CN100581355C - 精子的保藏和处理系统 - Google Patents

精子的保藏和处理系统 Download PDF

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CN100581355C
CN100581355C CN03821753A CN03821753A CN100581355C CN 100581355 C CN100581355 C CN 100581355C CN 03821753 A CN03821753 A CN 03821753A CN 03821753 A CN03821753 A CN 03821753A CN 100581355 C CN100581355 C CN 100581355C
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威廉·马克斯韦尔·奇泽姆·马克斯韦尔
费安娜·凯特·霍林斯墨德
贾斯蒂娜·凯利·奥布兰
加雷斯·埃文斯
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Abstract

本发明公开了精子的保藏和精子的其他处理。在一些实施方案中,本发明涉及精子的保藏,而精子的保藏可能进一步涉及人工授精、体外授精、超数排卵、哺乳动物的生产、通常的精子样品和精子处理。在一些实施方案中,本发明还涉及精子的处理特征,例如收集、处理、分选、储藏、运输、使用、受精或者授精技术。根据本发明,精子的保藏可以在于保持和改进精子质量,并且,在一些实施方案中,在于保持和改进一个或者一个以上的精子特征,例如生存力、运动力和功能性。受精率也得以保持和提高。一个实施方案可包括获取精子(10)。可将精子深低温保藏(20)和解冻(30)。可对精子进行处理(40)并对精子进行深低温保藏(50)。

Description

精子的保藏和处理系统
相关申请的交叉引用
本申请要求申请号为60/410,884,申请日为2003年9月13日的美国临时申请和申请号为10/340,881,申请日为2003年1月9日的美国非临时申请的优先权,在此将每个申请以参考的方式引入本文。
技术领域
本发明总体上涉及精子的保藏和精子的处理。本发明可应用于人工授精、体外受精、超数排卵、哺乳动物的生产、精子样品和精子的处理,所述处理包括收集、加工、分选、贮藏、运输、使用、受精或授精等特征。
背景技术
为了试图提高牲畜的生育率,已通过诸如人工授精或体外受精等技术来使哺乳动物的卵(有时指卵母细胞)受精。而且,随着安全而可靠的分选方法的发展,如普通或专门将精子分离成富含X染色体群体或Y染色体群体的方法的发展,已在许多种牲畜中有效地实现了性别的预先选择。通常,可以由本文和以下多个专利申请或专利公开的内容实现富含Y染色体精子与富含X染色体精子的分离,以及收集、加工、分选、贮藏、运输、使用、受精或授精等技术,这些技术统称精子和精液的处理;所述专利申请或专利例如:PCT/US99/17165、PCT/US01/45023、PCT/US01/15150、PCT/US98/27909、PCT/US01/45237、PCT/US01/18879、PCT/US00/30155、PCT/US01/02304、美国专利号6,071,689和专利号6,372,422、美国分案申请号10/081,955、美国临时申请号60/400,486和美国临时申请号60/400,971,在此将其以参考的方式引入本文。
尽管过去几年中,在精子的收集、加工、分选、贮藏、运输、使用、受精(统称精子的处理)中所用的各种各样的设备和方法有了很大的改进,但在保持精子的质量、精子的保藏性和刺激性方面,特别是在人工授精(体内)和体外受精(IVF)过程中,仍存在很大的问题,所述精子的质量例如生存力、运动力和功能性。一个可能的结果是受精率的降低。可以直接在体外(例如,在IVF过程中)和体内(例如人工授精过程中)比较精子质量,例如分成富含X染色体和Y染色体的精子的生存力,而在传统的精子处理中,精子质量通常都降低。更笼统一些,用于精子的生存力、运动力、功能性、保藏、刺激、受精和授精的传统处理技术可能没有产生较好的或足够的例如受精率或精子质量。
作为传统精子处理的一个例子,例如,在诸如精子的生存力、运动力、功能性等精子质量、受精率以及可能其它的有限特性或特征方面,用于繁殖哺乳动物的精子分选技术是受到限制的。例如,如果分选仪器与雄性个体,或者与需要用处理后的精子进行处理的待授精雌性受体或者待受精的卵距离较远时,精子的质量、受精率及其它特性或特征可能会受到负面影响。例如,当在将精子运输到分选仪器时可以使用一些以参考的方式引入本文的技术以及一些传统的技术进行保藏,这可以一定程度上获得精子的生存力、运动力、功能性以及理想的受精率。然而,特别是在任何一个精子的处理步骤中或多个步骤的组合中,很好地获得精子质量和其他特征的技术,可能是比较理想的,所述精子质量例如生存力、运动力和功能性,其他特征例如精子的刺激性、保藏性、保持或提高的受精率。所述处理特征可能包括例如收集、加工、分选、贮藏、运输、使用、受精或授精,特别是从分选仪器到受精地点(可能通过收集的个别样品或“授精管(straw)”)对分选出的精子进行保藏,或在任何其他精子处理阶段中对精子进行保藏。
