Recovery of COCs from ovaries with high follicle numbers enhances in vitro embryo yield in sheep

The aim of the present study was to investigate the correlation between the number of ovarian follicles and in vitro embryo development and quality in sheep. ewe ovaries were classified according to the number of follicles: ≤4 (Low), 5–7 (Intermediate), and ≥8 (High). IVM, IVF and IVC were performed under standard conditions. Cleavage rate and blastocyst development were assessed 48 h after fertilization and on Days 6, 7 and 8 of culture, respectively. Expanded blastocysts were vitrified; blastocoel re-expansion and hatching rates were assessed at 8, 16 and 72 h post-thawing and hatched blastocysts were analyzed with the TUNEL assay. In a subset of thawed blastocysts the incorporation of amino acids was evaluated. The proportion of ovaries varied significantly among the three groups (ANOVA F = 12.20, P = 0), and more ovaries (59%) were assigned to the Low group than to the Intermediate (28%; ANOVA F = 8.19, P = 0.009) and High group (13%; ANOVA F = 18.63, P = 0), (High vs. Intermediate F = 6.31, P = 0.020). The three groups statistically differed in the proportion of total blastocysts ( χ 2 2 = 22.616, P = 0.00), of blastocysts produced on Days 6 ( χ 2 2 = 6.829, P = 0.033) and 7 ( χ 2 2 = 6.810, P = 0.033), while no difference was found in the proportion of blastocysts obtained on Day 8 ( χ 2 2 = 3.874, P = 0.144) of culture after fertilization. A higher proportion of total blastocysts was obtained from the High (44%) compared with the other two groups (Low: 28%, χ 2 2 = 22.629, P = 0; Intermediate: 33%, χ 2 2 = 7.266, P = 0.007), while the Low and Intermediate groups did not statistically differ either in the total blastocyst output ( χ 2 2 = 3.384, P = 0.066), nor in the number of blastocysts produced on Days 6 (Low: 7%, Intermediate: 9%; χ 2 2 = 0.874, P = 0.35), 7 (Low: 14%, Intermediate: 16%, χ 2 2 = 1.256, P = 0.26) and 8 (Low: 6%, Intermediate: 7% χ 2 2 = 0.554, P = 0.45) of culture. The High group produced a significantly higher percentage of embryos on Days 6 (High: 13%, Low: 7%; χ 2 2 = 6.840, P = 0.009) and 7 (High: 21%, Low: 14%; χ 2 2 = 6.806, P = 0.009) of culture post-insemination than the Low group. ree categories did not differ in the blastocoel re-expansion ( χ 2 2 = 0.095, P = 0.95) and hatching rates ( χ 2 2 = 0.754, P = 0.68) after 72 h post-warming, in the total number of cells per blastocyst (ANOVA F = 1.12, P = 0.337) and in the (F = 0.46, P = 0.639) incorporation of amino acids. The number of TUNEL-positive cells per embryo was higher (ANOVA F = 4.32, P = 0.022) in the Low group compared to the other groups. In conclusion, high ovarian follicle number enhances in vitro embryo output in sheep, but has no effect on blastocyst quality.