Vermeirssen et al., 2000). In some fish, GnRHa treatment of male fish increased plasma levels of gonadal steroid hormones such as testosterone (T) and 11-ketotestosterone (11-KT) (Harmin and Crim, 1993; Pankhurst, 1994; Pankhurst and Poortenaar, 2000), but in others, it induced no clear changes (Clearwater and Crim, 1998) or even decreased the hormone levels (Vermeirssen et al., 2000). Progestogens such as 17,20a-dihydroxy-4-pregnene-3-one (17,20aP) and 17,20h-dihydroxy-4-pregnen-3-one (17-hydroxy-20h-dihydroprogesterone, 17,20hP) have been shown to respond to GnRHa treatment (Mylonas et al., 1997a,b; Vermeirssen et al., 1998; Vermeirssen et al., 2000). One of these progestogens, 17,20hP, has been suggested to play a key role in the process of spermiation in fish (Miura et al., 1991, 1992; Mylonas et al., 1997a,b; Vermeirssen et al., 1998). Recently, another progestogen, 17hydroxy-4-pregnen-3,20-dione (17-hydroxyprogesterone, 17P4) has also been suggested to be related to the process of spermiation in Atlantic salmon (Salmo salar), although the effect of this progestogen on milt production was attributed to its conversion to 17,20hP (King and Young, 2001). The changes of 17P4 level in response to GnRHa treatment, however, have not been studied in fish. The relationship between the level of 17P4 and milt production, therefore, has yet to be established. Few studies have, so far, measured sperm quality following GnRHa treatment (Mylonas et al., 1997a; Clearwater and Crim, 1998) despite the fact that the main interest of fish breeders is the viability of the fertilized eggs and embryos obtained using sperm produced by GnRHa treatment. In this study, we treated captive male starry flounder with GnRHa implants during their natural spawning season and investigated the individual response in milt volume, sperm count, sperm concentration, sperm motility and plasma T and 17P4. In addition, fertilization, hatching rate and larval growth were also studied in eggs inseminated with sperm produced by GnRHa-treated fish.
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2. Materials and methods 2.1. Fish Male starry flounder (caught from the wild) were kept in a circular tank (20 m3) for approximately 1 year at Uljin Hatchery, National Fisheries Research and Development Institute, Korea, until they were used for the experiment. At the beginning of their natural spawning season (January – March), 25 spermiating fish were randomly selected, placed into five plastic aquariums (200 l, five fish in each tank) with constant water flow and then subjected to GnRHa treatment. The size (means F S.D.) of fish used for the experiment was 551.7 F 109.4 g in body weight and 34.1 F 1.5 cm in total length. During the experiment, fish were kept under natural seawater temperature (means F S.D., 11.0 F 1.0 jC) and fed daily with frozen mackerel. All fish were tagged for identification with PIT tags (Pit-tag, Identification Devices). 2.2. GnRHa treatment The GnRH agonist, LHRHa (pGlu-His-Trp-Ser-Tyr-D-Ala-Leu-Arg-Pro-NHEt, Sigma) was dissolved in 50% ethanol (2 ml) and mixed with cholesterol powder (5.7 g). The mixture was dried, compounded with molten cocoa butter (0.3 g) and then compressed into pellets with a mold. Fish were weighed, and the pellets with different concentrations were prepared individually according to the weight of each fish, to achieve dosages of 50, 100 or 200 Ag GnRHa per kg of fish body weight. The pellets were implanted into the dorsal muscle. The fish (n = 5) received either no pellet (control), pellet that contains no GnRHa (sham), or a pellet containing GnRHa at three doses. 2.3. Sampling Fish were identified with the PIT tag before every sampling. Milt was collected individually from all fish in each group immediately before the implantation (0 day), and 1, 4, 9 and 14 days post implantation (dpi), and then every week up to 85 dpi when the majority of fish ceased spermiating. Milt was stripped into Eppendorf tubes by pressing the abdomen of the fish gently and was stored at 4jC until analysis. Blood samples were collected from three fish in each group every 2 weeks from 0 to 85 dpi and also at 4 dpi to see the response of T and 17P4 to the GnRHa treatment. Blood was taken from the vasculature at the caudal peduncle using a 3-ml heparinized syringe. The blood was centrifuged briefly and the plasma was stored at 73jC until analyzed for T and 17P4 using radioimmunoassay. 2.4. Milt variables Milt volume of individual fish was recorded after collection, and part of the milt was used to determine other variables. Sperm count and sperm concentration were measured using a haemocytometer after staining the sperm with 2% eosin solution. Sperm motility index was determined under a light microscope by the method of Stru¨ssmann et al. (1994)
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with slight modifications. Briefly, sperm with rapid movement, slow movement, moderate vibration and no movement were scored as 3, 2, 1 and 0, respectively. Milt sampled before (0 dpi) and soon after (1 dpi) the implantation was extremely viscous and small in quantity, causing difficulties in measuring sperm count, sperm concentration and sperm motility. Hence, the earliest data available for these parameters were from 4 dpi. The remaining milt was centrifuged at 5600 g for 30 min, and the pH was measured from the seminal fluid using an electronic pH meter (pH/Ion Meter EP-880).
