Addition of Macromolecules to PZM-3 Culture Medium on the Development and Hatching of In vitro Porcine Embryos

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 abattoirderived 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.

Relationship between Developmental Ability and Cell Number of Day 2 Porcine Embryos Produced by Parthenogenesis or Somatic Cell Nuclear Transfer

“…This study evaluated the developmental ability and quality of parthenogenetic and cloned porcine blastocysts produced by separate culture of 2-4 cell and 5-8 cell embryos on Day 2 of in vitro culture. Embryo quality was assessed based on blastocyst cell number, ICM rate and expression of apoptosis-related genes that are well known indicators of embryo quality (Yoshioka et al, 2002;Jang et al, 2004;Gupta et al, 2007a;Kim et al, 2007;Gupta et al, 2008b;Kim and McElroy, 2008;Zhang et al, 2009). Our results show that 2-4 cell parthenogenetic and cloned porcine embryos had higher development competence than those of 5-8 cell stage embryos and hence, morphological selection of such embryos may be a useful non-invasive tool to improve cloning efficiency after embryo transfer.…”

“…ICM rate is also a valuable indicator of embryo quality (Yoshioka et al, 2002;Kim et al, 2007;Kim and McElroy, 2008). While the proportion of ICM cells in blastocysts was suggested to be crucial for later postimplantation development (Iwasaki et al, 1990), an increased ICM rate was suggested to be associated with ''large offspring syndrome'' in cloned animals (Barcroft et al, 1998;Leese et al, 1998).…”

iSCNT embryo culture system for restoration of Cervus nippon hortulorum, presumed to be sika deer in the Korean Peninsula

“…Activated iSCNT-derived embryos were cultured in NCSU-23 [ 30 ], PZM-3 [ 11 ], or PZM-5 [ 23 ] droplets covered with mineral oil (10–15 embryos/droplets). These were cultured for 7 days in an incubator with an environment of 5% CO₂, 5% O 2 , and 90% N 2 at a temperature of 39°C.…”

The effect of macromolecule and growth factor combinations on in vitrodevelopment of bovine embryos

“…The serum contains not only a variety of energy sources (3,6) but also growth factors (8,13) needed for in vitro embryonic development. The beneficial effect of more complex protein supplements is evident after the activation of the embryonic genome and probably due to the presence of growth factors (10). One of the most commonly used supplements, BSA is an embryotrophic macromolecule used in embryo culture media, which is commonly replaced with synthetic compounds, such as PVA.…”