Maturation of captive male giant grouper Epinephelus lanceolatus in subtropical waters in Japan

Maturation of captive male giant grouper Epinephelus lanceolatus in subtropical waters in Japan | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Maturation of captive male giant grouper Epinephelus lanceolatus in subtropical waters in Japan Shukei Masuma, Ryuichiro Aoki This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5996034/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract This study aimed to determine the maturation process of captive male giant grouper Epinephelus lanceolatus (GG) in the subtropical waters of the Amami Islands, Japan. The experiment commenced in February 2019 using 134 GG (3 years and 5 months old; mean body weight [BW] = 6.0 kg) raised from hatchery-reared juveniles. Of those, 31 were administered 17α-methyltestosterone (MT) at a concentration of 2 mg/kg BW using cholesterol pellets, and the others were not. We assessed the spermiation response (SpR) of all individuals approximately once a month for over one year, from April 2019 to May 2020. Sperm motility (%SM) was also assessed when sufficient semen samples were obtained. The SpR was observed in 22.3% (23/103) untreated and 93.5% (29/31) of MT-treated GG. Some individuals from both groups showed consistent SpR throughout the year. The mean %SM for MT-untreated and treated GG were 49 and 56%, respectively, with no significant difference between the groups. Histological analysis of the gonads of the untreated GG was conducted on three fish harvested in July 2019. While two transitioned from female to male and contained immature oocytes, another possessed fully functional testis; this suggests that sex reversal in GG involves a direct transition from immature female to male. Our study demonstrated that hatchery-reared male GG mature year-round and could maintain motile spermatozoa of consistent quality in the subtropical waters of the Amami Islands. This result is critical for improving any hybridisation using GG males. This study is the first to report on the reproductive ecology of male GG in subtropical waters. Marine aquaculture Methyltestosterone Testis Sex reversal Spermiation response Sperm motility Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Epinepheline grouper species are widespread in temperate and tropical coastal waters (Heemstra and Randall 1993 ; Ma and Craig 2018 ). These species exhibit protogynous hermaphroditism, i.e., they change sex from female to male during their lifetime (Sadovy and Liu 2008). Among the epinepheline groupers species, Epinephelus spp. such as the orange-spotted grouper Epinephelus coioides , Malabar grouper E. malabaricus , Hong Kong grouper E. akaara , brown-marbled grouper E. fuscoguttatus , giant grouper E. lanceolatus (GG), and nine other species are highly valuable aquaculture species in Southeast and East Asia because of their good taste, cooking versatility, and high demand (Pierre et al. 2008 ). Along with aquaculture practices involving purebred groupers, Epinephelus species hybridisation has been successfully conducted to produce new groupers with desirable aquaculture traits (Bartley et al. 2001). Examples of such hybridisation include E. coioides × E. fuscoguttatus (Koh et al. 2008 ), E. coioides × E. lanceolatus (Koh et al. 2010 ), E. fuscoguttatus × E. lanceolatus (Ch’ng and Senoo 2008), E. fuscoguttatus × camouflage grouper E. polyphekadion (James et al. 1999 ), dusky grouper E. marginatus × white grouper E. aeneus (Glamuzina et al. 1999 ), longtooth grouper E. bruneus (LG) × E. lanceolatus (Murata et al. 2017 )d moara (synonym E. bruneus ) × E. lanceolatus (Chen et al. 2018 ). GG is commonly used as the sire in these cases due to its fast-growing trait (Liao et al. 2001 ). The GG is one of the largest epinepheline species, reaching 400 kg body weight (BW) in tropical and subtropical waters (Heemstra and Randall 1993 ). The GG is an important species in recreational and commercial fisheries, particularly in the live fish trade based in Hong Kong. However, it has been largely extirpated in heavily fished areas (Fennessy et al. 2018 ). To meet the growing demand for GG, larviculture technology was initiated in southern Taiwan in 1996 (Ho et al. 1997 ) and subsequently adopted by Asia-Pacific countries/regions, including China, Indonesia, Thailand, Vietnam, Malaysia, Hawaii, and Australia (Palma et al. 2019 ). Understanding the reproductive biology of GG is critical for improving captive breeding techniques (Palma et al. 2019 ). However, information on the maturation and reproduction of GG is primarily limited to tropical regions, and it remains uncertain whether GG can mature in subtropical regions. In Japan, the hybrid grouper Kue-tama (LGGG) E. bruneus × E. lanceolatus was recently established (Masuma 2018 ; Murata et al. 2017 ), and its aquaculture is spreading in subtropical areas due to its superior growth performance compared with LG (Masuma 2018 ). Currently, the production of LGGG relies on cryopreserved GG sperm imported from Malaysia or Taiwan (Murata et al. 2017 ). However, this practice limits the sustainable production of LGGG, as it depends heavily on external genetic resources. Therefore, understanding the maturation of male GG in subtropical regions is important for the sustainable aquaculture of LGGG and other promising hybrids using GG as a sire. We previously showed that 17α-methyltestosterone (MT)-treated and -untreated males showed a seasonal spermiation response (SpR) year-round in the subtropical Amami Islands of Japan (Masuma and Aoki 2024 ). In the present study, we aimed to determine the maturation of GG males under the same subtropical conditions and, therefore, assessed their reproductive biology by non-lethally monitoring the SpR (Ashley 2007 ; Masuma et al. 2021; McGarvey et al. 2020 ) and sperm motility (%SM) for over 1 year. In addition, we conducted anatomical and/or histological analyses of gonads obtained from GGs reared in a subtropical environment. Materials and Methods Profiles and treatments of studied fish GG juveniles were obtained from a commercial breeding company (Long-Diann Marine Boi Tech. Co., Led.) (22° 22′ N, 120° 35′ E) in Pingtung, Taiwan, in early September 2015 (exact date of birth unknown). Three hundred juvenile GGs weighing a mean of 2.0 g ( n = 12) were transported from Taiwan to the Amami Station, Aquaculture Research Institute, Kindai University (28° 23′ N, 129° 22′ E), in the subtropical waters of the Amami Islands, Kagoshima Prefecture, Japan on November 6 2015. The GG juveniles were reared in 1-m 3 FRP tanks indoors until 8 August 2016, after which 279 surviving juveniles [244.7 ± 92.9 g BW (mean ± sample standard deviation, n = 128)] were transported to an off-shore sea cage (5.6 × 5.6 × 4 m) and fed a commercially formulated feed (Kue-taro; Higashimaru Co., Ltd., Tokyo, Japan) twice or thrice a week. The GGs were continuously raised in the subtropical waters of the Amami Islands, except during the larval and early juvenile periods. On 27 February 2019, all 134 individuals of the surviving GGs (3 years and 5 months old; mean of 6.0 kg BW) were implanted with passive internal transponder tags (PIT; BIO12A; Biomark, Boise, ID, USA) in the abdominal cavity using an implanter (MK7; Biomark). Simultaneously, their body length (BL) was measured to the nearest 0.1 cm and BW to the nearest 0.1 kg, inadvertently without confirmation of the SpR, which indicates a functional male. During this procedure, 31 GGs (7 fish weighing approximately 8 kg, 12 fish approximately 6 kg, and 12 fish approximately 4 kg) were randomly selected and implanted once with a cholesterol pellet (Lee et al. 1986 ) containing MT (2 mg/kg BW) (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) into the abdominal cavity. At the time of MT implantation, the water temperature (WT) at a 3-m depth was 21.5°C. SpR and %SM We examined the SpR in all GGs from MT-untreated and -treated groups approximately once a month between 4 April 2019 and 16 May 2020 by gently applying abdominal pressure (Beirão et al. 2019 ). At the same time, BL and BW were measured, except on 23 April 2019, due to inclement weather. Individuals exhibiting SpR were numbered in order of SpR confirmation, starting with 1 for the untreated group and M1 for the MT-treated group; %SM was also assessed whenever sufficient semen was obtained, except on 2 May and 6 August 2019, when %SM assessment was not performed. To activate the sperm, 1 µL of fresh semen was diluted 1,000-fold with seawater and vortexed. Thereafter, a 10-µL aliquot of the diluted mixture was placed on a glass slide (Teflon Printed Glass Slide; 21 wells with 4 mm diameter; Funakoshi, Tokyo, Japan) and sperm movement immediately after activation was video recorded for 15 s using a camera (MC120 HD; Leica Microsystems, Wetzlar, Germany) attached to a stereomicroscope (M125; Leica Microsystems). These procedures were repeated twice for each sperm sample to obtain an average motility rate. The %SM 10 s after activation was determined by counting the number of motile spermatozoa among 50 randomly selected spermatozoa using the free software Tracker 5.0.7 (Copyright © 2019 Douglas Brown https://physlets.org/tracker ). Gonadal development Gonadal development was examined anatomically and/or histologically in 10 MT-untreated individuals after their harvest for trial sales at the market at 3 years and 7 months old to 4 years old. These individuals were harvested on April 4 (three fish), July 5 (three fish), and September 3 (four fish) in 2019. The July and September samples included one fish, each with a SpR history, while the others were haphazardly selected. For each specimen, gonads were removed by laparotomy after measuring BL and BW. Gonadal weight (GW) was measured (± 0.01 g) to calculate the gonadosomatic index [GSI = GW (g) × 100/BW (g) %]. Each gonad from the April and September specimens was checked for sex using gross appearance. If a gonad appeared as a testis, a small piece of gonadal tissue was microscopically examined with a drop of seawater to identify motile sperm. If sperm was confirmed, the gonad was identified as a testis. If sperm and oocyte were observed concurrently, the gonad was identified as a transitional phase. Furthermore, histological analysis was performed on the July specimens. A small piece of gonad from each specimen was fixed in Bouin’s solution for 24 h before preservation in 70% ethanol. The preserved tissues were then embedded in paraffin, sectioned transversely at 4 µm, and stained