CN1674780A - 低压精子分离系统 - Google Patents
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Abstract
用于生成精子授精样品的精子处理系统,所述精子授精样品的精子具有可控的精子生育力特征。
Description
本国际专利合作条约专利申请要求享有2002年8月1日提交的NO.60/400,971号美国临时专利申请的优先权。
技术领域
本发明涉及具有选择性可控的精子生育力特征的精子授精样品,该精子授精样品通过夹带在具有相应的选择性可调节流动性质的液流中产生;以及评价并比较精子授精样品生育力的方法。
背景技术
随着安全、可靠地将精子分离成富含X染色体的类群和富含Y染色体的类群的方法的发展,已经在许多种家畜中实现了性别预选。例如,可参见以下申请所公开的方法和装置:WO 00/06193、WO 02/043574、WO 01/85913、WO 99/33956、WO 01/40765、WO 98/34094、WO 99/42810和WO 02/043486。
关于经性别选择的精子的一个显著问题有可能是,按照常规方法将精子以足以产生经性别选择的授精样品或经性别选择的授精物的速率分离时,将迫使流式细胞计数器或流式分选装置的液流压力升高到每平方英寸约50磅,其中所述精子样品或授精物对于商业应用来说是可以存活或足以生育的。对于在因压力的应用而具有流动特征的液流中夹带的许多种哺乳动物的精子来说,其存活力、运动力或其它生育力特征已发生了改变。
关于经性别选择的精子授精物或经性别选择的精子授精样品的另一个显著问题是精子生育力特征的巨大差别,不同样品之间的生育力特征差别可能非常大。这样,在基本相同条件下成功实施的人工授精也会产生不同的妊娠率。
现有精子性别选择技术的另一显著问题是缺乏可以直接在体内(例如,与人工授精过程相关)和体外(例如,与IVF(体外受精)过程相关)对经性别选择的精子的生育力进行比较的测定方法。
本发明克服了与精子生育力下降等与精子相关的各种问题,所述精子已分离成富含X染色体和富含Y染色体的类群,并克服了缺乏异质精子(heterospermatic)测定方法的问题,所述异质精子测定方法是用于对经分离或分选的精子、尤其是流式分选的精子的功能和生育力进行比较的方法。
发明内容
因此,本发明的主要目的是提供控制精子的诸如运动力、存活力、受精率、卵裂率或胚囊率等精子生育力特征的装置和方法,所述精子是对得自哺乳动物雄性个体的精液进行分离而得到的。
根据本发明,提供可控的精子生育力可以用各种各样哺乳动物的精子来实现,所述哺乳动物包括但不限于牛、马、绵羊、犬、猫、猪、海洋动物、鹿、灵长类动物、山羊或由Wilson,D.E.和Reeder,D.M.,MammalSpecies of the World(世界哺乳动物种类),Smithsonian Institution Press,(1993)所列的哺乳动物,在此将其以参考的方式引入本文。
对于本发明的一些实施方案来说,可控的精子生育力特征包括在将精子从哺乳动物雄性动物的精液分离出来之前确定选择生育力特征,以及应用本发明来改变精子的生育力特征,以提供所需的生育力特征。对于本发明的其他实施方案来说,精子处理条件从更广泛的精子处理条件中选择,这些条件可用于处理特定哺乳动物的精子以获得具有可控精子生育力特征的精子。可控精子生育力特征可以包括能动精子、完整顶体、可存活精子在经处理精子群体中的所需比例,也可包括当将经处理的精子用于使卵母细胞体外受精时所需的卵母细胞卵裂率或胚囊形成率,也可包括当将经处理的精子用于人工授精时所需的妊娠率或子代性别比。对于本发明的一些实施方案来说,与对相同精子群的常规处理相比,在经处理的精子群内可以达到更高的能动精子比例、更高的可存活精子比例、更高的完整顶体比例或更多的可生育精子数。本发明的一些实施方案能提供具有可控精子生育力特征的精子,所述生育力特征与新鲜射精液中精子的生育力特征基本无差别或基本相当。在其他实例中,应用本发明的实施方案在必要时可以产生具有可控精子生育力特征的精子,所述生育力特征与新鲜射精液中精子的生育力特征基本不相同。具体地说,本发明的某些实施方案可以用于提供具有可控精子生育力特征的牛精子或用于提供具有可控精子生育力特征的马精子,如果必要的话所述精子的所述生育力特征可基本与新射出的牛精液或马精液中牛精子或马精子的生育力特征相当。
本发明的另一主要目的是提供具有可控精子生育力特征的精子授精样品,这些精子授精样品可以不加限制地应用于哺乳动物雌性个体的人工授精、卵母细胞的体外受精或精子的胞质内注射,等等。
本发明的另一主要目的是提供对精子进行性别选择的方法,其可以提供诸如运动力、存活力、受精率、卵裂率、胚囊率等生育力特征的确定控制。本发明此主要目的的一个方面是可提供流式细胞计数或细胞分选的装置,或细胞流式计数或细胞分选的方法,其可以对经性别选择的精子的生育力特征进行确定控制。
本发明的另一目的是提供经性别选择的、具有可控精子生育力特征的牛精子授精样品,这些牛精子授精样品专用于牛类哺乳动物雌性个体的人工授精,样品含有约100,000至约3,000,000个具有可控精子生育力特征的经性别选择的牛精子。
本发明的另一目的是提供经性别选择的、具有可控精子生育力特征的马精子授精样品,这些马精子授精样品专用于马类哺乳动物雌性个体的人工授精,样品含有约5,000,000至约50,000,000个具有可控精子生育力特征的经性别选择的马精子。
本发明的另一重要目的是提供在以下过程中保持精子的可控精子生育力特征的装置或方法,所述过程为精子的处理、储存和使用,包括但不限于雌性哺乳动物的授精或卵母细胞的受精。
本发明的另一重要目的是提供用具有可控精子生育力特征的精子授精样品进行雌性哺乳动物人工授精的方法。就本发明的某些实施方案而言,与此类人工授精方法中精子的常用数量或典型数量相比,无论是否将这些精子分成富含X染色体或富含Y染色体的精子群,该授精方法所使用的具有可控性生育力特征的精子数很低或更少。
本发明的另一主要目的是提供评价精子群的相对生育力的方法。本发明的一些实施方案提供了当对得自于哺乳动物不同雄性个体的精子各自进行了基本相同的流式计数处理时,对所述精子的相对生育力进行评价的方法。本发明的其他实施方案提供了对经过不同的流式计数处理的得自于哺乳动物相同雄性个体的精子群的相对生育力进行评价的方法。本发明的一些实施方案提供了显示具有可控性生育力特征的精子的相对生育力的方法。
本发明的更多重要目的在下面的发明说明中自然得以明确。
附图说明
图1为按照本发明所使用的流式计数仪分离技术的分选系统示意图。
图2为典型流式细胞计数仪自由下落区中夹带的细胞的图。
具体实施方式
本发明涉及精液或精子的处理系统,该系统可维持、增强、测定、检验或确定精子在采集、处理、储存、运输、分离或授精等各个过程中的生物学、化学、物理、生理或功能特性。
本发明的一个实施方案可以包括从上述广义界定的哺乳动物中获取精子。所述精子可以被夹带在具有流动特征的液流中。导管中液流的流动特征受以下因素影响:液流的流变学性质、导管(液流在其中流动)的构造或几何形状,以及施加于液流的外力,例如流体静力压、摆动振荡、压电振动、热振动等等。
