CN101496747B - 以少量精细胞对哺乳动物进行有性别选择的授精 - Google Patents

以少量精细胞对哺乳动物进行有性别选择的授精 Download PDF

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CN101496747B
CN101496747B CN200810128056XA CN200810128056A CN101496747B CN 101496747 B CN101496747 B CN 101496747B CN 200810128056X A CN200810128056X A CN 200810128056XA CN 200810128056 A CN200810128056 A CN 200810128056A CN 101496747 B CN101496747 B CN 101496747B
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乔治·E·赛德
丽沙·赫里克夫
约翰·申克
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Abstract

本发明提供一种以少量精细胞对哺乳动物进行有性别选择的授精技术，特别是以商业上可实施的方式并用以前认为在商业上不可能实现的精子剂量进行人工授精，繁殖性别选择的哺乳动物后代。本发明通过采用精子性别选择的分类技术，还实现了超数排卵、产生多个胚胎以得到选择性别的胚胎。本发明的授精技术结合了包括改进外鞘液的技术和其它使精细胞压力最小化的技术，改进了精子收集系统和收集技术，实现了精子样品及其所繁殖的动物在商业上的应用。

Description

以少量精细胞对哺乳动物进行有性别选择的授精
本申请是分案申请，其原申请的国际申请号为PCT/US98/27909，中国国家申请号为98813255.9，申请日为1998年12月31日，发明名称为“以少量精细胞对哺乳动物进行有性别选择的授精”。
技术领域
本发明涉及哺乳动物后代的性别选择。特别涉及低剂量人工授精和为繁殖所需性别的动物后代而增加产卵的方面。特别是本发明涉及无论使用何种分类技术，用低剂量实现性别选择的人工授精、涉及为性别选择经流式细胞计数术分类精子和低剂量人工授精系统，以及增加排卵结果的技术等。
背景技术
长期以来，人们一直希望能够选择动物后代的性别。除了明显的心理因素，当考虑到诸如牛的食用动物以及战利品动物，如马等类似动物时，实际的哺乳动物后代性别选择具有明显的经济学价值。由于存在这种巨大的需求，为获得性别选定的后代人们进行了各种大量的努力。似乎最可能实现所需结果的工作集中在授精前分类和选择X和Y精子。
尝试分类X和Y精子所面临的一个挑战是精子的数量很大。在自然授精中，有些动物种类产生数十亿的精子；在人工授精中虽然使用精子较少些，但仍然需很大数目。例如，人工授精技术通常使用1千万至5亿个精子(取决于物种)。因此即使在人工授精环境中也需要极大数目的精子。
已尝试了许多方法来实现分离携带X-和Y-染色体的精子。这些方法包括诸如在美国专利号4276139中公开的磁性分离技术，在美国专利号5514537中公开的柱式(columnar)分离技术，在美国专利号3894529，再审专利号32350，美国专利号4092229，4067965和4155831中讨论的重量分析技术。在US4083957中公开了对电学特性进行分析的尝试，在美国专利号4225405，4698142和4749458中讨论了结合电学和重力特性的分析。在美国专利号4009260和4339434中显示了尝试分析精子活力。诸如在美国专利号4511661和4999283(涉及单克隆抗体)和美国专利号5021244，5346990，5439362和5660997(涉及膜蛋白)，和美国专利号3687803，4191749，4448767，和4680258(涉及抗体)中显示的化学技术，以及在美国专利号4085205中显示的添加血清成分。尽管似乎提供了具有较高效力的各种技术，但事实上目前没有一种技术达到性别预选所需的水平。然而，不论最终使用何种分离技术，自然存在的大量精子的竞争性结合和分离携带X-和Y-染色体的精子的研究使开发以少量精子实现授精成为可能。
目前，用于实现分离携带X-和Y-染色体的精子的定量技术包括通过流式细胞计数术逐个辨别和分离精子。该技术随着技术进步和发现携带X-和Y-染色体的精子对不同的染色剂吸收显示出其可能性。这一点以前在美国专利号4362246中进行了讨论且通过Lawrence Johnson在美国专利号4135759中公开的技术进行了重要的阐述。Johnson的这种利用流式细胞计数术分离携带X-和Y-染色体的精子的技术非常重要和先进，因为它首次使分离该精细胞在商业上成为可能。尽管仍然是实验性的，但通过高速流式细胞计数器，如Cytomation公司生产的
Figure S200810128056XD00021
流式细胞计数器已显著增强了这种分离的效果且已在包括美国专利号5150313，5602039，5602349和5643796以及国际PCT专利申请WO96/12171的各种其它专利中进行了讨论。尽管利用Cytomation公司生产的
Figure S200810128056XD00022
细胞计数器极大地增加了速率，且尽管这种速率增加与常用的高数量的精细胞特别相关，但仍然存在一些问题。用流式细胞计数器尽管几乎可使速率增加10倍，但许多情况下需要越来越短的分类时间。首先，已发现作为实际问题，精子是时间关键性细胞。如果它们较长时间未用将失去效力。其次，收集、分类和授精的计时使速度在商业上有高度的重要性。因此，精细胞和该方法对时间要求的特性使速度成为实现高效率和成功率的至关重要因素。
从实践上到理论上还存在其它的问题。在实践方面，需要使用廉价的可大量使用的物质获得性别分类的精子样品。在费用方面，还需要能以尽可能有效的劳动实现分类(以及收集和授精)。因此，为了该领域成功商业生产，可能仅提高效率的改进仍然是有意义的。关于实际费用，是整个方法实践中最为棘手和敏感的方面。为此，需要简化过程使其程序尽可能粗略，以便操作错误或技巧的影响不大。结合这些情况更加需要用较小量的精子进行授精。
除了该方法的精密性外，已知精子本身是极其脆弱的细胞。尽管初看起来认为容易理解，但事实上该细胞的敏感性还没有被充分认识。一般在流式细胞计数术进行中，大多数分类的细胞或颗粒通常呈球形而且能经受各种物理方法处理。但精细胞却不是这样。事实上，正如本发明所公开的，采用通常的流式细胞计数术的方法对于在某些应用中进行精细胞的细胞计数术分类是不可行的。细胞的稀释、流式细胞计数仪需要逐个分离和鉴别细胞以及典型的流式细胞计数术在本身施加于被分类的细胞或其它物质的压力均可改变其敏感性。这也说明精细胞的特性，因为看起来即使精细胞通过流式细胞计数器并被分类时没有肉眼可辨别的副作用，但事实上这些细胞本身已经接受了压力，使它们在授精过程中没有达到最佳状况。因此，这些因素的相互作用对于精细胞分类并最终用于人工授精产生了一些特有的问题。
