Production of Piglets Derived from In Vitro-Produced Blastocysts Fertilized and Cultured in Chemically Defined Media: Effects of Theophylline, Adenosine, and Cysteine During In Vitro Fertilization1

Abstract

To further develop defined conditions for in vitro fertilization (IVF) and in vitro culture (IVC) of in vitro-matured porcine oocytes, we evaluated the effects of theophylline, adenosine, and cysteine in a chemically defined medium during IVF. Viability to full term of in vitro-produced blastocysts after IVF and IVC in chemically defined medium was also investigated by embryo transfer to recipients. A chemically defined medium, porcine gamate medium (PGM), was modified from porcine zygote medium (PZM-4), which was previously established. PGM was used as a basal medium for IVF and PZM-4 was for the culture of presumptive zygotes. Addition of 2.5 mM theophylline to PGM significantly increased the percentage of male pronuclear formation compared with controls (no addition). Addition of 1 microM adenosine to PGM supplemented either with or without 2.5 mM theophylline significantly reduced the number of penetrated spermatozoa compared with controls (no addition of adenosine). Supplementation with 0.2 microM cysteine in PGM containing both 2.5 mM theophylline and 1 microM adenosine further increased the percentage of development to the blastocyst stage, compared with no supplementation of cysteine, but there was no difference in fertilization parameters, such as monospermy and pronuclear formation, regardless of presence or absence of theophylline and adenosine. When Day 5 blastocysts were transferred into four recipients (20-25 blastocysts per recipient), all recipients became pregnant and farrowed a total of 21 live piglets. The present results clearly demonstrate that porcine blastocysts can be produced by IVF and IVC in chemically defined media and that they can develop to full term after embryo transfer.

Aberrant DNA methylation in porcine in vitro‐, parthenogenetic‐, and somatic cell nuclear transfer‐produced blastocysts

“…Fifty microliters of the diluted sperm was added to the oocytes and incubated for 5 hr in 5% CO 2 in air at 398C. The presumptive zygotes were then washed three times and incubated in PZM3 embryo culture medium (Yoshioka et al, 2003) in 5% CO 2 at 398C. Blastocysts were removed after 6 days of culture and the zonae pellucidae of the blastocysts were removed as described.…”

Male pronucleus formation after ICSI: effect of oocyte cysteine or sperm Triton X-100 treatments

“…The ability of porcine oocytes to adequately develop after in vitro maturation (IVM) and fertilization (IVF), leading to live piglets, has been largely demonstrated by several laboratories (Kikuchi et al 2002;Yoshioka et al 2002Yoshioka et al , 2003Suzuki et al 2004Suzuki et al , 2006Coy et al 2005;Garcia-Rosello et al 2006a;Yong et al 2006;Maehara et al 2012;Kaneko et al 2013). Despite this fact, the efficiency of IVP of blastocysts after intracytoplasmic sperm injection (ICSI) still remains low ).…”

“…In this regard, several IVM approaches have been applied, such as oocyte co-culture with follicular cells (Zheng and Sirard 1992;Ding and Foxcroft 1994;Agung et al 2010), or supplement addition to the IVM defined media with: porcine follicular fluid (Naito et al 1988;Yoshida et al 1992;Ka et al 1997), follicular fluid meiosis-activating sterol (Faerge et al 2006), bovine serum albumin (Zheng and Sirard 1992), fetal bovine serum (Kishida et al 2004), fetal calf serum (Naito et al 1988), epidermal growth factor (EGF) (Ding and Foxcroft 1994;Uhm et al 2010), β-mercaptoethanol (Abeydeera et al 1998), cysteine (CYS) (Yamauchi and Nagai 1999;Yoshioka et al 2003;Kobayashi et al 2007;Choe et al 2010), ascorbic acid (Tatemoto et al 2001), leptin (Craig et al 2005;Jin et al 2009) or lycopene (Watanabe et al 2010), among others.…”

Evaluation of Developmental Competence of In Vitro-produced Porcine Embryos Based on the Timing, Pattern and Evenness of the First Cleavage and Onset of the Second Cleavage

“…Birth of piglets has been accomplished from IVM-IVF embryos after IVC to the 2-to 4-cell stages [3][4][5] or 8-cell to morula stage [6]. Although transfer of in vitro-produced (IVP) blastocysts has also led to pregnancies and live births [7][8][9], utilization of early IVP embryos for transfer remains preferable because the viability of IVP porcine embryos is decreased by IVC after IVF [10]. High oxygen tension, imperfect culture media and polyspermy are thought to reduce the developmental competence of IVP porcine embryos [11].…”