301 EFFECT OF CO-CULTURE WITH DIFFERENT STAGE EMBRYOS ON DEVELOPMENT OF ALGINATE-ENCAPSULATED SMALL NUMBER BOVINE EMBRYOS

Abstract

Previous studies show that embryos cultured in large numbers have better developmental competence than those in small numbers in mice, sheep, and cattle. We have reported that co-culture of bovine embryos encapsulated in calcium-alginate gel (microcapsule) improves the development of embryos cultured in small numbers (Kobayashi et al. 2006 Reprod. Fertil. Devel. 18, 248). This method is beneficial for culture of small numbers of embryos such as OPU-derived embryos by recognizing the individual donor cows with abattoir-derived unidentified IVF embryos. In the previous study, we used the same stage embryos for co-culture of encapsulated embryos. However, in the case of unavailability of the same stage embryos, encapsulated embryos may be co-cultured with different stage embryos. Effect of different stage embryos on co-culture of encapsulated embryos is not clear. In the present study, we investigated the effect of co-culture of different stage embryos on development of encapsulated small number embryos. In vitro-matured and fertilized zygotes from abattoir derived ovaries were used for the experiment. Small numbers of zygotes were encapsulated by alginate-gel microcapsule to distinguish from co-cultured embryos. Encapsulation was carried out by putting the 1% sodium alginate solution containing zygotes slowly into 0.1% calcium chloride solution (microcapsule). The embryos used for co-culture were produced by IVF 1-3 days before preparation of encapsulated zygotes (Day 1, Day 2, and Day 3). Five encapsulated zygotes were cultured with 15 embryos for co-culture in one droplet (100 µL) made by CR1aa + 5% CS, at 38.5°C, CO₂ in air. Encapsulated zygotes co-cultured with the same stage of zygotes were assigned as a control (Day 0). The rates of cleavage on Day 2 and development to blastocyst stage on Day 9 were recorded. Data were analyzed by Student's t-test. No significant difference was observed in the rate of cleavage in all experimental groups compared with control (Day 1: 72.5% (n = 80) vs. control: 75.7% (n = 70); Day 2: 76.3% (n = 80) vs. control: 82.5% (n = 80); and Day 3: 78.7% (n = 75) vs. control: 70.8% (n = 65). There was not a significant difference in the rate of development to the blastocyst stage in all experimental groups compared with control (Day 1: 42.5% vs. control: 44.3%; Day 2: 43.8% vs. control: 38.8%; Day 3: 44.0% vs. control: 35.4%). These results indicate that co-culture of different stages of embryos can normally support the development of small numbers of encapsulated embryos. These methods are useful to improve the development of small numbers of embryos derived from OPU-IVF embryos without synchronization of the developmental stage of co-cultured embryos.