Study of genetic variability in the wildlife (Hokkaido brown bear)

1.北海道棕熊的精液采集与精子冷冻保存(1)采用电射精法成功采集了北海道棕熊的精液。获得的射精数据提供了熊精液的基线数据，如射精量。精子浓度，精子活力和精子异常率。(2)利用tris缓冲蛋黄膨化剂对熊精液进行冷冻技术研究，确定了最终甘油浓度、平衡周期、冷却速率等最佳冷冻条件。解冻后精液的特征表明，解冻后精液的平均精子活力和平均活精子率分别为36.0%和65.0%。(3)观察血清睾酮浓度、卵泡刺激素浓度和精液性状的年变化。血清睾酮浓度升高后，精子浓度随之升高。血清FSH浓度的年变化规律与睾丸体积变化规律相似。北海道棕熊的遗传变异(1)在北海道环境科学研究所收集了大约300个肝脏样本用于DNA研究。(2)虽然没有检测到任何单独变化的微卫星区域，但仍保持搜索微卫星区域的努力。先前开发的8组引物用于另一种熊的微卫星，应用于54只圈养的北海道棕熊。共检测到4 ~ 10个（6.5+1.9）等位基因，平均杂合度（基因多样性）为0.67+0.12。这些值与其他熊种的值相似，可能表明所应用的引物集被认为对评估北海道棕熊的遗传变异性是有效的。目前，从野生熊身上提取的DNA样本的分析是用这些引物进行的。(3)与北海道棕熊相同的研究，用这8组引物对日本黑熊进行了研究。

1.Semen collection and cryopreservation of spermatozoa in Hokkaido brown bear(1) Semen collection was succes using electroejaculation in Hokkaido brown bear. The obtained ejaculates provided baseline data of bear semen, such as ejaculate volume. sperm concentration, sperm motility, and sperm abnormality rate.(2) The study of freezing technique for bear semen using Tris-buffered egg yolk extender provided the optimal condition, such as final glycerol concentration, equilibration period, and cooling rate. The characteristics of post-thaw semen showed that the mean sperm motility and the mean percentage of live spermatozoa of post-thaw semen were 36.0% and 65.0%, respectively.(3) Annual changes in serum testosterone concentration, serum FSH concentration and seminal traits were investigated. The increase in serum testosterone concentration was followed by the increases of sperm concentration. The pattern of the annual change of serum FSH concentration was similar to that in testis volume.2.Genetic variability of Hokkaido brown bear(1) About 300 liver samples for DNA research were collected at the Hokkaido Institute of Environmental Science.(2) The effort of searching microsatellite region is maintained, though any individually variable microsatellite region has not been detected. Eight previously developed primer sets for microsatellites of another bear species were applied to 54 captive Hokkaido brown bears. As the result, 4 to 10 (6.5+1.9) alleles were detected, and the average heterozygosity (gene diversity) was 0.67+0.12. These values are similar to those of another bear species and may suggest that the applied primer sets were thought to be effective for evaluating the genetic variability of Hokkaido brown bears. At present, analysis of DNA samples extracted from wild bears is doing with those primer sets.(3) The same research as Hokkaido brown bear with those 8 primer sets are performing about the Japanese black bear.