- Email: [email protected]
In vitro culture of bovine nuclear transfer embryos in synthetic oviduct fluid medium (SOFM)
THERIOGENOLOGY
IN VITRO CULTURE OF BOVINE NUCLEAR TRANSFER EMBRYOS IN SYNTHETIC OVIDUCT FLUID MEDIUM (SOFM) K.J. McLaughlinl, D.M. McLean2 P A. Lewis2, L. Hicksl, G. Stevens2, B.D. Bartsch’ d.F. Seamark ‘Department of Obstetrics and Gynaecblogy, University of Adelaide, Adelaide, South Australia 5000. 2Flaxley Research Centre, Department of Agriculture, Flaxley, South Australia 5 151. A simple medium, SOFM, previously used for the culture of pronuclear bovine embryos (McLaughlin et al 1990, Therio. 33, 1191-l 199) was assessed for the culture of bovine nuclear transfer embryos. Oviductal oocytes were collected from superovulated donors 32 to 36 h post G&H. To obtain donor nuclei, embryos were collected 5.5 days post estrus. Oocytes and embryos were placed immediately into SOFM (supplemented with 10% heat inactivated sheep serum and 25mM HEPES) for two h prior to nuclear transfer. The oocyte nuclear material was removed by aspiration of cytoplasm proximal to the polar body. Donor nuclei blastomeres were isolated from the donor embryo and fused to the oocyte cytoplasm using a 10 second 500 V/cm AC alignment followed by a single 675 V/cm DC pulse of 100 usec duration with a subsequent reduction in the AC field strength to 0 V over 15 seconds. The treated embryos were cultured in the presence of 7.5 pg/ml cytochalasin B for 1 h prior to being placed into culture with SOFM supplemented with 20% heat inactivated human serum at 39OC in an atmosphere of 5% 02, 5% C02, 90% N , 95% relative humidity. From 19 successfully manipulated and fused embryos culture 3 for 144 h post fusion, 3 formed blastocysts with 25 + 6.2 cells per embryo. A total of 128 fused nuclear transfer embryos were also cultured for transfer to recipient cows and the cleavage rates recorded (Table). Twenty-six embryos (15 morulae, 11 blastocysts) were transferred to 17 recipient (l-3 embryos/recipient), resulting in 3 (17%) recipients with a progesterone level of >8nMol/l at day 23, with one (6%) delivering a viable calf aI term. Fused embryos
cleavage @ 24 hrs
8/16 cell @ 72 hrs
morula/ blastocyst @ 140hrs
Pregnancies day 23 term
128
83
32
26
3
1
(%)
(65)
(25)
(20)
(2)
(1)
Whilst SOFM supported preimplantation development of nuclear transfer embryos, the low cell numbers of blastocysts developed in vitro and the low pregnancy rate observed after transfer indicates limitations in the capacity of simple culture medium to maintain the full developmental potential of micromanipulated embryos. This research was supported Development Corporation.
JANUARY
1992 VOL. 37 NO. 1
in part by the Australian
Dairy
Research
and
255
IN VITRO CULTURE OF BOVINE NUCLEAR TRANSFER EMBRYOS IN SYNTHETIC OVIDUCT FLUID MEDIUM (SOFM) K.J. McLaughlinl, D.M. McLean2 P A. Lewis2, L. Hicksl, G. Stevens2, B.D. Bartsch’ d.F. Seamark ‘Department of Obstetrics and Gynaecblogy, University of Adelaide, Adelaide, South Australia 5000. 2Flaxley Research Centre, Department of Agriculture, Flaxley, South Australia 5 151. A simple medium, SOFM, previously used for the culture of pronuclear bovine embryos (McLaughlin et al 1990, Therio. 33, 1191-l 199) was assessed for the culture of bovine nuclear transfer embryos. Oviductal oocytes were collected from superovulated donors 32 to 36 h post G&H. To obtain donor nuclei, embryos were collected 5.5 days post estrus. Oocytes and embryos were placed immediately into SOFM (supplemented with 10% heat inactivated sheep serum and 25mM HEPES) for two h prior to nuclear transfer. The oocyte nuclear material was removed by aspiration of cytoplasm proximal to the polar body. Donor nuclei blastomeres were isolated from the donor embryo and fused to the oocyte cytoplasm using a 10 second 500 V/cm AC alignment followed by a single 675 V/cm DC pulse of 100 usec duration with a subsequent reduction in the AC field strength to 0 V over 15 seconds. The treated embryos were cultured in the presence of 7.5 pg/ml cytochalasin B for 1 h prior to being placed into culture with SOFM supplemented with 20% heat inactivated human serum at 39OC in an atmosphere of 5% 02, 5% C02, 90% N , 95% relative humidity. From 19 successfully manipulated and fused embryos culture 3 for 144 h post fusion, 3 formed blastocysts with 25 + 6.2 cells per embryo. A total of 128 fused nuclear transfer embryos were also cultured for transfer to recipient cows and the cleavage rates recorded (Table). Twenty-six embryos (15 morulae, 11 blastocysts) were transferred to 17 recipient (l-3 embryos/recipient), resulting in 3 (17%) recipients with a progesterone level of >8nMol/l at day 23, with one (6%) delivering a viable calf aI term. Fused embryos
cleavage @ 24 hrs
8/16 cell @ 72 hrs
morula/ blastocyst @ 140hrs
Pregnancies day 23 term
128
83
32
26
3
1
(%)
(65)
(25)
(20)
(2)
(1)
Whilst SOFM supported preimplantation development of nuclear transfer embryos, the low cell numbers of blastocysts developed in vitro and the low pregnancy rate observed after transfer indicates limitations in the capacity of simple culture medium to maintain the full developmental potential of micromanipulated embryos. This research was supported Development Corporation.
JANUARY
1992 VOL. 37 NO. 1
in part by the Australian
Dairy
Research
and
255