Abstract
In this study, we conducted various experiments in order to develop enhanced cultural conditions for in vitro-produced porcine embryos. All embryos were produced by in vitro maturation (IVM) and fertilization (IVF) of immature oocytes from abattoir- derived ovaries. In experiment 1, we cultured IVF embryos in 4 different groups, namely, 0% bovine serum albumin (BSA), 3% BSA, 0.05% Polyvinyl alcohol (PVA), and 0.5% Polyvinylpyrrolidone (PVP) added to the basal fluid cultural medium, Porcine zygote medium 3 (PZM-3). The rates of embryo development were higher in the group where the PZM-3 media had been supplemented with 3% BSA than the other groups. While not statistically significant, the percent of blastocysts and hatched blastocytes were 6.9% and 25.0% in the 3% BSA group vs. 1.2-6.4% and 0-16.7% in the other groups, respectively. In experiment 2, we added 10% fetal bovine serum (FBS) to PZM-3 on day 0 of culture and observed the development rate of blastocysts per day of culture from days 0 to 5. The development rate of blastocysts was higher at 15.6% on day 4 than on any other day, and was significantly higher than on day 0 or day 1 (p<0.05). The development rate of hatched blastocysts was 26.7% on day 4, and was higher than on any other day. In experiment 3, we cultured IVF embryos with different fluid culture media, grouped as 1) PZM-3+0.3% BSA (day0-day7); 2) PZM-3+0.3% BSA→(day-4) PZM-3+10% FBS; 3) PZM-3+0.3% BSA→PZM-3+0.3% BSA+(day-4) FBS 10%; and 4) PZM-3+0.3% BSA+10% FBS (day0-day7). The development rates of blastocysts and hatched blastocysts were 21.5% and 53.1% in group 3, respectively, which was significantly higher than group 4 with respect to blastocyst development (5.2%, p<0.05) but not hatched blastocysts (14.3%). The total cell number (TCN) of blastocysts in group 3 was higher at 37.8±16.1 than the other groups at 16.8±4.4 - 30.1±10.9; however, this was not significantly different. The results of this study showed that PZM-3 containing 0.3% BSA and supplemented with FBS during the later stage of culture on day 4 resulted in better TCNs and an increased rate of hatched blastocysts. (Key Words : Porcine Embryos, Macromolecule, Blastocysts, Cell Number)
Highlights
In vitro production (IVP) of porcine embryos is one of the major aspects of the study of mammalian embryogenesis and is widely used in veterinary medicine, animal husbandry, and biotechnology as a means for production of individuals with superior genetic characters by embryo transplantation, development of micromanipulation techniques (Lee et al, 2007), and stem cell studies.Many scientists have studied IVP of porcine embryos for a number of years. Iritani et al (1978) successfully performed in vitro fertilization (IVF) of porcine embryos in 1978 and Mattioli et al (1989) succeeded in birthing piglets by IVF embryo transplantation
In the present study, we researched the effects on the development and hatching of porcine in vitro embryos by addition of various macromolecules to Porcine Zygote Medium 3 (PZM-3) medium and serum addition at later stages of culture in order to establish enhanced culture conditions in order to better understand the process of basic molecular metabolism with respect to the IVP of porcine embryos
Solutions were expressed as percent dilutions (v:v) and all media used for in vitro matured (IVM), IVF and in vitro culture (IVC) were pre-warmed at 39°C in a 5% CO2 atmosphere at maximum humidity 4 h prior to use
Summary
In vitro production (IVP) of porcine embryos is one of the major aspects of the study of mammalian embryogenesis and is widely used in veterinary medicine, animal husbandry, and biotechnology as a means for production of individuals with superior genetic characters by embryo transplantation, development of micromanipulation techniques (Lee et al, 2007), and stem cell studies. One report indicated that addition of serum to later stage embryos produced in vitro could accelerate the development of blastocysts, resulting in an 80% hatch rate (Thompson et al, 1998; Choi et al, 2002). In the present study, we researched the effects on the development and hatching of porcine in vitro embryos by addition of various macromolecules to PZM-3 medium and serum addition at later stages of culture in order to establish enhanced culture conditions in order to better understand the process of basic molecular metabolism with respect to the IVP of porcine embryos
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