Effect of isolation methods on the recovery rates of pairs of follicles at less than 600 micro meter in diameter and their oocytes from bovine ovaries
2003
Sakakida, S. (Ishikawa Prefecture Coll. of Agriculture, Nonoichi (Japan)) | Izumi, T. | Matsumoto, Y. | Oonishi, M. | Nishikawa, T. | Miyamoto, H.
The objective of this study was to compare the mechanical method with a grating device and the enzymatic method on isolation of small follicles at less than 600 mm from bovine ovaries and on isolation of oocytes from these follicles for studying allometrically the relative growth relating the size of the follicle to its oocyte. Each ovary was cut into two halves, and the medulla was removed. The mechanical isolation of small follicles was performed by grating the ovarian cortex. The cortical fragments were then suspended in Hepes-buffered MEM 199 medium for loosing small follicles by pipetting. The enzymatic method was performed by using such proteolytic enzymes as collagenase I and DNase after mincing the ovarian cortex. The number of small follicles retrieved mechanically with the grating device per ovary within the limited time was an average =+- SE of 11.4 =+- 1.35 which was significantly higher than 7.5 =+- 0.81 obtained with enzymatic treatment (P < 0.05, Chi-square test). The recovery rates of oocytes from follicles isolated with the grating device and enzymatic treatment were 67.5% (135/200) and 60.8% (87/143), respectively. The difference between them was not significant, although it was suggested that the damage caused by residual enzyme after successive washes decreased the recovery rate of oocytes from small follicles.
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