Aquaculture performance of triploid barfin flounder Verasper moseri

ABSTRACT:   To evaluate the aquaculture performance of triploid barfin flounder Verasper moseri , the sex ratio, maturation, growth and the relative proportion of body parts were examined. The sex ratio of triploids was similar to diploids under communal rearing conditions, but the proportion of female diploids was higher than that of triploids under separate rearing conditions. The gonadosomatic index of triploid females was very low even during the spawning season, and the ovaries were rudimentary. These results suggest that triploid barfin flounder females were sterile. In addition, triploid males produced a small quantity of milt containing very few spermatozoa with abnormal shapes. Spermatozoa obtained from triploids were aneuploidies. When normal eggs were fertilized with sperm from triploid males, no fry developed. These results suggest that triploid barfin flounder males were functionally sterile. Triploid males grew more slowly than diploid males, and triploid females showed similar or slower growth than diploid females, whether reared separately (23 months) or communally (35 months). The ratios of visceral weight to the edible parts for triploid males were similar to those for diploid males, but ratios for triploid females were higher than for diploid females during the spawning period. In conclusion, a significant improvement of growth was not found in triploid barfin flounders.     

The timing of puberty in cultured female yellowtail flounder, Limanda ferruginea (Storer): Oogenesis and sex steroid production in vivo and in vitro

This study examines puberty in female yellowtail flounder, correlating macroscopic, histological and endocrine parameters at the gonadal level. Short-term ovarian tissue incubations were used to study estradiol-17β production in relation to oogenic stage. Examining 2 year classes of young flounder demonstrated that cultured females retain phenotypic plasticity in reproductive age with pubertal onset occurring in one-year, two-year and three-year-olds. Immature ovaries were steroidogenic and capable of responding to gonadotropic stimulation. Endocrine puberty in females was detected by a peak in estradiol-17β production during the cortical alveolar oocyte stage prior to any ovarian evidence of vitellogenesis. Puberty, once initiated, proceeded to ovulation within 8 to 12 months; vitellogenic oocyte development followed the group synchronous pattern. Estradiol-17β was clearly the dominant of the two measured plasma hormones during pubertal onset and throughout vitellogenesis. Plasma testosterone was consistently detectable at low levels by mid-to late-vitellogenesis. Ovaries showed the highest sensitivity to gonadotropic stimulation in vitro during late-vitellogenesis. Variable plasma levels in both estradiol-17β and testosterone occurred in preovulatory and ovulating females during the captive spawning period. Together the results show that yellowtail flounder can mature at a young age and small size when culture conditions permit. In addition, the early sensitivity to gonadotropin by the immature ovaries may be an important physiological determinant for the timing of puberty in this species.     

Assessment of sex and evaluation of ovarian maturation of fish using ultrasonography

Ultrasonography is a non‐invasive method that can be used to assess sex and maturity of fish. This study was conducted to examine the feasibility of using ultrasound images to sex juvenile and mature halibut Hippoglossus hippoglossus (Linnaeus), mature winter Pleuronectes americanus (Walbaum) and yellowtail flounder Pleuronectes ferruginea (Storer) and mature haddock Melanogrammus aeglefinus (Linnaeus). In females, both immature and mature ovaries were relatively easy to distinguish, but in males only mature testes were consistently distinguishable. Ovarian maturation of haddock and ovulatory cycles of halibut may also be assessed using ultrasound.     

Production of all-female triploid rainbow trout

Triploid rainbow trout were produced by heat shock treatment of eggs soon after fertilization with either normal sperm or sperm from masculinised females. The proportion of triploid fry, as judged from red blood cell nuclear volume, varied between 75% and 100% in three experiments using different batches of eggs from an autumn-spawning strain of trout while a single batch of eggs from a winter-spawning strain yielded 50% triploids.

A microscopic examination of the gonads was made on 5-month-old fish weighing between 1 and 3 g. In female controls the ovaries were packed with oocytes while those from female triploids, although showing the typical lamellar structure of an ovary, contained no oocytes, thus indicating that female triploids are sterile. The testes from triploid males appeared to be developing normally.

The use of masculinized females combined with heat shock treatment of eggs to produce triploids, allowed the production of sterile all-female triploids. This should have considerable potential for aquaculture.     