Fig. 1. Responses of plasma testosterone (A) and 17-hydroxyprogesterone (B) to GnRHa treatment during the natural spawning season in captive male starry flounder. Sham: treated with sham pellet. 50GnRHa, 100GnRHa and 200GnRHa: treated with the dosages of 50, 100 and 200 Ag GnRHa per kg fish, respectively. The data points underlined indicate that significant differences exist between treatments at each time point (Kruskal – Wallis test, P < 0.05). Different alphabetical superscripts indicate significant difference by multiple comparison between treatments (Tukey’s test, P < 0.05). *Significant difference from sham control (Dunnett’s t-test, P < 0.05).
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2.5. Steroid radioimmunoassay Testosterone and 17P4 were extracted from 250 Al of plasma using 2 ml of diethyl ether, and the hormone levels were determined after Aida et al. (1984), using tritiated T and 17P4 (Amersham Pharmacia, UK). Antibodies for both T and 17P4 were purchased from SigmaAldrich. The cross-reactivity of the T antibody with 17P4 was 0.5%, and of the 17P4 antibody with T was 0.2%. Extraction efficiencies, calculated as recovery of 3H-labeled steroids extracted with plasma, were 95% and 97% for T and 17P4, respectively. The intra-
Fig. 2. Effects of GnRHa treatment on milt volume (A, B). The data points underlined indicate that significant differences exist between treatments at each time point (one-way ANOVA, P < 0.05). Different alphabetical superscripts indicate significant difference by multiple comparison between treatments (Tukey’s test, P < 0.05). Sham: treated with sham pellet. 50GnRHa, 100GnRHa and 200GnRHa: treated with the dosages of 50, 100 and 200 Ag GnRHa per kg fish, respectively.
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and interassay coefficients of variation were 9.4% and 7.4% for T, and 9.7% and 8.8% for 17P4, respectively. 2.6. Sperm quality—fertilization, hatching and larval growth Sperm obtained from each group at 35 dpi was used to fertilize eggs from a female starry flounder that was also treated with GnRHa at the dosage of 200 Ag/kg. Sperm from each
Fig. 3. Effects of GnRHa treatment on sperm count (A) and sperm concentration (B). The data points underlined indicate that significant differences exist between treatments at each time point (one-way ANOVA, P < 0.05). Different alphabetical superscripts indicate significant difference by multiple comparison between treatments (Tukey’s test, P < 0.05). Sham: treated with sham pellet. 50GnRHa, 100GnRHa and 200GnRHa: treated with the dosages of 50, 100 and 200 Ag GnRHa per kg fish, respectively. nd: not determined.
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group was pooled (from all five fish) and 0.5 ml of the sperm was mixed with 20 ml of eggs (about 6000 eggs). Fertilized eggs from each group (n = 3) were placed in 50-l plastic tanks with gentle aeration. Water temperature and illumination were maintained at 12.5 F 1 jC and 130 – 150 lx, respectively. The seawater was partially exchanged everyday to maintain good water quality. Approximately 24 h after fertilization, eggs were sampled from each tank (in triplicate) and the number of fertilized eggs was counted under a microscope. By this time, the survived eggs reached the gastrula stage. Eggs showing clear cleavage were counted as ‘‘fertilized,’’ whereas eggs with no sign of cleavage were regarded as ‘‘unfertilized’’ (these eggs were mostly opaque). Five days after fertilization, hatching rate (%) was calculated by counting floating larvae and dead eggs. The hatched larvae were grown until metamorphosis (32 days post hatching, dph). They were fed with Chlorella spp. and rotifers (8 –10 individuals ml 1) from 4 to 24 dph and, thereafter, with rotifers and Artemia nauplii.
