Effects of melatonin on in vitro maturation of porcine oocyte and expression of melatonin receptor RNA in cumulus and granulosa cells.

Melatonin is a multifunctional molecule that mediates several circadian and seasonal processes in animal reproduction. Melatonin and its metabolites are antioxidants and free radical scavengers. We investigated the effects of melatonin on porcine oocyte maturation and embryo development. We then investigated the local expression of the melatonin receptor 1 (MT1) gene in cumulus cells, granulosa cells, and the oocytes with the reverse transcription-polymerase chain reaction (RT-PCR) method. We further evaluated the antioxidant effects [reactive oxygen species (ROS) levels in cumulus-oocytes complexes] of melatonin supplementation during in vitro maturation (IVM). Compared with control, melatonin supplementation (10 ng/mL) during IVM resulted in a greater proportion of oocytes extruding the polar body (75.6% versus 84.6%). Significantly greater proportion of parthenogenetically activated oocytes developed to blastocysts when the in vitro medium was supplemented with melatonin; however, cleavage frequency and blastocyst cell number were not affected by the treatment. RT-PCR analysis revealed the expression of MT1 gene in cumulus and granulosa cells but not in oocytes. Melatonin-treated oocytes had significantly lower levels of ROS than did control (untreated) oocytes. We conclude that exogenous melatonin has beneficial effects on nuclear and cytoplasmic maturation during porcine IVM. Some of the observed effects may be mediated by receptor binding and while others may have been receptor independent, e.g., direct free radical scavenging.

Temporal effects of alpha-tocopherol and L-ascorbic acid on in vitro fertilized porcine embryo development.

The susceptibility of embryos to reactive oxygen species (ROS) varies in different stages of embryo development. The present study evaluated temporal effects of alpha-tocopherol and L-ascorbic acid on the porcine embryo development, and investigated whether a single or twice supplements of these two antioxidants at a divided concentrations favors the embryo development. In order to determine temporal effects of alpha-tocopherol and/or L-ascorbic acid, 100 microM alpha-tocopherol or 200 microM L-ascorbic acid were supplemented to the North Carolina State University (NCSU)-23 embryo culture media at 0, 48, 96 and 120 h of culture. In another set of experiments, the concentration was divided into two equal halves, i.e., 50 microM alpha-tocopherol and 100 microM L-ascorbic acid, and supplemented twice at 0 and 48, 0 and 96, or 48 and 96 h of culture. Supplementing culture media with 100 microM alpha-tocopherol for the entire culture period of 168 h or starting from the 48 h of culture yielded higher blastocyst percentage compared with the control or starting from the 96 or 120 h of culture. L-Ascorbic acid (200 microM) alone or together with alpha-tocopherol (100 microM) with a single supplement did not affect the frequency of blastocyst formation or number of cells in blastocyst. L-ascorbic acid with a divided supplements yielded higher blastocyst percentage compared with the control. No synergistic effect was observed on embryo development at a single supplement of these antioxidants. Although, at divided supplements higher blastocyst percentage was observed compared with control group, no further beneficial effect was observed compared with alpha-tocopherol or L-ascorbic acid alone. Our results demonstrated that the embryotrophic effects of alpha-tocopherol and/or L-ascorbic acid, in terms of frequency of blastocyst formation and number of cells in blastocyst, depends on the concentration and supplementation timing.

Effect of potassium simplex optimization medium and NCSU23 supplemented with beta-mercaptoethanol and amino acids of in vitro fertilized porcine embryos.

The present study was conducted to examine the comparative efficacy of potassium simplex optimization medium (KSOM) and North Carolina State University (NCSU)-23 medium supplemented with beta-mercaptoethanol (beta-ME) and amino acids (AA) on the developmental competence of porcine in vitro fertilized (IVF) embryos. Four experiments were conducted. KSOM and NCSU-23 medium were used to culture porcine parthenogenetic (Exp. 1) and IVF (Exp. 2) embryos. KSOM and NCSU-23 were equally effective in supporting porcine parthenogenetic and IVF embryo development from the 1-cell stage to blastocysts. The NCSU-23 medium (Exp. 3) and KSOM (Exp. 4) were supplemented with amino acid (AA; 5 microl/ml non-essential amino acids + 10 microl/ml essential amino acids) and/or 10 microM beta-mercaptoethanol (beta-ME). The quality of blastocysts from Exp. 3 and 4 was evaluated by counting the number of total cells and determining the ratio of the inner cell mass (ICM) to trophoectoderm (TE) cells. Supplementing with AA and beta-ME or beta-ME alone in NCSU-23 produced significant (p<0.05) differences in terms of rate of cleavage to the 2- to 4- cell (80.8 to 85.4% vs. 73.6%) and blastocyst (30.4 to 30.5 vs. 23.5%) stages and the number of TE (51.4 to 53.8 vs. 35.8) and total cells (67.2 to 71.2 to 48.8) over the control group. On the other hand, supplementing KSOM with AA and/or beta-ME produced significant (p<0.05) differences in terms of rate of cleavage to the 2- to 4-cell (78.8% vs. 67.7%) and morula (57.8% vs. 46.3%) stages and the number of ICM (18.6 to 19.2 vs. 11.6) and total cells (62.8 to 70.6 vs. 42.8) over control group. In conclusion, our study demonstrates that both KSOM and NCSU-23 medium supplemented with AA and beta-ME and/or only beta-ME alone are superior to normal KSOM and NCSU-23 for porcine IVF embryo culture in terms of embryo developmental competence and quality.

Embryotrophic effects of ethylenediaminetetraacetic acid and hemoglobin on in vitro porcine embryos development.

This study investigated the embryotrophic effects of ethylenediaminetetraacetic acid (EDTA) and hemoglobin (Hb) on porcine preimplantation embryo development. Porcine embryos produced by in vitro maturation/fertilization were cultured for 6 days in modified North Carolina State University-23 medium (mNCSU-23) supplemented with EDTA and/or Hb. In Exp. 1, culturing porcine zygotes with 100 microM EDTA significantly increased cleavage frequencies (85.3%) at 48 h post insemination and the number of inner cell mass (ICM) (9.6+/-5.5) compared to the control (7.0+/-2.8). However, 100 microM EDTA did not improve blastocyst formation compared to 0, 1 or 10 microM EDTA. In Exp. 2, in vitro fertilized oocytes were cultured with 0, 1 or 10 microg/ml Hb. Culturing with Hb did not promote porcine embryo development, but significantly increased the cell numbers of blastocysts in 1 microg/ml Hb compared to 0 or 10 microg/ml Hb. In Exp. 3, culturing embryos with 100 microM EDTA+1 microg/ml Hb significantly improved frequencies of cleavage, blastocyst formation, and total cell numbers in blastocysts compared to the control. Moreover, 100 microM EDTA, 1 microg/ml Hb and their combination reduced reactive oxygen species (ROS) accumulation and decreased the incidence of apoptosis. In conclusion, the present study clearly demonstrated that the combining treatment of EDTA and Hb improved IVF porcine embryo development.