Actions of CSF2 and DKK1 on bovine embryo development and pregnancy outcomes are affected by composition of embryo culture medium

Abstract

Procedures for in vitro embryo production in cattle have not been optimized. In the current experiment, we utilized a 3 × 3 factorial design to test whether the proportion of embryos becoming blastocysts in culture and the pregnancy rate after embryo transfer are affected by type of serum in the medium [no serum; 3% (v/v) KnockOut Serum Replacement (SR); 3% (v/v) fetal bovine serum (FBS)] and addition of specific embryokines [vehicle; 10 ng/mL colony stimulating factor 2 (CSF2); 100 ng/mL dickkopf related protein 1 (DKK1)] at day 5 of culture. Embryos were produced using abattoir-derived ovaries and Y-sorted semen from two Angus sires. The percent of putative zygotes and cleaved embryos becoming blastocysts was improved by SR and FBS. Pregnancy rate at day 30 was determined for 1426 Nelore recipients and calving rate for 266 recipients. In the absence of CSF2 or DKK1, pregnancy rates were lower for embryos cultured with SR or FBS. CSF2 and DKK1 reduced pregnancy rate for embryos cultured without serum but had no detrimental effect in the SR or FBS groups. Indeed, CSF2 blocked the negative effect of FBS on pregnancy rate. Data on birth weights were available for 67 bull calves. There were no effects of treatment. The sire used to produce embryos had significant and large effects on development to the blastocyst stage, pregnancy rate at day 30, calving rate and pregnancy loss between day 30 and calving. Results indicate that (1) SR and FBS can improve embryonic development in vitro while also compromising competence of embryos to survive after transfer, (2) actions of CSF2 and DKK1 depend upon other characteristics of the embryo production system, and (3) sire can have a large effect on embryonic development before and after transfer.

Figure 1

Effects of medium and embryokine treatments on development of bovine embryos. Cleavage was measured at day 3 (A) and development to the blastocyst stage at day 6 (B,C) and 7 (D,E). Media treatments were no serum, serum replacement (SR) and fetal bovine serum (FBS). Embryokine treatments were added at day 5 and were vehicle, colony stimulating factor 2 (CSF2) and dickopf WNT signaling pathway inhibitor 1 (DKK1). Shown are least-squares means ± SEM. Cleavage rate was affected by medium (P = 0.029) and embryokine (P = 0.008). Using contrasts, cleavage was higher (P = 0.027) for FBS than SR. Cleavage was also lower (P = 0.012) for vehicle than for CSF2 or DKK1. The proportion of embryos that became blastocysts at day 6 (B,C) or day 7 (D,E), whether expressed as the percent of putative zygotes (B,D) or cleaved embryos (C,E), was affected by medium (P < 0.0001). Further analysis using orthogonal contrasts indicated that more embryos became blastocysts in media with SR or FBS than for no serum (P < 0.0001).The total number of putative zygotes/treatment were 945 for no serum—vehicle, 1165 for no serum—CSF2, 1216 for no serum DKK1, 1288 for SR—vehicle, 1416 for SR and CSF2, 1526 for SR and DKK1, 1288 for FBS and vehicle, 1551 for FBS and CSF2, and 1593 for FBS and DKK1.

Figure 2

Effects of medium and embryokine treatments on gestation length and calf birth weight. Embryokine treatments were added at day 5 of culture and were vehicle, colony stimulating factor 2 (CSF2) and dickopf WNT signaling pathway inhibitor 1 (DKK1). Gestation length (A) was affected by embryokine (P = 0.024) and the interaction between medium and embryokine (P = 0.031). Moreover, the contrast comparing vehicle vs DKK1 across media was significant (P = 0.017). Birth weights for individual male calves after adjustment for day of age at weighing and sire are shown in panel (B) while results of statistical analysis are in panel (C). There were no treatment effects on birth weight. Results in panels (A,C) are least-squares means ± SEM.