393
Fish were fed daily with fresh sardines, with half of the individuals treated with
the androgynous hormone 17- alpha-methyltestosterone (in capsules) with 1 mg per
kg of each individual. The other half received sardines with placebo, only to evaluate the effectiveness of hormonal treatment. The methodology adopted was successfully used in prior investigations of sexual inversion of the dusky grouper (Sanches
et al. 2009).
After 6 months applying the androgynous hormone, in an unprecedented way for
the species, it was confirmed the sexual inversion of the treated individuals, with
production of semen. Fish who were receiving placebo pills kept the ovotestis as
immature female, which was observed after the collection of oocytes, demonstrating their functional condition. Collecting semen from small-size individuals (one of
the fish had only 2.0 kg) was a great achievement – in Asia the males of other aquacultured Epinephelidae weight around 40 kg.
Through a partnership with researchers from Embrapa Tabuleiros Costeiros
(EMBRAPA, Brazilian Agricultural Research Corporation), the extracted semen
was characterized in its volume, sperm density, viability, motility rate, and duration
of motility using the Sperm Class Analyzer (SCA). This was the first time this system was used to analyze the semen of marine fish in Brazil, and the first time for
Goliath Grouper semen.
After the success in controlled sexual inversion with the acquisition of viable
sperm, experiments were performed to find a proper cryopreservation procedure.
The freezing preservation of semen brings countless advantages to the development
of a cultivation protocol. It allows the optimization for transportation, as well as
semen storage in gene banks. These banks present great benefits, such as: maintenance of semen stock of a large number of individuals in cryogenic tanks, which
guarantees the genetic viability and the exchange among many research institutes, a
continuous supply of semen throughout the reproductive season, and the reduction
of costs in maintenance of broodstocks.
The freezing protocol was a successful methodology for the E. marginatus. A
diluent, with pH 8.2 and 172 mOsm was used, together with the following chemical
compound (Diluent (g/L): NaCl, 7.89; KCl, 1.19; CaCl 2 , 0.22; MgC l2 , 0.72531;
NaH 2 PO 4 , 0.0805; NaHCO 3 , 0.84; pH 8.2; 172 mOsm). The ratio semen:diluent was
1:3 (v/v), with the cryoprotector (dimethyl sulfoxide) at 10%, equilibrium time of 1
min, and freezing speed of −60 °C/min. The freezing equipment used was used a
cryogenic tank with nitrogen vapor (CP 100 Taylor-Wharton – Harsco Corp.,
Theodore, AL, USA), at −196 °C. The cryopreserved semen was stored in a
Cryometal tank of 34 l.
The medium-size broodstocks (between 50 and 100 cm) used in experiments of
female gonadal maturation were collected in supply channels of two shrimp farms,
one in the Brazilian state of Pernambuco, and the other from Bahia. Fish were kept
in the laboratory in Ubatuba in recirculating aquaculture systems (RAS) of 3000 l,
with skimmers and water sterilization (using ultra-violet filters), with filtering rate
of 200% of the total volume per hour. The quality of the water, together with the
sterilization system permitted elevated survival rates and no report of diseases.
14 The Atlantic Goliath Grouper: Conservation Strategies for a Critically Endangered…
Fish were fed daily with fresh sardines, with half of the individuals treated with
the androgynous hormone 17- alpha-methyltestosterone (in capsules) with 1 mg per
kg of each individual. The other half received sardines with placebo, only to evaluate the effectiveness of hormonal treatment. The methodology adopted was successfully used in prior investigations of sexual inversion of the dusky grouper (Sanches
et al. 2009).
After 6 months applying the androgynous hormone, in an unprecedented way for
the species, it was confirmed the sexual inversion of the treated individuals, with
production of semen. Fish who were receiving placebo pills kept the ovotestis as
immature female, which was observed after the collection of oocytes, demonstrating their functional condition. Collecting semen from small-size individuals (one of
the fish had only 2.0 kg) was a great achievement – in Asia the males of other aquacultured Epinephelidae weight around 40 kg.
Through a partnership with researchers from Embrapa Tabuleiros Costeiros
(EMBRAPA, Brazilian Agricultural Research Corporation), the extracted semen
was characterized in its volume, sperm density, viability, motility rate, and duration
of motility using the Sperm Class Analyzer (SCA). This was the first time this system was used to analyze the semen of marine fish in Brazil, and the first time for
Goliath Grouper semen.
After the success in controlled sexual inversion with the acquisition of viable
sperm, experiments were performed to find a proper cryopreservation procedure.
The freezing preservation of semen brings countless advantages to the development
of a cultivation protocol. It allows the optimization for transportation, as well as
semen storage in gene banks. These banks present great benefits, such as: maintenance of semen stock of a large number of individuals in cryogenic tanks, which
guarantees the genetic viability and the exchange among many research institutes, a
continuous supply of semen throughout the reproductive season, and the reduction
of costs in maintenance of broodstocks.
The freezing protocol was a successful methodology for the E. marginatus. A
diluent, with pH 8.2 and 172 mOsm was used, together with the following chemical
compound (Diluent (g/L): NaCl, 7.89; KCl, 1.19; CaCl 2 , 0.22; MgC l2 , 0.72531;
NaH 2 PO 4 , 0.0805; NaHCO 3 , 0.84; pH 8.2; 172 mOsm). The ratio semen:diluent was
1:3 (v/v), with the cryoprotector (dimethyl sulfoxide) at 10%, equilibrium time of 1
min, and freezing speed of −60 °C/min. The freezing equipment used was used a
cryogenic tank with nitrogen vapor (CP 100 Taylor-Wharton – Harsco Corp.,
Theodore, AL, USA), at −196 °C. The cryopreserved semen was stored in a
Cryometal tank of 34 l.
The medium-size broodstocks (between 50 and 100 cm) used in experiments of
female gonadal maturation were collected in supply channels of two shrimp farms,
one in the Brazilian state of Pernambuco, and the other from Bahia. Fish were kept
in the laboratory in Ubatuba in recirculating aquaculture systems (RAS) of 3000 l,
with skimmers and water sterilization (using ultra-violet filters), with filtering rate
of 200% of the total volume per hour. The quality of the water, together with the
sterilization system permitted elevated survival rates and no report of diseases.
14 The Atlantic Goliath Grouper: Conservation Strategies for a Critically Endangered…