Duration of in vitro maturation of recipient oocytes affects blastocyst development of cloned porcine embryos.
This study investigated the effects of different incubation periods for oocyte maturation and contact inhibition of donor cells as well as different osmolarities for storage of recipient oocytes on fusion rates, cleavage rates, and blastocyst yields of porcine somatic nuclear transfer (SCNT) derived embryos. In addition, the in vivo developmental potential of cloned embryos derived from the most promising SCNT protocol was tested by transfer to recipient gilts. Storage of in vitro-matured oocytes for 7.5 h in calcium-free TL-HEPES medium at 295 or 320 mOsmol prior to activation yielded significantly (p < 0.05) higher parthenogenetic blastocyst rates compared to storage in TL-HEPES with an osmolarity of 270 mOsmol (24.4 +/- 3.0% and 26.2 +/- 4.3% vs. 18.3 +/- 6.4%, respectively, mean +/- SD) and improved the visibility of the polar body. Electrical fusion of fibroblasts to enucleated oocytes matured for 38, 40, or 42 h resulted in similar fusion and cleavage rates (74.8-84.4%). However, nuclear transfer with oocytes matured for 40 h in vitro yielded significantly higher (p < 0.05) development to the blastocyst stage after 7 days of culture (14.7 +/- 1.7%) than with oocytes matured for 38 h (9.5 +/- 2.1%) or 42 h (5.1 +/- 2.1%). Contact inhibition for 24, 48, or 72 h significantly (p < 0.05) increased the proportion of cells at G0/G1 compared with cycling fibroblasts. However, duration of contact inhibition of the donor cells for either 24, 48, or 72 h had no effect on blastocyst rates of SCNT embryos. Four gilts received an average of 150 SCNT embryos (range 138-161) reconstructed with oocytes matured for 40 h; two of these became pregnant; one of them went to term and farrowed four piglets on day 115 of pregnancy. Microsatellite analysis confirmed that the clones were genetically identical with the donor cells. These results show that changes of the in vitro maturation protocol may affect in vitro development of reconstructed porcine embryos, while duration of the contact inhibition period plays a minor role for the success of porcine SCNT. The effects on in vivo development are yet to be determined.
Department of Biotechnology, Institute for Animal Breeding, Mariensee, Germany.
Michael
M
Hölker
Björn
B
Petersen
Petra
P
Hassel
Wilfried A
WA
Kues
Erika
E
Lemme
Andrea
A
Lucas-Hahn
Heiner
H
Niemann
D000818:Animals; D001755:Blastocyst / Q000166:cytology / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D019976:Cloning, Organism / Q000379:methods*; D046508:Culture Techniques; D004624:Embryo Transfer; D004622:Embryo, Mammalian / Q000166:cytology; D005260:Female; D005347:Fibroblasts / Q000378:metabolism; D016193:G1 Phase; D018895:Microsatellite Repeats; D009865:Oocytes / Q000166:cytology* / Q000378:metabolism; D016192:Resting Phase, Cell Cycle; D013552:Swine; D013997:Time Factors
D002118:Calcium