Roth T L, Weiss R B, Buff J L, Bush L M, Wildt D E, Bush M
Conservation and Research Center/NOAHS, National Zoological Park, Smithsonian Institution, Front Royal, Virginia 22630, USA.
Biol Reprod. 1998 Feb;58(2):475-82. doi: 10.1095/biolreprod58.2.475.
Scimitar-horned oryx sperm function was studied using protocols developed for domestic cattle. Objectives were to assess sperm 1) viability and motility in vitro over time, 2) capacitation in heparin- or calcium-supplemented medium, and 3) function in an in vitro fertilization system using heterologous (domestic cow) oocytes. Seminal aliquots were washed, and sperm were resuspended in 1) Talp with 5% fetal calf serum (TALP), 2) TALP + 10 microM heparin, 3) TALP + 20 microM heparin, and 4) TALP + 10 mM CaCl. At 0, 3, and 6 h, aliquots were evaluated for sperm motility, viability (using Hoechst 33258), and ability to acrosome-react when exposed to lysophosphatidylcholine (LC). Sperm function was assessed by evaluating fertilization and embryo development after coculture of in vitro-matured domestic cow oocytes with oryx sperm. Overall mean percentages of motile and viable sperm remained high at 6 h (> 60% and > 70%, respectively). Fewer (p < 0.05) sperm incubated in TALP + 10 microM heparin for 6 h contained intact acrosomes after exposure to LC, but there were no differences between LC and control samples after incubation in TALP without heparin. LC-treated sperm in TALP + 10 mM CaCl contained fewer (p < 0.05) intact acrosomes at 3 and 6 h (52.6% and 31.2%, respectively) than paired controls (83.6% and 70.0%, respectively). Oryx sperm from all males were capable of fertilizing cow oocytes (range 17 of 26 [65.4%] to 25 of 26 [96.2%]). Of the 55 2-cell embryos produced, 34 (61.8%) developed to > or = 8 cells. Of the 24 uncleaved oocytes, 7 (29.2%) were polyspermic. These data demonstrate that processed sperm from the endangered scimitar-horned oryx remain vigorous in vitro for at least 6 h. Capacitation can be induced using cattle sperm-processing techniques, with sperm appearing most responsive to elevated CaCl concentrations. Most interesting was the successful production and development of hybrid embryos after coincubation of oryx sperm with cow oocytes, suggesting that the two bovid species have similar fertilization mechanisms.
使用为家牛开发的实验方案对弯角剑羚精子的功能进行了研究。目的是评估精子：1）体外随时间变化的活力和运动能力；2）在添加肝素或钙的培养基中的获能情况；3）在使用异种（家牛）卵母细胞的体外受精系统中的功能。对精液样本进行洗涤，然后将精子重悬于：1）含5%胎牛血清的台氏液（TALP）；2）TALP + 10微摩尔/升肝素；3）TALP + 20微摩尔/升肝素；4）TALP + 10毫摩尔/升氯化钙中。在0、3和6小时时，对样本进行评估，检测精子的运动能力、活力（使用Hoechst 33258）以及暴露于溶血磷脂酰胆碱（LC）时顶体反应的能力。通过将体外成熟的家牛卵母细胞与弯角剑羚精子共培养后评估受精和胚胎发育情况来评估精子功能。在6小时时，活动精子和存活精子的总体平均百分比仍然很高（分别> 60%和> 70%）。在TALP + 10微摩尔/升肝素中孵育6小时的精子，暴露于LC后含有完整顶体的精子较少（p < 0.05），但在不含肝素的TALP中孵育后，LC处理样本与对照样本之间没有差异。在TALP + 10毫摩尔/升氯化钙中，LC处理的精子在3小时和6小时时含有完整顶体的精子较少（分别为52.6%和31.2%）（p < 0.05），而配对对照分别为83.6%和70.0%。所有雄性弯角剑羚的精子都能够使家牛卵母细胞受精（范围从26个中的17个[65.4%]到26个中的25个[96.2%]）。在产生的55个2细胞胚胎中，34个（61.8%）发育到≥8细胞。在24个未受精卵母细胞中，7个（29.2%）是多精受精。这些数据表明，来自濒危弯角剑羚的处理后精子在体外至少6小时内仍保持活力。使用牛精子处理技术可以诱导获能情况，精子似乎对升高的氯化钙浓度反应最为敏感。最有趣的是弯角剑羚精子与家牛卵母细胞共孵育后成功产生并发育出杂交胚胎，这表明这两种牛科动物具有相似的受精机制。