传统精子处理的另一个局限性的例子是,由于处理相关的类型和程度,传统的处理技术在例如以下方面有些局限性:统称为精子质量的精子生存力、运动力、功能性等、精子刺激性、精子保藏性和受精率。已知的诸如保藏或分选这些处理技术会降低精子的质量、进而降低受精率。在有些需要进一步采取精子的保藏措施的情况下,已证明有精子质量和受精率降低的问题,所述情况例如在分选仪器与雄性个体或者与需要用处理后的精子进行处理的待授精或者待受精的雌性受体较远时。尽管人们或许已经认识到传统的保藏技术是给精子质量、受精率或其它相关特性或结果带来负面影响的因素,但是,迄今为止在精子保藏的过程中,或经确定为与精子保藏相关的过程中,或者总体上的精子处理过程中,没有克服可能确定的精子质量的降低和受精率的降低。
通常认为在对精子和富含精子的精液进行处理过程中,一些附加步骤会导致精子质量的降低和授精率的下降,以至于通常应避免附加的处理步骤。具体地,通常还认为处理过程例如保藏,特别是深低温保藏和分选可能会给精子带来过大破坏,特别是在既涉及深低温保藏,以及涉及分选的连续处理过程中。而且,传统的方法建议并教导:精子保藏,例如在前处理步骤后进行深低温保藏,以便保藏用于后续处理例如体内或体外技术的精子,在精子和技术本身没有损害到使后续处理的结果也受到负面影响的程度的情况下,则不能实施保藏。相应地,这些观点甚至已在传统的处理过程得到证明,并教导了不进行后续的处理步骤如分选和深低温保藏、或具有多步深低温保藏的处理步骤。
发明内容
本发明解决了与降低精子的特性或特征有关的多种以前已经明确的和可能未描述的问题,所述特性或特征为精子的质量(包括生存力、运动力和功能性)、刺激性、保藏性及精子的其他特性。并进一步研究了可能已经过或将经过收集、加工、分选、贮藏、运输、使用、受精或授精中的一个步骤或一个以上的步骤处理的精子以及可能的受精率降低。本发明还可以解决与精子质量有关的问题和可能造成的受精率的降低,所述精子为被分离成富含X染色体和Y染色体群体的精子,其可能为通过流体分选等技术分选出来的精子。更广泛地,本发明可解决在体内人工授精和体外受精中通常出现的授精率低问题,和精子的质量问题如精子的生存力、运动力和功能性等问题。本发明还解决了以下问题并得到了本领域技术人员预料不到的结果,所述问题如刺激性、保藏性、受精率以及提高或保持的生育力特征如胚泡发育率、在体内的发育能力、妊娠率和和细胞分裂。而且,本发明也描述了为获得期望的妊娠率所必须的经处理的精子的数量,以及进一步考虑分别由经分选保藏的精子和未经分选保藏的精子所获得的妊娠率。
自然的,在后续的描述中将会公开本发明的另一些重要目的和目标。
因此,为了达到本发明的多种目的和目标,在一些实施方式中本发明可包括一种处理精子样品的方法,其步骤包括获取精子,将精子深低温保藏、将精子解冻、将精子进行处理、将精子深低温保藏。还公开了相应的生产哺乳动物的方法和哺乳动物及精子样品。
此外,本发明的实施方案还可包括精子的处理方法,所提供的步骤为获取精子、将精子深低温保藏、将精子解冻、将精子进行处理以及将精子深低温保藏。本发明的另一实施方案可包括生产哺乳动物的方法,其包括以下方法步骤:获取精子、将精子深低温保藏、将精子解冻、分选精子、将精子深低温保藏、将精子解冻、给至少一个卵受精,从至少一个受精卵生产哺乳动物。
而且,为了达到本发明的多种目的和目标,本发明的一些实施方案是针对人工授精(体内)和体外受精过程的,也有一些实施方案是针对超数排卵过程的。另外,一些实施方案还可进一步涉及哺乳动物或者哺乳动物胚胎的生产,以及进一步涉及确定精子样品以及精液的处理,所述精子样品如授精管、丸状样品(pellet)、授精剂量或其它制备的精子样品。
发明的另一些实施方案会在本发明的说明书和下面的权利要求书中进行公开。
附图说明
正表A中每一个申请中包括的每一个图均以参考的方式引入本文作为本发明说明书的一部分。
图1是本发明一个实施方案的流程图。
图2是本发明又一个实施方案的流程图。
图3是本发明另一个实施方案的流程图。
图4是本发明一个实施方案的流程图。
图5是本发明另一个实施方案的流程图,在某些实施方案中,可能会结合图1的实施方案一起提供。
具体实施方式
本发明公开了精液和精子的处理和保藏技术,并且公开了保藏、刺激、受精和授精对一个或一个以上的精子特征或精子的质量、刺激性、保藏性和受精率的影响,所述精子质量例如细胞的生存力、运动力、功能性。本发明的实施方案进一步公开了增强或保持的授精特性,例如胚泡的发育率、体外发育能力和妊娠率或者细胞分裂。可以在各种各样的处理技术,例如收集、加工、分离、贮藏、运输、使用、受精或授精等处理技术中研究精子的特征或精子的质量。
精子的质量可能是以前提及的或在这里会进一步提及的精子的各种属性中的一种或多种,如:生存力、运动力、功能性;甚至可能被认为是精子的发育特性中的一种或多种。精子的特征可以指一个或一个以上的精子的生物学、化学、物理、生理学或功能属性等多种特征的一种或多种,例如:细胞携带的染色体的特性,在一些实施方案中可指以前描述的精子的质量。
生育特性能得以增强或保持,所述生育特征可以包括胚泡发育力、体内发育能力、妊娠率或细胞分裂。
精液或精子的处理技术可以指保藏、刺激、授精、受精、分选、选择、分离、解冻中的一种或多种的组合,也可能专门针对收集、加工、选择、贮存、运输、使用、受精或授精技术中的一种或多种的组合。
精子样品可以指含有精子的物质,可能带有精液、液体载体或其他物质,并可包括一个或多个的丸状样品、授精管、授精剂量或其他制备的精子样品。
精子可根据本发明得以保持或改良,在一些实施方案中也得以保持或改良,并且可根据受精及授精技术体现出来,所述实施方案例如精子的保藏,或更宽泛的精子的处理,例如通过在精子分选或精子保藏中的处理步骤。实施方案可以包括精子保藏的几个成分特性或其他的处理技术,这些在以前提及的专利和专利申请中公开过以及在下面会进一步描述,每一个以参考方式明确引用的参考文献均达到与本发明相符的程度。
