77 Monozygotic Twin Calves Production by Blastomere Separation Technique with Commercial Well-of-the-Well Culture Dish

Abstract

Monozygotic twin bovine embryos can be produced by blastomere separation of 2-cell embryos and commercial well-of-the-well (WOW) culture dish (Hashiyada et al. 2016 Reprod. Fertil. Dev. 28, 178) obtaining 60% and 48% of blastocyst formation and monozygotic blastocyst pairs, respectively. The present study was conducted to evaluate the fertility of blastocysts derived from this production system in Japanese Black beef cattle. Embryos were produced using oocytes collected by ovum pick-up technique. 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 for each culture step: in vitro maturation, fertilization, and culture (IVM,IVF, and IVC). Two-cell stage embryos were obtained 24 to 27 h post-insemination. Zonae pellucidae were removed by exposure to 0.25% pronase. Then, embryos were separated into blastomeres by gentle pipetting in IVC medium. Each blastomere was introduced into a single conical microwell of 25 wells, each having a diameter and depth of ~287 μm and 168 μm (Dai Nippon Printing, Tokyo, Japan). Blastomeres in wells were cultured covered with a droplet of 2.5 μL of IVC medium/well. The developed blastocysts in pairs on 7 days post-insemination were used for transfer. Single embryos of monozygotic twin embryos were transferred to Japanese Black cattle with a generally small body frame to produce twin calves from a set of recipients. Twin embryos were transferred in pairs to unilateral of uterus of non-lactating Holstein cows. Pregnancy and twin pregnancy were determined at 30 days of gestation by ultrasonography and were reconfirmed at 60 days with detection of fetal loss. Statistical significance was analysed by Fisher’s exact test. There was no significant difference in pregnancy rate or twin pregnancy rate between single embryo transfer (7/14, 50% and 2/7, 28.6%) and twin embryo transfer (9/21, 42.9% and 4/21, 19%). In either transfer method, fetal loss was not observed in diagnosis carried out at 60 days by ultrasonography. To date, 2 pairs of twin calves have been obtained from twin pregnant cows by twin embryo transfer within the normal range of gestation length (286 and 288 days) and birth weight (31-40 kg). These results indicate that blastocysts developed from blastomeres separated from 2-cell embryos by culturing with commercial WOW culture dish had fertility similar to that of intact embryos derived from standard in vitro culture and further demonstrate the possibility of production of normal twin calves.