尽管Johnson的专利和有关技术取得了巨大的进展，但仍然存在另一问题，即用性别选择的精子实现低剂量授精非常困难，无论使用何种分离技术。尽管有人曾经进行过低剂量授精，但似乎更多的是在理论上或实验室中而不是用于实践中或商业应用中。据此，不仅需要实现低剂量授精，同时要达到与目前不选择性别的，高剂量人工授精同样的受孕成功率。因此，本发明人在性别选择和低剂量人工授精两方面所取得的进展首次使商业应用成为可能。
同样，尽管Johnson的专利和有关技术取得了巨大的进展，但仍然面临另一问题，即高成功率的人工授精受众多因素相互影响，具有统计学特性。因此，建议的解决办法在某种程度上可能涉及结合一些因素，在非常精确的统计学研究中显示出某因素单独存在或者与其它因素结合是必须的。这一决定进一步结合如下事实，即，该结果本身可随物种而改变且由于在起初不可能有足够多的数据进行试验和统计学取样的事实而难以确定。由于这些原因，本发明还涉及结合多种因素，这些单独或结合的因素对特定的应用提供了合适的解决方法。因此认为本说明书是非常广义的，可实现所公开的技术的各种组合和渗透。可能存在与其它因素的未知协同效应。这些因素可包括在分类中，或者存在于流式细胞计数器内，或在收集步骤及授精步骤中所涉及。目前，研究主要在牛类中进行，然而，并不认为这些技术将局限于这类物种，因为这项技术只与精细胞相关。除了精细胞外，该技术可应用于其它敏感细胞的分类或在细胞进行分类时使流式细胞计数术造成的压力的影响减到最小。
令人感兴趣的是，当本发明采用的方法使分类的影响或对精细胞的压力最小化时，其他人实际上通过增加压力和对速度的需要及其它这种操作似乎采用的步骤偏离了这一方向。实质上，低剂量授精和高速处理的方式可能以单独或者互相联系的方式造成互相限制的问题。因此，尽管对高速度、低剂量选择性别的授精早有需要，而且认为技术不令人满意，且尽管实现的技术和环境早已可获得，但在本发明前这些技术的一些进展或者其组合被本领域的技术人员明显的忽略了。也许在某种程度上他们没有意识到这个问题涉及到多因素相互作用以及本领域涉及的细胞类型(精细胞或者也许是物种特异性精细胞)特有的必要性。有趣的是，正如本文中列举以前的工作所显示的，尽管进行了大量尝试，但显然其它人不理解这个领域固有的问题，如低剂量、选择性别的授精等。也许他们假定，由于自然交配也许涉及几十亿精子，因此用少4个数量级的数目可能对于实现人工授精产生实质性的限制。因此，在某种程度上偏离本发明技术方向的实际做法可能并不奇怪。由于本发明的结果表明选择性别、低剂量的人工授精的成功率与不选择性别的，高剂量的人工授精相当，因此，也许该结果在一定程度上会被认为是意想不到的。更令人惊奇的是，本发明的技术和进展事实上均达到了所示的重大结果。尽管每个技术单独看来不值得注意，但事实上，这些细微的改进导致在最终结果中产生了有意义的进展，无论是单独的改进还是与其它微小的改进相结合。
因此，本发明前的技术，对实现低剂量、选择性别的人工授精的成功率不可能达到在商业应用时所必需的操作或简化程序的水平。然而，除了实现在商业水平上的低剂量、选择性别的人工授精外，本发明还公开了这样的技术，该技术允许实现改进效果且因此有利于所需最终结果，即，在商业基础上的低剂量、选择性别的人工授精。
发明内容
因此，本发明要求在商业水平上实现低剂量授精和预定哺乳动物性别的结果。本发明还提供了改进的外鞘液和收集系统，用于通过流式细胞计数器分离技术分类精细胞以确定其性别。在这种分离技术中，用于流式细胞计数器的典型外鞘溶液被分类时对精细胞的压力最小化的液体取代。而且，改进了收集系统使精细胞受到的物理和化学压力最小。本发明提供了多种技术和物质，但正如本领域的技术人员容易理解的，可根据物种，分离技术，目的和在具体处理应用中涉及的其它参数可使用各种组合和变换方式，使技术达到最优化。
本发明的目的仅仅是实现用低剂量进行选择性别的授精，并在现实的商业环境下进行。还有一个目的是实现更好地分类诸如精细胞等物质。一个相关的目的是使分类功能本身对细胞或其它可能被分类的敏感性物质的影响减到最小。对于流式细胞计数分类技术，一个特别的目的是减小外鞘液对细胞的影响并有效提供使细胞耐受各种压力的外鞘液。一个类似的目的是提供特别适用于一般精细胞的物质和技术，并适用于牛精细胞及马精细胞，还适用于将精细胞分离成携带X-或Y-染色体的成分。相似的一个目的是将收集期(分类后)对细胞的影响，特别是对已进行性别分类的精细胞受到的理化影响减至最小。因此，本发明的一个目的是获得尽可能未受影响的分类结果。
本发明的另一目的是实现低剂量的，分类的授精，其水平和成功率与典型的不选择性别的高剂量的人工授精相当。围绕这一目的，一个目标是提供一个用于人工授精的总体系统，它能以商业上实际的方式达到该目的。因此，最小化对精细胞的压力或潜在损伤的前期目标是重要的。以既提供高速度，又具有低分类压力的方式进行分类也是一个重要的目标，特别适用于以低剂量的精细胞分类。提供对精子繁殖力无负面影响且适用于人工授精的外鞘液和其它液体，也是一个重要的目标。
本发明的说明书的其它部分和权利要求书公开了本发明进一步的目的。
附图说明
图1是用于本发明的流式细胞计数分离技术的分类器系统。
图2是典型的流式细胞计数器自由降落区的液流细胞图。
图3是粗略显示本发明结果差别的示意图。
图4是在收集区域收集的已被分类的细胞流。
具体实施方式
可见，本发明的基本概念可以结合并以各种方式体现。本发明仅仅涉及商业上实用的低剂量、选择性别的授精及其结果。对于流式细胞计数分离技术，本发明还涉及改进的流式细胞计数器系统以及用于产生性别特异性精子样品的系统，该样品可用于人工授精并以这种技术繁殖动物。本发明包括在商业条件下也可获得高成功率的总方法。而且，该技术是以通用的方式公开的，只要理解了其基本原则，可将其用于特定系统。尽管公开了装置的改进，但应理解这些改进不仅完成某些工作，而且装置可进行改变并以多种方式组合。重要的是，本说明书包括上述全部内容的各个方面。
如上所述，基本目的是分离携带X-染色体的精子与携带Y-染色体的精子。这通过分离两类精子以便分别进行包装和处理来实现。目前，这种分离优选通过使用流式细胞计数术进行。一般来说，流式细胞计数术是一种人们熟知的技术，其基本概念在Cytomation公司的许多专利，如美国专利和以前列出的其它文献中均有公开。这里引用各专利和参考文献以供参考；因此，本领域的技术人员容易理解其所涉及的基本原理。
基本上，流式细胞计数术包括分类物质，例如细胞，通过某种细胞源提供给流式细胞计数器。图1是该仪器示意图。该流式细胞计数器包括：细胞源(1)，用于产生或提供细胞或其它类型的物质供流式细胞计数器分析。细胞储存在喷嘴(2)内，以便细胞可被外鞘液(3)包围。外鞘液(3)通常由某种外鞘液源(4)供应，以便细胞源(1)供应细胞时，喷嘴(2)同时供应外鞘液(3)。以这种方式很容易理解外鞘液(3)如何形成围绕细胞的外鞘液体。由于以一定的压力给流式细胞计数器供应各种液体，因此它们流出喷嘴(2)并在喷嘴口(5)喷出。经过提供某种类型的振荡器(6)可在喷嘴(2)内形成压力波且在喷嘴口(5)处将液体传递出喷嘴(2)，其中该振荡器可通过振动控制器(19)极精确地控制。由于振荡器(6)作用于外鞘液(3)，最终流出喷嘴口(5)的液流(7)均匀地形成液滴(8)。由于细胞被外鞘液环绕，液滴(8)可含有分离的单独的细胞或其它物质。
