Influência da baixa temperatura e diferentes crioprotetores em oócitos e embriões de Colossoma macropomum e Piaractus brachypomus

Abstract

The aim of this work was to develop methods for cryopreservation of Colossoma macropomum oocytes and oocytes and embryos of Piaractus brachypomus, as well as to analyze the damages in oocytes and embryos after exposure to cryoprotectant solutions and cryopreservation. Oocytes of C. macropomum were distributed in six cryoprotectant solutions: T1 -methanol 1.6 M + sucrose 0.25 M, T2 - methanol 1.6 M + sucrose 0.5 M, T3 - methanol 1.6 M + trehalose 0.25 M, T4 - methanol 1.6 M + trehalose 0.5 M, T5 - methanol 1.6 M + fructose 0.25 M, T6 - methanol 1.6 M + fructose 0.5 M and T7 - outbreak control. Oocytes were kept in cryoprotectant solutions for 20 minutes at room temperature, fertilized and taken to incubators. A second portion of the oocytes were underwent to slow freezing, stored in liquid nitrogen, thawed, and a part was fixed while the other was fertilized and kept in incubators. The survival probability of oocytes subjected to T1 at room temperature was higher (15.8%). As for survival after hatched larvae, there was no difference between treatments. In the fertilization of oocytes that passed by slow freezing there was not hatching. Morphological preservation was observed in T1 and T2 with cryoprotectant solution containing methanol 1.6 M and sucrose 0.25 and 0.50 M, respectively. The oocytes of Piaractus brachypomus were distributed in eight cryoprotectant solutions: T1 - methanol 1.6 M + sucrose 0.25 M + 50 % L15, T2 -methanol 3.1 M + sucrose 0.25 M + 50 % L15, T3 - DMSO 0.7M + sucrose 0.25 M + 50% L15, T4 - DMSO 1.3 M + sucrose 0.25 M + 50% L15, T5 -methanol 1.6 M + sucrose 0.25 M + Hank, T6 - methanol 3.1 M + sucrose 0,25M + Hank, T7 -DMSO 0.7 M + sucrose 0.25 M + Hank, T8 - DMSO 1.3 M + sucrose 0.25 M + Hank. The embryos frozen at -13ºC were distributed in eight cryoprotectant solutions: T1 - methanol 3.1M + 0.15M PVP + Hank, xvi T2 - methanol 3.1 M + PVP 0.3 M + Hank, T3 - methanol 3.1 M + PVP 0,45M + Hank, T4 - methanol 3.1 M + PVP 0.6 M + Hank, T5 -methanol 3.1 M + sucrose 0.45 M + Hank, T6 - methanol 3.1 M + sucrose 0.63 M + Hank, T7 - methanol 3.1 M + sucrose 0.81 M + Hank, T8 - methanol 3.1 M + sucrose 1.0 M + Hank. Embryos frozen in liquid nitrogen were distributed in four cryoprotectant solutions: T1 - methanol 3.1 M + sucrose 0.45 M + Hank, T2 - methanol 3.1 M + sucrose 0.63 M + Hank, T3 - methanol 3.1 M + sucrose 0.81M + Hank, T4 - methanol 3.1 M + sucrose 1.0 M + Hank. For morphological analysis oocytes and embryos were processed in historesin and oocytes for scanning electron microscopy technique. For oocytes of P. brachypomus kept at room temperature and fertilized treatments that showed embryo development were T5 (methanol 1.5 M, sucrose 0.25 M solution and Hank), 17.3% and T7 (DMSO 0.7 M, sucrose 0.25 M and Hank) 1.8%. In fertilization of oocytes subjected to slow freezing curve there was not embryo development after fertilization. In the morphological analysis it was found that the oocytes of P. brachypomus when subjected to slow freezing in extender solution with methanol 1.6 M + sucrose 0.25 M + solution hank (T5) showed a full zona radiata with regular contour. From embryos that were underwent freezing at -13ºC in T5, 15.3% developed and was significantly higher than in T6 (4.6%), T3 (1.5%), T7 (1.8%) and T8 (1.9%) and there was also embryo development. In other treatments, all embryos subjected to cryoprotectant solutions at a temperature of -13ºC and embryos subjected to treatments in liquid nitrogen resulted in dead embryos. No treatment with oocysts of C. macropomum and oocytes and embryos of P. brachypomus provided maintenance of fertilizing capacity after freezing.