重要的是,液流的这些流动特征可以有助于使液流在导管中移动所需要的压力。作为非限制性的实例,在流式细胞计数中,液流在相对较大的横截面内移动,然后在相对较小的横截面内移动,并穿过分析界面到达最终收集处。
这种类型的构造或几何形状与液流的流变学性质一起可以产生局部力,如压缩力或偏转力等,这些力可以影响诸如在液流中夹带的精子等颗粒的物理完整性。
对于本发明的一些实施方案,图1显示了非限制性的概念性流式细胞计数仪或细胞分选仪器,所述实施方案包括以流式细胞计数或细胞分选为手段对精子进行分析、基于精子特性的分离、性别选择或进行其他处理的组件或步骤。
流式细胞计数仪或细胞分选仪器包括图1所示的全部或部分组件,包括但不限于精子源(1),其用于放置或供应精子以用于分析、分离、控制生育力特征或进行其他处理。
将精子以被鞘液(3)包围的方式沉积在喷嘴(2)内。本发明中可以利用与流式细胞计数仪或流式分选仪器兼容的任意鞘液,该鞘液可以提供流式分析或处理时精子可接受的环境,其包括但不限于含有磷酸缓冲盐水、柠檬酸盐溶液(如2.9%柠檬酸钠溶液)或HEPES(N-2-羟乙基哌嗪-N’-2-乙磺酸)缓冲液的鞘液,所述鞘液可以单独使用也可以进行各种组合后使用。
鞘液(3)通常由一些鞘液源(4)提供,以便当精子源(1)供应精子时,可以同时通过喷嘴(2)添加鞘液(3)。在这种方式下,鞘液(3)形成所述精子的鞘液环境。由于是在一定压力下将各种液流提供给流式细胞仪,所以它们在喷嘴口(5)流出喷嘴(2)。
通过提供某种可由振荡器控制器(7)非常精确地控制的振荡器(6),可在喷嘴(2)内形成压力波并将其传递给在喷嘴口(5)离开喷嘴(2)的鞘液。由于振荡器(6)如此对鞘液(3)进行作用,因此离开喷嘴口(5)的液流(8)最终并有规则地形成液滴(9)。因为精子由鞘液环境所包绕,所以液滴(9)可以在内部包含独立分开的精子(10)。
由于液滴(9)通常包含分离开的精子(10),所以流式计数仪或细胞分选仪器可以根据液滴(9)中所含有的精子的精子区别特征将液滴加以区分并分离出来。这通过精子传感系统(11)来完成。该精子传感系统至少包括某种检测器(12),所述检测器对包含于各液滴(9)中的精子产生反应。
授予Johnson的美国专利5,135,759中详细论述了一种精子传感系统(11),在此将其以参考的方式引入本文。按照Johnson专利对精子的解释,精子传感系统(11)可以基于特定染色剂的相对存在或相对缺失而产生反应,所述染色剂可以由诸如激光束(13)等刺激物所激发。尽管每种精子均可以被染色剂染色,但X染色体和Y染色体长度的不同使得染色的水平不同。因此,通过感知各精子中染色剂存在的程度,可以通过对不同发射水平进行区分而把含有X染色体的精子与含有Y染色体的精子区别开来。根据本发明也可以替换使用其他已知的光学、检测及精子分析系统,而Johnson的专利所提供的说明只是出于示例性的目的,以便可以理解本发明的大量而不同的用途。也可参见WO 01/85913,将其以参考的方式引入本文。
为了在流式细胞计数仪或细胞分选仪器分离技术中最终使细胞达到适当的分开和分离,将传感器(12)接收到的信号反馈给某种分选仪识别系统(14),其可以快速做出决定并且可以根据在所述液滴(9)中是否存在所需细胞而分别使各液滴(9)带有不同的电荷。按照这种方式,分选仪辨别系统(14)可以允许静电偏转板(15)根据液滴(9)是否含有具特定精子特征的精子而使液滴(9)发生偏转。结果是流式细胞计数仪或细胞分选仪器可以通过使细胞落入到一个或一个以上的收集容器(16)内,从而将细胞加以分离。这样,通过感知精子的某些性质,流式细胞计数仪或细胞分选仪器就可以根据特定特性将细胞区别开来,并将其置于合适的收集容器(16)中。在某些流式细胞计数仪或细胞分选仪器中,使含X染色体精子的液滴带正电荷,从而偏向一个方向,而使含Y染色体精子的液滴带负电荷,从而偏向另一个方向,废液(未分选细胞)不带电荷,从而以未偏转的液流被收集入抽吸管等。
现主要参考图2,则可进一步理解该过程。如该图所示,喷嘴(2)喷出液流(8),液流(8)因振荡器(7)(图2中未显示)而形成滴液(9)。由于细胞源(1)(图2中未显示)可以供应精子(10),所述精子可按如Johnson所述加以染色(或在本发明一些实施方案中使用DIC(微分干涉相衬法)技术时不染色),由激光(或使用DIC技术时照明光源)(13)照射染色剂(使用DIC技术时的精子头部)所产生的光束的光发射分别由传感器(12)测定,从而当将液滴(9)从液流(8)中分离出来时,可以由流式细胞计数仪控制各液滴(9)上是否有电荷的存在。这一控制依据液滴(9)中内含物的不同而使液滴(9)带上正电荷、负电荷,或不带电荷。如图2所示,某些液滴显示为偏转的液滴(17)。这些偏转的液滴(17)是含有精子(10)的液滴,该精子可以是一种性别或是另一种性别。然后它们沉积于合适的收集器(16)中,从而产生了经性别选择的精子群。
不管液流是否出现于流式细胞计数仪、细胞分选器或其他在液流中夹带精子的设备中,液流的流动特征均可以加以鉴定,并且可以引入用于改变液流的流动特征的调节设备以提高或降低诸如压缩力、偏转力等作用力,从而可以使液流中所夹带的颗粒发生物理、生理、功能或机械上的改变。
这样,利用调节设备可以产生流动路径中液流特征的选择性调节范围,并且其可以用递增测量值表示。例如,流式细胞计数仪或细胞分选仪器环境中液流特征的改变可以被递增调节的并以每平方英寸的磅数来衡量,与液流或鞘液速度的上升或下降相对应,通常液流压力可允许有每平方英寸约20至100磅的递增上升或下降。
根据本发明的一些实施方案,特定种类哺乳动物的精子在具有可调液流流动特征的液流中输送。接着可以对液流的流动特征在递增测量(incrementally measured)的范围内加以选择性调节,在此范围内流动的精子仍能存活。然后,按照测量范围内所产生的多种流动特征中各个流动特征对所采集的大量精子样品中每个精子样品的精子生育力特征进行评测。
随后,可以对精子的精子生育力特征进行控制,从而可以产生具有所需精子生育力特征的精子授精样品,所述精子来自于一种哺乳动物或一种哺乳动物的个体成员。
例如,将分别来自于6头公牛的精子用125μm的Hoechst 33342于34℃染色45分钟,并将其粗分选(经过流式细胞仪或细胞分选仪器而未分选成亚群)或用具有内径为70μm的喷嘴的流式细胞计数仪以约95%精确度和30磅/平方英寸(psi)、40psi或50psi的液流压力将其分选成含X染色体或含Y染色体(或二者均有)的精子群。将液流的压力从50psi降到30psi,分选率仅下降了2%至3%。
然后将精子冷却到5℃,通过离心加以浓缩,装入0.25ml吸管中,每100μl柱中共约有2×106个精子,用真空冷冻法与未经分选的对照一起冷冻。随后将管中的精子解冻。
然后由两位观察者对各种的精子生育力特征进行盲评:于解冻后30分钟和120分钟评估前进运动力,于解冻后150分钟用流式细胞计数加以评测,用PI(碘化丙啶)染色来评测活精子百分比,并用Hamilton Thorne系统于解冻后120分钟进行CASA分析(计算机辅助精液分析)。整个过程重复两次。
因子的方差分析(ANOVA)表明公牛和压力均具有显著的影响(p<0.005,表1)。
