Optimization of in vitro fertilization using cryopreserved sperm of Crassostrea angulata and establishment of a cryopreservation protocol for Chamelea gallina

• Autor(a) principal: Santos, Ana Luísa Lopes
• Data de Publicação: 2018
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10400.1/12500
• topic: Crassostrea angulata; Chamelea gallina; Criopreservação; Sémen; Fertilização

The development of methods that allow the conservation of genetic resources as sperm cryopreservation are necessary to avoid the pressure on the natural populations and the risk of extinction of Crassostrea angulata and Chamelea gallina, improving the reproductive strategies for eventually restocking programs and even aquaculture production with commercial purposes. Therefore, this work aimed to optimize the cryopreservation protocol for C. angulata testing different combinations of cryoprotectants, to assess the fertilization conditions for oyster and to assess the sperm quality parameters before and after exposure of C. gallina sperm to cryoprotectants solutions, determining the best cryopreservation conditions in terms of freezing rates. For optimization of a cryopreservation protocol for C. angulata, motility, viability and DNA integrity analysis were performed for cryopreserved sperm with dimethyl sulfoxide (DMSO), DMSO+trehalose and DMSO+sucrose. Fertilization trials with different sperm-to-egg ratios and volumes were tested and the success of fertilization trials was determined. For the assessment of sperm quality parameters of C. gallina, toxicity tests and cryopreservation assays were performed. For toxicity tests, fresh sperm was exposed to cryoprotectants (DMSO, ethylene glycol and methanol) and motility analysis were recorded. For cryopreservation assays, the same cryoprotectant conditions and two freezing rates were analysed. Motility and viability analysis were performed in post-thaw sperm. Supplementation of DMSO with sugars did not optimize motility, viability or decrease DNA fragmentation in Crassostrea angulata sperm. In fertilization trials, the best sperm-to-egg ratio using post-thawed sperm was 50,000 spermatozoa per egg. Trehalose did not improve the fertilization rate and no effect of fertilization volume was seen between different volumes. For Chamelea gallina, the toxicity trials demonstrated that DMSO was the most suitable cryoprotectant. However, post-thawed sperm showed no motility but DMSO showed the higher viability results, being the best conditions for sperm cryopreservation achieved with the fastest freezing rate. More studies are necessary to develop successfully a cryopreservation protocol for C. gallina.