104 Effects of the number of presumptive embryos in the culture environment on cleavage and blastocyst development rates for bovine in vitro embryos

In vitro production of bovine embryos is an important assisted reproductive technique that enables progressive cattle producers to reach their reproductive, financial, and genetic goals. The number of bovine embryos produced in vitro is increasing worldwide and will soon supplant the number of embryos produced via superovulation/embryo transfer. However, practitioners do not always have the luxury of determining how many ova to place into a culture environment, as that depends upon recovery from the donor female. The objectives of this experiment were to: (1) determine the optimal number of presumptive embryos per culture environment, and (2) evaluate the effects of conditioned media on presumptive embryo cleavage and development. During the multiple replicates of this project, follicles were aspirated from slaughterhouse ovaries, selected oocytes were matured (10 oocytes per 50-mL drop), fertilised (50 oocytes per 500-mL well), vortexed to remove cumulus, and randomly placed into culture groups (1, 5, 10, 20, or 50 per 50-mL drop). In vitro maturation (18–20 h), fertilisation (18–20 h), and culture were performed under an oil overlay at 38.5°C in a fully humidified 5% CO₂, 5% O₂, 90% nitrogen environment. On the seventh day of culture, the cleavage (≥2 cells) and development to the blastocyst rates were evaluated and analysed via chi square. In a separate experiment designed to enhance blastocyst development, conditioned media (25 mL) from the previous replicate was added to fresh media (25 mL) in the current replicate before culture for the 20- and 50-embryo culture groups. Results indicated that although cleavage rates (mean %) were similar among culture groups (1: 56.8%; 5: 61.3%; 10: 56.5%; 20: 58.2%; 50: 66.7%), the development to the blastocyst stage (mean %) was lower (P < 0.05) in the single-embryo culture group (7.2%) compared with the other culture groups (5: 19.3%; 10: 20.0%; 20: 19.4%; 50: 21.3%). Further, blastocyst development was similar in the original embryo culture groups (Group 20: 18.4%; 50: 16.9%) and the cultures using conditioned media (Group 20: 14.2%; 50: 16.9%). Similar cleavage rates but lower blastocyst development in the single presumptive embryo group compared with all other groups indicated that there is an apparent “helper effect” expressed in the culture environments associated with at least five ova and this “effect” occurs after cleavage but before blastocyst development. Future research will attempt to characterise this “helper effect” and what role, if any, it plays in the transition from maternal to embryonic control of the genome (a known arrest timepoint between cleavage and blastocyst development). Additionally, a novel in vitro culture system in which ova cultured in different groups may share growth factors/stimulants will be evaluated.