正如下面会进一步述及的那样,在一些实施方案中精子也能通过保藏和刺激技术得以保持和改良,与前边提及的专利申请和专利中的处理、刺激、保藏、受精和授精技术配合使用也会得以保持和改良。
因此,本发明可以体现为精子的处理方法。参见图1,在流程图中显示一些实施方案中的精子处理步骤。获取精子10并将精子深低温保藏20。然后将精子解冻14,接着进行后续的深低温保藏16步骤。这些特性中的每一个都将在下面进一步描述。图2显示了本发明的另一个精子处理实施方案,其与图1所示特征一致。该实施方案提供了步骤20、步骤22、步骤24和步骤26,这些与图1中的实施方案一致,该实施方案还进一步提供了确定至少一个精子样品的步骤。精子样品可以包括前边述及的任一精子样品,并与下边将进一步描述的一致。
本发明还可进一步体现为生产哺乳动物的方法,这和图1中的实施方案一致,这在图3的步骤30、步骤32、步骤34和步骤36可以很容易的辨别出来。来自于特征36的精子使至少一个卵受精38,并从至少一个的卵中生产出哺乳动物40。这些特性中的每一个同样都会在下面进一步描述。
图4显示了本发明的精子处理的实施方案。如图所示以及对前面描述的特征进行详细表述,可从哺乳动物源获取精子50,既可以直接获取该精子,也可以通过多种方法获取,如通过贮存设备。可将精子深低温保藏52。深低温保藏将在本发明的下面实施方案中进一步阐述。接着,可将精子解冻54。在再次对精子进行深低温保藏58前进一步对精子进行处理56。
总体上的精子处理和前述的过程可以独立于授精和受精技术而完成,或作为这些技术的一部分或与这些技术联合应用。上面过程中的每一个都可以分别在超数排卵技术中实施。因此,如图5所示,本发明的一些实施方案是生产哺乳动物的方法。经上述处理之后,或在一些实施方案中经过特征50至58后,可使至少一个卵受精62。在一个实施方案中,可将精子解冻60,并用精子使至少一个卵受精62。然后由处理后的精子受精的卵中生产哺乳动物64。在一些实施方案中描述了另外一些处理步骤例如精子的分选,优选在第一次解冻54后进行所述另外的处理步骤。
而且,例如如前所述的本发明的实施方案支持体内和体外技术。因此,雌性哺乳动物可以被从它的同类动物所获取的精子授精,所述精子例如前面已提到经处理的精子或经分选和深低温保藏的精子。授精技术可以包括用精子使至少一个雌性动物授精,提供的精子可能是解冻的、或有时是在未解冻的环境中的深低温保藏精子,这些精子可能采用丸状样品或是以前提及的其他精子样品形式。如果例如精子的质量在处理精子或生产哺乳动物中能够得以保持或改良的话,优选解冻后的精子。但是,在授精中也可以使用未解冻的、深低温保藏的精子,而且如果例如精子在雌性体内解冻是可接受的,且如果希望减少子宫外的步骤如精子处理,优选用未解冻的深低温保藏的精子。而且,总的来说可以用未解冻的深低温保藏的精子来完成本发明的人工授精和体外受精技术,所述未解冻的深低温保藏独立于其他处理特征或与其他处理特征配合,所述其他处理特征如解冻,后续的深低温保藏、分选、或本发明公开的其他步骤。也可完成如精子的分选这些处理步骤。
可用如前所述的至少一个精子样品进行授精。可使至少一个卵受精并从该至少一个受精卵中生产哺乳动物。可根据体外过程在体外使至少一个卵受精。可将这至少一个受精卵转移到雌性受体中,可生产哺乳动物胚胎,或在一些实施方案中,从该至少一个受精卵生产哺乳动物。
本发明还包括超数排卵技术。可使某种动物的雌性个体超数排卵,在一些实施方案中,超数排卵的雌性个体可由精子授精,并使至少一个卵受精。在另一些实施方案中,获取超数排卵雌性个体的至少一个卵,并在体外使该至少一个卵受精。在人工授精或体外受精技术中,可将这至少一个的受精卵转移到雌性受体中。
如前所述,精子的处理方法可包括确定精子的至少一个精子样品。因此,本发明的实施方案中可包括与前描述的实施方案相一致的精子处理的方法,如图2所示,包括确定精子的至少一个精子样品这个特征。在一些实施方案中,这确定的至少一个精子样品会成为后续深低温保藏步骤的对象,也就是说将该至少一个精子样品深低温保藏。
由于采用传统的处理技术,以前认为:这些处理的精子和确定的精子样品,不能得到足够的精子质量或受精能力。但是,本发明的一些优选的实施方案表明:如以下多个实施方案所述,精子样品能使雌性哺乳动物的至少一个卵受精;更进一步,用这至少一个精子样品使这种雌性个体授精,并在一些实施方案中,用这至少一个精子样品使雌性哺乳动物的至少一个卵受精。在一些实施方案中,精子样品的受精能力的特性延伸到了使某种超数排卵的雌性哺乳动物受精的能力,以及用至少一个精子样品来使这种超数排卵的雌体个体授精的能力。在一些实施方案中,精子样品的受精能力特性还延伸到了体外使至少一个卵受精的能力,以及用至少一个精子样品在体外使至少一个卵受精的能力。更进一步的实施方案提供确定至少一个能使至少一个雌性哺乳动物授精的精子样品以及用所说的至少一个精子样品使所述的至少一个雌体个体授精的步骤。与精子样品的受精能力和以上所述的其他特征一致,本发明提供大量卵的受精,甚至哺乳动物胚胎的生产,可被转移到雌性受体中的胚胎,以及广泛的哺乳动物的生产,例如哺乳动物的后代的生产。描述精子样品能力的本发明的实施方案可进一步包括利用所述经深低温保藏处理的精子生产至少一个预先选择性别的动物。再次说明,可相应地确定精子样品,使其成为授精管、丸状样品、授精剂量或其他制备的精子样品。
