96 EFFICIENT PRODUCTION OF MONOZYGOTIC TWIN BOVINE EMBRYOS USING BLASTOMERE SEPARATION TECHNIQUE WITH COMMERCIAL WELL OF THE WELL CULTURE DISH
Authors: Hashiyada Y. , Aikawa Y. , Sugimura S. , Matsuda H. , Ohtake M. , Yamanouchi T. , Goto Y. , Kobayashi S. , Imai K. 
Published: December 2015

Monozygotic twin embryos can be produced using the technique of blastomere separation and well of the well (WOW) dish having handmade micro-wells by the needle depression (Tagawa et al. 2008). We have recently reported that developed commercial WOW dish enhances embryo competence in individual culture (Sugimura et al. 2010). The present study was conducted to evaluate the availability of commercial WOW dish for production of monozygotic twin embryos in bovine. Embryos were produced using oocytes from ovaries collected at an abattoir by IVM, IVF, and IVC. For each culture, TCM-199 supplemented with 5% calf serum (CS), Brackett-Oliphant solution supplemented with 10 mg mL–1 BSA, and CR1aa containing 5% CS were used. To evaluate the adaptability of dishes on culture of isolated blastomeres from different cell stage, 2- (n = 63), 4- (n = 94), 8- (n = 137), and 10- to 14- (n = 116) cell stages were obtained on 24–27 h, 30–36 h, 48–54 h, and 48–54 h from the beginning of fertilization, respectively. The zona pellucida was removed by exposure of 0.25% pronase, followed by gentle pipetting by inspiration and expiration in the IVC medium. Then, two halves separated from the original number of blastomeres were randomly allocated to the conical micro-wells of commercial dish (Dai Nippon Printing, Tokyo, Japan) or created micro-wells by pressing the bottom of the dish with an eyeleteer (control). The approximate diameter and depth of each 25 wells in a commercial dish was 287 and 168 μm, and each 20 wells in the control were 800 and 600 μm. The blastomeres were cultured in wells covered with a droplet of 2.5 μL well–1 IVC medium until Day 8 (IVF = Day 0). Expanded blastocysts (n = 28) derived from tetra-blastomeres of 8-cell stage were stained to determine the number of the inner cell mass (ICM) and trophectoderm (TE) in each group. Statistical significance of the blastocyst formation rates and the number of cells were analysed by the chi-square test and the Student’s t-test, respectively. In the 2-cell stage, blastocyst formation rate in commercial dish tended to be higher than that in the control (60.0% v. 46.1%). The rate of monozygotic blastocyst pairs in commercial dish was higher compared with the control (48.0% v. 26.3%, P < 0.05). In the 4-cell stage, rates of blastocyst formation (50.0% v. 33.0%, P < 0.05) and the pairs (39.5% v. 12.5%, P < 0.01) in the commercial dish, both were higher compared with the control. In the 8-cell stage, there were no differences between two groups in rates of blastocyst formation (53.1% v. 59.0%) and the pairs (41.8% v. 48.7%), similarly in the 10- to 14-cell stage (47.9% v. 56.8% and 36.2% v. 40.9%, respectively). The ICM, TE, and total cell numbers were not different between the commercial and the control dish (28.0 ± 3.2 v. 26.0 ± 3.8, 64.6 ± 4.3 v. 76.0 ± 7.9, and 92.6 ± 6.2 v. 102.0 ± 11.0, respectively). These results indicate that separated blastomeres could be developed to blastocysts efficiently and stably regardless of embryo cell stage with a commercial WOW culture dish.