表1、精子解冻后在分选时对不同系统压力的响应a
压力(psi) | 未分选的对照 | |||
响应 | 50 | 40 | 30 | |
30分钟运动力(%)120分钟运动力(%)活精子(%)CASA总运动力(%)CASA ALH* | 44.734.551.725.16.0 | 48.640.855.737.27.6 | 49.642.757.840.97.8 | 52.140.858.534.88.8 |
a均有统计学显著性,p<0.005。
*侧向头部移位的振幅。
数字越大,表明僵硬程度越小,越具有正常的运动力。所有的响应中,在公牛之间存在典型的差异。但是,公牛-处理间相互作用除一个之外均非常小,表明该结果可类似地应用于群体中的大部分公牛。为提高压力递增调节液流的流动特征基本上可以影响所测量的所有精子生育力特征,但在表1中仅列出了有高度统计学显著性的那些。
可以理解,在约50psi和约40psi时提取的精子样品之间其精子生育力特征发生了显著的改变,而在约40psi和约30psi之间的改变要小得多,表明不能将对于精子生育力特征的影响假设为线性。对于暴露于30psi的所述流动特征的牛精子来说,如果将精子用于母牛的授精或人工授精,精子生育力特征基本与未经分选的对照相同或相当,而且许多响应还优于未经分选的对照。对取自公马的精子进行类似的过程,获得了类似的结果和结论。
可以对精子生育力特征进行控制,且对于取自哺乳动物的精子也是如此。对于大多数哺乳动物来说,改变液流特征以递增式地降低液流压力,不管是在流式细胞计数还是其他环境中均可得到具不同精子生育力特征的系列梯度的相应精子样品,这些精子样品可用于不同的过程,包括如下所述的人工授精、体外受精或胞质内注射。
本发明提供了其他检验来评估二项反应,如妊娠/未妊娠,除非处理间的差异非常大,否则这些反应通常均需要处理大量的动物以获得统计学显著性。在本发明的一个实施方案中包括竞争性(competitive)或异质精子的受精,授精前将不同处理组或雄性个体的精子进行混合并测定从各雄性个体或处理组得到的胚胎、胎儿或子代的比例,该方案可以扩大由于处理差异而产生的经经性别选择的精子在精子生育力特征方面的差异。
例如,通过在两个不同液流压力下利用流式细胞计数或细胞分选基于DNA含量对精子进行性别选择后,可以以性别为遗传学标记用异质精子授精来评估生育力。用被调节至30psi或50psi的液流压力在约95%的准确度下将来自两头公牛的精子各自分选为含X染色体或含Y染色体或同时含二者的精子群。分选后,通过离心浓缩精子,对于每头公牛将于30psi分选的1×106个含X染色体精子和于50psi分选的1×106个含Y染色体精子置于0.25ml的吸管内,在另一个吸管中则相反:为在30psi分选的1×106个含Y染色体精子和在50psi分选的1×106个含X染色体精子。然后将这些精子与未经分选的对照一起冷冻,数月后解冻,并在观察到发情期后12小时或24小时将其植入85个霍尔斯坦(Holstein)小母牛子宫体内,其中两个授精者间的亚组是平衡的。
受精后2个月,妊娠的43头小母牛中有81%胎的牛性别(由超声来确定)与在30psi时处理的精子的性别一致。如果认为在两种压力下分选的精子其精子生育力特征无差别,则上述结果与预期的50∶50的性别比率不同(p<0.01)。性别选定的精子在每剂量为2×106个精子时,妊娠率为51%(43/85),这与来自同一次射精液的每个剂量为20×106个未进行性别选择的精子的对照组的妊娠率类似,对照组的妊娠率为39%(9/23)。
本发明的另一个实施方案提供了利用具有可控精子生育力特征的精子来改变卵裂率和胚囊形成率的方法。通过对牛精子分别于40psi和50psi进行流式分选以产生各自具有可控生育力特征的两个牛精子样品。用来自各个精子样品的含X染色体精子和含Y染色体精子进行授精介质中的精子浓度的剂量效应试验。因此,可以进行包含2个液流压力、3个精子浓度(1×106、0.33×106和0.11×106个精子/ml)、6头公牛和2个性别的多因素过程。
从来自屠宰场的卵巢中约2mm至约8mm的卵泡中抽取约2,000个卵母细胞。按《Journal of Animal Sciences(动物科学杂志)》,78:152-157(2000)所述在整个过程中使用化学成分确定的培养基(CDM),将其以参考的方式引入本文。在含有0.5%FAF-BSA、15ng/mlNIDDK-oFSH-20、1μg/ml USDA-LH-B-5、0.1μg/ml E2、50ng/μl EGF和0.1mM半胱胺的M-CDM中于38.8℃和含5%CO2的空气中持续放置23小时使卵母细胞成熟。将每管2×106个精子的经分选的冷冻精子进行解冻,在400g(重力)下用2ml的45%和2ml的90%珀可(percoll)梯度液离心20分钟。弃去上层清液,在精子团粒中加入含有0.5%FAF-BSA、2mM咖啡因和0.02%肝素的FCDM,在500g(重力)下离心5分钟。弃去上层清液,留下约50μl精子悬液。将成熟的卵母细胞在FCDM中洗涤一次并以15个/5μl为一组转移到25μl的矿物油下的FCDM滴剂中。通过在每个滴剂中加入10μl精子悬液,于38.8℃、在含5%CO2的空气中放置18小时来完成受精。将假定的受精卵在38.5℃、在5%O2、5%CO2和90%N2(氮气)中在CDM1中培养2天并在CDM2中培养4.5天。在第7.5天时,评估胚囊的发育情况:将质量分成1到4(1为优秀,4为差),将发育阶段分成6到8(6为完整,6.5为扩张中,7为已扩张,7.5为孵化中,8为已孵化胚囊)。数据(表1)经反正弦转换后用ANOVA和第一偏差进行分析。
利用在约40psi时所获得的具有可控精子生育力特征的精子比在约50psi时所获得的精子的卵裂率(53.6%和43.6%)及胚囊率(18.2%和14.7%)要高一些(p<0.01)。剂量与压力之间无交互作用,因此各个精子浓度下对于更低的压力均有相似的优势。对于卵裂和胚囊产量可以发现明确的精子浓度的剂量效应(表2)。同样,对于两种反应在公牛间均存在巨大的差异(P<0.01),并且对于卵裂百分率来说存在着公牛-剂量的交互作用(P<0.01)。这些数据表明,某些公牛的精子剂量应大于1.0×106个精子/ml。Y-精子的胚胎质量优于X-精子的胚胎质量(1.12对1.57,p<0.01)。当对胚胎进行性别选择时,对于IVF胚胎,其他研究人员已经注意到了这一点,现在已经用有性别的精子确证了这种效果。
表2、用公牛表示的每个卵母细胞的卵裂率(%,C)和胚囊率(%,B)数据。
精子浓度(106) | 公牛 | |||||||||||||
H023 | H024 | H025 | H026 | H027 | H028 | 平均值 | ||||||||
C | B | C | B | C | B | C | B | C | B | C | B | C | B | |
0.110.331.0 | 184456 | 1418 | 6735 | 1214 | 47285 | 113143 | 366283 | 152134 | 202972 | 71127 | 546885 | 151829 | 30a47b69c | 8a14b28c |