with Mayer’s haematoxylin and 1% eosin Y. Subsequently, micrographs of histological gonad cross-sections captured using a digital slide scanner (NanoZoomer; HAMAMATSU Photonics, Shizuoka, Japan) were observed to determine the developmental stage according to Takano ( 1989 ) and Takahashi ( 1989 ). Statistical analysis Statistical analyses were performed using R version 4.0.5 (R Core Team 2021 ; R Foundation for Statistical Computing, Vienna, Austria). Welch’s two-sample t -test (Welch’s t-test) using the t -test function in the stats package (R Core Team 2021 ) was used to compare the differences in various parameters, such as BW and %SM, between MT-treated and -untreated groups. Fisher’s exact test for count data (Fisher’s test) with the fisher.test function in the same package was used to compare the mortality and SpR/non-SpR individuals between the two treatment groups and between size classes (8-, 6-, and 4-kg classes) of MT-treated GGs. For the %SM between months for sample sizes ≥ 3, the Kruskal–Wallis test (KW test), followed by pairwise comparisons using the Alexander–Govern test with Holm correction (PC test), was performed using the kw.test and paircomp functions in the one-way tests package (Dag et al. 2018 ). Statistical significance was set at p < 0.05. Values are given as mean ± standard deviation. Results The WT at a 3-m depth at the study site ranged from 19.9 to 28.6°C during the approximately 1-year study period, with a mean of 23.4°C. The WT increased from April, peaked around September, decreased gradually thereafter, and reached a minimum in February (Fig. 1). The initial BW and BL of the GGs of the MT-untreated group at the start of the study (4 April 2019) were 5.8 ± 1.4 kg and 54.0 ± 4.1 cm ( n = 103) at the start of the study and 8.0 ± 2.2 kg and 61.2 ± 5.1 cm ( n = 88) at the end of the study (16 May 2020), respectively. The BW and BL of the MT-treated group were 5.7 ± 1.7 kg and 53.3 ± 5.0 cm ( n = 31) and increased to 7.4 ± 2.4 kg and 59.8 ± 6.0 cm ( n = 30) at the end, respectively. No significant difference was detected in the BWs between the MT-untreated and -treated groups at the start of the experiment (Welch’s t-test, t = -0.477, df = 42.7, p = 0.636). On average, the MT-untreated GGs gained 2.1 ± 1.3 kg ( n = 88), while the MT-treated GGs gained 1.7 ± 1.1 kg ( n = 30) during the approximately 13-month study period. However, these values were not statistically different (Welch’s t-test, t = -1.27, df = 63.2, p = 0.208). The mortality rates were low in both groups, 4.9% (5/103 fish) in the MT-untreated GGs and 3.2% (2/31) in the MT-treated GGs, and MT treatment did not affect mortality (Fisher’s test, p = 1). SpR and %SM We confirmed the SpR in MT-untreated and -treated individuals on all examination days. In the MT-untreated group, 23 of the 103 individuals (22.3%) showed a SpR during the study period (Table 1). The MT-untreated GGs showing a SpR until the end of the study were numbered sequentially from 1 to 23. Only No. 1 showed a SpR at the first examination. The SpRs of Nos. 1, 2 and 4 were confirmed on all consecutive examination days for approximately 1 year after the first SpR, whereas the others individually showed sporadic or no SpR (Table 1). After excluding data for Nos. 6, 10, 12, and 13 due to harvesting or death, the proportion of SpR-positive, MT-untreated GGs increased from 1.0% (1/99 fish) to a peak of 13.0% (12/92) and then decreased to 8.2% (7/85) in January 2020. However, it increased again from the following March (10.6%, 9/85) (Fig. 2). The cumulative proportion of SpR-positive fish in the MT-untreated group gradually increased to 16.7% (16/96) by August 2019 and then to 26.1% (23/88) by May 2020 (Fig. 3). The BW of GGs at the onset of SpR in the MT-untreated group was 2.7–10.0 kg with an average of 6.9 kg; no apparent difference between them and the MT-untreated others without SpR were observed (Fig. 4). A SpR was confirmed in 29 31 (93.5%) MT-treated GGs by October 2019. These MT-treated GGs showing a SpR were numbered sequentially from M1 to M29, and the two remaining GGs without a SpR were assigned M30–M31 (Table 2). Of these, three fish (M1–M3) showed a SpR on the first examination day of the study on 4 April 2019, approximately 1 month after MT treatment. The SpR of M2, M3, M9, M10, M13, M15, M17, M18, and M24 were confirmed on all consecutive examination days after the first SpR, whereas the other individuals showed sporadic SpRs (Table 2). The proportion of SpR-positive, MT-treated GGs increased from 9.7% (3/31 fish) in April 2019 to a peak of 87.1% (27/31) in August 2019 and then decreased to 41.9% (13/31) in February 2020. However, it increased again from the following April 2020 (46.7%, 14/30) (Fig. 2). The seasonal fluctuation in the proportion of SpR-positive fish in the two groups year-round was generally similar (Fig. 2). The cumulative proportion of SpR-positive fish in the MT-treated group rapidly increased to 87.1% (27/31) by August 2019 and then to 93.5% (29/31) by October 2020 (Fig. 3), considerably higher than that of MT-untreated fish (26.1%, 23/88) (Fisher’s test, p < 0.001). Fish size at the time of MT treatment did not affect the onset of the first SpR: 11/12 fish in the 4 kg BW class, 12/12 in the 6 kg class, and 6/7 in the 8 kg class (Fisher’s test, p = 0.690). The mean %SMs for MT-untreated and -treated GGs were 49 ± 17% ( n = 23) and 56 ± 13% ( n = 29), respectively, with no significant difference between the groups (Fig. 5) (Welch’s t-test, t = 1.55, df = 38.8, p = 0.129). Statistical differences were detected across months in both groups (KW test, MT-untreated GG, χ 2 = 31.0, df = 11, p = 0.001; MT-treated GG, χ 2 = 23.7, df = 11, p = 0.014). Pairwise comparisons showed a significant difference between the following months: MT-untreated GGs, %SM in April 2020 was significantly lower than those in October and September 2019, February and May 2020 ( p < 0.05); MT-treated GGs, %SM in April 2020 was lower than that in October 2019 ( p = 0.030) (Fig. 6). %SM among the individuals did not significantly differ (KW test, MT-untreated GG, χ 2 = 19.4, df = 14, p = 0.151; MT-treated GG, χ 2 = 21.3, df = 23, p = 0.563). Gonadal developments The mean BW and GSI of the untreated GGs harvested in April, July, and September 2019 were 4.4 ± 0.4 kg and 0.29 ± 0.05% ( n = 3), 7.6 ± 1.1 kg and 0.34 ± 0.30% ( n = 3), and 5.7 ± 1.5 kg and 0.36 ± 0.09% ( n = 4), respectively. We microscopically examined %SM in portions of the gonads after dilution with seawater. Motile and immotile spermatozoa were observed in the gonads of two out of three fish examined in April (3 years and 7 months old), all three fish in July, and two out of four fish in September (4 years old). Before harvesting, two fish, No. 6 in July and No. 12 in September, showed three and one SpR event, respectively (Table 1). All seven individuals were microscopically identified as male or in the process of changing sex from female to male. Three gonads from July (3 years and 10 months old) were histologically examined (Fig. 7). The gonad shown in Fig. 7A (GSI 0.18%) comprised approximately one-third ovary and two-thirds testis, suggesting sex transition. Perinucleolar and oil droplet oocytes occupied the ovary, while testicular cysts contained spermatozoa and spermatocytes. Several spermatogonia were observed in the interstitial spaces between the cysts (Fig. 7a). Although several atretic oocytes remained in the periphery, a large part of the gonad, as shown in Fig. 7b (GSI 0.15%), was occupied by the testis with cysts containing several spermatozoa, indicating further progress in sex transition than that in the gonad in Fig. 7A (Fig. 7b). No oocytes were detected in the gonadal sections of No. 6 (Fig. 7C, GSI 0.69%), which displayed SpR from May to July (Table 2); therefore, this individual was considered to have changed sex to male (Fig. 7c). Discussion In this study, we evaluated the maturation of male GGs raised in a subtropical region of Japan by monitoring SpR, %SM, and gonadal development year-round. To the best of our knowledge, this is one of the few studies monitoring the SpR of groupers over 1 year. We demonstrated that male GGs could produce sperms year-round under the natural environmental conditions of the Amami Islands in the subtropics (WT 19.9–28.6°C) (Fig. 1 ). We previously reported that the SpR in LGGG, a hybrid between dam LG and sire GG, occurred year-round in the same area (Masuma and Aoki 2024 ); this suggests that the spermatogenesis and spermiation of GGs may also be well adapted to the subtropical conditions of the Amami Islands. Therefore, continuous SpR in LGGG is inherited from GG rather than LG, whose SpR was not observed between October and November and appears seasonal (Masuma et al. 2022 ). The duration of spermatogenesis in fish is affected by WT (Postingel Quirino et al. 2021 ). In the present study, the SpR activity of MT-untreated and -treated GGs tended to be low in the low WT January and February period (< 21°C). However, it was individually dependent (Fig. 2 , Tables 1 and 2 ). However, Schulz et al. ( 2010 ) indicated that some tropical fish do not display apparent seasonal variations in spermatogenic activity. The decrease in the SpR-positive rate observed in the current study may be specific to tropical fish reared in subtropical waters. Three MT-treated GGs (Nos M1–M3) exhibited a SpR at the first examination on 4 April 2019, despite it being only approximately 1 month after commencing MT treatment. Similarly, an MT-untreated GG (No. 1) displayed a SpR on the same day. Passini et al. ( 2018 ) suggested that MT treatment stimulates testicular development and growth, accelerating spermatogenesis in the common snook Centropomus undecimalis . In the present study, the proportion of SpR-positive GGs in the MT-treated group (93.5%) was significantly higher than in the untreated group (26.1%); this corroborates Passini et al.’s ( 2018 ) suggestion and indicates that MT treatment accelerated testicular development, leading to the early onset of the first SpR (Fig. 2 ). Twenty-three out of 111 MT-untreated GGs (≤ 10.0 kg BW) exhibited a SpR during this study, while a SpR was not observed in other individuals. In our previous study, MT-untreated LGGGs (4–5 years old; 6.0–13.7 kg, n = 9) without a SpR were histologically identified as female by having mature or immature ovaries (Masuma and Aoki 2024 ). Similarly, the gonads of the transitional stage were observed in GG in the present study. Liao et al. ( 2001 ) reported that the initial BW of GG spawners range