Culture medium composition affects the gene expression pattern and in vitro development potential of bovine somatic cell nuclear transfer (SCNT) embryos

Ricardo N Felnier,Pablo J Ross,María E Arias

doi:10.4067/s0716-97602013000400016

Abstract

Different culture systems have been studied that support development of somatic cell nuclear transfer (SCNT) embryos up to the blastocyst stage. However, the use of sequential and two-step culture systems has been less studied. The objective of the present study was to examine the developmental potential and quality of bovine SCNT embryos cultured in different two-step culture media based on KSOM, SOF and the macromolecules FBS and BSA (K-K/FBS, K-S/BSA and K-K/BSA, respectively). No differences were observed in the cleavage rate for any of the culture systems. However, there was a significant difference (P<0.01) in the rate of blastocyst development, with the K-K/ FBS culture system yielding a higher rate of blastocysts (28%) compared to other treatments (18 and 15%, for K-S/BSA and K-K/BSA, respectively). Although quality of embryos, as assessed by the total number of cells, was not different, the apoptosis index was significantly affected in the sequential culture system (K-S/BSA). Gene expression analysis showed alterations of DNMT1, IGF2, LIF, and PRDX6 genes in embryos cultured in K-S/FBS and of SOD2 in embryos cultured in K-K/BSA. In conclusion, we demonstrated that culture medium may affect not only the developmental potential of SCNT embryos but also, more importantly, the gene expression pattern and apoptotic index, presenting the possibility to manipulate the culture medium composition to modulate global gene expression and improve the overall efficiency of this technique.

Highlights

Potential applications of animal cloning in agriculture, biotechnology, biomedicine and basic research (Cibelli et al, 1998, Wells et al, 1999, Keefer 2004) have increased interest in this technology for many researchers around the world
A number of different culture media have been extensively evaluated in an effort to improve the quality of embryos produced in vitro and to mimic the physiological conditions that embryos would have in vivo
Most of this work has been carried out in embryos produced by in vitro fertilization (IVF) and only a few studies have focused on the evaluation of different culture media for embryos produced by somatic cell nuclear transfer (SCNT), presumably due to the early assumption that both embryo production methods would behave under the same culture conditions and because of the extra technical requirements involved in the generation of SCNT embryos

Summary

Introduction

Potential applications of animal cloning in agriculture, biotechnology, biomedicine and basic research (Cibelli et al, 1998, Wells et al, 1999, Keefer 2004) have increased interest in this technology for many researchers around the world. Nuclear transfer success rates in cattle are around 11% with adult somatic cells (Kubota et al, 2000, Gibbons et al, 2002, Panarace et al, 2007), in a few instances higher efficiencies have been described when blastomeres were used as nuclear donors (Peura and Trounson 1998). These data demonstrate that low efficiency is still a major drawback for widespread use of cloning technology. Culture conditions based on defined components free of serum, BSA or cell constituents which may introduce uncharacterized factors into the culture and to some extent have been attributed to problems associated with large offspring syndrome (Young et al, 1998), are being developed (Lim et al, 2007, Jang et al, 2011)

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