126 Embryo transfer medium supplemented with bovine plasminogen increases zona pellucida diameter and decreases thickness

Although multiple factors determine embryo transfer success in pregnancy establishment, transfer medium has an important role. Prior research in our laboratory demonstrated greater zona pellucida (ZP) diameter and reduced ZP thickness in embryos cultured for 2 h in medium containing bovine plasminogen. Thus, our hypothesis is supplementation of embryo transfer medium with plasminogen will soften the ZP, allowing for greater blastocyst expansion and hatching, potentially improving embryo transfer success. Two separate experiments were performed. In the first experiment, four crossbreed Angus cows were superovulated using a standard protocol and embryos were collected at Day 7. Bovine plasminogen was assayed to determine plasmin activity and 200 µg/mL were selected. Good to excellent embryos (n = 11) were cultured in either Ham’s F-12 with 1.5% bovine serum albumin (BSA), 1% antibiotic-antimycotic (AB/AM, 25 mM HEPES) and 200 µg/mL bovine plasminogen (enhanced transfer medium, ETM1) or Dulbecco’s phosphate-buffered saline with 10% heat-treated fetal calf serum (control transfer medium, CTM1) for 2 h at 38.5°C in a humidified atmosphere of 5% CO₂ in air. To assess hatching rates, embryos from both treatments were cultured for an additional 192 h in Ham’s F-12 with 1.5% BSA and 1% AB/AM and ZP were measured at the end of culture. In the second experiment, 21 Angus cows were superovulated using a standard protocol and collected embryos at Day 7. Bovine plasminogen was assayed for plasmin activity and demonstrated greater activity than experiment 1. To ensure no detrimental effects to the embryos, the concentration was halved with a shorter culture time compared to experiment 1. Good to excellent embryos (n = 114) were cultured in embryo transfer medium with 100 µg/mL bovine plasminogen (ETM2) or without (CTM1) for 1 h at 38.5°C, in a humidified atmosphere of 5% CO₂ in air. Embryos were cultured in a control culture medium for 168 h to monitor hatching rate and ZP were measured at the end of culture. Percent data were analysed using the GLIMMIX procedure in SAS with a logit function, and measurement data were analysed using the MIXED procedure in SAS. No differences were observed in hatching rates in either experiment (P > 0.1). ZP diameters were larger, and thicknesses were thinner in embryos cultured in ETM1 compared to CTM1 (P < 0.05) in the first experiment. In the second experiment, ZP diameters were larger (P < 0.05) and tended to be thinner (P = 0.1) in embryos cultured in ETM2 compared CTM2. Observations of thinner ZP with greater diameters in this research suggest transfer medium with bovine plasminogen promotes greater blastocyst expansion which may facilitate in utero hatching and improve overall pregnancy rates following transfer. Future research is warranted to investigate pregnancy success with in vivo and in vitro-derived embryos transferred in ETM2.

USDA is an equal opportunity employer.