from 20 to 40 kg. Similarly, Palma et al. ( 2019 ) reported that the onset of female sexual maturity occurs at 23.5 ± 1.5 kg in the Philippines and 33.5 ± 2.5 kg in Vietnam. Therefore, the occurrence of a SpR in the current study, presumably the first in the lifetime of the GGs, indicates a natural sex change from immature females to functional males. The mean BW of MT-untreated GGs showing a SpR was 6.9 kg (range 2.7–10.0 kg). Palma et al. ( 2019 ) reported that the maturity size of wild-caught GG males in the Philippines and Vietnam was 17.1 ± 2.1 kg and 34.3 ± 0.9 kg, respectively. Male GGs in the Philippines were smaller than females, but those in Vietnam exhibited overlapping sizes. In the present study, most MT-untreated males at their first SpR were smaller than wild-caught GGs. These results suggest that size does not play an important role in sex reversal in GGs. However, our results are based on hatchery-reared fish, and further study is needed to clarify if the same is true for wild GGs. Generally, testicular development during maturation is largely influenced by WT (Pino Martinez et al. 2022 ; Postingel Quirino et al. 2021 ). In the current study, the promotion of the first SpR (sex reversal) in MT-untreated GGs from April to early October could be attributed to increased WT (Fig. 1 ). The GG spawning season in tropical southern Taiwan is between May and October, with spawning peaks between August and September (Ho et al. 1997 ; Liao et al. 2001 ). Furthermore, Bright et al. ( 2016 ) reported that the natural spawning events of captive GGs occurred on July 22–29, September 14–19, and October 14–21, 2012, on a lunar cycle (six, six, and eight nights per batch, respectively) in tropical Cairns, Queensland, Australia. The sex reversal of captive GGs in this 1-year study occurred even before and/or during the spawning season. Such a sex change pattern may be rare in protogynous species where sex changes normally occur at the end of the breeding season (Masuma et al. 2022 ; Muncaster et al. 2013 ; Peng et al. 2020 ). Further studies are needed to assess the precise timing of sex change in GGs. The spermatozoa of GGs reared in subtropical waters exhibited high motility, with mean %SMs of 56% in the MT-treated and 49% in the untreated groups (Fig. 5 ). These values are similar to those of MT-treated and untreated LGGGs reared in the similar environment of the Amami Islands (46 and 58%, respectively) (Masuma and Aoki 2024 ). In both GGs and LGGGs, MT treatment did not affect %SM. Similarly, Viveiros et al. ( 2001 ) found that MT treatment did not affect the quality or quantity of semen of African catfish Clarias gariepinus . Conversely, in our study, statistical differences in %SM were detected between October and April in both groups (Fig. 6 ). Pairwise comparisons suggested that the %SM in April tended to be lower than that in the other months, suggesting potential seasonal fluctuation in sperm activity. However, our results do not allow us to infer the underlying cause of this phenomenon. Some environmental factors, such as WT or moon phase, may have affected sperm activity (Bright et al. 2016 ; Palma et al. 2019 ). Further research is needed to clarify this. Examining the gonads of randomly selected harvested fish identified individuals undergoing sex change with active motile spermatozoa in the testicular part. The gonads of two individuals histologically analysed in July were at the transitional stage with co-occurring immature oocytes and spermatozoa-containing cysts. Our findings demonstrate that hatchery-reared GGs change sex directly from immature females to males, and this sex transition can occur even during the spawning season in the tropical region between May and October (Ho et al. 1997 ; Liao et al. 2001 ). Palma et al. ( 2019 ) suggested that GGs are diandric protogynous hermaphrodites (Fennessy and Sadovy 2002 ), defined by males developing either from mature females or immature females. Our observations support this notion. Conclusions We demonstrated that regardless of MT treatment, male GGs can produce sperm year-round in the subtropic environment of the Amami Islands, Japan. MT treatment at a dose of 2 mg/kg BW effectively enhanced functional sex reversal and sperm production, irrespective of fish size at the treatment time. Monitoring the SpR for approximately 1 year with additional assessment of %SM and gonadal histology showed that GG males can mature in subtropical waters and maintain motile spermatozoa consistently throughout the year. Histological studies suggested that hatchery-reared GGs change sex directly from immature females to males, even during the spawning season. Our results are critical for improving any hybridisation using GG males. This study was the first to report on the reproductive ecology of male GGs in subtropical waters. Declarations Author contribution Shukei Masuma conceived and designed the study. Shukei Masuma and Ryuichiro Aoki conducted the investigation and formal analyses. Shukei Masuma wrote the first draft of the manuscript, and Aoki commented on it. Funding This study was supported by the Kindai University Fund for Domestic Research. Data availability All relevant data supporting the findings of this study are within the manuscript, if any, and are available from the corresponding author upon reasonable request. Competing interests The authors declare no competing interests. Ethics approval The study was approved by the Animal Ethics Committee of the Aquaculture Research Institute of Kindai University and was conducted according to the Guidelines for Animal Experimentation at the Aquaculture Research Institute of Kindai University. Acknowledgements The authors would like to thank Mr. Hiroki Yagi, Yoshiki Katsuda, and the staff of the Amami Station of the Aquaculture Technology and Production Center, Kindai University, who kindly assisted with monitoring. We are also grateful to Dr. Hiromi Ohta, Professor Emeritus at Kindai University, and Dr. Sho Shirakashi, Associate Professor at Kindai University, for their constructive advice. 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(in Chinese). https://en.tfrin.gov.tw/News_Content.aspx?n=349&s=226159 James CM, Al-Thobaiti SA, Rasem BM, Carlos MH (1999) Potential of grouper hybrid ( Epinephelus fuscoguttatus × E. polyphekadion ) for aquaculture. Naga, the ICLARM quarterly 22:19–23. http://worldfish.catalog.cgiar.org/naga/na_2187.pdf Koh ICC, Shaleh SRM, Akazawa N, Oota Y, Senoo S (2010) Egg and larval development of a new hybrid orange-spotted grouper Epinephelus coioides × giant grouper E. lanceolatus . Aquac Sci 58:1–10. https://doi.org/10.11233/aquaculturesci.58.1 Koh ICC, Shaleh SRM, Senoo S (2008) Egg and larval development of a new hybrid orange-spotted grouper Epinephelus coioides × tiger grouper E. fuscoguttatus . Aquac Sci 56:441–451. https://doi.org/10.11233/aquaculturesci.56.441 Lee C-S, Tamaru CS, Kelley CD (1986) Technique for making chronic-release LHRH-a and 17α-methyltestosterone pellets for intramuscular implantation in fishes. Aquaculture 59:161–168. https://doi.org/10.1016/0044-8486(86)90128-6 Liao IC, Su HM, Chang EY (2001) Techniques in finfish larviculture in Taiwan. Aquaculture 200:1–31. https://doi.org/10.1016/S0044-8486(01)00692-5 Ma KY, Craig MT (2018) An inconvenient monophyly: An update on the taxonomy of the groupers (Epinephelidae). Copeia 106:443–456. https://doi.org/10.1643/CI-18-055 Masuma S (2018) New fish for marine aquaculture by hybridization. Agricultural. Biotechnology. Hokuryukan, Tokyo 2:20–23. (in Japanese) Masuma S, Aoki R (2024) Maturation in a hybrid grouper, Kue-Tama, a cross between female longtooth grouper, Epinephelus bruneus , and male giant grouper, E. lanceolatus . Aquacult Int 32:3481–3498. https://doi.org/10.1007/s10499-023-01333-y Masuma S, Kusunoki Y, Aoki R (2022) Seasonal changes of plasma sex steroids in captive longtooth grouper Epinephelus bruneus (Bloch) in subtropical regions. Aquac Res 53:1268–1275. https://doi.org/10.1111/are.15660 McGarvey LM, Halvorson LJ, Ilgen JE, Guy CS, McLellan JG, Webb MAH (2020) Gametogenesis and assessment of nonlethal tools to assign sex and reproductive condition in burbot. Trans Am Fish Soc 149:225–240. https://doi.org/10.1002/tafs.10226 Muncaster S, Norberg B, Andersson E (2013) Natural sex change in the temperate protogynous Ballan wrasse Labrus bergylta . J Fish Biol 82:1858–1870. https://doi.org/10.1111/jfb.12113 Murata O, Itakura S, Yamamoto S, Hattori N, Kurata M, Ohta H, Masuma S (2017) Interspecific hybridization of longtooth grouper Epinephelus bruneus × giant grouper E. lanceolatus and growth of hybrid larvae and juveniles. Aquac Sci 65:93–95. (in Japanese with English abstract). https://doi.org/10.11233/AQUACULTURESCI.65.93 Palma P, Takemura A, Libunao GX, Superio J, de Jesus-Ayson EG, Ayson F, Nocillado J, Dennis L, Chan J, Thai TQ, Ninh NH, Elizur A (2019) Reproductive development of the threatened giant grouper Epinephelus lanceolatus . Aquaculture 509:1–7. https://doi.org/10.1016/j.aquaculture.2019.05.001 Passini G, Sterzelecki FC, de Carvalho CVA, Baloi MF, Naide V, Cerqueira VR (2018) 17α-methyltestosterone implants accelerate spermatogenesis in common snook, Centropomus undecimalis , during first sexual maturation. Theriogenology 106:134–140. https://doi.org/10.1016/j.theriogenology.2017.10.015 Peng C, Wang Q, Shi H, Chen J, Li S, Zhao H, Lin H, Yang J, Zhang Y (2020) Natural sex change in mature protogynous orange-spotted grouper (Epinephelus coioides): Gonadal restructuring, sex hormone shifts and gene profiles. J Fish Biol 97:785–793. https://doi.org/10.1111/jfb.14434 Pierre S, Gaillard S, Prévot-D’alvise N, Aubert J, Rostaing-Capaillon O, Leung-Tack D, Grillasca J-P (2008) Grouper aquaculture: Asian success and Mediterranean trials. Aquatic Conservation 18:297–308. https://doi.org/10.1002/aqc.840 Pino Martinez EP, Braanaas MF, Balseiro P, Kraugerud M, Pedrosa C, Imsland AKD, Handeland SO (2022) Constant high temperature promotes early changes in testis development associated with sexual maturation in male Atlantic salmon ( Salmo salar L.) post-smolts. Fishes 7:341. https://doi.org/10.3390/fishes7060341 Postingel Quirino P, da Silva Rodrigues M, da Silva Cabral EM, de Siqueira-Silva DH, Mori RH, Butzge AJ, Nóbrega RH, Ninhaus-Silveira A, Veríssimo-Silveira R (2021) The influence of increased water temperature on the duration of spermatogenesis in a Neotropical fish, Astyanax altiparanae (Characiformes, Characidae). Fish Physiol Biochem 47:747–755. https://doi.org/10.1007/s10695-020-00869-7 R Core Team (2021). R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria. Retrieved from URL. https://www.R-project.org/ Schulz RW, de França LR, Lareyre J-J, Le Gac F, Chiarini-Garcia H, Nobrega RH, Miura T (2010) Spermatogenesis in fish. Gen Comp Endocrinol 165:390–411. https://doi:10.1016/j.ygcen.2009.02.013. https://doi.org/10.1016/j.ygcen.2009.02.013 Takahashi Y (1989) Testicular structure and gametogenesis. In: Takashima F, Hanyu I (eds), Monographs on Aquaculture Science volume 4: Reproductive biology of fish and shellfish. Midori-shobo, Tokyo ISBN 4-89531-434-0, p 33–64. (in Japanese) Takano K (1989) Ovarian structure and gametogenesis. In: Takashima F, Hanyu I (eds), Monographs on Aquaculture Science volume 4: Reproductive biology of fish and shellfish. Midori-shobo, Tokyo ISBN 4-89531-434-0, p 3–34. (in Japanese) Viveiros ATM, Eding EH, Komen J (2001) Effects of 17α-methyltestosterone on seminal vesicle development and semen release response in the African catfish, Clarias gariepinus . Reproduction 122:817–827. https://doi.org/10.1530/rep.0.1220817 Tables Tables are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table12MaturationmalegiantgrouperMasumaAoki.