由于液滴(8)一般含有分离的细胞，流式细胞计数器根据是否含有某种细胞对液滴进行鉴定和分离。这通过细胞传感系统(9)实现。细胞传感系统至少包括某种类型的传感器(10)，它对各液滴(8)内的细胞发生反应，在Larry Johnson的初期工作，即美国专利5135759中已进行了详细的讨论。正如Johnson的专利对精细胞的描述，细胞传感系统(9)根据一种特定染色剂相对存在或不存在可产生反应。这种染色剂可被某些刺激剂激活，如激光激发器(11)。尽管各类精细胞均被该染色剂染色，但X-染色体和Y-染色体的不同长度使得染色的程度不同。因此，根据精细胞的染色程度，通过它们不同的发射水平可以区分携带X-染色体的精子和携带Y-染色体的精子。
为了实现用流式细胞计数器分离技术最终分离出合适的细胞，传感器(10)接受的信号提供给某种类型的分类器区别系统(12)，该系统极迅速地作出判断并根据液滴(8)内是否存在所需细胞而对各液滴(8)有区别地充电。分类器区别系统(12)以这种方式作用于静电偏转板(13)，根据液滴中是否含有合适的细胞或其它物质来偏转液滴(8)。结果，流式细胞计数器通过使细胞落入一个或多个收集器(14)中来分类细胞。因此，通过检测细胞或其它物质的某种特性，流式细胞计数器可根据具体特征区分细胞且将它们放入合适的收集器(14)中。在目前用于分类精子的系统中，携带X-染色体的精子的液滴被充正电且因此偏向一方，携带Y-染色体的精子的液滴被充负电且因此偏向另一边，废液流(未分类的细胞)未充电且因此以不偏转的液流收集进吸管等装置中。
参照图2，可更进一步了解该方法。如该图所示，由于有振荡器(6)(图2中未显示)，喷嘴(2)喷出液流(7)形成液滴(8)。由于细胞源(1)(图2未显示)可供应根据Johnson技术染色的精细胞(15)，传感器(10)测定到不同的经激光激发器(11)产生的光刺激，这样，当液滴(8)从液流(7)中分离时，流式细胞计数器就能控制每个液滴(8)存在或不存在电荷。根据其内容物分出带正电，带负电和不带电的液滴(8)。如图2所示，某些液滴显示为偏转的液滴(16)。这些偏转的液滴(16)含某种性别的精细胞(15)。然后将它们储存在合适的收集器(14)中备用。
对于精细胞分类应用特别重要的流式细胞计数术的一个方面是流式细胞计数器的高速运转。Cytomation公司商标为
Figure S200810128056XD00081
的流式细胞计数器取得了特别的进展。这些流式细胞计数器显著增加了分类速度且因此使得流式细胞计数术在精细胞分类的商业应用中(以及其它商业应用)成为可能。流式细胞计数器实现了高速分类，即速度比未用流式细胞计数器时显著增高。尤其是，Cytomation公司的流式细胞计数器使用的振荡器频率约大于5千赫兹，更具体地说可在10-30或甚至在50千赫兹的范围内运转。因此以极高的频率形成液滴且被外鞘液包围的细胞可非常迅速地从喷嘴(2)喷出。选择组成流式细胞计数器系统的每一部件如喷嘴(2)，振荡器(6)以产生高速细胞分类器。高速细胞分类器应用于分类精细胞细胞时，可实现大约每秒500次分类以上的分类速率。事实上，通过高速细胞分类器已实现了1000至1200次的分类速率。重要的是，应理解“高速”是一个相对的术语，随着流式细胞计数术的发展和实际应用，“高”是可变的或保持不变的。每种定义的一般原则是，当对特定的细胞，如精细胞进行分类时，分类是以流式细胞计数器的参数和物理性质对细胞本身有意义的速率进行的。
用流式细胞计数器分离技术分类精细胞时起作用的高速分类的一个方面是在流式细胞计数器内精细胞所受到的压力。例如，当高速运转时，流式细胞计数器可以每平方英寸50磅、60磅，甚至更高的压力运转。这样的压力认为是高的，因为对被分类的细胞可产生影响。对于这一方面本发明的关键技术在于特定细胞的压力阈值是决定的因素。而且，随着获得进一步的知识，将表明压力阈值是综合效应的函数如特定物种或对细胞进行的特定的预处理或后处理。关键是事实上细胞所受的压力改变了其活力和达到所需结果的能力。对于压力，当对精细胞施加更高压力时，可能导致流式细胞计数器分类细胞的效果下降。本发明使这些压力降到最小，从而得到了下文中提到的更高的效率和更低的剂量。
考虑到细胞的压力方面，本发明以使压力最小化的方式进行。可在整个周期中或在收集、分类甚至给动物授精的过程中的任意时间点这些压力可被最小化。重要的是，在流式细胞计数器内处理细胞而产生的压力对于这种应用似乎是明显的。在本发明的一个实施方案中，特别选择了外鞘液使其与分类前后的两种细胞液体环境(或之一)的压力一致。尽管可自然调节分类前或者分类后的液体，但在本发明的一个实施方案中调节外鞘液(3)，使对细胞产生的压力比以前产生的明显更小。在一个方面，本发明的意义在于它将全部注意力从对流式细胞计数器操作转移到处理和转移细胞本身的压力上。例如，尽管已知使用具有合适pH值或渗透压的液体，但本发明认识到可能存在对细胞反应过强的化学成分。可根据细胞或甚至在细胞处理前自然改变这些反应过强的化学成分。重要的是，目前对于精细胞，似乎某些代谢的化学成分，如柠檬酸盐可防止对细胞产生异常高的压力。因此，反应过强的化学成分可定义为在其功能性环境中和随后存在的处理技术中对细胞存在特定反应的成分。对于精细胞，似乎代谢成分，特别是柠檬酸盐对牛精细胞的稳定性和HEPES缓冲液对马精细胞的稳定性非常重要。因此，本发明通过选择操作类型或者选择使细胞经历的变化最小的物质，使细胞的改变最小。
根据本发明的一个实施方案选择一种物质使外鞘液在化学上协调使引起的变化最小。因此，经过选择合适的外鞘液，不仅流式细胞计数术的参数中，而且细胞参数的本身均改善了细胞的变化和分类的总体结果。图3中概念性地显示了这一点。图3显示了存在于该方法的各个时期的一些化学因素类型(例如柠檬酸盐或其它因素)，例如，显示的四个时期代表流式细胞计数术分离技术所显示的下列时期，但不受其限制：I期代表细胞源(1)内存在的细胞，II期表示在外鞘液环境中分类时存在的细胞，III期表示分类后收集的细胞，IV表示分类后在储存培养基中重建的细胞。现有技术所表示的这四个时期可以经历区别极大的化学因素环境。然而，如图所示，在本发明中细胞经历了极小的变化，最显著的是没有I和II期之间经历的溶液或液滴。这是选择合适的外鞘液的结果。因此，由于使用了合适的外鞘液，本发明的细胞受到的压力更低。
对于该现象可能存在的一个潜在的普遍性是某些化学成分比其它成分具有更高的敏感性。尽管根据精子的种类、操作方式，甚至是细胞的类型这种敏感性可自然变化，但用于所需目的(本文是人工授精)时细胞的活力改变极大，这种改变可以是自然的或者由于分类引起或两者都有，因此，细胞对该化学成分表现出敏感性。通过选择某些代谢的化学成分，最显著的是柠檬酸盐或柠檬酸循环中的化学物质，可出现极大的改进。因此，在牛精子应用中，选择并调配外鞘液(3)使其含有大约2.9％的柠檬酸钠成分。具体地说，2.9％的柠檬酸钠溶液制备方法如下：
1、将29.0克柠檬酸钠二水合物(Na3C6H5O7·2H2O)放入1000ml烧瓶中；
a将柠檬酸钠溶于3/4体积的水中，然后加水至总体积；
2、加入去离子水或纯净水至1000ml终体积；
3、转移到瓶中并在15 lbs压力下高压灭菌(245°F)至少30分钟；
