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.

中文翻译：

---

在零以下温度下冷却 pejerrey Odontesthes bonariensis (Teleostei, Atherinidae) 胚胎

Pejerrey 鱼 (Odontesthes bonariensis) 是季节性多重产卵者，具有重要的经济意义，是水产养殖的合适品种。由于这些原因，有必要应用生物技术来优化其圈养繁殖。在这种情况下，这项工作的目的是为低于零温度的 pejerrey 胚胎制定冷却方案。评估了两种冷冻保护溶液（CS：S1 和 S2）、两条冷却曲线（快速和慢速曲线）和两种储存温度（-14 和 -20 °C），持续 1 小时。在所有情况下都获得了高比例的胚胎存活 (80-100%)。特别是，对于 -14 °C 的冷却，最合适的方案是与 S1（2.5 M 甲醇、1.4 M Me2SO、0.3 M 蔗糖和 0.08 M NaCl）结合缓慢降温。孵化率（86.67±11。55%)，观察到的幼虫存活率与对照组没有差异，获得了约 30% 的外观正常的幼虫。此外，缓冷也是达到-20°C的最佳方式，孵化率在60%左右。然而，所有的幼虫都有不同类型的畸形。最后，为了改善在-20°C 下获得的结果，通过显微注射将 CSs 掺入胚胎中。在这种情况下，观察到最方便的组合是在卵周空间显微注射 S2（与 S1 的成分相同但不含 Me2SO），然后快速冷却。虽然孵化率没有提高 (67.93 ± 8.31%), 但显微注射允许获得至少 4.5% 的正常幼虫。这些结果表明，在 zub 零温度下冷却 pejerrey 胚胎是可行的。