Seleção de oócitos suínos através de Brilliant Cresyl Blue

Abstract

The production of swine embryos in vitro requires efficient in vitro maturation (IVM), which can be achieved by selection the most competent cumulus-oocyte complexes COC).The Brilliant Cresyl Blue (BCB) dye allows the selection of COC with complete growth by assessing their levels of the G6PDH enzyme. However, there is a possible negative effect of selection with BCB. This effect may be due to its intrinsic toxicity or to factors related to the composition of the media used during the test. This research had the objectives: to determine potential toxicity after exposure to BCB and to evaluate the effect of different medias for BCB staining on the ability to support oocyte development. On the first research, after BCB staining and after IVM, several tests were performed to evaluate the effects of their potential toxicity on mitochondrial activity and functionality: reactive oxygen species (ROS), ATP, mitochondrial membrane potential and the number of copies of mitochondrial DNA. The results showed that oocytes stained with BCB produced high levels of ROS, compared with control immediately after staining and after the IVM. The ATP and mitochondrial membrane potential showed similar results between groups after staining, however, after IVM oocytes BCB showed lower membrane potential and ATP. There was no difference in the number of copies of mtDNA in the evaluated groups. Already, on test of ATP content in early embryos, ATP was lower in BCB oocytes, however, there was no difference statistical. In second study, the most commonly used media, D-PBS, was compared with a more elaborate media for BCB called here: ReproPEL. The COC s submitted to both media were submitted to nuclear and cytoplasmatic maturation, parthenogenetic activation, and to the comet test. The great rates of nuclear IVM (P<0.05) were obtained for DPBS+ (63.1%), ReproPELc (55.1%) and ReproPEL+ (50.2%). The group with smaller area of CG (P<0.05), showing better migration, were ReproPELc, D-PBS+, D-PBS- and ReproPEL+. The parthenogenetic activation indicated that ReproPEL media presented satisfactory capacity of oocyte maintenance, resulting in acceptable rates of development to blastocyst stage: 13.0% for ReproPEL+; and 12.7% for ReproPELc. So, the ReproPEL media can be used for maintenance of swine oocytes, but it was not the most appropriate media for BCB staining. Moreover, after exposure to BCB and after IVM, BCB oocytes presented high toxicity at mitochondrial level, due to increased production of ROS, decreased membrane potential and compromised ATP production. However, the mitochondrial function was restored in early embryonic development. In conclusion, BCB was responsible for toxicity in immature swine oocytes, nevertheless, further studies must be performed to evaluate the changes caused by BCB in the embryonic level.