a在使溶液最少蒸发的条件下进行高压灭菌(未密闭时)；
b注意使水不沸腾掉；
4、在室温下慢慢冷却；
5、在5℃的冷环境中密封储存；
另外，对于外鞘溶液，可过滤柠檬酸钠溶液。
6、采用无菌技术用0.22微米滤器过滤。
有趣的是，对于马精细胞，上述溶液不合适。另外，已发现对于马精细胞，HEPES缓冲培养基，如HEPES牛配子培养基，特别是以前由J.J.Parrish为应用于牛而制备的HBGM3较合适。该培养基在文章“用肝素保存牛精子”，生殖生物学38，1171(1988)中进行了讨论，本文作为参考文献引用。这不仅是令人惊奇的，因为它与用于牛精子的物质不是相同的物质类型，而且实际上该缓冲液最初是为应用于牛而研制的。因此，用在马精子中，选择的外鞘液含有HEPES缓冲液。该溶液在室温下PH值约7.54(39℃时pH＝7.4)，其成分如下：
化学物质      干重(g/500ml)
CaCl2         0.145
KCl2          0.115
MgCl2·6H2O   0.004
NaH2PO4·H2O  0.018
NaCl          2.525
丙酮酸钠      0.011
乳酸(60％)    1.84ml
HEPES         4.765
NaHCO3        0.420
BSA(V部分)    3.0
可对本发明造成影响的另一方面是所用的细胞可能具有异常敏感性。一种考虑是，这可能是由于精细胞是一类不能修复的细胞。即，它们没有自我修复能力，因此，处理时它们可能需要比流式细胞计数器或其它处理装置处理一般细胞时更敏感。因此，当流式细胞计数器或其它分离装置建立精细胞源时，应当进行改进。精细胞这类细胞特有的另一潜在的相关方面是其DNA不能修复、不能复制且不能转录。上述每一种因素都可能起作用或单独或同时造成影响。因此，本发明的技术可应用于所有配子细胞或甚至是病毒及类似的不能修复、不能翻译、不能转录的细胞。
用流式细胞计数器处理重要方面是分类后应采用适当的化学和物理方法处理细胞。如图4所示，液滴(8)内的细胞进入收集器(14)时，重要的是收集器的容器大小要适当，以避免细胞和容器本身之间的影响。尽管已知将起始收集器液体(17)放入容器的底部，收集细胞时就不会冲撞容器的底部，但也可使用简单地加宽容器的方法起到这种作用，因为这样可以改变液流出现及细胞冲击容器时不可避免出现的飞溅。这样就起到了缓冲的作用，可以恰当地处理细胞，因为细胞对机械冲击十分脆弱，即它们受冲击易破裂或损伤。因此，当细胞计数器细胞源分离出细胞时，若被分类的细胞物事性质脆弱，重要的是提供某种缓冲因素，如加宽的收集管，其开口(18)宽敞以避免容器壁与细胞接触。这样，收集管的管壁不会靠的太近，被分类的细胞与管壁之间就不会存在任何明显接触。所以，除了要有收集器液体(17)外，还可以用粗的收集管。也许仅仅提供宽开口的容器用作收集器的部件就已足够。用于高速分类精细胞时，已发现开口内径至少15毫米的容器是足够的。特别是在该应用中使用14ml Falcon收集管时，发现该收集器(14)对细胞具有最小的物理损伤。
应注意即使是14ml Falcon收集管可能也不是最佳的。具体地说，据认为设计适合液流几何形状的收集容器(即“液流适配容器”)可能是最佳的。这种液流适配容器可具有任一或全部下列特征：相对较宽的喷嘴口，椭圆性的喷嘴口，高与宽的比率比现有技术的更小，成一定角度或与液流方向协调，例如侧壁与下落的液流平行等。还需要提供一种支架部件，如可动部件或类似于滚珠轴承的工具等，可以使收集管变化方向适于收集所需的下落的液流。另外，这类容器，例如，其中的收集管(以“Falcon-型”收集管为例)不仅要求有一定宽度，而且对制成收集管的材料也有要求(如用不粘附细胞的聚苯乙烯)等。(已知14ml Falcon收集管用该材料)。因此，该容器和其收集液对减小细胞的物理损伤也起到了缓冲作用，其大小也有利于收集足够的精子而无明显的稀释效应。
收集液(17)的另一作用是减小对细胞的化学压力。由于分类前和分类后给细胞提供营养是重要的，因此可选择同等营养水平的收集液使分类前和分类后的营养平衡。对于牛精子，使用含2％卵黄的卵黄柠檬酸盐作为营养成分，已发现使用6％的卵黄柠檬酸(即在柠檬酸盐溶液中有6％的卵黄含量)效果很好。这是因为细胞分类前后体积不同。收集器液体(17)开始(分类前)体积约为2ml。细胞分类后可增加大约2倍的体积(最终是起始体积的3倍)，溶液中卵黄柠檬酸盐极少(由于阻塞流式细胞计数器的其它原因)。因此，卵黄柠檬酸盐含量水平的最终结果相当于起始结果，即按体积在柠檬酸盐溶液中有2％的卵黄含量。因此选择收集液(17)的最佳营养成分应与起始的营养成分或其它液体环境平衡。这种方式使细胞分类的时间最短，液体成分改变最小。自然，这种液体环境可在流式细胞计数器内提供或在分类前提供给细胞，重要的是使细胞在其生命周期中任意时间所受的压力都最小。而且，由于可改变化学物质的起始含量(例如，可增大或减小柠檬酸盐中的卵黄含量百分数)，同样也可改变起始收集液环境或体积使最终结果相同。因此，在开始分类前，收集器液体为含6％的卵黄的柠檬酸盐溶液，分类后，含有性别特异性精子的收集器液体是含2％卵黄的柠檬酸盐溶液，与起始营养成分含量相当。
应注意由于其它原因后来的使用中可将这些精细胞在有20％卵黄含量的柠檬酸盐溶液中处理，但这些改变并不对细胞产生压力，因为它们仅仅是总体授精过程的一个已知部分。本领域的技术人员容易理解可自然改变该含量，20％的卵黄柠檬酸盐缓冲液的组成如下；
I最终组成：
80％柠檬酸钠溶液(72mM)
20％(体积/体积)卵黄
II制备1升溶液：
A柠檬酸钠溶液
1将29.0克柠檬酸钠二水合物(Na3C6H5O7·2H2O)放入1000ml烧瓶中。
2加入去离子水或纯净水至1000ml终体积。
3转移到瓶中并在15 lbs压力下高压灭菌(245°F)至少30分钟：
a在使溶液最小蒸发的条件下(覆盖不严时)进行高压灭菌；
b注意使水不沸腾掉。
4在室温下慢慢冷却。
5在5℃的冷环境中密封储存。
B鸡蛋的准备
1采购新鲜鸡蛋。
2洗去鸡蛋的脏物(不用过多去污剂)并漂洗。
3将鸡蛋浸入70％乙醇中2-5分钟。
4取出鸡蛋晾干(或擦干)，置于干净的毛巾上。
C制备扩充液
1使用无菌，干净的玻璃制品。
2A-组分(无甘油组分)
a将800ml 2.9％柠檬酸钠溶液放入1000ml量筒中。
b扩充液无甘油组分(A-组分)的抗生素含量为：
i泰乐菌素＝100μg/ml
ii庆大霉素＝500μg/ml
iii林可霉素-奇霉素＝300/600μg/ml
c加入200mlD部分新制备的卵黄
i彻底混合
d这样提供了以2.9％柠檬酸钠，20％卵黄和抗生素为基础的A-组分扩充液，(已知抗生素浓度对牛精子无毒性)。
e扩充液可在5℃储存过夜。
f第二天倾析上清液(800ml以上)。
g第二天用前加温到37℃。
D向缓冲溶液中加入卵黄，可使用下列程序。
1洗涤鸡蛋并使其干燥(见上面B)。
2打开鸡蛋并用卵黄分离器分离卵黄和蛋清。或者，可在两个打开的半壳中来回倾倒卵黄。不要使卵黄周围的膜破裂。
3将卵黄放在15cm的无菌滤纸上。
4将滤纸片放到含有缓冲液的量筒上并挤压卵黄(使膜破裂)使卵黄过滤进量筒中。一般一个鸡蛋可获得大约12-15ml卵黄。
