Effects of 17[alpha]-methyltestosterone and aromatase inhibitor letrozole on sex reversal, gonadal structure, and growth in yellow catfish pelteobagrus fulvidraco.
Yellow catfish (Pelteobagrus fulvidraco Richardson, 1846) has become a commercially important edible fish with high market demand and high price during the past decade in China, and has promising market potential in Japan, South Korea, East and South Asia (Wang et al., 2006). The species exhibits a sexually dimorphic growth pattern, with males growing faster and reaching larger ultimate size than females (Park et al., 2004; Liu et al., 2007; Wang et al., 2009). Several efforts have been made to produce successive all-male populations by combining induction of sex reversal and gynogenesis (Liu et al., 2007, 2013) as well as application of sex-linked markers (Wang et al., 2009; Dan et al., 2013). Theoretically, the proportion of male progeny of sex-reversed YY females and YY males should be 100%. Actually, however, the proportion of males ranged from 75.9% to 100% under normal rearing temperatures (24-28 [degrees]C; Liu et al., 2007). The contradiction between theory and practice raised the question of what other factors or mechanisms caused the sex reversal of genotypic male individuals. Other than minor genetic sex factors, temperature is of the most concern and the most investigated environmental factor that can influence sex differentiation (Penman and Piferrer, 2008; Shen and Wang, 2014). In a closely related species of yellow catfish, Pseudobagrus vachelli, temperature treatment during the labile period of sex differentiation dramatically altered the population sex ratio: 20 [degrees]C, 24 [degrees]C, and 32 [degrees]C treatments produced a balanced sex ratio (45.3%, 43.3%, and 50.7% males, respectively), while 30 [degrees]C and 34 [degrees]C treatments produced male-skewed (83.3% males) and female-skewed (26.4% males) sex ratios, respectively (Chen, 2007). As far as we know, the effects of temperature on sex differentiation in yellow catfish have not been reported. It is worth mentioning that the name Pseudobagrus fulvidraco (English name, bagrid/bullhead catfish), which was used at one time, is a synonym for Pelteobagrus fulvidraco; the taxonomic revision was made in 1990 (Lee and Kim, 1990). The fish (bagrid catfish Pseudobagrus fulvidraco) used in Park et al. (2003, 2004), in which sex differentiation was systematically studied, actually is Pelteobagrus fulvidraco.
Sex-determining mechanisms in fish are quite complex, including genetic sex determination, temperature-dependent sex determination, and the transitional form called genetic sex determination plus temperature effects (Devlin and Nagahama, 2002; Penman and Piferrer, 2008; Pandian, 2011; Shen and Wang, 2014). Sex differentiation in fish is also complex and labile, with the potential to be affected by rearing temperature, exogenous hormones or hormonal-pathway-related chemicals (e.g., aromatase inhibitor [AI], an estrogen/androgen receptor antagonist), social factors, and epigenetic modulation (Piferrer and Guiguen, 2008; Wu et al., 2012, unpubl. abstract; Piferrer, 2013). The complexity and lability of the mechanisms and pathways involved in the final determination of an individual's sex, therefore, hamper both the production of a monosex population and the clarification of the mechanisms involved.
Returning to the question raised earlier: Why do a proportion of XY or YY genotypic males develop into females? And what about the opposite: If genotypic females from an all-female population develop into phenotypic males, what is the proportion and what is the mechanism? Undoubtedly, an all-female population would be one of the best resources for investigating sex-determining mechanisms, sex differentiation, related gene expression, sex-linked markers, and associated epigenetic mechanisms (Kitano et al., 1999; D'Cotta et al., 2001; Shen and Wang, 2014). In an attempt to produce an all-female population, masculinization of a normal mixed-sex population will be the initial step according to the laws of Mendelian inheritance (the offspring of an XX female and an XX male will be all female).
Hence, the objectives of our present study are to evaluate the effectiveness of various doses of two hormonal treatments on masculinization, as well as growth performance and changes in gonadal structure after the termination of treatments.
Materials and Methods
Fish obtained from controlled reproduction were fed rotifers starting from 3 days post-hatching (DPH) and gradually weaned to a microbound diet by 8 DPH. Afterward, fry were fed only the microbound diet (series of sizes, Shengsuo Fishery Feed Research Centre of Shandong Province, PR China; main nutrient components: crude protein [greater than or equal to] 50%, crude fat [greater than or equal to] 8%, raw fiber [less than or equal to] 3%, ash [less than or equal to] 16.5%, moisture [less than or equal to] 12%).
