Effect of Growth Factors on Bovine Blastocyst Development in a Serum-Free Medium

Abstract

To investigate the effect of growth factors on pre-implantation development, bovine zygotes, produced by in vitro fertilization (IVF) of in vitro-matured (IVM) oocytes, were cultured in a serum-free medium to which the following growth factors were added one at a time: epidermal growth factor (EGF), acidic fibroblast growth factor (a-FGF), insulin-like growth factor-II (IGF-II), platelet-derived growth factor from human platelets (PDGF), and platelet-derived growth factor-AB, human, recombinant (PDGF-AB). All growth factors were added at a dose of either 10 or 50 ng/ml, except PDGF which was added at a dose of either 5 or 15 ng/ml. The control medium was TCM 199 supplemented with sodium pyruvate (0.25 mmol/1), BSA (10 mg/ml), insulin (5 μg/ml), transferrin (5 μg/ml), and sodium selenite (5 ng/ml). Embryos were cultured for 8 days (day of insemination = Day 0). The mean percentages of first cleavage on Day 2 varied from 67% to 86% and the differences between the 2 doses, or between the control and growth factor- treated groups were not significant (p≥0.13). The effects of the two doses on subsequent development up to the blastocyst stage did not differ either (p≥0.12). There was no stimulatory effect of any of the used exogenous growth factors on embryo development up to the morula or blastocyst stage on Day 7, or blastocyst stage on Day 8. Moreover, medium supplemented with PDGF had fewer blastocysts than the control (p≤0.03). The results indicate that growth factor supplementation may not necessarily increase the yield of blastocysts from bovine IVM-IVF oocytes in a serum-free medium.

Sammanfattning

For att undersoka effekten av tillvaxtfaktorer på embryoutvecklingen fore implantationen användes notkreatursembryon i en-cellstadiet, framtagna genom in vitro befruktning (IVF) av in vitro mognade (IVM) oocyter. Embryonerna odlades i ett serum-fntt medium till vilket en av foljande tillvaxtfaktorer tillsattes: epidermal growth factor (EGF), acidic fibroblast growth factor (a-FGF), insulin-like growth factor-II (IGF-II), platelet-derived growth factor från humana trombocyter (PDGF) och human rekombinant platelet-derived growth factor-AB (PDGF-AB). Alia tillvaxtfaktorer tillsattes i doser av 10 eller 50 ng/ml, utom PDGF som tillsattes i an-tingen 5 eller 15 ng/ml. Som kontrollmedium anvandes TCM 199 till vilket tillsatts natriumpyru-vate (0.25 mmol/1), BSA (10 mg/ml), insulin (5 μg/ml), transferrin (5 μg/ml) och natrium- selenit (5 ng/ml). Embryonerna odlades vid 39°C och 5% CO₂ i fuktmattad luft under 8 dagar (dag 0 = dagen for be-fruktningen). Den genomsnittliga andelen oocyter som genomgått forsta delningen på dag 2 varierade från 67% till 86%. Skillnaderna mellan de 2 doserna eller mellan kontrollgrupp och behandlingsgrupper var lnte signifikant (p≥0.13). Det var lnte heller någon skillnad i effekt av de två doserna på utveck-lingen till blastocyststadiet (p≥0.12). Ingen av de exogent tillforda tillvåxtfaktorerna hade någon sti-mulerande effekt på embryoutvecklingen till morula eller blastocyststadiet på dag 7 eller till blastocyststadiet på dag 8. Fårre blastocyster utvecklades ι medium med tillsats av PDGF an ι kontrollmediet (p≤0.03). Resultaten lndikerar att tillsattande av tillvaxtfaktorer till ett serum-fritt medium inte sti-mulerar utvecklingen av IVM-IVF oocyter till blastocyster.