可在本发明的各种因素中互相影响的另一方面是使用低剂量的精子进行人工授精等。在Rupert P.Amman和George E.Seidel，Jr.，Colorado联合大学出版社(1982)的“哺乳动物精子分类的前景”中可发现关于选择性别的人工授精的其它背景。正如所述，自然授精涉及的精子数为几十亿。目前典型的人工授精，牛用几百万的精子进行，马用几亿精子进行。术语“低剂量”指进行授精比典型的人工授精所用的精子剂量不足其一半，甚至不足其10％的数目。因此，“低剂量”是根据典型的人工授精剂量对比，或者也可以看作是绝对数目小。目前牛的剂量是1百万到1千万精子，低剂量可认为是绝对数目大约500,000精子或者低至300,000精子甚至更低。事实上，通过应用本发明的技术，以100,000和250,000个精子进行的人工授精已显示出成功率较高(怀孕率分别为41％和50％)，正如48 Theriogenology 1255(1997)文章“用极低数目的非冷冻的及选择性别的精子对小母牛进行子宫角授精”所示。由于精细胞似乎显示出对稀释有敏感性，这些结果特别显示了本发明的各种技术互相依赖于使用低剂量的精子样品。绝对数目根据不同物种，对于马，只需约不足二千五百万、或一千万、或五百万，甚至一百万个精子，这被认为是低剂量方法。
另外一个重要方面是用本发明的技术选择性别的精子被用于人工授精系统。因此对于流式细胞计数技术，当收集器(14)用于提供人工授精的精子时，本发明的技术特别适用。而且可将人工授精用途与在低剂量环境中的用途结合起来产生使本发明的各种技术非常协调的协同效应。自然，选择性别的精子不仅在人工授精方式中应用而且在其它技术，例如体外授精等中应用。
用流式细胞计数术或其它分离技术收集、分类和最终给动物授精的方法包括各种步骤。给牛授精时，首先用人工阴道收集牛的精液，每毫升大约15亿个精子。用分光光度计检查干净的精液以测定浓度并通过显微镜检评估以保证满足合适的游动性和活力标准。然后加入抗生素。每次射精产生大约60-70％活跃精子的起始样品。用某种类型的TALP(tyrode白蛋白乳酸丙酮酸)稀释可得到约1亿/ml操作浓度的精子数(用于流式分析)。TALP不仅给精细胞提供营养，而且可使它们在染色步骤中高度活化。在某些物种，如马精液中，染色前可进行离心。根据复染或单染方法进行染色，后者是Johnson的专利和有关技术的主题。也可调配扩充液以形成合适的营养环境同时进行染色。在牛应用中，可在染色后立即在柠檬酸盐溶液中加入大约20％的卵黄成分。而且，在精细胞染色中，已发现使用较高的染色量可达到比预期更好的结果。高浓度染色包括使用数十微摩尔浓度的染色剂，如在下面实施例中所讨论的，使用了浓度为38微摩尔的Hoechst 33342染色剂。
加入染色剂后，培养一段时间，如在34℃下培养1小时以加速染色剂吸收，浓度为每毫升大约1亿个精细胞。然后进行过滤以去掉精细胞团块，随后进行稀释或扩充到每毫升大约1亿个精细胞的所需分类浓度。然后用上述各种技术进行分类，在收集期回收精细胞。如上所述，收集可产生大约含2％卵黄柠檬酸盐的样品(对于牛物种)。离心将样品浓缩到每毫升大约3-5百万个精细胞，之后去掉外鞘液和保护液。然后用20％卵黄柠檬酸盐或康奈尔通用扩充液(Cornell Universal Extender，缩写为CUE)等进行最终扩充。康奈尔通用扩充液每1000ml具有如下组成：
14.5g二水柠檬酸钠
2.1gNaHCO3
0.4gKCl
3.0g葡萄糖
9.37g甘氨酸
0.87g柠檬酸
对于20％的卵黄组合物，可使用800mlCUE和大约200ml卵黄。
最后这次扩充后，得到每毫升3-5百万个精子(牛)。然后冷却该样品以减缓精子的代谢，使其可较长期限内使用。在马物种中，可在输卵管中或按本领域的技术人员均理解的其它授精方法使用该样品。在牛精子中，该样品可被再稀释一次至所需剂量浓度。已发现，稀释可对精细胞活力造成影响，因此可用较少量样品授精，以避免过分稀释。目前不论使用何种分离技术，可获得每0.184ml约300,000个精子的低剂量。而且，需要维持大约5％的精浆浓度，目前已满足了该要求。然后可将精细胞样品放在细管中用于人工授精和运输给待授精的奶牛或小母牛。
为了实现便利的定时人工授精，可使用已知技术同步小母牛或奶牛的发情期，例如根据本领域众所周知的技术使用前列腺素F2α。这种物质特别有价值，已报道它能有效增强母牛的生育力，文章“前列腺素F2α-一种奶牛的生殖药物？”Theriogenology，18，245(1982)在此所为参考。尽管最近的研究结果未强调这一理论，但本发明证实在选择性别的低剂量授精情况下特别有价值。对于牛，通过使用胚胎移植装置将精细胞深深地植入子宫角内可实现人工授精。授精时间可不在典型用于人工授精的峰值时间进行，而晚一些进行，如晚12小时后进行，因为选择性别的人工授精可能在稍晚发生。也可使用胚胎移植装置，因为子宫壁对这种低剂量、选择性别的授精具有较高的敏感性。
还可以结合该技术实现高效繁殖。特别是，现在发明的允许高速分类和选择性别的胚胎低剂量授精的方法也可用于超排卵的动物。使用超排卵药物或任何其它技术可实现超排卵。超排卵药物可直接或间接作用，例如，通过反应链实现高于正常产卵。精子与超排卵结合是令人惊奇的，因为以前认为超排卵阻碍了该结合。在超排卵牛中精子传输受到影响，因此，对动物进行人工授精往往需要进行多次和/或用成倍的剂量。另外，以前确定精子性别的过程相对较慢；因此，测定用超排卵药物(如FSH即卵泡刺激素)处理的母牛一次人工受精后的受精率是有意义的，所用的精子是上述新技术的组合而得到的性别选择的未冷冻的精子，总数仅600,000个。
例如，用如下标准方法使12个Angus杂种小母牛超排卵：在发情期第9天和第12天之间以半天的间隔肌肉内注射6，6，4，4，2，2，2和2mg FSH；第6和第7次FSH注射时同时肌肉内注射25和12.5mg前列腺素F-2α。繁殖力未知的公牛精子用Hoechst 33342染色然后用流式细胞计数器/分类器分类每秒产生每种性别700-800活精子。平均分类纯度为所需性别的89％。经过650g离心10分钟将分类的精子浓缩到3.36×106个精子/ml，冷却到5℃，并储存4小时。然后将184ul装入0.25ml塑料细管中；使用自动侧向开口的胚胎移植鞘在发情后20-24小时将一半的剂量注入各子宫角中。发情期后7或16天以标准的非外科手术方法收集胚胎。在第7天和第16天之间收集的结果相似，分类的X-及Y-精子数量也相似。从9只小母牛回收胚胎。有52个正常发育的胚胎(平均，4.3±5.3个/供体)，13个发育延缓的胚胎和3 1个未受精的卵。使用Y-染色体特异性的DNA序列的引物经PCR测定46个胚胎的性别；43个(93％)为所需性别。尽管该研究只用了少量动物，令人惊奇的是，对排卵过多的小母牛仅用总数为600,000个(活的)选择性别的未冷冻精子授精，得到与常规方法相似的结果。也可对上述方法进行改变，包括通过非流式细胞计数术方法分类精子，以其它方式实现超排卵，以其它方式增加受精等，但不限于这些方法。
