Blastocysts’ production after long-term delayed shipment (72h) of cumulus oocyte complexes (COCs) in holding medium

Abstract

In regular practice, after oocyte pick-up (OPU) the recovered cumulus-oocytes-complexes (COCs) may be held in holding medium during transportation. This methodology is included in our SOP, in which COCs are shipped to our facilities, in a homemade holding medium (M199-Hanks/HEPES + M199-Earle's supplemented with 20% FBS), and are later transferred into in vitro maturation (IVM) dishes. Here, we report an OPU session involving four mares with a total of 45 COCs recovered, in which, due to external causes, the shipment of the COCs was extended for 72 hours. Upon arrival at our facilities, the COCs’ temperature was measured (20.2°C) and based on previous publications (Josson AR et al. J Equine Vet Sci. 2020; 89:103063; Foss R et al. Equine Vet J. 2013; 45(S45):39-43), it was decided to continue with the cycles. COCs were transferred to the IVM dishes following our standard methodology. After 31 hours in IVM medium (M199-Earle's medium supplemented with FSH, EGF and 10% FBS) at 38.2°C, 7% CO2 and atmospheric %O2, oocytes were denuded; a total of 21/45 (46.7%) oocytes showed a first polar body, and were available to perform ICSI. This ratio was slightly lower than that observed in previous shipments from the same OPU center (63.7% maturation, n=267 COCs), but not significantly different (p>0.05, Fisher's exact test). ICSI was performed using a piezo-actuator, and theinjected oocytes were put in culture in a mix of Global-DMEM/F12 media supplemented with 6% FBS, in humid benchtop incubators at 38.2°C, 7% CO2 and 7% O2. On D5 of development, embryos were evaluated: an average of 4 (±3.4) embryos cleaved per case (76.2% cleavage rate), which again did not differ significantly from our mean cleavage rate under regular conditions (82.4%, n= 170 oocytes, p>0.05, Fisher's exact test). Non-cleaved and degenerated embryos were discarded, while the developing ones were transferred to a new dish prepared with fresh culture medium. Embryo development was followed daily from D7 to D10 to assess the formation of blastocysts. A total of 10 blastocysts were obtained, with an average of 2.5 blastocysts per OPU and a total blastocyst rate of 47.6%. These outcomes suggest that a longer holding period of COCs after OPU may not be detrimental to the subsequent in vitro embryo development and may produce results similar to those observed in regular standard cycles. However, more studies should be performed to confirm these findings and the implantation potential of the blastocysts produced. In addition, long-term shipment should be avoided as a standard practice, due to the difficulties in keeping a stable temperature during such long periods of time during shipping.