The effect of ploidy on the fatty acid profile during the reproductive cycle of female rainbow trout (Oncorhynchus mykiss)

Rainbow trout Oncorhynchus mykiss triploids are regularly produced in fish farms to improve growth because the triploid females do not develop ovaries during the reproductive cycle. In this study, the tissue fatty acid allocations in triploid and diploid females were compared during the reproductive cycle to determine whether the ploidy influences the fatty acid profile of fish produced in aquaculture. The ovaries, liver, and white muscle fatty acid contents of diploid and triploid females were analyzed during the reproductive cycle. Diploid females tend to accumulate more polyunsaturated fatty acids than triploids during some phases of the reproductive cycle, and this profile was compensated by an increase in saturated and monounsaturated fatty acids in triploid females. Arachidonic acid (C20:4n6) was the main n6 polyunsaturated fatty acid in the ovaries of diploid females during the most advanced phases of the reproductive cycle, and docosahexaenoic acid (C22:6n3) was the main n3 polyunsaturated fatty acid. In triploid females, the percentage of both of these polyunsaturated fatty acids was lower than in diploid females during the most advanced phases of the reproductive cycle. In general, the lack of ovary development altered the hepatic synthesis of some fatty acids, mainly monounsaturated and polyunsaturated fatty acids, decreasing the content of the main fatty acids in the white muscle and, consequently, the mobilization of these specific fatty acids to the ovaries.     

Production of meiotic gynogenetic and triploid sea bass, Dicentrarchus labrax L.: 1. Performances, maturation and carcass quality

Meiotic gynogenetic and triploid sea bass were produced by pressure shocks according to a previously published protocol. Pressure-treated groups did not survive as well as controls during early development and larval rearing. Performances, sexual maturation and carcass quality were examined over a period of 34–45 months. At the age of 34 months, growth of the gynogenetic fish was comparable to that of the control but inferior in the triploid fish. A predominance of male fish was found within the triploid groups, while diploid and meiotic gynogenetic fish showed equal proportions of the sexes. Gonadal maturation in triploid fish was significantly impaired, particularly in the females that showed rudimentary ovaries. Triploid males exhibited primary maturation but proved to be gametically sterile. Pressure-induced triploids did not grow as well as diploids, but these results might be ascribed to specific on-growing conditions (communal rearing). The performance of gynogenetic sea bass was comparable to that of control. The superiority of diploid fish over their triploid counterparts was confirmed during the final growing period and more clearly so in females. Performances of triploids varied according to their maternal origin. Overall, striking qualitative differences between diploid and triploid fish were found at the age of 34 and 45 months, although the results varied in a gender-specific manner. A strong maternal effect was also observed. The potential advantages of triploid sea bass for aquaculture purposes are discussed.     

Induction of triploidy in mud loach (Misgurnus mizolepis) and its effect on gonad development and growth

Triploidy was induced in mud loach (Misgurnus mizolepis) by cold shocking fertilized eggs 5 min post-fertilization at 2°C for 15 to 60 min. Best results were obtained when eggs were shocked for 60 min; 98% of fish examined in that treatment were triploids. Triploidy was confirmed by erythrocyte measurements and chromosome counts. Diploids had 48 chromosomes, while triploids had 72. Histological analysis of 9-month-old triploid ovaries showed an appreciable number of oocytes at the chromatin nucleolus stage with considerable interstitial tissue. However, diploids had well developed oocytes. Diploid testes from diploid males exhibited normal spermatids and spermatozoa, while a few were seen in triploid males. Growth rate was evaluated over a 9-month growth trial. Although male and female triploids were slightly heavier than their diploid counterparts from the third to the ninth month, their growth rates were not significantly different compared to their diploid controls.     

The reproductive physiology of three age classes of adult female diploid and triploid brook trout (Salvelinus fontinalis)

Triploid female fish show impaired gametogenesis and are unable to produce viable offspring. The reproductive physiology of artificially-induced triploid female salmonids has been well described up until the time of first sexual maturation in diploids, but few reports exist for older triploids. This study reports the influence of triploidy on growth, ovarian development and reproductive endocrinology among three age classes of female brook trout (Salvelinus fontinalis) in comparison to sibling diploids. Triploids were larger than diploids for most of the study period, but the difference was statistically significant only during maturation and spawning of 2⁺ diploids. Plasma estradiol-17 (E₂), testosterone (T) and vitellogenin (VTG) levels in triploids were generally lower than in diploids, and VTG was the only parameter to show seasonal fluctuations resembling those of diploids. Triploids showed significantly lower GSI and total oocyte number than diploids of similar age, and only half of all triploids sacrificed during the study (n=56) had developing oocytes in their ovaries. At age 3⁺, 13 of 19 triploid females had oocytes at various stages of development, including perinucleolar, yolk vesicle and yolk globule stages. In addition, three of these fish had collectively produced 72 mature stage oocytes. Thus, whereas diploid brook trout can produce mature oocytes as two-year-olds, triploids cannot do so until four years of age, with the number of mature oocytes being greatly reduced.     