a,b,c带不同上标的值在组内有差别,p<0.01。
**经分选的精子的生育力低于未经分选的对照精子的生育力,部分原因是由于在用流式细胞技术进行精子分选过程中所受到的机械损伤。降低系统压力可以同时改善IVF中的精子质量和生育力。本研究评估了精子分选时系统压力的影响,并评估了延长卵母细胞的成熟时间对ICSI(胞浆内精子注射)后胚胎发育的影响。将分别来自3头公牛的精子用125μM Hoechst 33342于34℃染色45分钟,用压力为40psi或50psi的SX MoFlo分选仪以约95%的精确度将其分选成含X染色体的精子群和含Y染色体的精子群,然后低温保存。将得自屠宰场的卵巢的50个牛卵母细胞放到各孔中,孔内含有1ml的CDM1,在CDM1中含有0.5%的无脂肪酸-牛血清白蛋白(FAF-BSA)、2mM葡萄糖、50ng/ml EGF(表皮生长因子)、15ng/ml NIDDK-oFSH(羊促滤泡激素)-20、1μg/mlUSDA-LH-B-5、1μg/ml E2(雌二醇)和0.1mM半胱胺的,然后于38.5℃和含5%CO2的空气中使卵母细胞成熟24小时或30小时。通过涡旋将成熟卵母细胞的卵丘细胞除去,选取带有极体的卵母细胞。将得自经分选的冷冻-解冻精子的能动精子分别用2ml的45%和90%的Percoll液通过离心进行回收,并将浓度调到4×106个精子/ml。将成熟的卵母细胞分成两个注射组:使用Piezo注射系统的ICSI组和假注射组。精子注射管的外径为8μm~10μm。所有操作均在室温(24℃~25℃)下进行。注射后,卵母细胞用5μM离子霉素活化4分钟,在50μl的CDM1中于38.5℃、5%CO2、5%O2和90%N2中进行培养,在注射72小时后对卵裂进行评估。对ICSI组和假注射组中未分裂的卵母细胞用地衣红染色,然后评价受精状态。进一步培养分裂的胚胎,并在注射后7.5天评价胚囊的发育情况。对数据进行方差分析,对百分率数据进行反正弦转换。
对于成熟24小时的卵母细胞,在40psi和50psi下分选的精子对于卵裂率或胚囊率没有差别(p>0.1),压力和公牛之间也没有交互作用。公牛对所研究的全部反应均有显著的影响(p<0.05)。用40psi分选的精子注射时,成熟30小时的卵母细胞可获得比成熟24小时的卵母细胞更高的卵裂率(22.9%对12.2%,P<0.05),而胚囊率则无差异(p>0.1)。ICSI组的全部胚囊发育情况比假注射组的好(7.5%对1.3%,P<0.05)。当用成熟24小时的未分裂卵母细胞进行受精状态评估时,ICSI组含有2个极体和/或解聚精子的百分率要高于假注射组(15.7%对1.7%,P<0.05)。对于成熟30小时的卵母细胞,两组间的受精状态无差异。我们得出结论,使用于40psi和50psi分选的能动精子进行ICSI后,胚囊的分裂或发育没有差别。
在本发明的另一实施方案中,使用性别作为遗传学标记的异质精子授精可以用于对雄性的生育力进行分级,以及用于对处理中不涉及精子性别选择的精子的生育力进行分级。目前体外精子功能试验与雄性生育力之间没有很好的相关性,同质精子授精每次处理需要进行数百次授精以获得精确的生育力数据。异质授精通过将2个或2个以上的雄性的精子混合起来,实现了对大部分受检物种的相对生育力的精确估计。
将来自4组(每组4头公牛)的冷冻的经流式分选的精子解冻后,以组内3头公牛的所有组合(ABC、ABD、ACD、BCD)将其植入发情期开始后12小时或24小时的小母牛中。用来自各公牛的等量的可前进运动精子进行授精,解冻后总共有600,000个能动精子。将各个授精物的一半放置在各个子宫角中。发情后14.5至20天不做手术而收集胚胎。从332头小母牛中收集到了165个(48%)正在生长的胚胎。用多态性DNA标记对胚胎进行基因型分析,以确定每个胚胎活检物的雄性亲代。基因型分析后,可将165个胚胎中的118个归属到具体的雄性亲代。利用最大可能性分析法则来计算用于对公牛组进行分级的异质精子指数。根据这些指数对组内的每头公牛进行分级(表1)。第1组中,最差的公牛其生育力显著低于其他两头公牛(P<0.05)。第2组中,占优势的公牛具有最高的指数(p<0.05)。在第3组和第4组也可发现类似的差别。但是,其中三个组的某些公牛的生育力高低是不明确的(P>0.05)。
表1、组内公牛个体的异质精子指数±SE(标准误差)
第1组 | 第2组 | 第3组 | 第4组 |
1.47±.41a0.44±.27a,b1.84±.46a0.25±.17b | 2.43±.43a0.22±.15b0.90±.35b0.45±.23b | 1.68±.44a1.09±.39a,b0.83±.31a,b0.40±.22b | 0.92±.36a,b0.46±.20a2.02±.40b0.59±.24a |
a,b带不同上标的指数具有差别,P<0.05。
使用这些方法,每组4头公牛平均有30个基因型胚胎,使得可以对生育力具有明显差异的公牛进行检测。可用此技术对精子处理进行评估。该体内试验需要的雌性个体少,且快速提供了有关哪个公牛具有相对较高或较低的生育力的信息。
通过用本发明的性别选定的精子使雌性个体人工授精而得到的小牛群基本与用未经性别选择的精子所得到的对照小牛是一样的。而且,用性别选定的精子使雌性个体人工授精所获得的小牛约有90%的性别与预计的一样。如上所述,精子可以根据DNA的含量在以H33342染色后用流式细胞计数或细胞分选进行性别选择。性别选择后的精子可以如《Theriogenology(动物生殖学)》,52:1375中所述在低温保存,在此将其以参考的方式引入本文。
各种食用牛及奶牛中的小母牛和母牛,其发情期可以通过下列方法得到同步:每日喂饲0.5mg醋酸美仑孕酮(MGA),持续14天,然后在17至19天后肌肉注射(im)25mg前列腺素F2(PGF2);或以12天为间隔肌肉注射25mg的前列腺素F2。不管使用冷冻-解冻的性别选定授精样品还是使用来自于同次射精液的冷冻-解冻精子,均在首次观察到发情后12小时或24小时完成授精。对每一饲养组,约有2/3的授精是使用经性别选择的精子,余下的使用对照精子。2个月后用超声诊断妊娠情况及胎儿的性别。
在产仔及断奶期间在不同管理水平下在13个农场中对牛进行管理(每个农场的数量为49至228个)。收集的数据包括妊娠时间、出生时体重,产仔难度(1=正常至4=剖腹产)、断奶时体重、新生牛死亡数以及出生至断奶期间的死亡数。并非所有的农场所都记录了出生时体重和断奶时体重。将数据进行包含下列因素的方差分析:管理组别、分选与对照精子、产仔的性别。对百分率数据进行反正弦转换。表1列出了最小二乘平均数。
表1、性别分选小牛和对照小牛的产仔结果
处理 | 数目 | 妊娠时间(天) | 新生牛死亡率(%) | 产仔难度 | 出生体量(kg) | 断奶时存活率(%) | 断奶时体重(kg) |
经性别分选a | 574 | 279 | 3.9 | 1.31 | 34.3 | 92.0 | 239 |
对照 | 385 | 279 | 5.9 | 1.30 | 34.1 | 88.9 | 239 |
a对任何响应均无显著性差异(P>0.1)。
