114 DEVELOPMENT OF AN OPTIMIZED WELL IN WELL CULTURE SYSTEM: EFFECT OF MINIWELL CALIBER AND CULTURE DROP VOLUME ON DEVELOPMENTAL COMPETENCE OF BOVINE EMBRYOS

Abstract

To overcome developmental problems due to the scientific need to track single embryos or due to embryo culture in small groups, the Well in Well culture system has been developed previously. Here we aimed to examine the effects of different MiniWell caliber and different embryo densities on developmental rates. Cumulus oocyte complexes (COC) aspirated from small follicles (2–8 mm) were cultured in modified TCM (TCM199, Sigma, Taufkirchen, Germany) supplemented with 12% heat inactivated oestrus cow serum and 10 μg mL^–1 FSH (FSH-p, Sheering, Kenilworth, NJ, USA) for 24 h at 39°C in a humidified atmosphere with 5% CO₂ in air. Fertilization was performed in Fert-TALP medium. After 20 h coincubation with sperm cells, presumptive zygotes were allocated into MiniWells of different diameter (0.3, 0.4, 0.5, and 0.7 mm) and depth (0.3, 0.5, and 0.7 mm) to investigate the effect of MiniWell diameter and depth on further development of the embryos. To investigate the effect of embryo density we compared the development of zygotes placed in MiniWells with different total volumes of culture medium per well. For all experiments bovine embryos cultured in groups of 16 and groups of 50 served as controls. Developmental rates of in vitro-produced embryos were analysed by chi-square test. Differences of P < 0.05 were considered to be significant. When embryos were cultured in a 4 × 3 factorial design (n = 240 per treatment group in 16 replicates), MiniWell diameter (0.3 mm v. 0.4 mm v. 0.5 mm v. 0.7 mm) significantly affected developmental rates to the blastocyst stage (21.5% v. 26.9% v. 32.5% v. 31.3%, respectively) and MiniWell depth (0.3 mm v. 0.5 mm v. 0.7 mm) influenced development (24.4% v. 27.4% v. 29.3%, respectively). When embryos (n = 160 per treatment group in 10 replicates) were cultured in different total volumes of culture media per Well (0 μL, v. 150 μL v. 500 μL) development of embryos to the blastocyst stage (3.1% v. 13.1% v. 33.1%, respectively) differed significantly. When a total of 240 embryos cultured in 15 replicates in group of 16 were compared with a total of 750 embryos cultured in 15 replicates in group of 50, embryos cultured in group of 16 reached the blastocyst stage at a significantly lower level than zygotes cultured in the group of 50 (22.2% v. 30.3%), whereas zygotes cultured in MiniWells were able to compensate against low embryo densities reaching a blastocyst rate as high as embryos cultured in group of 50 (31.3 v. 30.3%). The best developmental rate of bovine zygotes to the blastocyst stage was observed in MiniWells with a diameter of 0.7 mm and 0.7 mm depth in 500 μL culture medium per well. In conclusion, we successfully optimized the Well in Well culture system by exploring the most suitable MiniWell properties supporting improved developmental rates to the blastoyst stage by compensating against negative effects of low embryo densities allowing to track each individual embryo over the complete culture period.