有人会认为前述的每一个实施方案和下面将进一步描述的实施方案偏离传统的精子处理、哺乳动物的生产、哺乳动物胚胎的生产和精子样品的确定。传统认为这些处理精子的方法不能有效地确保精子的质量,或不能提供足够高的授精率、受精率或妊娠率。但是,正如在多种引用的参考文献中所教导的那样,实际上为了以下目的可对精子进行处理,所述目的例如是为了分选精子,可能是去区分精子是含X染色体的精子还是含Y染色体的精子,或在一些情况下对哺乳动物或哺乳动物胚胎进行性别的预先选择。本发明也为这些处理过程,而且在一些优选的实施方案中,提供额外的保藏特性,这些特征是迄今为止传统认为商业上不可行的,或不可能的,并且迄今为止本发明为那些以前已确定但仍未解决的问题提供了解决方案。
迄今为止,精液,特别是精子,可由本发明的哺乳动物获取或者处理,这些哺乳动物包括马科动物、牛科动物、猫科动物、羊科动物(ovids)、犬科动物、水牛、牛、麋鹿、猪或其它哺乳动物种类。更进一步,一些实施方案可提供从珍奇哺乳动物种类、濒危哺乳动物种类、稀有哺乳动物种类、甚至动物学样品或个体中获取和处理精液。并可根据前述及下面详细描述的技术,由此生产哺乳动物或哺乳动物的胚胎。如前所述,在一些实施方案中,确定了精子样品。
例如可以通过冷冻将精子样品深低温保藏,深低温保藏可以使用多种保藏技术,例如用Hepes(N-2-羟乙基哌嗪-N’-2-乙烷磺酸)缓冲的冷藏稀释剂(crydiluent)冷藏。精子可以丸状样品或授精管的形式提供,而且例如公羊精子可用多种解冻技术解冻。可在对精子进行处理的过程中提供其它精子样品,并且当该精子样品被确定为精子样品时或将其用于授精或使卵受精时可以经深低温保藏也可以不经深低温保藏。
可以在精液、精子或精子样品中单独加入或联合加入一种或一种以上的添加物。在一个实施方案中,可向精子样品中加入冷藏稀释剂以用来保藏精子。向精子样品中加入一种或多种添加物,在一些深低温保藏步骤或具有深低温保藏(有时称冷冻)步骤的实施方案中,所述添加剂例如为冷藏稀释剂。添加物的加入可以保持或改良精子,可进一步保持或改良精子的质量,所述质量例如精子的生存力、运动力和功能性,和可保持或刺激性、受精率及其它一个或一个以上的可能的精子特性。在本发明的另一些实施方案中,可以从精子样品中去除单独加入或联合加入的一种或多种诸如冷藏稀释剂等添加物。在一些具有深低温保藏的实施方案中从精子样品中去除冷藏稀释剂或其他添加物,还可以保持或改良精子,可更进一步保持或改良精子的质量、受精率及其他可能的一个或一个以上的精子特性,所述保持或改良精子的质量例如保持或改良精子的生存力、运动力、功能性。
作为所公开的技术的一部分,在本发明的一些实施方案中精子得以保持或改良,并且精子的质量和其它可能的一个或一个以上的精子特性可进一步得以保持和提高,所述精子的质量例如生存力、运动力、功能性。而且,根据本发明的技术特征和下面将进一步描述的内容,受精率至少可得以保持,甚至有了提高。受精特性也可得以保持或提高,在一些实施方案中,胚泡发育力可得以提高,单精子受精作用至少得以保持或提高,分裂率至少得以保持或提高。在本发明的一些实施方案中,如前所述,精子样品可通过以下方法来保藏,所述方法如深低温保藏(例如冰冻),或前面提及的、以参考的方式引入本文专利申请和专利中所公开的其他保藏技术。在如下描述的一些实施方案中精子质量得以保持。然而,精子质量的保持应解释为包含精子特性的改良。例如,本发明可使精子的运动力、生存力、功能性得以提高,并且在一些实施方式中,可使精子样品中精子的运动力、生存力、功能性得以提高。
因此,可向精子样品中单独加入或联合加入冷藏稀释剂或其他添加物来保藏或刺激精子。在一个优选实施方案中,向精子样品中加入冷藏稀释剂或其它添加物,这种加入有利于通过深低温保藏即冷冻精子样品来保藏精子,从而保持或改良精子,进而保持或提高精子的质量及其他可能的一个或一个以上的精子特性,所述保持或提高的精子质量例如至少保持或提高精子的生存力、运动力、功能性。如前所述,可在样品保藏前或样品保藏过程中向精子样品中加入冷藏稀释剂或其它添加物。然后,可通过例如前面提及的专利申请及专利中所公开的收集、加工、分选、贮藏、运输、使用、受精或授精等技术对所述样品进行处理。
在一个实施方案中,向预先收集的精子样品中单独加入或联合加入冷藏稀释剂或其它添加物,随后将所述样品例如通过冷冻深低温保藏,接着将样品解冻,然后进行处理,该处理包括收集、加工、分选、贮藏、运输、使用、受精或授精等处理技术。一个这种处理步骤可以是分选,在一些实施方案中将含Y染色体精子与含X染色体精子分离,可能会分选出一个或一个以上的高纯度样品。通过这种处理技术有很多好处,其中包括精子质量的保持。例如,如前所述的专利申请和专利中描述的多种分选方法,能将含Y染色体精子与含X染色体精子分离,同时最大限度地降低了对可生存的精子的损害。更进一步,不能生存的精子、污染物、冷藏稀释剂或其它添加物、可通过这些分离技术除去。而且,精子质量的其它方面也可得以保持或改良,特别是精子的生存力、运动力和功能性方面。可在处理过程中进一步对精子样品进行刺激以保持或改良前面描述的精子质量。美国专利号5,135,759中了描述本领域已知这样一种精子处理的方法,其公开了一种流式细胞计数分选技术,将其以参考的方式引入本文。
可通过前述的分选精子的方法对精子进行处理,或在一些实施方案中,根据以下方法进行处理。在一些实施方案中,分选可能包括根据至少一个理想的特征来选择精子,所述理想的特征可能是精子的质量特性,如生存力、运动力、功能性、刺激性和保藏性,或是一个或一个以上的精子的生物学、化学、物理、生理学、功能等属性中的一种或一种以上的特性的组合,例如精子所携带的染色体的特性。可对精子进行染色,优选用Hoechst 33342或类似的染料或染色剂,或用这些染料或染色剂的组合,并根据所述染色情况和选择来区分精子。然后根据精子的差别特征来分离精子并随后进行收集。可以通过引用的参考文献或以参考的方式作为引用的多种方法对精子进行处理,在一些优选实施方案中,所述处理包括精子质量的保持。
