.2. Broodstock management for the first trial (2009–2010 season)
Yearling ELTs were caught by set net or hook and line, and were held in a net pen located in Kushimoto, Wakayama Prefecture, Japan. On 22 July 2009, 32 of the 1-year-old ELTs were transferred to Tateyama Station and used as broodstock. To maintain the rearing water temperature during the winter season in 2009–2010, seawater was heated by passing it through a boiler (23 °C) from 31 December 2009 to 2 April 2010. During this period, the average and minimum water temperatures in the tank were 16.2 ± 0.9 °C and 14.2 °C, respectively, whereas the average and minimum temperatures of natural seawater were 14.5 ± 0.9 °C and 12.5 °C, respectively. Eastern little tuna broodstocks were maintained under a long-day photoperiod (16 h of light/8 h of dark) throughout the year using a 480-lux metal halide lamp, while a 12-lux incandescent lamp was used as a nightlight to prevent the fish from colliding with the tank wall, because ELTs maintained in tanks often collide in the dark.
The ELTs were fed primarily defrosted sand lance (Ammodytes personatus) and krill (Euphausia superb) at 5–10% of their body weight (BW) per day. As is customary with tunas maintained in tanks, throughout the year some fish collided with the tank walls and either died immediately or they were seriously injured. When fish died of such accidental causes during the rearing period, their TL, BW, and gonadal weight were measured. The gonadosomatic index (GSI) was calculated according to the following formula: GSI (%) = (gonadal weight [g]/BW [g]) × 100. To investigate gonadal development in tank-reared ELTs during the first trial (2009–2010 season), the gonads of ELTs that died of accidental causes were removed and examined histologically, as described below.
Fourteen of the 32 ELTs survived 1 year of rearing in a 70-m3 land-based tank, and these fourteen 2-year-old ELTs were kept in the same tank and used as broodstocks for the first spawning induction trial in the 2010 spawning season. As the broodstocks did not spawn between May and September 2010, the first implantation of GnRHa-containing cholesterol pellets was carried out with 11 (7 females and 4 males) of the 14 broodstocks on 17 September 2010 in the 70-m3 land-based tank. The second GnRHa implantation was carried out on 6 October 2010 with 7 (4 females and 3 males) of 12 broodstocks (2 female broodstock had died by the time of the second GnRHa administration). Six of the 7 broodstocks used for the second GnRHa administration had also been subjected to GnRHa implantation in the first trial on 17 September 2010 (these fish were administered GnRHa twice in 2010). During the spawning induction experiments, the water temperature ranged from 23.7 to 26.6 °C. Both GnRHa administration experiments were performed in the same 70-m3 land-based tank. Because all broodstocks could not be captured for the implantation due to technical difficulties, GnRHa was not administered to 3 fish in the first implantation and 5 fish in the second implantation.