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Parthenogenetic development of porcine oocytes activated with multiple sets of electrical pulses
Theriogenology
356
PARTHENOGENETIC DEVELOPMENT OF PORCINE OOCYTES ACTIVATED WITH MULTIPLE SETS OF ELECTRICAL PULSES
C.G. Grupen, Z.T. Du, P.J. Verma, S.M. Mcllfatrick, R.J Ashman and M.B. Nottle
BresaGen Limited, P.O. Box 259, Rundle Mall, Adelaide, South Australia, 5000, Australia
Nuclear transfer embryo development requires activation of the enucleated recipient oocyte or of the reconstructed embryo itself. At fertilization, multiple intracellular calcium oscillations signal the oocyte to resume meiosis following sperm penetration. When oocytes are artificially activated with a single electrical pulse only one calcium oscillation is observed (Sun et al., Development, 115:947-956, 1992). In the present study we examined whether two or three sets of electrical pulses improves parthenogenetic development of oocytes compared with one set. Oocytes were collected from superovulated Large White x Landrace donor pigs 50 h posthCG and denuded of cumulus cells by hyaluronidase treatment and pipetting. Oocytes were activated with two DC pulses (1.5 kV/cm, 60 ~tsec) 1 sec apart in 0.3 M mannitol solution containing 0.1 mM CaC12, 0.1 mM MgSO 4 and 0.01% PVA. Additional sets of pulses were given 30 and 60 minutes after the first set. Oocytes were cultured in NCSU23 for 7 days with 10% FBS being added at 96h. Blastocysts were differentially stained and trophectoderm (TE) and ICM cell numbers were determined. The results of four replicates are shown in table 1.
Table 1. Development of oocytes activated with 1, 2 or 3 sets of electrical pulses. I
Number developing to (%)
Cell number (mean + SEM)
Sets of pulses
n
2-cell
4-cell
1
45
43 (96)
43 (96)
23 (51) "b 16 (35f b 25.8+2.7 13.0+2.1
2
49
46 (94)
42 (85)
33 (67) a
morula blastocyst
Total
TE
ICM 12.7+2.6
24 (49) a 25.9+2.4 12.9+2.2 12.9+2.4
3 47 42 (89) 37 (78) 20(43) b 11 (23)b 30.0+1.7 14.6+2.2 15.4+2.2 *Values with different superscripts within columns are significantly different (P<0.05).
Activation with two or three sets of pulses did not affect parthenogenetic development, blastocyst cell number or cell allocation compared with one set of pulses. However, parthenogenetic development to the momla and blastocyst stages was greater with two sets of pulses than with three sets. In conclusion, the results suggest that activation of porcine oocytes using multiple sets of pulses is unlikely to improve the development of nuclear transfer embryos.
356
PARTHENOGENETIC DEVELOPMENT OF PORCINE OOCYTES ACTIVATED WITH MULTIPLE SETS OF ELECTRICAL PULSES
C.G. Grupen, Z.T. Du, P.J. Verma, S.M. Mcllfatrick, R.J Ashman and M.B. Nottle
BresaGen Limited, P.O. Box 259, Rundle Mall, Adelaide, South Australia, 5000, Australia
Nuclear transfer embryo development requires activation of the enucleated recipient oocyte or of the reconstructed embryo itself. At fertilization, multiple intracellular calcium oscillations signal the oocyte to resume meiosis following sperm penetration. When oocytes are artificially activated with a single electrical pulse only one calcium oscillation is observed (Sun et al., Development, 115:947-956, 1992). In the present study we examined whether two or three sets of electrical pulses improves parthenogenetic development of oocytes compared with one set. Oocytes were collected from superovulated Large White x Landrace donor pigs 50 h posthCG and denuded of cumulus cells by hyaluronidase treatment and pipetting. Oocytes were activated with two DC pulses (1.5 kV/cm, 60 ~tsec) 1 sec apart in 0.3 M mannitol solution containing 0.1 mM CaC12, 0.1 mM MgSO 4 and 0.01% PVA. Additional sets of pulses were given 30 and 60 minutes after the first set. Oocytes were cultured in NCSU23 for 7 days with 10% FBS being added at 96h. Blastocysts were differentially stained and trophectoderm (TE) and ICM cell numbers were determined. The results of four replicates are shown in table 1.
Table 1. Development of oocytes activated with 1, 2 or 3 sets of electrical pulses. I
Number developing to (%)
Cell number (mean + SEM)
Sets of pulses
n
2-cell
4-cell
1
45
43 (96)
43 (96)
23 (51) "b 16 (35f b 25.8+2.7 13.0+2.1
2
49
46 (94)
42 (85)
33 (67) a
morula blastocyst
Total
TE
ICM 12.7+2.6
24 (49) a 25.9+2.4 12.9+2.2 12.9+2.4
3 47 42 (89) 37 (78) 20(43) b 11 (23)b 30.0+1.7 14.6+2.2 15.4+2.2 *Values with different superscripts within columns are significantly different (P<0.05).
Activation with two or three sets of pulses did not affect parthenogenetic development, blastocyst cell number or cell allocation compared with one set of pulses. However, parthenogenetic development to the momla and blastocyst stages was greater with two sets of pulses than with three sets. In conclusion, the results suggest that activation of porcine oocytes using multiple sets of pulses is unlikely to improve the development of nuclear transfer embryos.