实施方案1
在一个关于保藏和处理技术的实施方案中,将200μl的解冻后的精子放置在2ml的PurespermTM、人工制备物和基于Tris(三(羟甲基)氨基甲烷)的稀释液的分离梯度(90%∶45%)上。然后将梯度制备物在1000g离心15分钟,去除PurespermTM离心后的沉淀,缓慢地利用温的基于Tris的稀释液稀释4倍,然后在650g离心3分钟。除去上清液,然后根据以前描述的办法对精子进行染色、温育和分选。将基于Tris稀释液用作染色介质,将
Figure C0382175300151
(minitub,德国)和20%的蛋黄用作收集介质。
上述的例子是本发明技术的各种实施方案之一,其提供例如通过深低温保藏或者冷冻对精子进行保藏;将精子解冻;例如通过染色鉴定精子;将精子分选为例如含X染色体精子和含Y染色体精子。
虽然上面的例子提供了精子保藏技术和精子收集、保藏、解冻和分离等处理技术,本发明还包含或明确公开了其他的技术。例如,收集、加工、分离、储藏、运输、使用、受精或者授精等多种技术的一种或者多种结合可以作为本发明的有创造性的技术的一部分进行实施。在一个实施方案中,可以收集精子,然后将含Y染色体精子与含X染色体精子分离。在某些环境,例如,在收集精子之后可以方便地使用分选器设备的环境中,在分离精子之前可能不需要保藏步骤。然后作为附加的步骤,可以将经分选的精子样品或样品保藏起来,并且如上文所述,可能用于进一步的加工、分离、储藏、运输、利用、受精或者授精中。可以在处理过程中对分选后的精子样品进行刺激以保持和提高如前所述的精子质量。下一个例子描述了处理技术的一个例子。
实施方案2
将分选后的样品在室温(24℃)于700g离心6分钟,去掉上清液,分选后的精子可以被“新鲜”地用于AI(人工授精)或者IVF系统;或者使用以Hepes为基础的冷藏稀释剂将剩余的沉淀稀释4倍,该稀释液可以与对精子进行最初深低温保藏使用的稀释液相同,以及使用各种各样的技术,例如用那些以前描述过的和下面描述的技术进行冷冻。使用以下方法对重新冷冻和分选后的精子进行解冻,然后可用于AI或者IVF系统中,所述解冻方法例如将玻璃试管在37℃的水浴中进行摇晃。
实施方案3
将从冷冻-解冻沉淀中分选出的精子用于羊科哺乳动物的体外受精系统中。用0.5ml的IVF介质将用于羊科IVF系统中的冷冻-分选和冷冻-分选-冷冻精子缓慢稀释,并且在一些实施方案中,使用羊科IVF的标准程序,放入具有严密的盖子的Falcon试管中,在300g离心6分钟。除去上清液,然后将剩余精子以浓度为1,000,000个有运动力精子/ml的浓度快速放在IVF池中。优选高标准的介质制品(即,使用不足24小时的卵、超速离心蛋黄稀释液和精细过滤)和需要对样品进行处理(即常温)。
其他的保藏、加工、受精和授精技术不需要包括分离步骤。例如,可以收集精液,然后进行保藏步骤和后续的加工、储藏、运输、使用、受精或者授精。
此外,作为保藏、加工、受精和授精技术或者是它们的联合的一部分,可以进行一个或一个以上的保藏步骤,在一些实施方案中这些保藏包括例如将精子样品进行冷冻的深低温保藏和后续的解冻步骤,所述保藏可以在进行一个或者一个以上的其他处理技术之前、同时或者之后进行。
实施方案4
如前所述,当分选器离雄性个体距离较远时,在繁殖家畜和野生动物中使用的精子分选方法可能会受到限制,但是如果分选深低温保藏和解冻后的精子,例如冷冻-解冻精子(Lu KH等,Theriogenology 1999:52:1393~1405),然后深低温保藏或者重新冷冻它就便利多了。可以在处理除去冷藏稀释液之后用质量得以保持的冷冻-解冻公羊精子进行高纯度的分选。本研究的目的在于评估分选以及第二次深低温保藏/解冻步骤后的冷冻-解冻精子的功能。整个试验中使用两个公羊的冷冻精液(n=每只公羊的2次射精量)。解冻后的精子处理组包括(i)未分选(对照);(ii)分选(冷冻-分选)和(iii)分选后重新冷冻(冷冻-分选-冷冻)。在利用Hoechst 33342和食用色素温育除去不能存活的精子后,利用高速分选器(SX MoFlo(R),Cytomation,CO,美国)分离X和Y精子。重新分析显示所有的处理组均为富含X和Y的高纯度样品(87.0%±4.5%)。对于IVF,用1×106个能动精子/mL对472个IVM(体外成熟)卵母细胞进行授精。在SOF(合成输卵液)介质中培养3小时后,将卵母细胞转移到Sydney(悉尼)IVF分裂介质(Cook(R)QLD,澳大利亚)中培养4天,然后利用Sydney IVF胚泡介质(Cook(R))再在5%O2∶5%CO2∶90%N2培养3天。在授精后(p.i.)24和48小时测定卵母细胞的分裂。在授精后52小时,利用苔红素对未裂变的卵母细胞进行染色,以确定成熟和受精。可以通过方差分析(ANOVA)、卡方检验和费歇尔精确试验(FisherExact Test)对三次重复的数据进行分析。在授精时,冷冻-分选(85.8%±2.4%)和冷冻-分选-冷冻(66.7%±7.7%)的可运动精子%(±SEM(标准平均误))比对照组(36.7%±2.1%)的要高(P<0.001)。成熟率是95.6%(451/472)。单性生殖对照组(没有精子)的卵母细胞的分裂很低(2/56;3.6%)。多精子受精很低(9/451;2.0%),并且处理组之间没有差异。
