Possible Application of Mibolerone for Induced Sex Inversion of Grouper Epinephelus Coioides

FISHERIES SCIENCE 2001; 67: 232–237

Original Article

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

G F QUINITIO,* J D TAN-FERMIN AND A NAGAI

Aquaculture Department, Southeast Asian Fisheries Development Center, Tigbauan, 5021 Iloilo, The Philippines

SUMMARY: Thirty immature juvenile grouper Epinephelus coioides (19Ð168 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 mg 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 Thereafter, the hormone treatment was withdrawn and the experiment was terminated at Week 24.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 mg/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 mg/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 mg/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 However, the presence of few primary oocytes was observed when mibolerone was withdrawn suggesting that sex-inversed fish reverted back to a female condition.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.

KEY WORDS: Epinephelus coiodes, grouper, mibolerone, protogynous hermaphrodite, sex inversion, sex reversal.

INTRODUCTION fario,8 and by injection in juvenile E. suillus (= E. coioides).9 Other hormones whether alone or in Grouper has become a very important culture combination with MT have also been tested in an species due to its good taste and high price. attempt to induce sex change in grouper.8 However, one hindrance in seed production of Cheque Drops is the brand name of mibolerone groupers is their protogynous hermaphrodite which is produced by the Upjohn Company characteristic. In general, there are few males avail- (Kalamazoo, MI, USA). Its active ingredient, able. To solve this problem, induction of sex 17-b-hydroxy-7a,17-dimethyl-estr-4-en-3-one, is change in groupers has been carried out using a non-progestational steroid recommended for social control1 or hormones. 17a-Methyltestos- oral use to prevent estrus in adult female dogs. terone (MT) is effective in inducing sex inversion Mibolerone has been tested for induction of sex of different groupers either by oral administration reversal in tilapia10,11 and immersion of tilapia fry in Epinephelus tauvina,2,3 Mycteroperca microlepis4 in 0.6 p.p.m. mibolerone for 5 weeks appears to and E. fario,5,6 pellet implant in E. tauvina7 and E. be effective.11 Oral administration has also been tested but data were limited although the tech- nique has great potential.11 *Corresponding author: Tel: 63(33)335-1009. Fax: 63(33)335- In this study, we tested mibolerone to ﬁnd out if 1008. Email: [email protected] it could also be effective for inducing sex inversion Received 19 April 2000. Accepted 6 November 2000. in grouper when incorporated into the feeds. Induced sex inversion in grouper FISHERIES SCIENCE 233

MATERIALS AND METHODS the urinary bladder. Serial sections of 5 mm thick were made, stained with Mallory’s trichrome12 and Test animals examined using a light microscope.

A total of 120 juvenile grouper E. coioides with initial bodyweight (BW) of 19–168 g and total Relative counts of germ cells length (TL) of 11.5–23.2 cm were used as test animals. These were randomly stocked in four 2 ¥ The relative abundance of stromal cells and germ 3 ¥ 1.3 m concrete tanks. Each tank had a water cells observed in all gonad sections were arbitrarily volume of 6 t and was provided with aeration and designated as none (-), few (+), moderate (++), and ﬂow-through ﬁltered seawater. abundant (+++).

