Effects of activation methods and culture conditions on development of parthenogenetic porcine embryos

Abstract

The effects of different activation methods and culture conditions on early development of porcine parthenotes were examined. Three different activation methods were tested: (1) electroporation; (2) electroporation followed by incubation in the presence of butyrolactone I, an inhibitor of cdc2 and cdk2 kinases; and (3) electroporation followed by a treatment with cycloheximide, a blocker of protein synthesis. The activated oocytes were cultured in two different media, NCSU-23 and PZM-3 under 5% CO 2 in air. In a separate experiment, the effects of high (∼20%) or low (5%) O 2 tension on early embryo development were also evaluated. The average pronuclear formation was less ( p < 0.05) in the electroporated oocytes (83.9 ± 1.7%) compared with those activated by electroporation and butyrolactone I or electroporation plus cycloheximide (92.8 ± 0.8 and 93.0 ± 1.0%). In PZM-3 medium, the average frequencies of blastocyst formation (59.7 ± 3.6%) and hatching (10.6 ± 1.3%) were greater than those in NCSU-23 medium (39.9 ± 3.1% blastocyst formation, p < 0.05; and 0.2 ± 0.2% hatching; p < 0.001). Furthermore, the average nuclear number was also greater ( p < 0.001) in blastocysts developed in PZM-3 (50.2 ± 1.3) than in those developed in NCSU-23 (35.3 ± 1.1). Blastocyst formation was similar ( p > 0.10) among the three activation procedures when parthenotes were cultured in NCSU-23, while in PZM-3 more ( p < 0.05) parthenotes produced by electroporation plus butyrolactone or electroporation plus cycloheximide developed into blastocysts compared to electroporation alone (64.9 ± 5.2 and 68.6 ± 3.5% compared with 45.6 ± 4.7%). Incidences of apoptotic nuclei were similar ( p > 0.10) among all treatments. No difference in development was found between parthenotes that developed under high versus low O 2 tension ( p > 0.10). These results demonstrate that activation methods targeting the calcium signaling pathway at several points trigger embryonic development more efficiently than electroporation alone. The data also imply that the PZM-3 medium provides for enhanced culture conditions for the early development of parthenogenetic porcine embryos than NCSU-23.