在所研究的任何响应中经性别选择组和对照组的小牛之间没有差异(P>0.1),也没有发现显著的交互作用。对于所研究的全部反应,管理组均存在显著的效应(除断奶时存活率为P<0.02外,其余均为P<0.001)。同样雌性和雄性小牛之间在以下方面均有显著的差异(所有的P均小于0.001):出生时体重(32.2kg和35.5kg)、断奶时体重(232kg和246kg)、产仔难度(1.20和1.42)和妊娠时间(278天和280天)。
对照组小牛的性别比率是雄性占51.0%(N=382)。X染色体分选精子产生的雌性占87.7%,而Y染色体分选精子产生的雄性占93.6%(N=94)。有少数小牛出生时即死亡,未记录这些小牛的性别并且未包含在内。
公马精子流式细胞分离的最新发展,使得可以产生预定性别的正常小马驹(Lindsey A.C.,Morris L.H.,Allen W.R.,Schenk J.L.,Squires E.L.,Bruemmer J.E.Equine vet.J.2002,34:128-132)。为实现此技术,需要将精液从流式细胞仪转至母马处。该研究对经性别预选后的新鲜公马精子的寿命和顶体状态进行了检验。对7匹公马每匹用人造阴道收集三次射精液,并置于脱脂乳-葡萄糖稀释剂(体积比为1∶1)内,于20℃在2~6小时内运到实验室。将精液以400g离心10分钟,除去精液浆。将精子团粒重悬浮于Kenneys改良台氏培养基(Kenneys modified tyrodesmedium,KMT)中,浓度为100×106个精子/ml,用Hoechst 33342(5mg/ml,Sigma-Aldrich,St.Louis MO)染色,孵育30分钟,进行流式细胞计数。将分选出来的精子离心,重悬于KMT中,浓度为40×106个精子/ml,以250μl的等分液于4℃或20℃储存48小时。在分选前以及分选后0、2、12、24、36和48小时评估精子的总前进运动力(TPM)和顶体状态。TPM用显微镜进行评估,顶体用异硫氰酸荧光素-花生凝集素(FITC-PNA)(Sigma-Aldrich)染色并分成完整、部分缺损或完全缺失。公马、时间及储存温度的影响用Proc GLM程序进行分析,并进行最小平均数比较(SAS研究所)。
公马对随时间的精子运动力和完整顶体比率有影响(P=0.03)。用Hoechst 33342染色和孵育使完整顶体的比率下降(表1)。但是,分选后所观察到的完整顶体比率高于分选前精子群中的完整顶体比率。在分选后的48小时内,随着完全缺失顶体的增加(P<0.0001),完整顶体的比率下降(P<0.0001),但时间对部分缺损顶体的比率没有影响。分选后精子储存温度具有显著影响,使得在20℃储存12小时的精子的运动力高于在4℃储存12小时的精子的运动力。通过流式细胞计数对精子进行性别分选筛选出了可以使顶体完整性保持24小时的精子群,其相当于新鲜精子。经FACS(荧光激活的细胞分选法)分离后精子寿命可以维持12小时,能使经性别选择后的精子被运送到位于距流式细胞计数仪处有一定距离的母马处。
表1、流式分选的精子随时间变化的运动力和顶体状态
a,b同一栏内带不同上标的值具有显著性差别(p<0.05)。
在已进行的研究中已经精确和可靠地产生了预定性别的小马驹(Lindsey等.,Equine Vet.J.2002,34:128-132)。但是,如果可以得到冷冻/解冻的经性别分选的精子,则经性别分选的精子可以在工业上得到更有效的应用。本研究的目的是比较两类精子的运动特征,其中一类精子于15℃储存18小时、流式分选,然后冷冻,另一类精子于15℃运输18小时后直接低温保存。对于5匹公马中每匹均使用两次射精液。在收集之后,两种处理所用的精子均用Kenney改良台氏培养基(KMT)稀释到25×106个精子/ml,并在15℃的水浴中储存18小时。储存之后,使精子温度达到周围环境的温度(约22℃),然后以600g离心10分钟。弃去精液浆,在KMT中将精子沉淀重悬浮到500×106个精子/ml。从该样品移去一等分精子(作为对照),用脱脂乳、蛋黄冷冻稀释剂(4%甘油,FR5)稀释到87×106个精子/ml,使其在90分钟内缓慢冷却至5℃,然后冷冻于液氮蒸气中。精子的另一等分液(流式分选组)用KMT稀释到100×106个精子/ml,用Hoechst 33342(5mg/ml,Sigma-Aldrich,St.LouisMO)染色,孵育30分钟,然后进行流式细胞计数分选。分选的精子以850g离心20分钟,在FR5中重悬浮到87×106个精子/ml,使其在90分钟内缓慢冷却至5℃,然后低温保存。将用于两种处理的精子装在0.25ml的吸管内,每管含有20×106个精子。在解冻后30分钟和90分钟时(由2位观察人员)对可见(visual)前进运动力进行评估(盲评)。每管的等分精子均用KMT和含2mM咖啡因的KMT稀释。可使样品于37℃平衡5~10分钟后再进行评估。每一组的第二吸管用Hamilton-Thorn运动力分析仪(CASA)进行评估(含或不含咖啡因)。结果如表1所示。运动参数的差别由方差分析来确定。根据大部分经测量的反应,流式分选对精子运动力有损害。另外,2mM咖啡因可以对许多精子反应进行改善。这里存在着交互作用,因此与对照精子相比,咖啡因可以使分选精子的某些反应得到更大的改善。因此,分选所带来的损伤可以部分地由咖啡因来补偿。母马的生殖道中也可能存在类似的补偿。目前研究仍在进行,以对分选过的、低温保存的公马精子的生育力与在低温保存前已储存并分选过的精子的生育力进行比较。
处理 | 可见30 | 可见90 | CASA总 | CASA前进 | VAP | VSL | VCL | ALH | BCF | STR | LIN |
C-对照对照C-分选分选 | 50a45a32b18c | 47a40b31c16d | 64a50b32c24d | 60a44b21c12d | 94a82a48b39b | 80a69a38b30b | 164a144a98b80b | 6.19a5.73a4.75b4.51b | 33a33a41b37c | 83a82a73b69b | 50a50a39b38b |
a,b,c,d同一栏内不同上标的值具有差别(P,0.05)。
C-用2mM咖啡因刺激的处理组。
