208 EFFECT OF GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR ON BLASTOCYST DEVELOPMENT AND POST-TRANSFER SURVIVAL OF IN VITRO-PRODUCED BOVINE EMBRYOS

Abstract

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine that has been implicated in preimplantation embryo development. Granulocyte-macrophage-CSF improves the proportion of bovine embryos that become blastocysts in vitro (Moraes and Hansen 1997 Biol. Reprod. 57, 1060–1065) and increases blastocyst cell numbers in mice (Robertson et al. 2001 Biol. Reprod. 64, 1206–1215). The long-term goal of the present research was to evaluate the effects of GM-CSF on post-transfer survival of bovine embryos. The experiments used recombinant ovine GM-CSF produced in transfected Chinese hamster ovary (CHO) cells or an equivalent volume of cytokine-free CHO cell supernatant (control). The objective of the first study was to evaluate the effects of GM-CSF on post-transfer survival. Embryos were cultured with 10 ng mL^–1 of either GM-CSF or cytokine-free CHO cell supernatant added to culture medium at Day 1 after insemination. Embryos were transferred at Day 7 to lactating dairy cows according to a timed embryo transfer protocol. Pregnancy was evaluated at approximately Day 45 of gestation. There was no significant difference in the proportion of embryos becoming blastocysts at Day 7 after insemination (34.8 v. 37.5% for the control and GM-CSF; SEM = 2.4%). There was also no difference in pregnancy rates between cows receiving control embryos (6/24; 25%) and cows receiving embryos treated with GM-CSF (8/35; 23%). A second study determined the effects of various concentrations of GM-CSF on the development of in vitro-produced embryos to the blastocyst stage. Embryos were cultured in 5% (v/v) oxygen (low oxygen) or atmospheric oxygen (21%, w/v; high oxygen) in the presence of 0, 1, 10, or 100 ng mL^–1 of GM-CSF or an equivalent volume of cytokine-free CHO cell supernatant (control). The GM-CSF was added on either Day 1 or Day 5 after insemination. Cleavage rate was accessed on Day 3 after insemination. Stage of development was recorded at Day 7 and Day 8 after insemination. There was no effect of GM-CSF on cleavage rate. Addition of GM-CSF at Day 5 to embryos cultured in low or high oxygen increased the percentage of oocytes that became blastocysts at Day 7 (P < 0.01) and Day 8 (P < 0.01), but addition at Day 1 did not have a significant effect on blastocyst development. The greatest effects of GM-CSF occurred at a concentration of 10 ng mL^–1. At this concentration, least squares means for the percentage of oocytes that became blastocysts at Day 7 were 13.9 v. 21.6% (control v. GM-CSF) when GM-CSF was added at Day 5, and 19.5 v. 21.5% when GM-CSF was added at Day 1. The percentage of blastocysts at Day 8 was 20.9 v. 28.7% when GM-CSF was added at Day 5, and 26.7 v. 27.5% when GM-CSF was added at Day 1. In conclusion, GM-CSF can affect the competence of embryos to develop to the blastocyst stage, but at the concentrations and times given, there was no evidence that GM-CSF enhanced embryo survival after transfer.