17[alpha]-methyltestosterone (MT) or letrozole (LZ) were dissolved in 95% ethanol, and then thoroughly mixed with the microbound diet (nearly 4 ml ethanol per 10 g diet). Ethanol was removed by evaporation at 40 [degrees]C for about 4 h. The control diet was prepared in the same manner using 95% ethanol without hormones. The treated microbound diet was stored at 4 [degrees]C.
The experiment consisted of seven oral administration treatments containing 0, 20, 50, and 100 mg [kg.sup.-1] MT or LZ. During the experiment, fish were kept in 0.86-[m.sup.3] net enclosures suspended in cement tanks (49.5 [m.sup.3]). There were seven cement tanks, each containing three replicate enclosures of the same treatment. Each enclosure contained 150 fry, 10 days old with a mean total length of 12.9 mm and mean body weight of 17.0 mg (n = 100 fish taken from the stocking population for measurement).
A triangular floating plastic feeding frame (0.3 m X 0.3 m X 0.3 m) was fixed at one corner of each net cage. During the experiment, water temperature ranged between 25.5 and 28.3 [degrees]C and dissolved oxygen ranged between 5.2 and 6.5 mg [1.sup.-1] . Net enclosures were cleaned biweekly to maintain good water exchange, and debris accumulated on the bottom of tanks was siphoned on 25 and 61 DPH. Water in each cement pool was completely renewed each week with a water flow rate of about 4.9 1 [min.sup.-1]. In an attempt to cover the labile period of sex differentiation (8 to 30 DPH) determined by Park et al. (2004), and considering larvae's acceptance of formula diet, fish were successfully fed with the experimental diet starting from 10 DPH to the end of drug treatments at 59 DPH. The larvae were fed three times a day between 10 and 23 DPH and twice a day between 24 and 59 DPH. The survival rate was evaluated on 25 and 55 DPH. The amount of feed fed to each cement tank was recorded daily.
Twenty fish were sampled randomly from each cage on 14, 19, 24, 29, 34, 39, 44, and 51 DPH to be measured, after being slightly anesthetized with MS-222 (70 mg [l.sup.-1]), for total length (precision 0.1 mm) and body weight (precision 0.01 g). Fish were returned to their original cages after measurement. On 60 DPH, 16 fish from each cage were fixed in Bouin's solution (whole body) for histological examination after measurement of total length and body weight. Mortality was evaluated at 25 DPH and 55 DPH.
Growth retarding rate (GRR) in MT treatments from 29 to 105 DPH was calculated to explore the growth profile versus that of the control group during and after MT treatment by using the following formula,
GRR = (BWc-BWt)/BWc
where BWc = mean body weight in the control group and BWt = mean body weight in the MT-treated group. The means used for GRR were from the pooled data of the replicates. In other words, there were no replicates for GRR in each group.
The remaining juveniles were fed with an untreated commercial expanded yellow catfish diet (Yueyang Zhanxiang Biological Science and Technology Co., Ltd, China; 40.89% crude protein, 4.50% crude fat, 3% raw fiber, and 19.05% ash) twice per day from 61 DPH to 105 DPH. Sixteen juveniles at 75 DPH and fifteen juveniles at 105 DPH were sampled randomly in each cage and fixed in Bouin's solution (whole body) after measurement.
Separated gonad connected with kidney from each fish was processed for histological sectioning (6-8 [micro]m for each section) after routine dehydration and paraffin embedding. For each fish, successive cross-sections covering the whole gonad tissue were cut using a microtome. At least 10 sections of each fish were stained with hematoxylin and eosin. The slides were examined and photographed with an optical microscope to determine their phenotypic sex. Each fish was categorized as male, female, or intersex. Fish were classified as intersex when both female and male germ cells were seen in the same gonad slice.
Data of survival rates and growth performances (except GRR) were analyzed with ANOVA followed by Duncan's post hoc multiple comparisons. Deviation of sex ratios in treatment groups from the control group were examined by means of the chi-square test. When required, normality was ensured after logarithmic, sinusoidal, or arcsine transformation of data. Differences were considered significant when P < 0.05.
Survival and growth performance
Effects of 17[alpha]-methyltestosterone (MT) and letrozole (LZ) treatments on the survival rate at 25 and 55 days post-hatching (DPH), as well as on total length at the termination of treatments (60 DPH) and 45 days after cessation of the treatments (105 DPH) are presented in Table 1. There was no significant survival difference on any given day, and no mortality occurred in any treatment after 55 DPH.