将科罗拉多州和明尼苏达州的年龄为3~6岁的母麋鹿,在9月份通过将孕酮CIDR(含有孕酮并缓慢释放的阴道栓)植入阴道12~14天以使它们发情期同步。移去CIDR后,给麋鹿肌肉内施用200IU的eCG,在60小时后使其同步授精。通过电刺激射精从5岁大的公麋鹿收集新鲜的精液,缓慢冷却4小时以上,直到约20℃,作为纯射精液运输到精子分选试验室。将1.5ml等分液于15,000g离心10秒,使射精液浓缩到1×109个精子/mL以用于染色。将精液在112μM的Hoechst 33342中,在TALP液中浓度为200×106个精子/mL的条件下,于34℃孵育45分钟,然后稀释到100×106个精子/mL用于分选。根据含有X染色体的精子与含有Y染色体的精子其DNA含量不同来分选精子。含有X染色体的麋鹿精子DNA含量比含有Y染色体的麋鹿精子DNA含量多3.8%。使用MoFloSX,以50psi操作,使用基于TRIS的鞘液,对精子进行4小时以上的流式分选。用发射功率为150mW的氩激光的351及364波段来激发与DNA结合的Hoechst 33342染色剂。收集含有X染色体和含有Y染色体的精子(通过对用声波降解的精子等分液进行DNA重分析,验证其纯度约为92%),将其以约4700个精子/秒的速度收集到含有2ml的20%蛋黄-TRIS稀释剂的试管中。连续收集15ml的分选体积。对于每一性别,分选得到约110×106个精子,冷却90分钟以上使之达到5℃。于5℃将等体积的含甘油(12%)的稀释剂加到经分选的体积中。通过将含30ml的分选精子等分液于4℃以850×g离心20分钟来进行浓缩。汇集精子沉淀,调节到21.7×106个精子/mL,并装到0.25ml的吸管内。每根管中均含有总量为5×106的精子,将其在液氮蒸气中进行冷冻。作为对照,同时将来自相同射精液的总量为5×106个精子象经性别分选的精子一样在0.25ml的管中冷冻。于37℃解冻30秒后,通过目测估计,分别有65%和60%的精子(分别为对照和经性别分选)具有前进运动力。用常规的子宫体内经子宫颈精液沉积法,对三匹来自不同地方及不同饲养条件的母麋鹿进行授精。授精后40天,通过检测血液中妊娠特异性的蛋白B(Bio Tracking,Moscow,Idaho)来确定妊娠情况。位于一个地方的10匹母麋鹿授精时状态不好,用经性别分选的或对照精子均未能妊娠。在其他地区用经性别分选的精子所获得的妊娠率(61%,11/18)与用对照授精物获得的妊娠率(50%,3/6)类似。得到这些妊娠率(经性别分选的和对照)所使用的精子少于正常的麋鹿人工授精所使用的精子。11只用经性别分选的精子所得到的小仔中有9只(82%)为预期的性别。
本发明还可包括根据本发明上述任意实施方案所产生的哺乳动物;或者可以包括根据本发明各种实施方案所产生的具有预定性别的哺乳动物,所述实施方案提供了富含X染色体精子群或富含Y染色体精子群的精子授精样品;或者包括根据本发明任意实施方案产生的所哺乳动物,在所述实施方案中精子授精样品所含有的精子数量少于特定哺乳动物授精时通常所使用的精子数量;或者包括上述根据本发明所产生的麋鹿子代。
从上文中可以很容易地理解,本发明的基本思想可以用不同途径来具体实施。本发明涉及精子处理系统,该系统包括进行精子处理的技术及装置。在本申请中,通过所述各种装置所得到的部分结果和所采用的固有步骤而公开了各种细胞处理技术。它们仅仅是利用所述预期装置的自然结果。另外,当部分装置被披露时,应该理解,这些装置不仅完成了某些方法,并且还可以用多种方式加以改变。重要的是,对于所有的前述内容,应当理解所有这些方面均包含在本发明所披露的内容之中。
这份非临时申请中所包括的讨论,其目的是为了提供基本的描述。读者应知道,具体的讨论可能并非明确地描述所有可能的实施方案,许多替代选择是隐含的。它可能没有充分地解释本发明的普遍性质,也可能没有明确地显示各个性质或元件实际上如何代表大量不同功能或大量替代方案或等同元件。同样,这些也隐含地包括在本发明的公开内容中。当从装置术语的角度描述本发明时,装置的每一元件隐含地执行一种功能。装置权利要求不仅可包括所述的装置,还可以包括用于实现本发明的功能或每个元件所执行的功能的方法或过程的权利要求。所有描述及术语均并非是对权利要求范围的限制,权利要求的范围包含在整个专利申请中。
同时应当理解的是,可以实施各种变更而不会背离本发明的本质。这些变更也暗含地包括在本说明中。它们仍属于本发明的范围。广泛的公开内容包括于本发明的公开内容之内,其中包括已经展示的显式实施方案、大量隐含的替代实施方案及广泛的方法或过程。
而且,本发明及权利要求的每个不同元件还可以用多种方式获得。本公开内容应理解为包括每一个这样的变化,不论它是任意的装置的实施方案、方法或过程的实施方案的变更,还是甚至仅仅是其中任意元件的变更。特别是,应该理解,由于本方面公开内容涉及本发明的元件,每个元件的文字可以用等同的装置术语或方法术语来表达——甚至仅仅是其具有相同的功能或结果。应认为这些等同的、更广泛的甚至是更上位的术语包含于各元件或动作的描述中。当需要对本发明所包括的隐含的广泛范围进行明确时,这些术语可加以替换。仅举一个例子来说,应该理解的是,所有的动作均可以用执行该动作的装置或促使产生该动作的元件来表达。类似地,每一公开的物理元件应理解为包含了对所述物理元件执行的动作的公开。就此最后一方面来说,仅举一个例子,“流式分选仪”的公开内容应理解为包含了“流式分选”动作的公开内容,无论其是否明确讨论过;反过来说,当有“流式分选”动作的有效公开时,这样的公开内容应理解为包括了“流式分选仪”,甚至包括了“流式分选方式”的公开。这些变化及替代术语应理解为明确包括在说明书内。
本专利申请中提及的任何法律、法令、规章或准则的条款,或此专利申请中提及的专利、出版物或其他参考文献均以参考的方式引入本文。另外,对于所用的每一术语,应当理解,除非其在本申请中的应用与通用词典中的解释不一致,否则应当认为,本文引入了通用词典中对各条术语的定义。在此以参考的方式引入例如Random House Webster’s UnabridgedDictionary(《兰登书屋韦氏大词典》)第二版的所有定义、替代术语和同义词。最后,以参考方式引入该临时专利申请的参考文献列表中所列出的所有文献或其他与该申请同时提交的信息声明均在此附上,并以参考的方式引入;但是,对于上述每一项来说,这些以参考方式引入的信息或声明可被认为与此/这些发明的专利权不一致,很清楚这样的声明不能认为是本申请人所做出的。
因此,应理解为申请人至少要求如下:i)本文所公开和描述的各种精子处理装置;ii)所公开和描述的相关方法;iii)这些装置和方法的类似、等同甚至是隐含的变化;iv)可以完成所公开和描述的所示各个功能的替代设计;v)完成每一所示功能的替代设计和方法,其为隐含的完成所公开和描述的功能的替代设计和方法;vi)作为分开的和独立的发明所显示的各个性质、元件及步骤;vii)由所公开的不同系统或组分而改进的申请;viii)由此类系统或元件所生成的最终产品;以及ix)基本在上文中并参考任何所附实施例所描述的方法和装置,X)所公开元件的各种组合及排列,及xi)从属于每个或其中任何一个独立权利要求或概念的每个从属的潜在的从属权利要求或概念。就这点上,应理解的是出于实践的原因及为了避免增加数以百计权利要求的可能性,申请人最终仅提交了具有最初的从属权利要求的权利要求。支持应当被理解为达到有关新实体法规(new matterlaws)所要求的程度,以便能够在一项独立权利要求或概念之下增加各种的从属权利要求或其他要素,作为任何其他独立权利要求或概念的从属权利要求或要素,上述法规包括但不限于《欧洲专利公约》第123(2)条和美国专利法35 USC 132或其他此类法律。而且,如果使用或当使用表示转接关系的措词“包含”时,根据习惯上对权利要求的解释,使用该词是为了保持本文权利要求为“开放式”。因此,除非上下文需要,术语“包含”或诸如“包括“或“含有”等变化形式应理解为,它们意指包括所声明的要素或步骤或者要素或步骤的组,而不排除任何其他的要素或步骤或者要素或步骤的组。这样的术语应以其最广泛的形式来解释,以使申请人获得法律许可的最广泛的范围。