Genetic characterization, morphometrics and gonad development of induced interspecific hybrids between yellowtail flounder, Pleuronectes ferrugineus (Storer) and winter flounder, Pleuronectes americanus (Walbaum)

Viable interspecific hybrids between yellowtail flounder (Pleuronectes ferrugineus, Storer) and winter flounder (Pleuronectes americanus, Walbaum) were produced by artificial insemination of yellowtail flounder eggs with winter flounder sperm. However, mean fertilization rate, hatching success and early survival up to 3 weeks post hatch were significantly lower than those of parental pure cross controls (P < 0.01). Overall, cytogenetic traits (karyological analysis and estimation of cellular DNA contents using flow cytometry) of hybrid flounder were intermediate between the two parental species. Microsatellite assay was used to distinguish the parental genomes in the hybrids; in most cases, one allele was specific to each of the parents. Morphometrics assessed by body proportions indicated that hybrids generally displayed a morphology intermediate between the maternal and paternal species. Interspecific hybrids exhibited abnormal and retarded gonad development in both sexes based on histological analysis of gonads from adult fish. The sterility of the hybrids presents a significant advantage for their use in aquaculture, as potential escapees would not be capable of reproducing in the wild and contaminating natural stocks.     

Growth performance, morphometric traits and gonad development of induced reciprocal diploid and triploid hybrids between the mud loach (Misgurnus mizolepis Günther) and cyprinid loach (Misgurnus anguillicaudatus Cantor)

Two species of loach, the mud loach (Misgurnus mizolepis Günther) and the cyprinid loach (M. anguillicaudatus Cantor), are commercially important in Korea both for food and ceremonial purposes. The mud loach has superior potential for aquaculture in terms of growth, whereas the cyprinid loach has a more desirable body shape and colour. This study was conducted to produce reciprocal diploid and triploid hybrids and to evaluate their potential benefits in culture by examining growth performance, morphometrics and gonad development. Reciprocal diploid and triploid hybridization was performed by artificial insemination without or with the induction of triploidy. The successful formation of diploid or triploid karyogamy was verified by flow cytometric analysis. Body weights of induced reciprocal diploid hybrids were intermediate between those of the two parental species, i.e. hybrids were heavier than cyprinid loach but lighter than mud loach. In contrast, the growth performance of triploid hybrids was similar to that of their corresponding maternal parents. Diploid hybrids showed intermediate morphometric traits between the two species; however, the body proportions of triploid hybrids were more similar to those of the maternal species than the paternal species. Histological analyses indicated that reciprocal diploid hybrids of both sexes were able to reach maturity, as evidenced by the presence of mature oocytes or spermatozoa in the gonad tissues. However, triploid hybrids showed stringent sterility at the gonadic level; the sizes of ovaries and testes were much smaller, and gonad development was abnormal and significantly retarded.     

Ovarian maturation stages of the mud crab Scylla serrata

Ovarian maturation in adult wild‐sourced and pond‐grown Scylla serrata (Forsskål) was determined based on gross morphology and histological appearance. There were no significant differences noted in the histological features of both wild and pond‐reared S. serrata females. Ovarian maturation was classified into five stages: immature, early maturing, late maturing, fully mature and spent. The immature ovaries are thin and translucent to off white and contain oogonia, primary oocytes with large nuclei. The follicle cells were found around the periphery of the lobes and an area among groups of oogonia and oocytes. The follicle cells gradually enclosed the oocytes. The early‐maturing ovaries were yellow and small yolk globules started to appear in larger oocytes. In late‐maturing ovaries, the colour became light orange and lobules were apparent. Yolk globules occurred in the cytoplasm with larger globular inclusions towards the periphery, while follicle cells were hardly recognizable. Fully mature ovaries were orange to deep orange and had swollen lobules. Large yolk globules were apparent in the entire cytoplasm. Follicle cells were hardly seen. Spent ovaries were similar to the early‐maturing and late‐maturing stage in partially spawned females. The ovarian development was correlated closely to the gonadosomatic index, oocyte diameter, and ovarian histology. The classification of ovarian maturation provides baseline information for further studies on reproductive biology. Likewise, the information provides a guide for broodstock management in the hatchery.     