Fig. 4. Relationships between milt volume ( Y) and the levels of plasma testosterone (X) (A) and 17hydroxyprogesterone (X) (B) that were elevated by GnRHa treatment. r = Pearson’s Correlation Coefficient.
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2.7. Statistics All data were expressed as means F S.E. Data for steroid hormones were analyzed using Kruskal – Wallis test, followed by nonparametric Tukey-type multiple comparisons ( P < 0.05) (Zar, 1984) and Dunnett’s t-test to show difference between sham groups and treatments ( P < 0.05). Data for milt variables, fertilization, hatching and larval growth were analyzed using one-way ANOVA, followed by Tukey’s test ( P < 0.05). Additionally, Pearson’s Correlation Coefficient (r) was calculated to find out the relationships between variables measured. The data for milt volume and sperm count were subjected to regression analysis to determine whether the main response to GnRHa is the increase of milt hydration or actual sperm production.
3. Results 3.1. Response of plasma T and 17P4 to GnRHa treatment Implantation with GnRHa induced increases of both plasma T and 17P4 levels (Fig. 1A and B). The increase of plasma T level was faster and stronger at higher doses, showing dose-dependency in responding to GnRHa. Plasma 17P4 levels increased with a similar tendency, but with greater individual variation among fish. High levels of both hormones after the implantation also appeared to last longer at higher GnRHa doses. In control and sham groups, T level increased slightly in the middle of the experiment, but 17P4 level remained lower than 0.5 ng/ml throughout the experiment without any surge. 3.2. Effects of GnRHa treatment on milt volume, sperm count, sperm concentration All milt variables studied here showed either dose-dependent increase or decrease after the implantation with GnRHa (Figs. 2 and 3). The treatment also advanced the timing of the major milt production within the period studied (Fig. 2A and B). The peak of milt
Fig. 5. Regression analysis of milt volume (X) and sperm count ( Y). r = Pearson’s Correlation Coefficient.
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production was observed at around 21 dpi in all treated groups and at 56 dpi in control groups. In treated groups, the milt production started to increase sharply from 9 dpi and became significantly different from control groups mainly between 14 and 28 dpi at higher doses (100GnRH and 200GnRH) ( P < 0.05, Fig. 2A and B). In control and sham groups, the milt production was low throughout the experiment. The number of spermatozoa produced during the experiment responded to the GnRH treatment, but the differences were statistically significant at 9, 14 and 21 dpi only at the highest dose ( P < 0.05, Fig. 3A). In contrast, sperm concentration decreased significantly in all treated groups at 14 and 21 dpi ( P < 0.05, Fig. 3B). The concentration in treated groups tended to be constantly lower than that in sham groups throughout the experiment. Sperm motility index was high from 4 to 49 dpi in all experimental groups including controls, and it declined after 49 dpi in all groups, except the 200GnRH group where it
Fig. 6. Relationship between sperm motility and the level of 17-hydroxyprogesterone (A) and seminal plasma pH (B).