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5996034","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":414317121,"identity":"beb5e4a8-be83-4bfe-b4aa-72767d983ef9","order_by":0,"name":"Shukei Masuma","email":"","orcid":"","institution":"Kindai University","correspondingAuthor":false,"prefix":"","firstName":"Shukei","middleName":"","lastName":"Masuma","suffix":""},{"id":414317122,"identity":"e665e2fb-1a68-4ac2-8b2e-104c74b4d086","order_by":1,"name":"Ryuichiro Aoki","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABE0lEQVRIiWNgGAWjYJCCA4wNEmAGY4OBjQEbA4MBQwJEgigtacRpASqF04cNGEBa8AHzGTmGB37usMjnZ2B++HBGwXljPunmjR8eMNjJMzCexWqNzI0cg4O9ZyQsZzawGRtuMLhtxiZzrFgigSHZsIHhXAI2LRISOQaHGdskDAwOMJhJPjC4bcMGFAFqYQYqP4PVhUha2L8BtZwDaTH+kcBQT4wWHjPJDQYHzIBazIC2HMathedZwcFeoBbJZp5iwxkGycZsEmllFgkGxw3bcPmFPXnzh59tdQb87O0bH/b8sTOcPyN5880fFdXy/BLYQ4xBAGYSM4ow0ElsEmew6mDgx24SWKoHp9QoGAWjYBSMKAAA0lNbgO15g9QAAAAASUVORK5CYII=","orcid":"","institution":"Kindai University","correspondingAuthor":true,"prefix":"","firstName":"Ryuichiro","middleName":"","lastName":"Aoki","suffix":""}],"badges":[],"createdAt":"2025-02-10 06:08:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5996034/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5996034/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":76138008,"identity":"4cdcee9e-f000-4fab-b535-a2a3136407e2","added_by":"auto","created_at":"2025-02-12 16:48:12","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":326832,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in seawater temperature during the study period at the sea cage location at the study site\u003c/p\u003e","description":"","filename":"Fig.1MasumaandAoki.png","url":"https://assets-eu.researchsquare.com/files/rs-5996034/v1/07bf242ca911cb6ddfdf6f70.png"},{"id":76138005,"identity":"695d2d42-1c80-4240-85fb-21c887fb96dc","added_by":"auto","created_at":"2025-02-12 16:48:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":432401,"visible":true,"origin":"","legend":"\u003cp\u003eSeasonal changes in the proportions of individuals showing a spermiation response (SpR) in methyltestosterone (MT)-untreated (open circles) and -treated (black circles) giant grouper \u003cem\u003eEpinephelus lanceolatus\u003c/em\u003e (GG)\u003c/p\u003e","description":"","filename":"Fig.2MasumaandAoki.png","url":"https://assets-eu.researchsquare.com/files/rs-5996034/v1/81fe11447c1aa7710dca07a0.png"},{"id":76138003,"identity":"36e22954-4c62-4a6e-9e35-1ae6deaa9fbf","added_by":"auto","created_at":"2025-02-12 16:48:12","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":425132,"visible":true,"origin":"","legend":"\u003cp\u003eCumulative proportions of individuals showing a spermiation response (SpR) in methyltestosterone (MT)-untreated (open circles) and -treated (black circles) giant grouper \u003cem\u003eEpinephelus lanceolatus \u003c/em\u003e(GG)\u003c/p\u003e","description":"","filename":"Fig.3MasumaandAoki.png","url":"https://assets-eu.researchsquare.com/files/rs-5996034/v1/f6492a448354f7357ddb7195.png"},{"id":76138931,"identity":"8ad745d4-401a-4a3b-8211-ade1eb81a145","added_by":"auto","created_at":"2025-02-12 16:56:13","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":318957,"visible":true,"origin":"","legend":"\u003cp\u003eBody weight (closed circles, ⬤) at the first spermiation response in methyltestosterone-untreated giant grouper \u003cem\u003eEpinephelus lanceolatus \u003c/em\u003e(GG). The boxes represent the 25 and 75% quartiles; horizontal lines within boxes represent the median; cross marks represent the average; and vertical broken lines show the range of all methyltestosterone-untreated GGs\u003c/p\u003e","description":"","filename":"Fig.4MasumaandAoki.png","url":"https://assets-eu.researchsquare.com/files/rs-5996034/v1/68267f4596b9caba92253676.png"},{"id":76138011,"identity":"8c52da1b-3c43-41b1-a6e7-3b98bcad1ecf","added_by":"auto","created_at":"2025-02-12 16:48:12","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":126030,"visible":true,"origin":"","legend":"\u003cp\u003eSperm motility of methyltestosterone (MT)-untreated and -treated giant grouper\u003cem\u003eEpinephelus lanceolatus\u003c/em\u003e (GG). The boxes represent the 25 and 75% quartiles; cross marks within boxes indicate the average; horizontal lines within boxes represent the median; and vertical broken lines show the range\u003c/p\u003e","description":"","filename":"Fig.5MasumaandAoki.png","url":"https://assets-eu.researchsquare.com/files/rs-5996034/v1/55ee0e0727fc30101e18f93a.png"},{"id":76138924,"identity":"d5f81221-a5fb-4f34-9790-bc20390def12","added_by":"auto","created_at":"2025-02-12 16:56:13","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":487132,"visible":true,"origin":"","legend":"\u003cp\u003eSeasonal changes in the sperm motility of methyltestosterone (MT)-untreated (lower figure) and -treated (upper figure) giant grouper \u003cem\u003eEpinephelus lanceolatus\u003c/em\u003e. The boxes represent the 25 and 75% quartiles; horizontal lines represent the median; cross marks within boxes represent the average; and vertical lines show the range. Different superscript letters indicate significant differences (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05) between months\u003c/p\u003e","description":"","filename":"Fig.6MasumaandAoki.png","url":"https://assets-eu.researchsquare.com/files/rs-5996034/v1/668cc42b1b97a862e78b5f8d.png"},{"id":76138935,"identity":"a037b3a4-a13b-4d4e-8bba-9fe7799185f6","added_by":"auto","created_at":"2025-02-12 16:56:14","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":7847336,"visible":true,"origin":"","legend":"\u003cp\u003ePhotomicrographs of three gonadal sections of \u003cem\u003eEpinephelus lanceolatus\u003c/em\u003e harvested in July 2019. Images with uppercase letters (A, B, C) show gonadal cross-sections; lowercase-lettered panels (a, b, c) enlarge the rectangle within the left side images. AO, atretic oocyte; PN, perinucleolar stage oocyte; SG, spermatogonia; SC, spermatocytes; ST, spermatids; SZ, spermatozoa. Scale bars of left side = 5 mm; right side = 250 μm\u003c/p\u003e","description":"","filename":"Fig.7MasumaandAoki.png","url":"https://assets-eu.researchsquare.com/files/rs-5996034/v1/2e9c6bcc0717df3e8115b904.png"},{"id":81593054,"identity":"13bc9771-49f5-4451-83d6-0f64bb346886","added_by":"auto","created_at":"2025-04-29 01:31:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10785059,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5996034/v1/2646d8db-d287-44ea-b59d-19341ac32816.pdf"},{"id":76138013,"identity":"a9ee0048-f425-46e5-b07a-c43ef5c7773c","added_by":"auto","created_at":"2025-02-12 16:48:13","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":40177,"visible":true,"origin":"","legend":"","description":"","filename":"Table12MaturationmalegiantgrouperMasumaAoki.docx","url":"https://assets-eu.researchsquare.com/files/rs-5996034/v1/87844297bd702ab4fb43cc70.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Maturation of captive male giant grouper Epinephelus lanceolatus in subtropical waters in Japan","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEpinepheline grouper species are widespread in temperate and tropical coastal waters (Heemstra and Randall \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Ma and Craig \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). These species exhibit protogynous hermaphroditism, i.e., they change sex from female to male during their lifetime (Sadovy and Liu 2008). Among the epinepheline groupers species, \u003cem\u003eEpinephelus\u003c/em\u003e spp. such as the orange-spotted grouper \u003cem\u003eEpinephelus coioides\u003c/em\u003e, Malabar grouper \u003cem\u003eE. malabaricus\u003c/em\u003e, Hong Kong grouper \u003cem\u003eE. akaara\u003c/em\u003e, brown-marbled grouper \u003cem\u003eE. fuscoguttatus\u003c/em\u003e, giant grouper \u003cem\u003eE. lanceolatus\u003c/em\u003e (GG), and nine other species are highly valuable aquaculture species in Southeast and East Asia because of their good taste, cooking versatility, and high demand (Pierre et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAlong with aquaculture practices involving purebred groupers, \u003cem\u003eEpinephelus\u003c/em\u003e species hybridisation has been successfully conducted to produce new groupers with desirable aquaculture traits (Bartley et al. 2001). Examples of such hybridisation include \u003cem\u003eE. coioides\u003c/em\u003e \u0026times; \u003cem\u003eE. fuscoguttatus\u003c/em\u003e (Koh et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), E. \u003cem\u003ecoioides\u003c/em\u003e \u0026times; \u003cem\u003eE. lanceolatus\u003c/em\u003e (Koh et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), E. \u003cem\u003efuscoguttatus\u003c/em\u003e \u0026times; \u003cem\u003eE. lanceolatus\u003c/em\u003e (Ch\u0026rsquo;ng and Senoo 2008), \u003cem\u003eE. fuscoguttatus\u003c/em\u003e \u0026times; camouflage grouper \u003cem\u003eE. polyphekadion\u003c/em\u003e (James et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1999\u003c/span\u003e), dusky grouper \u003cem\u003eE. marginatus\u003c/em\u003e \u0026times; white grouper \u003cem\u003eE. aeneus\u003c/em\u003e (Glamuzina et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1999\u003c/span\u003e), longtooth grouper \u003cem\u003eE. bruneus\u003c/em\u003e (LG) \u0026times; \u003cem\u003eE. lanceolatus\u003c/em\u003e (Murata et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2017\u003c/span\u003e)d \u003cem\u003emoara\u003c/em\u003e (synonym \u003cem\u003eE. bruneus\u003c/em\u003e) \u0026times; \u003cem\u003eE. lanceolatus\u003c/em\u003e (Chen et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). GG is commonly used as the sire in these cases due to its fast-growing trait (Liao et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2001\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe GG is one of the largest epinepheline species, reaching 400 kg body weight (BW) in tropical and subtropical waters (Heemstra and Randall \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). The GG is an important species in recreational and commercial fisheries, particularly in the live fish trade based in Hong Kong. However, it has been largely extirpated in heavily fished areas (Fennessy et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). To meet the growing demand for GG, larviculture technology was initiated in southern Taiwan in 1996 (Ho et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1997\u003c/span\u003e) and subsequently adopted by Asia-Pacific countries/regions, including China, Indonesia, Thailand, Vietnam, Malaysia, Hawaii, and Australia (Palma et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Understanding the reproductive biology of GG is critical for improving captive breeding techniques (Palma et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). However, information on the maturation and reproduction of GG is primarily limited to tropical regions, and it remains uncertain whether GG can mature in subtropical regions.\u003c/p\u003e \u003cp\u003eIn Japan, the hybrid grouper Kue-tama (LGGG) \u003cem\u003eE. bruneus\u003c/em\u003e \u0026times; \u003cem\u003eE. lanceolatus\u003c/em\u003e was recently established (Masuma \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Murata et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), and its aquaculture is spreading in subtropical areas due to its superior growth performance compared with LG (Masuma \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Currently, the production of LGGG relies on cryopreserved GG sperm imported from Malaysia or Taiwan (Murata et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). However, this practice limits the sustainable production of LGGG, as it depends heavily on external genetic resources. Therefore, understanding the maturation of male GG in subtropical regions is important for the sustainable aquaculture of LGGG and other promising hybrids using GG as a sire. We previously showed that 17α-methyltestosterone (MT)-treated and -untreated males showed a seasonal spermiation response (SpR) year-round in the subtropical Amami Islands of Japan (Masuma and Aoki \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the present study, we aimed to determine the maturation of GG males under the same subtropical conditions and, therefore, assessed their reproductive biology by non-lethally monitoring the SpR (Ashley \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Masuma et al. 2021; McGarvey et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and sperm motility (%SM) for over 1 year. In addition, we conducted anatomical and/or histological analyses of gonads obtained from GGs reared in a subtropical environment.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eProfiles and treatments of studied fish\u003c/h2\u003e \u003cp\u003eGG juveniles were obtained from a commercial breeding company (Long-Diann Marine Boi Tech. Co., Led.) (22\u0026deg; 22\u0026prime; N, 120\u0026deg; 35\u0026prime; E) in Pingtung, Taiwan, in early September 2015 (exact date of birth unknown). Three hundred juvenile GGs weighing a mean of 2.0 g (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;12) were transported from Taiwan to the Amami Station, Aquaculture Research Institute, Kindai University (28\u0026deg; 23\u0026prime; N, 129\u0026deg; 22\u0026prime; E), in the subtropical waters of the Amami Islands, Kagoshima Prefecture, Japan on November 6 2015. The GG juveniles were reared in 1-m\u003csup\u003e3\u003c/sup\u003e FRP tanks indoors until 8 August 2016, after which 279 surviving juveniles [244.7\u0026thinsp;\u0026plusmn;\u0026thinsp;92.9 g BW (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;sample standard deviation, \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;128)] were transported to an off-shore sea cage (5.6 \u0026times; 5.6 \u0026times; 4 m) and fed a commercially formulated feed (Kue-taro; Higashimaru Co., Ltd., Tokyo, Japan) twice or thrice a week. The GGs were continuously raised in the subtropical waters of the Amami Islands, except during the larval and early juvenile periods.\u003c/p\u003e \u003cp\u003eOn 27 February 2019, all 134 individuals of the surviving GGs (3 years and 5 months old; mean of 6.0 kg BW) were implanted with passive internal transponder tags (PIT; BIO12A; Biomark, Boise, ID, USA) in the abdominal cavity using an implanter (MK7; Biomark). Simultaneously, their body length (BL) was measured to the nearest 0.1 cm and BW to the nearest 0.1 kg, inadvertently without confirmation of the SpR, which indicates a functional male. During this procedure, 31 GGs (7 fish weighing approximately 8 kg, 12 fish approximately 6 kg, and 12 fish approximately 4 kg) were randomly selected and implanted once with a cholesterol pellet (Lee et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1986\u003c/span\u003e) containing MT (2 mg/kg BW) (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) into the abdominal cavity. At the time of MT implantation, the water temperature (WT) at a 3-m depth was 21.5\u0026deg;C.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSpR and %SM\u003c/h3\u003e\n\u003cp\u003eWe examined the SpR in all GGs from MT-untreated and -treated groups approximately once a month between 4 April 2019 and 16 May 2020 by gently applying abdominal pressure (Beir\u0026atilde;o et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). At the same time, BL and BW were measured, except on 23 April 2019, due to inclement weather. Individuals exhibiting SpR were numbered in order of SpR confirmation, starting with 1 for the untreated group and M1 for the MT-treated group; %SM was also assessed whenever sufficient semen was obtained, except on 2 May and 6 August 2019, when %SM assessment was not performed. To activate the sperm, 1 \u0026micro;L of fresh semen was diluted 1,000-fold with seawater and vortexed. Thereafter, a 10-\u0026micro;L aliquot of the diluted mixture was placed on a glass slide (Teflon Printed Glass Slide; 21 wells with 4 mm diameter; Funakoshi, Tokyo, Japan) and sperm movement immediately after activation was video recorded for 15 s using a camera (MC120 HD; Leica Microsystems, Wetzlar, Germany) attached to a stereomicroscope (M125; Leica Microsystems). These procedures were repeated twice for each sperm sample to obtain an average motility rate. The %SM 10 s after activation was determined by counting the number of motile spermatozoa among 50 randomly selected spermatozoa using the free software Tracker 5.0.7 (Copyright \u0026copy; 2019 Douglas Brown \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://physlets.org/tracker\u003c/span\u003e\u003cspan address=\"https://physlets.org/tracker\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eGonadal development\u003c/h3\u003e\n\u003cp\u003eGonadal development was examined anatomically and/or histologically in 10 MT-untreated individuals after their harvest for trial sales at the market at 3 years and 7 months old to 4 years old. These individuals were harvested on April 4 (three fish), July 5 (three fish), and September 3 (four fish) in 2019. The July and September samples included one fish, each with a SpR history, while the others were haphazardly selected. For each specimen, gonads were removed by laparotomy after measuring BL and BW. Gonadal weight (GW) was measured (\u0026plusmn;\u0026thinsp;0.01 g) to calculate the gonadosomatic index [GSI\u0026thinsp;=\u0026thinsp;GW (g) \u0026times; 100/BW (g) %]. Each gonad from the April and September specimens was checked for sex using gross appearance. If a gonad appeared as a testis, a small piece of gonadal tissue was microscopically examined with a drop of seawater to identify motile sperm. If sperm was confirmed, the gonad was identified as a testis. If sperm and oocyte were observed concurrently, the gonad was identified as a transitional phase.\u003c/p\u003e \u003cp\u003eFurthermore, histological analysis was performed on the July specimens. A small piece of gonad from each specimen was fixed in Bouin\u0026rsquo;s solution for 24 h before preservation in 70% ethanol. The preserved tissues were then embedded in paraffin, sectioned transversely at 4 \u0026micro;m, and stained with Mayer\u0026rsquo;s haematoxylin and 1% eosin Y. Subsequently, micrographs of histological gonad cross-sections captured using a digital slide scanner (NanoZoomer; HAMAMATSU Photonics, Shizuoka, Japan) were observed to determine the developmental stage according to Takano (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1989\u003c/span\u003e) and Takahashi (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1989\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using R version 4.0.5 (R Core Team \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; R Foundation for Statistical Computing, Vienna, Austria). Welch\u0026rsquo;s two-sample \u003cem\u003et\u003c/em\u003e-test (Welch\u0026rsquo;s t-test) using the \u003cem\u003et\u003c/em\u003e-test function in the stats package (R Core Team \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) was used to compare the differences in various parameters, such as BW and %SM, between MT-treated and -untreated groups. Fisher\u0026rsquo;s exact test for count data (Fisher\u0026rsquo;s test) with the fisher.test function in the same package was used to compare the mortality and SpR/non-SpR individuals between the two treatment groups and between size classes (8-, 6-, and 4-kg classes) of MT-treated GGs. For the %SM between months for sample sizes\u0026thinsp;\u0026ge;\u0026thinsp;3, the Kruskal\u0026ndash;Wallis test (KW test), followed by pairwise comparisons using the Alexander\u0026ndash;Govern test with Holm correction (PC test), was performed using the kw.test and paircomp functions in the one-way tests package (Dag et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Statistical significance was set at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Values are given as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eThe WT at a 3-m depth at the study site ranged from 19.9 to 28.6°C during the approximately 1-year study period, with a mean of 23.4°C. The WT increased from April, peaked around September, decreased gradually thereafter, and reached a minimum in February (Fig.