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Hormonal sex reversal has been extensively used for sex determination and producing monosex fish for aquaculture purposes. Steroid hormones or hor- mone analogues as well as non-steroid compounds (Table 7.3) are commonly used for producing monosex tilapia. The hormones are generally incorporated into larval feeds and administered to undifferentiated larvae at very early larval stages (preferably at first feeding) for sufficient time to enable sex reversal. The use of hormones has been under increasing public criticism due to their possi- ble health and environmental impacts. As a result, the use of hormones for sex reversal of tilapia is either licensed (in USA) or banned (in Europe) (Penman and McAndrew, 2000).

7.8.2.1. All-male production

ORAL ADMINISTRATION. Many steroid hormones, steroid analogues or non-steroid compounds have been widely used in male sex reversal of various tilapia species. The percentage of sex-reversed males depends mainly on the type of hormones,

Hormone (compound) Abbreviation

17_{α-methyldihydrotestosterone} MDHT 17α-methyltestosterone MT 17α-ethynyltestosterone ET 17α-methyl-5-androsten-3- 17β-diol Androstenedione AN 17α-ethymyloestradiol EE Oestradiol-17β E2 Fadrozole F

Trenbolone acetate TBA

11β-hydroxyandrostenedione 11β-OHA4 Aromatase inhibitor AI Diethylstilboestrol DES Tamoxifen Acriflavine Mibolerone MI

Table 7.3. Steroid hormones, hormone analogues and non-steroid compounds used for producing monosex tilapia.

dose, timing and duration of administration, fish species and size/age of larvae, as indicated in Table 7.4. Available data indicate that 17α-methyltestosterone (MT) hormone is the most common and successful hormone used for tilapia sex reversal (Guerrero and Guerrero, 1988; Ridha and Lone, 1990; Lone and Ridha, 1993; Penman and McAndrew, 2000; Beardmore et al., 2001). The doses of steroid hormones tested for sex reversal of tilapia ranged from < 30 to 100 mg/kg food, provided for the fish for < 15–> 60 days. However, the best results have been reported at a dose of 30–60 mg/kg adminis- tered for about 25–30 days (Guerrero and Guerrero, 1988; Vera-Cruz and Mair, 1994; Green et al., 1997). Guerrero (1975) found that the administration of 30 mg 17α-ethynyltestosterone (ET)/kg feed for 18 days produced 98% males in

O. mossambicus, while 60 mg/kg produced 100%

males. MT produced 98 and 85% males at 30 and 60 mg/kg. Excessive doses of hormones may therefore reduce the percentage of sex-reversed fish, increase fish mortality and cause sterility or paradoxical sex reversal (Green et al., 1997; Beardmore et al., 2001).

The use of hormones for sex reversal has been under increasing public criticism due to their potential health and environmental impacts. Therefore, recent studies have considered the use of non-steroidal compounds to manipulate sex inversion. Non-steroidal compounds may exhibit antagonistic or agonistic effects to sex steroids that are generally involved in sexual differentiation in fish. When tilapia hybrids (O. niloticus× O. aureus) were fed tamoxifen-treated diets, 100% males were produced at a dose of 100 mg/kg feed (Hines and Watts, 1995). When acriflavine was used, 89% and 85% males were produced at 15 and 50 mg/kg diet.

IMMERSION TECHNIQUES. Oral administration of hormones for sex reversal of tilapia is generally safe and successful; however, hormone traces from uneaten food and metabolites are often a major environmental concern. Immersing fish fry in hormone solution for short periods of time has attracted attention as a successful alternative tool to overcome this problem. One of the major advantages of the immersion technique is the sub- stantial decrease in treatment period and the reduction of possible effects of the hormones on the workers (Gale et al., 1999). This technique is

also characterized by minimal intervention, which is usually reduced to the time when the fish is most sensitive to the treatments. In spite of these attrib- utes, the use of the immersion technique for masculinization of tilapia has not been developed for practical and commercial use.

