Morphologic changes of porcine cumulus-oocyte complexes (COCs) after preservation for 3 d.

<p>a1, a2, b1, b2, c1, c2, When preserved in TCM-199, pFF and FCS at 4°C or 27.5°C, cumulus cells attached tightly to oocytes and displayed no expansion; a3, c3, After preservation in TCM-199 and FCS at 38.5°C, almost all of the cumulus have shed from the oocytes; b3, After pFF preservation at 38.5°C, the whole COCs degenerated and showed black appearance; b2, c2, After preservation in pFF and FCS at 27.5°C, oocytes displayed dark ooplasm with compact cumulus cells. Scale bar  = 200 µm.</p

The GVBD inhibition after preservation for different times in different media at 27.5°C.

<p>Data are presented as means ± SEM from three replicated experiments.</p>1<p>Means oocytes treated in every group.</p>2<p>Percentages are based on the total number of oocytes examined in every group.</p>A, B<p>Values with different superscripts are significantly different in each column (P<0.05).</p>a, b<p>Values with different superscripts are significantly different in each line (P<0.05).</p

Maturation of oocytes after preservation at 27.5°C in different media.

<p>Data are presented as means ± SEM from three replicated experiments.</p>a, b, c<p>Values with different superscripts are significantly different in each column (P<0.05).</p

Effects of different preservation media on GSH level after preservation at 27.5°C for 3 d.

<p>Data are presented as means ± SEM from three replicated experiments.</p>a, b, c<p>Values with different superscripts are significantly different in each column (P<0.05).</p

Effect of different media on CG distribution in porcine oocytes after preservation.

<p>A. a, b, CGs perinuclear area distribution within the porcine oocytes; a, most of the CGs aggregate in the perinuclear area, with a few migrating to the central inner cytoplasm or cortical area; b is intermediate pattern between a and c, with most of the CGs still remaining in perinuclear area and some migrating to the central cytoplasm; c, CGs distributed uniformly in the inner cytoplasmic area, and a few migrated to the cortical area to form a discontinuous ring; d, CGs cortical area distribution; almost all of the CGs have migrated into the cortical area, and a few still remained in the central cytoplasm. Green, cortical granules. Scale bar = 10 µm. (White arrows pointing at the white circle denotes the germinal vesicle). B. Ratios of different CG distribution patterns within the porcine oocytes after preservation in different media. Porcine oocytes were preserved in TCM-199, pFF and FCS at 27.5°C for 3 d, and then CG distribution was stained with FITC-labeled peanut agglutinin. Three categories of CG distribution were detected: perinuclear area; inner cyoplasmic area; cortical area. The graph shows the mean ± SEM of three independent experiments. The number “n” in the bracket means the total treated oocyte in every group. The superscripts ^{a, b} over the bars represent values that differ significantly in every categories of CG distribution (P<0.05).</p

Effect of preserving medium on early development of IVM-IVF porcine oocytes after preservation at 27.5°C for 3 d.

<p>Data are presented as means ± SEM from three replicated experiments.</p><p>The values “n” in bracket represent the number of the oocytes cleaved at 48 hr and forming blastocysts at 7 d after IVF.</p>1<p>Percentages are based on the total number of oocytes examined.</p>a, b, c<p>Values with different superscripts are significantly different in each column (P<0.05).</p

Effects of different preservation media on spindle configuration in porcine oocytes after IVM.

<p>A. a, represents a normal spindle in porcine oocyte. b and c represent abnormal spindle. In b1, c1 arrows indicate abnormal spindle organization distribution. In b2, arrows indicate fragmented and displaced chromosomes. Green, tubulin; Red, chromatin. Scale bar  = 10 µm. B. Normal ratios of spindle configuration in porcine oocytes after preservation in different media following IVM. Porcine oocytes were preserved in TCM-199, pFF and FCS at 27.5°C for 3 d, then spindle was stained with FITC-α-tubulin after culture for 44 h <i>in vitro</i>. Normal and abnormal spindle configuration were detected. The graph shows the mean ± SEM of three independent experiments. The number “n” over the bars means the total treated oocyte in every group. The superscripts ^{a, b} over the bars represent values of normal tubulin configuration that differ significantly between groups (P<0.05).</p

Effect of different media on actin configuration in porcine oocytes after preservation.

<p>A. a, represents the normal actin pattern in porcine oocytes (white arrow pointing at the white circle denotes the germinal vesicle). b, c, and d represent abnormal actin patterns. In b, actin fragments formed an incomplete ring with large punctiform aggregation. In c, small punctiform actin aggregated as an incomplete ring. In d, only some actin aggregated near oocyte membrane. Green, microfilaments; Red, chromatin. Scale bar  = 10 µm. B. Ratios of oocytes with normal actin configuration in control group and different preservation groups. Porcine oocytes were preserved in TCM-199, pFF and FCS at 27.5°C for 3 d, then the actin configuration was stained with FITC-phalloidine. Normal and abnormal actin configuration were detected. The graph shows the mean ± SEM of three independent experiments. The number “n” over the bars means the total treated oocyte in every group. The superscripts ^{a, b} over the bars represent values of normal actin configuration that differ significantly (P<0.05).</p

Effects of different media on mitochondria distribution in porcine oocytes after preservation.

<p>A. a, peripheral mitochondrial distribution, with no distribution at the center of the oocytes; b, semi-peripheral mitochondrial distribution; c, mitochondrial relocation and uniform distribution in the inner region of oocyte cytoplasm. Red, Mito Tracker Red staining. Scale bar  = 10 µm. B. Ratios of different mitochondria distribution patterns within the porcine oocytes after preservation with different media. Porcine oocytes were preserved in TCM-199, pFF and FCS at 27.5°C for 3 d, and then mitochondria distribution was stained with MitoTracker Red CMXRos. Three categories of mitochondria distribution were detected: peripheral; semi-peripheral; diffuse. The graph shows the mean ± SEM of three independent experiments. The number “n” in the bracket means the total treated oocyte in every group. The superscripts ^{a, b, c} over the bars represent values that differ significantly in every categories of mitochondrial distribution (P<0.05).</p

Effects of temperatures and media on GVBD inhibition of porcine oocytes after preservation for 3 d^*.

<p>Data are presented as means ± SEM from three replicated experiments.</p>*<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014242#pone-0014242-t001" target="_blank">Table1</a> was analyzed with double factor variance analysis, because the effects of different media, time, and the interaction are all significant. So we did not consider the main effects.</p>1<p>Means oocytes treated in every group.</p>2<p>Percentages are based on the total number of oocytes examined in every group.</p>A, B, C, D<p>Values with different superscripts are significantly different in each column (P<0.05).</p>a, b<p>Values with different superscripts are significantly different in each line (P<0.05).</p