15. Can post-thaw sperm quality affect progeny from European seabass?

The European sea bass (Dicentrarchus labrax) represents one of the most popular fish species in fisheries and aquaculture all over Europe. In order to pursue more effective production or genetic selection, the cryopreservation of fresh semen assumes an important role. However for both applications, cryopreserved sperm must guarantee the conservation of genetic material avoiding progeny alterations. Functional genomics offers the possibility to discover the molecular mechanisms underlying such changes, and thus markers for assisting selection methods and cryopreservation protocols. In the present work, sperm samples (n = 6) were collected using abdominal massage and sperm was immediately diluted 1:3 (v/v) in NAM media. Two extenders were used: NAM + 10% Me2SO and NAM + 10% Me2SO containing as supplements 0.25 mM vit E, 5 mM vit C, 0.25 mM DHA and 12 mM Se. For cryopreservation, diluted sperm was diluted again (1:3, v/v) in each extender, loaded into 0.5 mL straws and placed at 6.5 cm over the N2 surface for 15 min. Straws were plugged in the N2 and stored until used. For thawing, straws were immersed in a water bath at 35 °C for 15 s. Post-thaw sperm quality was assessed using motility analysis (CASA software), cell viability (IP/SYBR-14), DNA integrity and fertilization ability (hatching rate). Progeny profile gene expression, both from larvae sired with fresh and cryopreserved sperm was performed using a custom oligo DNA microarray for D. labrax comprising 19,048 unique transcripts. Results demonstrated a decrease in sperm quality after cryopreservation using both extenders. In around 1% of the genes, 149 transcripts were significantly regulated between individuals originating from fresh semen when compared with cryopreserved. Individuals originating from cryopreserved semen with supplements just showed 77 differential expressed genes when compared with cryopreserved semen without supplements. The differences between fresh semen and cryopreserved with supplements involved a larger number of differential expressed genes. A set of genes were selected for quantitative RT-PCR validation and fold change correlation between microarray and qPCR data was always significant. Despite the well-known limitations of annotation in non-model species, important information was obtained to identify biological processes that are significantly enriched among differentially expressed genes. New insights were obtained on gene related mechanisms involved in stress and primary metabolism, suggesting that proper cryopreservation conditions can be achieved following clues provided by gene expression profiles.