Claims (62)
1、一种生成精子授精样品的方法,该方法包括:
a.从哺乳动物的雄性个体获取精液;
b.生成具有流动特征的液流;
c.改变所述液流的流动特征以调节液流的压力;
d.将精子夹带入所述液流中;
e.通过调节所述液流的压力来控制精子的生育力特征;及
f.产生具有可控精子生育力特征的精子授精样品。
2.如权利要求1所述的生成精子授精样品的方法,其中所述哺乳动物选自牛、马、绵羊、犬、猫、猪、海洋动物、鹿、灵长类动物或山羊。
3.如权利要求1所述的生成精子授精样品的方法,其中所述液流包含鞘液流。
4.如权利要求3所述的生成精子授精样品的方法,其中所述鞘液流包括含有磷酸缓冲盐溶液的鞘液。
5.如权利要求3所述的生成精子授精样品的方法,其中所述鞘液流包括含有柠檬酸盐缓冲液的鞘液。
6.如权利要求5所述的生成精子授精样品的方法,其中所述柠檬酸盐缓冲液包含约2.9%的柠檬酸钠。
7.如权利要求3所述的生成精子授精样品的方法,其中所述鞘液流包括含有HEPES缓冲液的鞘液。
8.如权利要求1所述的生成精子授精样品的方法,其中所述液流在流式细胞计数仪或细胞分选仪中产生。
9.如权利要求1所述的生成精子授精样品的方法,其中改变所述液流的流动特征以调节液流压力的步骤包括改变所述流动特征以将所述液流的压力调节到每平方英寸约20磅至每平方英寸约60磅之间。
10.如权利要求1所述的生成精子授精样品的方法,其中从哺乳动物的雄性个体获取精液的步骤包括从公牛获取牛精液;通过调节所述液流的压力来控制精子的生育力特征的步骤包括确定被夹带入所述液流中的牛精子的生育力特征,该牛精子的生育力特征与所述精液中的所述牛精子的生育力特征之间无实质性差别。
11.如权利要求1所述的生成精子授精样品的方法,其中从哺乳动物的雄性个体获取精液的步骤包括从公牛获取牛精液;通过调节所述液流的压力来控制精子的生育力特征的步骤包括确定被夹带入所述液流中的所述牛精子的生育力特征,该牛精子的生育力特征与所述精液中的所述牛精子的牛精子生育力特征基本相当。
12.如权利要求1所述的生成精子授精样品的方法,其中从哺乳动物的雄性个体获取精液的步骤包括从公牛获取牛精液;产生具有可控精子生育力特征的精子授精样品的步骤包括产生具有牛精子生育力特征的牛精子授精样品,该牛精子授精样品的牛精子生育力特征与所述精液中的所述牛精子的生育力特征之间无实质性差别。
13.如权利要求1所述的生成精子授精样品的方法,其中从哺乳动物的雄性个体获取精液的步骤包括从公牛获取牛精液;产生具有可控精子生育力特征的精子授精样品的步骤包括产生具有牛精子生育力特征的牛精子授精样品,该牛精子授精样品的牛精子生育力特征与所述精液中的所述牛精子的生育力特征基本相当。
14.如权利要求1所述的生成精子授精样品的方法,其中从哺乳动物的雄性个体获取精液的步骤包括从公牛获取牛精液;所述改变液流的流动特征以调节液流的压力的步骤包括改变所述液流的所述流动特征以将液流压力调节到每平方英寸约30磅至每平方英寸约50磅之间。
15.如权利要求1所述的生成精子授精样品的方法,其中从哺乳动物的雄性个体获取精液的步骤包括从公牛获取牛精液;所述改变液流的流动特征以调节液流的压力的步骤包括改变所述液流的所述流动特征以将液流压力调节到每平方英寸约30磅至每平方英寸约40磅之间。
16.如权利要求1所述的生成精子授精样品的方法,其中从哺乳动物的雄性个体获取精液的步骤包括从公牛获取牛精液;所述改变液流的流动特征以调节液流的压力的步骤包括改变所述液流的所述流动特征以将液流压力调节到每平方英寸约40磅。
17.如权利要求1所述的生成精子授精样品的方法,其中从哺乳动物的雄性个体获取精液的步骤包括从公马获取马精液;通过调节所述液流的压力来控制精子的生育力特征的步骤包括确定被夹带入所述液流中的马精子的生育力特征,该马精子的生育力特征与所述精液中的所述马精子的生育力特征之间无明显差别。
18.如权利要求1所述的生成精子授精样品的方法,其中从哺乳动物的雄性个体获取精液的步骤包括从公马获取马精液;通过调节所述液流的压力来控制精子的生育力特征的步骤包括确定被夹带入所述液流中的马精子的生育力特征,该马精子的生育力特征与所述精液中的所述马精子的生育力特征基本相当。
19.如权利要求1所述的生成精子授精样品的方法,其中从哺乳动物的雄性个体获取精液的步骤包括从公马获取马精液;产生具有可控精子生育力特征的精子授精样品的步骤包括产生具有所述马精子生育力特征的马精子授精样品,该马精子授精样品的马精子生育力特征与所述精液中的所述马精子的生育力特征之间无实质性差别。
20.如权利要求1所述的生成精子授精样品的方法,其中从哺乳动物的雄性个体获取精液的步骤包括从公马获取马精液;产生具有可控精子生育力特征的精子授精样品的步骤包括产生具有马精子生育力特征的马精子授精样品,该马精子授精样品的马精子生育力特征与所述精液中的所述马精子的生育力特征基本相当。
21.如权利要求1所述的生成精子授精样品的方法,其中从哺乳动物的雄性个体获取精液的步骤包括从公马获取马精液;所述改变液流的流动特征以调节液流的压力的步骤包括改变所述液流的所述流动特征以将液流压力调节到每平方英寸约30磅至每平方英寸约50磅之间。
22.如权利要求1所述的生成精子授精样品的方法,其中从哺乳动物的雄性个体获取精液的步骤包括从公马获取马精液;所述改变液流的流动特征以调节液流的压力的步骤包括改变所述液流的所述流动特征以将液流压力调节到每平方英寸约30磅至每平方英寸约40磅之间。
23.如权利要求1所述的生成精子授精样品的方法,其中从哺乳动物的雄性个体获取精液的步骤包括从公马获取马精液;所述改变液流的流动特征以调节液流的压力的步骤包括改变所述液流的所述流动特征以将液流压力调节到每平方英寸约40磅。
24.如权利要求1所述的生成精子授精样品的方法,其中所述精子的生育力特征包括精子的运动力。
25.如权利要求1所述的生成精子授精样品的方法,其中所述生育力特征包括牛精子的运动力。
26.如权利要求1所述的生成精子授精样品的方法,其中所述生育力特征包括马精子的运动力。
27.如权利要求1所述的生成精子授精样品的方法,其中所述生育力特征包括精子的存活力。
28.如权利要求1所述的生成精子授精样品的方法,其中所述生育力特征包括牛精子的存活力。
29.如权利要求1所述的生成精子授精样品的方法,其中所述生育力特征包括马精子的存活力。
30.如权利要求1所述的生成精子授精样品的方法,其中所述生育力特征包括所述哺乳动物雌性个体的妊娠率,该雌性个体已用所述具有可控精子生育力特征的精子授精样品进行授精。
31.如权利要求1所述的生成精子授精样品的方法,其中所述生育力特征包括母牛的妊娠率,该母牛已用具有可控精子生育力特征的牛精子授精样品进行授精。
32.如权利要求1所述的生成精子授精样品的方法,其中所述生育力特征包括母马的妊娠率,该母马已用具有可控精子生育力特征的马精子授精样品进行授精。
33.如权利要求1所述的生成精子授精样品的方法,其中所述生育力特征包括卵母细胞的卵裂率,该卵母细胞已用所述具有可控精子生育力特征的精子授精样品进行受精。
34.如权利要求1所述的生成精子授精样品的方法,其中所述生育力特征包括牛卵母细胞的卵裂率,该牛卵母细胞已用所述具有可控精子生育力特征的牛精子授精样品受精。