In the MT treatment groups, growth suppression started from 29 DPH and difference from control groups became significant after 44 DPH (Fig. 1). Apparently, compensatory growth occurred after cessation of MT administration, because there were no significant size differences on 105 DPH. Similarly, the growth retarding rate (GRR) increased during the treatment period and decreased after termination of MT administration (Fig. 2). In contrast to MT treatments, LZ treatment at the lowest dose promoted growth, and higher doses did not affect growth (Fig. 3). Yet the size of fish 45 days after termination of the LZ treatments was similar to that of the control group.
MT treatments at various dosages had no masculinization effect. However, all MT treatments produced low levels of intersex fish, and no intersex fish were observed in the control treatment (Table 2). LZ treatments at all dosages significantly increased male ratios compared to the control group, and intersex fish were also observed in all LZ treatments.
Changes of sex ratios including male percentage and intersex percentage, as well as female percentage in the [L.sub.20], [L.sub.50], and [L.sub.100] treatments are presented in Figure 4 (A, B, C). No significant difference was observed at 75 DPH compared with 60 DPH, including male percentage, intersex percentage, and female percentage in each corresponding LZ treatment. However, male percentage decreased at 105 DPH compared with 60 DPH in the corresponding [L.sub.20] and [L.sub.50] treatments. Intersex percentage increased at 105 DPH compared with 60 DPH in the [L.sub.100] treatment.
Histological structure of gonads
Representative gonadal sections of yellow catfish at 60 DPH from this study are shown in Figure 5. The testicular structures of male yellow catfish exposed to MT were affected notably in a dose-dependent manner. Histological evaluation of the testes from fish treated with the lower dose of MT (20 mg [kg.sup.-1]) revealed that the density of spermatozoa was increased, and a large amount of spermatozoa was present in the tubule lumens; in addition, the lumens showed a remarkable enlargement (Fig. 5A) compared with the control group (Fig. 5B). Enlarged lumens were also found in [M.sub.50] (Fig. 5C) and [M.sub.100] (Fig. 5D) treatments. However, it seems that some spermatozoa were released from the body, or degenerated. Especially in the [M.sub.100] treatment, all that remained of the testicular structures were vacuolated seminiferous tubules; in some of the testes examined, no male germ cells were observed (Fig. 5D). Intersex gonads were found in all MT groups. The developmental stage of testes in the intersex fish was similar to those in control males, except that some vacuolated seminiferous tubules were observed in intersex gonads. Oocytes in intersex gonads showed no remarkable difference when compared with control females and phenotypic females in MT treatments.
When compared to the control group, male yellow catfish exposed to LZ for 50 days showed testicular development. Histologically, testes from fish treated with LZ showed abundant spermatozoa in the lobule lumens, as well as enlarged lumens (Fig. 5E) compared with the control group (Fig. 5B); this is similar to the observations in [M.sub.20] treatment (Fig. 5a). Vacuolated tissues were also observed in intersex gonads in [L.sub.50] and [L.sub.100] treatments (Fig. 5F). However, it seems that these were degenerated oocytes (similar shape and size), rather than vacuolated seminiferous lobules. Structures as well as developmental stage of testes in the intersex fish were similar to those in control males in all LZ treatments. Oocytes in intersex gonads showed no remarkable difference compared with control females and normal females in LZ treatments.
is interesting that histological evaluation of ovaries from normal females treated with MT and LZ for 50 days failed to reveal any remarkable change when compared with the control group.
Figure 6 shows representative histological samples from fish treated with LZ and from the control group at 105 DPH, 45 days after the cessation of treatments. Increased density of spermatozoa and enlarged lumens were observed in normal males in the LZ-treated groups (Fig. 6A), if compared with control and intersex gonads. Developmental stage in intersex testes (Fig. 6B) lagged behind the normal males in LZ-treated fish; however, this is similar to the control group (Fig. 6C). No visible difference in ovary structure was observed among normal females in the control group, intersex, or normal females in LZ treatments.
