66 Lipid profiling of bovine blastocysts produced invitro with and without a stimulator of cyclic guanosine monophosphate synthesis by multiple reaction monitoring profiling

Cryopreservation of oocytes and embryos is an essential technique for invitro-produced cattle worldwide. One of the great difficulties of cryopreservation of oocytes and blastocysts is the accumulation of lipids in the cytoplasm when produced invitro. The lipid metabolism of oocytes and embryos is classically regulated by the cAMP pathway. Furthermore, previous studies have suggested that the cyclic guanosine monophosphate (cGMP) pathway may also be involved in modulating lipid metabolism through protein kinase G activation. The objective of this study was to investigate the lipid profile of bovine blastocysts produced invitro when stimulated by specific stimulator of cGMP synthesis (NPPB). Pools of oocytes were matured invitro for 24h in tissue culture medium 199, with 15% bovine serum, 0.5µgmL−1 FSH, 5µgmL−1 LH, 0.8mM L-glutamine, and 50µgmL−1 gentamicin at 38.5°C and 10−6 M NPPB. The control group was matured without NPPB. After 22h, the oocytes were fertilised invitro with frozen sperm. The IVM oocytes were fertilised and cultured according standard procedures (Rubessa et al. 2011 Theriogenology 76, 1347-1355). After the 7 days (Day 7), the blastocysts (from early blastocyst to expanded blastocyst) were collected, washed in methanol:water (vol/vol) 1:3, and frozen at −80°C. The lipid extraction of the samples was performed based on the standard protocol (Bligh and Dyer 1959 Can. J. Biochem. Physiol. 37, 911-917) but adapted for small samples. The samples were diluted and analysed in the Agilent 6410 QQQ (Agilent Technologies) mass spectrometer and analysed according to the multiple reaction monitoring method described by de Lima et al. (2018 J. Mass Spectrom. 53, 1247-1252). Data for 3 replicates/group were normalized and then submitted to t-test statistical analysis and principal component analysis, by Metaboanalyst 4.0, with a significance level of 5%. The rates of cleavage and blastocysts were not affected when we used the mGC stimulator presenting a 61% rate of cleavage for both groups, and 24.4% and 25% of blastocyst rate for control and NPPB, respectively (P<0.05). The results, regarding 164 lipids analysed, showed that the lipid profile was not affected when we used NPPB, maintaining the same profile of lipid classes. When we observe the quantitative values, we see a nonsignificant decrease in the lipid classes sphingomyelin, phosphatidylcholine, and triacylglycerol. The values for each class for control and NPPB, respectively, were 0.70 and 0.64 ng/blastocyst for sphingomyelin, 6.45 and 6.07 ng/blastocyst for phosphatidylcholine, and 11.82 and 10.51 ng/blastocyst for triacylglycerol (P<0.05). For the other classes of phospholipids (PE, PG, and PI), we observed a small increase when treated with NPPB, also not significant. We conclude that although we do not have significant differences between the control and the treatment, each class of lipid can respond differently when stimulated with cGMP synthesis.