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Method of oocyte activation affects cloning efficiency in pigs
Author(s) -
Whitworth Kristin M.,
Li Rongfeng,
Spate Lee D.,
Wax David M.,
Rieke August,
Whyte Jeffrey J.,
Manandhar Gaurishankar,
Sutovsky Miriam,
Green Jonathan A.,
Sutovsky Peter,
Prather Randall S.
Publication year - 2009
Publication title -
molecular reproduction and development
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.745
H-Index - 105
eISSN - 1098-2795
pISSN - 1040-452X
DOI - 10.1002/mrd.20987
Subject(s) - somatic cell nuclear transfer , biology , andrology , blastocyst , embryo , cell fusion , oocyte activation , embryo transfer , cloning (programming) , microbiology and biotechnology , oocyte , embryogenesis , cell culture , genetics , medicine , computer science , programming language
Abstract The following experiments compared the efficiency of three fusion/activation protocols following somatic cell nuclear transfer (SCNT) with porcine somatic cells transfected with enhanced green fluorescent protein driven by the chicken β‐actin/rabbit β‐globin hybrid promoter (pCAGG‐EGFP). The three protocols included electrical fusion/activation (NT1), electrical fusion/activation followed by treatment with a reversible proteasomal inhibitor MG132 (NT2) and electrical fusion in low Ca 2+ followed by chemical activation with thimerosal/dithiothreitol (NT3). Data were collected at Days 6, 12, 14, 30, and 114 of gestation. Fusion rates, blastocyst‐stage mean cell numbers, recovery rates, and pregnancy rates were calculated and compared between protocols. Fusion rates were significantly higher for NT1 and NT2 compared to NT3 ( P < 0.05). There was no significant difference in mean nuclear number. Pregnancy rate for NT2 was 100% (n = 19) at all stages collected and was significantly higher than NT1 (71.4%, n = 28; P < 0.05), but was not significantly higher than NT3 (82.6%, n = 23; P < 0.15). Recovery rates were calculated based on the number of embryos, conceptuses, fetuses, or piglets present at the time of collection, divided by the number of embryos transferred to the recipient gilts. Recovery rates between the three groups were not significantly different at any of the stages collected ( P > 0.05). All fusion/activation treatments produced live, pCAGG‐EGFP positive piglets from SCNT. Treatment with MG132 after fusion/activation of reconstructed porcine embryos was the most effective method when comparing the overall pregnancy rates. The beneficial effect of NT2 protocol may be due to the stimulation of proteasomes that infiltrate donor cell nucleus shortly after nuclear transfer. Mol. Reprod. Dev. 76: 490–500, 2009. © 2008 Wiley‐Liss, Inc.