表1、卵母细胞的受精和早期胚胎发育,所述卵母细胞分别与冷冻-解冻的未分选(对照组)、冷冻-解冻和分选(冷冻-分选)、冷冻-解冻和分选然后再冷冻-解冻(冷冻-分选-冷冻)的公羊精子温育。括号中的数值是百分数。
d单精受精,各竖栏中,具有不同上标的数值有差异(p<0.05)
所有处理组的受精率和分裂率都一致很高,使用冷冻-分选-冷冻精子受精的卵母细胞胚泡的发育率比使用对照精子受精的卵母细胞胚泡的发育率高。这个结果证明了冷冻-解冻公羊精子在重新深低温保藏后可以立即进行或者将来在IVF系统中进行性别分选。
上面的例子是本发明技术的各种各样的实施方案中的一个,其提供例如通过深低温保藏或者冷冻对精子进行保藏;将精子解冻;将精子分选为例如含X染色体精子和含Y染色体精子;通过深低温保藏或者冷冻对所述分选后的精子进行保藏;进一步包括将精子样品解冻精子以用于例如受精或者授精。
应当考虑到保藏和处理过程的各自生育率,所述过程例如下一个例子所描述的对性别分选和深低温保藏的精子样品进行分离,以及与本发明所公开的深低温保藏、解冻,加工和随后的深低温保藏结合起来。
实施方案5
利用少量(2×106~4×106)的深低温保藏的经分选性别的精子进行人工授精(AI)后生产了羔羊。使用分选后含X或者Y染色体的冷冻-解冻精子进行人工授精后的母羊的妊娠率(分别为25%和15%)比采用商业剂量的未分选过的冷冻-解冻精子进行人工授精后的母羊的妊娠率(54%)低。本研究的目的在于确定与未分选的精子获得相同妊娠率的所需的经分选的、冷冻-解冻精子的最低量。
如前所述,对来自2只公羊的单次射精液的精子样品进行染色、温育、分析并采用改进的高速细胞分选器(
Figure C0382175300191
Cytomation,Fort Collins,CO,美国)进行分选。将未经性别分选的精子在15,000~18,000/serc处理放入离心管中,该离心管预先用鞘液中1%的BSA(牛血清白蛋白)浸泡过,所述鞘液含有0.2ml的Tris-缓冲液和20%的蛋黄(体积比)。对于每一个样品,对1.3×106个精子进行性别分选并进行分析以确定纯度。使用含有13.5%的蛋黄和6%的甘油的两性离子缓冲稀释液对分选和未分选(对照)样品进行稀释,并且冷冻成250μl含有5×106个精子的小丸,利用孕激素棉球剂(sponge)(FGA,Vetrepharm A/Asia,Sydney)插入阴道12天控制144只美利奴(Merino)羊的发情时间,在除去棉球剂(SR)时注射400I.U的PMSG(孕马血清促性腺激素,Pregnecol,VetrepharmA/Asia),在除去棉球剂后的36小时,给134只母羊注射了40μg的GnRH(促性腺素释放激素,
Figure C0382175300192
Intervet)以控制排卵的时间。在除去棉球剂57~60小时后,利用5×106、10×106、20×106、40×106个分选后或未分选的冷冻-解冻精子,对111只母羊在子宫中通过腹腔镜检法进行了人工授精。13只没有给予GnRH的母羊利用商业剂量的未分选的冷冻-解冻精子进行了授精。13只没有给予GnRH的母羊在除去棉球剂后57~58小时利用商业剂量的未分选的冷冻-解冻精子进行了授精。可以通过超声波在第53天诊断妊娠状况。通过卡方分析数据。
解冻后的精子运动力是37.8%±1.78%(经分选)的和42.9%±0.93%(未经分选的)。7/13(53.8%)的没有给予GnRH的母羊怀孕,对于给予GnRH的母羊,妊娠率受授精用精子数量的影响(p<0.05),但是不受公羊或者精子类型的影响(p>0.05)。对于利用分选或者未分选(对照)的冷冻-解冻精子进行授精的母羊,使用授精量为10×106和40×106的妊娠率高于使用授精量为5×106和20×106的妊娠率(表1)。这个结果表明在接近排卵期的授精时,为达到商业上可接受的妊娠率,所需的最小量是40×106个分选后的冷冻-解冻精子。
表1利用冷冻-解冻后的对照组和分选后的公羊精子对母羊进行子宫内授精后的妊娠状况。
Figure C0382175300201
ab在同一个竖列,不同的上标有差异(p<0.05)
a本研究得到XY,Inc;CO,美国;Australian Research Council,Vetrephann A/Asia的支持。
实施方案6
能够对冷冻-解冻精子进行分选和重新冷冻将显著提高精子处理技术例如精子的性别分离技术的潜在应用,例如用于品种管理。根据本发明的某些实施方案,冷冻-解冻、分选、重新冷冻然后解冻的公羊精子在体外表现出更全的功能,其产生胚泡的能力比冷冻-解冻、未分选的精子(Hollingshead FK等,2003 Theriogenology 59 209)的更强。一些实施方案对于评价体外生成胚胎的体外能力非常有用,所述胚胎由经分选和第二次深低温保藏/解冻步骤后,冷冻-解冻的精子进行处理而来。
本发明的一个技术方案的整个过程使用2只公羊的冷冻精液(n=每只公羊的三次射精量)。解冻后的精子处理组包括(i)未分选(对照);(ii)经分选(冷冻-分选)和(iii)分选然后重新冷冻(冷冻-分选-冷冻)。在利用Hoechst 33342和食用色素进行温育以除去没有生存力的精子后,将X和Y精子利用高速分选器进行分离(
Figure C0382175300211