17[alpha]-methyltestosterone (MT) is considered to be an effective androgen in inducing sex inversion in teleosts and is widely used for this purpose. Functional males with spermiation have been obtained by administration of MT in several fish species, including Japanese flounder, Paralichthys olivaceus (Kitano et al., 2000), and grouper, Epinephelus septemfasciatus (Tanaka et al., 1999). However, high-dose or long-term treatment with MT can result in feminization effects (Piferrer and Donaldson, 1991; Piferrer et al., 1994; Ankley et al., 2001; Parrott and Wood, 2002; Zerulla et al., 2002). Explanation of this phenomenon may be (1) aromatization of exogenous androgen to estrogen, and (2) inhibition of the biosynthesis of the endogenous androgen in genotypic males (Piferrer and Donaldson, 1991; Hornung et al., 2004). In the present study, failure of masculinization of yellow catfish by administration of MT at variable doses may involve the former mechanism given that testicular development was advanced to some extent in histological examination. High levels of estrogens may repress spermatogenesis and cause testicular degeneration (Piferrer and Donaldson, 1991). In high-dose treatments ([M.sub.50] and [M.sub.100]), a large amount of vacuolated seminiferous lobules was observed, which may be due to the degeneration of testes caused by high levels of estrogens that come from aromatization of MT. Similar structure and developmental stage of ovaries between MT treatments and the control group may provide support for the speculation, given that high levels of androgens might delay ovary development (Scholz and Kluver, 2009), which was not the case in the present work. The results of exposure of yellow catfish to MT in our study are very similar to those of Arslan et al. (2009), who found that MT was ineffective in altering phenotypic sex when largemouth bass were administered 60 mg [kg.sup.-1] MT for 30, 45, or 60 days, and produced a few intersex fish (2.2%-3.6%). Similarly, Rinchard et al. (1999) found that oral administration of MT at a dose of 15 mg [kg.sup.-1] of food for 60 days did not significantly alter sex ratios and did not increase growth during or after MT treatment. In addition, the male gonads consisted mainly of vacuolized connective tissues, which was also observed in the present study after MT treatment (Fig. 5C, D). Hornung et al. (2004) reported that aromatization of MT to ME2 (17[alpha]-methylestradiol) contributes to the estrogenic effects in fathead minnows following exposure to MT, and found that plasma vitellogenin levels were significantly higher in both male and female fish exposed to 200 [micro]g [l.sup.-1] MT than to 20 [micro]g [l.sup.-1] MT, indicating that MT treatment resulted in higher plasma estrogen levels. This may explain the differences in testes structure between variable doses of MT treatments, [M.sub.20] and [M.sub.50] or [M.sub.100] in the present study. We coined a new term, growth retarding rate (GRR), deduced from the growth difference between MT-treated and control groups to explore the growth profile during and after MT treatment (Fig. 2). Examination of the growth profile in Figure 1 leads to the intuitive impression that the gap of growth between MT-treated and control groups increases with time from 29 DPH to 60 DPH and decreases from 60 DPH to 105 DPH, and the calculation of GRR (Fig. 2) shows that this is actually the case. Furthermore, the growth inhibition effects were temporary rather than permanent according to Figure 2. Growth suppression with exposure to MT in the present study may also involve the higher levels of estrogen compared with the control group to some extent, as we mentioned earlier. Physiological and molecular biological analyses in the future will be helpful to uncover the players involved in the process of natural sex differentiation or artificial sex reversal.
Aromatase is responsible for the final step of estrogen biosynthesis, catalyzing the aromatization of androstenedione and testosterone into estrone and estradiol, respectively (Haynes et al., 2003); its activity determines the androgen-to-estrogen ratio in developing gonads (Navarro-Martin et al., 2009). Consequently, aromatase activity is crucial for establishing the final sex phenotype, with high and low expression levels of cyp19a closely associated with ovarian and testicular differentiation, respectively (Nakamura et al., 2003; Blazquez et al., 2008). Blockade of estrogen synthesis via administration of an aromatase inhibitor (AI) resulted in complete functional masculinization in fish (Piferrer et al., 1994; Guiguen et al., 1999; Kitano et al., 2000; Kwon et al., 2000; Ankley et al., 2002; Kajiura-Kobayashi et al., 2003; Suzuki et al., 2004; Uchida et al., 2004). In the present work, 83.3% males were produced at the middle dose treatment ([L.sub.50]). Park et al. (2004) reported that oral administration with estradiol-17[beta] and tamoxifen (an antiestrogen) are effective for sex reversal in yellow catfish (100% female and 90% male, respectively), and the labile period of sex differentiation falls between the 8th and 30th day post-hatching. The masculinization experiment conducted during 10 DPH to 59 DPH in our present study almost covered this labile period under similar culture conditions. Therefore, our results showed that the LZ dosages applied in this study may be not enough to induce 100% masculinization. Remarkably, growth promoting effects of the lowest dose treatment ([L.sub.20]) were observed compared with the control group, suggesting that this compound may also possess anabolic growth stimulative potential in fish. Uchida et al. (2004) found that total length and body weight of zebrafish (Danio rerio) in fadrazole (a non-steroidal AI) treatments were longer and heavier than those of non-treated fish. Growth-promoting effects were also observed in yellow catfish through administration of an AI tamoxifen (Park et al., 2003, 2004). In tilapia (Oreochromis niloticus), growth of juvenile and mature females was increased by 32% with AI treatment, along with suppressed aromatase gene expression (cypl9ala) and reduced plasma estrogen level. On the other hand, estrogen-treated juveniles showed reduced growth with high levels of estrogen in plasma (Baroiller et al., 2014). These data strongly suggest that the growth-promoting effects of low-dose LZ in the present work were attributable to reduced plasma estrogen. However, it remains unclear why high-dose LZ treatments did not display growth-promoting effects in the present work. Further studies are required to unveil the growth-promoting mechanisms of LZ not only in fish but in higher vertebrates as well (Zhou et al., 2005).
