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Two groups of female eels were used in the present study. The first consisted of wild female eels, A. japonica [Silver eel; body weight (BW): 570–840 g] that were caught from the Nakdong River (Busan, Korea) during October and November 2011. The second comprised cultivated female A. japonica (BW: 550–720 g) that were feminized by estradiol-17ß administration (Kim et al., 2013) and were reared at the Inland Aquaculture Research Center for 3 years. Male eels (BW: 250–420 g) that were reared at the Inland Aquaculture Research for 2 years were also transported to the National Institute of Fisheries Science (NIFS) along with the feminized eels as a resource for subsequent experiments. All experimental eels were acclimated to seawater and kept without feeding in 1000 L flow-through tanks under a water temperature of 20°C.

Hormonal treatment was carried out for artificial maturation as described previously (Kim et al., 2006, Kim et al., 2007). Female eels were repeatedly injected with salmon pituitary extract (20 mg acetone-dried pituitary powder/individual), followed by injection with 17α, 20ß-dihydroxy-4-pregnene-3-one (2 μg/g BW, Sigma, St. Louis, MO, USA), and male eels were repeatedly injected with human chorionic gonadotropin (hCG; 1 IU/g BW/week, Dae-Seung Microbiology Co. Ltd., Seoul, Korea). After 8–10 hormone injections, eggs and semen were obtained from matured female and male eels, respectively.

The eggs used in the present study were ovulated from a single wild female eel (ID:1072） and a single feminized eel (ID:5444) that had been matured at the same time. From these, 2 g of ovulated eggs was inseminated with 1 mL pre-diluted milt (sperm motility >80 %) for the determination of egg fertility and hatchability in micro-plates; the majority of ovulated eggs were inseminated with enough volume of pre-diluted milt for the mass production of larvae (Ohta et al., 1997).

The flotation, hatching, and survival rates of the inseminated eggs were determined using a micro-plate method (Unuma et al., 2004). Just after insemination in sea water, 3 mL seawater containing over 300 eggs was transferred to a dish and the number of floating and sinking eggs was counted for flotation rate count. Approximately 100 floating eggs were transferred individually into the wells of 48-well micro-plates (SPL, Seoul, Korea) that were filled with 1 mL filtered (pore size, 0.2-μm) seawater containing 100,000 IU/L penicillin G (Janssen Pharmaceutical, Geel, Belgium), 0.1 g/L streptomycin sulfate (USB Corporation, Cleveland, OH, USA), and 1 μg/mL poly-ethylene glycol 6,000 (PEG 6,000; Calbiochem, Darmstadt, Germany). The plates were maintained at 23°C in an incubator with humidity maintained at 100% to avoid evaporation of the rearing water. Fertilization rate was checked at 6 h after insemination. The flotation rates of the fertilized eggs obtained from the wild female and feminized eels were 97.7% and 92.6%, respectively. The fertilization rates at 6 h after insemination were 68.7% and 67.6% respectively(<Table 1>). The number of hatched larvae at 3 days after insemination and surviving larvae at 8 dah in each 48-well plate were counted. The hatching rates at 3 days after artificial insemination in the two broodstock groups were 42.3% in the wild female and 45.5% in the feminized eel. The survival rates at 8 days after hatching in were 20% and 22%, respectively. The number of final metamorphosed larvae was compared between the two separate 20 L aquariums (about 1500 larvae/aquarium, 4 aquariums for each group). The final metamorphosis rate was converted into percentages; the assessment period lasted until 14 months from hatching. After 14 months following hatching, the final metamorphosed rates were 0.1% in the wild female aquarium and 0.15% in the feminized aquarium.

Mass fertilized and hatching larvae were first kept in 500 L polycarbonate tanks supplied with filtered seawater (Cartridge filter pore size: 10-μm) at 23°C for 8 dah until functional mouth development occurred. Afterward, approximately 1500 larvae at initial densities were transferred to one round-type 20 L aquarium using a silicon tube as a siphon. Filtered seawater was supplied at a rate of 0.9–1.0 L/min. The larvae were fed a shark egg-based slurry-type diet that was modified from Tanaka et al.(2003) five times a day at 2 h intervals from 9 dah (Kim et al., 2014). The water supply to the rearing tanks was stopped when the diet was added. After feeding (for 20 min), the water supply was restarted to flush out any uneaten diet. After the final flush, each rearing tank was connected to a clean tank with a polycarbonate tube to transfer the living eel larvae by siphoning. In this study, two separate aquariums were observed. One aquarium contained larvae from the wild female eel and the other held larvae from the feminized eel.

To assess larva size, 5–10 larvae were randomly sampled from each aquarium on Days 30 and 60 after hatching and then fixed in 5% formalin. However, as the remaining populations were below 80 to 10 larvae in each aquarium by 120–257 dah, respectively, the mean sizes estimated for these days might be biased. The total length (TL) was measured under a binocular microscope after anesthetized with 100 ppm 3-aminobenzoic -acid ethyl ester (MS-222; Sigma). Photographs of the larvae were taken using an Axio Imager A1 microscope (Carl Zeiss, Oberkochen, Germany) and images were captured using an Axio Cam MRC5 camera (Carl Zeiss) and AxioVision software (Axiovs40, v 4.6.3.0).

Eel larvae from the wild female and feminized eels were photographed at 30, 60, 120, 150, 170, 192, and 212 dah. The total body length gradually increased prior to metamorphosis; quantification of the daily growth is displayed in [Fig. 1] as divided between 0–120 and 121–212 dah. The growth rates of the eel larvae from the wild female eel were 0.2500 mm/day during 0–120 dah and 0.1718 mm/day during 121–212 dah. The body shape became elongated and compressed and the body depth gradually increased. Upon reaching approximately 30 mm in TL, the body shape appeared as a leaf shape. This tendency progressed until metamorphosis ([Fig. 1, 2]).

[Fig. 1] 
Growth in total length of eel larvae from wild female (A) and feminized (B) eels. TL: total length (mm).

[Fig. 2] 
Morphological changes in larvae from the wild female (A) and feminized (B) eels from 30–212 dah.

In comparison, the growth rates of the eel larvae from the feminized eels were 0.2690 mm/day during 0–120 dah and 0.2053 mm/day during 121–212 dah. The leptocephali from both the wild female and feminized eels grew in body length and width until metamorphosis, with a similar tendency of change in body shape between both groups ([Fig. 1, 2]).

Metamorphosis in the eel post-larvae occurred after approximately 245 dah (from 245–260 dah); the patterns of morphological changes were similar between the two groups. The head and tail regions were slenderized, the body changed to a cylindrical shape, and the membranous caudal fin was elongated toward the anterior ([Fig. 2]). The position of the anus gradually moved to the anterior region and the body length began to shorten somewhat as the individual began the process of metamorphosis ([Fig. 3]).

[Fig. 3] 
Morphological changes in post-larvae from the wild female (A) and feminized (B) eel during metamorphosis (wild eel, 248 and 256 dah; feminized eel, 247 and 256 dah). Arrows: position of the anus.

As the first step of metamorphosis, the width of the eye shortened ([Fig. 4]). The teeth had degenerated by the completion of metamorphosis and gill development was predominant, such that the increase of length and density of the gill filaments could be readily observed([Fig. 4]).

[Fig. 4] 
Morphological changes from the wild female in the head region in post-larvae during metamorphosis. From 247–256 dah are shown.