DakoCytomation,Fort Collins,CO,美国)。在一些实施方案中,重新分析表明在所有的处理组为高纯度的富含X染色体和富含Y染色体的样品(89%±1.2%)。在本发明实施的一个技术方案中,在受精后的第6天,将2个胚胎(胚泡阶段或者更大)转移给每一个受体。用卡方和费歇尔精确试验对数据进行分析。在本实施方案中,在体内,胚胎的成活率在整个精液的处理组中都是相同的(28/64,全部为43.8%),并且20/23(87.0%)经性别选择的羔羊为预先确定的性别(表2)。本发明实施方案的这些结果可以证明在体外生产的经性别分离的胚胎在体内的发育能力很高,由此增加了诸如性别分选技术等精子处理技术的潜在应用,所述胚胎由经过分选和第二次深低温保藏/解冻步骤后的冷冻-解冻公羊精子进行处理而来。
表2:将体外生产的胚胎转移到体内的存活率,所述胚胎由冷冻-解冻的未分选(对照)、冷冻-解冻并分选(冷冻-分选)和冷冻-解冻,分选然后冷冻-解冻(冷冻-分选-冷冻)的公羊精子进行处理而来。
  精子处理组   受体数量   第20天的妊娠数目(%)<sup>a</sup>   第60天的妊娠数目(%)<sup>b</sup>   在第20天和第60天之间失去妊娠的数目(%)   生产的羔羊数目/转移的胚胎数目(%)
  对照   8   6(75.0)   5(62.5)   1(16.7)   5/16(31.3)
  冷冻-分选   9   7(77.8)   5(55.6)   2(28.6)   6/18(33.3)
  冷冻-分选-冷冻   17   14(82.4)   14(82.4)   0(0)   17/34(50.0)
a通过血液孕酮化验进行诊断。b.通过超声波检查法进行诊断。
本发明可以进一步包括如前文定义的精子样品,例如根据本发明中任何上述的实施方案中生产的授精管和在一些实施方案中用于IVF中的授精管,所述实施方案例如任何处理、哺乳动物的生产、哺乳动物胚胎的生产、刺激、保藏、受精和授精技术,并且精子质量得以保持或者增强,例如授精管具有理想的生存力、运动力和功能性,或者其他性质,或者诸如本发明所公开的性质等性质的组合,在一些实施方案中,特别是对于马科动物而言可能会导致理想水平的受精率。精子样品例如至少一个或者多个授精管可能特别适合单个胚胎的生产。
本发明可以进一步包括根据本发明中任意一种上述实施方案所生产哺乳动物;或者可以包括根据本发明的各种各样提供精子授精样品的实施方案获得的预先确定性别的哺乳动物,所述精子授精样品具有富含X染色体的精子群体或者富含Y染色体的精子群体;或者根据本发明中任意一个实施方案生产的哺乳动物,其中精子授精样品中含有精子比用于使该特定种类哺乳动物授精的典型量少;或者根据上述本发明公开的内容生产的麋鹿后代。
本发明进一步包括本发明所公开的、和前面提及的专利申请和参考文献所公开的组合特征中可能包含的各种各样的处理、保藏、刺激、受精、授精、和哺乳动物或者哺乳动物胚胎的生产技术。相应的,由保藏、刺激、受精、授精、和哺乳动物或者哺乳动物的胚胎生产系统的实施方案形式提供的精液和精子的各种各样处理特征,表明了与前述专利申请和参考文献相一致的精子质量,例如,诸如生存力、运动力、功能性或者受精率等一个或者一个以上的精子的特征。此外,可以在以下条件下关注精子特性,所述条件为各种各样的收集、处理、分离、储藏、运输、使用、受精或者授精技术,并且在那些或者其他的各种各样的实施方案中在分析、试验、精子的生物学,化学、物理、生理学或者功能等属性的条件下关注精子特性。因此,本发明的系统可以提供精子处理、刺激、保藏、受精和授精,例如引入流式分选技术、高纯度分离技术、低剂量受精和授精技术,异精(heterospermic)受精过程,例如根据仅有几个例子所示,评价在分选器中的不同压力环境下处理的相对生存力、运动力、功能性或者生育力。
无论是设备、方法、还是其他,以参考的方式引入本文的公开内容,例如所提供的各种各样将Y染色体精子与X染色体精子分离的各种例子,以及公开的其他有关收集、处理、分离、储藏、运输、使用、受精和授精技术,并不是意味着将本发明限定到任何特定的实施方案。以参考方式引入的描述和各种各样的例子不能解释为将本发明仅限定至精子分选技术或者仅为精子保藏技术。然而,应理解,公开内容将各种各样的技术并入本发明的各种实施方案中。此外,本发明应当理解为包含了与公开的特征相一致的精子的处理、保藏、刺激、受精和授精这些技术。
从上文中,可以很容易理解本发明的基本观点,这些观点可以采用多种方式体现。它包括精子的保藏、精子的处理、精子样品、包括实现精子处理的技术和设备在内的哺乳动物或者哺乳动物胚胎生产系统。在本申请中,各种精子处理技术以所描述的各种各样的设备所取得的部分成果和以固有的应用步骤的形式得以公开。它们仅仅是使用预期和描述的设备的自然结果。此外,当一些设备公开后,应当理解这些设备不但可以成功实施一些确定的方法,而且可以以多种形式加以变化。重要的是,关于上文的所有内容,所有的方面都应理解为包括在本公开内容的范围内。
而且,本发明及权利要求的每一不同元件还可以多种方式获得。本公开内容应理解为包括每一个这样的变化,不论它是任意装置实施方案、方法或过程实施方案的变更,还是甚至仅仅是其中任意元件的变更。特别是,应该理解,由于本公开内容涉及本发明的元件,每一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。当需要使本发明所包括的隐含的广泛范围明确时,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,“深低温保藏元素”或者“深低温保藏设备”或者类似的公开应当理解为包括了“深低温保藏”的动作的公开——无论是否明确的讨论过——并且,相反的,如果有效地公开了“深低温保藏”的动作,这样的公开应当理解为包括“深低温保藏器”和甚至“深低温保藏方式”的公开。这样的变化和可替换的术语可以被理解为清楚的包括在说明书中。