Triploid Induction in the Yellowtail Tetra,Astyanax altiparanae,Using Temperature Shock: Tools for Conservation and Aquaculture

Triploidization is an interesting tool to produce sterile fish. In the yellowtail tetra, Astyanax altiparanae, this can be applied for aquaculture and surrogate technologies. In this study, we compared the efficacy of cold (2 C) or heat shock (38 C, 40 C, and 42 C) on triploid induction in the yellowtail tetra. The eggs were treated with cold or heat shock, 2 min postfertilization (30 min in cold shock or 2 min in heat shock). Intact embryos served as the control group. Ploidy status was confirmed by karyotyping, flow cytometry, and nuclear diameter of erythrocytes. The hatching rate decreased after cold shock (12.69 ± 15.76%) and heat shock at 42 C (0.35 ± 0.69%) in comparison with the control group (63.19 ± 16.82%). At 38 C and 40 C, hatching rates (61.29 ± 17.73% and 61.75 ± 22.1%, respectively) were not decreased. Only one triploid arose at 38 C (1/80). At 40 C, a high number of triploids arose (72/78). At 42 C, very few embryos developed into the hatching stage. A large number of haploid individuals arose after cold shock (61/75), with only one triploid. Our results indicate that heat shocking of embryos at 40 C is optimum for triploid production in the yellowtail tetra.     

Ovaprim treatment promotes oocyte development and milt fertilization rate in diploid and triploid African catfish (<Emphasis Type="Italic">Clarias gariepinus)</Emphasis>

Triploid fish are increasingly used in aquaculture because they are generally unable to reproduce successfully. Energy is channeled into somatic growth rather than gonadal development, and in the event of escape, the animals are unlikely to breed successfully among themselves or with wild conspecifics. This study tested the ability of recently matured triploid African catfish (Clarias gariepinus) to produce and fertilize eggs with and without ovaprim treatment. Triploid females did not show the increase in ovary size observed in diploid members of the same cohort between 8 and 9 months of age, or the coincident decrease in visceral fat deposits, and this was unaffected by up to 5 weekly i.m. injections of 0.5 ml kg⁻¹ Ovaprim. However, we observed advanced vitellogenin (Vtg) sequestration in oocytes of triploid females, albeit to a lesser degree and with lesser cortical alveoli, compared to oocytes from diploid cohort members. Histological sections revealed a positive trend of oocyte development up to the third weekly ovaprim injection followed by a negative gonadal development in weeks four and five. Milt from triploid males injected 9–12 h earlier with 0.25 ml kg⁻¹ ovaprim i.m. fertilized more diploid eggs than milt from untreated triploid males (30 vs. 20%), but none of the developing embryos of triploid paternity survived to hatch. In contrast, milt of diploid males fertilized 49% of eggs, and 20% of the developing embryos hatched successfully. These rates were improved in ovaprim-injected diploid males to 70% fertilization and 33% hatch. This study demonstrates potential of overcoming non-viability of eggs from triploid female African catfish, and enhancing the ability of triploid milt to fertilize eggs.     

Effect of dietary sodium chloride acclimation on growth and plasma thyroid hormones in tilapia Oreochromis niloticus (L.) in relation to sex

This trial was conducted in order to test the effect of supplemental dietary sodium chloride on growth and plasma thyroid hormone levels of male and female monosex groups of tilapia Oreochromis niloticus (L.) reared in fresh water and 10‰ brackish water. At the end of the experiment. the gonadal development of females was observed. A high‐salt diet (8% NaCl) was fed to female and male tilapia for a period of 3 weeks. After this time. both groups were transferred directly to fresh water and 10‰ brackish water, and a growth trial was conducted over 90 days. The results indicate that dietary supplementation with NaCl improved the growth of females and males at 30 days, except for females maintained in fresh water. Females reared in 10‰ brackish water showed an increase in growth rates that was accompanied by the highest plasma T3 levels. In contrast, males did not exhibit the same relationship in 10‰ brackish water, showing lower plasma T3 levels than in fresh water. Histological sections of O. niloticus ovaries revealed that female groups contained oocytes at different stages of development. At 90 days, females reared in 10‰ brackish water showed oocytes in maturation, while in fresh water, they showed the highest proportion of vitellogenic oocytes and no mature oocytes. The highest proportion of mature oocytes observed in brackish water was associated with the better growth rate in relation to fresh water. These results confirm that tilapia breed freely in low‐salinity brackish water as well as in fresh water.     

