Blash S, Melican D, Gavin W
Genzyme Transgenics Corporation, Framingham, MA 01701, USA.
Theriogenology. 2000 Oct 1;54(6):899-905. doi: 10.1016/S0093-691X(00)00400-3.
In the field of transgenic production, the ability to carry a male's genetic contribution beyond its natural life span is remarkably important. The ability to successfully collect and cryopreserve sperm from the epididymis at necropsy may prove to be a useful technique for preserving valuable genes. Thirty-two bucks ranging in age from 13 days to 7 years were examined in this study and 25 had epididymal sperm extracted at necropsy. Seven bucks yielded clear fluid with no spermatozoa; all were under four months of age. Testes were removed from the scrotal sac, small lateral incisions made across the convoluted tubules, pressure applied to the tail of the epididymis and small droplets of sperm pipetted into equilibrated extender. The average initial analysis of wave motion (0 to 5, 5 being rapid wave motion), live/dead sperm percentage and acrosomal integrity of 25 fresh epididymal samples were 5.0, 92%, and 100%, respectively. By comparison, the same parameters obtained from 206 fresh ejaculated samples were 3.0, 86%, and 95%, respectively. After being cryopreserved in liquid nitrogen, one straw from each sample was thawed after 3 to 60 days of cryostorage. Results of post-thaw analysis of 25 cryopreserved epididymal sperm samples for live/dead percentage and acrosomal integrity were 82% and 84%, respectively. By comparison, results of post-thaw analysis of 206 cryopreserved ejaculated sperm samples for live/dead percentage and acrosomal integrity were 60% and 89%, respectively. To assess the competence of the frozen epididymal sperm, IVF and AI were performed. In parallel IVF experiments, 40% of the oocytes showed cleavage patterns, with 6% developing to the blastocyst stage using frozen epididymal sperm, while 37% of the oocytes showed cleavage patterns and 4% developed into blastocysts using frozen ejaculated sperm. One artificial insemination out of 20 resulted in a pregnancy using frozen epididymal sperm, while 7 of 18 artificial inseminations resulted in a pregnancy using frozen ejaculated sperm. This data documents the successful collection and cryopreservation of epididymal sperm from the goat and its use for in vitro fertilization and artificial insemination.
在转基因生产领域，使雄性的遗传贡献超越其自然寿命的能力非常重要。在尸检时成功从附睾采集并冷冻保存精子的能力可能被证明是保存珍贵基因的一项有用技术。本研究检查了32只年龄从13天到7岁的公羊，其中25只在尸检时采集了附睾精子。7只公羊产生的是无精子的清亮液体；它们均未满四个月大。从阴囊囊中取出睾丸，在盘曲的小管上做小的侧面切口，对附睾尾部施加压力，并用移液器将小滴精子移入平衡的稀释液中。对25份新鲜附睾样本的平均初始波动分析（0至5级，5级为快速波动）、活/死精子百分比和顶体完整性分别为5.0、92%和100%。相比之下，从206份新鲜射精样本获得的相同参数分别为3.0、86%和95%。在液氮中冷冻保存后，每个样本的一根细管在冷冻保存3至60天后解冻。对25份冷冻保存的附睾精子样本进行解冻后活/死百分比和顶体完整性分析的结果分别为82%和84%。相比之下，对206份冷冻保存的射精精子样本进行解冻后活/死百分比和顶体完整性分析的结果分别为60%和89%。为评估冷冻附睾精子的受精能力，进行了体外受精和人工授精。在平行的体外受精实验中，使用冷冻附睾精子时，40%的卵母细胞呈现出分裂模式，其中6%发育到囊胚阶段；而使用冷冻射精精子时，37%的卵母细胞呈现出分裂模式，4%发育成囊胚。20次人工授精中有1次使用冷冻附睾精子使母羊怀孕，而18次人工授精中有7次使用冷冻射精精子使母羊怀孕。这些数据证明了成功从山羊采集并冷冻保存附睾精子及其用于体外受精和人工授精。
