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1、中国奶牛 2012年13期转基因牛与非转基因牛性控精液分离及配种效果初探吕小青1朱玉林1,*李艳华1薛建华1 杜欣悦1 王彦平1 戴云平2（1、北京奶牛中心 北京 100192 2、中国农业大学 北京 100193）摘要：本研究对转基因奶牛以及非转基因奶牛精液经流式细胞仪分离后精子活力、分离准确率进行了比较分析，同时对分离的性控精液进行了人工输精，对受体牛的情期受胎率进行了统计分析。结果表明，转基因牛与非转基因牛在冷冻解冻后活力以及分离准确率方面并无明显差异；转基因牛与非转基因牛的性控精液的情期受胎率分别为57.4%（转基因牛）和59.3%（非转基因牛），二者之间无显著差异(P0.05)。关键
2、词：转基因牛 性控精液 流式细胞仪利用流式细胞仪分离精子的技术已经成功应用于许多哺乳动物，如牛、猪、羊、狗、马、猫等1，其原理主要是根据X、Y 精子的DNA含量的差异对X、Y 精子进行分离。SX-MoFlo 是由美国DaKa公司和XY公司共同设计的生产精子的专用设备，经过技术的不断改善，最明显的是陶瓷喷嘴和激光器的改进，精子分离速度从2000年的平均每秒2000个到目前每秒45005000个或更高2，随着分离速率的不断提高，性控精液的品质也得以改进。性控精液通过提高母犊牛出生率，可成倍提高母牛的繁殖效率，对加速高产奶牛的繁育以及缩短奶牛世代间隔具有重要的意义，其应用前景广阔3。本研究对转基因牛
3、与非转基因牛的性控精液分离效果进行了初步分析，同时对分离性控精液的配种效果进行了统计分析，旨在探讨转基因是否影响种公牛的繁殖性能。1. 材料与方法1.1 种牛来源 非转基因荷斯坦种公牛10头来源于北京奶牛中心种公牛站，转基因牛10头来源于中国农业大学培育的转基因荷斯坦公牛，目前饲养于北京市种公牛站。人工授精实验在北京奶牛中心良种场进行。1.2 原精液 本实验所用公牛健康、性欲旺盛，用假阴道采集精液。采集后观察精液的颜色，合格鲜精的色泽为乳白色或稍带淡黄色。选择活力0.75，密度6亿/mL的鲜精用于本实验。1.3性控精液的染色、分离、冷冻及保存采集的原精液经品质鉴定（活力、密度、色泽）后进行染色
4、，根据精液密度加入染色用的5mg/mL的HO33342，34水浴锅内暗室孵育45min后，加入2mL 4% egg yolk再用50m过滤器过滤到新的上样管上机分离。性控精液利用流式细胞仪SXMoFlo精子专用分离设备进行分离，按照90%以上的准确率对定向正确的X精子和Y精子设门分离，本实验中只收集X精子，Y精子和死精子废弃。将分离收集的精子经过4平衡和离心，精确测定精液的体积和计算精子浓度，再经过稀释、细管包装和冷冻降温，最后将0.25mL细管（每管约200万个精子）投入液氮保存备用。1.4性控精液解冻及质检 用流式细胞仪分离的转基因牛与非转基因性控精液, 在38C水浴解冻15s，用手帕将解
5、冻后的精液细管擦干，取20l解冻的精液样品滴到预热的载玻片上，并加盖玻片37在相差显微镜下检测精子的活力，每个样片观察5个视野，并观察不同液层内的精子运动状态，进行全面评价，精子活力以直线运动精子占总精子数的比例来评价。X/Y分离准确率通过SX-MoFlo带有的一个专用程序进行检测，每次可取精子样本5000-10000个，重复测定3次，并计算其平均值。1.5 人工输精将精液从液氮中取出，轻轻甩掉细管棉塞端液氮，经37.5水浴解冻后输精。青年受体母牛发情结束后4-6h采用直肠把握输精法将性控精液输送到子宫角内，输精1次，一次用一支精液。输精60d后进行直肠检查妊娠诊断。1.6 统计分析本实验数据
6、用Excel 2007进行单因素方差分析，P0.05为差异不显著；P0.05为差异显著；P0.01为差异极显著。2. 结果2.1 转基因牛与非转基因牛精液经分离冷冻后对活力、分离准确率等相关参数的比较本实验通过比较转基因牛与非转基因牛性控精液在冷冻解冻后的活力以及X/Y分离准确率，分析转基因牛与非转基因牛性控精液之间的差异。结果表明，转基因牛与非转基因牛性控精液在冷冻解冻后活力以及分离准确率方面并无明显差异（见表1）。表1 转基因牛与非转基因牛精液经分离冷冻后对活力、分离准确率等相关参数的比较头数原精活力冷冻解冻后活力分离准确率非转基因牛10783（40.81.6）a（91.81.2）a转基因
7、牛10783（41.13.9）a（92.22.0）a注：同列内肩标字母相同表示差异不显著（P0.05），下同2.2 转基因牛与非转基因牛性控精液移植受胎率的比较分析本实验通过对青年牛进行人工输精，结果表明转基因牛与非转基因牛性控精液的情期受胎率分别为57.4%（转基因牛）和59.3%（非转基因牛），经统计分析，二者之间无显著差异（见表2），表明转基因牛X性控冷冻精液同样可应用于人工授精。表2转基因牛与非转基因牛性控精液移植受胎率的比较分析受体牛输精母牛头数指检妊娠母牛数（率）非转基因牛青年牛216128(59.3)a转基因牛青年牛5431(57.4)a3. 讨论转基因技术是将外源基因导入到某个
8、生物体基因组中，通过导入基因（转基因）的表达修饰生物体性状，含有转基因的生物体被称为“遗传修饰过的生物体”（GMO）。从1980年世界上第一只转基因小鼠诞生到如今4，科学家相继培育成功了转基因牛、绵羊、猪、鱼、昆虫、牛、鸡、山羊、大鼠、小鼠等转基因动物5 6，转基因动物的研究发展迅速，其种类、数量均急剧增加。然而公众对转基因动物本身及其产品的安全性问题普遍持保留态度，存在各种各样的疑问，因此转基因安全评价显得尤为重要，迫切需要建立相关的检验机构和法规标准。本研究通过对转基因牛以及非转基因牛性控冷冻精液活力等相关参数的比较，结果显示二者之间没有明显的差异，同时通过对获得的性控精液进行人工授精雌性
9、准确率均在90%以上，青年牛情期受胎率达58%。本实验结果显示转基因表达对转基因牛的繁殖性能没有产生明显的影响，预示着转基因牛本身及其产品是相对安全的。目前，流式细胞仪分离的性控精液已经在生产实践中获得了广泛应用，本实验初步分析了转基因牛与非转基因牛精液经流式细胞仪分离后活力、分离准确率等的差异，可以为进一步研究转基因种公牛的繁殖性能提供借鉴。参考文献1.马利兵，潘建刚，王凤等，流式细胞技术在哺乳动物性别控制中的研究进展，黑龙江畜牧兽医，2009,11（2）：22-23.2.吕自力，帅志强，张云梅等，SX-MoFlo流式细胞仪分离牛精子的特征及生产效率分析，黑龙江动物繁殖学，16（3）：25-
10、27.3.阚远征，郑维韬，奶牛性控精液的经济价值分析，中国奶牛，2010（9）：23-25.4.Gordon J W, Scangos G A, Plotkin D J, et al.Genetic transformation of mouse embryos by microinjection of purified DNA. Proc Natl Acad Sci U S A. 1980 Dec;77(12):73807384.5.李想，逍遥，张鑫等，转基因动物研究进展，中国奶牛，2009（9）：25-29.6. 林莉 胡佐忠，转基因动物技术的研究进展，畜禽业，2005（5）： 20-23