长鳍篮子鱼繁殖季节性腺的组织学研究

采用常规组织切片法研究了长鳍篮子鱼繁殖季节各期卵巢和精巢的组织学结构特征。在所获得样本中,未发现Ⅰ期、Ⅱ期的卵巢和精巢。Ⅲ期卵巢以第Ⅲ时相卵母细胞为主,这时期液泡、卵黄颗粒出现,同时含有少量Ⅰ时相和Ⅱ时相的卵母细胞;Ⅳ期卵巢以第Ⅳ时相卵母细胞为主,Ⅳ时相晚期卵母细胞开始出现油球,细胞核偏移和变形,放射带明显;Ⅴ期卵巢的卵细胞游离,卵细胞的外层分别有胶膜、放射带和质膜;Ⅵ期卵巢主要由Ⅲ时相卵母细胞和大量的空滤泡外膜组成。长鳍篮子鱼精巢为辐射型,精巢内生殖细胞分为初级精母细胞、次级精母细胞、精子细胞和精子,各期生殖细胞和支持细胞组成了精小管,同一精小管中的生精细胞发育不同步。精子成熟后,充满整个精小管,完成发育过程。     

Characterization of triploid hybrid groupers from interspecies hybridization (Epinephelus coioides ♀ × Epinephelus lanceolatus ♂)

Interspecies hybridization is widely used in aquaculture as a beneficial strategy. Diploid and triploid hybrids have been detected from the interspecies hybridization of Epinephelus coioides ♀ × Epinephelus lanceolatus ♂. This is the first report of triploidization through hybridization in grouper. Confirmation has been obtained through flow cytometry, karyotyping and erythrocyte nuclei measurement. The chromosome numbers of E. coioides, E. lanceolatus, diploid hybrid grouper are 48 and triploid hybrid grouper are 72. Measurements of erythrocyte nuclei indicate that triploid fish have a larger nuclear surface than the diploid groupers, and the average ratio of triploid to diploid surface area is 1.59. During the first 1.5 years, triploid hybrid groupers grow faster than diploid hybrid groupers or either parent species. The average growth rate of triploid hybrids is 1.61 times greater than that of diploid hybrids at 6 months of age and 1.43 times greater at 18 months of age. The triploid hybrid groupers are inferior in gonadal development, with no primary‐growth‐stage oocytes appearing in the gonads at 18 months of age. Morphological studies indicate that triploid hybrid groupers have distinctive differences in snout length, eye diameter, body trunk shape, and tail shape development compared with diploid hybrid groupers. Triploid hybrid groupers have an advantage in growth ability, and artificial breeding of triploid groupers might be of great potential use in the grouper aquaculture industry.     

Oogenesis and lipids in gonad and liver of daubed shanny (Leptoclinus maculatus) females from Svalbard waters

Oocyte and liver histomorphology of the daubed shanny (Leptoclinus maculatus) from Isfjorden and Kongsfjorden in Svalbard were investigated during three Arctic seasons: summer (July), autumn (October) and winter (April). Three oocyte developmental phases were observed: primary growth phase, secondary growth phase and maturation phase. We observed four different developmental stages: (1) perinucleolus stage with cortical alveolus formation, (2) lipid droplets formation, (3) vitellogenesis stage and (4) maturation. Late maturation stage of oocytes in the ovaries was from the autumn season. Females accumulated lipids in liver (up to 35.2?% dw) and deposited large amounts of lipids into gonads (up to 52.2?% dw) during maturation. Lipid classes in female gonads changed seasonally, with relative increase in cholesterol during summer and depletion of storage lipids (triacylglycerols and wax esters/cholesterol esters) during the winter. Lipid composition in liver changed during oocyte development and spawning, as neutral lipids were transferred to developing oocytes during summer to autumn. During winter, storage lipids were depleted during starvation. Based on the increase in gonadosomatic index (GSI) with length and observed maturation stages, females seem to mature at a length of about 125-130?mm. The GSI and hepatosomatic index of large females sampled in autumn (September-October) were significantly higher than for females in late winter (April) and spring (May). These results indicate that spawning takes place during winter in Isfjorden and that energy reserves stored in the liver are utilized by females during gonadal development and reproduction.     