\u0026nbsp;1).\u003c/p\u003e\n\u003cp\u003eThe initial BW and BL of the GGs of the MT-untreated group at the start of the study (4 April 2019) were 5.8 ± 1.4 kg and 54.0 ± 4.1 cm (\u003cem\u003en\u003c/em\u003e = 103) at the start of the study and 8.0 ± 2.2 kg and 61.2 ± 5.1 cm (\u003cem\u003en\u003c/em\u003e = 88) at the end of the study (16 May 2020), respectively. The BW and BL of the MT-treated group were 5.7 ± 1.7 kg and 53.3 ± 5.0 cm (\u003cem\u003en\u003c/em\u003e = 31) and increased to 7.4 ± 2.4 kg and 59.8 ± 6.0 cm (\u003cem\u003en\u003c/em\u003e = 30) at the end, respectively. No significant difference was detected in the BWs between the MT-untreated and -treated groups at the start of the experiment (Welch’s t-test, \u003cem\u003et\u003c/em\u003e = -0.477, \u003cem\u003edf\u003c/em\u003e = 42.7, \u003cem\u003ep\u003c/em\u003e = 0.636). On average, the MT-untreated GGs gained 2.1 ± 1.3 kg (\u003cem\u003en\u003c/em\u003e = 88), while the MT-treated GGs gained 1.7 ± 1.1 kg (\u003cem\u003en\u003c/em\u003e = 30) during the approximately 13-month study period. However, these values were not statistically different (Welch’s t-test, \u003cem\u003et\u003c/em\u003e = -1.27, \u003cem\u003edf\u003c/em\u003e = 63.2, \u003cem\u003ep\u003c/em\u003e = 0.208). The mortality rates were low in both groups, 4.9% (5/103 fish) in the MT-untreated GGs and 3.2% (2/31) in the MT-treated GGs, and MT treatment did not affect mortality (Fisher’s test, \u003cem\u003ep\u003c/em\u003e = 1).\u003c/p\u003e\n\u003cdiv id=\"Sec8\"\u003e\n \u003ch2\u003eSpR and %SM\u003c/h2\u003e\n \u003cp\u003eWe confirmed the SpR in MT-untreated and -treated individuals on all examination days. In the MT-untreated group, 23 of the 103 individuals (22.3%) showed a SpR during the study period (Table 1). The MT-untreated GGs showing a SpR until the end of the study were numbered sequentially from 1 to 23. Only No. 1 showed a SpR at the first examination. The SpRs of Nos. 1, 2 and 4 were confirmed on all consecutive examination days for approximately 1 year after the first SpR, whereas the others individually showed sporadic or no SpR (Table 1). After excluding data for Nos. 6, 10, 12, and 13 due to harvesting or death, the proportion of SpR-positive, MT-untreated GGs increased from 1.0% (1/99 fish) to a peak of 13.0% (12/92) and then decreased to 8.2% (7/85) in January 2020. However, it increased again from the following March (10.6%, 9/85) (Fig. 2). The cumulative proportion of SpR-positive fish in the MT-untreated group gradually increased to 16.7% (16/96) by August 2019 and then to 26.1% (23/88) by May 2020 (Fig. 3). The BW of GGs at the onset of SpR in the MT-untreated group was 2.7–10.0 kg with an average of 6.9 kg; no apparent difference between them and the MT-untreated others without SpR were observed (Fig. 4).\u003c/p\u003e\n \u003cdiv\u003eA SpR was confirmed in 29 31 (93.5%) MT-treated GGs by October 2019. These MT-treated GGs showing a SpR were numbered sequentially from M1 to M29, and the two remaining GGs without a SpR were assigned M30–M31 (Table 2). Of these, three fish (M1–M3) showed a SpR on the first examination day of the study on 4 April 2019, approximately 1 month after MT treatment. The SpR of M2, M3, M9, M10, M13, M15, M17, M18, and M24 were confirmed on all consecutive examination days after the first SpR, whereas the other individuals showed sporadic SpRs (Table 2). The proportion of SpR-positive, MT-treated GGs increased from 9.7% (3/31 fish) in April 2019 to a peak of 87.1% (27/31) in August 2019 and then decreased to 41.9% (13/31) in February 2020. However, it increased again from the following April 2020 (46.7%, 14/30) (Fig. 2). The seasonal fluctuation in the proportion of SpR-positive fish in the two groups year-round was generally similar (Fig. 2). The cumulative proportion of SpR-positive fish in the MT-treated group rapidly increased to 87.1% (27/31) by August 2019 and then to 93.5% (29/31) by October 2020 (Fig. 3), considerably higher than that of MT-untreated fish (26.1%, 23/88) (Fisher’s test, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001). Fish size at the time of MT treatment did not affect the onset of the first SpR: 11/12 fish in the 4 kg BW class, 12/12 in the 6 kg class, and 6/7 in the 8 kg class (Fisher’s test, \u003cem\u003ep\u003c/em\u003e = 0.690).\u003c/div\u003e\n \u003cdiv\u003eThe mean %SMs for MT-untreated and -treated GGs were 49 ± 17% (\u003cem\u003en\u003c/em\u003e = 23) and 56 ± 13% (\u003cem\u003en\u003c/em\u003e = 29), respectively, with no significant difference between the groups (Fig. 5) (Welch’s t-test, \u003cem\u003et\u003c/em\u003e = 1.55, \u003cem\u003edf\u003c/em\u003e = 38.8, \u003cem\u003ep\u003c/em\u003e = 0.129). Statistical differences were detected across months in both groups (KW test, MT-untreated GG, \u003cem\u003eχ\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e = 31.0, \u003cem\u003edf\u003c/em\u003e = 11, \u003cem\u003ep\u003c/em\u003e = 0.001; MT-treated GG, \u003cem\u003eχ\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e = 23.7, \u003cem\u003edf\u003c/em\u003e = 11, \u003cem\u003ep\u003c/em\u003e = 0.014). Pairwise comparisons showed a significant difference between the following months: MT-untreated GGs, %SM in April 2020 was significantly lower than those in October and September 2019, February and May 2020 (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05); MT-treated GGs, %SM in April 2020 was lower than that in October 2019 (\u003cem\u003ep\u003c/em\u003e = 0.030) (Fig. 6). %SM among the individuals did not significantly differ (KW test, MT-untreated GG, \u003cem\u003eχ\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e = 19.4, \u003cem\u003edf\u003c/em\u003e = 14, \u003cem\u003ep\u003c/em\u003e = 0.151; MT-treated GG, \u003cem\u003eχ\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e = 21.3, \u003cem\u003edf\u003c/em\u003e = 23, \u003cem\u003ep\u003c/em\u003e = 0.563).\u003c/div\u003e\n\u003c/div\u003e\n\u003ch3\u003eGonadal developments\u003c/h3\u003e\n\u003cp\u003eThe mean BW and GSI of the untreated GGs harvested in April, July, and September 2019 were 4.4 ± 0.4 kg and 0.29 ± 0.05% (\u003cem\u003en\u003c/em\u003e = 3), 7.6 ± 1.1 kg and 0.34 ± 0.30% (\u003cem\u003en\u003c/em\u003e = 3), and 5.7 ± 1.5 kg and 0.36 ± 0.09% (\u003cem\u003en\u003c/em\u003e = 4), respectively.\u003c/p\u003e\n\u003cp\u003eWe microscopically examined %SM in portions of the gonads after dilution with seawater. Motile and immotile spermatozoa were observed in the gonads of two out of three fish examined in April (3 years and 7 months old), all three fish in July, and two out of four fish in September (4 years old). Before harvesting, two fish, No. 6 in July and No. 12 in September, showed three and one SpR event, respectively (Table\u0026nbsp;1). All seven individuals were microscopically identified as male or in the process of changing sex from female to male.\u003c/p\u003e\n\u003cp\u003eThree gonads from July (3 years and 10 months old) were histologically examined (Fig.\u0026nbsp;7). The gonad shown in Fig.\u0026nbsp;7A (GSI 0.18%) comprised approximately one-third ovary and two-thirds testis, suggesting sex transition. Perinucleolar and oil droplet oocytes occupied the ovary, while testicular cysts contained spermatozoa and spermatocytes. Several spermatogonia were observed in the interstitial spaces between the cysts (Fig.\u0026nbsp;7a). Although several atretic oocytes remained in the periphery, a large part of the gonad, as shown in Fig.\u0026nbsp;7b (GSI 0.15%), was occupied by the testis with cysts containing several spermatozoa, indicating further progress in sex transition than that in the gonad in Fig.\u0026nbsp;7A (Fig.\u0026nbsp;7b). No oocytes were detected in the gonadal sections of No. 6 (Fig.\u0026nbsp;7C, GSI 0.69%), which displayed SpR from May to July (Table\u0026nbsp;2); therefore, this individual was considered to have changed sex to male (Fig.\u0026nbsp;7c).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we evaluated the maturation of male GGs raised in a subtropical region of Japan by monitoring SpR, %SM, and gonadal development year-round.\u003c/p\u003e \u003cp\u003eTo the best of our knowledge, this is one of the few studies monitoring the SpR of groupers over 1 year. We demonstrated that male GGs could produce sperms year-round under the natural environmental conditions of the Amami Islands in the subtropics (WT 19.9\u0026ndash;28.6\u0026deg;C) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). We previously reported that the SpR in LGGG, a hybrid between dam LG and sire GG, occurred year-round in the same area (Masuma and Aoki \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2024\u003c/span\u003e); this suggests that the spermatogenesis and spermiation of GGs may also be well adapted to the subtropical conditions of the Amami Islands. Therefore, continuous SpR in LGGG is inherited from GG rather than LG, whose SpR was not observed between October and November and appears seasonal (Masuma et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe duration of spermatogenesis in fish is affected by WT (Postingel Quirino et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In the present study, the SpR activity of MT-untreated and -treated GGs tended to be low in the low WT January and February period (\u0026lt;\u0026thinsp;21\u0026deg;C). However, it was individually dependent (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). However, Schulz et al. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) indicated that some tropical fish do not display apparent seasonal variations in spermatogenic activity. The decrease in the SpR-positive rate observed in the current study may be specific to tropical fish reared in subtropical waters.\u003c/p\u003e \u003cp\u003eThree MT-treated GGs (Nos M1\u0026ndash;M3) exhibited a SpR at the first examination on 4 April 2019, despite it being only approximately 1 month after commencing MT treatment. Similarly, an MT-untreated GG (No. 1) displayed a SpR on the same day. Passini et al. (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) suggested that MT treatment stimulates testicular development and growth, accelerating spermatogenesis in the common snook \u003cem\u003eCentropomus undecimalis\u003c/em\u003e. In the present study, the proportion of SpR-positive GGs in the MT-treated group (93.5%) was significantly higher than in the untreated group (26.1%); this corroborates Passini et al.