In vivo Ultrasonography and Ovariectomy for Evaluating Individual Follicles Superovulated on Different Stage of Estrous Cycles in Japanese Black Cows

为了解不同发情周期应用卵泡刺激素(FSH)超排治疗的卵巢反应，分别于第7天(3头奶牛)和第10天(4头奶牛)开始超数排卵治疗，同时进行卵巢切除和卵泡形态观察。在发情前注射前列腺素F_(2a)和前列腺素T_(2a)，每隔12小时用超声监测一次卵巢，直到超数排卵治疗结束。如前所述，所有直径超过4 mm的卵泡都在冷冻超声图像中使用内置卡尺进行监测。两组的优势卵泡直径(DF)均明显大于最大从属卵泡直径(LS…在第7天组，DF在第7天达到最大直径(7.4 mm)，而第10天组在第8天达到最大直径(12.7 mm)，第10天停止生长(12.4 mm)。在第7天组，S大小的卵泡数从第一次排卵时的30.7个减少到末次排卵时的17.0个，而L大小的卵泡数从1 0个增加到15.7 mm。第10天组，S体型由32.8 mm降至17.5 mm，L体型增大1.3 mm至31.5 mm。在第7天组，对FSH注射有反应的卵泡(直径4 mm)在第一次注射FSH后48小时达到最多(5.0个卵泡)，而第10天组在24小时(8.0个卵泡)出现最多。在超排过程中，第10天组的血浆E_2水平显著高于第7天组。在超排过程中超声观察到223个卵泡(直径4 mm)中，170个卵泡被单独解剖(73%的回收率)，123个卵母细胞在体视显微镜下采集(72%的回收率)。第7天组与第10天组卵泡液中类固醇激素浓度差异无统计学意义，但较晚出现的卵泡有增加卵泡液中类固醇E/P比值的趋势。对92个卵母细胞进行荧光显色处理后，卵母细胞呈钙黄素-AM阳性。不同处理的卵泡形态特征没有显著差异。这些结果表明，在DF丧失其显性作用后第10天开始的超排处理比在DF仍处于优势阶段的第7天执行的处理更容易提高卵巢反应。较少

In order to clarify the ovarian response to the superovulation treatment carried out with FSH in different estrous cycles, a consecutive ultrasonography for tracing the individual follicular development and an ovariectomy for follicular morphology were performed.The superovulation treatment accompanied with multiple intramuscular injections of p-FSH (28 AU) executed in decreasing doses at 12 hr intervals for 4 days, was initiated on Day 7 (3 cows) and on Day 10 (4 cows). Ovaries were monitored with ultrasonography at 12 hr intervals after an estrus previously synchronized estrus by PGF_<2a> injection until the end of superovulation treatment. All the follicles more than 4mm in diameter were monitored in freezing ultrasonography images with built-in calipers as previously described. And then, the ovariectomy was carried out at 1OO hr after the 1^<st> FSH injection.In both groups, the diameter of dominant follicle (DF) became significantly larger than the largest subordinate follicle (LS … More F) since Day 4. In Day 7 group, DF reached the maximum diameter (7.4 mm) on Day 7, while in Day 10 group it reached that (12.7 mm) on Day 8, ceasing to grow on Day 10 (12.4 mm). In Day 7 group, the number of S-size follicles decreased from 30.7 at the first FSH to 17.0 at the last FSH, while L-sized ones increased from 1.0 to 15.7 mm. In Day 10 group, S-sized ones decreased from 32.8 mm at first FSH to 17.5 mm at last FSH, and L-sized ones increased 1.3 mm to 31.5 mm. In Day 7 group, the number of follicles (>4mm in diameter) recruited in response to FSH injections showed maximum at 48 hr (5.0 follicles) after first FSH injection, while it showed that earlier at 24 hr (8.0 follicles) in Day 10 group. The plasma E_2 profile showed significantly higher levels at and after 36 hr, during superovulation treatment in Day 10 group, compared those in Day 7 group.Out of 223 follicles (>4 mm in diameter) observed with ultrasonography during the superovulation treatment, 170 follicles were individual dissected (73% recovery), 123 oocytes were collected under stereomicroscope (72%o recovery). Between the Day 7 and Day 10 groups, there was no statistic difference in the follicular fluid steroid concentration, however the follicles that emerged later tended to increase the E/P ratio of follicular fluid steroids. A total of 92 oocytes subjected for fluorochroming showed a positive viable color against Calcein-AM.There was no significant difference in the follicular morphological characteristics depending on the treatment.These results indicate that the ovarian responses were apt to be made more increased by the superovulation treatment initiated at Day 10 after DF loss its dominance effect than by the treatment executed at Day 7 when DF was still in dominant phase. Less