而且，根据DNA成分确定精子性别的准确性，高速流式细胞计数器/细胞分类器，以及以低于500,000个的总精子数授精母牛而不影响其繁殖力的方法使得几年内确定牲畜性别的活精液在产业上成为可能。以＞85％的精确率选择性别的精子将具有非常广阔的应用前景。也许最明显的是为得到雌性后代，给一种牛(既产奶又产肉型)授精，为得到肉用雄性牛，使另一种牛(既产奶又产肉型)繁殖出完全不同的公牛类型。上述的一种极重要的牛是用携带X-染色体的精子授精母牛产生雌性牛犊，其难产发生率比雄性牛犊低，主要因为其体积较小。而且，母牛犊出生率的数量优势证明了雄性年轻种奶牛的效率更高。具有85％以上的母奶牛也使经营奶牛成为可能。因为奶牛在其寿命中平均繁殖不超过两只存活牛犊，这是具有吸引力的，因为减少了与妊娠和分娩有关的问题。经营单一性别的肉牛繁殖系统也将成为可能，每只雌性牛用于繁殖并在2至3岁间屠宰，因此，在该系统中大量的营养用于肉牛生长，较少量用于维持种群。性别选择的精液可用于体外授精和对超排卵的奶牛用于胚胎移植。常常是某一种性别的牛犊比另一种性别的更有价值，尽管已有准确定胚胎性别的方法，但既费时，且产生了半数是性别价值不高的胚胎。据推测如果选择性别附加的费用较低且生育力受到的影响很小，精确选择性别的精液将广泛用于牛的人工授精。随着性别选择的精液的出现，人工授精的肉牛会大量增加。
令人感兴趣的是，不在通常植入子宫体内进行授精，而是在子宫角内深处授精可获得更好的结果。还有，由此进一步研究的样品显示在子宫角内深处同侧和对侧授精没有区别。用胚胎移植设备可很好地放入子宫角。由于在同侧和对侧授精的结果没有明显差异，因此本发明人提出在双侧都可进行人工授精，不必去鉴别合适的子宫角。
进行人工授精当然希望繁殖出所需性别的动物。根据上述方法通过使用选择性别的精液就可以达到此目的。还应理解本发明的技术还可应用于其它技术，例如，腹腔镜授精，输卵管授精等。
下面的实验作为实施例。尽管此处未全部描述本发明的每个方面，但通过介绍本发明不同方面的内容，确实显示出效果改进，而且，一些实验小结已写入参考文献“用极少量的未冷冻且选择性别的精细胞对小母牛进行子宫角授精”中。该文章归纳的一些数据表明了本发明的效果。关于实验，如下所述，用选择性别的、未冷冻的精细胞进行的人工授精具有较高的成功率：
实施例1
用25毫克前列腺素F-2α以12天的间隔给月龄13-14，中等大小的Angus母牛用药，使其同步发情，并且在观察到出现发情期后6-26小时授精。将从3个14-26个月龄的公牛新收集的精液加入到38微摩尔/升的Hoechst33342中，在TALP培养基中以75×106个精子/毫升在34℃温育1小时。使用在50psi下操作的
Figure S200810128056XD00191
流动细胞计/细胞分类仪，并且用2.9％柠檬酸钠作为外鞘液，在来自150mW下以351和364纳米的激光激发的落射荧光的基础上分类性染色体而分类精子。以约500个活精子/秒的速度收集带X染色体的精子(经再分类超声波处理的精子等分试样证实，纯度约90％)至2毫升含100微升Cornell UniversalExtender(CUE)和20％卵黄的Eppendorf管。将收集的精子以600×g离心10分钟，并且在CUE中再悬浮到1.63×106活精子/毫升。对于液体精液性别未分类的对照，将Hoechst 33342染色的精子用外鞘液稀释到9×105精子/毫升和离心并且在CUE中再悬浮到1.63×106个游动的精子/毫升。将性别选择的精液和液体对照精液在75分钟之内冷却到5℃，加载到0.25毫升的细管中(184微升/细管)。将细管置于3～5℃控制温度的液体冷却器中运送到240km之外，用于在分类后5到9小时授精。用侧开口的蓝鞘(IMV)进行性别选择的精液和液体对照精液授精，每个细管的一半进入子宫角(3×105活精子/母牛)。作为标准对照，将来自同样的公牛的精液在0.5厘米的细管中用标准方法冷冻(解冻后平均15.6×106个能动精子/剂量)，在35℃解冻30秒，并且授精到子宫体中。2个授精者轮流用3只公牛精液进行操作，对于性别选择的精液和两种对照精液以3∶2∶2母牛数的比例授精。在授精后31-34天用超声波确定受孕，在64-67天可分辩出胎牛性别时进一步证实。数据表示在表中。
 处理     母牛数  31-41天受孕数  64-67天受孕数   雌性胎儿数
 性别选择的精液     45  20(44％)  19(42％)   18(95％)a
 液体对照     28  15(54％)  15(54％)   8(53％)b
 冷冻对照     29  16(55％)  15(52％)   12(80％)c
a、b间具有显著性差异(P＜0.02)。
虽然性别选择的精液的怀孕比例只是对照组的80％，这一差别不具有统计学意义(＞0.1)。性别选择和冷冻对照组中的受孕数在64-67天时各减少了一个；在性别选择组中19个胎牛中有18个是雌性(95％)，而对照组中30个胎牛中仅有20个是雌性(67％)。液体精液对照组怀孕率与含有高50多倍能动精子(总精子超过120倍)的冷冻精液对照组有实质上相同的怀孕率，证明了在子宫角中低剂量授精的效率。利用流式细胞计技术和人工授精，我们已经明显改变了牛的性别比例。
同样，用来进行性别选择的非冷冻精细胞进行了实验，报道如下：
实施例2
实验目的是在理想的实地条件下，确定用极低数目的冷冻精子对母牛授精时的受孕率。将生殖力高于平均数的三只Holstein公牛的精液加入匀浆的牛奶、7％的甘油(CSS)扩充液中，再加5％的同源精液浆液中扩充，制成每0.25毫升French细管中含2×105、5×105、或10×106(对照)总精子的三种授精样品，然后在流动的液氮蒸发器中冷冻。在37℃水中解冻精液20秒。利用25毫克前列腺素F-2-α(
Figure S200810128056XD00211
)在12天的间隔注射月龄13-15，体重350-450公斤的Holstein母牛两次，在检测到发情后12或24小时，利用胚移植细管枪和侧开口的鞘授精，将一半精液深入到每个子宫角中。在5个月中重复实验5次，在两个授精技术人员交替进行。孕育的环境温度往往是-10到-20℃，所以必须小心保持授精仪器温暖。在发情后40-44天超声波检测到能成活的胎牛确定受孕，在发情后55-62天进一步确认，在这段时间202个受孕数少了4个。55-62天时每次授精2×105，5×105和10×106个总精子的受孕比例是55/103(53％)，71/101(70％)，和72/102(71％)(P＜0.1)。与母牛在公牛中自然怀孕的比例是不同的(P＜0.05)(59，62和74％)，但两个技术人员进行的操作结果无差别(64％和65％)，在发情期后的不同时间授精，结果也无差别(12小时65％，24小时64％，每次N＝153)。用上述方法，每次授精用5×105和10×106总精子，母牛怀孕的比例是相同的。
对已选择性别、未冷冻的精子细胞也进行了实验，报道如下；
实施例3
