Culture of preantral follicles has important biotechnological implications through its potential to produce large quantities of oocytes for embryo production and transfer. The objective of this study was to determine the comparison of different isolation method of mouse preantral follicles, and to examine in vitro development of mouse preantral follicles isolated by different method. Preantral follicles were mechanically or enzymatically extracted from mouse ovaries. Mechanical isolation method used fine gauge needles and enzymatic method of isolating follicles used 1 mg/ml collagenage (Type IA) and 0.2 mg/ml DNase Ⅰ in Leibovitz L-15 medium. The solution containing Leibovitz L-15 medium, enzyme and ovary fragments was incubated at 37℃ for 30 min. The selection criteria are as follows: primary follicle of 75 to 99 μm, early secondary follicle of 100 to 125 μm and late secondary follicle of 126 to 150 μm in diameter. The recovered preantral follicles were cultured for 10 days in alpha-minimal essential medium (α-MEM) + 5% FBS + ITS + 100 mIU/ ml FSH. The collected primary follicles by enzymatic treatment were higher than mechanical method. Others stage preantral follicle by mechanical isolation were higher than enzymatic method. After 10 days of culture, no statistical differences were shown in survival rates of preantral follicle among the 2 culture groups. The metaphase Ⅱ rates of the oocytes were significantly higher (p<0.05) in mechanical method (17.8%) than in enzymatic method (5.1%). These results suggest that the isolation method of choice depends on the target stage preantral follicles and mechanical isolation is an optimal method of preantral follicles in a culture of mouse preantral follicle.

The objective of this study was to determine effects of different culture media. Preantral follicles were mechanically extracted from bovine ovaries and cultured for 16 days in tissue culture medium (TCM)‐199, DMEM or alphaminimal essential medium (α‐MEM) + 10% FBS + 0.1 mg/ml sodium pyruvate + 100 mIU/ml FSH. The collected primary follicles from ovary were higher than the primary and secondary follicles. The survival rates of the follicles in TCM‐199 were significantly higher (p<0.05) than those in DMEM and α‐MEM. The diameter of the follicles progressively increased during 12 days of culture. The maximum size (139.1±5.4 μm) reached on Day 12 of the in vitro culture and decreased on Day 16. These results suggest that in a culture of bovine preantral follicles, TCM‐199 is an optimal medium and a longer‐term culture of preantral follicles (>12 days) may be needed to form antra.

The purpose of this study was to assess for re-breeding concentrate period in postpartum in milking cows. The 48 cows aged 3.5～5.5 years and of 400～600 kg body weight were examined every 3rd day from 15 to 36 day postpartum. Blood samples for progesterone and estradiol 17 β hormone analyses were withdrawn from the coccygeal vein every third day until the end of the experiment. The ovarian follicular numbers were verified and measured using a multi frequency probe. The least squares means are presented for each day by GLM of SAS. The results showed that ovary lengths (right ovary; 1.64±0.62 cm, left ovary; 1.44±0.46 cm) were similar in right and left ovary activity level during estrous cycle of postpartum cows. We were judged completed uterus on day at 2.31±0.17 cm level of cervix diameter. And we were monitoring started at 6.44±2.03 cm from day 15 after postpartum. The results showed that mean plasma concentration of progesterone (3.28 ng/ml) in large follicle gradually increased days 30 in postpartum. And, monitoring of estradiol 17β (22.18 pg/ml) hormone during postpartum period would be useful to predict the ovarian and uterus activity for re-breeding in postpartum milking cows. From these results, we conclude that cervix diameter (mean: 2.31 cm) was very important for reproductive organ recovery standard level of postpartum milking cows, hormone secretion level (P4: 3.28 ng/ml, E2: 22.18 pg/ml) and body condition score (2.5～2.75) level about 30 days in postpartum period.