153 SUPPLEMENTATION WITH LINOLENIC ACID AND L-CARNITINE DURING IVM REDUCED THE EXPRESSION OF GENES RELATED TO LIPOGENESIS BUT DID NOT ALTER THE LIPID CONTENT AND CRYOTOLERANCE OF IN VITRO-PRODUCED EMBRYOS
Authors: Leao B. C. S. , Rocha Frigoni N. A. S. , Dall'Acqua P. C. , Ambrogi M. , Nunes G. B. , Rodrigues L. T. , Silva M. I. A. , Mingoti G. Z. 
Published: December 2016

This study was conducted to evaluate the impact of supplementation during in vitro maturation (IVM) with linolenic acid (ALA), l-carnitine (L-car), or the combination of both supplements on the embryo intracellular lipid content and cryotolerance, as well as in the embryo expression of genes involved in lipid metabolism (lipogenesis regulation: SCD1, FASN, and SREBP1; and β-oxidation pathway: CPT1B and CPT2). Cumulus-oocyte complexes (n = 1076) were IVM for 22 h at 38.5°C and 5% CO2 in air, in TCM-199 medium with bicarbonate, hormones, and 10% FCS (control group), supplemented with 100 μM ALA (ALA group), 5 mM L-car (L-car group), or a combination of 100 μM ALA + 5 mM L-car (ALA + L-car group). After IVF, presumptive zygotes were in vitro cultured in SOFaa medium supplemented with 5 mg mL−1 BSA and 2.5% FCS, at 38.5°C and 5% CO2 in air during 7 days. Cleavage and blastocyst rates were evaluated on Day 3 and 7, respectively (IVF = Day 0). At Day 7, the blastocysts were stained with the lipophilic dye Sudan Black B (n = 60), vitrified/warmed (n = 260; Ingámed® protocol, Maringa-PR, Brazil), or collected for analysis of gene expression (n = 180). Embryonic development were analysed by ANOVA and the multiple comparisons of means were determined by Tukey’s test. The embryonic re-expansion data were subjected to chi-square test and the differences in gene expression among groups were evaluated by Duncan’s multiple range test (P < 0.05). Data are presented as means ± standard error means. There was no effect (P > 0.05) of the supplements used during IVM on cleavage (79.54 ± 2.76% to 82.16 ± 1.13%) and blastocyst rates (29.03 ± 3.07% to 30.46 ± 2.01%). Similarly, the intracellular lipid content in Day-7 blastocysts (1.03 ± 0.04 to 1.15 ± 0.07 pixels) and the embryonic cryotolerance, assessed by the re-expansion rates after 24 h (67.3 to 78.3%) hatching rates after 48 h (11.5 to 25.5%) of post-warming culture, were unaffected (P > 0.05) by the supplements of IVM medium. Although the treatments did not alter (P > 0.05) the expression of CPT1B and CPT2 genes, the expression of FASN gene was decreased (P < 0.05) in the ALA group and the expression of SREBP1 gene was decreased (P < 0.05) in the ALA and L-car groups. The expression of the gene SCD1 was reduced (P < 0.05) in all treatments compared with the control group. Thus, despite the lack of effects of the treatments performed during IVM on the intracellular lipid content and cryotolerance of the embryos derived from the treated oocytes, a reduction in the expression of genes related to lipogenesis was observed in Day-7 blastocysts. These results suggest that treatments performed in the oocytes during IVM may have prolonged effects, affecting the subsequent expression of genes in embryos. Further studies are needed to determine the mechanisms related to the differentiation of the oocyte machinery during maturation.
Financial support was provided by FAPESP (#2012/10084–4 and #2013/07382–6).