318 EFFECT OF PIG FOLLICLE FLUID AND FETAL CALF SERUM ON PORCINE OOCYTE MATURATION AND SUBSEQUENT DEVELOPMENT AFTER ACTIVATION AND SOMATIC CELL NUCLEAR TRANSFER

Abstract

In vitro maturation of porcine oocytes is very important for understanding porcine somatic cell nuclear transfer (SCNT). In order to develop an in vitro maturation system that can provide more high quality oocytes, the effect of porcine follicle fluid (pFF) (gathered from 3–5-mm porcine follicles) and fetal calf serum (FCS: Sigma, St. Louis, MO, USA), as an important additional component of a chemically-defined medium was studied. Cumulus-oocyte complexes (COC) derived from follicles 3–5 mm in diameter were cultured in three different media: a chemically-defined medium (CDM: TCM-199 with 0.1 mg mL⁻¹ cysteine, 10 ng mL⁻¹ EGF, 0.5 μg mL⁻¹ LH and 0.5 μg mL⁻¹ FSH); CDM with 10% pFF (CDM + p); and CDM with 10% FCS (CDM + F). After 42–44 h of maturation, oocytes with a clear polar body were classified as matured oocytes. Matured oocytes stimulated by electric pulse (120 v, 30 μs, 2 pulse), or enucleated and fused with fibroblasts to construct SCNT embryos by using the same electrical parameters. All of these parthenogenetic and SCNT embryos were cultured in Porcine Zygote Medium-3. The blastocyst rate was assessed under a stereomicroscope on Day 6, and the number of nuclei in the blastocysts was counted under a fluorescent microscope after staining with 5 μg mL⁻¹ of Hoechst 33342. All data were subjected to a Generalized Linear Model Procedure (PROC-GLM) of Statistical Analysis System (SAS). The maturation rates of porcine oocytes in CDM and CDM + p were 53.2 ± 3.8% (539/1050) and 69.7 ± 3.8% (587/847), respectively;; in CDM and CDM + F, 61.1 ± 3.1% (471/776) and 70.2 ± 3.7% (577/844), respectively. Oocytes matured in CDM + p and CDM + F showed a higher (P < 0.05) maturation rate than those in CDM. The percentages of parthenogenetic blastocysts of oocytes matured in CDM and CDM + p were 13.9 ± 2.1% (35/250) and 20.2 ± 5.3% (64/300), and the numbers of nuclei in these blastocysts were 25.8 ± 2.3 and 25.8 ± 1.4, respectively. The blastocyst rate from CDM- and CDM + F-matured oocytes were 20.1 ± 2.0% (53/272) and 22.2 ± 4.7%(71/298), and the numbers of nuclei in these blastocysts were 24.7 ± 1.5 and 25.3 ± 1.5, respectively. There were no significant (P > 0.05) differences in the percentages of parthenogenetic blastocysts and nuclei numbers between CDM and CDM + p, or CDM and CDM + F. The percentages of blastocysts in SCNT embryos derived from CDM and CDM + p were 8.1 ± 1.5% (14/192) and 12.3 ± 1.9% (24/192), while the nuclei numbers in these blastocysts were 26.6 ± 1.2 and 34.5 ± 2.2, respectively. The percentages of blastocysts after SCNT from oocytes matured in CDM and CDM + F were 24.3 ± 4.9% (35/139) and 27.1 ± 5.5% (45/176), while the numbers of nuclei were 29.8 ± 2.5 and 32.2 ± 1.9, respectively. There were no significant (P > 0.05) differences between CDM and CDM + p, or CDM and CDM + F in SCNT embryo blastocyst rate, but the SCNT embryos derived from CDM + p showed a higher (P < 0.05) nuclear number. In conclusion, these results indicate that 10% pFF or FCS in CDM can promote a higher maturation rate of porcine oocytes. As recipient cytoplasm for SCNT, oocytes matured in CDM + p can support development of blastocysts that contain more nuclei than those matured in CDM alone. Supported in part by Food for the 21st Century and RR13438.