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。
这样,应当理解申请人至少要求以下权利:i)这里公开和描述的每一个精子处理方法、哺乳动物生产方法、精子样品、哺乳动物和哺乳动物胚胎;ii)所公开和描述的相关的系统、装置和多个设备;iii)这些各系统和方法的类似、等同甚至是隐含的变化;iv)完成公开和描述的所示各功能的替代设计;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示的每一性质、元件及步骤;vii)由所公开的不同系统或组分改进的申请;viii)由此类系统或元件所生成的终产品;ix)基本在上文和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)作为从属于所述独立权利要求或概念的各潜在从属权利要求或概念。
申请人可提交具有最初一系列从属权利要求的权利要求。应当理解的是,应当达到有关新物质法规(new matter laws)所要求的支持程度,以便能够在一项独立权利要求或概念之下增加任何不同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)条款和美国专利法35 USC 132或其他此类法律。
此外,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一组要素或步骤,而不排除任何其他的要素或步骤或一组要素或步骤。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 (28)

1.一种精子处理方法,该方法包括以下步骤:
获取精子;
将所述的精子深低温保藏;
将所述的精子解冻;
对所述的精子进行处理;以及
将所述的精子深低温保藏。
2.如权利要求1所述的精子处理方法,其中所述的处理步骤包括对所述的精子进行分选。
3.如权利要求2所述的精子处理方法,其中所述的分选步骤包括根据至少一个所需的精子特征选择所述的精子。
4.如权利要求3所述的精子处理方法,其中所述的选择步骤包括根据至少一个所需的精子特征对所述的精子进行染色。
5.如权利要求4所述的精子处理方法,其中所述的选择步骤还包括根据至少一个所需的精子特征区分所述的精子。
6.如权利要求5所述的精子处理方法,其中所述的区分步骤包括区分所述的精子是含X染色体的精子还是含Y染色体的精子。
7.如权利要求5所述的精子处理方法,其中所述的选择步骤还包括分离具有所述至少一个所需的精子特征的所述精子。
8.如权利要求5或7所述的精子处理方法,该方法还包括收集所述的精子的步骤。
9.如权利要求8所述的精子处理方法,其中所述的收集所述的精子的步骤包括保持精子质量的步骤。
10.如权利要求2所述的精子处理方法,其中所述的分选步骤包括流式细胞仪分选。
11.如权利要求1所述的精子处理方法,该方法还包括从所述的经处理精子确定精子样品的步骤。
12.如权利要求11所述的精子处理方法,其中所述的第二个深低温保藏步骤包括将所述的精子样品深低温保藏。
13.如权利要求11所述的精子处理方法,其中所述的确定精子样品的步骤包括确定多个精子样品,并且其中所述的第二个深低温保藏步骤包括将所述的多个样品深低温保藏。
14.如权利要求1所述的精子处理方法,该方法还包括从所述的处理后深低温保藏的精子确定精子样品的步骤。
15.如权利要求1所述的精子处理方法,该方法还包括将所述的处理后深低温保藏的精子解冻的步骤。
16.如权利要求15所述的精子处理方法,该方法还包括从所述的经处理、深低温保藏、解冻的精子确定精子样品的步骤。
17.如权利要求15所述的精子处理方法,其中所述的确定精子样品的步骤包括确定多个的精子样品。
18.如权利要求16所述的精子处理方法,其中所述的确定精子样品的步骤包括确定多个的精子样品。
19.如权利要求1所述的精子处理方法,其中所述的获取步骤包括获取具有大量精子的精液,其中所述的深低温保藏步骤包括将所述的精液深低温保藏,并且所述的解冻步骤包括将所述的深低温保藏后的精液解冻。
20.如权利要求1所述的精子处理方法,其中所述的获取步骤包括从动物学样品获取精子。
21.如权利要求1所述的精子处理方法,该方法还包括保持所述的处理后深低温保藏的精子的精子质量的步骤。
22.如权利要求21所述的精子处理方法,其中所述的保持精子质量的步骤包括提高所述的处理后深低温保藏的精子的运动力。
23.如权利要求21所述的精子处理方法,其中所述的保持精子质量的步骤包括提高所述的处理后深低温保藏的精子的生存力。
24.如权利要求21所述的精子处理方法,其中所述的保持精子质量的步骤包括改进所述的处理后深低温保藏的精子的功能性。
25.如权利要求1所述的精子处理方法,该方法还包括利用所述的处理后深低温保藏的精子来保持受精率的步骤。
26.如权利要求1所述的精子处理方法,该方法还包括利用分选后深低温保藏的精子来增强胚泡的发育率的步骤。
27.如权利要求1所述的精子处理方法,该方法还包括利用分选后深低温保藏的精子来保持单精受精的步骤。
28.如权利要求1所述的精子处理方法,该方法还包括利用分选后深低温保藏的精子来保持分裂率的步骤。
CN03821753A 2002-09-13 2003-09-15 精子的保藏和处理系统 Expired - Lifetime CN100581355C (zh)

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