Feed and feeding Water parameters

The fish were initially weaned to feed on artificial Water parameters were measured daily at 1000 h. feed for about 2 weeks before the start of the treat- During the entire experimental period ranges ment. The artificial feed given to the fish con- were: water temperature, 25.8–30.0°C; salinity, tained: tuna fish meal, 55%; meat and bone meal, 27–32 p.p.t.; pH, 7.54–8.64; and dissolved oxygen, 15%; shrimp head meal, 10%; rice bran, 5%; wheat 6.2–8.8 p.p.m. flour, 10%; soybean oil, 2%; cod liver oil, 2%; and vitamin mix, 1%. The feeding rate used was 4% BW per day. Each RESULTS feed ration was mixed with 70% water to become moist and then made into about 1.5 cm balls just Table1 shows the range of bodyweight and gives before feeding. Feeding was done every morning at a summary of the relative abundance of stromal about 0900 h by giving the feed balls one at a time. and germ cells in the gonads of juvenile groupers Sampling for total biomass was done every 2 weeks fed diets containing different concentrations of to adjust the feeding rate. Raw fish was added to mibolerone during the experimental period. At the the feed at 10% of the ration on Week 9 because the start of the experiment (Week 0), nine of the 10 fish fish seemed to be losing appetite at Week 8. Bacte- sampled still had very immature gonads (Fig. 1a) rial infection was affecting the fish so, starting on with a moderate number of stromal cells and the 11th week, 0.2% oxytetracyline (Sigma Chemi- gonia, consisting of oogonia and spermatogonia, cals, St Louis, MO, USA) was added to the artificial and no primary oocytes. Primary oocytes referred feed. On Week 14, the fish were injected with 25 mg to include both oocytes in the chromatin and oxytetracycline/kg BW to prevent further mortality. perinucleolus stage. The 10th fish had abundant The feed given to the fish was shifted to 100% raw primary oocytes with a moderate number of fish a day before Week 16. stromal cells and few gonia (Fig. 1b). During the entire experimental period, fish fed the diet that did not contain mibolerone had immature ovaries Treatments similar to those of the initial control, except for an increase in the number of primary oocytes in Ten fish were killed at the time of stocking which the gonads of fish that were killed at Week 24. served as an initial control. Incorporation of Moreover, gonad of one fish was even noted mibolerone at 0, 50, 100, and 200 mg/kg feed was to contain atretic oocytes (Fig. 2a) during this initiated 1 week after stocking. The Cheque Drops period. used contained 100 mg mibolerone dissolved in At Weeks 8 and 16, most of the fish treated propylene glycol to make 1 mL solution. The feed with mibolerone at 50 mg/kg feed had few to a mod- and hormone were mixed just before feeding the erate number of stromal cells, with a moderate fish. After Week 16, the hormone treatment was abundance of gonia. No primary oocytes and withdrawn and the experiment was terminated other spermatogenic cells were generally observed on Week 24. At Week 8, 16, and 24, three to five except at Week 8 when few spermatocytes occurred fish were killed and the gonads were removed, while gonads of one fish contained abundant sper- preserved in Bouin’s solution, and embedded in matocytes. During the experimental period, fish paraffin. Adjacent muscle tissues were taken and treated with mibolerone at 100 and 200 mg/kg feed preserved in the fixative when the gonads were too had few or no stromal cells, moderate number small to be distinguished from the intestine and of gonia, no primary oocytes, few to moderate 234 FISHERIES SCIENCE GF Quinitio et al.

Table 1 Relative abundance of stromal and germ cells in juvenile Epinephelus coioides during oral administration of different dosages of mibolerone for 16 weeks and after its withdrawal Week Treatment No. of Body- Stromal Germ cells Remarks (mg/kg fish weight cells G PO SC ST SZ feed) sampled (g) 0 Initial 10 17–66 ++ ++ - - - - Immature gonads except for one fish with abundant PO. 8 0 4 46–122 ++ + + - - - Immature ovaries. 50 3 42–80 +++-+--Gonads undergoing spermatogenesis. One already had abundant SC. 100 4 24–111 -++-++-Gonads undergoing spermatogenesis. One fish had few PO while one already had moderate number of ST. 200 3 101–141 -++-+++-Gonads undergoing spermatogenesis. One fish had few PO. 16 0 4 62–148 ++ + - - - - Immature gonads. 50 3 91–131 ++ ++ - - - - Only one fish had few SC and ST. 100 4 34–190 -++-+++-Gonads undergoing spermatogenesis. One fish had few PO. 200 4 50–134 +++-+++-Gonads undergoing spermatogenesis. 24 0 5 72–168 +++++---Relatively few GC were observed in most fish. One fish had atretic oocytes. 50 5 138–236 ++ + + - - - Proliferation of stromal cells and occurrence of a few PO were observed. 100 5 77–338 ++ ++ + - - - Proliferation of stromal cells and occurrence of a few PO were observed. 200 5 115–208 ++ + + - - - Proliferation of stromal cells and occurrence of a few PO were observed.

