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

• Published in: Animal reproduction science 2008-03, Vol.104 (2), p.264-274
• Main Authors: Nánássy, László, Lee, Kiho, Jávor, András, Macháty, Zoltán
• Format: Article
• Language: English
• Subjects: 4-Butyrolactone - analogs & derivatives; 4-Butyrolactone - pharmacology; Animals; apoptosis; blastocyst; butyrolactone l; Cell Culture Techniques - methods; Cell Culture Techniques - veterinary; Culture Media; cycloheximide; Cycloheximide - pharmacology; electroporation; Electroporation - veterinary; Embryo; embryo (animal); embryo culture; embryo transfer; embryogenesis; Embryonic Development - drug effects; Embryonic Development - physiology; Female; in vitro fertilization; in vitro maturation; nuclear transplantation; Oocyte; Oocyte activation; oocytes; Oocytes - physiology; optimization; oxygen; Oxygen - pharmacology; parthenogenesis; Parthenogenesis - drug effects; Parthenogenesis - physiology; Pig; Preimplantation development; Protein Kinase Inhibitors - pharmacology; Protein Synthesis Inhibitors - pharmacology; sows; Swine - embryology; Swine - physiology
• ISSN: 0378-4320; 1873-2232
• DOI: 10.1016/j.anireprosci.2007.01.019

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.