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The meiotic spindle in the oocyte is composed of microtubules and plays a key role in normal chromosome alignment and segregation during meiosis. In oocytes from farm animals, the meiotic spindle cannot be detected by conventional light microscopy due to the characteristic of their cytoplasm. Conventional methods to image the meiotic spindle rely on fixation of the oocytes. Polarized light microscopy (PLM) allows noninvasive evaluation of the meiotic spindle of metaphase oocytes. The aim of this study was to assess the efficiency of polarized light microscopy to detect microtubule-polymerized protein within in vitromatured prepubertal sheep and goat oocytes. We carried out 2 studies. In the first one, cumulus–oocyte complexes from slaughterhouse sheep ovaries were matured in vitrofor 27h. After in vitromaturation, oocytes (n=77) were denuded of cumulus cells and placed individually in 10-μL drops of TCM-199-HEPES-BSA in a glass Petri dish. Polarized light microscopy was used to detect the presence of polymerized protein, which could be associated with the forming of a meiotic spindle. To confirm the presence of the polymerized protein and the meiotic spindle, each individual oocyte was subjected to immunostaining and chromatin detection as described by (Morató et al.2008 Mol. Reprod. Dev. 75, 191–201). The experiment was replicated 4 times. The correlation analysis was performed using the Proc Corr procedure of SAS. There was a positive correlation (r=0.87; P<0.001) between the signal obtained by PLM and the presence of microtubule-polymerized protein as confirmed by immunostaining. A positive PLM signal was detected in 87.0% of the oocytes, and 69.0% of the oocytes reached the metaphase II (MII) stage after in vitromaturation. A barrel-shaped spindle was observed in 77.3% of the MII oocytes. In the second study, we performed a similar experiment but used goat oocytes. A total of 78 oocytes were used, and PLM and immunostaining were performed in each individual oocyte as it was described with sheep oocytes. There was also a positive correlation (r=1; P<0.001) between the signal obtained by PLM and the presence of microtubule-polymerized protein. A positive PLM signal was detected in 98.7% of the oocytes, and 80.7% of the oocytes reached the MII stage after in vitromaturation. A barrel-shaped spindle was observed in 92.0% of the MII oocytes. These results indicate that PLM is an efficient system to detect polymerized protein in in vitromatured sheep and goat oocytes. This work was supported by the following grant: INIA: RZ2007-00013-00-00. M. Muñoz and D. Martín are sponsored by RYC08-03454 and PTA2007-0268-I, respectively. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate the utility of the brilliant cresyl blue (BCB) test as an indirect measure of oocyte growth to select competent prepubertal sheep oocytes for in vitroembryo production. The BCB stain allows the determination of glucose–6-phosphate dehydrogenase (G6PD) activity, an enzyme with decreased activity in oocytes that have finished their growth phase. Oocytes were obtained after slicing the surface of lamb ovaries (2–5 months old) obtained from a local abattoir. Oocytes with more than 3 compact cumulus layers and homogeny cytoplasm were selected and stained with different concentrations of BCB diluted in PBS (13-, 26-, 39-, and 52-μM BCB) during 60min at 38.5°C in a humidified air atmosphere. Oocytes were classified into groups depending on their cytoplasm coloration: oocytes with blue cytoplasm or grown oocytes (BCB+) and oocytes without blue coloration or growing oocytes (BCB–). Oocytes were matured in an enriched TCM-199 medium for 24h at 38.5°C and 5% CO2in a humidified atmosphere. Oocyte diameter was also measured. Matured oocytes were partially denuded and transferred to fertilization medium (SOF) supplemented with 10% of oestrous sheep serum. Fresh semen was kept at room temperature (25°C) for 1h. Highly motile spermatozoa were selected by using Ovipure density gradient (Nidacon EVB S.L.), and oocytes were fertilized with 1×106spmL-1. After 20h postinsemination, presumptive zygotes were cultured for 8 days in SOF with 10% of fetal bovine serum at 38.5°C, 5% CO2, and 90% N2. Data was analysed by performing Fisher’s exact test for blastocyst production and ANOVA with Tukey’s post-test for oocyte diameter. Table 1 shows the percentage of BCB-stained oocytes and their embryo development. In this study oocytes exposed during 60min to 13-μM BCB showed a higher percentage of embryos reaching blastocyst stage than did those in the control group (≤0.01). In other species such as goats (Rodriguez-Gonzalez et al.2002 Theriogenology 57(5), 1397–1409) and cattle (Alm et al.2005 Theriogenology 63(8), 2194–2205), the best protocol for the oocyte selection was the use of 26-μM BCB during 90min. Oocyte diameter showed significant differences between BCB– with BCB+ and control group (110μm, 134μm, and 121μm, respectively, ≤0.001). In conclusion, using 13μM of BCB during 60min is a suitable technique for increasing embryo blastocyst rates using lamb oocytes. Table 1.Effect of BCB1concentration on embryo development of lamb oocytesThe grant sponsor was the Spanish Ministry of Science and Innovation, Code: AGL2007-60227-CO2-01. [ABSTRACT FROM AUTHOR]

