159 Effect of Commercial Embryo Holding Medium on Development and Quality of Immature Bovine Oocytes

Abstract

Bovine in vitro embryo production following ovum pick-up (OPU) in the field is hampered due to large time gaps between first and last OPU sessions. As oocytes will start immediate maturation, scheduling further manipulations makes the laboratory work laborious. There is a need for an easy and low-cost method that conserves the oocytes with full developmental capacity and allows scheduling laboratory work. In this regard, a commercial embryo holding medium (EHM; Syngro®; Bioniche Inc., WA, USA) was evaluated for conserving immature oocytes. Bovine immature oocytes (n = 2160) were collected from slaughterhouse animals by follicle aspiration and grouped per 60 and stored in 1 mL of EHM in 1-mL sterile glass osmometer tubes. Then, different temperatures [4°C, room temperature (RT), and 38.5°C] and different storage times (6, 10, 14 h) were assessed. After storage, oocytes were matured in TCM-199 supplemented with epidermal growth factor and gentamycin for 22 h. Fertilization was performed and zygotes (n = 1786) were cultured per 25 in 50 µL of SOF medium supplemented with 0.4% BSA and insulin, transferrin, and selenium. Control groups were included: immature oocytes (n = 1080) were not stored in EHM but immediately matured; fertilized and zygotes (n = 896) were cultured. Further, differential apoptotic staining was performed on a random subgroup of blastocysts to assess quality. Generalized fixed effect models were computed using R studio. Storage for 6 h showed a decrease in cleavage and blastocyst rate at 38.5°C (50 ± 3.9%; 11 ± 1.8%) compared with the control (78 ± 3.0%; 36 ± 2.8%). When increasing storage time, 38.5°C was not included; here, 4°C had a lower cleavage and blastocyst yield (47 ± 2.9%; 20 ± 3.3%) compared with the control (75 ± 2.5%; 41 ± 4.6%). For both 6 and 10 h, storage at RT resulted in similar cleavage (76 ± 3.4%; 74 ± 2.6%) and blastocyst rates (35 ± 2.7%; 40 ± 4.5%) as the control (P > 0.05). However, increasing storage to 14 h at RT decreased cleavage (61 ± 2.8%) and blastocyst yield (26 ± 2.5%) compared with the control (78 ± 2.4%; 39 ± 2.8%; P < 0.05). Evaluating embryo quality in all groups, no significant differences were found for any holding time or temperature of the EHM. To simulate OPU settings, EHM was also tested in 38 small groups of 10 immature oocytes that were subsequently matured, fertilized, and cultured. Based on the previous results, EHM storage was performed for 6 and 10 h at RT. Blastocyst development was not different between RT (19.8 ± 3.5%; 18.8 ± 3.6%) and the control (20.6 ± 3.6%; 18.3 ± 3.4%; P ≥ 0.05). To conclude, a commercial EHM can be used to conserve immature bovine oocytes without losing developmental capacity. Storage is recommended for no longer than 10 h and at RT in EHM. It opens new perspectives for practitioners, because this method is simple and low-cost; moreover, the start of maturation and subsequent in vitro embryo production process can be scheduled to avoid evening or night work at the laboratory.