Testicular development was stimulated in the LZ treatment groups, which was also reported in other species from previous studies. After LZ treatment in lizards, Podarcis sicula (Cardone et al., 2002), histological sections showed that the diameter of seminiferous tubules was increased, and increases in the number of germ cells and spermatozoa were observed. Afonso et al. (2000) reported that a treatment group of adult male coho salmon, Oncorhynchus kisutch, injected with AI (fadrozole) spermiated earlier than the control group at 16 days after treatments, with the inhibition of 17[beta]-estradiol production causing a premature increase in 17, 20[beta]-P (17, 20[beta]-dihydroxy-4-pregnen-3-one) levels. Several studies have shown that in male fish, plasma 17, 20[beta]-P levels remain low during the period of testicular development (spermatogenesis) and then increase dramatically coincidentally with spermiation (Ueda et al., 1983; Scott and Sumpter, 1989; Planas and Swanson, 1995). Premature development of testes and extraordinarily early spermiation in the present study was the first report for 2-monold yellow catfish (7.81 cm total length, 4.83 g body weight). In a chronological study of sex differentiation in yellow catfish (Park et al., 2004), histological sections showed that spermatozoa began to enter the growth stage on 100 DPH (9.48 cm total length, 12.79 g body weight). The development-promoting effect on testis may be due to the inhibition of 17[beta]-estradiol production and prematurely increased plasma 17, 20[beta]-P levels in the present study, and these results may be useful in modulating spermatogenesis in fish and higher vertebrates so as to induce artificial reproduction.
Yamamoto (1969) reported that complete and functional sex reversal should occur if the administration of sex hormones is started at the undifferentiated gonad stage and is continued through the subsequent stages of sexual differentiation. However, in the present study, male percentage decreased in lower dose treatments ([L.sub.20] and [L.sub.50]) and intersex ratio increased in the high-dose treatment ([L.sub.100]) 45 days after the masculinization experiment. At 60 DPH in our study, histological sections showed perinucleolus-stage oocytes in ovaries. It could be concluded that female germ cells can retain a certain level of bipotentiality after sex differentiation in yellow catfish if the two results are combined. Several previous studies reported the sexual bipotentiality of germ cells into adulthood even in gonochoristic fish species (Pandian and Sheela, 1995; Baroiller et al., 1999; Striissmann and Nakamura, 2002). The results may be helpful in artificial inducing of dedifferentiation and redifferentiation of differentiated oocytes.
To conclude, this study demonstrated that the non-steroidal aromatase inhibitor letrozole has the ability to induce masculinization and can promote growth performance, advance testicular development, and promote spermiation during early gonadal development in yellow catfish. It also provides the first evidence that female germ cells in yellow catfish possess a certain level of bipotentiality after sex differentiation. In the present study, three dosages of MT failed to cause masculinization in yellow catfish, while inducing intersex. A lower dose of MT (20 mg [kg.sup.-1]) advanced testicular development; however, a comparative high dose resulted in reproductive toxicity such as vacuolated seminiferous lobules. Further study on short-term, effective production of all-female populations will be beneficial in exploring the complexity of sex determination and differentiation, associated gene expression, sex-linked markers, etc. The mechanisms of the growth-promoting effects of LZ treatment and its advancement of testicular development remain to be elucidated.
We thank Mr. Fan Qizhi and Mr. Wang Xiaojun for preparing the experiment and providing fish care. We are indebted to Prof. Bangke Zhu, Dr. Zexia Gao, and Ms. Joy Bauman for reviewing the manuscript and providing valuable comments.
