Development and Application of a Chemically Defined Medium for the In Vitro Production of Porcine Embryos

Abstract

Abstract. Recent advances in systems for in vitro production (IVP) of porcine embryos, including in vitro oocyte maturation, fertilization and embryo culture, have enabled us to generate viable embryos that can develop to full term after transfer into recipients. This technology is being applied now to developments in gamete/embryo biology and agriculture, as well as in producing cloned and genetically modified pigs. Chemically defined media for IVP of embryos are useful for a precise analysis of the physical action of substances on gametogenesis and early embryogenesis, because they eliminate undefined factors present in biological materials, such as serum or serum albumin. Use of a chemically defined medium also improves the reliability of media formulations, yields a higher reproducibility of results and ensures biosafety of culture media by eliminating protein preparations, which may be contaminated with pathogens. Therefore, it has certain advantages for research and for commercial purposes. We have recently developed a defined IVP system for porcine embryos using a single basic medium based on the composition of porcine oviductal fluid. This paper discusses the developmental ability and normality of porcine IVP embryos, and limitations and advancements in this system. Key words: Chemically defined medium, Developmental competence, In vitro production, Piglet (J. Reprod. Dev. 57: [9][10][11][12][13][14][15][16] 2011) standard in vitro production (IVP) system for preimplantation embryos includes three technological steps: the in vitro maturation (IVM) of immature oocytes, in vitro fertilization (IVF) and in vitro culture (IVC) of zygotes. Successful large-scale IVP of porcine embryos will enable us to reduce the cost and time required and will be valuable for research in reproductive physiology, agriculture and biotechnology. The first successful production of piglets from in vitro-matured and in vitro-fertilized oocytes was reported by Mattioli et al. in 1989 [1], in which 2-to 4-cell embryos at 44 h after IVF were transferred into recipients. Since then, some laboratories have succeeded in producing piglets from cleaved embryos at the 2-to 4-cell stage cultured for 24-36 h [2][3][4] and from 8-cell to morula-stage embryos cultured for 96 h [5] following IVM and IVF. Moreover, in the present decade it has been demonstrated that porcine in vitro-produced blastocysts cultured for 5 or 6 days after IVF could develop to full term [6,7]. However, despite recent improvements in porcine IVP techniques, the developmental rate of embryos matured and fertilized in vitro to the blastocyst stage and their quality are still low compared with in vivo-derived embryos [8]. The low developmental competence of porcine in vitro-produced embryos might be caused by several factors, including a reduced incidence of male pronuclear formation, a high incidence of polyspermy and suboptimal conditions for embryo culture [9,10].Numerous media, such as modified Whitten's medium [11], North Carolina State University (NCSU) 23 medium...

Phosphorylated histone H2A.x in porcine embryos produced by IVF and somatic cell nuclear transfer

“…Because pigs are physiologically similar to humans, there is growing interest in the use of these technologies to create special animal models for the study of physiopathological processes and xenotransplantation (Kues & Niemann 2004, Kuwaki et al 2005, Aigner et al 2010. However, the development of IVF and SCNT porcine embryos is influenced by different factors, including oocyte quality, polyspermy, abnormal cell cycle interactions, deficient epigenetic reprogramming, and altered gene expression (Abeydeera et al 2000, Bortvin et al 2003, Lucifero et al 2006, Niemann et al 2008, Nascimento et al 2010, Whitworth & Prather 2010, Yoshioka 2011.…”

Current and Future Assisted Reproductive Technologies for Mammalian Farm Animals

“…Two important components to achieving this success are the conditions for oocyte maturation and embryo culture. A variety of modifi cations of culture media have been evaluated for optimizing oocyte maturation and embryonic development (see reviews by Hinrichs 2010 ;Vajta et al 2010 ;Yoshioka 2011 ;Block et al 2011 ). Data in cattle suggest that a period of withdrawal of FSH support for the follicle (termed FSH coasting) is benefi cial for subsequent in vitro maturation (Nivet et al 2012 ).…”

39 Factors Influencing the Efficiency of Generating Genetically Engineered Pigs by Nuclear Transfer: Multi-Factorial Analysis of a Large Data Set

“…This is possible since the in vitro culture systems for pig embryos have been markedly improved within the last decade [27,28]. Indeed, culture of embryos for two days and selection of 2-cell to 4-cell stage embryos for ET resulted in the highest proportion of offspring per SCNT embryos transferred.…”

“…However, the majority of these studies have addressed only single factors, e.g. SCNT procedure [23][24][25][26], oocyte and embryo culture systems [27,28], donor cell type [29,30], and the method of genetic modification [31,32]. Combined assessment of multiple factors and comparative analysis of their relative contribution to cloning efficiency have not yet been performed to our knowledge.…”