Cooling of Siamese fighting fish Betta splendens (Teleostei, Osphronemidae) embryos at low temperatures.

The Siamese fighting fish (Betta splendens) has great importance as an ornamental aquarium fish as well as laboratory model species. Due to its rapid development, a cooling-embryo protocol could provide some advantages in their transportation, embryonic synchronization, and optimization of hatcheries. In this context, this work aimed to develop a protocol to storage B. splendens embryos at two temperatures (5 and 14 °C), testing three cryoprotective solutions (S1: 0.5 M sucrose, 1.5 M methanol; S2: 0.25 M sucrose, 0.75 M methanol; and S3: 0.125 M sucrose, 0.375 M methanol) and evaluating the quality of the larvae obtained. Moreover, a method to isolate the embryos from the bubble nest constructed by the male and to incubate them without parental care was applied in this study. The cooling assays were done using embryos of 24-h-post-fertilization at 26 °C and the results demonstrated that it is possible to store these embryos deprived of cryoprotectants at 5 °C for at least 6-h without negative effects. Meanwhile, S2 and S3 were the most suitable solutions for its storage for 9-h at 5 °C or 24-h at 14 °C, obtaining 77% hatching and 52% normal larvae in the first case or 88% hatching and 81% larvae with mild abnormalities in the second one. Indeed, type and frequency of larval abnormalities were evaluated and, remarkably, a partial recovery was described on malformed larvae from embryo cooled at 14 °C. Finally, this work is the first report about the cooling of B. splendens embryos and establishes the conditions for further studies on this field with this species.

Cooling of pejerrey Odontesthes bonariensis (Teleostei, Atherinidae) embryos at sub-zero temperatures.

Pejerrey fish (Odontesthes bonariensis) is a seasonal multiple spawner with great economic importance and an adequate species for Aquaculture. For these reasons, it is necessary to apply biotechnologies to optimize its reproduction in captivity. In this context, the aim of this work was to develop a cooling protocol for pejerrey embryos at sub-zero temperatures. Two cryoprotective solutions (CSs: S1 and S2), two cooling curves (a fast and a slow one) and two storage temperatures (-14 and -20 °C) were evaluated for 1 h. High percentages of embryo survival (80-100%) were obtained in all cases. In particular, for cooling at -14 °C, the most suitable protocol was the slow temperature decrease in combination with S1 (2.5 M methanol, 1.4 M Me2SO, 0.3 M sucrose, and 0.08 M NaCl). The hatching rate (86.67 ± 11.55%) and the larval survival observed did not differ from those of the control group, and about 30% of normal-looking larvae were obtained. Besides, the slow cooling was also the best way to reach -20 °C, obtaining a hatching rate of around 60%. However, all the larvae had different kind of malformations. Finally, in order to improve the results obtained at -20 °C, the CSs were incorporated into the embryos by microinjection. In this case, it was observed that the most convenient combination was the microinjection of S2 (same composition as S1 but without Me2SO) in the perivitelline space followed by rapid cooling. Although the hatching rate was not improved (67.93 ± 8.31%), the microinjection allowed to obtain at least 4.5% normal-looking larvae. These results showed that the cooling of pejerrey embryos at zub-zero temperatures was feasible. Moreover, the microinjection of cryoprotectants within the pejerrey O.bonariensis embryos was employed for the first time in this species.