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remained high until 85 dpi. No clear changes in sperm motility or seminal plasma pH were observed between experimental groups (data not shown). 3.3. Correlation between milt variables and plasma T and 17P4 To identify any correlation between variables measured in this study, the data from individual fish were further analyzed by calculating Pearson’s Correlation Coefficient (r). Plasma T level was not correlated with milt volume (r = 0.250, Fig. 4A) or any other milt variables (data not shown). Interestingly, however, a strong positive correlation was found between plasma 17P4 level and milt volume (r = 0.690, P < 0.01, Fig. 4B). Plasma 17P4 levels were also positively correlated with spermatozoa number (r = 0.470, P < 0.01) (data not shown). Milt volume showed a very strong positive correlation with sperm count (r = 0.860, P < 0.01, Fig. 5), indicating that the increase of milt production after GnRHa treatment was due to not only increasing milt volume but also increasing spermatozoa production. Regression analysis of the correlation between sperm count ( Y) and milt volume (X) projected a model ( Y = 0.5598X + 0.1507), where the increase of sperm number is predicted to be nearly 50% slower than the increase of milt volume in response to GnRHa treatment, indicating the decrease of the density of spermatozoa. Sperm motility index measured from individual fish did not show a linear correlation with plasma 17P4 level, but did show a very interesting relationship. Notably, when the plasma 17P4 level was high, the sperm motility index was almost always the highest (Fig. 6A). Additionally, when the individual data for sperm motility index were plotted against seminal plasma pH, the pH tended to be high when the motility index is high (r = 520, P < 0.01, Fig. 6B). 3.4. Sperm quality—fertilization, hatching and larval growth No differences were found in fertilization rate and larval growth between different groups (Table 1). Hatching rate in the 100GnRHa group was higher than that in control
Table 1 Effect of GnRHa treatment to male broodstock on fertilization and hatching rates and growth of the resultant larva Treatments
Control Sham pellet 50GnRHa 100GnRHa 200GnRHa
Fertilization rate (%)
Hatching rate (%)
Total length (mm) 18 dph
32 dph
59.1 F 1.4 60.5 F 2.6 52.9 F 2.7 60.2 F 2.8 52.8 F 2.5
32.0 F 1.3 41.1 F 2.8 38.9 F 2.6 48.7 F 3.8* 37.7 F 2.8
6.11 F 0.11 6.60 F 0.09 6.14 F 0.19 6.14 F 0.15 6.39 F 0.18
10.12 F 0.16 10.18 F 0.08 10.01 F 0.12 10.08 F 0.23 10.32 F 0.19
No significant differences were observed between treatments (Tukey’s test, P>0.05) except the hatching rate of 100GnRHa group that was significantly different only from control (marked by *) ( P < 0.05). dph: days post hatching. Each data is mean F S.E. (n = 3).
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group ( P < 0.05). Overall, no negative effects on sperm quality were found after GnRHa treatment up to the stage of metamorphosis.
4. Discussion GnRHa treatment increased milt production in a dose dependent manner and elevated the levels of plasma T and 17P4 in starry flounder male during the natural reproductive season. The increase of milt volume by GnRHa has been consistent in all previous studies on flatfish species (Harmin and Crim, 1993; Clearwater and Crim, 1998; Pankhurst and Poortenaar, 2000; Vermeirssen et al., 2000) and also non-flatfish species (Garcia, 1993; Garcia-Abiado et al., 1996; Mylonas et al., 1997a,b; King and Young, 2001). Increased milt volume induced by GnRHa, sometimes resulted from increased milt fluidity. In many studies, however, the increase of milt volume induced by GnRHa was not only caused by an increase in milt fluidity, but also in sperm production (Harmin and Crim, 1993; Mylonas et al., 1997a; Clearwater and Crim, 1998; Pankhurst and Poortenaar, 2000). Clearwater and Crim (1998) observed a decrease in spermatocrit from yellowtail flounder after GnRHa treatment, but explained it as the result of a seasonal decrease in sperm concentration rather than a GnRHa effect. On the contrary, a significant decrease of spermatocrit in response to GnRHa treatment has been reported in rabbitfish (Siganus guttatus) (Garcia, 1993), plaice and Atlantic halibut (Vermeirssen et al., 1998, 2000). Supporting the latter findings, the results from the present study also demonstrate that GnRHa treatment decreases sperm concentration. The regression analysis of sperm count and milt volume indicates that sperm count increases as milt volume increases, but the increase of milt volume is nearly twice as rapid as the increase of sperm count. This suggests that GnRHa treatment can be an effective way to ease the problem of extremely viscous milt in the captive male starry flounder. The effect of GnRHa on sperm motility is not clear in this study, since the motility measured was high in all groups regardless of GnRHa treatment from the early