The percentage of sex-reversed males pro- duced by the immersion technique ranges from < 60% to 100%, depending on fish species, type and dose of hormone and immersion period (Table 7.4). Varadaraj and Pandian (1987) found that the immersion of O. mossambicus in 5 or 10 µg/l of 17α-methyl-5-androsten-3-17β-diol for 10 days post-hatch (dph) caused 100% masculinization. Similar results were reported with O. spilurus immersed in MT (2.5 mg/l) for 4 days in brackish water, followed by oral adminis- tration of the hormone at 50 mg/kg for 35 days (Lone and Ridha, 1993). The exposure of Nile tilapia fry to 500 µg/l 17α-methyldihydro- testosterone (MDHT) for 3 h on days 10 and 13 post-fertilization (dpf) also resulted in 83–100% males (Gale et al., 1999), while low concentration of MDHT and MT (100 µg/l) caused a lower male percentage. Afonso and Leboute (2003) also inves- tigated the masculinizing potency of ET, MT and MDHT applied to Nile tilapia larvae at 14 dph, in two consecutive trials. In the first trial, the fish were subjected to a single 4 h immersion dose at concentrations ranging from 200 to 1800 µg/l. In the second trial, the fish were immersed in these androgens at 1800 µg/l for 4 h either as a single immersion at 14 days or as double immersions at 10 and 14 dph. The authors found that, when different concentrations were applied, the best male proportion was achieved at 1800 µg/l, but none of the treatments produced 100% male (86–90%). The results of the second trial con- firmed those of the first trial. However, two immersions slightly improved the male propor- tion, especially in the MT-treated group, where the male proportion increased from 92 to 98%, but two immersions decreased the male percent- age in the MDHT group to 94% compared to 100% at one immersion.

The exposure of fish larvae to ultrasound is believed to increase the transport of hormone from the water into the fish body, leading to a higher masculinization rate (Bart, 2002). Bart (2002) stud- ied the effects of ultrasound exposure time (1 and 2 h) on sex reversal of Nile tilapia, using four differ- ent hormones (androstenedione (AN), MDHT,

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Species Hormone Route Optimum dose Timing Duration % Males Remarks Reference

O. n F Food 200–500 7 dph 30 days 92.5–96 Genetically females Kwonet al. (2000)

F Food 75–100 Fry 30 days 100 Afonsoet al. (2001)

ET Food 60 Fry 25–28 days 91–99.4 Vera-Cruz and Mair (1994)

MT Food 30 Fry 21 days 99 Guerrero and Guerrero (1988)

MT Food 60 10.4 mm 14–28 days 82–92 Smith and Phelps (1997)

TBA Immersion 250µg/l 10 dpf 2 h 98–100 Exposed to ultrasound for 2 h Bart (2002)

AN Immersion 100–250µg/l 10 dpf 2 h 92 Exposed to ultrasound for 2 h Bart (2002)

MT Immersion 50µg/l 10 dpf 2 h 98 Exposed to ultrasound for 2 h Bart (2002)

MDHT Immersion 250µg/l 10 dpf 2 h 98–100 Exposed to ultrasound for 2 h Bart (2002)

MT Food 60 10 dpf 10–30 dpf 92 Galeet al. (1999)

MT Immersion 500µg/l 10 and 13 dpf 3 h 87 Galeet al. (1999)

MDHT Immersion 500µg/l 10 and 13 dpf 3 h 83–100 Galeet al. (1999)

MDHT Immersion 1800µg/l 14 dph 4 h 100 Single immersion Wassermann and Afonso (2003)

MT Immersion 1800µg/l 10 and 14 dph 4 h 98.3 Two immersions Wassermann and Afonso (2003)

ET Immersion 1800_µg/l 14 dph 4 h 86.7 Two immersions Wassermann and Afonso (2003)

O. m MDHT Immersion 5–10_µg/l 10 dph 10 days 100 Varadaraj and Pandian (1987)

MT Food 30 18 days 98 Guerrero (1975)

ET Food 60 9–11 mm 18 days 100 Guerrero (1975)

Continued

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Chapter 7

Species Hormone Route Optimum dose Timing Duration % Males Remarks Reference

O. a MI Immersion 0.6 ppm 9–11 mm 35 days 82. Torranset al. (1988)

MT Food 50 9 mm 42 days 100 Hines and Watts (1995)

ET Food 60 Fry 25–28 days 83–97 Mélard and Ducarme (1993),

Mélardet al. (1995)

TBA Food 25 9.1 mm 28 days 98.3 Galvezet al. (1996)

O. s MT Food 70 Fry 38 days 90 Ridha and Lone (1990)

MT Immersion 2.5 ppm Yolk-sac fry 4 days 100 In BW, later fed MT at

50 mg/kg feed

Lone and Ridha (1993) Red

tilapia

11β-OHA4 Food 50 10 dpf 28 days 99.1 Desprezet al.(2003)

O.n× O. a

Tamoxifen Food 100 9 mm 42 days 100 Hines and Watts (1995)

O.n× O. a

ET Food 60 Fry 25–28 days 96–100 Rothbardet al.(1983)

O. a,O. aureus;O. m,O. mossambicus;O. n,O. niloticus;O. s,O. spilurus; dph, days post-hatch; dpf, days post-fertilization; BW, brackish water.

