Cross-validation of techniques for measuring lipid content of bovine oocytes and blastocysts.
The main objective was to test and validate a fluorescence approach to quantify lipid content of individual bovine oocytes and blastocysts. For Experiment 1, denuded oocytes were evaluated, as well as in vitro-produced blastocysts in a factorial design: cows versus feedlot heifers; three additives during Days 2.5-7.5 of culture (Control; 10% FCS; 0.3 μM phenazine ethosulfate (PES), an electron acceptor that oxidizes NADPH); and two blastocyst stages (early versus expanded). All blastocysts were graded subjectively for darkness (1 = clear … 4 = dark). In Experiment 2, denuded oocytes were used to measure lipid content in a factorial design of: cows versus heifers and four subjective darkness grades (1 = clear … 4 = dark). To quantify lipids, oocytes and 7.5 d blastocysts were fixed and then stained with 1 μg/mL Nile Red dye in mPBS overnight. A digital photograph of the equatorial part of the oocyte and embryo was taken at 200×, and fluorescence intensity (Arbitrary Fluorescence Units, AFU) was measured with Image Pro software. Reverse images of the same photographs were used to count numbers of cytoplasmic lipid droplets of various sizes (LC). The linear regression equation of LC with AFU in oocytes had an r(2) = 0.84, and for blastocysts r(2) = 0.91. The LC and AFU also had similar coefficients of variation from the ANOVA for blastocysts (38 vs 44%, respectively). Treatment differences were of similar magnitude with both procedures: lipid content in oocytes and blastocysts from heifers and cows was similar (P > 0.1); PES reduced lipid accumulation, and FCS increased it relative to the Control for AFU (18.6 vs 46.6 vs 36.9 units, respectively), and LC (1763 vs 4081 vs 3310, respectively; all, P < 0.01). Early blastocysts resulted in more lipid accumulation per unit area than expanded ones based on AFU (41.5 vs 26.6) and LC (3519 vs 2583; both P <0.01). There was a strong relationship (P < 0.01) between subjective oocyte and blastocyst darkness and lipid content. The less labor intensive fluorescence staining was a reliable technique for quantifying lipid droplets in oocytes and blastocysts.
Animal Reproduction and Biotechnology Laboratory, Colorado State University, Fort Collins, CO 80523-1683, USA.
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Barceló-Fimbres
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Seidel
D000818:Animals; D001755:Blastocyst / Q000737:chemistry*; D002417:Cattle; D002478:Cells, Cultured; D046149:Embryo Culture Techniques / Q000662:veterinary; D005260:Female; D005307:Fertilization in Vitro / Q000662:veterinary; D005456:Fluorescent Dyes; D008055:Lipids / Q000032:analysis*; D008297:Male; D008856:Microscopy, Fluorescence / Q000379:methods*; D009865:Oocytes / Q000737:chemistry*; D010078:Oxazines
D005456:Fluorescent Dyes; D008055:Lipids; D010078:Oxazines; C044808:nile red