Effects of platelet-derived growth factor (PDGF) on the in vitro production of bovine embryos in protein-free media

Abstract

The purpose of our experiments was to explore the effects of platelet-derived growth factor (PDGF)-supplementation at the various steps of in vitro production of bovine embryos using protein-free media. Cumulus-oocyte-complexes (COC) were collected by slicing abattoir ovaries and then dividing the COC into 2 morphological categories. After maturation for 24 h in TCM-199 supplemented with hormones and either 20% estrous cow serum (ECS) or 1 mg/ml polyvinyl-alcohol (PVA), oocytes were co-incubated for 19 h with frozen/thawed spermatozoa from bull of proven fertility. The semen was diluted in Fert-Talp supplemented with heparin, hypotaurine and epinephrine and either 6 mg/ml bovine serum albumin (BSA) or 1 mg/ml PVA. Presumptive zygotes were transferred into embryo culture medium containing either 20% ECS or 1 mg/ml PVA for a total of 10 d. The PDGF was added at concentrations of 1, 10 or 100 ng/ml to the maturation medium (Experiment 1), fertilization medium (Experiment 2) or culture medium from Day 1 on (Experiment 3), respectively, or at 1 ng/ml PDGF to both the fertilization and culture medium from Day 3 on (Experiment 4), with each medium supplemented with PVA. Oocytes/embryos incubated in the absence of PDGF in media supplemented with either ECS or PVA served as controls. An average of 20 COC was incubated in 1 droplet under silicone oil, and each experiment contained 4 to 6 replicates. No significant differences were found among the various concentrations of PDGF, nor did PDGF-supplementation during maturation (Experiment 1) or embryo culture on Day 1 (Experiment 3) significantly affect development of oocytes/embryos (34.7 ± 3.5 to 40.4 ± 2.5% morulae, 11.9 ± 2.4 to 18.8 ± 2.5% blastocysts; and 23.2 ± 2.3 to 27.5 ± 3.4% morulae, 11.5 ± 2.6 to 12.7 ± 2.3% blastocysts, respectively; x ± SEM). In the presence of 10 ng/ml PDGF in the fertilization medium development to morulae and blastocysts was similar to that of the ECS-group, and was higher (P < 0.05) than that of the PVA-control (ECS: 32.1 ± 4.6 and 13.8 ± 2.7%; PVA: 17.5 ± 0.8 and 6.1 ± 1.3%; PDGF: 30.6 ± 3.0 and 14.0 ± 2.2%, respectively). Development to morulae/blastocysts was increased, and was at the same level as in the ECS-group when the fertilization and/or embryo culture medium on Day 3 contained PDGF compared with the PVA-control group (morulae: ECS 25.3 ± 4.4%, PVA 13.9 ± 2.2% [P < 0.05], PDGF 16.7 ± 3.2 to 19.1 ± 1.1%; blastocysts: ECS 5.3 ± 2.1%, PVA 5.0 ± 1.7%, PDGF 7.1 ± 1.6 to 9.1 ± 1.7%, respectively). These results indicate that under our laboratory conditions PDGF can elevate low rates of development and the addition of PDGF to the fertilization medium enhances bovine preimplantation embryonic development. Thus, PDGF can be potentially an important factor in a completely defined medium to substitute the effects of serum.