将从大西洋育种公司收集的公牛精液，用HEPES缓冲的扩充液+0.1％BSA以1∶4比例稀释，约用2小时运到160km处的Beltsville，Maryland，在那里用前述的方法，在环境气温下用流式细胞计数仪进行分类至TEST生产的20％扩充液中(Boil Reprod 41：199)。每种性别，每5-6小时分类速度高达2×106个精子，纯度约为90％。经离心(300克4分钟)浓缩精子到2×106精子/毫升。将一些精子分类到含有同源精液浆液的扩充液中(终浓度为5％)。空运分类的精子到克罗拉多(2,600km)，并且分别在大气温度或5℃储藏(运载6小时中用Equitainer冷却，即一种带有冰室的隔热装置)。在精子分类后9到29小时内对发情11到36小时授精检测到的小母牛或无奶母牛进行授精。将精子(0.1毫升中1到2×105)置于授精时超声波确定的最大卵泡的卵巢一侧的子宫角的深处。
用运送并储藏在大气温度的精子授精，10只母牛中没有一个受孕。用运送过程中冷藏在5℃的精子授精，29只母牛在第4周有14个受孕，第8周12个(41％)受孕。分类后10小时内授精的22只母牛，8周时受孕11只，但在分类后17到24个小时授精的7只牛中只有1只怀孕。加入精液浆液没有明显的效果。在怀孕的60-70天时超声波成象确定，12个胎牛中只有1只不是预期的性别，一只性别不清楚，10只是预期的性别。
随后，将0.05毫升上述扩充的精液植入另外33个母牛子宫角；在授精后4个星期只有3个授孕，8周时只有一只仍怀孕(未用超声波成象确认)。但是，用前面组的不同公牛，所有授精在分类后18到29小时进行。用另一只公牛的精子对距我们实验室200km处的另38只母牛进行了类似的人工授精，授精在分类后约22小时进行。这些牛没有一个在授精后8星期怀孕。
总之，用经流式细胞计数进行性染色体分类的精子进行人工授精，使牛怀孕是可能的，并且胎牛的性别比例接近于90％再分析分类的精子的DNA含量预测。但是，在这些需要长距离运送精子的初步实验中受孕率变化很大。分类这17小时后生殖力明显下降，但是其中有一些混乱，因为不同时间用了不同的公牛。需要进行进一步研究，以确定所观察到的受孕率的变化是否由于公牛的不同、授精技术、精子分类与授精时间的间隔或其它原因造成的。
最后，用未进行性别选择并未冷冻的精子细胞进行了实验，报告如下：
实施例4
实验目的是确定在理想的实验条件下，用极低数目精子对母牛进行授精的受孕率，来自三个Holstein公牛的精液加入到Cornell UniversalExtender，加5％的同源精液浆液，扩充到每0.1毫升中1×105或2.5×105个精子；用每0.25毫升2.5×106个总精子的精液作对照。将扩充完全的精液装入改进的0.25毫升塑料French细管，可每次释放出0.1或0.25毫升授精剂量。将精液冷却到5℃，在收集后26-27小时使用。每次25毫克前列腺素F-2α(
Figure S200810128056XD00221
)以12天的间隔注射月龄13-15，体重350-450公斤的Holstein小母牛，用胚移植细管枪和侧开口鞘在检测到发情后24小时在一侧子宫角授精。授精在与发情后12小时超声波确定的最大卵泡同侧；在发情后7-9天超声波检测到黄体证实了排卵的一侧。在发情后42-45天超声波检测到胎牛确定怀孕。实验重复了四次，由三个授精技术人员轮流进行。在225个小母牛中有205个准确地确定了排卵侧(91％)；惊人的是，同侧和对侧授精怀孕的比例几乎相同。对于1×105，2.5×105和2.5×106个精子/授精(P＞0.1)怀孕比例是38/93(41％)，45/87(52％)，和25/45(56％)。不同技术人员操作，母牛怀孕比例有明显的差异(P＜0.05)，但在用不同公牛精液没有差异。用上述方法，减少每次授精的精子数，使用流式细胞计数仪分类的精子用于商业是可能的。
从上述各种实验可以看出，实施结论根据实地结果统计得出。因此还可设计一些实验方案，对上述实验方案进行适当的综合与限制。这样，可以使各种影响因素起到进一步协同作用。例如，可以研究染色剂和与激光激发结合的染色剂的影响。
本发明申请所讨论的只是基本的说明。读者应理解特定的实验并不能清楚叙述所有可能实施的方案，本发明这些实验包含了许多可以替代的情况。本申请也没有完全解释本发明的全部性质，不可能明确显示每个特征或要素如何代表了更广的功能范围或多种可替代或作用相同的要素。实际上，这些都包含在本发明中。在本发明着重叙述某设备时，该设备的每个部件都包含着一种功能。有关设备的权利要求不仅包括设备本身，也包括方法、步骤以说明本发明及每个部件的功能。
说明及术语都不限于权利的范围，应理解在未脱离本发明的精神下可作各种改变，这些改变都包含在说明书中，仍落入本发明的范围。本发明公开的范围既包括了直接的实施例，大量可替代的隐含的实施方案，也包括了方法和步骤等。
此外，本发明中的每一部分也可用多种方法去实现。应理解为包括每种改变，任一设备方案的改变、方法的改变，或这些要素中的任一改变。尤其是，在本发明公开的申请文件中，每项内容都可以用意义相同的仪器术语或方法术语表示，即使仅有功能或结果相同。这种意义相同，范围宽甚至更概括的术语应认为包含于说明书中每项内容中。在本发明所包括的广义的范围中需要明确之处，可以替换这些术语。应该理解，一切行动都可以用完成这个行动的手段或引起这个行动产生的因素来表示。同样，所公开的每项实际内容应理解为包括了该实际内容所导致产生的行动。例如，公开了“收集器”，应该理解为包括了“收集”这个行为，无论文中是否已直接讨论过这种行为。反之，如果仅公开了“收集”，应理解为也公开了“收集器”。这种变化和可替代的术语应理解为已清楚的包括在说明书中。
另外，应该理解，除了最初提出的权利要求，权利要求可扩展为更广泛的范围，至少：i)如本文公开和叙述的装置，ii)公开和叙述的有关方法，iii)与每个装置和方法相似的、相同的和暗含变化的装置、方法，iv)完成公开和叙述的每个功能的那些可取代的设计，v)隐含了完成公开和叙述的每个功能的那些可取代的设计和方法，vi)分开和独立的发明所示的每个特征，组成和步骤，和vii)上面每项的各种结合和变换。
为有助于理解本发明，各种公开的参考文献可能是有帮助的。这些参考文献在下面列出了，并且引入本文作为参考；但是，在叙述与这些发明的专利不一致的情况下，可以认为这些陈述明显不出自该申请人。可能有帮助的参考文献包括：美国专利5660997；5589457；5514537；5439362；5346990；5135759；5021244；4999283；4749458；4688142；4680258；4511661；4448767；4362246；4339434；4276139；4225405；4192749；4155831；4092229；4085205；4083957；4067965；4009260；3894529；3687806；RE32350。有帮助的参考文献可以包括下面出版物：“利用非常少的非冷冻精子授精Holstein小母牛”G.E.Seidel，Jr.，C.H.Allen，Z，Brink，J.K.Graham，和M.B.Cattell，克罗拉多州立大学，Fort Collins，大西洋育种公司，Lancaster，PA.，DUO Dairy，Loveland，CO.1995年7月；“用X和Y小牛精子人工授精”，G.E.Seidel，Jr.