G, gonia; PO, primary oocytes; SC, spermatocytes; ST, spermatids; SZ, spermatozoa. -, none; +, few; ++, moderate; +++, abundant.

Table 2 Total number of groupers stocked, sampled, recorded mortality, unaccounted, and remaining during the mibolerone experiment Treatment Fish stocked Fish sampled Recorded mortality Unaccounted Remaining (mg/kg feed) 03013 4 94 50 30 14 7 3 6 100 30 13 3 12 2 200 30 12 7 10 1

number of spermatocytes and spermatid (Fig. similarly showed a proliferation of stromal cells, 2b,c). However, spermatozoa were not observed. few to moderate gonia, and few primary oocytes When the treatments had been withdrawn for (Fig. 2d). No spermatogenic cells could be 8 weeks at Week 24, the gonads of ﬁsh previously observed at this stage. given mibolerone at 50, 100, and 200 mg/kg feed During the experiment, mortalities caused by Induced sex inversion in grouper FISHERIES SCIENCE 235

bacterial infection were observed. The first occurrence was on the 4th week wherein a fish was found dead in the group fed diet that did not contain mibolerone. On Week 11, mortalities were also observed in fish given mibolerone at 50 mg/kg (1 fish) and 200 mg/kg (2 fish). Mortalities were observed until Week 16. The total recorded mortality and unaccounted fish are summarized in Table 2. The mean BW and relative weight gain of fish during the sampling period are shown in Table 3. In general, the fish grew during the experimental period. The slowest growth was observed from Week 8 to Week 16 wherein the range of the relative weight gain among treatments was from 9.5 to 21.2%. From Week 16 to Week 24, growth of the fish was fastest (191.9–201.9%).

DISCUSSION

Results of the present study, showed that juvenile E. coioides, despite its small size (19–168 g initial BW), can be inverted into males as early as 7 weeks after the start of treatment by incorporating mibolerone into the diet. Although spermatogenesis was activated, no spermatozoa were observed Fig. 1 Cross-section of grouper gonads sampled as initial control. G, gonia; PO, primary oocytes. (a) Section in the gonad sections of ﬁsh treated with any showing the presence of stromal cells and gonia in cysts. dose of mibolerone. Spermatogenesis was more Scale bar represents 20 mm. (b) Immature ovary showing advanced in 100 and 200 mg/kg feed compared to abundant primary oocytes. Scale bar represents 50 mm. 50 mg/kg feed which lasted from Week 8 to Week 16.

Fig. 2 Cross-section of grouper gonads during the mibolerone treatment and after withdrawal. G, gonia; PO, primary oocyte; SC, spermatocytes; ST, spermatids. (a) Section showing atretic oocytes from a ﬁsh given 0 mg/kg feed at Week 24. Scale bar represents 150 mm. (b) Section showing different spermatogenic cells. Scale bar represents 50 mm. (c) A closer view of gonia and spermatocytes. Scale bar represents 20 mm. (d) Section showing proliferation of stromal cells after the withdrawal of treatment (Week 24). Scale bar represents 20 mm. 236 FISHERIES SCIENCE GF Quinitio et al.

