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Treatment of porcine donor cells and reconstructed embryos with the antioxidant melatonin enhances cloning efficiency

Authors

  • Yun-Wei Pang,

    1. Key Laboratory of Animal Genetics, Breeding and Reproduction of the Ministry of Agriculture, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing, China
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    • These authors contributed equally to this work
  • Lei An,

    1. Key Laboratory of Animal Genetics, Breeding and Reproduction of the Ministry of Agriculture, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing, China
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    • These authors contributed equally to this work
  • Peng Wang,

    1. Key Laboratory of Animal Genetics, Breeding and Reproduction of the Ministry of Agriculture, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing, China
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  • Yong Yu,

    1. Key Laboratory of Animal Genetics, Breeding and Reproduction of the Ministry of Agriculture, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing, China
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  • Qiu-Dan Yin,

    1. Key Laboratory of Animal Genetics, Breeding and Reproduction of the Ministry of Agriculture, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing, China
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  • Xiao-Hong Wang,

    1. Key Laboratory of Animal Genetics, Breeding and Reproduction of the Ministry of Agriculture, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing, China
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  •   Xin-Zhang,

    1. Key Laboratory of Animal Genetics, Breeding and Reproduction of the Ministry of Agriculture, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing, China
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  •   Qian-Zhang,

    1. Key Laboratory of Animal Genetics, Breeding and Reproduction of the Ministry of Agriculture, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing, China
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  • Mei-Ling Yang,

    1. Key Laboratory of Animal Genetics, Breeding and Reproduction of the Ministry of Agriculture, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing, China
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  •   Min-Guo,

    1. Key Laboratory of Animal Genetics, Breeding and Reproduction of the Ministry of Agriculture, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing, China
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  • Zhong-Hong Wu,

    Corresponding author
    • Key Laboratory of Animal Genetics, Breeding and Reproduction of the Ministry of Agriculture, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing, China
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  • Jian-Hui Tian

    Corresponding author
    • Key Laboratory of Animal Genetics, Breeding and Reproduction of the Ministry of Agriculture, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing, China
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Address reprint requests to Jian-Hui Tian or Zhong-Hong Wu, College of Animal Science and Technology, China Agricultural University, Yuanminyuan West Road 2#, 100193, Beijing,China. E-mails: tianjh@cau.edu.cn;wuzhh@cau.edu.cn

Abstract

This study was conducted to investigate the effect of melatonin during the culture of donor cells and cloned embryos on the in vitro developmental competence and quality of cloned porcine embryos. At concentrations of 10−6 m or 10−8 m, melatonin significantly enhanced the proliferation of porcine fetal fibroblasts (PFFs), and the blastocyst rate was significantly increased in the 10−10 m melatonin-treated donor cell group. Cloned embryo development was also improved in embryo culture medium that was supplemented with 10−9 m or 10−12 m melatonin. When both donor cells and cloned embryos were treated with melatonin, the cleavage rate and total cell number of blastocysts were not significantly affected; however, the blastocyst rate was increased significantly (20.0% versus 11.7%). TUNEL assays showed that combined melatonin treatment reduced the rate of apoptotic nuclei (3.6% versus 6.1%). Gene expression analysis of the apoptosis-related genes BAX, BCL2L1, and p53 showed that the expression of BCL2L1 was significantly elevated 2.7-fold relative to the control group, while the expression of BAX and p53 was significantly decreased by 3.7-fold and 23.2-fold, respectively. In addition, we detected the expression of two melatonin receptors (MT1 and MT2) in PFFs but not in porcine cloned embryos. We conclude that exogenous melatonin enhances the development of porcine cloned embryos and improves embryo quality by inhibiting p53-mediated apoptotic pathway. The proliferation of PFFs may be mediated by receptor binding, but the beneficial effects of melatonin on embryonic development may be receptor-independent, possibly through melatonin's ability to directly scavenge free radicals.

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