70 Does the addition of docosahexaenoic acid to in vitro systems during culture improve the quality of bovine embryos?

Abstract

The aims of this study were to decrease the apoptotic index and increase cryotolerance of bovine embryos produced in vitro with the addition of 1 µM docosahexaenoic acid (DHA). On Day 1, presumed zygotes were cultivated with 1 µM DHA (Sigma, St. Louis, MO, USA; n = 437), and without the agonist (control group; n = 450). The cleavage rate (%) was evaluated on Day 2 and the development of blastocysts on Day 7. Embryos before and after vitrification were fixed for the TUNEL trial. After vitrification, the embryos were heated and re-cultivated to evaluate the hatching rate at 12, 24, 36, 48, 60, and 72 h. A sample of re-cultivated embryos at 12 h of DHA (n = 5), and without the agonist (control group; n = 6), was frozen for mass spectrometry (MALDI-MS). Statistical analyses of deviance were carried out considering generalized mixed linear models, and the effect of the collection day (block) was considered as random. For the count variables, the Poisson distribution and the log link function were considered. In the cases of variables represented by rates, binomial distribution and the logit link function were used. In the study of cryotolerance, ANOVA of the hatching rate for each one of the times evaluated was carried out. In cases of significance of the effect of treatments, the Dunnett test was applied to compare treatments. Multivariate and univariate statistical models were used for analysis of MALDI-MS. All analyses were made using the GLIMMIX procedure of the SAS software (SAS Institute Inc., Cary, NC, USA). The cleavage rate was not different between the groups (P > 0.05) and the production of blastocysts was lower in the DHA group (P < 0.05). The number of cells per embryo was higher (P < 0.05) by the addition of 1 μM DHA in blastocysts pre- and post-vitrification. The rate of total and internal cell mass apoptosis in fresh embryos (11.73 and 15.98%) increased compared with the control group (9.62% and 11.03%, respectively). The proportion of internal cell mass in fresh embryos decreased in the DHA group (39.93%) compared with the control group (57.00%). Hatching rates at 48, 60, and 72 h after devitrification in the group treated with 1 μM DHA were not different (P > 0.05) compared with the control group. Phosphatidylcholine [phosphatidylcholine (32: 0) + H] was more abundant (P < 0.05) in embryos cultured with DHA, and thus was considered as a negative apoptosis biomarker. In conclusion, the use of 1 μM DHA in vitrification of bovine embryos impairs embryonic quality and development under the conditions of the present study.

Research was supported by CAPES, FAPEMIG, PPGCV/UFLA, EVUFMG, CENATTE EMBRIÕES.