stages of the experiment. Differences were only noticeable at the highest dose of GnRHa near the end of the spawning season when the motility declined in all other groups. Seminal plasma pH was also little affected by the treatment. However, sperm motility appears to be positively related with seminal plasma pH when individual data were examined. Morisawa and Morisawa (1988) suggested that fish spermatozoa acquire the mobility during the transition from the testis to sperm duct where the pH is high. In support of this, a positive linear relationship between seminal plasma pH and percentage of motile spermatozoa has been shown in a cyprinid fish Alburnus alburnus (Lahnsteiner et al., 1996). Clearwater and Crim (1998) has also shown that GnRHa treatment increase both sperm motility and seminal plasma pH. The increased level of plasma T in response to GnRHa in this study is in agreement with results from other fish species, including winter flounder (Pseudopleuronectes americanus) (Harmin and Crim, 1993), plaice (Vermeirssen et al., 1998), and greenback flounder (Rhombosolea tapirina) (Pankhurst and Poortenaar, 2000). These results, however, contradict with the results from other studies where the GnRHa induced no clear changes in plasma T level as in striped bass (Morone saxatilis) (Mylonas et al., 1997b), white bass (Morone chrysops) (Mylonas et al., 1997a) and yellowtail flounder (Clearwater and Crim,
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1998); or decreased the hormone levels as in Atlantic halibut (Vermeirssen et al., 2000) and striped bass (Mylonas et al., 1998). It has been suggested that plasma T is more likely associated with spermatogenesis than spermiation in fish (Borg, 1994; Kokokiris et al., 2000). In the present study, T was elevated in response to GnRHa, but was not correlated with any milt variables, implying that the spermiation process in this species may not be dependent on plasma T. Plasma 17P4 has not been measured previously in response to GnRHa treatment in male fish. However, other progestogens such as 17,20aP and/or 17,20hP have been shown to increase after GnRHa treatment in other fish species (Mylonas et al., 1997a,b; Vermeirssen et al., 1998; Vermeirssen et al., 2000). It has also been shown that, in an in vitro study, testicular somatic cells produce 17P4 in response to gonadotropin, and this 17P4 is converted to 17,20hP by sperm 20h-hydroxysteroid dehydrogenase (Sakai et al., 1989). Ueda et al. (1985) suggested that the effect of 17P4 on milt volume is not direct, but via its conversion to 17,20hP. King and Young (2001) have shown that injection of GnRHa in combination with 17P4 to Atlantic salmon males resulted in a consistent increase of milt volume. The present finding also demonstrates a strong positive correlation between plasma 17P4 level and milt production (both milt volume and sperm count). Taken together, these data suggest that 17P4 is in the pathway of GnRH stimulation of milt production in fish. Earlier studies demonstrated that spermiation in teleosts involves a hormone-dependent hydration of seminal plasma (Clemens and Grant, 1965; Billard et al., 1982). This indicates that one of the most likely functions of the increased progestogens in response to GnRHa or gonadotropin is stimulating this hydration process. In other studies, it has been suggested that 17,20hP stimulates the ‘‘sperm duct’’ to increase seminal plasma pH by which sperm acquires motility (Miura et al., 1991, 1992; Yaron, 1995). In accordance with this, plasma 17P4 level in the present study was shown to be almost always high when the motility of sperm was highest. Together, these data suggest that progestogens increased after GnRHa treatments, may stimulate sperm duct to induce the hydration of semen and to increase the pH, resulting in increased milt volume and sperm motility. No negative effects of GnRHa treatment on fertilization and hatching rate, and subsequent growth of the larva were found in this study. In other studies, the sperm produced from pike (Esox lucius) treated with either gonadotropin or pituitary extracts has also been proven to have good fertilizing ability (Billard and Marcel, 1980). In other flatfish species, GnRHa treatment did not cause a negative effect on the fertilizing ability of sperm (Clearwater and Crim, 1998). These data, including the present findings, indicate that GnRHa treatment during the natural spawning season has no negative effect on sperm quality and subsequent larval growth in fish. In conclusion, GnRHa treatment during the natural spawning season, especially by implantation, can stimulate milt hydration and sperm production in captive male flounder, and 17P4 may be in the pathway of the GnRH action on this process.
Acknowledgements This study was funded in part by the Fisheries and Marine Bioresources Development Center, Pukyong National University, Korea.
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