In mature female grouper, MT has only been reported to be successful in inducing sex inversion by oral administration.2–4,6 Chao and Chow3 also observed sex inversion in immature female E. tauvina (1100–2600 g initial BW) given oral administration of MT at 1 mg/kg BW three times per 17.6 84.5 13.3 81.9 39.9 122.0 35.4 105.2 ± ± ± week. Occurrence of spermatogenic cells and ± atretic previtellogenic oocytes was observed as early as 2 weeks after treatment. Using injection 24 Week as a mode of administration, Tan-Fermin et al.9 were also able to sex inverse 2-year-old juvenile E. coioides using MT at 0, 0.5, or 1.0 mg/kg BW every 15 days for 6 months. Regardless of treatment, gonad sections of fish with a minimum bodyweight sampled of 1.2 kg showed the presence of spermatozoa and milt was expressed in some of the bigger fish. However, Tan-Fermin et al.9 did not observe spermatogenesis in fishes less than 1.2 kg BW thereby concluding that induction of sex inversion in juvenile grouper is probably synergistic with age and 8.9 12.1 9 121.6 7.3 21.2 11 201.9 14.1 9.5 6 201.6 size. Our results showed that mibolerone can 13.3 15.4 7 191.9 ± ± ± ± induce sex change even in grouper as small as 25 g during the mibolerone experiment

BW (given 100 mg/kg feed at Week 8). This shows 16 Week that mibolerone is a very potent agent in inducing sex change in E. coioides. Meriwether and Torrans10 observed that mibolerone required only about 1% of the androgen ethynyl testosterone to achieve similar results in sex reversal of tilapia. sampled When the mibolerone treatment was terminated, the gonads of grouper in all treatments coioides Epinephelus reverted back to female. Sex reversal to female was also observed in male E. coioides that were sex inversed from immature female13 and in E. tauvina that were inversed from immature, mature, and mature resting female3 after termination of 6.9 38.4 15 65.9 6.3 27.8 15 111.0 8.9 42.7 12 90.8 3 8.5 23.9 13 93.5 ± ± ± hormone treatment. Chao and Chow have attrib- ± uted this reversal to the absence of Leydig or inter- Week 8 Week stitial cells in the gonads of MT-treated groupers. Leydig cells are involved in the production of androgens so that without these cells the withdrawal of androgen treatment caused the reversion to female. sampled The permanence of sex inversion in groupers

may be inﬂuenced by factors such as age, size, and weight gain of juvenile SEM) and relative

1,9,14,15 1 ± 4.6 24 58.8 4.5 29 91.6 6.4 26 82.9 social aspects. Quinitio et al. clearly demon- 6.3 28 81.0 ± ± ± strated that size plays a role in sex inversion when ± mature female groupers are isolated from a group under captive conditions.

In the present study, we have shown that 0 Week mibolerone which is recommended for prevention 30 42.5 30 71.7 30 58.1 30 65.4 of estrus in adult female dogs is also very effective fish (g) weight fish (g) weight gain (%) fish (g) weight gain (%) fish (g) weight gain (%) in inducing sex inversion in immature juvenile sampled groupers. Therefore, it is also highly possible to apply this hormone to mature female groupers to bodyweight (mean Mean produce functional males. However, tests still have to be conducted to determine the required dosage ( m g/kgfeed) of No. Body- of No. Body- Weight of No. Body- Weight of No. Body- Weight Treatment 0 0.5 Table 3 2.0 of mibolerone. It is also interesting to note that the 1.0 Induced sex inversion in grouper FISHERIES SCIENCE 237