The aim of this study was to assess the effect of an in vitrogrowth medium (De Wu et al.2006 Biol. Rep. 75, 547-554) in prepubertal ewe oocytes selected by the brilliant cresyl blue (BCB) test. Prepubertal ewe oocytes were recovered by slicing ovaries of slaughtered animals and immediately exposed during 1 h to 13 μM BCB and classified according to their cytoplasm coloration (Rodriguez-Gonzalez E et al.2002 Theri- ogenology 57(5), 1397-1409): BCB+ (blue cytoplasm, hypothetically grown oocytes) and BCB- (uncolored cytoplasm, hypothetically growing oocytes). Uncolored oocytes (BCB-) were matured using three culture media: growth medium (GM: TCM-199, 0.04 μg mL-1FSH, 0.04 μg mL-1LH, 0.004 μg mL-1estradiol, 100 μg mL-1ascorbic acid, and 5 μL mL-1ITS: insulin transferrin selenium), conventional maturation medium (CM: TCM-199, 10 μg mL-1FSH, 10 μg mL-1LH and 1 μg mL-1estradiol) and modified maturation medium (MM: CM with the addition of 100 μg mL-1ascorbic acid and 5 μL mL-1ITS). Oocytes were matured in GM for 12 h and then separated into 2 groups, CM (GM+CM) and MM (GM+MM) for another 12 h of maturation. Two extra groups of BCB- oocytes were directly cultured for 24 h in CM or MM media (BCB-/CM and BCB-/MM). Colored oocytes (BCB+) and a control group (oocytes not exposed to BCB) were matured for 24 h in CM. All groups were cultured at 38.5°C and 5% CO2in humidified atmosphere. Fertilization took place in SOF medium supplemented with 10% of estrous sheep serum during 20 h with a sperm concentration of 1 × 106spermatozoa/mL. Presumptive zygotes were cultured for 8 days in SOF with 10% FCS at 38.5°C, 5% CO2and 5% O2. Results are shown in Table 1. The percentage of morula plus blastocyst obtained from BCB - oocytes was significantly increased in oocytes exposed to growth medium (containing ITS, ascorbic acid and low hormone concentrations; groups GM+CM and GM+MM) for the first 12 h. An increasing tendency has also been observed in blastocyst yield in these two groups. Regarding maturation rate, no differences were found in all groups (data not shown). In conclusion, as De Wu et al.(2006) showed in prepubertal gilts, we also achieved some improvements in embryo development of growing oocytes when the first 12 h of maturation took place in a growth medium. However, embryo developmental potential of BCB- oocytes is still lower compared with that of BCB+ oocytes. Table 1.Effect of GM on embryo development of BCB- oocytesGrant sponsor Spanish Ministry of Science and Innovation.Code: AGL2007-60227-CO2-01 [ABSTRACT FROM AUTHOR]

An abstract of the study "Relative mRNA Expression of 4 Candidates in Lamb Oocytes Selected by Brilliant Cresyl Blue Staining," by M. G. Catalá et al. is presented.

The prepubertal goat ovary presents a large number of small oocytes with a compromised competence to develop up to blastocyst stage. In pigs (Wu et al.2006), using growth medium (GM) composed by low hormone concentrations, ascorbic acid, and insulin transferrin selenium (ITS) during the first 24h of in vitromaturation (IVM) improved embryo development of small oocytes. The aim of this study was to test the GM in small prepubertal goat oocytes in order to increase blastocyst yield. The cumulus–oocyte complexes (COC) were recovered from prepubertal (1–2 months old) goat ovaries by slicing. The COC with a compact cumulus and homogeneous cytoplasm were selected and classified into 2 categories based on oocyte diameter: <125μm and ≥125μm. The ≥125μm oocytes were matured in groups of 25 to 30 COC/100μL drops of conventional IVM medium covered with mineral oil for 24h (Treatment A). This medium was TCM-199 supplemented with 10% donor bovine serum, 10μgmL-1FSH, 10μgmL-1LH, 1μgmL-117β-oestradiol, and 100μM cysteamine. The <125μm oocytes were distributed into 3 experimental groups: Treatment B, COC matured in the conventional IVM medium; Treatment C, COC cultured in GM (TCM-199, 10% donor bovine serum, 0.04μgmL-1FSH, 0.04μgmL-1LH, 0.004μgmL-117β-oestradiol, 100μM cysteamine, 100μgmL-1ascorbic acid, and 5μLmL-1ITS) for 12h before placement for other 12h in the conventional IVM medium, all drops of growth or maturation medium were covered with mineral oil; Treatment D, COC cultured during the first 12h in GM and other 12h into the conventional medium supplemented with 100μgmL-1ascorbic acid and 5μLmL-1ITS. After IVM, oocytes were fertilized for 24h with a sperm concentration of 4×106spzmL-1. Presumptive zygotes were cultured in SOF for 9 days. The cleavage rate was evaluated at 48h post-insemination and blastocyst percentages at the final in vitroembryo culture (treatments A, B, C: 5 replicates; treatment D: 4 replicates). The results are shown in the Table 1. Cleavage and embryo development did not show different results when we compared small oocytes matured in GM to those matured in conventional IVM medium. However, the biggest oocytes (≥125μm) showed the highest percentage of blastocyst development. The current study shows that the culture of small prepubertal goat oocytes in GM does not improve blastocyst yield. Table 1.Effect of growth medium on embryo development of small oocytes (<125μm) from prepubertal goats [ABSTRACT FROM AUTHOR]