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ZHI-GANG SHEN (1), Ql-XUE FAN (1*), WEI YANG (1), YUN-LONG ZHANG (1), AND HAN-PING WANG (2)
(1) College of Fishery, Huazhong Agricultural University, Wuhan, Hubei 430070, PR China; and (2) Aquaculture Genetics and Breeding Laboratory, The Ohio State University South Centers, 1864 Shyville Road, Piketon, Ohio 45661, USA
Received 8 September 2014; accepted 30 January 2015.
(*) To whom correspondence should be addressed. E-mail: firstname.lastname@example.org
Table 1 Effects of various doses of 17[alpha]-methyltestosterone (M) and letrozole (L) on survival rate of yellow catfish at 25 and 55 days post hatching (DPH), as well as mean total length on 60 DPH (the end of treatments) and 105 DPH Survival (%) Drug doses (mg [kg.sup.-1]) 25 DPH 55 DPH 0 (control) 80.00 [+ or -] 4.58 55.33 [+ or -] 4.33 [M.sub.20] 79.17 [+ or -] 8.90 53.67 [+ or -] 5.49 [M.sub.50] 84.13 [+ or -] 4.73 55.67 [+ or -] 1.20 [M.sub.100] 83.07 [+ or -] 2.41 49.67 [+ or -] 9.49 0 (control) 80.00 [+ or -] 4.58 55.33 [+ or -] 4.33 [L.sub.20] 81.83 [+ or -] 4.85 49.00 [+ or -] 5.20 [L.sub.50] 85.50 [+ or -] 1.40 60.67 [+ or -] 1.45 [L.sub.100] 87.77 [+ or -] 6.53 63.33 [+ or -] 9.33 Mean total length (mm) 60 DPH 105 DPH 78.10 [+ or -] [0.13.sub.a] 98.37 [+ or -] 4.32 63.87 [+ or -] [4.86.sub.b] 93.33 [+ or -] 5.79 60.23 [+ or -] [3.64.sub.b] 92.21 [+ or -] 4.71 57.33 [+ or -] [2.02.sub.b] 91.34 [+ or -] 5.21 78.10 [+ or -] [0.13.sub.B] 98.37 [+ or -] 4.32 81.69 [+ or -] [1.87.sub.A] 100.06[+ or -] 5.99 76.25 [+ or -] [0.40.sub.B] 96.72 [+ or -] 4.52 76.36 [+ or -] [0.83.sub.B] 97.02 [+ or -] 5.43 Note: Means ([+ or -]SD) within a column followed by different subscript letters were significantly different (P < 0.05). Sample sizes of growth performance in each group were 20 x 3 individuals every time. Table 2 Effects of various doses of 17[alpha]-methyltestosterone (M) and letrozole (L) on sex ratios of yellow catfish at 60 days post-hatching Drug doses (mg [kg.sup.-1]) Male (%) Intersex (%) 0 (control) 37.5 [+ or -] 0.0 0.0 [M.sub.20] 45.8 [+ or -] 11.0 4.2 [+ or -] 1.2 [M.sub.50] 33.3 [+ or -] 11.0 4.2 [+ or -] 0.0 [M.sub.100] 50.0 [+ or -] 12.5 8.3 [+ or -] 2.1 0 (control) 37.5 [+ or -] [0.0.sub.a] 0.0 [L.sub.20] 75.0 [+ or -] [0.0.sub.b] 8.3 [+ or -] 1.2 [L.sub.50] 83.3 [+ or -] [4.2.sub.b] 8.3 [+ or -] 2.1 [L.sub.100] 75.0 [+ or -] [7.2.sub.b] 12.5 [+ or -]2.1 Female (%) 62.5 [+ or -] 0.0 50.0 [+ or -]12.5 62.5 [+ or -] 7.2 41.7 [+ or -]18.2 62.5 [+ or -][0.0.sub.A] 16.7 [+ or -][8.3.sub.B] 8.4 [+ or -][4.2.sub.B] 12.5 [+ or -][9.5.sub.B] Note: Means ([+ or -]SD) within a column followed by different subscript letters were significantly different (P < 0.05). Intersex, female, and male germ cells simultaneously existed in the same gonad slice, n, the number of sexed fish, = 48.
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|Author:||Shen, Zhi-Gang; Fan, Qi-Xue; Yang, Wei; Zhang, Yun-Long; Wang, Han-Ping|
|Publication:||The Biological Bulletin|
|Date:||Apr 1, 2015|
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