EFFECTS OF EXTRACELLULAR MATRICES AND GROWTH-FACTORS ON THE DEVELOPMENT OF ISOLATED PORCINE BLASTOMERES

The effects of extracellular matrices and growth factors on the development of isolated blastomeres derived from intact 4-,8-, and 16-cell porcine embryos (termed, respectively, 1/4, 1/8 and 1/16 blastomeres) were investigated in vitro and in vivo. Blastomeres were incubated in extracellular matrix components fibronectin (FIN) or swine skin gelatin (SSG)-precoated culture dishes containing either modified Krebs' Ringer Bicarbonate solution (mKRB) supplemented with 10% heat-inactivated lamb serum, or Hanks' solution supplemented with 10% heat-inactivated newborn calf serum (NBCS) or Waymouth medium supplemented with 10% NBCS or in noncoated dishes in mKRB supplemented with either insulin (10, 100, or 1000-mu-g/ml), transferrin (10, 100, or 1000-mu-g/ml), or cAMP (0.2 or 2.0-mu-g/ml). Cultures were observed at 24-h intervals and morphological development was recorded. Blastomeres were classified into three categories according to their morphology: (1) regular blastocysts, (2) trophectodermal vesicles, or (3) no development. After 96 h, culture was terminated; the overall diameter of the blastocysts was determined and the nuclei were counted. Blastomeres/blastocysts did not adhere to the bottom of the culture dishes coated with extracellular matrices. Blastocyst formation rate was highest when FIN/mKRB was used and reached 44.3%, 41.8%, and 36.5% for 1/4, 1/8, and 1/16 blastomeres, respectively. The respective blastocysts contained an average of 31.2 +/- 5.8, 58.2 +/- 8.4, and 18.5 +/- 3.5 nuclei and had an overall diameter of 250.0 +/- 10.1, 235.0 +/- 12.8, and 172.5 +/- 13.7-mu-m. 1/8 blastomeres displayed a better (p < 0.05) growth rate than 1/4 and 1/16 blastomeres, and 1/8 blastomeres in FIN/mKRB grew better (p < 0.01) when cultured in an open system than in a microdrop under oil (36.5% vs 5.0% blastocysts). Neither cAMP nor transferrin had a significant stimulating effect on blastocyst development of 1/8 blastomeres when mKRB plus serum was used as the medium. Insulin supplementation (10 or 100-mu-g/ml) to mKRB plus lamb serum significantly (p < 0.05) stimulated blastocyst formation rate compared with controls (58.6% and 38.9% vs. 17.7%, respectively; 32.6 +/- 2.5, 58.5 +/- 11.8, and 45.1 +/- 4.6 nuclei, respectively). Transfer of 457 blastocysts grown for 24 h in FIN/mKRB to 16 recipient gilts led to three pregnancies and two litters were born from 1/8 blastomere-derived blastocysts following 116 days of gestation. It is concluded that (1) 1/8 blastomeres have a greater developmental potential in vitro than 1/4 and 1/16 blastomeres, (2) FIN coating of the culture dishes significantly enhances blastocyst development of 1/8 and 1/16 blastomeres, (3) insulin supplementation of serum-enriched culture medium improves blastocyst development from 1/8 blastomeres, and (4) at least some 1/8 porcine blastomeres are capable of full-term development.