Reproduction and Seed Production 129

MT and trenbolone acetate (TBA)). He found that subjecting the fry (10 dpf) to ultrasound for 2 h resulted in a significant increase in the male percentage, compared to 1 h exposure. In addi- tion, TBA (250 µg/l) produced 98–100% males and was not significantly different from 100 µg/l. A lower percentage of males was produced with the MDHT treatment (90.5%).

The immersion of tilapia eggs, instead of fry, has recently emerged as an effective tool for inducing sex reversal. When fertilized eggs are immersed in the hormone solution, the hormone is absorbed through passive diffusion across the lipid membrane of the egg. One major advantage of this technique is the substantial reduction in the amount of hormone used, since small amounts of water are generally used for egg incu- bation compared to the amounts used in larval holding units. As yet, little information is avail- able on the sex reversal of tilapia using the immersion technique. Only two studies have been reported in this regard. The first study was reported in Thailand, where the immersion of 2-day-old Nile tilapia eggs in MT at 500 µg/l, for 24 h resulted in 88% males (Anon., 2002, cited in Cagauan et al., 2004).

More recently, Cagauan et al. (2004) evalu- ated the sex reversal of Nile tilapia in the Philippines, by immersing fertilized eggs (3–4 days old) in different concentrations of MT (0, 200, 400, 600 and 800 µg/l) for 24, 48, 72 and 96 h. The highest male proportion of 91% was obtained at 800 µg/l for 96 h. However, the results of this study are questionable. At 72 and 96 h, it is very likely that some of the eggs (which were already 3–4 days old) had hatched into sac fry. It is possi- ble, therefore, that, at long immersion times (72 and 96 h), the authors may have immersed sac fry rather than eggs. Further investigations are there- fore urgently needed to improve the immersion technique in order to increase the percentage of sex-reversed tilapia to 100% or close to 100%.

Oestrogen biosynthesis is mediated by the steroidogenic enzyme cytochrome P450 aromatase, which converts androgens to oestrogens (Afonso

et al., 2001). Non-steroidal aromatase inhibitors

(AI) have been shown to alter the sex of several animals, including tilapia. Afonso et al. (2001) fed Nile tilapia larvae with diets containing 0, 50, 75 and 100 mg/kg of the aromatase inhibitor Fadrozole (F) for 15 and 30 days, starting 9 days after hatching. Regardless of the feeding period,

the proportion of males was significantly higher in the treated groups, and 100% males were pro- duced at the high doses (75 and 100 mg/kg) for 30 days. This result means that 100% Nile tilapia males can be produced by suppressing aromatase activity.

7.8.2.2. All-female production

In many fish species, females grow at higher rates and attain larger sizes than males. Males may also mature before females reach marketable size, leading to dispersion of sizes and reduction in production. The production of all-female fish is an effective way of solving these problems and controlling reproduction of cultured tilapia. There- fore, the interest in producing an all-female popula- tion for aquaculture purposes has increased. As for males, several natural or synthetic steroids, mainly oestrogens or other chemicals with oestrogenic capacity, have been used to feminize tilapia. Both oral administration and immersion protocols have been used with varying degrees of success (Table 7.5). Synthetic chemicals have been used for tilapia feminization at larger scales than natu- ral steroids, with 17α-ethynyloestradiol (EE) and diethylstilboestrol (DES) being the most common and the most effective.

The relative effectiveness of steroids on feminization of tilapia depends on the type of hor- mone, fish species, larval stage, dose and treatment timing and duration. For example, Rosenstein and Hulata (1994) found that DES was more effective in feminizing blue tilapia (O. aureus) than EE, while the opposite was found in the case of Nile tilapia (Gilling et al., 1996). Moreover, Rosenstein and Hulata (1992) failed to feminize O. mossambicus and

O. mossambicus × Oreochromis urolepis hornorum

hybrids by immersing eggs and fry in oestradiol-17β (E2), progesterone, flutamide and progesterone+ flutamide at different solutions and durations. However, these authors reported a 100% feminization when oestrogens (DES and EE) were orally administered to these fish. On the other hand, EE, DES and E2 successfully feminized Nile tilapia fry using the immersion technique (Gilling et al., 1996). It appears from these results that more work is needed to verify whether oral administration or the immersion technique is more effective and would produce higher all-female populations.