35.如权利要求1所述的生成精子授精样品的方法,其中所述生育力特征包括马卵母细胞的卵裂率,该马卵母细胞已用所述具有可控精子生育力特征的马精子授精样品进行受精。
36.如权利要求1所述的生成精子授精样品的方法,其中所述生育力特征包括卵母细胞的胚囊率,该卵母细胞已用所述具有可控精子生育力特征的精子授精样品进行受精。
37.如权利要求1所述的生成精子授精样品的方法,其中所述生育力特征包括牛卵母细胞的胚囊率,该牛卵母细胞已用所述具有可控精子生育力特征的牛精子授精样品进行受精。
38.如权利要求1所述的生成精子授精样品的方法,其中所述生育力特征包括马卵母细胞的胚囊率,该马卵母细胞已用所述具有可控精子生育力特征的马精子授精样品进行受精。
39.如权利要求31所述的生成精子授精样品的方法,其中所述具有可控精子生育力特征的牛精子授精样品含有约1×105至2×107个所述的牛精子。
40.如权利要求31所述的生成精子授精样品的方法,其中所述具有可控精子生育力特征的牛精子授精样品含有约1×106至3×106个所述的牛精子。
41.如权利要求1所述的生成精子授精样品的方法,该方法还包括将所述精子染色的步骤。
42.如权利要求41所述的生成精子授精样品的方法,其中所述将精子染色的步骤包括用Hoechst 33342对所述精子进行染色。
43.如权利要求1所述的生成精子授精样品的方法,该方法还包括在所述液流中产生液滴,其中在一些所述液滴中含有一个精子,该精子为任何一种所述精子。
44.如权利要求43所述的生成精子授精样品的方法,该方法还包括根据性别特征区分所述精子。
45.如权利要求44所述的生成精子授精样品的方法,其中根据性别特征区分所述精子的步骤包括根据DNA含量区分所述精子。
46.如权利要求44所述的生成精子授精样品的方法,其中根据性别特征区分所述精子的步骤包括根据精子头部的体积区分所述精子。
47.如权利要求44所述的生成精子授精样品的方法,该方法还包括根据所述性别特征分离精子的步骤。
48.如权利要求47所述的生成精子授精样品的方法,该方法还包括将所述具有可控精子生育力特征的精子授精样品收集到收集容器中的步骤。
49.如权利要求48所述的生成精子授精样品的方法,其中所述将具有可控精子生育力特征的所述精子授精样品收集到收集容器中的步骤包括将经性别选择的精子授精样品收集到所述收集容器中。
50.如权利要求49所述的生成精子授精样品的方法,其中所述经性别选择的精子授精样品包括用于人工授精的经性别选择的精子授精样品。
51.如权利要求49所述的生成精子授精样品的方法,其中所述经性别选择的精子授精样品包括用于体外受精的经性别选择的精子授精样品。
52.如权利要求49所述的生成精子授精样品的方法,其中所述经性别选择的精子授精样品包括用于胞质内注射的经性别选择的精子注射样品。
53.一种对经性别选择的精子的生育力进行评价的方法,该方法包括:
a.从哺乳动物的雄性个体获取精液;
b.提供经过第一处理条件处理的含Y染色体的经性别选择的精子;
c.提供经过第二处理条件处理的含X染色体的经性别选择的精子;
d.产生授精样品,该授精样品中的所述经过第一处理条件处理的含Y染色体的经性别选择的精子和所述经过第二处理条件处理的含X染色体的经性别选择的精子成一定比率;
e.用所述授精样品使所述哺乳动物的至少一个雌性个体授精,所述授精样品中的经过第一处理条件处理的含Y染色体的精子和所述经过第二处理条件处理的含X染色体的精子成所述比率;
f.由所述哺乳动物的至少一个雌性个体产生子代;
g.通过对以下两种比率进行比较来评价所述经性别选择的精子的生育力,一种比率为所述哺乳动物的至少一个雌性个体所产生的子代的性别比率,另一种比率为所述经过第一处理条件处理的含Y染色体的精子与所述经过第二处理条件处理的含X染色体的精子的比率。
54.如权利要求53所述的对经性别选择的精子的生育力进行评价的方法,其中所述经过第一处理条件处理的含Y染色体的精子与经过第二处理条件处理的含X染色体的精子的所述比率包括基本等量的经过第一处理条件处理的含Y染色体的精子和经过第二处理条件处理的含X染色体的精子。
55.如权利要求53所述的对经性别选择的精子的生育力进行评价的方法,其中所述哺乳动物的所述至少一个雌性个体的子代的所述性别比率包括胚胎的性别比率。
56.如权利要求53所述的对经性别选择的精子的生育力进行评价的方法,其中所述哺乳动物的所述至少一个雌性个体的子代的所述性别比率包括胎仔的性别比率。
56.如权利要求53所述的对经性别选择的精子的生育力进行评价的方法,其中所述哺乳动物的所述至少一个雌性个体的子代的所述性别比率包括已出生后代的性别比率。
57.如权利要求53所述的对经性别选择的精子的生育力进行评价的方法,其中所述哺乳动物的所述至少一个雌性个体包括数量足够多的所述哺乳动物的雌性个体,以产生数量足够多的子代来确定子代的所述性别比率。
58.一种对来自于一种哺乳动物的雄性个体的精子的生育力进行评价的方法,该方法包括:
a.从一种哺乳动物的至少两个雄性个体获取精子;
b.对所述至少两个雄性个体的所述精子进行基本相同的流式细胞计数处理;
c.用精子混合物使所述哺乳动物的至少一个雌性个体授精,所述精子混合物由所述哺乳动物的至少两个雄性个体中的每一个个体的所述精子以基本等量混合而成,所述精子经过基本相同的流式细胞计数处理;
d.从所述哺乳动物的至少一个雌性个体收集胚胎;
e.测定所述哺乳动物的所述至少两个雄性个体中的哪一个为每个胚胎的雄性亲代;及
f.根据以所述哺乳动物的至少两个雄性个体中的每一个个体作为雄性亲代的相对胚胎数,对所述至少两个雄性个体的相对生育力进行分级。
59.如权利要求58所述的对来自于一种哺乳动物的雄性个体的精子的生育力进行评价的方法,其中所述流式细胞计数处理包括根据性别特征对来自所述至少两个雄性个体的每一个个体的所述精子进行分离。
60.如权利要求58所述的对来自于一种哺乳动物的雄性个体的精子的生育力进行评价的方法,其中所述流式细胞计数处理包括对来自于所述至少两个雄性个体的每一个个体的精子进行性别选择。
61.一种对来自于一种哺乳动物的雄性个体的精子的生育力进行评价的方法,该方法包括:
a.从一种哺乳动物的至少两个雄性个体获取精子;
b.将所述至少两个雄性个体的精子进行基本相同的流式细胞计数处理;
c.用精子混合物使卵母细胞体外受精,所述精子混合物由所述哺乳动物的至少两个雄性个体中的每一个个体的所述精子以基本等量混合而成,所述精子经过基本相同的流式细胞计数处理;
d.收集通过使所述卵母细胞体外受精而产生的胚胎;
e.测定所述哺乳动物的所述至少两个雄性个体中哪一个为每个胚胎的雄性亲代;及
f.根据以所述哺乳动物的至少两个雄性个体中的每一个个体作为雄性亲代的相对胚胎数,对所述至少两个雄性个体的相对生育力进行分级。
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2010
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2013
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