loss of appetite after feeding the fish artificial feed ration, spawning and embryogeny of the protogynous only for 8 weeks or even thereafter when raw grouper, Mycteroperca microlepis. J. World Maricult. Soc. fish was added to the artificial feed. Based on 1983; 14: 639–649. this observation, it may be best to inject the 5. Yeh S-L, Luo W-S, Ting Y-Y. Studies on the sexual conversion mibolerone into the raw fish that is to be fed since of grouper with hormonal treatment. Bull. Taiwan Fish. Res. Inst. 1986; 41: 241–258 (in Chinese). the groupers had good appetite when given raw 6. Kuo C-M, Ting Y-Y, Yeh S-L. Induced sex reversal and spawn- fish alone. ing of blue-spotted grouper, Epinephelus fario. Aquaculture Another question that may be considered is the 1988; 74: 113–126. effect of mibolerone on growth of grouper. It seems 7. Chao TM, Lim LC. Recent developments in the breeding of that the mibolerone treatment did not affect their grouper (Epinephelus spp.) in Singapore. Singap. J. Prim. growth because the highest weight gain was Ind. 1991; 19: 78–93. observed in 200 mg/kg at Week 8 while at Week 16 8. Yeh S-L, Ting Y-Y, Kuo C-M. Induced sex reversal of grouper the highest was at 50 mg/kg. However, this observa- (Epinephelus salmonoides; Epinephelus fario) after implan- tion is not conclusive since no proper replication tation of pelleted androgen. Bull. Taiwan Fish. Res. Inst. was done. 1988; 45: 103–114 (in Chinese). 9. Tan-Fermin JT, Garcia LMB, Castillo Jr AR. Induction of sex inversion in juvenile grouper, Epinephelus suillus (Valenci- ennes) injections of 17alpha-methyltestosterone. Jpn. J. ACKNOWLEDGMENTS Ichthyol. 1994; 40: 413–420. 10. Meriwether FH, Torrans EL. Evaluation of a new androgen (mibolerone) and procedure to induce functional sex rever- The authors thank Mary Ann Garcia, Nora B. sal in tilapia. In: Maclean JL, Dizon LB, Hosillos LV (eds). The Caberoy, and Deogracias Reyes, Jr for their techni- First Asian Fisheries Forum. Asian Fisheries Society, Manila. cal assistance. We are also grateful to Dr Rafael 1986; 675–678. Guerero III who provided the mibolerone used at 11. Torrans L, Meriwether F, Lowell F. Sex-reversal of Ore- the start of the study and the remaining require- ochromis aureus by immersion in mibolerone, a synthetic ments were kindly provided by Animal Health steroid. J. World Aquacult. Soc. 1998; 19: 97–102. Division, Upjohn Inc. (The Philippines). 12. Humason GL. Animal Tissue Techniques, 3rd edn. W.H. Freeman, San Francisco. 1972. 13. Tan-Fermin JD. Withdrawal of exogenous 17-alpha methyltestosterone causes reversal of sex-inversed male grouper REFERENCES Epinephelus suillus (Valenciennes). Philipp. Sci. 1992; 29: 33–39. 1. Quinitio GF, Caberoy NB, Reyes Jr DM. Induction of sex 14. Shapiro DY. Sexual differentiation, social behavior and sex change in female Epinephelus coioides by social control. reversal in coral reef fishes. Bioscience 1987; 37: 490–497. Isr. J. Aquacult. Bamidgeh 1997; 49: 77–83. 15. Debas L, Fostier A, Fuchs J, Weppe M, Nedelec G, Benett A, 2. Chen FY, Chow M, Chao TM, Lim R. Artificial spawning and AQUACOP, Cauty C, Jalabert B. The sexuality of cultured her- larval rearing of the grouper, Epinephelus tauvina (Forskal) maphroditic fish species: Analysis of morphological and in Singapore. Singap. J. Prim. Ind. 1977; 5: 1–21. endocrinological features in a protogynous hermaphrodite, 3. Chao TM, Chow M. Effect of methyltestosterone on gonadal Epinephelus microdon, as a basis for research to control development of Epinephelus tauvina (Forskal). Singap. J. reproduction in the grouper. In: Barrett J (ed.). Advances Prim. Ind. 1990; 18: 1–14. in Tropical Aquaculture. AQUACOP, IFREMER Actes de 4. Roberts Jr DE, Schlieder RA. Induced sex inversion, matu- Colloque 9, Tahiti. 1990; 545–559.