title: Effects of extracellular matrices and growth factors on the development of isolated porcine blastomeres.
abstract: 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 1,000 micrograms/ml), transferrin (10, 100, or 1,000 micrograms/ml), or cAMP (0.2 or 2.0 micrograms/ml). Cultures 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 determined; 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 microns, 1/8 blastomeres displayed a better (p less than 0.05) growth rate than 1/4 and 1/16 blastomeres, and 1/8 blastomeres in FIN/mKRB grew better (p less than 0.01) when cultured in an open system than in a microdrop under oil (35.5% 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 micrograms/ml) to mKRB plus lamb serum significantly (p less than 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 recipients gilts led to three pregnancies, and two litters were born from 1/8 blastomere-derived blastocysts following 116 days of gestation.4+ is
authors.affiliation: Institut für Tierzucht und Tiervehalten, (FAL), Neustadt, F.R.G.
authors.forename: S
authors.initials: S
authors.lastname: Saito
authors.forename: H
authors.initials: H
authors.lastname: Niemann
mesh_terms: D000818:Animals; D001757:Blastomeres / Q000166:cytology* / Q000187:drug effects; D003470:Culture Media; D000242:Cyclic AMP / Q000494:pharmacology; D005109:Extracellular Matrix / Q000378:metabolism; D005260:Female; D005353:Fibronectins / Q000494:pharmacology; D005780:Gelatin / Q000494:pharmacology; D006133:Growth Substances / Q000494:pharmacology*; D066298:In Vitro Techniques; D007328:Insulin / Q000494:pharmacology; D011247:Pregnancy; D013552:Swine; D014168:Transferrin / Q000494:pharmacology
chemical_list: D003470:Culture Media; D005353:Fibronectins; D006133:Growth Substances; D007328:Insulin; D014168:Transferrin; D005780:Gelatin; D000242:Cyclic AMP