79 Cytokine supplementation to improve developmental competence of bovine embryos following slow-rate freezing
K. S. Stoecklein A , N. J. Drum A , A. García-Guerra B , B. J. Duran B , J. G. N. Moraes C , L. D. Spate A , R. S. Prather A and M. S. Ortega AA Division of Animal Sciences, University of Missouri, Columbia, MO, USA
B Department of Animal Sciences, The Ohio State University, Columbus, OH, USA
C Department of Animal and Food Sciences, Oklahoma State University, Stillwater, OK, USA
Reproduction, Fertility and Development 34(2) 276-276 https://doi.org/10.1071/RDv34n2Ab79
Published: 7 December 2021
© 2022 The Author(s) (or their employer(s)). Published by CSIRO Publishing on behalf of the IETS
Cryopreservation of bovine embryos is an important component as it relates to the use of in vitro embryo production. Specifically, cryopreservation of embryos by slow-rate freezing is the most efficient and practical approach for the adoption of this technology. Currently, in vitro-produced embryos cryopreserved using slow-rate freezing have reduced survival compared to those that are transferred fresh. Previously, the addition of fibroblast growth factor 2 (FGF2; 40 ng mL−1), leukemia inhibitory factor (LIF; 20 ng mL−1), and insulin-like growth factor 1 (IGF1; 20 ng mL−1), termed FLI, to an in vitro culture system improved bovine embryo development to the blastocyst stage, from 31% to 42%, as well as re-expansion following cryopreservation, from 39% to 82%. Thus, the objective of this study was to determine how the addition of FLI influences embryo elongation and pregnancy establishment following cryopreservation by slow-rate freezing and direct transfer. Embryos were produced in vitro by using abattoir-derived oocytes and fertilised using standard procedures. Following fertilization, embryos were cultured to the blastocyst stage either with or without FLI supplementation (± FLI). Blastocyst stage (stage 6) embryos with a quality grade of 1 were loaded into straws with ethylene glycol and sucrose, cryopreserved by slow-rate freezing, and stored in liquid nitrogen until transfer. For experiment 1, a total of 55 −FLI and 50 + FLI embryos were transferred into synchronised nonlactating recipient beef cows (n = 21; 5 embryos each) 7 days after the last gonadotrophin-releasing hormone (GnRH) of a 5-day CO-Synch protocol. Eight days later, females were killed, uteri were collected and flushed, and conceptuses were recovered. In experiment 2, a single frozen-thawed embryo was transferred to recipient females (n = 18) 7 days following detection of oestrus, and pregnancy diagnosis was performed on Day 30 using transrectal ultrasonography. Data on embryo length and average embryos recovered were analysed by ANOVA using the Proc GLM procedure of SAS version 9.4 (SAS Institute Inc.). Data for embryo recovery rate and Day 30 pregnancy were analysed by using a logistic regression model in Proc Glimmix within SAS. In experiment 1, embryo recovery was 30.9 ± 6.2% in the −FLI and 32.0 ± 6.6% in the + FLI group (P = 0.90). On average, 1.89 ± 0.34 −FLI and 2.29 ± 0.38 + FLI embryos were recovered per pregnant cow (P = 0.45). The length of elongated conceptuses was not different (P = 0.24) between the −FLI (3.5 cm ± 0.75) and + FLI (4.9 cm ± 0.83) groups. In experiment 2, Day 30 pregnancies per ET was 30.0 ± 14.5% in the −FLI group and 50.0 ± 17.7% in the + FLI group (P = 0.4). We conclude that embryos cultured with or without FLI and cryopreserved by slow-rate freezing have similar developmental competence up to Day 30 of pregnancy. Further research and a larger sample size are needed to confirm these findings and elucidate the implications of culturing embryos in the presence of FLI.
This research was supported by the National Needs Fellowship funded by USDA NIFA Grant 2019-38420-28972 and the Clifton Murphy Scholarship Fund.