11、.注：本文受国家科技重大专项项目“高品质转基因奶牛新品种培育(2011ZX 08007-001) ”资助。作者简介：吕小青（1981-），女，主要从事奶牛胚胎工程技术研究工作Email: 13810787287通讯作者: 朱玉林（1964-），男，主要从事奶牛胚胎工程技术研究工作Preliminary analysis of sexed semen separation and breedingresults of transgenic cattle and non-transgenic cattleLv Xiaoqing1 , Zhu Yulin1,* , Li yanhua1 , Xue
12、Jianhua1 , Xinyue Du1,Yanping Wang1, Yunping Dai 2(1. Beijing Dairy Cattle Center, Beijing ,100192 ; 2. China Agricultural University, Beijing , 100193)Abstract: In this study, we analysed the difference of sperm motility and X/Y ratio of sorted sperm between transgenic and non-transgenic dairy cow
13、sperm separation after freezing and thawing, and also analysed the recipient cow pregnancy rate after sorted semen artificial insemination. The results show that the sperm motility and X/Y ratio of sorted sperm did not differ significantly between transgenic cattle and non-transgenic cattle; the pre
14、gnancy rate of non-transgenic cattle and transgenic cattle sexed semen were 59.3% (non-transgenic cattle) and 57.4 % (transgenic cattle), there were no significant differences (P 0.05).Keywords: transgenic cattle sexed semen flow cytometryEditors note: Judson Jones is a meteorologist, journalist and
15、 photographer. He has freelanced with CNN for four years, covering severe weather from tornadoes to typhoons. Follow him on Twitter: jnjonesjr (CNN) - I will always wonder what it was like to huddle around a shortwave radio and through the crackling static from space hear the faint beeps of the worl
16、ds first satellite - Sputnik. I also missed watching Neil Armstrong step foot on the moon and the first space shuttle take off for the stars. Those events were way before my time.As a kid, I was fascinated with what goes on in the sky, and when NASA pulled the plug on the shuttle program I was heart
17、broken. Yet the privatized space race has renewed my childhood dreams to reach for the stars.As a meteorologist, Ive still seen many important weather and space events, but right now, if you were sitting next to me, youd hear my foot tapping rapidly under my desk. Im anxious for the next one: a spac
18、e capsule hanging from a crane in the New Mexico desert.Its like the set for a George Lucas movie floating to the edge of space.You and I will have the chance to watch a man take a leap into an unimaginable free fall from the edge of space - live.The (lack of) air up there Watch man jump from 96,000
19、 feet Tuesday, I sat at work glued to the live stream of the Red Bull Stratos Mission. I watched the balloons positioned at different altitudes in the sky to test the winds, knowing that if they would just line up in a vertical straight line we would be go for launch.I feel this mission was created
20、for me because I am also a journalist and a photographer, but above all I live for taking a leap of faith - the feeling of pushing the envelope into uncharted territory.The guy who is going to do this, Felix Baumgartner, must have that same feeling, at a level I will never reach. However, it did not