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Species Hormone Route Optimum dose (mg/kg) Fish age/size Duration (days) % Females Reference

O. niloticus EE Immersion 170–200µg/l Fry 18 100 Gillinget al. (1996)

DES Food 400 8.7 mm 28 80 Potts and Phelps (1995)

EE Food 100 7–12 mm 40 91 Mohamedet al. (2004)

O. mossambicus DES Food 100 10 days old 11 100 Varadaraj (1989)

EE Food 100 Yolk-sac fry 10 100 Rosenstein and Hulata (1994)

O. aureus EE Food 100 Yolk-sac fry 10 100 Rosenstein and Hulata (1994)

EE_{+ Methallib Food} 100 Fry 42 95 Mairet al. (1987)

EE Food 100–200 Fry 40 93–98 Mélard (1995)

O. spilurus EE Food 100 9 mm 42 92 Ridha and Lone (1995)

Reproduction and Seed Production 131

7.8.3. Hybridization

Hybridization of two different species has been extensively and successfully applied to produce monosex populations. There are several condi- tions affecting the success of hybridization of related fish taxa, including: (i) external fertiliza- tion; (ii) unequal abundance of two parental spe- cies; (iii) weak behavioural isolating mechanisms; (iv) competition for limited spawning habitat; and (v) decreasing habitat complexity (Scribner et al., 2001). These factors are very likely to fit with most tilapias; therefore, tilapia have received a good deal of attention, and have become the best-known example of hybrid production in aquaculture, since they are characterized with short life cycles and frequent spawning.

A substantial amount of information is now available on all-male and all-female tilapia hybrids (Tables 7.6 and 7.7). Most of the crosses carried out for the production of monosex tilapia have been

between maternal mouthbrooders (Wohlfarth and Hulata, 1983; Beardmore et al., 2001). Most of crosses between pure mouthbrooding tilapias result in all-male or nearly all-male hybrids (Lovshin, 1982; McAndrew, 1993; Penman and McAndrew, 2000) (Table 7.6). All-male progenies have been produced from the following crosses: male

O. hornorum× female O. niloticus (Lee, 1979), male O. aureus× female O. niloticus (Wohlfarth, 1994),

male O. mossambicus × female O. aureus (Pierce, 1980), male O. hornorum× female O. mossambicus (Hickling, 1960) and male O. mossambicus× female

O. spilurus niger (Pruginin, 1967). Other crosses

resulted in 50 to 98% males (Table 7.6). The failure to produce all-male progenies, in many cases, has been attributed to poor segregation of broodfish by sex and species, and also to the introduction of hybrids into broodstock ponds (Beardmore et al., 2001). Tilapia strains may also influence the results of tilapia hybridization, as reported by Marengoni

et al. (1998). These authors found that, when Nile

Species hybridized

% Males Reference

Male Female

O. niloticus O. aureus 75–95 Pruginin (1967)

O. spilurus niger 93–98 Pruginin (1967)

O. hornorum 75 Pruginin (1967)

O. aureus O. niloticus 50–100 Lee (1979), Wohlfarth

(1994) O. niloticus (Stirling strain) 100 Marengoniet al. (1998) O. niloticus (Uganda strain) 96–100 Prugininet al. (1975) O. mossambicus 100 Beardmoreet al. (2001)

Tilapia vulcani 93–98 Prugininet al. (1975)

O. mossambicus O. aureus 89 Pierce (1980)

O. hornorum O. niloticus 100 Lee (1979), Wohlfarthet al.

(1990)

O. mossambicus 100 Hickling (1960)

O. aureus 90–100 Pruginin (1967), Lee (1979)

O. spilurus 100 Pruginin (1967)

O. macrochir O. mossambicus 100 Majumdaret al. (1983)

O. spilurus 97.9 Majumdaret al. (1983)

O. niloticus 100 Pruginin (1967)

tilapia females were bred with blue tilapia males, only the Stirling strain of Nile tilapia produced 100% males, while a Japan strain produced only 91% males.