Effect of pronase treatment, microdissection, and zona pellucida removal on the development of porcine embryos and blastomeres in vitro

The in vitro development of porcine blastomeres and the effects of pronase treatment, microdissection, and zona pellucida removal used in the isolation procedure were investigated. Seven hundred and forty-nine two to eight-cell embryos were collected from 11 sows and 74 gilts. Zona-free porcine blastomeres (ISOL BL) were obtained by treating embryos with 2.5 or 5.0% pronase for 3.0 min and microdissecting with finely drawn siliconized glass pipettes. The effect of the pronase treatment on subsequent in vitro development was evaluated by treating two to eight-cell embryos with 5.0% pronase for 3.0 min (PTD EMB). The effect of pronase treatment and microdissection on in vitro development was evaluated by microdissecting PTD EMB, leaving one blastomere bounded by the zona pellucida (BL ZP). Untreated two to eight-cell embryos were cultured as controls (CONTROLS). Embryos and blastomeres were cultured individually in microdrops of Whitten's medium with 15 mg/ml bovine serum albumin (WM + BSA) under paraffin oil in a humidified atmosphere of 5% CO2 in air at 37 degrees C. Observations were conducted at 24-h intervals and at the cessation of division embryos were fixed, stained, nuclei enumerated, and cleavage indices assigned. Blastocysts and vesiculated embryos which developed were measured using an ocular micrometer. The incidence of blastocyst formation was greater (P less than 0.05) for ISOL BL from four-cell than from two or eight-cell embryos. The presence of the zona pellucida did not significantly affect the incidence of blastocyst formation by single blastomeres. Although ISOL BL did not develop as well as CONTROLS or PTD EMB (P less than 0.05), development of BL ZP was not significantly different from the respective PTD EMB. Blastocysts developing from blastomeres had fewer cells and were smaller than CONTROLS or PTD EMB (P less than 0.05). Although development of ISOL BL may have been impaired by the isolation procedures employed, BL ZP are capable of in vitro development comparable to their respective PTD EMB.