This study evaluated the effect of the cryoprotectants and the low temperatures on the embryonic development of Prochilodus lineatus, describing their main morphological alterations. On chilling sensitivity test, the survival rates at the twenty somites stage (20S) were 53.6% at 0ºC, and 100% in 5ºC. To test toxicity, the embryos were exposed to a graded series of 1,2-Propanediol (PROP), dimethyl sulfoxide (Me 2 SO 4 ) and glycerol (GLY), terminating in a solution of high osmolarity. There was no significant difference in the embryos survival of toxicity test between series of PROP and Me 2 SO 4 in the 6S and 20S. In the cooling protocols, were evaluated the effects of low temperature associated with cryoprotectants. At 5ºC, PROP showed survival rates above 75% in the gastrula stage (G) and above 90% in the 6S and 20S stages. High rates of abnormalities were observed, and the most recurrent were: small bodies, fins presenting uncontrolled cell growth, membrane rupture, and retraction. These results demonstrate the need to use cryoprotectant solutions, even when there is no ice nucleation, and, on the other hand, shows that high cryoprotectant concentrations promote numerous morphological lesions, compromising normal embryonic development.Keywords: Cooling, Cryopreservation, Injuries, Morphology, Toxicity.Este estudo avaliou os efeitos dos crioprotetores e das baixas temperaturas no desenvolvimento embrionário de Prochilodus lineatus, descrevendo suas principais alterações morfológicas. Nos testes de sensibilidade ao frio, as taxas de sobrevivência, no estágio de vinte somitos (20S), foram de 53,6% a 0ºC e 100% a 5ºC. Nos testes de toxicidade, os embriões foram expostos a uma série graduada dos crioprotetores: 1,2-Propanediol (PROP), dimetilsulfóxido (Me 2 SO 4 ) e glicerol (GLY), terminando em uma combinação de alta osmolaridade. Não houve diferença significativa na sobrevivência dos embriões nas séries do PROP e Me 2 SO 4 nos estágios de 6S e 20S. Nos protocolos de resfriamento, foram avaliados os efeitos da baixa temperatura associados as séries crioprotetoras. A 5ºC, o PROP apresentou taxas de sobrevivência acima de 75% no estádio de gástrula (G) e acima de 90% nos estádios 6S e 20S. Foram observadas altas taxas de anormalidades sendo as mais recorrentes: corpos pequenos, nadadeiras apresentando crescimento celular descontrolado, ruptura da membrana e retração vitelínica. Estes resultados demonstram a necessidade do uso das soluções crioprotetoras, mesmo não havendo nucleação de gelo e, em contrapartida, demonstra que as elevadas concentrações crioprotetoras promovem numerosas lesões morfológicas, comprometendo o desenvolvimento embrionário. Palavras-chave:Criopreservação, Injúria, Morfologia, Resfriamento, Toxicidade.

The objective of our work was to describe a low toxicity cryoprotectant solution that allowed vitreous solid formation. Embryos of Prochilodus lineatus were submitted to sensitivity evaluations of six internal cryoprotectants (dimethyl sulphoxide -glycerol -GLY and 1,2-propanediol -PROP) at concentrations of 1-6 M; and two external cryoprotectants (sucrose -SUC and glucose -GLU) at concentrations of 0.1-1 M for 20 min. The capacity of the cryoprotectant solutions to exchange heat with the medium and to form glassy solids was evaluated by immersing 10 ll of cryoprotectant in liquid nitrogen. The PROP had a high survival rate at all concentrations evaluated, and was the only substance that allowed a vitreous solid formation.Thus, it is concluded that the PROP-6 M was the most adequate solution for embryonic vitrification processes, because heat exchange between the system (PROP 6 M/embryos/liquid nitrogen) was faster than for other cryoprotectants and combinations thereof; has low toxicity, promote high rates of dehydration in short periods, and reach the vitreous state, being a good candidate to be used in the tests of embryonic vitrification.

Summary Aiming to provide data on the biology of Leiarius marmoratus, which will aid in its production in captivity, as well as in studies for its preservation in the environment, this work had as objectives: analyze and describe main morphological alterations during larval ontogeny of the species. We analyzed 205 individuals, obtained by induced reproduction (Colpani Pisciculture) and kept in CEPTA/ICMBIO, Pirassununga, São Paulo, Brazil. Analyses were performed from hatching moment to 30th day. The specimens were classified into two periods: larval (Stages: vitelline, pre‐flexion, flexion, post‐flexion) and juvenile. Hatched larvae showed ident chromatophores only at anterior and posterior extremities of yolk sac. The standard length ranged from 2.16 mm (yolk) to 28.84 mm (Youth). Dorsal fin rays were initially observed at flexion stage (12–14 rays). Major alterations occurred during post‐flexion/juvenile stage, when dorsal, pectoral, pelvic, anal, and caudal fins were observed and pigmentation intensified throughout the lateral region, forming bands in the body, one between the end of the head and beginning of dorsal to pelvic fin, and another one beginning at dorsal to caudal peduncle and four longitudinal at the head.

This study aimed to evaluate the vitellogenic transference and incorporation of long-chain polyunsaturated fatty acids (LC-PUFA) into the membranes of Prochilodus lineatus embryos, aiming to increase the permeability to cryoprotectants and resistance to electric fields. One hundred thirty broodstock of P. lineatus were fed with control (C) or fish oil-supplemented diets (FO) for 12 months. The fatty acid (FA) profle was determined using gas chromatography. For the neutral fraction, the FO group had a decrease in monounsaturated fatty acids (MUFA) and an increase in n3PUFA and, n6PUFA. To test for cryoprotectant toxicity, embryos were exposed for 20 min to a cryoprotectant solution of 1,2-Propanediol (Prop) at a concentration of 5 or 6 molar (M). For FO, a reduction in survival of 33.1% was observed in 5 M, and no survival was observed at 6 M. Embryo samples were exposed the six polarized electric fields (3.4-51.6 joules), and with 11.2 J of energy, the control group exhibited reduced survival in 98.3% of the fish, whereas the FO presented superior resistance, exhibiting a survival similar to that of the OJ up to 40.2 J. We conclude that FA were transferred between P. lineatus broodstock to the embryos, with an increase in LC-PUFA resulting in lower survival rates in the cryoprotectant test in the FO group and a greater physical plasticity of FO embryos to electrical field tests. K E Y W O R D Sbiotechnology, cryoprotectants, electric field, fatty acids composition, Prochilodus lineatus, toxicity