Stage-specific effects of the osmolarity of a culture medium on the development of parthenogenetic diploids in the pig.

The objective of this study was to investigate the effects of osmolarity of culture media on the development of porcine parthenogenetic diploids. Oocyte-cumulus-granulosa cell complexes were collected from ovaries and then in vitro-cultured for 48 h. The mature oocytes were subjected to a single electro-stimulation (El-St; 100 micros, 1500 V/cm), treated with 5.0 microg/ml Cytochalasin B for 4h and then cultured under various conditions as described below. In Experiment 1, the diploids were cultured for 168 h after El-St in modified Whitten's medium with 256 mOsmol (mWM256), mKRB with 309 mOsmol, and mWM with 309 mOsmol (mWM309), in which the osmolarity was adjusted by addition of NaCl or mannitol, or by reduction of distilled water. In Experiment 2, the diploids were cultured in the five media used in Experiment 1 for the first 48 h, and then in mWM256 until 168 h after El-St. In Experiment 3, the diploids were cultured for the first 48 h in mWM with osmolarity adjusted from 256 to 330 mOsmol by addition of NaCl for the first 48 h and then in mWM256 until 168 h after El-St. In Experiment 4, the diploids were cultured in mWM with 290 mOsmol (mWM290) for the first period of 24, 48, or 72 h, and then in mWM256 until 168 h after El-St. In Experiment 5, after diploids were cultured in mWM290 for the first 48 h, the obtained 4-cell diploids were transferred to mWM with osmolarity adjusted from 200 to 310 mOsmol by addition of NaCl, then cultured until 168 h after El-St. All media were supplemented with 0.5mg/ml hyaluronic acid and 4.0mg/ml bovine serum albumin. The results obtained in Experiments 1-5 indicate that the osmolarity of a medium, but not the Na(+)/K(+) ratio, exerts effects on the development of diploids to the blastocyst stage. The change of osmolarity of the culture media after the 4-cell stage increased the rate of expanded blastocyst formation in porcine diploids. The optimal osmolarities of culture medium for the first 48 h after El-St (before the 4-cell stage) were 290 and 280-320 mOsmol, and those for the later period (after the 4-cell stage) were 256 and 220-270 mOsmol, respectively.