圆斑星鲽mtDNA控制区结构

对圆斑星鲽mtDNA控制区序列及鲽形目鱼类鲽科的条斑星鲽、大西洋黄盖鲽、马舌鲽，美洲拟庸鲽和鲆科的牙鲆以及鳎科的欧洲鳎、塞内加尔鳎、沙鳎控制区进行了比较分析。结果表明，圆斑星鲽的控制区序列可分为扩展终止相关序列区（ETAS）、中央保守区（CSB—A、B、C、D、E、F）、保守序列区（CSB1、CSB2、CSB3）和串联重复序列区（Tandem repeat region）。通过与其他脊椎动物线粒体控制区序列的比较，发现鲽形目的鲆、鲽类和鳎类与两栖纲的无尾类在CSB-3之后存在相似的串联重复序列。     

A description of the ovarian development stages of penaeid shrimps from the coast of Pakistan

In this study, four species of penaeid shrimp from Pakistan's inshore waters were studied. These were Penaeus penicillatus (Alcock), Penaeus merguiensis (de Man), Metapenaeus affinis (Milne Edwards) and Parapenaeopsis stylifera (Milne Edwards). During maturation, shrimp ovaries pass through a series of colour changes. The various colours observed in the ovaries of the four species of penaeid shrimps were translucent, white, cream, yellow, green‐yellow, green‐white, light green and dark green. The different type of oocytes observed in histological sections of the penaeid ovaries are as follows: chromatin nucleolar oocytes, perinucleolar oocytes, yolkless oocytes, yolky oocytes and oocytes with cortical bodies. The ovarian developmental stages recognized in these shrimps are: undeveloped, developing, nearly ripe, fully ripe, resorbing and resorbing/ developing. The ovarian developmental stages were similar in all the four species with the exception of the fully ripe stage. In the fully ripe stage of P. penicillatus and P. merguiensis, the cortical bodies were present at the periphery in two shapes; spherical as well as rod‐like. On the other hand, in the fully ripe stage of M. affinis and P. stylifera, the cortical bodies were present only at the periphery and were only spherical. A relationship between colour of ovaries and histological stages of ovarian development was established. A female shrimp with a dark‐green ovary is an indication of the ready‐ to‐spawn condition. In P. merguiensis and Penaeus penicillatus, respectively, 100% and 88.1% females with dark‐green ovaries had oocytes with cortical bodies, whereas in M. affinis and P. stylifera, respectively, 50% and 28.6% females with dark‐green ovaries had oocytes with cortical bodies.     

Possible application of mibolerone for induced sex inversion of grouper Epinephelus coioides

SUMMARY: Thirty immature juvenile grouper Epinephelus coioides (19–168 g bodyweight, BW) were randomly stocked in four units 6 t tanks to determine if mibolerone can be used to induce sex inversion in groupers. After acclimatization and weaning to artificial feed, the feed given daily (4% BW/day) was supplemented with 0, 50, 100, and 200 μg mibolerone/kg feed for about 18 weeks. Thereafter, the hormone treatment was withdrawn and the experiment was terminated at Week 24. Ten fish were killed for gonad histology at stocking to serve as an initial control while about three to five fish were killed every 8 weeks. In general, ovaries of initial controls showed the presence of moderate stromal cells and gonia and few primary oocytes. At Weeks 8 and 16, ovaries of the control fish (0 μg/kg) were similar to that of the initial control except that primary oocytes increased at Week 24. Gonads of fish fed diets containing 100 and 200 μg/kg had none to moderate spermatocytes and few spermatids at Week 8 and 16, although spermatozoa were not observed, indicating that the fish were undergoing spermatogenesis. Spermatogenesis at 50 μg/kg was not as advanced since only few spermatocytes occurred at Weeks 8 followed by moderate gonia and no spermatocytes and spermatids at Week 16. However, the presence of few primary oocytes was observed when mibolerone was withdrawn suggesting that sex-inversed fish reverted back to a female condition. These results show that sex inversion in juvenile grouper can be induced by oral administration of mibolerone and may have possible application on mature females to produce functional males.