Blastocyst development of oocytes recovered from slaughterhouse ovaries (CONT) and by ovum pick-up (OPU) from the ovaries of non-superstimulated females (IMA), superstimulated females (FSH) and superstimulated females that received an ovulation inducer (MII) that were vitrified (VIT) at the metaphase II stage.

<p>^a,b,c,d Values with different superscripts in the same column are different at P<0.05.</p><p>* Hatched blastocyst at D8 as a percentage of oocyte number.</p><p>Blastocyst development of oocytes recovered from slaughterhouse ovaries (CONT) and by ovum pick-up (OPU) from the ovaries of non-superstimulated females (IMA), superstimulated females (FSH) and superstimulated females that received an ovulation inducer (MII) that were vitrified (VIT) at the metaphase II stage.</p

Experimental design flow diagram.

<p>Flow diagram of experimental design for the different treatments. Oocytes recovered from slaughterhouse ovaries (CONT), obtained by OPU from non-superstimulated females (IMA) and from superstimulated females (FSH) were matured in vitro. In vivo-matured oocytes were obtained by OPU from superstimulated females that received an ovulation inducer 24 hours previously (MII). A sample of matured oocytes from each of four groups was used to study the composition of plasma membrane phospholipids using MALDI-TOF. The remaining oocytes were divided in half, one half consisting of non-vitrified fresh oocytes (CONT, IMA, FSH and MII) and other of vitrified/ warmed oocytes (CONT Vit, IMA Vit, FSH Vit and MII Vit). At the end of the warming process, the eight groups were used for in vitro fertilization and culture. Cleavage at D2 and blastocyst development at D7 and D8 were evaluated. At D8, all of the blastocysts were measured, and those larger than 160 μM in diameter were stained for total cell number counting.</p

Percentage, mean (μm) and standard deviation (SD) of size (μm) and total cell number of D8 blastocyst with diameters > 160 μm derived from oocytes of different maturation conditions: slaughterhouse ovaries (CONT) and by OPU, from ovaries of non-superstimulated females (IMA), superstimulated females (FSH) and superstimulated females that had received a ovulatory inducer (MII).

<p>^a,b,c,d Values with different superscripts in the same column are significantly different by Kruskall-Wallis test (P < 0.05).</p><p>CONT = oocytes from slaughterhouse ovaries that were matured in vitro; IMA = OPU oocytes from non-stimulated animals matured in vitro; FSH = OPU oocytes from FSH simulated animal and matured in vitro; MII = OPU oocytes after in vivo maturation.</p><p>*Represents to the quantity of embryos that was able to be evaluated in the counting of total cell numbers because some embryos were lost during staining or could not be observed.</p><p>Percentage, mean (μm) and standard deviation (SD) of size (μm) and total cell number of D8 blastocyst with diameters > 160 μm derived from oocytes of different maturation conditions: slaughterhouse ovaries (CONT) and by OPU, from ovaries of non-superstimulated females (IMA), superstimulated females (FSH) and superstimulated females that had received a ovulatory inducer (MII).</p

3D PCA plot for MALDI-TOF data of individual oocytes from different maturation systems.

<p>Shown is a 3D PCA plot for the MALDI-TOF data of single oocytes. a) Red (n = 12), blue (n = 13), pink (n = 13) and dark yellow (n = 10). Each point indicates the 3D PCA plot of an oocyte based on its phospholipid composition. The following four fresh oocyte experimental groups are represented: immature and in vitro-matured oocytes recovered from slaughter house ovaries (CONT), oocytes obtained by OPU from non-superstimulated females (IMA), superstimulated females (FSH) and in vivo-matured oocytes obtained by OPU from superstimulated females that received an ovulation inducer (MI). b) indicates the main ions represented, 760.6[PC (34:1) + H]⁺ and 782.6 [PC (34:6) + H]⁺ or [PC (34:1) + Na]⁺, are responsible for the most variability between the treatments. The three principal components explain >73% of the variability of the data.</p

Comparison of the relative intensity of most dispersed ions after mass spectrometry (MALDI-TOF) analyses, of oocytes from different maturation systems.

<p>The 760.6 ion corresponds to [PC (34:1) + H] ⁺ and 782.6 to [PC (34:6) + H] ⁺ or [PC (34:1) + Na], both phosphatidylcholines (PC).</p><p>^ab Different letters in the same column indicates statically differences among the treatments after ANOVA according to Tukey’s test (P<0.05).</p><p>CONT = oocytes from slaughterhouse ovaries and matured in vitro; IMA = OPU oocytes from non-stimulated animals and matured in vitro; FSH = OPU oocytes from FSH simulated animal and matured in vitro; MII = OPU oocytes after in vivo maturation.</p><p>The data are expressed as arbitrary unit intensity and standard deviation (±SD), and each ion represents a different phospholipid.</p

Total number (N), mean and standard deviation (±SD) of follicles per female evaluated and classified by the color Doppler, after nine replicates, as having intense, moderate or absent blood vascularization in the ovaries of non-superstimulated (IMA), superstimulated (FSH) and superstimulated females that received an ovulation inducer (MII).

<p>^a,b,c Values with different superscripts in the same column are significantly different by Kruskall-Wallis test (P < 0.05).</p><p>Total number (N), mean and standard deviation (±SD) of follicles per female evaluated and classified by the color Doppler, after nine replicates, as having intense, moderate or absent blood vascularization in the ovaries of non-superstimulated (IMA), superstimulated (FSH) and superstimulated females that received an ovulation inducer (MII).</p

Total number (N) and percentage (%) of viable and nonviable oocytes recovered by ovum pick-up, after nine replicates, from the ovaries of non-superstimulated (IMA), superstimulated (FSH) and superstimulated females that received an ovulation inducer (MII).

<p>^a,b,c Values with different superscripts in the same column are significantly different, by to Chi—square test (P < 0.05).</p><p>* Only those oocytes presenting less than three cumulus cell layers or heterogeneous cytoplasm were classified as non-viable for IMA and FSH groups. For the MII group, only oocytes with heterogeneous cytoplasm or without the first polar body extrusion and/or cumulus cell expansion were considered non-viable. The percentage is expressed as the ratio to the total number of recovered oocytes.</p><p>**Oocytes with heterogeneous cytoplasm and presenting vacuolization at the three experimental groups were classified as degenerated. The percentage is expressed as the ratio of the total number of non-viable oocytes.</p><p>Total number (N) and percentage (%) of viable and nonviable oocytes recovered by ovum pick-up, after nine replicates, from the ovaries of non-superstimulated (IMA), superstimulated (FSH) and superstimulated females that received an ovulation inducer (MII).</p