\u0026rsquo;s (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) suggestion and indicates that MT treatment accelerated testicular development, leading to the early onset of the first SpR (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTwenty-three out of 111 MT-untreated GGs (\u0026le;\u0026thinsp;10.0 kg BW) exhibited a SpR during this study, while a SpR was not observed in other individuals. In our previous study, MT-untreated LGGGs (4\u0026ndash;5 years old; 6.0\u0026ndash;13.7 kg, \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;9) without a SpR were histologically identified as female by having mature or immature ovaries (Masuma and Aoki \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Similarly, the gonads of the transitional stage were observed in GG in the present study. Liao et al. (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2001\u003c/span\u003e) reported that the initial BW of GG spawners range from 20 to 40 kg. Similarly, Palma et al. (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) reported that the onset of female sexual maturity occurs at 23.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5 kg in the Philippines and 33.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5 kg in Vietnam. Therefore, the occurrence of a SpR in the current study, presumably the first in the lifetime of the GGs, indicates a natural sex change from immature females to functional males.\u003c/p\u003e \u003cp\u003eThe mean BW of MT-untreated GGs showing a SpR was 6.9 kg (range 2.7\u0026ndash;10.0 kg). Palma et al. (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) reported that the maturity size of wild-caught GG males in the Philippines and Vietnam was 17.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1 kg and 34.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9 kg, respectively. Male GGs in the Philippines were smaller than females, but those in Vietnam exhibited overlapping sizes. In the present study, most MT-untreated males at their first SpR were smaller than wild-caught GGs. These results suggest that size does not play an important role in sex reversal in GGs. However, our results are based on hatchery-reared fish, and further study is needed to clarify if the same is true for wild GGs.\u003c/p\u003e \u003cp\u003eGenerally, testicular development during maturation is largely influenced by WT (Pino Martinez et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Postingel Quirino et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In the current study, the promotion of the first SpR (sex reversal) in MT-untreated GGs from April to early October could be attributed to increased WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The GG spawning season in tropical southern Taiwan is between May and October, with spawning peaks between August and September (Ho et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Liao et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Furthermore, Bright et al. (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) reported that the natural spawning events of captive GGs occurred on July 22\u0026ndash;29, September 14\u0026ndash;19, and October 14\u0026ndash;21, 2012, on a lunar cycle (six, six, and eight nights per batch, respectively) in tropical Cairns, Queensland, Australia. The sex reversal of captive GGs in this 1-year study occurred even before and/or during the spawning season. Such a sex change pattern may be rare in protogynous species where sex changes normally occur at the end of the breeding season (Masuma et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Muncaster et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Peng et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Further studies are needed to assess the precise timing of sex change in GGs.\u003c/p\u003e \u003cp\u003eThe spermatozoa of GGs reared in subtropical waters exhibited high motility, with mean %SMs of 56% in the MT-treated and 49% in the untreated groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). These values are similar to those of MT-treated and untreated LGGGs reared in the similar environment of the Amami Islands (46 and 58%, respectively) (Masuma and Aoki \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In both GGs and LGGGs, MT treatment did not affect %SM. Similarly, Viveiros et al. (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2001\u003c/span\u003e) found that MT treatment did not affect the quality or quantity of semen of African catfish \u003cem\u003eClarias gariepinus\u003c/em\u003e. Conversely, in our study, statistical differences in %SM were detected between October and April in both groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Pairwise comparisons suggested that the %SM in April tended to be lower than that in the other months, suggesting potential seasonal fluctuation in sperm activity. However, our results do not allow us to infer the underlying cause of this phenomenon. Some environmental factors, such as WT or moon phase, may have affected sperm activity (Bright et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Palma et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Further research is needed to clarify this.\u003c/p\u003e \u003cp\u003eExamining the gonads of randomly selected harvested fish identified individuals undergoing sex change with active motile spermatozoa in the testicular part. The gonads of two individuals histologically analysed in July were at the transitional stage with co-occurring immature oocytes and spermatozoa-containing cysts. Our findings demonstrate that hatchery-reared GGs change sex directly from immature females to males, and this sex transition can occur even during the spawning season in the tropical region between May and October (Ho et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Liao et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Palma et al. (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) suggested that GGs are diandric protogynous hermaphrodites (Fennessy and Sadovy \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), defined by males developing either from mature females or immature females. Our observations support this notion.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eWe demonstrated that regardless of MT treatment, male GGs can produce sperm year-round in the subtropic environment of the Amami Islands, Japan. MT treatment at a dose of 2 mg/kg BW effectively enhanced functional sex reversal and sperm production, irrespective of fish size at the treatment time. Monitoring the SpR for approximately 1 year with additional assessment of %SM and gonadal histology showed that GG males can mature in subtropical waters and maintain motile spermatozoa consistently throughout the year. Histological studies suggested that hatchery-reared GGs change sex directly from immature females to males, even during the spawning season. Our results are critical for improving any hybridisation using GG males. This study was the first to report on the reproductive ecology of male GGs in subtropical waters.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eShukei Masuma conceived and designed the study. Shukei Masuma and Ryuichiro Aoki conducted the investigation and formal analyses. Shukei Masuma wrote the first draft of the manuscript, and Aoki commented on it. \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e \u003c/p\u003e\n\u003cp\u003eThis study was supported by the Kindai University Fund for Domestic Research.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll relevant data supporting the findings of this study are within the manuscript, if any, and are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was approved by the Animal Ethics Committee of the Aquaculture Research Institute of Kindai University and was conducted according to the Guidelines for Animal Experimentation at the Aquaculture Research Institute of Kindai University.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank Mr. Hiroki Yagi, Yoshiki Katsuda, and the staff of the Amami Station of the Aquaculture Technology and Production Center, Kindai University, who kindly assisted with monitoring. We are also grateful to Dr. Hiromi Ohta, Professor Emeritus at Kindai University, and Dr. Sho Shirakashi, Associate Professor at Kindai University, for their constructive advice. We would like to thank Editage (www.editage.jp) for English language editing.\u0026nbsp;\u003cbr\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAshley PJ (2007) Fish welfare: Current issues in aquaculture. Appl Anim Behav Sci 104:199\u0026ndash;235. https://doi.org/10.1016/j.applanim.2006.09.001\u003c/li\u003e\n\u003cli\u003eBartley DM, Rana K, Immink AJ (2000) The use of interspecific hybrids in aquaculture and fisheries. Rev Fish Biol Fish 10:325\u0026ndash;337. https://doi.org/10.1023/A:1016691725361\u003c/li\u003e\n\u003cli\u003eBeir\u0026atilde;o J, Boulais M, Gallego V, O\u0026rsquo;Brien JK, Peixoto S, Robeck TR, Cabrita E (2019) Sperm handling in aquatic animals for artificial reproduction. Theriogenology 133:161\u0026ndash;178. https://doi.org/10.1016/j.theriogenology.2019.05.004\u003c/li\u003e\n\u003cli\u003eBright D, Reynolds A, Nguyen NH, Knuckey R, Knibb W, Elizur A (2016) A study into parental assignment of the communal spawning protogynous hermaphrodite, giant grouper (\u003cem\u003eEpinephelus lanceolatus\u003c/em\u003e). Aquaculture 459:19\u0026ndash;25. https://doi.org/10.1016/j.aquaculture.2016.03.013\u003c/li\u003e\n\u003cli\u003eChen Z-F, Tian Y-S, Wang P-F, Tang J, Liu J-C, Ma W-H, Li W-S, Wang X-M, Zhai J-M (2018) Embryonic and larval development of a hybrid between kelp grouper \u003cem\u003eEpinephelus moara\u003c/em\u003e ♀ \u0026times; giant grouper \u003cem\u003eE. lanceolatus\u003c/em\u003e ♂ using cryopreserved sperm. Aquac Res 49:1407\u0026ndash;1413. https://doi.org/10.1111/are.13591\u003c/li\u003e\n\u003cli\u003eDag O, Dolgun A, Konar NM (2018) onewaytests: An R package for One-way tests in independent groups designs. R J 10:175\u0026ndash;199. https://doi.org/10.32614/RJ-2018-022\u003c/li\u003e\n\u003cli\u003eDe Mitcheson YS, Liu M (2008) Functional hermaphroditism in teleosts. Fish Fish (Oxf) 9:1\u0026ndash;43. https://doi.org/10.1111/j.1467-2979.2007.00266.x\u003c/li\u003e\n\u003cli\u003eCh\u0026rsquo;ng CL (2008) Senoo S Egg and larval development of a new hybrid grouper, tiger grouper \u003cem\u003eEpinephelus fuscoguttatus\u003c/em\u003e \u0026times; giant grouper \u003cem\u003eE. lanceolatus\u003c/em\u003e. Aquacult Sci, vol 56, p 505\u0026ndash;512. https://doi.org/10.11233/aquaculturesci.58.1\u003c/li\u003e\n\u003cli\u003eFennessy S, Pollard DA, Samoils M (2018) \u003cem\u003eEpinephelus lanceolatus\u003c/em\u003e. 