21、 stop me from feeling his pain when a gust of swirling wind kicked up and twisted the partially filled balloon that would take him to the upper end of our atmosphere. As soon as the 40-acre balloon, with skin no thicker than a dry cleaning bag, scraped the ground I knew it was over.How claustrophobi
22、a almost grounded supersonic skydiverWith each twist, you could see the wrinkles of disappointment on the face of the current record holder and capcom (capsule communications), Col. Joe Kittinger. He hung his head low in mission control as he told Baumgartner the disappointing news: Mission aborted.
23、The supersonic descent could happen as early as Sunday.The weather plays an important role in this mission. Starting at the ground, conditions have to be very calm - winds less than 2 mph, with no precipitation or humidity and limited cloud cover. The balloon, with capsule attached, will move throug
24、h the lower level of the atmosphere (the troposphere) where our day-to-day weather lives. It will climb higher than the tip of Mount Everest (5.5 miles/8.85 kilometers), drifting even higher than the cruising altitude of commercial airliners (5.6 miles/9.17 kilometers) and into the stratosphere. As
25、he crosses the boundary layer (called the tropopause), he can expect a lot of turbulence.The balloon will slowly drift to the edge of space at 120,000 feet (22.7 miles/36.53 kilometers). Here, Fearless Felix will unclip. He will roll back the door.Then, I would assume, he will slowly step out onto s
26、omething resembling an Olympic diving platform.Below, the Earth becomes the concrete bottom of a swimming pool that he wants to land on, but not too hard. Still, hell be traveling fast, so despite the distance, it will not be like diving into the deep end of a pool. It will be like he is diving into
27、 the shallow end.Skydiver preps for the big jumpWhen he jumps, he is expected to reach the speed of sound - 690 mph (1,110 kph) - in less than 40 seconds. Like hitting the top of the water, he will begin to slow as he approaches the more dense air closer to Earth. But this will not be enough to stop
28、 him completely.If he goes too fast or spins out of control, he has a stabilization parachute that can be deployed to slow him down. His team hopes its not needed. Instead, he plans to deploy his 270-square-foot (25-square-meter) main chute at an altitude of around 5,000 feet (1,524 meters).In order
29、 to deploy this chute successfully, he will have to slow to 172 mph (277 kph). He will have a reserve parachute that will open automatically if he loses consciousness at mach speeds.Even if everything goes as planned, it wont. Baumgartner still will free fall at a speed that would cause you and me t
30、o pass out, and no parachute is guaranteed to work higher than 25,000 feet (7,620 meters).It might not be the moon, but Kittinger free fell from 102,800 feet in 1960 - at the dawn of an infamous space race that captured the hearts of many. Baumgartner will attempt to break that record, a feat that boggles the mind. This is one of those monumental moments I will always remember, because there is no way Id miss this.
