Cryopreservation toxicity and morphological outcomes in Piaractus brachypomus oocytes and embryos

This study evaluated the toxicity of cryoprotectants and the performance of low-rate freezing protocols for oocytes and embryos of Piaractus brachypomus, a Neotropical fish of increasing relevant for Brazilian aquaculture and genetic conservation. Eight cryoprotectant solutions based on methanol (MeOH) or dimethyl sulfoxide (Me₂SO), combined with 0.25 M sucrose in L-15 or HBSS media, were tested for oocytes toxicity at 28 °C and post-freezing viability. In spite of histological and scanning electron microscopy (SEM) analyses indicated preservation of gross morphological features, none of the cryopreserved oocytes supported embryonic development, indicating loss of functional viability following cryoprotectant exposure and freezing. For embryos, two low-rate freezing protocols were evaluated: Protocol 1 (P1E), based on gradual cooling to -13 °C, and Protocol 2 (P2E), involving linear cooling to -60 °C followed by storage in liquid nitrogen. In P1E, eight treatments using 3.1 M MeOH combined with different concentrations of polyvinylpyrrolidone (PVP) or sucrose were tested. The highest proportion of morphologically viable embryos (15.3%) was obtained with MeOT + 0.45 M sucrose (SC5), which different significantly from the other treatments. In contrast, no morphologically viable embryos were recovered after P2E, likely due to inadequate dehydration and intracellular ice formation. Overall, high concentrations of permeant cryoprotectants and prolonged equilibration times were detrimental to oocyte and embryo viability, while sucrose showed better cryoprotective performance than PVP. Even though protocols tested were insufficient to ensure consistent post-thaw viability, the partial success observed in P1E under MeOH and sucrose combinations provides a relevant experimental basis for future refinement of conservation strategies and contributes to development of ex situ germoplasm conservation approaches for P. brachypomus and other Neotropical species.