141 Positive effects of supplementation in bovine culture medium with 3 cytokines

Abstract

Recently, our laboratory has identified that supplementation of 3 cytokines, 40 ng mL⁻¹ FGF2, 20 ng mL⁻¹ LIF, and 20 ng mL⁻¹ IGF1 (termed FLI), to porcine oocyte maturation medium leads to an increase in maturation percentage and embryo development to blastocyst. Similarly, addition of FLI to porcine embryo culture at Day 2 after fertilization increased the number of embryos that became blastocyst. The objective of this study was to determine whether the effect of FLI on embryonic development was maintained in the bovine. Two variations of the bovine culture medium, basic SOF and SOF-BE2 (containing 0.4 mM trisodium citrate and 2.56 mM myo-inositol), were supplemented with FLI at the beginning of culture, and development to the blastocyst stage on Day 8 was determined. Embryos were produced from abattoir-derived oocytes from Bos taurus breeds and fertilized with sperm from a single Holstein bull proven of high fertility under in vitro conditions. All statistical analyses were completed by logistic regression using the Genmod procedure of SAS v. 9.4 (SAS Institute Inc., Cary, NC, USA). In the first experiment, zygotes (oocytes exposed to sperm) were placed in culture in basic SOF with or without supplementation with FLI. After 4 replicates containing 90 control embryos and 93 FLI-treated embryos we found an increase (P < 0.05) in blastocyst percentages of 21.1 ± 0.2% for the control compared with 40.9 ± 0.2% for the FLI-treated embryos. In a second experiment, zygotes were cultured in SOF-BE2 with or without supplementation with FLI. A total of 679 zygotes (332 control and 347 FLI-treated zygotes) across 4 replicates were evaluated. Percentage of embryos developing to the blastocyst stage increased (P < 0.05) from 33.2 ± 0.07% in the control group to 42.6 ± 0.07% in the FLI-supplemented group. Next, blastocysts derived from the second experiment were vitrified for subsequent transfer to synchronized Angus recipient heifers. Re-expansion 16 h after thawing was recorded before transfer. For the control group, 10 out of 24 embryos (41.7%), and 28 out of 41 embryos (68.3%) of the FLI-treated embryos, re-expanded. Three re-expanded blastocysts were then nonsurgically transferred to the uterine horn of each synchronized heifer (N = 3 control embryo containing heifers, 9 FLI embryo containing heifers). The number of viable fetuses (presence of a heartbeat) per heifer will be determined via ultrasound around Day 35. Although more vitrification and transfer experiments are being completed, preliminary data suggest that supplementation of FLI during culture might improve of the embryos to freezing. Furthermore, supplementation of FLI improves development to the blastocyst stage in bovine embryos under different conditions.

This work was supported in part by funds from the Agriculture and Food Research Initiative Competitive Grant no. 2013-68004-20365 from the United States Department of Agriculture National Institute of Food and Agriculture and National Institutes of Health Grant R01 HD072898, Food for the 21st Century, and the Clifton Murphy Scholarship Fund.