302 REPLACEMENT OF FETAL BOVINE SERUM WITH KNOCKOUT SERUM REPLACER AND STEM CELLS CONDITIONED MEDIUM DURING IN VITRO CULTURE OF BOVINE EMBRYOS

Abstract

The use of fetal bovine serum (FBS) as protein supplementation in IVP of bovine embryos has presented difficulties because it can introduce a number of pathogenic components in culture systems, can be related to the birth of calf with abnormal growth and development, and precludes the establishment of the actual nutritional needs of the embryo, because it contains an unlimited variety of substances. This study evaluated the replacement of the FBS in the medium of in vitro culture (IVC) of bovine embryos, using the knockout serum replacer (KSR) as protein supplementation and culture medium conditioned with stem cells. Therefore, bovine oocytes from ovaries of slaughterhouse were selected and matured in vitro in TCM-199 medium supplemented with 10% FBS (Crypion), 1.0 μg mL^-1 FSH (Pluset^®, Calier, Barcelona, Spain), 50 μg mL^-1 hCG (Profasi^®, Serono, Geneva, Switzerland), 1.0 μg mL^-1 estradiol (Sigma E-2758, Sigma Chemical, St. Louis, MO, USA), 0.2 mM sodium pyruvate, and 83.4 μg mL^-1 amikacin for 24 h. After that, 1144 oocytes were fertilized in IVF-TALP medium containing 6 mg mL^-1 of BSA. After 18 to 22 h, the zygotes were cultured in SOF + 5% FBS (group 2); SOF + 5% KSR (group 3); SOF (5% FBS) + 10% SOF (5% FBS) conditioned by stem cells (group 4); or SOF (5% KSR) + 10% SOF (5% KSR) conditioned by stem cells (group 5), in an atmosphere of 5% O² at 38.5°C for 8 days. A control group outside the controlled atmosphere was added, supplemented with 5% FBS (group 1). The SOF medium supplemented with 5% FBS or KSR was conditioned by stem cells and added to SOF medium for the culture of embryo at a concentration of 10%. The rates of cleavage and production of blastocysts were assessed 48 hours and 7 days after IVF, respectively, and analyzed by chi-square test, with a significance level of 5% in the statistical program Minitab^® (release 14.1, Minitab, State College, PA, USA). On the eighth day, the TUNEL test for determination of the percentage of apoptosis and the differential staining technique for determination of inner cell mass (ICM) and trophoblast (TF) were performed. The results were submitted to ANOVA, followed by comparing the means by Tukey’s test using the program GraphPad Prism (GraphPad, San Diego, CA, USA). The treatments did not differ in the production of embryos, being similar to the control group: G1 = 31.75% (74/233), G2 = 35.26% (79/224), G3 = 32.70% (74/226), G4 = 28.76% (63/219), and G5 = 26.85% (65/242). With regard to the assessment of embryonic quality, the treatments showed similar results to the control groups. No differences were observed among groups both in color and ICM/TF ratio (G1 = 0.60, G2 = 0.62, G3 =0.65, G4 = 0.60, and G5 = 0.60). Furthermore, the TUNEL showed no significant difference in the percentage of apoptosis among groups (G1 = 7.10%, G2 = 3.76%, G3 = 5.58%, G4 = 4.50%, and G5 = 4.11%). The data obtained so far indicate that it is possible to produce embryos in vitro by replacing the FBS in the culture, achieving results similar to those obtained with serum.

Financial support: FAPESP 2007/58506-6.