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FAO Fish Synop 125:69\u0026ndash;293. http://www.fao.org/3/a-t0540e.pdf\u003c/li\u003e\n\u003cli\u003eHo YS, Chen WY, Liao IC (1997) Experiments on the artificial propagation of giant grouper \u003cem\u003eEpinephelus lanceolatus\u003c/em\u003e. J Taiwan Fish Res 5:129\u0026ndash;139. (in Chinese). https://en.tfrin.gov.tw/News_Content.aspx?n=349\u0026amp;s=226159\u003c/li\u003e\n\u003cli\u003eJames CM, Al-Thobaiti SA, Rasem BM, Carlos MH (1999) Potential of grouper hybrid (\u003cem\u003eEpinephelus fuscoguttatus\u003c/em\u003e \u0026times; \u003cem\u003eE. polyphekadion\u003c/em\u003e) for aquaculture. Naga, the ICLARM quarterly 22:19\u0026ndash;23. http://worldfish.catalog.cgiar.org/naga/na_2187.pdf\u003c/li\u003e\n\u003cli\u003eKoh ICC, Shaleh SRM, Akazawa N, Oota Y, Senoo S (2010) Egg and larval development of a new hybrid orange-spotted grouper \u003cem\u003eEpinephelus coioides\u003c/em\u003e \u0026times; giant grouper \u003cem\u003eE. lanceolatus\u003c/em\u003e. Aquac Sci 58:1\u0026ndash;10. https://doi.org/10.11233/aquaculturesci.58.1\u003c/li\u003e\n\u003cli\u003eKoh ICC, Shaleh SRM, Senoo S (2008) Egg and larval development of a new hybrid orange-spotted grouper \u003cem\u003eEpinephelus coioides\u003c/em\u003e \u0026times; tiger grouper \u003cem\u003eE. fuscoguttatus\u003c/em\u003e. Aquac Sci 56:441\u0026ndash;451. https://doi.org/10.11233/aquaculturesci.56.441\u003c/li\u003e\n\u003cli\u003eLee C-S, Tamaru CS, Kelley CD (1986) Technique for making chronic-release LHRH-a and 17\u0026alpha;-methyltestosterone pellets for intramuscular implantation in fishes. Aquaculture 59:161\u0026ndash;168. https://doi.org/10.1016/0044-8486(86)90128-6\u003c/li\u003e\n\u003cli\u003eLiao IC, Su HM, Chang EY (2001) Techniques in finfish larviculture in Taiwan. Aquaculture 200:1\u0026ndash;31. https://doi.org/10.1016/S0044-8486(01)00692-5\u003c/li\u003e\n\u003cli\u003eMa KY, Craig MT (2018) An inconvenient monophyly: An update on the taxonomy of the groupers (Epinephelidae). Copeia 106:443\u0026ndash;456. https://doi.org/10.1643/CI-18-055\u003c/li\u003e\n\u003cli\u003eMasuma S (2018) New fish for marine aquaculture by hybridization. Agricultural. Biotechnology. Hokuryukan, Tokyo 2:20\u0026ndash;23. (in Japanese)\u003c/li\u003e\n\u003cli\u003eMasuma S, Aoki R (2024) Maturation in a hybrid grouper, Kue-Tama, a cross between female longtooth grouper, \u003cem\u003eEpinephelus bruneus\u003c/em\u003e, and male giant grouper, \u003cem\u003eE. lanceolatus\u003c/em\u003e. Aquacult Int 32:3481\u0026ndash;3498. https://doi.org/10.1007/s10499-023-01333-y\u003c/li\u003e\n\u003cli\u003eMasuma S, Kusunoki Y, Aoki R (2022) Seasonal changes of plasma sex steroids in captive longtooth grouper \u003cem\u003eEpinephelus bruneus\u003c/em\u003e (Bloch) in subtropical regions. Aquac Res 53:1268\u0026ndash;1275. https://doi.org/10.1111/are.15660\u003c/li\u003e\n\u003cli\u003eMcGarvey LM, Halvorson LJ, Ilgen JE, Guy CS, McLellan JG, Webb MAH (2020) Gametogenesis and assessment of nonlethal tools to assign sex and reproductive condition in burbot. Trans Am Fish Soc 149:225\u0026ndash;240. https://doi.org/10.1002/tafs.10226\u003c/li\u003e\n\u003cli\u003eMuncaster S, Norberg B, Andersson E (2013) Natural sex change in the temperate protogynous Ballan wrasse \u003cem\u003eLabrus bergylta\u003c/em\u003e. J Fish Biol 82:1858\u0026ndash;1870. https://doi.org/10.1111/jfb.12113\u003c/li\u003e\n\u003cli\u003eMurata O, Itakura S, Yamamoto S, Hattori N, Kurata M, Ohta H, Masuma S (2017) Interspecific hybridization of longtooth grouper \u003cem\u003eEpinephelus bruneus\u003c/em\u003e \u0026times; giant grouper \u003cem\u003eE. lanceolatus\u003c/em\u003e and growth of hybrid larvae and juveniles. Aquac Sci 65:93\u0026ndash;95. (in Japanese with English abstract). https://doi.org/10.11233/AQUACULTURESCI.65.93\u003c/li\u003e\n\u003cli\u003ePalma P, Takemura A, Libunao GX, Superio J, de Jesus-Ayson EG, Ayson F, Nocillado J, Dennis L, Chan J, Thai TQ, Ninh NH, Elizur A (2019) Reproductive development of the threatened giant grouper \u003cem\u003eEpinephelus lanceolatus\u003c/em\u003e. Aquaculture 509:1\u0026ndash;7. https://doi.org/10.1016/j.aquaculture.2019.05.001\u003c/li\u003e\n\u003cli\u003ePassini G, Sterzelecki FC, de Carvalho CVA, Baloi MF, Naide V, Cerqueira VR (2018) 17\u0026alpha;-methyltestosterone implants accelerate spermatogenesis in common snook, \u003cem\u003eCentropomus undecimalis\u003c/em\u003e, during first sexual maturation. Theriogenology 106:134\u0026ndash;140. https://doi.org/10.1016/j.theriogenology.2017.10.015\u003c/li\u003e\n\u003cli\u003ePeng C, Wang Q, Shi H, Chen J, Li S, Zhao H, Lin H, Yang J, Zhang Y (2020) Natural sex change in mature protogynous orange-spotted grouper (Epinephelus coioides): Gonadal restructuring, sex hormone shifts and gene profiles. J Fish Biol 97:785\u0026ndash;793. https://doi.org/10.1111/jfb.14434\u003c/li\u003e\n\u003cli\u003ePierre S, Gaillard S, Pr\u0026eacute;vot-D\u0026rsquo;alvise N, Aubert J, Rostaing-Capaillon O, Leung-Tack D, Grillasca J-P (2008) Grouper aquaculture: Asian success and Mediterranean trials. Aquatic Conservation 18:297\u0026ndash;308. https://doi.org/10.1002/aqc.840\u003c/li\u003e\n\u003cli\u003ePino Martinez EP, Braanaas MF, Balseiro P, Kraugerud M, Pedrosa C, Imsland AKD, Handeland SO (2022) Constant high temperature promotes early changes in testis development associated with sexual maturation in male Atlantic salmon (\u003cem\u003eSalmo salar\u003c/em\u003e L.) post-smolts. Fishes 7:341. https://doi.org/10.3390/fishes7060341\u003c/li\u003e\n\u003cli\u003ePostingel Quirino P, da Silva Rodrigues M, da Silva Cabral EM, de Siqueira-Silva DH, Mori RH, Butzge AJ, N\u0026oacute;brega RH, Ninhaus-Silveira A, Ver\u0026iacute;ssimo-Silveira R (2021) The influence of increased water temperature on the duration of spermatogenesis in a Neotropical fish, \u003cem\u003eAstyanax altiparanae\u003c/em\u003e (Characiformes, Characidae). Fish Physiol Biochem 47:747\u0026ndash;755. https://doi.org/10.1007/s10695-020-00869-7\u003c/li\u003e\n\u003cli\u003eR Core Team (2021). R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria. Retrieved from URL. https://www.R-project.org/\u003c/li\u003e\n\u003cli\u003eSchulz RW, de Fran\u0026ccedil;a LR, Lareyre J-J, Le Gac F, Chiarini-Garcia H, Nobrega RH, Miura T (2010) Spermatogenesis in fish. Gen Comp Endocrinol 165:390\u0026ndash;411. https://doi:10.1016/j.ygcen.2009.02.013. https://doi.org/10.1016/j.ygcen.2009.02.013\u003c/li\u003e\n\u003cli\u003eTakahashi Y (1989) Testicular structure and gametogenesis. In: Takashima F, Hanyu I (eds), Monographs on Aquaculture Science volume 4: Reproductive biology of fish and shellfish. Midori-shobo, Tokyo ISBN 4-89531-434-0, p 33\u0026ndash;64. (in Japanese)\u003c/li\u003e\n\u003cli\u003eTakano K (1989) Ovarian structure and gametogenesis. In: Takashima F, Hanyu I (eds), Monographs on Aquaculture Science volume 4: Reproductive biology of fish and shellfish. Midori-shobo, Tokyo ISBN 4-89531-434-0, p 3\u0026ndash;34. (in Japanese)\u003c/li\u003e\n\u003cli\u003eViveiros ATM, Eding EH, Komen J (2001) Effects of 17\u0026alpha;-methyltestosterone on seminal vesicle development and semen release response in the African catfish, \u003cem\u003eClarias gariepinus\u003c/em\u003e. Reproduction 122:817\u0026ndash;827. https://doi.org/10.1530/rep.0.1220817\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Marine aquaculture, Methyltestosterone, Testis, Sex reversal, Spermiation response, Sperm motility","lastPublishedDoi":"10.21203/rs.3.rs-5996034/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5996034/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study aimed to determine the maturation process of captive male giant grouper \u003cem\u003eEpinephelus lanceolatus\u003c/em\u003e (GG) in the subtropical waters of the Amami Islands, Japan. The experiment commenced in February 2019 using 134 GG (3 years and 5 months old; mean body weight [BW]\u0026thinsp;=\u0026thinsp;6.0 kg) raised from hatchery-reared juveniles. Of those, 31 were administered 17α-methyltestosterone (MT) at a concentration of 2 mg/kg BW using cholesterol pellets, and the others were not. We assessed the spermiation response (SpR) of all individuals approximately once a month for over one year, from April 2019 to May 2020. Sperm motility (%SM) was also assessed when sufficient semen samples were obtained. The SpR was observed in 22.3% (23/103) untreated and 93.5% (29/31) of MT-treated GG. Some individuals from both groups showed consistent SpR throughout the year. The mean %SM for MT-untreated and treated GG were 49 and 56%, respectively, with no significant difference between the groups. Histological analysis of the gonads of the untreated GG was conducted on three fish harvested in July 2019. While two transitioned from female to male and contained immature oocytes, another possessed fully functional testis; this suggests that sex reversal in GG involves a direct transition from immature female to male. Our study demonstrated that hatchery-reared male GG mature year-round and could maintain motile spermatozoa of consistent quality in the subtropical waters of the Amami Islands. This result is critical for improving any hybridisation using GG males. This study is the first to report on the reproductive ecology of male GG in subtropical waters.\u003c/p\u003e","manuscriptTitle":"Maturation of captive male giant grouper Epinephelus lanceolatus in subtropical waters in Japan","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-12 16:48:07","doi":"10.21203/rs.3.rs-5996034/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5c32c254-4b75-4569-9255-f3f7f34d8d28","owner":[],"postedDate":"February 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-04-29T01:23:30+00:00","versionOfRecord":[],"versionCreatedAt":"2025-02-12 16:48:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5996034","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5996034","identity":"rs-5996034","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}