131 Fatty acid binding protein inhibition as a strategy to reduce the lipid content of in vitro-produced bovine embryos

Abstract

Lipid accumulation decreases cryopreservation survival of in vitro-produced embryos, reducing pregnancy rate after embryo transfer. Fatty acid binding protein 3 (FABP3) plays a role in lipid transport from cumulus cells to the oocyte during maturation. Blocking this transport could reduce lipid content in the oocyte and embryo and increase cryopreservation survival. This preliminary study aimed to test the effect of α-truxillic acid (FABP-I), a chemical molecule that inhibits FABP3/5 action by receptor competition, on lipid content of matured oocytes and blastocysts after culture. Slaughterhouse-derived cumulus-oocyte complexes were matured with 0 (control), 10, 50, 100 and 500nM FABP-I for 22h. In Experiment 1, 346 oocytes in 3 replicates were fixed following maturation and stained with 1μg mL−1 Nile Red to evaluate total lipid content; maturation was assessed by nuclear staining with 10μg mL−1 Hoechst 33342[ACD1]. In Experiment 2, 876 cumulus-oocyte complexes in 5 replicates were matured for 22h under the same concentrations of FABP-I, then fertilized for 18h and cultured for 7 days. Cleavage and blastocyst development were evaluated on Day 2 and 7, respectively. Blastocysts were fixed at Day 7 and stained with Nile Red. Fluorescence intensity was measured in arbitrary units using ImageJ (NIH), and data was analysed using GLM procedure of SAS (SAS Institute Inc., Cary, NC, USA). In Experiment 1, maturation rate did not differ among treatments (70.2±8.7; P=0.7). There were significant effects of treatment, replicate and interaction of treatment by replicate on fluorescence intensity. Compared with control (23.6±0.6), intensity was lowest in oocytes matured with 500nM FABP-I (21.2±0.6; P<0.01) and highest in the 10nM group (26.5±0.6; P<0.01). Staining intensity tended to decrease in the 100nM group (22.1±0.6; P=0.09) and was not different in the 50nM group (24.0±0.7; P=0.6). In Experiment 2, cleavage rate (75.8±2.9; P=0.3) did not differ and blastocyst development tended to be different among treatments (P=0.06). Compared with the control (33.3±4.8), the 500nM group had lower development (17.0±4.8; P<0.03); 10 and 50nM groups had numerically lower (24.7 and 24.0±4.8) and the 100nM group had the highest development rate (37.3±4.8), although either was significant. Treatment tended to affect fluorescence intensity of blastocysts (P=0.07; n=209), and there were significant effects of replicate and interaction between replicate and treatment. Compared with the control (11.7±1.3), fluorescence intensity was lower in the 50nM group (6.8±1.3; P<0.01), whereas 10nM had a tendency for lower intensity (8.3±1.2; P=0.06). Groups 100 and 500nM were not significantly different from controls (9.4±0.9 and 10.7±1.5, respectively). In conclusion, addition of FABP-I up to 500nM did not affect maturation or embryo cleavage but altered blastocyst development. Exposure to 50nM reduced staining intensity in blastocysts without significant decrease in development, whereas 100nM resulted in numerically lower oocyte staining intensity and higher blastocyst development. Future experiments will evaluate cryopreservation survival of embryos treated with FABP-I, and embryo transfer.