，L.A.Johnson，C.A.Allen，G.R.Welch，M.D.Holland，Z.Brink和M.B.Cattell，动物繁殖和生物技术实验室，克罗拉多州立大学，Fort Collins，CO；Germplasm和种质和配子生理学实验室，ARS，USDA，Beltsville，MD；大西洋育种公司，Lancaster，PA；DUO Diary，Loveland，CO，USA 1996年1月；“利用非常少的冷冻精子授精小母牛”G.E.Seidel，Jr.，C.H.Allen，Z.Brink，M.D.Holland，M.B.Cattell，克罗拉多州立大学，Fort Collins，大西洋育种公司，Lancaster，PA，DUO Dairy，Loveland，CO，1996年7月；“利用流式细胞计分类和未分类的精液低剂量子宫内授精繁殖山羊”，D.G.Cran，W.A.C.McKelvey，M.E.King，D.F.Dolman，T.G.McEvoy，P.J.Broadbent和J.J.Robinson，Mastercalf，Craibstone，Bucksburn，Aberdeen，AB21 9TN，UKScottish农业学院，Craibstone，Bucksburn，Aberdeen。AB21 9YA，UK，兽类遗传学，第267页；“利用非常少的非冷冻和性别选择的精子子宫角授精小母牛”，G.E.Seidel，Jr.，C.H.Allen，L.A.Johnson，M.D.Holland，Z.Brink，G.R.Welch，J.K.Graham和M.B.Cattell，克罗拉多州立大学动物繁殖和生物技术实验室，大西洋育种公司，Lancaster，PA 17601，种质和配子生理学实验室ARS，USDA，Beltsville，MD20705，DUO Diary，Loveland，CO 80538，兽类遗传学48：1255-1264，1997；“利用Hepqrin的是小牛精子获能”，J.J.Parrish，J.Susko-Parrish，M.A.Winer，和N.L.First，威斯康星大学肉类和动物科学系，麦迪生，WI53706，  繁殖生物学38，117-1180(1988)；“前列腺素F2a-一种乳牛的繁殖药物？”，K.L.Macmillan和A.M.Day，Ruakura动物研究站，Private Bag，Hamilton，新西兰，兽类遗传学，1982年9月，18卷，第3期，245-253页；“选择性别的哺乳动物精子的远景”，克罗拉多联合大学出版社，克罗拉多州立大学，兽医和生物医学科学动物繁殖实验学院，Fort Collins，CO，80523Rupert P.Amann和George E.Seidel，Jr.，编辑，1982年；“卵黄柠檬酸盐和牛奶扩充液对冷藏公牛精子的染色质结构和存活率的影响”，乳品科学杂志74：3836，D.S.Karabinus和D.P.Evenson和M.T.Kaproth；“在流动细胞计分类细胞过程中测定公羊和公猪精子”，Reprod.Dom Anim32：251；“利用限量pLH补充的纯化pFSH进行山羊超排卵”，兽类学，43：797，M.A.Nowshari，J.F.Beckers和W.Holtz；“综述：在哺乳动物中预选择性别”，Dtsch.tierarztl.Wschr.103：285，L.A.Johnson。
在整个说明书中，除非本文需要，词“含有”或“包括”、“包含”，将理解为包括所述的部分或整体，或包括了几个部分、几个整体的组合，但并不表示排斥任何其它部分、整体或部分、整体的组合。

Claims (11)

1.产生性别选择的非人类胚胎的方法，包括：
a.收集一种雄性非人类哺乳动物的精子细胞，所述精子细胞为牛精子细胞；
b.测定所述雄性非人类哺乳动物精子细胞的性别，该测定过程包括以下步骤：
i.建立一种提供待分类的所述细胞的细胞源；
ii.建立一种用于所述细胞的外鞘液；
iii.对所述外鞘液进行化学配制以创造一种所述细胞的外鞘液环境，使分类前后所述细胞的液体环境一致，其中所述外鞘液包含柠檬酸盐；
iv.检测所述细胞的性质；
v.辩认出所需性别的所述细胞；
c.根据所述精子细胞的性别分类所述精子细胞；
d.在体外用性别分类的精子使多个卵受精，
其中所述性别选择的非人类胚胎不是体内胚胎。
2.权利要求1所述的产生性别选择的非人类胚胎的方法，还包括：
a.将所述雄性非人类哺乳动物的精子细胞染色；
b.用高速流式细胞计数仪根据所述精子细胞的性别分类细胞；
c.浓缩所述已分类的精子细胞。
3.权利要求1所述的产生性别选择的非人类胚胎的方法，其中将所述性别选择的胚胎冷冻以用于运输。
4.权利要求1所述的产生性别选择的非人类胚胎的方法，还包括以高速分类所述细胞。
5.权利要求1所述的产生性别选择的非人类胚胎的方法，其中所述精子细胞对在外鞘液环境中的化学成分是反应过强的。
6.权利要求1所述的产生性别选择的非人类胚胎的方法，其中所述使多个卵受精的步骤使用低剂量的性别分类的精子。
7.权利要求6所述的产生性别选择的非人类胚胎的方法，其中所述低剂量的精子包括低于典型剂量的10%的剂量。
8.权利要求6所述的产生性别选择的非人类胚胎的方法，其中所述低剂量的精子包括低于500,000精子的剂量。
9.权利要求6所述的产生性别选择的非人类胚胎的方法，其中所述低剂量的精子包括低于300,000精子的剂量。
10.权利要求1所述的产生性别选择的非人类胚胎的方法，其中化学配制的外鞘液包括包含2.9%柠檬酸钠的溶液。
11.权利要求1所述的产生性别选择的非人类胚胎的方法，其中所述对所述外鞘液进行化学配制以创造一种所述细胞的外鞘液环境，使分类前后细胞的液体环境一致的步骤还包括建立一种含有2.9%柠檬酸钠的外鞘液。
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CN101502450B (zh) 2012-08-22
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AR057750A2 (es) 2007-12-12
US20130150663A1 (en) 2013-06-13
US6149867A (en) 2000-11-21
US20070026379A1 (en) 2007-02-01
US20130149737A1 (en) 2013-06-13
US6524860B1 (en) 2003-02-25
US9365822B2 (en) 2016-06-14
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US7195920B2 (en) 2007-03-27
US9422523B2 (en) 2016-08-23
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US20070099260A1 (en) 2007-05-03
US20030129091A1 (en) 2003-07-10
CN101504405B (zh) 2016-02-17
UY25333A1 (es) 1999-07-19
CN101504407B (zh) 2013-09-11
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